CN117255679A

CN117255679A - Regulatory T cell (TREG) extracellular vesicle compositions and methods

Info

Publication number: CN117255679A
Application number: CN202280030298.3A
Authority: CN
Inventors: 斯坦利·赫什·阿佩尔; 亚伦·德鲁·托姆; 詹森·罗伯特·索恩霍夫
Original assignee: Methodist Hospital
Current assignee: Methodist Hospital
Priority date: 2021-02-26
Filing date: 2022-02-25
Publication date: 2023-12-19

Abstract

The present disclosure provides anti-inflammatory Extracellular Vesicles (EVs) derived from ex vivo expanded human suppressive immune cells, e.g., regulatory T cells (tregs). These EVs are useful in the treatment of diseases such as Amyotrophic Lateral Sclerosis (ALS), alzheimer's disease, and other neurological diseases, as well as inflammatory and autoimmune diseases or dysfunctions.

Description

Regulatory T cell (TREG) extracellular vesicle compositions and methods

RELATED APPLICATIONS

The present application claims the benefit of U.S. provisional patent application Ser. No.63/208,395, filed on 6 months 08 of 2021, and U.S. provisional patent application Ser. No.63/154,449, filed on 26 months 2 of 2021, each of which is incorporated herein by reference in its entirety.

1. Technical field

The present disclosure provides anti-inflammatory and restorative Extracellular Vesicles (EVs) derived from ex vivo expanded human suppressor immune cells, e.g., regulatory T cells (tregs) and useful in the treatment of diseases such as Amyotrophic Lateral Sclerosis (ALS), alzheimer's disease, and other neurological diseases, as well as inflammatory, metabolic, and autoimmune diseases or dysfunctions.

2. Background art

Inflammatory and neuroinflammatory mechanisms contribute to a variety of devastating diseases including neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS), parkinson's disease and multiple sclerosis. Neurodegenerative diseases like this lead to a great health and economic burden, which is only further exacerbated over time.

Currently, there is no disease modifying treatment available for these diseases. Anti-inflammatory treatments have been used for decades in an attempt to ameliorate a variety of neurodegenerative diseases. However, little progress has been made by single drug/target approaches.

More and more studies have shown that the immune system is involved in the etiology of diseases like this and that immune cells are dysfunctional as mediators of the pathogenesis of the main disease. Complex signaling mechanisms and intrinsic redundancy of the immune system and its components can help explain the ineffectiveness of this single drug/single target anti-inflammatory approach.

Recently, tremendous promise has been demonstrated by regulatory T cell (Treg) cell therapies that may represent a more global approach to inhibiting immune system dysfunction that contributes to the disease. For example, clinical trials involving the administration of expanded autologous tregs to ALS patients reported that Treg therapy slowed the rate of progression during early and late stages of disease, and that the suppressive function of tregs was associated with a slowing of disease progression (Thonhoff, j.r. et al, 2018, neurology-Neuroimmunology Neuroinflammation (4)).

Nevertheless, there remains a need for the development of other therapies that can inhibit inflammation and/or promote anti-inflammatory immune system components and that can exert such effects in the pro-inflammatory, toxic microenvironment of the disease condition.

3. Summary of the invention

Provided herein are Extracellular Vesicles (EVs) that exhibit impressive anti-inflammatory activity in vitro and in vivo. The EVs provided herein are derived from ex vivo expanded human suppressor immune cells, e.g., regulatory T cells (tregs). As demonstrated herein, EVs described in the present disclosure retain immunosuppressive activity of the cells from which they are derived. Furthermore, since EVs are not themselves cells, they avoid potential cell-based problems such as immune rejection and the possibility of polarization towards pro-inflammatory cell types. As such, the anti-inflammatory EVs provided herein are particularly useful for the treatment of a variety of diseases, such as, for example, neurodegenerative disorders, such as Amyotrophic Lateral Sclerosis (ALS).

The results provided herein demonstrate that EVs described in the present disclosure are capable of effectively inhibiting T-responsive cell proliferation and pro-inflammatory spinal cord activity, e.g., macrophage activity, in vitro and also exerting an effective in vivo anti-inflammatory effect via intravenous or intranasal administration. For example, the in vivo results provided herein using the anti-inflammatory Treg EV compositions disclosed herein demonstrate anti-inflammatory effects in a model of inflammation and a model of motor neuron degenerative disease that mimics ALS. For example, the results provided herein demonstrate that EVs are capable of inhibiting brain and peripheral inflammation in a model of neurogenic inflammation in vivo, and are also capable of inhibiting inflammation and prolonging survival in a model of Amyotrophic Lateral Sclerosis (ALS) in vivo. The results presented herein also demonstrate that Treg EVs have greater inhibition of pro-inflammatory immune cells than EVs derived from Mesenchymal Stem Cells (MSCs).

In addition, the anti-inflammatory EVs provided herein exhibit significant dimensional consistency, stability and activity from batch to batch and exhibit unique structural features, for example, structural features characterized by Treg EV surface markers and RNA profiles. Still further, as demonstrated herein, the methods provided herein result in effective anti-inflammatory EVs that exhibit similar structural and inhibitory activity characteristics, whether the original Treg starting material is obtained from a healthy subject or an ALS patient.

In one aspect, provided herein is an isolated, cell-free, population of anti-inflammatory Extracellular Vesicles (EVs), wherein the anti-inflammatory EVs are derived from ex vivo expanded human suppressor immune cells, wherein: i) The population exhibits a size diameter distribution of about 50nm to about 150 nm; ii) the population comprises EV surfaces CD2, CD25 and HLA-DRDPDQ; iii) The population comprises hsa-miR-1290, hsa-miR-146a-5p and hsa-miR-155-5p microRNAs (miRNAs); and iv) the population exhibits the ability to inhibit bone marrow cells, e.g., macrophages, as measured by the ability to reduce pro-inflammatory cytokine production by bone marrow cells (e.g., exhibit the ability to reduce expression of IL-6, IL-8, IL1 beta, or interferon-gamma in bone marrow cells) and the ability to increase expression of one or more anti-inflammatory markers in bone marrow cells (e.g., the ability to increase expression of IL-10, arg1, and/or CD206 in bone marrow cells), or as measured by the ability to inhibit proliferation of responsive T cells; and wherein the human suppressive immune cells are regulatory T cells (tregs). In certain embodiments, the human tregs are from a healthy human subject. In certain embodiments, the human tregs are from a human subject diagnosed with or suspected of having Amyotrophic Lateral Sclerosis (ALS).

In certain embodiments, the anti-inflammatory EV population further comprises EV surfaces CD44, CD29, CD4, and CD45. In certain embodiments, the anti-inflammatory EV population further comprises EV surfaces CD44, CD29, CD4, and CD45. In certain embodiments, the anti-inflammatory EV population further comprises EV surfaces CD9, CD63, and CD81. In certain embodiments, the anti-inflammatory EV population is substantially devoid of EV surfaces CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD c, CD40, MSCP, CD146, CD86, CD326, CD133, CD142, CD31, and CD14. In certain embodiments, the anti-inflammatory EV population further comprises EV surfaces CD44, CD29, CD4, and CD45. In certain embodiments, the anti-inflammatory EV population further comprises EV surfaces CD44, CD29, CD4 and CD45, CD9, CD63 and CD81, and ii) lacks significantly EV surfaces CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP, CD146, CD86, CD326, CD133, CD142, CD31 and CD14.

In certain embodiments, the ratio of hsa-miR-146a-5p to hsa-miR-155-5p present in the anti-inflammatory EV population is about 2 to about 3. In certain embodiments, the abundance of hsa-miR-1290 in the anti-inflammatory EV population is at least 2-fold greater than hsa-miR-155-5 p. In particular embodiments, the ratio of hsa-miR-146a-5p to hsa-miR-155-5p present in the anti-inflammatory EV population is about 2 to about 3 and the abundance of hsa-miR-1290 in the anti-inflammatory EV population is at least 2-fold greater than hsa-miR-155-5 p.

In a specific embodiment, at least about 90% of the EVs of the anti-inflammatory population exhibit a dimensional diameter of about 50nm to about 150 nm. In certain embodiments, the anti-inflammatory EV population exhibits an average size diameter of about 80nm to about 110 nm. In certain embodiments, the anti-inflammatory EV population exhibits a median size diameter of about 70nm to about 110 nm. In certain embodiments, the anti-inflammatory EV population exhibits a mode size diameter of about 65nm to about 95 nm. In particular embodiments, at least about 90% of the EVs in the anti-inflammatory EV population exhibit a size diameter of about 50 to about 150nm, and the population exhibits an average size diameter of about 80nm to about 110nm, a median size diameter of about 70nm to about 110nm, and a mode size diameter of about 65nm to about 95 nm.

Provided herein are isolated, cell-free, anti-inflammatory EV populations, wherein the anti-inflammatory EVs are derived from ex vivo expanded human suppressor immune cells, e.g., regulatory T cells (tregs). Also provided herein are pharmaceutical compositions and cryopreserved compositions comprising the isolated, cell-free, anti-inflammatory EV populations described herein, methods of producing the EV populations and methods of using the EVs to treat diseases, such as neurodegenerative diseases, e.g., ALS.

In one aspect, provided herein is an isolated, cell-free, population of anti-inflammatory Extracellular Vesicles (EVs), wherein the anti-inflammatory EVs are derived from ex vivo expanded human inhibitory immune cells. In some embodiments, the human suppressive immune cells are regulatory T cells (tregs). In some embodiments, the tregs are from a healthy human subject.

In some embodiments, the tregs are from a human subject diagnosed with or suspected of having a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is alzheimer's disease. In some embodiments, the neurodegenerative disorder is Amyotrophic Lateral Sclerosis (ALS). In some embodiments, the neurodegenerative disease is Multiple Sclerosis (MS). In some embodiments, the neurodegenerative disease is parkinson's disease.

In some embodiments, the tregs are from a human subject diagnosed with or suspected of having a stroke.

In some embodiments, the tregs are from an elderly subject.

In some embodiments, the tregs are from multiple human subjects. In some embodiments, the tregs are from a plurality of unrelated human subjects.

In some embodiments, the anti-inflammatory EV exhibits the ability to increase expression of one or more anti-inflammatory markers in inflammatory cells. In some embodiments, the inflammatory cell is a bone marrow cell. In some embodiments, the anti-inflammatory EV exhibits the ability to increase expression of IL-10, arg1, and/or CD206 in inflammatory cells.

In some embodiments, the anti-inflammatory EV exhibits the ability to inhibit inflammatory cells as measured by pro-inflammatory cytokine production by the inflammatory cells. In some embodiments, the inflammatory cell is a bone marrow cell. In some embodiments, the bone marrow cells are monocytes, macrophages or microglia. In some embodiments, the macrophage is an M1 macrophage. In some embodiments, the M1 macrophage is an Induced Pluripotent Stem Cell (iPSC) -derived M1 macrophage.

In some embodiments, the ability to inhibit an inflammatory cell is measured by IL-6, IL-8, TNF alpha, IL1 beta, and/or interferon gamma production by the inflammatory cell.

In some embodiments, the anti-inflammatory EV exhibits the ability to increase expression of IL-1, arg1 and/or CD206 and the ability to inhibit production of IL-6, IL-8, TNF alpha, IL1 beta and/or interferon gamma in inflammatory cells, e.g., bone marrow cells, e.g., macrophages.

In some embodiments, the anti-inflammatory EV exhibits an inhibitory function, as determined by inhibition of responsive T cell proliferation. In some embodiments, proliferation of the responsive T cells is determined by flow cytometry or thymidine incorporation.

In some embodiments, the population is an anti-inflammatory EV population containing saline. In some embodiments, the population is an anti-inflammatory EV population containing physiological saline. In some embodiments, the population is an anti-inflammatory EV population containing phosphate buffered saline.

In some embodiments, the anti-inflammatory EV population comprises exosomes and microbubbles. In some embodiments, a majority of EVs are exosomes. In some embodiments, at least about 80%, about 90%, or about 95% of the EVs are exosomes. In some embodiments, a majority of EVs are microbubbles. In some embodiments, at least about 80%, about 90%, or about 95% of the EVs are microbubbles.

In some embodiments, the anti-inflammatory EV population comprises at least about 50% exosomes. In some embodiments, at least about 60% of EVs are exosomes. In some embodiments, at least about 70% of EVs are exosomes.

In some embodiments, the anti-inflammatory EV population comprises at least about 50% microbubbles. In some embodiments, at least about 60% of EVs are microbubbles. In some embodiments, at least about 70% of the EVs are microbubbles.

In some embodiments, a majority of EVs in the anti-inflammatory EV population provided herein have diameters of about 30nm to about 1000 nm. In some embodiments, a majority of the EVs have diameters of about 30nm to about 100nm, about 30nm to about 150nm, about 30 to about 200nm, about 40 to about 100nm, about 80 to about 110nm, about 80 to about 125nm, or about 100 to about 120 nm. In some embodiments, a majority of the EVs have diameters of about 60nm to about 1000nm, about 70nm to about 1000nm, about 80nm to about 1000nm, 100 to about 1000nm, about 200 to about 1000nm, or about 300 to about 1000 nm. In some embodiments, a majority of the EVs have diameters of about 20nm to about 300nm, about 20nm to about 275nm, about 20 to about 250nm, about 20 to about 200nm, or about 20nm to about 175 nm.

In another aspect, provided herein are pharmaceutical compositions comprising the isolated, cell-free anti-inflammatory EV populations provided herein. In certain embodiments, the pharmaceutical composition comprises an isolated, cell-free, anti-inflammatory EV population provided herein in saline. In some embodiments, the anti-inflammatory EV population comprises about 1 x 10 ⁶ Up to about 1X 10 ¹⁴ EV, about 1×10 ⁸ Up to about 1X 10 ¹⁴ EV, about 1×10 ⁸ Up to about 1X 10 ¹² EV, about 1×10 ⁸ Up to about 1X 10 ¹⁰ EV, about 1×10 ¹⁰ Up to about 1X 10 ¹⁴ EV or about 1×10 ¹⁰ Up to about 1X 10 ¹² And (5) EV. In some embodiments, the anti-inflammatory EV population comprises about 1 x 10 ⁹ EV, about 5×10 ⁹ EV, about 1×10 ¹⁰ EV, about 5×10 ¹⁰ EV, about 1×10 ¹¹ EV, about 5×10 ¹¹ EV or about 1×10 ¹² And (5) EV. In some embodiments, the anti-inflammatory EV population comprises about 1 x 10 ⁶ Up to about 1X 10 ¹⁴ EV/ml, about 1X 10 ⁸ Up to about 1X 10 ¹⁴ EV/ml, about 1X 10 ⁸ Up to about 1X 10 ¹² EV/ml, about 1X 10 ⁸ Up to about 1X 10 ¹⁰ EV/ml, about 1X 10 ¹⁰ Up to about 1X 10 ¹⁴ EV/ml or about 1X 10 ¹⁰ Up to about 1X 10 ¹² EV/ml. In some embodiments, the anti-inflammatory EV population comprises about 5 x 10 ⁸ EV/ml, about 1X 10 ⁹ EV/ml, about 2.5X10 ⁹ EV/ml, about 5X 10 ⁹ EV/ml, about 1X 10 ¹⁰ EV/ml, about 2.5X10 ¹⁰ EV/ml, about 5X 10 ¹⁰ EV/ml, about 1X 10 ¹¹ EV/ml, about 2.5X10 ¹¹ EV/ml, about 5X 10 ¹¹ EV/ml or about 1X 10 ¹² EV/ml.

In some embodiments, the anti-inflammatory EV population in the pharmaceutical compositions provided herein comprises about 1 μg to about 200mg EV. In some embodiments, the anti-inflammatory EV population comprises about 1 μg to about 15mg EV. In some embodiments, the anti-inflammatory EV population comprises about 1 μg to about 15mg EV/ml.

In some embodiments, the pharmaceutical composition is a cryopreserved pharmaceutical composition. In some embodiments, the pharmaceutical composition is previously cryopreserved.

In another aspect, provided herein are cryopreserved compositions comprising the isolated, cell-free anti-inflammatory EV populations provided herein.

In another aspect, provided herein is a method of producing an isolated, cell-free, anti-inflammatory Extracellular Vesicle (EV) population, the method comprising the steps of: (a) Expanding a population of human suppressive immune cells ex vivo in a medium to produce a culture comprising cells, medium and anti-inflammatory EV; and (b) isolating the anti-inflammatory EV from the culture. In some embodiments, the population of human suppressive immune cells is a population of regulatory T cells (tregs).

In some embodiments, step (b) comprises removing cells from the culture, followed by precipitation of the culture with polyethylene glycol. In some embodiments, step (b) comprises: (i) Removing cells from the culture to produce a solution containing cell-free, anti-inflammatory EV; and (ii) isolating the anti-inflammatory EV from the cell-free, anti-inflammatory EV-containing solution in (i).

In some embodiments, step (i) comprises flowing the culture through a filter, thereby retaining cells through the filter and thereby removing cells from the culture. In some embodiments, step (i) comprises microfiltration.

In some embodiments, step (ii) comprises step (ii-a): the solution containing the cell-free, anti-inflammatory EV is flowed through a filter, thereby retaining the anti-inflammatory EV through the filter. In some embodiments, the filter has a molecular weight cut-off (MWCO) of about 200 kilodaltons (kDa) to about 600 kDa. In some embodiments, the filter has an MWCO of about 500 kDa.

In some embodiments, step (ii) comprises ultrafiltration. In some embodiments, step (ii) further comprises step (ii-b): buffer exchange is performed such that the resulting isolated, cell-free anti-inflammatory EV population is an isolated, cell-free anti-inflammatory EV population containing buffer. In some embodiments, the buffer is a saline-containing buffer. In some embodiments, the saline-containing buffer is physiological saline. In some embodiments, the saline-containing buffer is PBS.

In some embodiments, step (ii-b) comprises diafiltration.

In some embodiments, steps (ii-a) and (ii-b) are performed simultaneously.

In some embodiments, step (b) comprises tangential flow filtration.

In some embodiments, the medium in step (a) is serum-free. In some embodiments, the medium in step (a) comprises serum. In some embodiments, the serum is human AB serum. In some embodiments, serum is consumed for serum-derived EVs.

In some embodiments, the method of producing an isolated, cell-free, anti-inflammatory EV population further comprises, prior to step (a), the step of enriching tregs from a cell sample suspected of containing tregs to produce a baseline population of Treg cells that is the population of tregs that is then expanded in step (a). In some embodiments, the cell sample is a leukocyte-depleted cell sample. In some embodiments, the method further comprises obtaining the cell sample from the donor by a leukapheresis method. In some embodiments, the cell sample is not stored overnight or frozen prior to performing the enrichment step. In some embodiments, the cell sample is obtained within 30 minutes before the enrichment step begins. In some embodiments, the enriching step comprises depleting cd8+/cd19+ cells, followed by enriching for cd25+ cells. In some embodiments, step (a) is performed within 30 minutes of the enriching step.

In some embodiments, step (a) of the method of producing an isolated, cell-free anti-inflammatory EV population comprises culturing the Treg in a medium comprising anti-CD 3 antibody and anti-CD 28 antibody coated beads. In some embodiments, the beads are first added to the culture medium within about 24 hours of the start of the culture. In some embodiments, the anti-CD 3 antibody and anti-CD 28 antibody coated beads are added to the culture medium about 14 days after the anti-CD 3 antibody and anti-CD 28 antibody coated beads are first added to the culture medium.

In some embodiments, step (a) further comprises adding IL-2 to the medium within about 6 days of the start of the culture. In some embodiments, step (a) further comprises supplementing the medium with IL-2 about every 2-3 days after first adding IL-2 to the medium.

In some embodiments, step (a) further comprises adding rapamycin to the medium within about 24 hours of the start of the culturing. In some embodiments, step a) further comprises supplementing the culture medium with rapamycin every 2-3 days after first adding rapamycin to the culture medium.

In some embodiments, step (a) is automated. In some embodiments, step a) is performed in a bioreactor.

In some embodiments, step (b) of the method of producing an isolated, cell-free anti-inflammatory EV population may begin at any point during step a).

In some embodiments, the tregs enriched in step (a) are from a healthy human subject. In some embodiments, the tregs are from a human subject diagnosed with or suspected of having a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is alzheimer's disease, amyotrophic Lateral Sclerosis (ALS), multiple Sclerosis (MS), or parkinson's disease. In some embodiments, the tregs are from a human subject diagnosed with or suspected of having a stroke. In some embodiments, the tregs are from an elderly subject. In some embodiments, the tregs are from multiple human subjects.

In some embodiments, the population of human suppressive immune cells expanded in step (a) is a population of genetically engineered human suppressive immune cells.

In some embodiments, the population of tregs expanded in step (a) is a population of genetically engineered tregs.

In another aspect, provided herein are pharmaceutical compositions comprising an isolated, cell-free, anti-inflammatory EV population, wherein the population is prepared by any one of the methods described herein.

In some embodiments, the method of producing an isolated, cell-free anti-inflammatory EV population further comprises (c) cryopreserving the isolated, cell-free anti-inflammatory EV population, thereby producing a cryopreserved isolated, cell-free anti-inflammatory EV population. Also provided herein are cryopreserved compositions comprising isolated, cell-free anti-inflammatory EV populations, wherein the cryopreserved compositions are prepared using these methods.

In some embodiments, the method further comprises thawing the cryopreserved, isolated cell-free anti-inflammatory EV population after about 1 week, 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, or about 24 months of cryopreservation. Also provided herein are compositions, e.g., pharmaceutical compositions, comprising isolated, cell-free, anti-inflammatory EV populations, wherein the compositions, e.g., pharmaceutical compositions, are prepared using these methods.

In another aspect, provided herein is an isolated, cell-free population of anti-inflammatory EVs, wherein the anti-inflammatory EVs are derived from an ex vivo expanded population of Treg cells that exhibit the ability to inhibit inflammatory cells, as measured by pro-inflammatory cytokine production by inflammatory cells, wherein the inflammatory cells are macrophages or monocytes from a human donor or produced from induced pluripotent stem cells, wherein the ex vivo expanded population of Treg cells has been expanded from a baseline Treg, and wherein the ex vivo expanded population of Treg cells is expanded in the ex vivo expanded population of Treg cells: (a) Reduced expression of baseline signature gene products for one or more of the dysfunctions listed in table 3 and/or table 4 relative to expression of one or more gene products in baseline tregs; (b) Reduced expression of one or more dysfunctional baseline signature gene products listed in table 5 relative to expression of one or more gene products in baseline tregs; (c) Elevated expression of one or more Treg-related signature gene products listed in table 6 relative to expression of one or more gene products in baseline tregs; (d) Elevated expression of one or more mitochondrial tag gene products listed in table 7 relative to expression of one or more gene products in baseline tregs; (e) Elevated expression of one or more cell proliferation tag gene products listed in table 8 relative to expression of one or more gene products in baseline tregs; or (f) increased expression of one or more highest protein expression signature gene products listed in table 9 relative to expression of one or more gene products in a baseline Treg. In some embodiments, provided herein are pharmaceutical compositions comprising the isolated, cell-free anti-inflammatory EV population.

In another aspect, provided herein are methods of treating a condition associated with Treg dysfunction, comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein.

In another aspect, provided herein are methods of treating a condition associated with Treg deficiency comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein.

In another aspect, provided herein are methods of treating a disorder associated with excessive activation of the immune system, the method comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein.

In another aspect, provided herein is a method of treating an inflammatory condition driven by a T cell response, the method comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein.

In another aspect, provided herein is a method of treating an inflammatory condition driven by a bone marrow cell response, the method comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein. In some embodiments, the bone marrow cells are monocytes, macrophages or microglia.

In another aspect, provided herein is a method of treating a neurodegenerative disorder in a subject in need thereof, the method comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein. In some embodiments, the neurodegenerative disease is ALS, alzheimer's disease, parkinson's disease, frontotemporal dementia, or huntington's disease.

In some embodiments, the neurodegenerative disease is ALS, alzheimer's disease, parkinson's disease, frontotemporal dementia, multiple sclerosis, or huntington's disease.

In another aspect, provided herein is a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein. In some embodiments, the autoimmune disease is polymyositis, ulcerative colitis, inflammatory bowel disease, crohn's disease, celiac disease, systemic sclerosis (scleroderma), multiple Sclerosis (MS), rheumatoid Arthritis (RA), type I diabetes, psoriasis, dermatomyositis, lupus, e.g., systemic lupus erythematosus or cutaneous lupus, myasthenia gravis, autoimmune kidney disease, autoimmune hemolytic anemia, autoimmune cytopenia, autoimmune encephalitis, autoimmune hepatitis, autoimmune uveitis, alopecia, thyroiditis, or pemphigus.

In another aspect, provided herein is a method of treating graft versus host disease in a subject in need thereof, the method comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein. In some embodiments, the subject has received a bone marrow transplant, a kidney transplant, or a liver transplant.

In another aspect, provided herein is a method of improving islet transplantation survival in a subject in need thereof, the method comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein.

In another aspect, provided herein is a method of treating cardiac inflammation in a subject in need thereof, the method comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein. In some embodiments, the cardiac inflammation is associated with atherosclerosis, myocardial infarction, ischemic cardiomyopathy, or heart failure.

In another aspect, provided herein is a method of treating a neurological inflammation in a subject in need thereof, the method comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein. In some embodiments, the neurogenic inflammation is associated with stroke, acute disseminated encephalomyelitis, acute optic neuritis, acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, gillin-barre syndrome, transverse myelitis, optic neuromyelitis, epilepsy, traumatic brain injury, spinal cord injury, encephalitis, central nervous system vasculitis, nervous system sarcoidosis, autoimmune or post-infection encephalitis, or chronic meningitis.

In another aspect, provided herein is a method of treating Treg lesions (tregolopathy) in a subject in need thereof, the method comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein. In some embodiments, the Treg lesions are caused by FOXP3, CD25, cytotoxic T lymphocyte-associated antigen 4 (CTLA 4), LPS-reactive beige anchor-like protein (LRBA) or BTB domain and CNC homologous gene 2 (BACH 2) loss of function mutations or signal transduction and activator of transcription 3 (STAT 3) function gain mutations.

In some embodiments, the anti-inflammatory EV administered to the subject is derived from tregs autologous to the subject. In some embodiments, the anti-inflammatory EV is derived from tregs that are allogeneic to the subject.

In some embodiments, the pharmaceutical composition is administered via intranasal administration. In some embodiments, the intranasal administration is via a spray method or a nasal drip method. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered by local injection.

In some embodiments, the methods of treatment provided herein further comprise administering to the subject a pharmaceutical composition comprising a population of therapeutic tregs, wherein the tregs have been expanded ex vivo and cryopreserved, and wherein the tregs have not been further expanded prior to administration. In some embodiments, the population of therapeutic tregs is autologous to the subject. In some embodiments, the population of therapeutic tregs is allogeneic to the subject. In some embodiments, the pharmaceutical composition comprising the therapeutic Treg population is administered intravenously. In some embodiments, the pharmaceutical composition comprising the anti-inflammatory EV and the pharmaceutical composition comprising the population of therapeutic tregs are administered to the patient on the same day.

In certain embodiments, the methods of treatment provided herein comprise administering to a subject in need of treatment a pharmaceutical composition comprising an isolated, cell-free population of anti-inflammatory EVs, wherein the EVs have been cryopreserved and thawed prior to administration to the subject. In certain embodiments, the methods of treatment provided herein comprise administering to a subject in need of treatment a pharmaceutical composition comprising an isolated, cell-free population of anti-inflammatory EVs, wherein the EVs are stored at 4 ℃, e.g., overnight at 4 ℃, prior to administration to the subject. In particular embodiments, the methods of treatment provided herein comprise administering to a subject in need of treatment a pharmaceutical composition comprising an isolated, cell-free, anti-inflammatory EV population, wherein the EV has been cryopreserved, thawed, and stored at 4 ℃, e.g., overnight at 4 ℃, prior to administration to the subject.

In certain embodiments, the methods of treatment provided herein comprise administering to a subject in need of treatment a pharmaceutical composition comprising an isolated, cell-free population of anti-inflammatory EVs, wherein the EVs have undergone at least 2 freeze/thaw cycles prior to administration to the subject, e.g., the EVs have undergone from about 2 to about 20 freeze/thaw cycles prior to administration to the subject.

Other illustrative embodiments are shown below:

1. an isolated, cell-free population of anti-inflammatory Extracellular Vesicles (EVs), wherein the anti-inflammatory EVs are derived from ex vivo expanded human suppressor immune cells,

wherein:

i) The population exhibits a size diameter distribution of about 50nm to about 150 nm;

ii) the population comprises EV surfaces CD2, CD25 and HLA-DRDPDQ;

iii) The population comprises hsa-miR-1290, hsa-miR-146a-5p and hsa-miR-155-5p microRNAs (miRNAs);

iv) the population exhibits the ability to inhibit bone marrow cells as measured by the ability to reduce the production of pro-inflammatory cytokines by bone marrow cells and the ability to increase the expression of one or more anti-inflammatory markers in bone marrow cells, or as measured by the ability to inhibit proliferation of responsive T cells; and is also provided with

Wherein the human suppressive immune cells are regulatory T cells (tregs).

2. The anti-inflammatory EV population of embodiment 1 wherein at least about 90% of EVs in said population exhibit a dimensional diameter of about 50nm to about 150 nm.

3. The anti-inflammatory EV population of embodiment 1 or 2 wherein said population exhibits an average size diameter of from about 80nm to about 110 nm.

4. The anti-inflammatory EV population of any one of embodiments 1-3 wherein said population exhibits a median size diameter of from about 70nm to about 110 nm.

5. The anti-inflammatory EV population of any one of embodiments 1-4 wherein said population exhibits a mode size diameter of about 65nm to about 95 nm.

6. The anti-inflammatory EV population of embodiment 1 wherein at least about 90% of EVs in the population exhibit a size diameter of about 50 to about 150nm and the population exhibits an average size diameter of about 80nm to about 110nm, a median size diameter of about 70nm to about 110nm, and a mode size diameter of about 65nm to about 95 nm.

7. The anti-inflammatory EV population of any one of embodiments 1-6 wherein said population further comprises EV surfaces CD44, CD29, CD4, and CD45.

8. The anti-inflammatory EV population of any one of embodiments 1-7 wherein said population further comprises EV surfaces CD9, CD63, and CD81.

9. The anti-inflammatory EV population of any one of embodiments 1-8 wherein the population is substantially devoid of EV surfaces CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP, CD146, CD86, CD326, CD133, CD142, CD31, and CD14.

10. The anti-inflammatory EV population of embodiments 1 or 6, wherein the population further comprises EV surfaces CD44, CD29, CD4, CD45, CD9, CD63, and CD81, and wherein the population is substantially devoid of EV surfaces CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP, CD146, CD86, CD326, CD133, CD142, CD31, and CD14.

11. The anti-inflammatory EV population of any one of embodiments 1-10 wherein the ratio of hsa-miR-146a-5p to hsa-miR-155-5p in the population is from about 2 to about 3.

12. The anti-inflammatory EV population of any one of embodiments 1-11 wherein hsa-miR-1290 is at least 2-fold more abundant than hsa-miR-155-5 p.

13. The anti-inflammatory EV population of any one of claims 1-12 wherein the tregs are from a healthy human subject.

14. The anti-inflammatory EV population of any one of embodiments 1-12 wherein the tregs are from a human subject diagnosed with or suspected of having a neurodegenerative disorder.

15. The anti-inflammatory EV population of any one of embodiments 1 to 14 wherein said anti-inflammatory EVs exhibit the ability to increase expression of IL-10, arg1, and/or CD206 in bone marrow cells.

16. The anti-inflammatory EV population of any one of embodiments 1 to 15 wherein said anti-inflammatory EV exhibits the ability to reduce expression of IL-6, IL-8, IL1 β, or interferon- γ in bone marrow cells.

17. The anti-inflammatory EV population of embodiment 1 wherein proliferation of said responsive T cells is determined by flow cytometry or thymidine incorporation.

18. The anti-inflammatory EV population of any one of embodiments 1-17 wherein said population is an anti-inflammatory EV population containing saline.

19. An isolated, cell-free population of anti-inflammatory Extracellular Vesicles (EVs), wherein the anti-inflammatory EVs are derived from ex vivo expanded human inhibitory immune cells.

20. The anti-inflammatory EV population of claim 19 wherein the human suppressor immune cells are regulatory T cells (tregs).

21. The anti-inflammatory EV population of embodiment 20 wherein the tregs are from healthy human subjects.

22. The anti-inflammatory EV population of claim 21 wherein the tregs are from a human subject diagnosed with or suspected of having a neurodegenerative disorder.

23. The anti-inflammatory EV population of embodiment 22 wherein said neurodegenerative disorder is alzheimer's disease.

24. The anti-inflammatory EV population of embodiment 22 wherein the neurodegenerative disorder is Amyotrophic Lateral Sclerosis (ALS).

25. The anti-inflammatory EV population of embodiment 22 wherein the neurodegenerative disease is Multiple Sclerosis (MS).

26. The anti-inflammatory EV population of embodiment 22 wherein said neurodegenerative disease is parkinson's disease.

27. The anti-inflammatory EV population of embodiment 20 wherein the tregs are from a human subject diagnosed with or suspected of having a stroke.

28. The anti-inflammatory EV population of embodiment 20 wherein the tregs are from an elderly subject.

29. The anti-inflammatory EV population of any one of claims 20-28 wherein the tregs are from multiple human subjects.

30. The anti-inflammatory EV population of claim 29 wherein the tregs are from a plurality of unrelated human subjects.

31. The anti-inflammatory EV population of any one of embodiments 19-30 wherein said anti-inflammatory EVs exhibit the ability to increase expression of one or more anti-inflammatory markers in inflammatory cells.

32. The anti-inflammatory EV population of embodiment 31 wherein said inflammatory cells are bone marrow cells.

33. The anti-inflammatory EV population of embodiment 31 or 32 wherein said anti-inflammatory EVs exhibit the ability to increase expression of IL-10, arg1, and/or CD206 in inflammatory cells.

34. The population of anti-inflammatory EVs of any one of embodiments 19-33, wherein the anti-inflammatory EVs exhibit the ability to inhibit inflammatory cells as measured by pro-inflammatory cytokine production by the inflammatory cells.

35. The method of embodiment 34, wherein the inflammatory cell is a bone marrow cell.

36. The anti-inflammatory EV population of embodiment 35 wherein the bone marrow cells are monocytes, macrophages or microglia.

37. The anti-inflammatory EV population of embodiment 36 wherein the macrophages are M1 macrophages.

38. The anti-inflammatory EV population of embodiment 37 wherein the M1 macrophages are Induced Pluripotent Stem Cell (iPSC) -derived M1 macrophages.

39. The anti-inflammatory EV population of any one of embodiments 31-38 wherein the ability to inhibit inflammatory cells is measured by IL-6, IL-8, tnfa, IL1 β, and/or interferon- γ production by said inflammatory cells.

40. The anti-inflammatory EV population of any one of embodiments 19-39 wherein said anti-inflammatory EVs exhibit inhibitory function as determined by inhibition of responsive T cell proliferation.

41. The anti-inflammatory EV population of embodiment 40 wherein proliferation of said responsive T cells is determined by flow cytometry or thymidine incorporation.

42. The anti-inflammatory EV population of any one of embodiments 19-41 wherein the population is an anti-inflammatory EV population that contains saline.

43. The anti-inflammatory EV population of any one of embodiments 19-41 wherein the population is an anti-inflammatory EV population containing physiological saline.

44. The anti-inflammatory EV population of any one of embodiments 19-41 wherein the population is an anti-inflammatory EV population containing phosphate buffered saline.

45. The anti-inflammatory EV population of any one of embodiments 19-44 wherein the anti-inflammatory EV population comprises exosomes and microbubbles.

46. The anti-inflammatory EV population of embodiment 45 wherein a majority of EVs are exosomes.

47. The anti-inflammatory EV population of embodiment 46 wherein at least about 80%, about 90%, or about 95% of the EVs are exosomes.

48. The anti-inflammatory EV population of embodiment 47 wherein a majority of EVs are microbubbles.

49. The anti-inflammatory EV population of embodiment 48 wherein at least about 80%, about 90%, or about 95% of the EVs are microbubbles.

50. The anti-inflammatory EV population of embodiment 45 wherein a majority of EVs have diameters from about 30nm to about 1000 nm.

51. The anti-inflammatory EV population of embodiment 45 wherein a majority of EVs have diameters of about 30nm to about 100nm, about 30nm to about 150nm, about 30 to about 200nm, about 40 to about 100nm, about 80 to about 110nm, about 80 to about 125nm, or about 100 to about 120 nm.

52. The anti-inflammatory EV population of embodiment 25 wherein a majority of EVs have diameters from about 60nm to about 1000nm, from about 70nm to about 1000nm, from about 80nm to about 1000nm, from 100 to about 1000nm, from about 200 to about 1000nm, or from about 300 to about 1000 nm.

53. A pharmaceutical composition comprising the isolated, cell-free anti-inflammatory EV population of any one of embodiments 1-52.

54. The pharmaceutical composition of embodiment 53, wherein the anti-inflammatory EV population comprises about 1 x 10 ⁶ Up to about 1X 10 ¹⁴ EV, about 1×10 ⁸ Up to about 1X 10 ¹⁴ EV, about 1×10 ⁸ Up to about 1X 10 ¹² EV, about 1×10 ⁸ Up to about 1X 10 ¹⁰ EV, about 1×10 ¹⁰ Up to about 1X 10 ¹⁴ EV or about 1×10 ¹⁰ Up to about 1X 10 ¹² And (5) EV.

55. The pharmaceutical composition of embodiment 53, wherein the anti-inflammatory EV population comprisesAbout 1X 10 ⁶ Up to about 1X 10 ¹⁴ EV/ml, about 1X 10 ⁸ Up to about 1X 10 ¹⁴ EV/ml, about 1X 10 ⁸ Up to about 1X 10 ¹² EV/ml, about 1X 10 ⁸ Up to about 1X 10 ¹⁰ EV/ml, about 1X 10 ¹⁰ Up to about 1X 10 ¹⁴ EV/ml or about 1X 10 ¹⁰ Up to about 1X 10 ¹² EV/ml.

56. The pharmaceutical composition of embodiment 53, wherein the anti-inflammatory EV population comprises about 1 μg to about 200mg EV.

57. The pharmaceutical composition of embodiment 53, wherein the anti-inflammatory EV population comprises about 1 μg to about 15mg EV.

58. The pharmaceutical composition of embodiment 53, wherein the anti-inflammatory EV population comprises about 1 μg to about 15mg EV/ml.

59. The pharmaceutical composition of any one of embodiments 53-58, wherein the pharmaceutical composition is a cryopreserved pharmaceutical composition.

60. The pharmaceutical composition of any one of embodiments 53-58, wherein the pharmaceutical composition was previously cryopreserved.

61. A cryopreserved composition comprising the isolated, cell-free anti-inflammatory EV population of any one of embodiments 1-53.

62. A method of producing an isolated, cell-free, anti-inflammatory Extracellular Vesicle (EV) population, the method comprising the steps of:

a. expanding a population of human suppressive immune cells ex vivo in a medium to produce a culture comprising cells, medium and anti-inflammatory EV; and

b. isolating the anti-inflammatory EV from the culture.

63. The method of embodiment 62, wherein the population of human suppressive immune cells is a population of regulatory T cells (tregs).

64. The method of embodiment 62 or 63, wherein step b) comprises removing cells from the culture, followed by precipitation of the culture with polyethylene glycol.

65. The method of embodiment 62 or 63, wherein step b) comprises:

i) Removing cells from the culture to produce a solution containing cell-free, anti-inflammatory EV; and

ii) isolating the anti-inflammatory EV from the cell-free, anti-inflammatory EV-containing solution in i).

66. The method of embodiment 65, wherein step i) comprises flowing the culture through a filter, thereby retaining cells through the filter and thereby removing cells from the culture.

67. The method of embodiment 65 or 66, wherein step i) comprises microfiltration.

68. The method according to any of embodiments 65-67, wherein step ii) comprises step ii-a): the solution containing the cell-free, anti-inflammatory EV is flowed through a filter, thereby retaining the anti-inflammatory EV through the filter.

69. The method of embodiment 68, wherein the filter has a molecular weight cut-off (MWCO) of about 200 kilodaltons (kDa) to about 600 kDa.

70. The method of embodiment 69, wherein the filter has a MWCO of about 500 kDa.

71. The method of any of embodiments 65-70, wherein step ii) comprises ultrafiltration.

72. The method according to any of embodiments 68-71, wherein step ii) further comprises step ii-b): buffer exchange is performed such that the resulting isolated, cell-free anti-inflammatory EV population is an isolated, cell-free anti-inflammatory EV population containing buffer.

73. The method of embodiment 72, wherein the buffer is a saline-containing buffer.

74. The method of embodiment 73, wherein the saline-containing buffer is physiological saline.

75. The method of embodiment 74, wherein the saline-containing buffer is PBS.

76. The method of any one of embodiments 73-75, wherein step ii-b) comprises diafiltration.

77. The method according to any one of embodiments 73-76, wherein steps ii-a) and ii-b) are performed simultaneously.

78. The method of any of embodiments 62-77, wherein step b) comprises tangential flow filtration.

79. The method according to any one of embodiments 62-78, wherein the medium in step a) is serum-free.

80. The method according to any one of embodiments 62-79, wherein the medium in step a) comprises serum.

81. The method of embodiment 80, wherein the serum is human AB serum.

82. The method of embodiment 80 or 81, wherein serum is consumed for serum-derived EVs.

83. The method of any one of embodiments 62-82, further comprising, prior to step a), the step of enriching tregs from the cell sample suspected of containing tregs to produce a baseline population of Treg cells that is the population of tregs subsequently expanded in a).

84. The method of embodiment 83, wherein the cell sample is a leukocyte-removed cell sample.

85. The method of embodiment 83 or 84, wherein the method further comprises obtaining the cell sample from a donor by a leukocyte removal method.

86. The method of any one of embodiments 83-85, wherein the cell sample is not stored overnight or frozen prior to performing the enriching step.

87. The method of any one of embodiments 83-86, wherein the cell sample is obtained within 30 minutes before the enrichment step begins.

88. The method of any one of embodiments 82-87, wherein the enriching step comprises depleting cd8+/cd19+ cells and then enriching cd25+ cells.

89. The method of any one of embodiments 62-88, wherein step a) is performed within 30 minutes of the enriching step.

90. The method of any one of embodiments 62-89, wherein step a) comprises culturing the Treg in a medium comprising anti-CD 3 antibody and anti-CD 28 antibody coated beads.

91. The method of embodiment 90, wherein the beads are first added to the culture medium within about 24 hours of the start of the culture.

92. The method of embodiment 90 or 91, wherein the anti-CD 3 antibody and anti-CD 28 antibody coated beads are added to the culture medium about 14 days after the first adding the anti-CD 3 antibody and anti-CD 28 antibody coated beads to the culture medium.

93. The method of any one of embodiments 90-92, wherein step a) further comprises adding IL-2 to the culture medium within about 6 days of the start of the culture.

94. The method of embodiment 93, wherein step a) further comprises supplementing the medium with IL-2 about every 2-3 days after first adding IL-2 to the medium.

95. The method according to any one of embodiments 90-94, wherein step a) further comprises adding rapamycin to the medium within about 24 hours of the start of the culturing.

96. The method according to embodiment 95, wherein step a) further comprises supplementing the culture medium with rapamycin every 2-3 days after first adding rapamycin to the culture medium.

97. The method of any of embodiments 62-96, wherein step a) is automated.

98. The method of any of embodiments 62-97, wherein step a) is performed in a bioreactor.

99. The method according to any of embodiments 62-98, wherein step b) may be initiated at any point during step a).

100. The method of any one of claims 63-99, wherein the tregs are from a healthy human subject.

101. The method of any one of embodiments 63-99, wherein the tregs are from a human subject diagnosed with or suspected of having a neurodegenerative disorder.

102. The method of embodiment 101, wherein the neurodegenerative disorder is alzheimer's disease, amyotrophic Lateral Sclerosis (ALS), multiple Sclerosis (MS), or parkinson's disease.

103. The method of any one of claims 63-102, wherein the tregs are from a human subject diagnosed with or suspected of having a stroke.

104. The method of any one of claims 63-102, wherein the tregs are from an elderly subject.

105. The method of any one of claims 63-104, wherein the Treg is from a plurality of human subjects.

106. The method of embodiment 62, wherein the population of human suppressive immune cells is a genetically engineered population of human suppressive immune cells.

107. The method of any one of claims 63-106, wherein the population of tregs is a population of genetically engineered tregs.

108. A pharmaceutical composition comprising an isolated, cell-free, anti-inflammatory EV population, wherein the population is prepared by any one of the methods according to embodiments 62-107.

109. The method of any of embodiments 62-107, further comprising: c) Cryopreserving the isolated, cell-free anti-inflammatory EV population, thereby producing a cryopreserved, isolated, cell-free anti-inflammatory EV population.

110. The method of embodiment 109, further comprising thawing the cryopreserved, isolated cell-free anti-inflammatory EV population after about 1 week, 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, or about 24 months of cryopreservation.

111. A pharmaceutical composition comprising the isolated, cell-free anti-inflammatory EV population of embodiment 110.

112. An isolated, cell-free population of anti-inflammatory EVs, wherein the anti-inflammatory EVs are derived from a population of ex vivo expanded Treg cells that exhibit the ability to inhibit inflammatory cells, as measured by pro-inflammatory cytokine production by inflammatory cells, wherein the inflammatory cells are macrophages or monocytes that are derived from a human donor or are produced from induced pluripotent stem cells, wherein the population of ex vivo expanded Treg cells has been expanded from a baseline Treg, and wherein the population of ex vivo expanded Treg cells is expanded in the population of ex vivo expanded Treg cells:

a) Reduced expression of baseline signature gene products for one or more of the dysfunctions listed in table 3 and/or table 4 relative to expression of one or more gene products in baseline tregs;

b) Reduced expression of one or more dysfunctional baseline signature gene products listed in table 5 relative to expression of one or more gene products in baseline tregs;

c) Elevated expression of one or more Treg-related signature gene products listed in table 6 relative to expression of one or more gene products in baseline tregs;

d) Elevated expression of one or more mitochondrial tag gene products listed in table 7 relative to expression of one or more gene products in baseline tregs;

e) Elevated expression of one or more cell proliferation tag gene products listed in table 8 relative to expression of one or more gene products in baseline tregs; or alternatively

f) The expression of one or more highest protein expression signature gene products listed in table 9 is increased relative to the expression of one or more gene products in baseline tregs.

113. A pharmaceutical composition comprising the isolated, cell-free anti-inflammatory EV population of embodiment 112.

114. A method of treating a disorder associated with Treg dysfunction, the method comprising administering to a subject in need of such treatment a composition according to any one of embodiments 53-60, 108, 111 or 113.

115. A method of treating a condition associated with Treg deficiency, the method comprising administering to a subject in need of such treatment a pharmaceutical composition according to any one of embodiments 53-60, 108, 111 or 113.

116. A method of treating a disorder associated with excessive activation of the immune system, the method comprising administering to a subject in need of such treatment the pharmaceutical composition according to any one of embodiments 53-60, 108, 111 or 113.

117. A method of treating an inflammatory condition driven by a T cell response, the method comprising administering to a subject in need of such treatment the pharmaceutical composition according to any one of embodiments 53-60, 108, 111 or 113.

118. A method of treating an inflammatory condition driven by a bone marrow cell response, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of embodiments 53-60, 108, 111, or 113.

119. The method of embodiment 118, wherein the bone marrow cells are monocytes, macrophages or microglia.

120. A method of treating a neurodegenerative disorder in a subject in need thereof, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of embodiments 53-60, 108, 111, or 113.

121. The method of embodiment 120, wherein the neurodegenerative disease is ALS, alzheimer's disease, parkinson's disease, frontotemporal dementia, or huntington's disease.

122. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of embodiments 53-60, 108, 111, or 113.

123. The method of embodiment 122, wherein the autoimmune disease is polymyositis, ulcerative colitis, inflammatory bowel disease, crohn's disease, celiac disease, systemic sclerosis (scleroderma), multiple Sclerosis (MS), rheumatoid Arthritis (RA), type I diabetes, psoriasis, dermatomyositis, systemic lupus erythematosus, cutaneous lupus, myasthenia gravis, autoimmune kidney disease, autoimmune hemolytic anemia, autoimmune cytopenia, autoimmune encephalitis, autoimmune hepatitis, autoimmune uveitis, alopecia, thyroiditis, or pemphigus.

124. A method of treating graft versus host disease in a subject in need thereof, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of embodiments 53-60, 108, 111, or 113. The method of embodiment 106, wherein the subject has received a bone marrow transplant, a kidney transplant, or a liver transplant.

125. A method of improving islet transplantation survival in a subject in need thereof, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of embodiments 53-60, 108, 111, or 113.

126. A method of treating cardiac inflammation in a subject in need thereof, the method comprising administering to a subject in need of such treatment the pharmaceutical composition according to any one of embodiments 53-60, 108, 111 or 113.

127. The method of embodiment 126, wherein the cardiac inflammation is associated with atherosclerosis, myocardial infarction, ischemic cardiomyopathy, or heart failure.

128. A method of treating neurogenic inflammation in a subject in need thereof, the method comprising administering to a subject in need of such treatment the pharmaceutical composition according to any one of embodiments 53-60, 108, 111 or 113.

129. The method of embodiment 128, wherein the neurogenic inflammation is associated with stroke, acute disseminated encephalomyelitis, acute optic neuritis, acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, gill-barre syndrome, transverse myelitis, optic neuromyelitis, epilepsy, traumatic brain injury, spinal cord injury, encephalitis, central nervous system vasculitis, nervous system sarcoidosis, autoimmune or post-infection encephalitis, or chronic meningitis.

130. A method of treating Treg lesions in a subject in need thereof, comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of embodiments 53-60, 108, 111 or 113.

131. The method of embodiment 130, wherein the Treg lesions are caused by FOXP3, CD25, cytotoxic T lymphocyte-associated antigen 4 (CTLA 4), LPS-reactive beige anchor-like protein (LRBA) or BTB domain and CNC homologous gene 2 (BACH 2) loss of function mutations or signal transduction and activator of transcription 3 (STAT 3) function gain mutations.

132. The method of any one of embodiments 114-131, wherein the anti-inflammatory EV is derived from tregs autologous to the subject.

133. The method of any one of embodiments 114-131, wherein the anti-inflammatory EV is derived from tregs allogeneic to the subject.

134. The method of any of embodiments 114-133, wherein the pharmaceutical composition is administered via intranasal administration.

135. The method of embodiment 134, wherein the intranasal administration is via spray or nasal drip.

136. The method of any one of embodiments 114-135, wherein the pharmaceutical composition is administered intravenously.

137. The method of any one of embodiments 114-135, wherein the pharmaceutical composition is administered by local injection.

138. The method of any one of embodiments 114-137, wherein the method further comprises administering to the subject a pharmaceutical composition comprising a population of therapeutic tregs, wherein the tregs have been expanded ex vivo and cryopreserved, and wherein the tregs have not been further expanded prior to administration.

139. The method of claim 138, wherein the population of therapeutic tregs is autologous to the subject.

140. The method of claim 138, wherein the population of therapeutic tregs is allogeneic to the subject.

141. The method of any one of embodiments 138-140, wherein a pharmaceutical composition comprising the population of therapeutic tregs is administered intravenously.

142. The method of any of embodiments 138-141, wherein the pharmaceutical composition comprising the anti-inflammatory EV and the pharmaceutical composition comprising the population of therapeutic tregs are administered to the patient on the same day.

143. The method of any one of embodiments 114-140, wherein the isolated, cell-free anti-inflammatory EV population has been cryopreserved and thawed prior to administration to the subject.

144. The method of any one of embodiments 114-140, wherein the isolated, cell-free anti-inflammatory EV population is stored overnight at 4 ℃ prior to administration to the subject.

145. The method of embodiment 144, wherein the isolated, cell-free anti-inflammatory EV population has been cryopreserved prior to administration to the subject, then thawed and stored overnight at 4 ℃.

146. The method of any one of embodiments 114-140, wherein the isolated, cell-free anti-inflammatory EV population has undergone at least 2 freeze/thaw cycles prior to administration to the subject.

147. The method of embodiment 146, wherein the isolated, cell-free anti-inflammatory EV population has undergone from about 2 to about 20 freeze/thaw cycles prior to administration to the subject.

4. Description of the drawings

Fig. 1 a process flow diagram of an exemplary method of treg isolation, enrichment and ex vivo amplification.

Fig. 2A-2K fig. 2A: the 2 EV populations produced (mixed Treg-derived EV and enriched or pure Treg EV) are illustrated and the names of the populations used in the experiment are shown in fig. 2B-2K. The use of the improved Treg ex vivo expansion protocol described in example 1 resulted in a mixed EV population from Treg cultures. Tregs are obtained from ALS patients and the medium used during this amplification process contains 5% human AB serum. Thus, anti-inflammatory EVs isolated from this culture are present with the medium serum EV. Treg-derived anti-inflammatory EV populations are estimated to be about 20-30% of the total EV population. A second population of tregs was collected from a healthy patient sample and amplified using the improved Treg ex vivo amplification procedure described in example 1, but using medium containing exogenously depleted Fetal Bovine Serum (FBS) in place of human AB serum. Thus, the anti-inflammatory EV population produced from this culture constitutes an isolated sourcePure batches of EV of amplified human tregs. FIGS. 2B-2F and 2I-2K: the experiment used EV populations isolated using PEG. Fig. 2G-2H: the experiment used EV populations isolated using Tangential Flow Filtration (TFF). Fig. 2B: 1X 10 at every 50,000M 1 cells stimulated overnight with LPS/IFNgamma ⁸ After co-cultivation of the Treg EVs, treg-mixed EVs reduced iPSC-derived M1 IL-6 protein by-70%. Fig. 2C: mixed Treg EVs are able to inhibit Tresp proliferation at progressively higher doses. Fig. 2D: pure Treg EV batches demonstrated the ability to inhibit IL-6 transcripts. Fig. 2E: after overnight stimulation, pure Treg EV inhibited the M1 IL-6 protein. Fig. 2F: pure Treg EV inhibits Tresp proliferation at progressively higher doses. Fig. 2G: it was shown that mixed Treg EVs were able to inhibit M1 IL-6 protein production irrespective of whether isolation was performed via PEG precipitation or TFF (n=3; PEG and TFF isolation procedure was performed on expanded tregs from the same three patients in clinical trials). Fig. 2H: it was shown that mixed Treg EVs were able to suppress Tresp proliferation irrespective of whether isolation was performed via PEG precipitation or TFF (n=3; PEG and TFF isolation procedure was performed on expanded tregs from the same three patients). Fig. 2I: an exemplary size spectrum of Treg EVs as described in this example and produced demonstrates a single peak distribution within 20-200 nm. Fig. 2J: graphs showing Miltenyi MACSPlex exosome kit (Miltenyi Biotec) analysis of Treg (mixed) EVs and medium EVs. Fig. 2K: graphs showing Miltenyi MACSPlex exosome kit (Miltenyi Biotec) analysis of Treg (mixed) EVs and medium EVs. ALS Treg evn=7; culture medium evn=3. Values are shown as mean +/-SEM of analysis by one-way analysis of variance and Tukey post-hoc test. * Represents p-value less than 0.01; * Represents p-values less than 0.001.

FIGS. 3A-3D. Anti-inflammatory effects of Treg EV were evaluated in LPS-induced neurogenic inflammation models. Briefly, 2mg/kg LPS was injected intraperitoneally. 2 hours after injection, pure Treg EV was administered intranasally. After 12 hours post intranasal administration, brain and spleen cd11b+ bone marrow cells were isolated. The pro-inflammatory transcripts were analyzed to evaluate anti-inflammatory effects. Fig. 3A: the graph depicts the LPS-induced neurogenic inflammation model and Treg EV treatment paradigm. Fig. 3B: intranasal Treg EV reduced IL-6 and IL-1 beta transcripts in hippocampus. Fig. 3C: IL-6 transcripts reduced following intranasal Treg EV treatment in mouse cortex. Fig. 3D: peripheral bone marrow cell activation was reduced following intranasal treatment with Treg EV; reduced IL-6 and TNF transcripts in spleen-derived cd11b+ bone marrow cells were demonstrated. p-values are p <0.05 and p <0.01.

Figures 4A-4F. Starting at day 90 (about 20 days after onset of symptoms in the model) Treg EVs were intranasally administered every two weeks in SOD1 mice to evaluate the mouse clinical benefit of multiple rounds of intranasal Treg EVs. After mice were sacrificed, inflammatory markers in the lumbar segment of spinal cord inflammation were assessed by RNA analysis. Fig. 4A: the graph shows the intranasal Treg EV treatment paradigm of SOD1 mouse model of ALS. Fig. 4B: intranasal Treg EV treatment increased survival probability compared to intranasal PBS treatment. Fig. 4C: treg EV treatment slowed disease progression as defined by a modified scoring system for assessing ALS progression in mice; the effect is more pronounced when mice experience a rapid phase of their disease progression. Fig. 4D: treatment significantly extends disease duration. Fig. 4E: mean life span is increased in animals receiving Treg EV treatment. Fig. 4F: after the animals reached their ethical endpoint, the lumbar spinal cord was dissected. RNA analysis was performed using spinal cord tissue to examine inflammatory markers. Reduced levels of inflammatory markers were observed in the spinal cord, while increased Treg (FOXP 3) and anti-inflammatory M2 macrophage (CD 206) signals were observed in the treated animals. Values are shown as mean +/-SEM by one-factor analysis of variance, turkey post-hoc test (PBS n=3, lps+pbs n=4, lps+treg evn=4, trev evn=3 only). * Represents a p-value of less than 0.05; * Represents p-values less than 0.001.

Fig. 5A-5C fig. 5A: at 1X 10 ⁸ Dose sum of individual EV 1X 10 ⁷ At the dose of each EV, treg EVs were able to inhibit M1 pro-inflammatory IL-6 protein by 46% and 30.6%, respectively, as compared to 13.7% and 3.3% for MSC EVs, respectively. Fig. 5B: at 1X 10 ⁸ The sum of the doses of (2) is 1×10 ⁷ Treg EV inhibited M1-derived pro-inflammatory IL-8 protein by 60% and 50% respectively compared to MSC EV at 1X 10 ⁸ Shows 20% inhibition at the dose. "control exosomes" in fig. 5A and 5B: EV derived from non-exosome-depleted medium of cell-free culture. Fig. 5C: in comparative studyIn the middle, treg EV inhibited T cell proliferation more than MSC EV.

Figures 6A-6℃ Treg EV stability and function were evaluated after 1 to 20 freeze/thaw cycles and after 3, 6 or 12 months of storage at-20 ℃. Fig. 6A: after multiple (up to 20) freeze/thaw cycles, no loss in Treg EV particle numbers was observed. Fig. 6B: no significant deviation in Treg EV particle size was observed during the same freeze/thaw cycle. Fig. 6C: treg EV inhibition of T cell proliferation did not decrease over time in frozen storage at-20 ℃.

Fig. 7A-7B fig. 7A: treg EV concentration after EV isolation from the amplification medium (EV pellet/ml medium). EV concentrations from the medium alone ("medium") are also shown. Fig. 7B: fold increase in EV from ex vivo expanded Treg cell populations compared to EV from medium alone.

Fig. 8 an exemplary size spectrum of Treg EVs isolated using TFF protocol. The spectrum shows EV average values of 92.1 nm.+ -. 4.2nm and modes of 73.3 nm.+ -. 6.1 nm.

Fig. 9A-9B fig. 9A: treg EV derived from ex vivo expanded ALS patient tregs induces M1 cells to increase Arg1 mRNA expression in an EV concentration-dependent manner. Fig. 9B: treg EV derived from ex vivo expanded ALS patient tregs induces M1 cells to increase CD206 mRNA expression in an EV concentration-dependent manner.

Fig. 10A-10C fig. 10A: treg EVs were shown to be significantly more effective than MSC EVs in inhibiting M1 pro-inflammatory IL-6 protein production (n=3; for each group, indicating a p-value of less than 0.001 compared to the corresponding MSC EV). Fig. 10B: treg EVs were shown to be significantly more effective than MSC EVs in inhibiting T cell proliferation (n=3 for each group, meaning that the p-value is less than 0.001 compared to the corresponding MSC EV). Fig. 10c: treg EVs were shown to be significantly more effective than MSC EVs in inhibiting M1 pro-inflammatory IL-8 protein production (n=3; for each group, indicating a p-value of less than 0.001 compared to the corresponding MSC EV). The Treg EVs used for these experiments were pure Treg EVs and the MSC EVs used for these experiments were pure MSC EVs.

Fig. 11A-11B fig. 11A: average particle size of EV isolated via TFF. Fig. 11B: mode of particle size of EV isolated via TFF.

Fig. 12 recovery of EV isolated via TFF (n=6, results reported as mean ± SD).

Fig. 13 is a flow chart of a process for producing a population of tregs in a bioreactor.

Fig. 14A-14F fig. 14A: the graph depicts a model of LPS-induced acute inflammation, in which WT mice were administered LPS via intraperitoneal injection and subsequently treated by single tail vein (IV) injection of Treg EV at different doses. After overnight treatment, mice were sacrificed and peripheral immune cells were isolated from the mice spleens for subsequent inflammatory transcript analysis. Fig. 14B: the graph depicts a model of LPS-induced acute inflammation, in which WT mice were administered LPS via intraperitoneal injection and subsequently treated by single tail vein (IV) injection of Treg EV at different doses. After overnight treatment, mice were sacrificed and brain tissues (hippocampus and cortex) were isolated and analyzed for neuroinflammatory marker transcripts. Fig. 14C: the graph shows the fold change in proinflammatory transcripts of IL6 and iNOS in cd11b+ bone marrow cells from spleen following IV treatment of Treg EV. Fig. 14D: the graph shows the fold change in proinflammatory transcripts of IL1b and ifnγ in cd11b+ bone marrow cells from spleen following IV treatment of Treg EV. Fig. 14E: the graph shows fold changes in anti-inflammatory related transcripts of CD206 (MRC 1) and CD163 in cd11b+ bone marrow cells following Treg EV treatment. Fig. 14F: the graph shows fold changes in FOXP3 and IL2RA (CD 25) in fresh spleen isolated cd4+cd25+ tregs after Treg EV treatment. Data are shown as mean ± SEM and were statistically analyzed by one-way anova and Tukey post-hoc test (PBS n=5, lps n=5, lps+treg ev1×10 ⁹ n=5 (peripheral tissue), 4-5 (brain tissue), lps+tregev1×10 ¹⁰ n=5 (peripheral tissue), 4-5 (brain tissue), lps+tregev1×10 ¹¹ n=5 (peripheral tissue), 4-5 (brain tissue)). p-value is p<0.05，**p<0.01 and p<0.001。

Fig. 15A-15B fig. 15A: the graph shows the fold change in IL-6, IL1b and TNF RNA in hippocampus following IV Treg EV treatment. Fig. 15B: the graph shows the fold change in IL-6, IL1b and TNF in cortex after Treg EV treatment. Data are shown as mean ± SEM and were performed by one-way analysis of variance and Tukey post-hoc test (PBS n=5, lpS n＝5，LPS+Treg EV 1×10 ⁹ n＝4-5，LPS+Treg EV 1×10 ¹⁰ n＝4-5，LPS+Treg EV 1×10 ¹¹ n＝5)。

Fig. 16A-16B fig. 16A: particle size distribution of Treg EVs isolated via nanoparticle tracking analysis of TFF produced. Fig. 16B: particle size data from TFF isolated Treg EVs generated via nanoparticle tracking analysis.

Figure 17 in vitro Treg EV inhibition of t cell proliferation (n=6).

Figure 18 quantification of Treg functional protein in Treg EV using ELISA.

Figures 19A-19B. Quantification of residual IL2 and albumin concentrations as a percentage of the original total amount in concentrated batches after TFF and in final exemplary dosage formulations.

Fig. 20A-20E fig. 20A: stability of Treg EV at room temperature and at 4 ℃. Results sets of Treg EVs run BioR4-6 using bioreactors from time points of 8 hours and BioR5 and BioR6 for all time points are provided. Fig. 20B: stability of Treg EV at room temperature and at 4 ℃ shows interruption of individual sample results among the results shown in fig. 20A. Fig. 20C: stability of the particle size distribution of Treg EV at room temperature and at 4 ℃. As in fig. 20A, a set of results is provided for running BioR4-6 using bioreactors from 8 hour time points and Treg EVs for BioR5 and BioR6 at all time points. Fig. 20D: stability of Treg EV after prolonged storage at-20 ℃ and-80 ℃. For each time point, n=3 (Treg EV from BioR 4-6). Fig. 20E: stability of particle size distribution of Treg EV after prolonged storage at-20 ℃ and-80 ℃. For each time point, n=3 (Treg EV from BioR 4-6).

Figure 21 ability of treg EV to inhibit activated pro-inflammatory M1 cells. * Represents p-value less than 0.01; * Represents p-values less than 0.001.

Ev surface protein characterization. Top plate: bioreactor TFF Treg EV. Bottom plate: culture medium EV.

ALS patient Treg EV surface protein characterization.

Figures 24A-24B, these figures show the exosome marker levels associated with ALS-expanded Treg EVs and control-expanded Treg EVs (n=3 for ALS-expanded Treg EVs and n=6 for control-expanded Treg EVs).

5. Detailed description of the invention

Described herein are anti-inflammatory EV populations derived from ex vivo expanded human suppressive immune cells, e.g., regulatory T cells (tregs). The EVs provided herein exhibit impressive anti-inflammatory activity in vitro and in vivo. For example, the results provided herein demonstrate that EVs described in the present disclosure are capable of effectively inhibiting T-responsive cell proliferation and pro-inflammatory spinal cord activity, e.g., macrophage activity, in vitro and also exert an effective in vivo anti-inflammatory effect. Briefly, the results provided herein demonstrate that EVs are capable of inhibiting brain and peripheral inflammation in a model of neurogenic inflammation in vivo, and are also capable of inhibiting inflammation, prolonging survival, and slowing the progression of advanced disease in a model of Amyotrophic Lateral Sclerosis (ALS) in vivo. The anti-inflammatory EV shows inhibition of the pro-inflammatory response via intravenous or intranasal administration. The results presented herein demonstrate that Treg EVs have greater inhibition of pro-inflammatory immune cells than EVs derived from Mesenchymal Stem Cells (MSCs).

Without wishing to be bound by theory or mechanism, it appears that EVs described in the present disclosure retain the immunosuppressive activity of the cells from which they are derived. Furthermore, since EVs are not themselves cells, they avoid potential cell-based problems such as immune rejection and the possibility of polarization towards pro-inflammatory cell types. As such, the anti-inflammatory EVs provided herein are particularly useful for the treatment of a variety of diseases, such as, for example, neurodegenerative disorders, such as Amyotrophic Lateral Sclerosis (ALS).

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

The terms "a" and "an" as used herein are to be understood as singular or plural and mean "one or more" unless specifically stated or apparent from the background.

The terms "comprising," "such as," and the like are intended to mean including, unless otherwise specifically indicated.

The terms "or" and "may be used interchangeably and they may be understood to mean" and/or ".

Unless otherwise indicated herein or clearly contradicted by context, the use of terms such as "comprising," "having," "including" or "containing" with respect to one or more elements in the description of any aspect or embodiment of the invention is intended to provide support for "consisting of," "consisting essentially of, or" consisting essentially of the particular one or more elements (e.g., unless otherwise indicated or clearly contradicted by context, a composition described herein as comprising the particular element should be understood to also describe a composition consisting of the element).

As used herein, the terms "about" and "approximately" are interchangeable and will generally be understood to refer to a range of numbers surrounding a given number, and to refer to all numbers within the range of numbers recited (e.g., "about 5 to 15" means "about 5 to about 15" unless otherwise indicated). Furthermore, all numerical ranges herein should be understood to include each and every integer within the range. In particular, unless otherwise indicated, the terms mean within plus or minus 10% of a given value or range. In the case where integers are required, the term means rounded up or down to the nearest integer within plus or minus 10% of a given value or range.

5.1 anti-inflammatory Extracellular Vesicles (EV)

Provided herein are isolated, cell-free, populations of anti-inflammatory Extracellular Vesicles (EVs), wherein the anti-inflammatory EVs are derived from ex vivo expanded human inhibitory immune cells. In certain embodiments, the anti-inflammatory EV is derived from human regulatory T cells (tregs).

In certain embodiments, an isolated, cell-free, anti-inflammatory EV population is produced by a method described herein, e.g., as described in section 5.2 below.

For ease of reference, the terms "extracellular vesicle", "EV", "extracellular vesicle particle" and "EV particle" are used interchangeably herein unless otherwise indicated. Furthermore, as should be self-evident, it is to be understood that references to "anti-inflammatory extracellular vesicles," "anti-inflammatory EVs," "anti-inflammatory exosomes," and the like, as used herein, include Treg EVs and Treg exosomes described in detail herein. Although for ease of description not all embodiments provided herein are reproduced to enumerate, for example, both "anti-inflammatory EVs" and "Treg EVs", it should be understood that each of the embodiments listing such "anti-inflammatory" embodiments includes and may be replaced by a respective "Treg EV" and "Treg exosome" as these are described herein.

EV is a membrane-bound particle released by cells. Typically, EVs comprise one or more components from the cells that release them, e.g., one or more DNA, RNA (e.g., coding and/or non-coding RNA, e.g., mRNA microrna, and/or long non-coding RNA), protein (e.g., signal transduction proteins, receptors, other surface proteins, glycoproteins, and/or enzymes), or non-protein, e.g., lipid components. The dimensions of the EV are typically in the range of about 30nm to about 1000nm in diameter. Larger EVs are sometimes referred to as "microbubbles". In general, microbubbles have a size diameter greater than about 200nm. Sometimes, a smaller EV is referred to as "exosome". Generally, the exosomes have a dimensional diameter in the range of about 30-40nm to about 150-200 nm. Methods for determining EV particle size and concentration are well known to those skilled in the art.

Methods for determining EV particle size, concentration and purity are well known and include determination methods using dynamic light scattering or single particle trajectory analysis or using techniques such as flow cytometry, ELISA or electron microscopy. See, e.g., balaj et al, (2015) Sci Rep 5,10266, nakai et al (2016) Sci Rep 6,33935 and Carnino et al (2019) Respiratory Research 20:240. In particular embodiments, conventional determinations may be made using a nanoparticle analyzer, such as a NanoSight (Malvern Panalytical) nanoparticle analyzer.

In some embodiments, western blotting, ELISA, and other protein-related assays may be used, or commercially available arrays, such as Exo-Check ^TM Protein analysis of exosome antibody array (System Biosciences) and/or MACSPlex exosome kit (Miltenyi Biotec), EV was analyzed for the presence of exosome markers and/or Treg markers (e.g., CD 25). In certain embodiments, the EV population may be analyzed for the presence of serum-related proteins. In particular embodiments, the EV population described herein is substantially free of serum-related proteins.

In certain embodiments, an anti-inflammatory EV as described herein exhibits the ability to increase expression of one or more anti-inflammatory markers in inflammatory cells. For example, in particular embodiments, the anti-inflammatory EVs described herein exhibit the ability to increase transcription and/or mRNA expression levels of one or more genes encoding anti-inflammatory proteins in inflammatory cells. In another example, in a particular embodiment, an anti-inflammatory EV as described herein exhibits the ability to enhance translation, processing, secretion and/or activation of one or more anti-inflammatory proteins produced by inflammatory cells.

In specific embodiments, the anti-inflammatory marker is IL-10, arg1 and/or CD206. In specific examples, the inflammatory cell is a bone marrow cell, e.g., a monocyte, macrophage, or microglial cell, e.g., a human inflammatory cell, e.g., a human monocyte, macrophage, or microglial cell.

In certain embodiments, an anti-inflammatory EV described herein exhibits the ability to inhibit inflammatory cells. For example, in certain embodiments, the anti-inflammatory EVs described herein exhibit the ability to inhibit inflammatory cells, as measured by pro-inflammatory cytokine production by the inflammatory cells.

In some embodiments, the ability to inhibit inflammatory cells is measured by IL-6, tnfα, il1β, IL8, and/or interferon- γ production by the inflammatory cells. In some embodiments, the ability to inhibit inflammatory cells is measured by IL-6 production by the inflammatory cells.

In particular embodiments, the inflammatory cell is a bone marrow cell, e.g., a monocyte, macrophage or microglial cell, e.g., a human inflammatory cell, e.g., a human bone marrow cell, such as a human monocyte, macrophage or microglial cell. In specific examples, the bone marrow cells, e.g., monocytes, macrophages or microglia, are derived from a human donor or are produced from induced pluripotent stem cells. In certain embodiments, the macrophage is an M1 macrophage, such as an Induced Pluripotent Stem Cell (iPSC) -derived M1 macrophage.

In certain embodiments, an anti-inflammatory EV described herein exhibits the ability to inhibit pro-inflammatory M1 cells. In some embodiments, the ability to inhibit a pro-inflammatory M1 cell is measured by IL-6 production by the pro-inflammatory M1 cell.

In certain embodiments, the anti-inflammatory EVs described herein exhibit the ability to inhibit IL-6 protein production by pro-inflammatory M1 cells by about 20% to about 70%, about 20% to about 50%, about 25% to about 50%, about 30% to about 50%, or about 25% to about 45%.

In certain embodiments, the anti-inflammatory EVs described herein exhibit the ability to inhibit inflammatory cells, as determined by responding to T cell proliferation inhibition. In specific embodiments, proliferation of responsive T cells is determined by flow cytometry or thymidine incorporation, e.g., tritiated thymidine incorporation.

In certain embodiments, an anti-inflammatory EV described herein exhibits the ability to inhibit inflammatory cells (e.g., as measured by pro-inflammatory cytokine production and/or in response to T cell proliferation) and to increase expression of one or more inflammatory markers in inflammatory cells.

In certain aspects, the anti-inflammatory EV population as described herein comprises exosomes. In other aspects, the anti-inflammatory EV population described herein comprises microbubbles. In other aspects, the anti-inflammatory EV population as described herein comprises exosomes and microbubbles.

In certain embodiments, a majority of EVs of an anti-inflammatory EV population as described herein are exosomes. For example, in certain embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more of the EVs of the anti-inflammatory exosome populations described herein are exosomes.

In certain embodiments, a majority of EVs of the anti-inflammatory EV population as described herein are microbubbles. For example, in certain embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more of the EVs of the anti-inflammatory exosome populations described herein are microbubbles.

In certain embodiments, the EV of the anti-inflammatory EV population as described herein has a dimensional diameter of about 5nm to about 1000nm. In certain embodiments, the EV of the anti-inflammatory EV population as described herein has a dimensional diameter of about 10nm to about 1000nm. In certain embodiments, the EV of the anti-inflammatory EV population as described herein has a dimensional diameter of about 15nm to about 1000nm. In certain embodiments, the EV of the anti-inflammatory EV population as described herein has a dimensional diameter of about 20nm to about 1000nm. In certain embodiments, the EV of the anti-inflammatory EV population as described herein has a dimensional diameter of about 30nm to about 1000nm. In certain embodiments, the EV of the anti-inflammatory EV population as described herein has a dimensional diameter of about 20nm to about 300nm. In certain embodiments, the EV of the anti-inflammatory EV population as described herein has a dimensional diameter of about 20nm to about 275nm. In certain embodiments, the EV of the anti-inflammatory EV population as described herein has a dimensional diameter of about 20nm to about 250nm. In certain embodiments, the EV of the anti-inflammatory EV population as described herein has a dimensional diameter of about 20nm to about 200nm. In certain embodiments, the EV of the anti-inflammatory EV population as described herein has a dimensional diameter of about 20nm to about 175nm. In certain embodiments, the EV of the anti-inflammatory EV population as described herein has a dimensional diameter of about 50nm to about 200nm. In certain embodiments, the EV of the anti-inflammatory EV population as described herein has a dimensional diameter of about 50nm to about 175nm. In certain embodiments, the EV of the anti-inflammatory EV population as described herein has a dimensional diameter of about 50nm to about 150nm.

In certain embodiments, a majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of the anti-inflammatory EV population as described herein has a size diameter of about 5nm to about 1000nm, about 10nm to about 1000nm, about 15nm to about 1000nm, about 20nm to about 1000nm, or about 30nm to about 1000nm.

In certain embodiments, a majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of the anti-inflammatory EV population as described herein has a size diameter of about 20nm to about 300nm, about 20nm to about 275nm, about 20nm to about 250nm, about 20nm to about 200nm, or about 20nm to about 175nm.

In certain embodiments, a majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of the anti-inflammatory EV population as described herein has a size diameter of about 20nm to about 200nm.

In certain embodiments, a majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of the anti-inflammatory EV population as described herein has a size diameter of about 50nm to about 200nm, about 50nm to about 175nm, or about 50nm to about 150nm.

In certain embodiments, a majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of the anti-inflammatory EV population as described herein has a size diameter of about 50nm to about 150nm.

In certain embodiments, a majority of EVs of an anti-inflammatory EV population as described herein have a dimensional diameter of less than about 300nm, less than about 200nm, less than about 150nm, or less than about 100nm. For example, in certain embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more of the anti-inflammatory exosome populations described herein have an EV size diameter of less than about 300nm, less than about 200nm, less than about 150nm, or less than about 100nm.

In certain embodiments, the anti-inflammatory EV population as described herein has a EV size diameter of about 30nm to about 300nm, about 30nm to about 250nm, about 30nm to about 200nm, about 30nm to about 160nm, about 30nm to about 150nm, about 30nm to about 100nm, about 40nm to about 300nm, about 40nm to about 200nm, about 40nm to about 160nm, about 40nm to about 150nm, about 40nm to about 100nm, about 60nm to about 300nm, about 60nm to about 200nm, about 60nm to about 160nm, about 60nm to about 150nm, about 60nm to about 125nm, about 60nm to about 110nm, about 60nm to about 100nm, about 60nm to about 80nm, about 70nm to about 300nm, about 70nm to about 200nm, about 70nm to about 160nm, about 70nm to about 150nm, about 70nm to about 125nm, about 110nm, about 70nm to about 100nm, about 80nm to about 300nm, about 80nm to about 80nm, about 80nm or about 80nm to about 80 nm.

In certain embodiments, a majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of the anti-inflammatory EV population as described herein has a size diameter of about 30nm to about 300nm, about 30nm to about 250nm, about 30nm to about 200nm, about 30nm to about 160nm, about 30nm to about 150nm, about 30nm to about 100nm, about 40nm to about 300nm, about 40nm to about 200nm, about 40nm to about 160nm, about 40nm to about 150nm, about 40nm to about 100nm, about 60nm to about 300nm, about 60nm to about 200nm, about 60nm to about 160nm, about 60nm to about 125nm, about 60nm to about 110nm, about 60nm to about 100nm, about 60nm to about 80nm, about 70nm to about 70nm, about 70nm to about 200nm, about 70nm to about 80nm, about 80nm or about 80nm to about 80 nm.

In certain embodiments, a majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of the anti-inflammatory EV population as described herein has a EV dimension diameter of about 30nm, about 40nm, about 50nm, about 60nm, about 65nm, about 70nm, about 75nm, 80nm, about 85nm, about 90nm, about 95nm, about 100nm, about 110nm to about 120nm, about 150nm, about 175nm, about 200nm, about 250nm or about 300nm.

In certain embodiments, a majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of the anti-inflammatory EV population as described herein has a size diameter of EV greater than about 300nm, greater than about 400nm, greater than about 500nm, greater than about 700nm, or greater than about 800nm.

In certain embodiments, the anti-inflammatory EV population as described herein has an EV size diameter of about 200nm to about 1000nm, about 300nm to about 1000nm, about 400nm to about 1000nm, about 500nm to about 1000nm, about 600nm to about 1000nm, about 700nm to about 1000nm, about 800nm to about 1000nm, about 200nm to about 800nm, about 300nm to about 800nm, about 400nm to about 800nm, about 500nm to about 800nm, about 600nm to about 800nm, about 200nm to about 600nm, about 300nm to about 600nm, about 400nm to about 600nm, about 200nm to about 500nm, or about 300nm to about 500nm.

In certain embodiments, the anti-inflammatory EV population as described herein for a majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) has a dimensional diameter of about 200nm to about 1000nm, about 300nm to about 1000nm, about 400nm to about 1000nm, about 500nm to about 1000nm, about 600nm to about 1000nm, about 700nm to about 1000nm, about 800nm to about 1000nm, about 200nm to about 800nm, about 300nm to about 800nm, about 400nm to about 800nm, about 500nm to about 800nm, about 600nm to about 800nm, about 200nm to about 600nm, about 300nm to about 600nm, about 400nm to about 600nm, about 200nm to about 500nm, or about 300nm to about 500nm.

In certain embodiments, a majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of the anti-inflammatory EV population as described herein has a size diameter of about 400nm, about 450nm, about 500nm, about 600nm, about 650nm, about 700nm, about 750nm, 800nm, about 850nm, about 900nm, about 950nm or about 1000nm.

In certain embodiments, the average size diameter of EVs of an anti-inflammatory EV population as described herein is from about 30nm to about 1000nm. In certain embodiments, the average size diameter of EVs of an anti-inflammatory EV population as described herein is less than about 300nm, less than about 200nm, less than about 150nm, or less than about 100nm. In certain embodiments, the anti-inflammatory EV population as described herein has an EV average dimensional diameter of about 30nm to about 300nm, about 30nm to about 250nm, about 30nm to about 200nm, about 30nm to about 160nm, about 30nm to about 150nm, about 30nm to about 100nm, about 40nm to about 300nm, about 40nm to about 200nm, about 40nm to about 160nm, about 40nm to about 150nm, about 40nm to about 100nm, about 60nm to about 300nm, about 60nm to about 200nm, about 60nm to about 160nm, about 60nm to about 150nm, about 60nm to about 125nm, about 60nm to about 110nm, about 60nm to about 100nm, about 60nm to about 80nm, about 70nm to about 300nm, about 70nm to about 200nm, about 70nm to about 160nm, about 70nm to about 150nm, about 70nm to about 125nm, about 70nm to about 110nm, about 70nm to about 100nm, about 80nm to about 80nm, about 80nm or about 80nm to about 80 nm.

In certain embodiments, the anti-inflammatory EV population as described herein has an EV average dimensional diameter of about 30nm, about 40nm, about 50nm, about 60nm, about 65nm, about 70nm, about 75nm, 80nm, about 85nm, about 90nm, about 95nm, about 100nm, about 110nm to about 120nm, about 150nm, about 175nm, about 200nm, about 250nm, or about 300nm.

In certain embodiments, the anti-inflammatory EV population as described herein has an EV average dimensional diameter of about 80nm to about 110nm, about 80nm to about 100nm, about 80 to about 95nm, about 80-90nm, about 85nm to about 110nm, about 85nm to about 100nm, about 85 to about 95nm, about 90nm to about 110nm, about 90nm to about 100nm, or about 90 to about 95nm.

In certain embodiments, the anti-inflammatory EV population as described herein has an EV average dimensional diameter greater than about 300nm, greater than about 400nm, greater than about 500nm, greater than about 700nm, or greater than about 800nm. In certain embodiments, the anti-inflammatory EV population as described herein has an EV average dimensional diameter of about 200nm to about 1000nm, about 300nm to about 1000nm, about 400nm to about 1000nm, about 500nm to about 1000nm, about 600nm to about 1000nm, about 700nm to about 1000nm, about 800nm to about 1000nm, about 200nm to about 800nm, about 300nm to about 800nm, about 400nm to about 800nm, about 500nm to about 800nm, about 600nm to about 800nm, about 200nm to about 600nm, about 300nm to about 600nm, about 400nm to about 600nm, about 200nm to about 500nm, or about 300nm to about 500nm.

In certain embodiments, the anti-inflammatory EV population as described herein has an EV average dimensional diameter of about 400nm, about 450nm, about 500nm, about 600nm, about 650nm, about 700nm, about 750nm, 800nm, about 850nm, about 900nm, about 950nm, or about 1000nm. In certain embodiments, the average size diameter of EVs of an anti-inflammatory EV population as described herein is from about 80nm to about 110nm. In certain embodiments, the average size diameter of EVs of an anti-inflammatory EV population as described herein is about 85nm to about 100nm. In certain embodiments, the average size diameter of EVs of an anti-inflammatory EV population as described herein is from about 80nm to about 100nm. In certain embodiments, the average size diameter of EVs of an anti-inflammatory EV population as described herein is about 85nm to about 95nm.

In certain embodiments, the median size diameter of the EVs of the anti-inflammatory EV population as described herein is from about 30nm to about 1000nm. In certain embodiments, the median size diameter of the EVs of the anti-inflammatory EV population as described herein is less than about 300nm, less than about 200nm, less than about 150nm, or less than about 100nm. In certain embodiments, the anti-inflammatory EV population as described herein has a median size diameter of EV of about 30nm to about 300nm, about 30nm to about 250nm, about 30nm to about 200nm, about 30nm to about 160nm, about 30nm to about 150nm, about 30nm to about 100nm, about 40nm to about 300nm, about 40nm to about 200nm, about 40nm to about 160nm, about 40nm to about 150nm, about 40nm to about 100nm, about 60nm to about 300nm, about 60nm to about 200nm, about 60nm to about 160nm, about 60nm to about 150nm, about 60nm to about 125nm, about 60nm to about 110nm, about 60nm to about 100nm, about 60nm to about 80nm, about 70nm to about 300nm, about 70nm to about 200nm, about 70nm to about 160nm, about 70nm to about 150nm, about 70nm to about 125nm, about 70nm to about 110nm, about 70nm to about 100nm, about 75nm to about 75nm, about 80nm to about 80nm, about 80nm or about 80nm to about 80 nm. In certain embodiments, the median size diameter of the EVs of the anti-inflammatory EV population as described herein is from about 30nm to about 1000nm.

In certain embodiments, the median size diameter of the EVs of the anti-inflammatory EV population as described herein is from about 70nm to about 100nm. In certain embodiments, the median size diameter of the EVs of the anti-inflammatory EV population as described herein is from about 75nm to about 100nm. In certain embodiments, the median size diameter of the EVs of the anti-inflammatory EV population as described herein is from about 75nm to about 95nm.

In certain embodiments, the anti-inflammatory EV population as described herein has an EV median size diameter of about 30nm, about 40nm, about 50nm, about 60nm, about 65nm, about 70nm, about 75nm, 80nm, about 85nm, about 90nm, about 95nm, about 100nm, about 110nm to about 120nm, about 150nm, about 175nm, about 200nm, about 250nm, or about 300nm.

In certain embodiments, the anti-inflammatory EV population as described herein has an EV median size diameter greater than about 300nm, greater than about 400nm, greater than about 500nm, greater than about 700nm, or greater than about 800nm. In certain embodiments, the anti-inflammatory EV population as described herein has a median size diameter of EV from about 200nm to about 1000nm, from about 300nm to about 1000nm, from about 400nm to about 1000nm, from about 500nm to about 1000nm, from about 600nm to about 1000nm, from about 700nm to about 1000nm, from about 800nm to about 1000nm, from about 200nm to about 800nm, from about 300nm to about 800nm, from about 400nm to about 800nm, from about 500nm to about 800nm, from about 600nm to about 800nm, from about 200nm to about 600nm, from about 300nm to about 600nm, from about 400nm to about 600nm, from about 200nm to about 500nm, or from about 300nm to about 500nm.

In certain embodiments, the anti-inflammatory EV population as described herein has an EV median size diameter of about 400nm, about 450nm, about 500nm, about 600nm, about 650nm, about 700nm, about 750nm, 800nm, about 850nm, about 900nm, about 950nm, or about 1000nm.

In certain embodiments, the mode size diameter of the EVs of the anti-inflammatory EV population as described herein is about 30nm to about 1000nm. In certain embodiments, the mode size diameter of the EV of the anti-inflammatory EV population as described herein is less than about 300nm, less than about 200nm, less than about 150nm, or less than about 100nm. In some embodiments of the present invention, in some embodiments, the mode-size diameter of the EVs of the anti-inflammatory EV population as described herein is about 30nm to about 300nm, about 30nm to about 250nm, about 30nm to about 200nm, about 30nm to about 160nm, about 30nm to about 150nm, about 30nm to about 100nm, about 40nm to about 300nm, about 40nm to about 200nm, about 40nm to about 160nm, about 40nm to about 150nm, about 40nm to about 100nm, about 60nm to about 300nm, about 60nm to about 200nm, about 60nm to about 160nm, about 60nm to about 150nm, about 60nm to about 125nm, about 60nm to about 110nm, about 60nm to about 100nm, about 60nm to about 80nm, about 70nm to about 300nm, about 70nm to about 200nm, about 70nm to about 160nm, about 70nm to about 150nm, about 125nm, about 70nm to about 110nm, about 70nm to about 100nm, about 80nm to about 300nm, about 80nm to about 200nm, about 80nm to about 80nm, about 80nm to about 125nm, about 80nm to about 80nm, about 80nm or about 80nm to about 120nm. In certain embodiments, the mode size diameter of the EV of the anti-inflammatory EV population as described herein is about 65nm to about 85nm, about 65nm to about 80nm, about 65nm to about 75nm, about 70nm to about 85nm, about 70nm to about 80nm, or about 70nm to about 75nm.

In certain embodiments, the mode size diameter of the EVs of the anti-inflammatory EV population as described herein is about 65nm to about 95nm. In certain embodiments, the mode size diameter of the EVs of the anti-inflammatory EV population as described herein is about 75nm to about 85nm. In certain embodiments, the mode size diameter of the EVs of the anti-inflammatory EV population as described herein is about 75nm to about 85nm.

In certain embodiments, the mode size diameter of the EV of the anti-inflammatory EV population as described herein is about 30nm, about 40nm, about 50nm, about 60nm, about 65nm, about 70nm, about 75nm, 80nm, about 85nm, about 90nm, about 95nm, about 100nm, about 110nm to about 120nm, about 150nm, about 175nm, about 200nm, about 250nm, or about 300nm.

In certain embodiments, the mode size diameter of the EV of the anti-inflammatory EV population as described herein is greater than about 300nm, greater than about 400nm, greater than about 500nm, greater than about 700nm, or greater than about 800nm. In certain embodiments, the mode-sized diameter of the EV of the anti-inflammatory EV population as described herein is from about 200nm to about 1000nm, from about 300nm to about 1000nm, from about 400nm to about 1000nm, from about 500nm to about 1000nm, from about 600nm to about 1000nm, from about 700nm to about 1000nm, from about 800nm to about 1000nm, from about 200nm to about 800nm, from about 300nm to about 800nm, from about 400nm to about 800nm, from about 500nm to about 800nm, from about 600nm to about 800nm, from about 200nm to about 600nm, from about 300nm to about 600nm, from about 400nm to about 600nm, from about 200nm to about 500nm, or from about 300nm to about 500nm.

In certain embodiments, the mode size diameter of an EV of an anti-inflammatory EV population as described herein is about 400nm, about 450nm, about 500nm, about 600nm, about 650nm, about 700nm, about 750nm, 800nm, about 850nm, about 900nm, about 950nm, or about 1000nm.

In certain aspects, the anti-inflammatory EV population as described herein is an anti-inflammatory EV population containing a buffer. The anti-inflammatory EVs described herein are derived from ex vivo expanded human suppressive immune cells, e.g., tregs. As explained in detail below, these populations of anti-inflammatory EVs can be isolated from cultures comprising ex vivo expanded human suppressor immune cells, e.g., tregs and culture medium. In some cases, as part of the method of isolating an EV from a culture, the medium may be replaced with a buffer, e.g., a sterile buffer, e.g., a buffer suitable for administration to a human, such as suitable for administration to a human for therapeutic use. In these cases, the resulting isolated, cell-free anti-inflammatory EV population may be referred to as an anti-inflammatory EV population containing a buffer.

Similarly, in certain embodiments, the anti-inflammatory EV population as described herein is an anti-inflammatory EV population containing saline. In a specific embodiment, the anti-inflammatory EV population as described herein is an anti-inflammatory EV population containing physiological saline. In a specific embodiment, the anti-inflammatory EV population as described herein is an anti-inflammatory EV population containing 0.9% saline. In a specific embodiment, the anti-inflammatory EV population as described herein is an anti-inflammatory EV population containing phosphate buffered saline.

The isolated, cell-free, anti-inflammatory EV populations described herein are substantially free of cellular material, particulates, or other contaminants (e.g., organelles, lipids, cholesterol) from a cell or tissue source from which the EV is derived, e.g., from human inhibitory immune cells from which the EV is derived, e.g., tregs. For example, the isolated, cell-free anti-inflammatory EV populations described herein typically contain less than about 5 weight percent, less than about 1 weight percent, less than about 0.5 weight percent, less than about 0.1 weight percent, or less than about 0.01 weight percent of cellular material, microparticles, or other contaminants (e.g., organelles, lipids, cholesterol) from the EV-derived cell or tissue source, e.g., from the EV-derived human inhibitory immune cell, e.g., treg.

In certain embodiments, the isolated, cell-free, anti-inflammatory EV population described herein is present in a composition that is substantially free of other EVs. For example, in certain embodiments, the isolated, cell-free, anti-inflammatory EV population described herein is present in a composition that contains less than about 20%, less than about 10%, less than about 5%, or less than about 1% of other EVs.

In certain embodiments, the isolated, cell-free anti-inflammatory EV population described herein is present in a composition comprising other EVs, wherein the isolated, cell-free anti-inflammatory EV population comprises about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or greater than about 95% of EVs in the composition. In specific embodiments, the other EV is a serum EV, e.g., a bovine serum EV or a human serum EV.

In certain embodiments, the cell-free anti-inflammatory EV population consists of anti-inflammatory EVs comprising one or more cargo heterologous to the EV, e.g., a drug, a detectable label, a protein, a nucleic acid, e.g., RNA, such as mRNA and/or miRNA. The resulting anti-inflammatory EV may have one or more cargo present within the EV and/or on the EV surface.

In certain embodiments, the cell-free anti-inflammatory EV population has been generated from Treg cells that have been genetically engineered, e.g., genetically engineered, to express cargo loaded into the EV at the time of their production. Alternatively, one or more cargoes (for example) may be introduced into the culture medium during the EV production process and loaded into the EV as it is produced. In other alternatives, one or more cargo may be introduced into the EV via these techniques, such as electroporation or sonication. The resulting anti-inflammatory EV may have one or more cargo present within the EV and/or on the EV surface.

5.1.1 Gene product expression profiling

The anti-inflammatory EV populations provided herein can be characterized by their gene product expression profile. For example, if the anti-inflammatory EV population is derived from a population of tregs cultured in a serum-containing medium that contains a serum EV (also referred to as a "medium EV"), the gene product expression profile of the anti-inflammatory EV population can be compared to that of the medium EV.

The change in gene product expression can be expressed as a "fold increase" or a "fold decrease" or as a log (or "log 2") of the fold change that is base two. In some embodiments, gene product expression is increased by at least about 4-fold. In some embodiments, the gene product expression is increased by about 5-10 fold, about 10-15 fold, about 15-20 fold, about 20-25 fold, about 25-30 fold, about 30-35 fold, about 35-40 fold, about 40-45 fold, about 45-50 fold, about 50-60 fold, about 60-70 fold, about 70-80 fold, about 80-90 fold, about 90-100 fold, or at least about 100 fold. In some embodiments, gene product expression is reduced by at least about 4-fold. In some embodiments, the gene product expression is increased by about 5-10 fold, about 10-15 fold, about 15-20 fold, about 20-25 fold, about 25-30 fold, about 30-35 fold, about 35-40 fold, about 40-45 fold, about 45-50 fold, about 50-60 fold, about 60-70 fold, about 70-80 fold, about 80-90 fold, about 90-100 fold, or at least about 100 fold.

In some embodiments, the anti-inflammatory EV population provided herein is characterized by its gene product expression profile as shown in table 10. In some embodiments, an anti-inflammatory EV population provided herein is characterized by detectable expression of at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or all 191 gene products listed in table 10. In specific embodiments, these gene products include at least one of MIF, LGALS3 and S100A4. In other specific embodiments, these gene products include at least MIF, LGALS3 and S100A4.

In some embodiments, an anti-inflammatory EV population provided herein is characterized by an increase in expression (e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or all 191 gene products) of at least 5, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold expression, at least 0.5, at least 1, at least 2, at least 3, at least 5, at least 7, at least 8, at least 9, or at least 10-fold log 2-fold expression and/or a statistically significant increase in expression (e.g., < 0.0.1, <0.05 or <0.001 >). In specific embodiments, these gene products include at least one of MIF, LGALS3 and S100A4. In other specific embodiments, these gene products include at least MIF, LGALS3 and S100A4.

By any method known in the art, e.g., real-time quantitative PCR, fluidigm ^TM Chip assays, RNA sequencing, or proteomic analysis (e.g., single-shot proteomics) determines gene product expression. In particular embodimentsIn this manner, gene product expression is determined by proteomic analysis (e.g., single shot proteomics). In particular embodiments, the proteomic analysis is performed by mass spectrometry.

Similarly, in this context, the population of tregs from which anti-inflammatory EVs provided herein are derived may be characterized by their gene product expression profile. For example, the gene product expression profile of the population of tregs provided herein can be compared to tregs at baseline. In this regard, the term "baseline" or "baseline population of Treg cells" means a population of tregs that has been enriched from a patient sample, but has not yet been expanded. In this aspect of Treg gene product expression as discussed herein, the 2 values are considered to be substantially identical when the difference between them is statistically insignificant (i.e., p > 0.05) and/or if the fold difference between the 2 values is less than about 2 times (high or low) as the base 2 logarithm.

The level of gene product expression may be above or below the detection limit of the method used to measure gene product expression. In some embodiments, the expression levels of the gene products listed in any one of tables 3-7 may be undetectable (i.e., below the detection level of the method used, such as single shot proteomics) in the Treg population from which the anti-inflammatory EVs provided herein are derived. In some casesIn embodiments, the expression level of the gene products listed in any one of tables 3-7 may be detectable (i.e., higher than the detection level of the method used, such as single shot proteomics) in the Treg population from which the anti-inflammatory EVs provided herein are derived. In some embodiments, the expression levels of the gene products listed in any one of tables 3-7 may become detectable or undetectable in the Treg population from which the anti-inflammatory EVs provided herein are derived by enrichment or expansion of the Treg population. By any method known in the art, e.g., real-time quantitative PCR, fluidigm ^TM Chip assays, RNA sequencing, or proteomic analysis (e.g., single-shot proteomics) determines gene product expression. In particular embodiments, gene product expression is determined by proteomic analysis (e.g., single shot proteomics). In particular embodiments, the proteomic analysis is performed by mass spectrometry.

In some embodiments, expression of one or more gene products listed in table 3 and/or table 4 is reduced in tregs from which anti-inflammatory EVs provided herein are derived after expansion compared to tregs at baseline. In some embodiments, expression of one or more gene products listed in table 3 is reduced in tregs from which anti-inflammatory EVs provided herein are derived after expansion compared to tregs at baseline. In some embodiments, expression of one or more gene products listed in table 4 is reduced in tregs from which anti-inflammatory EVs provided herein are derived after expansion compared to tregs at baseline.

In some embodiments, expression of one or more of the gene products listed in tables 5-9 is increased in tregs from which anti-inflammatory EVs provided herein are derived after expansion compared to tregs at baseline. In some embodiments, expression of one or more gene products (e.g., one or more of the gene products listed in table 3) associated with a dysfunctional Treg phenotype is reduced in tregs derived from an anti-inflammatory EV provided herein after expansion compared to tregs at baseline. Dysfunctional Treg phenotypes include, for example, deregulated calcium dynamics, loss of MeCP2 binding to 5-hydroxymethylcytosine (5 hmC) -DNA, dysregulation of MeCP2 expression or activity, and loss of MeCP2 regulation, phosphorylation, or binding capacity.

5.1.2 Treg EV surface marker profile

The anti-inflammatory EV populations provided herein can be characterized by their Treg EV surface marker profile. If the anti-inflammatory EV population is derived from a population of tregs cultured in a serum-containing medium that contains a serum EV (also referred to as a "medium EV"), the Treg EV surface marker profile of the anti-inflammatory EV population can be compared to the surface marker profile of the medium EV.

In particular embodiments, EV surface marker profiles may be generated by using antibody-based methods that detect epitopes on the EV surface. For example, well-known techniques using a mixture of multiple fluorescently labeled bead populations (each coated with a specific antibody that binds to a surface epitope of interest) can be used to generate EV surface marker profiles in conjunction with flow cytometry methods. For example, MACPlex exosome kit (Miltenyi Biotec) uses this technique. In a specific embodiment, the anti-inflammatory EV population provided herein is characterized by a Treg surface marker profile generated using the MACSPlex exosome kit (Miltenyi Biotec).

For example, the EV surface marker spectrum may be represented using relative intensity units, e.g., median Fluorescence Intensity (MFI) units, such that the EV surface marker spectrum represents the detectable presence or absence of the marker being assayed and additionally represents the relative amounts of these markers.

In specific examples, an anti-inflammatory EV population as described herein shows a Treg EV surface marker profile as shown in example 21 below ("Treg EV surface marker signature"). In specific examples, the anti-inflammatory EV population as described herein shows Treg EV surface marker profiles as shown in example 21 ("Treg EV surface marker features") below generated using the techniques described in example 21, e.g., using the MACSPlex exosome kit (Miltenyi Biotec).

Each surface marker (e.g., CD2, CD25, etc.) is well known, as are antibodies specific for epitopes present on these markers. It should be noted that "HLA-DRDPDQ" refers to HLA class II molecules HLA-DR, HLA-DP and HLA-DQ. Since Treg EVs described herein are typically derived from ex vivo expanded human Treg cells, in particular embodiments, these surface markers are human (e.g., human CD2, human HLA-DRDPDQ, CD25, etc.).

In certain embodiments, the anti-inflammatory EV populations provided herein are characterized by Treg EV surface marker profiles comprising at least one, two, or all three of CD2, HLA-DRDPDQ, and/or CD 25. In a specific embodiment, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising CD2, HLA-DRDPDQ, and CD 25. In a specific embodiment, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising CD2, HLA-DRDPDQ, and CD25, wherein CD2 is present in an amount at least 1-fold, 2-fold, or 5-fold higher than HLA-DRDPDQ and/or CD25, as measured using an assay for measuring or identifying Treg EV surface marker profiles, e.g., a MACSPlex exosome kit assay.

As used herein, when describing an anti-inflammatory EV population provided herein as characterized by a Treg surface marker profile comprising one or more of the listed markers, this means that the anti-inflammatory EV population comprising a detectable amount of the markers is using an assay for measuring or identifying Treg EV surface marker profiles. Such assays are well known in the art and, for example, antibody-based detection assays, e.g., antibody-based detection techniques comprising detection of antibodies specific for a surface marker epitope, either directly or indirectly labeled, e.g., fluorescently labeled. Such an assay may, for example, comprise a bead coated with an antibody specific for a surface marker epitope in combination with flow cytometry using a differential label. In particular embodiments, such assays can be assayed using the MACSPlex exosome kit, as described, for example, in example 21 ("Treg EV surface marker features") below.

In certain embodiments, the anti-inflammatory EV population provided herein is characterized by comprising at least one, two, or all three of CD2, HLA-DRDPDQ, and/or CD25, and substantially lacking at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all of the Treg EV surface marker profile of: CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31 and/or CD14.

In certain embodiments, the anti-inflammatory EV population provided herein is characterized by comprising CD2, HLA-DRDPDQ, and/or CD25 and is substantially devoid of Treg EV surface marker profiles of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all of the following: CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31 and/or CD14.

In certain embodiments, the anti-inflammatory EV population provided herein is characterized as comprising CD2, HLA-DRDPDQ, and CD25, and is substantially devoid of the following Treg EV surface marker profile: CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31 and CD14.

In particular embodiments, "substantial absence" as used herein may mean a level below the detection level using an assay for measuring or identifying Treg EV surface marker profiles, e.g., a MACSPlex exosome kit assay. In particular embodiments, "substantial absence" as used herein may mean a level at least 5-fold, 10-fold, 15-fold, or 20-fold lower than the level of CD2, HLA-DRDPDQ, and/or CD25 as measured using an assay for measuring or identifying Treg EV surface marker profiles, e.g., a MACSPlex exosome assay, if these markers are part of the Treg EV surface marker profile. In particular embodiments, CD2 is part of a Treg EV surface marker profile, and "substantially absent" as used herein means at least 5-fold, 10-fold, 15-fold, or 20-fold lower levels than CD2 as measured using an assay for measuring or identifying Treg EV surface marker profile, e.g., a MACSPlex exosome kit assay.

In certain embodiments, the anti-inflammatory EV populations provided herein are characterized by a Treg EV surface marker profile comprising at least one, two, or all three of CD2, HLA-DRDPDQ, and/or CD25 and further comprising at least one of CD44, CD29, CD4, and/or CD 45. In certain embodiments, the anti-inflammatory EV populations provided herein are characterized by a Treg EV surface marker profile comprising CD2, HLA-DRDPDQ, and CD25 and further comprising at least one of CD44, CD29, CD4, and/or CD 45. In certain embodiments, the anti-inflammatory EV populations provided herein are characterized by Treg EV surface marker profiles comprising CD2, HLA-DRDPDQ, and CD25 and further comprising CD44, CD29, CD4, and CD 45. In certain embodiments, the anti-inflammatory EV populations provided herein are characterized by a Treg EV surface marker profile comprising CD2, HLA-DRDPDQ, and CD25 and further comprising CD44, CD29, CD4, and CD45, wherein CD2 is present in an amount at least 1-fold, 2-fold, or 5-fold higher than HLA-DRDPDQ and/or CD25, and CD44, CD29, CD4, and CD45, as measured using an assay for measuring or identifying Treg EV surface marker profile, e.g., a MACSPlex exor kit assay.

In any of the embodiments of such anti-inflammatory EV populations described directly above, such populations are further characterized by a substantial absence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or all of the Treg EV surface marker profiles of: CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31 and/or CD14. In any of the embodiments of such anti-inflammatory EV populations described directly above, such populations are further characterized by the substantial absence of the following Treg EV surface marker profile: CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31 and CD14.

In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) At least one, two or all three of CD2, HLA-DRDPDQ and/or CD25, ii) further comprises at least one of CD44, CD29, CD4 and/or CD45, and iii) further comprises at least one of the following: HLA-ABC, CD24, CD69, CD41b and/or CD42a. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises at least one of CD44, CD29, CD4 and/or CD45, and iii) further comprises at least one of: HLA-ABC, CD24, CD69, CD41b and/or CD42a, e.g., at least one of HLA-ABC, CD24 and/or CD 69. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45 and iii) further comprises at least one of the following: HLA-ABC, CD24, CD69, CD41b and/or CD42a, e.g., at least one of HLA-ABC, CD24 and/or CD 69. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45 and iii) further comprises HLA-aBC, CD24 and CD69 and optionally CD41b and CD42a. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45 and iii) further comprises HLA-aBC, CD24 and CD69, and optionally CD41b and CD42a, wherein CD2 is present in an amount at least 1, 2 or 5 times higher than HLA-DRDPDQ and/or CD25, and CD44, CD29, CD4, CD45, HLA-aBC, CD24, CD69 and (if present) CD41b and CD42a, as measured using an assay for measuring or identifying Treg EV surface marker profile, e.g., a MACSPlex exosome assay.

In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) At least one, two or all three of CD2, HLA-DRDPDQ and/or CD25, ii) further comprises at least one of CD44, CD29, CD4 and/or CD45, iii) further comprises at least one of the following: HLA-ABC, CD24, CD69, CD41b and/or CD42a, e.g., at least one of HLA-ABC, CD24 and/or CD 69; and iv) further comprising at least one exosome or EV marker, e.g., at least one, two or all three of: CD81, CD63 and/or CD9. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises at least one of CD44, CD29, CD4 and/or CD45, iii) further comprises at least one of the following: HLA-ABC, CD24, CD69, CD41b and/or CD42a, e.g., at least one of HLA-ABC, CD24 and/or CD 69; and iv) further comprising at least one exosome or EV marker, e.g., at least one, two or all three of: CD81, CD63 and/or CD9. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45, iii) further comprises at least one of the following: HLA-ABC, CD24, CD69, CD41b and/or CD42a, e.g., at least one of HLA-ABC, CD24 and/or CD 69; and iv) further comprising at least one exosome or EV marker, e.g., at least one, two or all three of: CD81, CD63 and/or CD9. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45, iii) further comprises at least one of the following: HLA-ABC, CD24, CD69, and optionally CD41b and CD42a; and iv) further comprising at least one exosome or EV marker, e.g., at least one, two or all three of: CD81, CD63 and/or CD9. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45, iii) further comprises at least one of the following: HLA-ABC, CD24, CD69, and optionally CD41b and CD42a; and iv) further comprising at least one, two or all three of the following: CD81, CD63 and/or CD9. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45, iii) further comprises HLA-aBC, CD24, CD69, and optionally CD41b and CD42a; and iv) further comprising at least one, two or all three of the following: CD81, CD63 and CD9. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45, iii) further comprises HLA-aBC, CD24, CD69, and optionally CD41b and CD42a; and iv) further comprises CD81, CD63 and CD9. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45, iii) further comprises HLA-aBC, CD24, CD69, CD41b and CD42a; and iv) further comprises CD81, CD63 and CD9. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45, iii) further comprises HLA-aBC, CD24, CD69, CD41b and CD42a; and iv) further comprising CD81, CD63 and CD9, wherein CD2 is present in an amount at least 1-fold, 2-fold or 5-fold higher than HLA-DRDPDQ and/or CD25, and CD44, CD29, CD4, CD45, HLA-aBC, CD24, CD69, CD42a, CD81, CD63 and CD9, as measured using an assay for measuring or identifying Treg EV surface marker profile, e.g., a MACSPlex exosome kit assay.

In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45, iii) further comprises HLA-aBC, CD24, CD69, CD41b and CD42a; iv) also comprises CD81, CD63 and CD9; and wherein the Treg EV surface marker profile is characterized by the substantial absence of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31 and CD14.

In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45, iii) further comprises HLA-aBC, CD24, CD69, CD41b and CD42a; iv) further comprising CD81, CD63 and CD9, wherein CD2 is present in an amount at least 1-fold, 2-fold or 5-fold higher than HLA-DRDPDQ and/or CD25, and CD44, CD29, CD4, CD45, HLA-aBC, CD24, CD69, CD42a, CD81, CD63 and CD9, as measured using an assay for measuring or identifying Treg EV surface marker profile, e.g., a MACSPlex exosome kit assay; and wherein the Treg EV surface marker profile is characterized by the substantial absence of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31 and CD14.

In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) At least one, two or all three of CD2, HLA-DRDPDQ and/or CD25, ii) further comprises at least one of CD44, CD29, CD4 and/or CD45, and iii) further comprises at least one of the following: HLA-ABC, CD24 and/or CD69. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises at least one of CD44, CD29, CD4 and/or CD45, and iii) further comprises at least one of: HLA-ABC, CD24 and/or CD69. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45 and iii) further comprises at least one of the following: HLA-ABC, CD24 and/or CD69. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45 and iii) further comprises HLA-aBC, CD24 and CD69.

In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) At least one, two or all three of CD2, HLA-DRDPDQ and/or CD25, ii) further comprises at least one of CD44, CD29, CD4 and/or CD45, iii) further comprises at least one of HLA-aBC, CD24 and/or CD69; and iv) further comprising at least one exosome or EV marker, e.g., at least one, two or all three of: CD81, CD63 and/or CD9. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises at least one of CD44, CD29, CD4 and/or CD45, iii) further comprises at least one of HLA-aBC, CD24 and/or CD69; and iv) further comprising at least one exosome or EV marker, e.g., at least one, two or all three of: CD81, CD63 and/or CD9. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprising CD44, CD29, CD4 and CD45, iii) further comprising at least one of HLA-aBC, CD24 and/or CD69; and iv) further comprising at least one exosome or EV marker, e.g., at least one, two or all three of: CD81, CD63 and/or CD9. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45, iii) further comprises at least one of the following: HLA-ABC, CD24 and CD69; and iv) further comprises at least one exosome or EV marker, e.g., at least one, two or all three of: CD81, CD63 and/or CD9. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45, iii) further comprises at least one of the following: HLA-ABC, CD24 and CD69; and iv) further comprising at least one, two or all three of the following: CD81, CD63 and/or CD9. In certain embodiments, the anti-inflammatory EV population provided herein is characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ and CD25, ii) further comprises CD44, CD29, CD4 and CD45, iii) further comprises at least one of the following: HLA-ABC, CD24 and CD69; and iv) further comprises CD81, CD63 and CD9.

5.1.3Treg EV RNA Spectrum

The anti-inflammatory EV populations provided herein can be characterized by their Treg EV RNA profile, characterized by the microrna (miRNA) profile of the anti-inflammatory EV population as described herein.

The Treg EV RNA profile may be generated using well known techniques, for example, those techniques as described in example 22 below ("Treg EV RNA profile"). For example, RNAs can be isolated from an EV population, small RNAs can be enriched, e.g., for RNAs about 15-200, e.g., 17-200 nucleotides long, and sequenced, wherein the sequences are counted when they are identified as corresponding to sequences associated with known small mirnas. Such "read counts" can be used to evaluate the abundance of a given miRNA within an EV population.

In one embodiment, the anti-inflammatory EV population provided herein is described as having the following characteristics: treg RNA profile comprising three, four or all five of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and/or hsa-miR-21-5p. In certain embodiments, the anti-inflammatory EV population provided herein is described as having the following characteristics: a Treg RNA profile comprising each of: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p.

In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-155-5 p. In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-1290 and hsa-miR-155-5p and this population comprises hsa-miR-1290 in an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than hsa-miR-155-5p.

In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Three, four or all five of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and/or hsa-miR-21-5p; and ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p. In certain embodiments, the anti-inflammatory EV population provided herein is described as having the following characteristics: a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p and ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p.

In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p. In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-1290 and hsa-miR-155-5p and this population comprises hsa-miR-1290 in an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than hsa-miR-155-5p. In other embodiments of any of these anti-inflammatory EV populations described directly above, the population comprises at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than any of the mirnas of those listed at ii). In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-1555-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-155-5 p. And the population comprises hsa-miR-1290 and hsa-miR-146a-5p in an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold greater than any of the miRNAs of those listed at ii).

In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Three, four or all five of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and/or hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; and iii) any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p. In certain embodiments, the anti-inflammatory EV population provided herein is described as having the following characteristics: a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; and iii) any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p. In certain embodiments, the anti-inflammatory EV population provided herein is described as having the following characteristics: a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; and iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p.

In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p. In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-1290 and hsa-miR-155-5p and this population comprises hsa-miR-1290 in an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than hsa-miR-155-5p. In other embodiments of any of these anti-inflammatory EV populations described directly above, the population comprises at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than any of the mirnas listed at ii) and iii). In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p; and the population comprises hsa-miR-1290 and hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold greater than any of the miRNAs of those listed at ii) and iii).

In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Three, four or all five of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and/or hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; and iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; and any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; and any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; and any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; and each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p.

In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p. In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-1290 and hsa-miR-155-5p and this population comprises hsa-miR-1290 in an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than hsa-miR-155-5p. In other embodiments of any of these anti-inflammatory EV populations described directly above, the population comprises at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than any of the mirnas listed at ii) and iii). In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p; and the population comprises hsa-miR-1290 and hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold greater than any of the miRNAs of those listed at ii) to iv).

In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Three, four or all five of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and/or hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; and v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; and v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; and v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; and v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; and v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; and v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p.

In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p. In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-1290 and hsa-miR-155-5p and this population comprises hsa-miR-1290 in an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than hsa-miR-155-5p. In other embodiments of any of these anti-inflammatory EV populations described directly above, the population comprises at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than any of the mirnas listed at ii) and iii). In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p; and the population comprises hsa-miR-1290 and hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold greater than any of the miRNAs of those listed at ii) to v).

In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Three, four or all five of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and/or hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; and vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; and vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; and vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; and vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; and vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; and vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; and vi) each of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p.

In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p. In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-1290 and hsa-miR-155-5p and this population comprises hsa-miR-1290 in an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than hsa-miR-155-5p. In other embodiments of any of these anti-inflammatory EV populations described directly above, the population comprises at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than any of the mirnas listed at ii) and iii). In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p; and the population comprises hsa-miR-1290 and hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold greater than any of the miRNAs of those listed at ii) to vi).

In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Three, four or all five of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and/or hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; and vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; and vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; and vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; and vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; and vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; and vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; and vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; and vii) each of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and hsa-miR-103a-3p.

In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p. In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-1290 and hsa-miR-155-5p and this population comprises hsa-miR-1290 in an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than hsa-miR-155-5p. In other embodiments of any of these anti-inflammatory EV populations described directly above, the population comprises at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than any of the mirnas listed at ii) and iii). In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p; and the population comprises hsa-miR-1290 and hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold greater than any of the miRNAs of those listed at ii) to vii).

In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Three, four or all five of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and/or hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; and viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; and viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; and viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; and viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; and viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; and viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; and viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; vii) each of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and hsa-miR-103a-3p; and viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; vii) each of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and hsa-miR-103a-3p; and viii) each of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and hsa-miR-625-5p.

In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p. In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-1290 and hsa-miR-155-5p and this population comprises hsa-miR-1290 in an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than hsa-miR-155-5p. In other embodiments of any of these anti-inflammatory EV populations described directly above, the population comprises at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than any of the mirnas listed at ii) and iii). In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p; and the population comprises hsa-miR-1290 and hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold greater than any of the miRNAs of those listed at ii) to viii).

In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Three, four or all five of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and/or hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; and ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; and ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; and ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; and ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; and ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; and ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; and ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of 1-5 of: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; vii) each of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; and ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; and ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of 1-5 of: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; vii) each of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and hsa-miR-103a-3p; viii) each of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and hsa-miR-625-5p; and ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of 1-5 of: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; vii) each of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and hsa-miR-103a-3p; viii) each of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and hsa-miR-625-5p; and ix) each of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and hsa-miR-98-5p.

In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p. In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-1290 and hsa-miR-155-5p and this population comprises hsa-miR-1290 in an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than hsa-miR-155-5p. In other embodiments of any of these anti-inflammatory EV populations described directly above, the population comprises at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than any of the mirnas listed at ii) and iii). In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p; and the population comprises hsa-miR-1290 and hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold greater than any of the miRNAs of those listed at ii) to ix).

In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Three, four or all five of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and/or hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p; and x) any 1 to 5 of the following: hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p and/or hsa-miR-342-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) any 1-5 of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and/or hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p; and x) any 1 to 5 of the following: hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p and/or hsa-miR-342-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Any 1-5 of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and/or hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p; and x) any 1 to 5 of the following: hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p and/or hsa-miR-342-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) any 1-5 of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and/or hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p; and x) any 1 to 5 of the following: hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p and/or hsa-miR-342-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) any 1-5 of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and/or hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p; and x) any 1 to 5 of the following: hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p and/or hsa-miR-342-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) any 1-5 of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and/or hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and hsa-miR-625-5p; ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and hsa-miR-98-5p; and x) any 1 to 5 of the following: hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p and hsa-miR-342-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; vii) any 1-5 of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and/or hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p; and x) any 1 to 5 of the following: hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p and/or hsa-miR-342-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; vii) each of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and hsa-miR-103a-3p; viii) any 1-5 of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and/or hsa-miR-625-5p; ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p; and x) any 1 to 5 of the following: hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p and/or hsa-miR-342-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; vii) each of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and hsa-miR-103a-3p; viii) each of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and hsa-miR-625-5p; ix) any 1-5 of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and/or hsa-miR-98-5p; and x) any 1 to 5 of the following: hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p and/or hsa-miR-342-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; vii) each of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and hsa-miR-103a-3p; viii) each of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and hsa-miR-625-5p; ix) each of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and hsa-miR-98-5p; and x) any 1 to 5 of the following: hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p and/or hsa-miR-342-5p. In one embodiment, the anti-inflammatory EV population provided herein is described as characterized by a Treg RNA profile comprising: i) Each of the following: hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p and hsa-miR-21-5p; ii) each of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p and hsa-miR-320a-3p; iii) Each of the following: hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-26a-5p; iv) each of the following: hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p and hsa-miR-342-3p; v) each of the following: hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p and hsa-miR-181a-5p; vi) each of the following: hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p and hsa-miR-17-5p; vii) each of the following: hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p and hsa-miR-103a-3p; viii) each of the following: hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p and hsa-miR-625-5p; ix) each of the following: hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p and hsa-miR-98-5p; and x) each of the following: hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p and hsa-miR-342-5p.

In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p. In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-1290 and hsa-miR-155-5p and this population comprises hsa-miR-1290 in an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than hsa-miR-155-5p. In other embodiments of any of these anti-inflammatory EV populations described directly above, the population comprises at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than any of the mirnas listed at ii) and iii). In certain embodiments of any such anti-inflammatory EV population described directly above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a ratio of about 1-10, e.g., 1.5-6, e.g., about 2, about 3, about 2 to about 4, or about 5 hsa-miR-146a-5p/hsa-miR-15-5 p; and the population comprises hsa-miR-1290 and hsa-miR-146a-5p at an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold greater than any of the miRNAs of those listed at ii) to x).

5.2 methods of producing anti-inflammatory EV

In certain aspects, provided herein are methods of producing an isolated, cell-free, anti-inflammatory EV population.

In certain embodiments, provided herein are methods of producing an isolated, cell-free, anti-inflammatory EV population, wherein the method comprises: a) Expanding a population of human suppressive immune cells ex vivo in a medium to produce a culture comprising the population of human suppressive immune cells, the medium and an anti-inflammatory EV (without wishing to be bound by theory or mechanism, it is believed that during ex vivo expansion, the EV is released into the culture by the human suppressive immune cells), and b) isolating the anti-inflammatory EV from the culture.

In certain embodiments, provided herein are methods of producing an isolated, cell-free, anti-inflammatory EV population, wherein the method comprises: a) Expanding a population of human suppressive immune cells ex vivo in a medium to produce a culture comprising the population of tregs, a medium, and an anti-inflammatory EV, wherein the population of human suppressive immune cells is a population of regulatory T cells (tregs), and b) isolating the anti-inflammatory EV from the culture.

Methods for obtaining, optionally enriching and ex vivo expanding, human suppressive immune cells, e.g. tregs, are well known. An exemplary method is provided below.

For ease of description, the method of producing an isolated, cell-free, anti-inflammatory EV population may be expressed herein as comprising: a) Expanding a population of human suppressive immune cells ex vivo in a medium to produce a culture comprising the population of human suppressive immune cells, the medium and an anti-inflammatory EV, and b) isolating the anti-inflammatory EV from the culture. However, it will be appreciated that isolation of the anti-inflammatory EV from the culture may be performed at any point once ex vivo amplification begins or may be repeated during the period of ex vivo amplification.

For example, the ex vivo amplification may comprise multiple rounds of amplification. In one non-limiting example, the EV-containing medium from which the EV can be isolated may be collected at the end of one or more rounds of amplification. In another non-limiting example, the culture medium comprising EV may be collected at a point during amplification when the culture medium is replenished or changed. In another non-limiting example, the EV may be isolated at the end of the ex vivo amplification process. In another non-limiting example, the EV may be isolated at multiple points during the ex vivo amplification process, for example, at one or more of the points mentioned above.

In certain embodiments, the ex vivo amplification period lasts about 24 hours, 48 hours, or 72 hours. In certain embodiments, the ex vivo amplification period lasts for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 10 days, 14 days, 2 weeks, 3 weeks, or more.

In some embodiments, the EV may be isolated after about 24 hours, 48 hours, or 72 hours of amplification. In some embodiments, the EV may be isolated about 24 hours, about 48 hours, or about 72 hours after the medium is replenished or changed. In some embodiments, the EV is isolated every 2, 3, 4, or 5 days.

In certain embodiments, the culture medium comprising the EV may be collected at one or more points during the amplification process and isolation of the EV is initiated at the time the culture medium is collected, e.g., within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, or overnight after the culture medium is collected. In particular embodiments, the culture medium comprising the EV may be collected at one or more points during the amplification process and stored at 4 ℃ prior to EV isolation. In particular embodiments, for example, the culture medium comprising the EV may be collected at one or more points during the amplification process and may be stored at 4 ℃ for about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, or overnight prior to isolation of the EV from the culture medium.

In certain embodiments, the culture medium comprising the EV may be collected at one or more points during the amplification process and stored, e.g., frozen, prior to EV isolation.

In particular embodiments, the EV may be isolated from the cell culture by centrifugation, e.g., differential centrifugation. In certain embodiments, differential centrifugation may be used to isolate the desired EV subpopulation. For example, differential centrifugation can be used to isolate EV subpopulations enriched for smaller particle size sizes (e.g., exosomes; EVs with particle sizes less than about 300nm, less than about 200nm, less than about 160nm, less than about 150nm, less than about 130nm, less than about 100nm, or less than about 80 nm). In a specific non-limiting example, a step of centrifugation at 2,000g (3,000 rpm) for 20min can be used to remove cell debris and dead cells, and centrifugation at 16,500g (9,800 rpm) for 45min or at 100,000g (26,450 rpm) for 2h to specifically isolate exosomes.

The EVs can also be purified using gradient density centrifugation, which separates EVs from the culture based on their buoyant density in sucrose, iohexol or iodixanol solutions.

Other examples of methods for isolating EVs include precipitation with organic solvents (e.g., polyethylene glycol, sodium acetate, or protamine), immunoprecipitation, separation using antibody-coated magnetic beads, microfluidic devices, and ultrafiltration, which are described, for example, in Carnino et al Respiratory Research (2019) 20:240 and Momen-Heravi et al biol. Chem.2013;394 (10):1253-1262. Other exemplary methods are isolation using heparin-conjugated agarose beads (see, e.g., balaj et al (2015) Sci Rep 5,10266) and purification using Tim 4-affinity purification (see, e.g., nakai et al (2016) Sci Rep 6,33935).

Commercial kits for EV isolation are also available. Non-limiting examples include the exoEasy kit (Qiagen),Kit (Systems Bioscience) and easy Sep ^TM Human ubiquitin extracellular vesicles positive selection kit (Stem Cell Technologies).

In some embodiments, a method of producing an isolated, cell-free anti-inflammatory EV population provided herein comprises the steps of: (a) Expanding a population of human suppressive immune cells (e.g., a population of Treg cells) ex vivo in a medium to produce a culture comprising cells, medium, and an anti-inflammatory EV; and (b) isolating the anti-inflammatory EV from the culture.

In certain embodiments, the method of generating an isolated, cell-free population of anti-inflammatory EVs, isolating an anti-inflammatory EV from the culture comprises polyethylene glycol (PEG) precipitation. In a specific embodiment, PEG is added to the culture, thereby precipitating EV from the culture. After removing the EV-containing precipitate from the culture, the EV is washed to produce an isolated, cell-free, anti-inflammatory EV population.

An exemplary, non-limiting procedure for isolating EVs from cells (e.g., tregs) using PEG precipitation may include the steps of: (i) Centrifugation of medium from cell cultures (e.g., ex vivo expanded human suppressor immune cells, e.g., tregs, cell cultures) at 3000×g for 15 minutes to remove cells and debris; (ii) adding a PEG reagent to the supernatant, e.g., in PEG: supernatant 1:5 ratio; (iii) thoroughly mixing; (iv) cold storage overnight at 4 ℃; (v) centrifugation at 1500 Xg for 30 minutes; (vi) aspirating the supernatant; (vii) centrifuging again at 1500 Xg for 10 minutes; (viii) Removing the supernatant, e.g., via aspiration; and (ix) resuspending the resulting EV particles in a sterile buffer, e.g., sterile PBS.

Filtration, e.g., tangential Flow Filtration (TFF), may also be used to isolate EVs from cell cultures. For example, TFF can be used to effectively isolate and concentrate EV populations in a scalable and reproducible manner, even when starting with large culture volumes.

In particular embodiments, for example, the isolating step (b) comprises removing cells from the culture to produce a cell-free, anti-inflammatory EV population.

In a specific embodiment, for example, the separation step (b) comprises the steps of: (i) Removing cells from the culture to produce a solution containing cell-free, anti-inflammatory EV; and (ii) isolating the anti-inflammatory EV from the cell-free, anti-inflammatory EV-containing solution of (i). Steps (i) and (ii) may be performed separately as separate steps, e.g. sequentially, or may be accomplished as a single step.

In some embodiments, step (b) comprises filtering, e.g., one or more filtering steps. In particular embodiments, the filtering comprises TFF. For example, some or all of the filtering may use TFF. For example, filtration may be used to remove cells and debris from the culture. Filtration may also be used to isolate and concentrate EVs, for example, EVs having a particular size or range of sizes. In certain embodiments, removal of cells and debris and separation of EVs, e.g., EVs of a particular size or range of sizes, may be achieved using a single filtration step. In other embodiments, for example, a series of (two or more) filtration steps may be used to remove cells and debris and isolate EVs, isolating EVs of a particular size range. For example, one or more filtration steps may be used to first remove cells and debris to produce a solution containing EVs, followed by one or more filtration steps to separate and concentrate EV populations from the solution, e.g., to separate and concentrate EVs of a particular size or size range from the solution.

In some embodiments, step (b), e.g., step (i), comprises filtration, e.g., microfiltration (e.g., microfiltration by TFF). For example, the culture may be passed through a filter, e.g., a 0.05 μm, 0.1 μm, 0.2 μm, 0.45 μm, 0.65 μm, or 0.8 μm filter, to remove cells and any debris from the culture to produce a solution containing a cell-free anti-inflammatory EV that includes the anti-inflammatory population. In particular embodiments, the culture may be passed through a 0.65 μm filter to remove cells and any debris from the culture to produce a solution containing a cell-free anti-inflammatory EV that includes the anti-inflammatory population. In particular embodiments, TFF may be used to circulate the culture through a filter, e.g., a 0.05 μm, 0.1 μm, 0.2 μm, 0.45 μm, 0.65 μm, or 0.8 μm filter, to remove cells and any debris from the culture to produce a solution containing a cell-free anti-inflammatory EV including the anti-inflammatory EV population. In particular embodiments, TFF may be used to circulate the culture through a 0.65 μm filter to remove cells and any debris from the culture to produce a solution containing cell-free anti-inflammatory EVs including the anti-inflammatory EV population. In a specific embodiment, the filter used in step (i) has a length of 85cm ² Is a membrane area of (a). In a specific embodiment, the filter used in step (i) is a hollow fiber filter. In a specific embodiment, the filter used in step (i) is a hollow fiber filter with a fiber diameter of 0.75 mm. One or more rounds of filtering may be used. One or more filter sizes may be used. In addition to the removal of cells and debris, it is understood that such filtration may also be used to isolate EVs of a particular size or range of sizes.

In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is performed at a flow rate of 20-1000 mL/min. In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is performed at a flow rate of 50-500 mL/min. In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is performed at a flow rate of 100-200 mL/min. In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is performed at a flow rate of about 100 mL/min. In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is performed at a flow rate of about 150 mL/min. In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is performed at a flow rate of about 200 mL/min.

In a specific embodiment, at about 2,000 to 5,000 seconds ^-1 The microfiltration in step (i) is performed using a hollow fiber filter (e.g., microfiltration by TFF). In a specific embodiment, at about 2,000 to 3,000 seconds ^-1 The microfiltration in step (i) is performed using a hollow fiber filter (e.g., microfiltration by TFF). In a specific embodiment, at about 3,000 to 4,000 seconds ^-1 The microfiltration in step (i) is performed using a hollow fiber filter (e.g., microfiltration by TFF). In a specific embodiment, at about 4,000 to 5,000 seconds ^-1 The microfiltration in step (i) is performed using a hollow fiber filter (e.g., microfiltration by TFF). In a specific embodiment, at about 2,000s ^-1 The microfiltration in step (i) is performed using a hollow fiber filter (e.g., microfiltration by TFF). In a specific embodiment, at about 3,000 seconds ^-1 The microfiltration in step (i) is performed using a hollow fiber filter (e.g., microfiltration by TFF). In a specific embodiment, at about 4,000s ^-1 The microfiltration in step (i) is performed using a hollow fiber filter (e.g., microfiltration by TFF). In a specific embodiment, at about 5,000 seconds ^-1 The microfiltration in step (i) is performed using a hollow fiber filter (e.g., microfiltration by TFF). Shear rate is a term used for hollow fiber membranes and is affected by flow rate and fiber radius. Although typical shear rate values are 2000-12000s ^-1 But preferably in step (i)Maintaining a shear rate of about 2,000 to 5,000 seconds ^-1 (and not higher) to avoid EV fragmentation and to result in high EV recovery efficiency (e.g., recovery of EV greater than 90% or greater than 95%). In a specific embodiment, the shear rate maintained in step (i) is from about 2,000 to about 5,000 seconds ^-1 The flow rate was 100-200mL/min and a hollow fiber filter with a fiber diameter of 0.75mm was used.

In certain embodiments, the hold-up hydraulic pressure of step (i) is maintained at about 5psi. In a specific embodiment, the shear rate maintained in step (i) is from about 2,000 to about 5,000 seconds ^-1 A flow rate of 100-200mL/min and using a hollow fiber filter with a fiber diameter of about 0.75mm resulted in a retained hydraulic pressure of about 5psi.

In some embodiments, step (b) comprises step (ii), and step (ii) may comprise filtration, e.g., ultrafiltration (e.g., ultrafiltration by TFF). In a specific embodiment, step (ii) comprises the step of passing the solution containing the cell-free, anti-inflammatory EV through a filter, thereby retaining the anti-inflammatory EV through the filter, e.g., an anti-inflammatory EV of a particular size or size range. In a specific embodiment, step (ii) comprises the step of circulating a solution containing the cell-free, anti-inflammatory EV through a filter using TFF, thereby retaining the anti-inflammatory EV through said filter. One or more rounds of filtering may be used. One or more filter sizes may be used. Step (ii) may also be used to concentrate EV. In a specific embodiment, the final volume of the EV-containing solution after concentration is about 5-200mL. In a specific embodiment, the final volume of the EV-containing solution after concentration is about 10-100mL. In a specific embodiment, the final volume of the EV-containing solution after concentration is about 10-50mL. In a specific embodiment, the final volume of the EV-containing solution after concentration is about 10mL. In a specific embodiment, the final volume of the EV-containing solution after concentration is about 15mL. In a specific embodiment, the final volume of the EV-containing solution after concentration is about 20mL. In a specific embodiment, the final volume of the EV-containing solution after concentration is about 25mL. In a specific embodiment, the final volume of the EV-containing solution after concentration is about 30mL.

In some embodiments, at least one filter used in step (ii) has the following molecular weight cut-off (MWCO): about 50 kilodaltons (kDa) to about 750kDa, about 100kDa to about 750kDa, about 300kDa to about 750kDa, or about 300kDa to about 500kDa. In some embodiments, the filter has the following MWCO: about 50kDa, about 60kDa, about 70kDa, about 80kDa, about 90kDa, about 100kDa, about 110kDa, about 120kDa, about 150kDa, about 200kDa, about 300kDa, about 400kDa, about 500kDa, about 600kDa, about 700kDa or about 750kDa. In one embodiment, the filter has an MWCO of about 500kDa. In some embodiments, the filter used in step (ii) has a pore size of about 0.3 μm, about 0.22 μm, about 0.2 μm, or about 0.1 μm. In a specific embodiment, the filter used in step (ii) has a length of 115cm ² Is a membrane area of (a). In a specific embodiment, the filter used in step (ii) is a hollow fiber filter. In a specific embodiment, the filter used in step (ii) is a hollow fiber filter with a fiber diameter of 0.5 mm.

In certain embodiments, step (ii) is designed to retain an EV having a particular particle size or size range, e.g., to retain an EV of greater than about 50nm to about 60nm, about 60nm to about 80nm, about 60nm to about 70nm, about 70nm to about 80nm, about 100nm, about 150nm, or about 200 nm.

In certain embodiments, step (ii) is designed to preserve EVs of greater than about 50nm to about 60nm and includes the use of filters with MWCO of about 300 kDa. In certain embodiments, step (ii) is designed to preserve EVs greater than about 50nm and includes the use of filters with MWCO of about 300 kDa. In certain embodiments, step (ii) is designed to preserve EVs of greater than about 70nm to about 80nm and includes the use of filters with MWCO of about 500 kDa. In certain embodiments, step (ii) is designed to preserve EVs greater than about 70nm and includes the use of filters with MWCO of about 500 kDa. In certain embodiments, step (ii) is designed to preserve EVs greater than about 80nm and includes the use of filters with MWCO of about 500 kDa. In certain embodiments, step (ii) is designed to preserve EVs greater than about 60nm and includes the use of filters with MWCO of about 500 kDa.

In some embodiments, step (b), e.g., step (b) (ii), comprises performing a buffer exchange, such that the resulting isolated, cell-free anti-inflammatory EV population is an isolated, cell-free anti-inflammatory EV population containing a buffer. In a specific embodiment, the buffer exchange comprises diafiltration. In particular embodiments, buffer exchange includes TFF and diafiltration. In a specific embodiment, diafiltration is effected at 2X-100X. In a specific embodiment, diafiltration is effected at 5X-50X. In a specific embodiment, diafiltration is effected at 5X-20X. In a specific embodiment, diafiltration is effected at 5X. In a specific embodiment, diafiltration is effected at 10X. In a specific embodiment, diafiltration is effected at 15X. In a specific embodiment, diafiltration is effected at 20X.

For example, in some embodiments, step (b) comprises step (ii), and step (ii) may comprise the step of circulating a solution containing a cell-free, anti-inflammatory EV through a filter using TFF, thereby retaining the anti-inflammatory EV through the filter, wherein the circulating comprises incorporating a suitable buffer into the solution, whereby during the treatment the buffer replaces the solution, thereby resulting in the production of an isolated, cell-free, anti-inflammatory EV population containing the buffer.

In certain embodiments, the buffer is a sterile buffer. In certain embodiments, the buffer is a sterile buffer suitable for administration to a human, e.g., suitable for administration to a human for therapeutic use. In a specific embodiment, the buffer is a saline-containing buffer. In one embodiment, the buffer is saline. In one embodiment, the buffer is physiological saline (physiological saline). In one embodiment, the buffer is normal saline (normal saline). In one embodiment, the buffer is 0.9% saline. In one embodiment, the buffer is Phosphate Buffered Saline (PBS).

In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) is performed at a flow rate of 20-1000mL/min (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) is performed at a flow rate of 50-500mL/min (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) is performed at a flow rate of 80-200mL/min (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) is performed at a flow rate of 80-175mL/min (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) is performed at a flow rate of about 80mL/min (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) is performed at a flow rate of about 100mL/min (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) is performed at a flow rate of about 125mL/min (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) is performed at a flow rate of about 150mL/min (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) is performed at a flow rate of about 175mL/min (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) is performed at a flow rate of about 200mL/min (e.g., ultrafiltration by TFF, and optionally diafiltration).

In a specific embodiment, at about 2,000 to 8,000 seconds ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, about 2,000 to 7,500 seconds ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, at about 2,000 to 7,000 seconds ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In particular embodimentsWherein, the reaction time is about 2,000 to 3,000s ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, at about 3,000 to 4,000 seconds ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, at about 4,000 to 5,000 seconds ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, at about 5,000 to about 6,000 seconds ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, about 6,000 to 7,000 seconds ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, at about 7,000 to 8,000 seconds ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, at about 2,000s ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, at about 3,000 seconds ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, at about 4,000s ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, at about 5,000 seconds ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, at about 6,000 seconds ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, at about 7,000 seconds ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, at about 7,500s ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). In a specific embodiment, at about 8,000 seconds ^-1 The ultrafiltration (and optionally diafiltration) in step (ii) is performed using a hollow fiber filter (e.g., ultrafiltration by TFF, and optionally diafiltration). Shear rate is a term used for hollow fiber membranes and is affected by flow rate and fiber radius. Although typical shear rate values are 2000-12000s ^-1 Preferably, however, the shear rate maintained in step (ii) is from about 2,000 to about 8,000s ^-1 About 2,000 to 7,500s ^-1 Or about 2,000 to 7,000s ^-1 (and not higher) to avoid EV fragmentation and to result in high EV recovery efficiency (e.g., recovery of EV greater than 90% or greater than 95%). In a specific embodiment, the shear rate maintained in step (ii) is from about 2,000 to 7,500s ^-1 A hollow fiber filter having a fiber diameter of 0.5mm was used at a flow rate of 80 to 200 mL/min.

In certain embodiments, the transmembrane pressure of step (ii) is maintained at about 10psi. In a specific embodiment, the shear rate maintained in step (ii) is from about 2,000 to 7,500s ^-1 A flow rate of 80-200mL/min and using a hollow fiber filter with a fiber diameter of 0.5mm resulted in a transmembrane pressure of about 10psi.

In certain embodiments, the separating step (b) comprises: step (i) comprising microfiltration as described above, and step (ii) comprising ultrafiltration as described above and optionally diafiltration as described above.

In certain embodiments, step (i) described above is performed using one or more pumps (e.g., one or more automated pumps), such as a main pump and an auxiliary pump. In certain embodiments, step (ii) described above is performed using one or more pumps (e.g., one or more automated pumps), such as a main pump and an auxiliary pump. In certain embodiments, step (i) and step (ii) described above are performed using one or more pumps (e.g., one or more automated pumps), such as a main pump and an auxiliary pump, respectively.

In a specific non-limiting example, the Repligen KR2i TFF system can be used to isolate, concentrate and diafiltrate EVs from cell cultures into buffers suitable for therapeutic use. For example, EV separation using TFF may include the steps of: (i) Using TFF and membrane area 85cm ² And a Midi 20cm0.65 μm Spectrum mPES hollow fiber filter (D02-E65U-07-N) with a fiber diameter of 0.75mm circulates the medium to filter out cells and debris (e.g., using a flow rate of 100-200mL/min, which results in about 2,000-5,000 s) ^-1 While maintaining a different transmembrane pressure (TMP) driven by a 5psi hold-up hydraulic pressure) and (ii) using the permeate of the process to concentrate and diafiltrate the EV product. For example, the process may use a TFF system and have a length of 115cm ² Midi 20cm 500kD Spectrum mPES hollow fiber filters (D02-E500-05-N) of filter membrane area and 0.5mm fiber diameter to retain/concentrate particles greater than about 60-80nm into the retention solution by continuous circulation (e.g., using a flow rate of 80-200mL/min, which results in 2,000-7,500 s) ^-1 While maintaining and driving filtration at 10psi TMP). Incorporation of a suitable buffer into the circulation (e.g., sterile saline or sterile PBS) can be performed to diafiltrate and replace the existing solution so that the EV is ultimately in a sterile solution acceptable for therapeutic use.

In certain embodiments, the isolated EV may be stored at-20 ℃. In particular embodiments, the isolated EV may be stored at-20℃while limiting freeze/thaw cycles.

In certain embodiments, the isolated EV may be stored at about 2 ℃ to about 8 ℃ (e.g., at about 4 ℃), e.g., may be stored at about 2 ℃ to about 8 ℃ (e.g., at about 4 ℃) for up to about 1 week, e.g., may be stored at about 2 ℃ to about 8 ℃ (e.g., at about 4 ℃) for about overnight, up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, or up to about 7 days. In certain embodiments, the isolated EV is stored at 4 ℃ for less than about 2 weeks, e.g., at about 2 ℃ to about 8 ℃ (e.g., at about 4 ℃) for less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days.

In certain embodiments, the methods provided herein for generating an isolated, cell-free, anti-inflammatory EV population result in about 1 x 10 ⁸ Up to about 1X 10 ¹⁰ Yields of individual EV/ml media. In certain embodiments, the methods provided herein for generating an isolated, cell-free, anti-inflammatory EV population result in about 5 x 10 ⁸ Up to about 1X 10 ¹⁰ Yields of individual EV/ml media. In certain embodiments, the methods provided herein for generating an isolated, cell-free, anti-inflammatory EV population result in about 1 x 10 ⁹ Up to about 1X 10 ¹⁰ Yields of individual EV/ml media. In certain embodiments, the methods provided herein for generating an isolated, cell-free, anti-inflammatory EV population result in about 5 x 10 ⁹ Up to about 1X 10 ¹⁰ Yields of individual EV/ml media. In certain embodiments, the methods provided herein for generating an isolated, cell-free, anti-inflammatory EV population result in about 1 x 10 ⁹ EV/ml, about 2X 10 ⁹ EV/ml, about 3X 10 ⁹ EV/ml, about 4X 10 ⁹ EV/ml, about 5X 10 ⁹ EV/ml, about 6X 10 ⁹ EV/ml, about 7X 10 ⁹ EV/ml, about 8X 10 ⁹ EV/ml, about 9X 10 ⁹ EV/ml or about 1X 10 ¹⁰ Yield of EV/ml media.

Anti-inflammatory EVs provided herein can be derived from ex vivo expanded human suppressor immune cells, e.g., tregs. Provided herein are exemplary methods for expanding tregs.

In some embodiments, an anti-inflammatory EV provided herein is derived from ex vivo expanded human suppressive immune cells, e.g., tregs. Provided herein are exemplary methods for expanding tregs.

5.2.1 cultivation, enrichment and expansion of human suppressive immune cells

The isolated, cell-free anti-inflammatory EV populations provided herein are derived from ex vivo expanded human suppressive immune cells. In certain aspects, the isolated, cell-free, anti-inflammatory EV populations provided herein are derived from ex vivo expanded human tregs.

In some embodiments, the human suppressive immune cells, e.g., tregs, are from a healthy human subject. In some embodiments, the human suppressive immune cells, e.g., tregs, are from more than one healthy human subject. In specific embodiments, for example, a human inhibitory immune cell, e.g., treg, is from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or more healthy human subjects. In other specific embodiments, for example, a human suppressive immune cell, e.g., treg, is from 2-50, 2-5, 2-10, 5-50, 5-25, 10-15, 10-50, 10-25, 15-25, 25-30, 30-35, 35-40, 40-45, or 45-50 subjects. In some embodiments, the subject is related. In some embodiments, the subject is not related.

In embodiments in which human suppressive immune cells, e.g., tregs, are from more than one human subject, a method of generating an isolated, cell-free, anti-inflammatory EV population can include mixing cells from more than one human subject together prior to expanding the cells ex vivo. In other specific embodiments in which human suppressive immune cells, e.g., tregs, are from more than one human subject, a method of generating an isolated, cell-free, anti-inflammatory EV population may comprise expanding cells from one or more human subjects separately and mixing anti-inflammatory EVs generated from each culture.

In some embodiments, the human suppressive immune cells, e.g., tregs, are from a donor subject diagnosed with or suspected of having a disorder associated with Treg dysfunction. In some embodiments, the donor subject is diagnosed with or suspected of having a disorder associated with Treg deficiency. In some embodiments, the donor subject is diagnosed with or suspected of having a condition driven by a T cell response.

In some embodiments, the human suppressive immune cells, e.g., tregs, are from a donor subject diagnosed with or suspected of having a neurodegenerative disease. In some embodiments, the donor subject is diagnosed with or suspected of having alzheimer's disease, amyotrophic lateral sclerosis, multiple Sclerosis (MS), parkinson's disease, huntington's disease, or frontotemporal dementia.

In some embodiments, the human suppressive immune cells, e.g., tregs, are from a donor subject diagnosed with or suspected of having a disorder that would benefit from down-regulation of the immune system.

In some embodiments, the human suppressive immune cells, e.g., tregs, are from a donor subject diagnosed with or suspected of having an autoimmune disease. The autoimmune disease may be, for example, systemic sclerosis (scleroderma), polymyositis, ulcerative colitis, inflammatory bowel disease, crohn's disease, celiac disease, multiple Sclerosis (MS), rheumatoid Arthritis (RA), type I diabetes, psoriasis, dermatomyositis, lupus, for example, systemic lupus erythematosus or cutaneous lupus, myasthenia gravis, autoimmune kidney disease, autoimmune hemolytic anemia, autoimmune cytopenia, autoimmune hepatitis, autoimmune uveitis, alopecia, thyroiditis, or pemphigus.

In some embodiments, the human suppressive immune cells, e.g., tregs, are from a donor subject diagnosed with or suspected of having heart failure or ischemic cardiomyopathy. In some embodiments, the donor subject is diagnosed with or suspected of having a graft versus host disease, e.g., after undergoing an organ transplant (e.g., a kidney transplant or a liver transplant), or after undergoing a stem cell transplant (e.g., a hematopoietic stem cell transplant).

In some embodiments, the human suppressive immune cells, e.g., tregs, are from a donor subject diagnosed with or suspected of having neurogenic inflammation. The neurogenic inflammation may be associated with, for example, stroke, acute disseminated encephalitis, acute optic neuritis, transverse myelitis, optic neuromyelitis, epilepsy, traumatic brain injury, spinal cord injury, encephalitis, central Nervous System (CNS) vasculitis, nervous system sarcoidosis, autoimmune or post-infection encephalitis, or chronic meningitis.

In some embodiments, the human inhibitory immune cells, e.g., tregs, are from a donor subject diagnosed with or suspected of having Chronic Inflammatory Demyelinating Polyneuropathy (CIDP). In some embodiments, the donor subject is diagnosed with or suspected of having Acute Inflammatory Demyelinating Polyneuropathy (AIDP). In some embodiments, the donor subject is diagnosed with or suspected of having a gehlrabi syndrome (GBS).

In some embodiments, the human suppressive immune cells, e.g., tregs, are from a donor subject diagnosed with or suspected of suffering from cardiac inflammation, e.g., cardiac inflammation associated with myocardial infarction, ischemic cardiomyopathy, heart failure.

In some embodiments, the human suppressive immune cells, e.g., tregs, are from a donor subject with stroke.

In some embodiments, the human suppressive immune cells, e.g., tregs, are from a donor subject diagnosed with or suspected of having cancer, e.g., blood cancer.

In some embodiments, the human suppressive immune cells, e.g., tregs, are from a donor subject diagnosed with or suspected of having asthma.

In some embodiments, the human suppressive immune cells, e.g., tregs, are from a donor subject diagnosed with or suspected of having eczema.

In some embodiments, the human suppressive immune cells, e.g., tregs, are from a donor subject diagnosed with or suspected of having a disorder associated with excessive activation of the immune system.

In some embodiments, the human suppressive immune cells, e.g., tregs, are from a donor subject diagnosed with or suspected of having a Treg lesion (tregolopathia). Treg lesions may be caused, for example, by FOXP3, CD25, cytotoxic T lymphocyte-associated antigen 4 (CTLA 4), LPS-reactive beige anchor-like protein (LRBA) or BTB domain and CNC homologous gene 2 (BACH 2) gene loss-of-function mutations or signal transduction and activator of transcription 3 (STAT 3) function gain-of-function mutations.

In some embodiments, the human suppressive immune cells, e.g., tregs, are from one or more adult subjects, e.g., one or more healthy adult subjects. In certain embodiments, one or more subjects are at least 18, 20, 25, 30, 35, 40, 45, 50, or 55 years old. In specific embodiments, for example, a human suppressive immune cell, e.g., treg, is from one or more adult subjects, wherein the one or more healthy adult subjects are about 18-55, about 18-50, about 18-45, about 18-40, about 18-35, about 18-30, about 18-25, about 20-55, about 25-55, about 30-55, about 35-55, about 40-55, about 25-50, about 30-50, about 35-45, about 25-45, about 40-50 years old.

In some embodiments, the human suppressive immune cells, e.g., tregs, are from an elderly subject, e.g., a healthy elderly subject, e.g., a subject at least 65, at least 70, at least 75, at least 80, at least 85, or at least 90 years old.

In some embodiments, the anti-inflammatory EVs provided herein are derived from a genetically engineered population of human suppressive immune cells, e.g., tregs.

5.2.1.1 methods for improving in vitro expanded tregs

In some embodiments, the isolated, cell-free, anti-inflammatory EV population provided herein is derived from an ex vivo expanded population of human tregs. Methods for expanding tregs are well known. This section describes an improved method of expanding tregs ex vivo.

In certain embodiments, a biological donor sample containing or suspected of containing tregs, e.g., a peripheral blood sample or thymus tissue, may be obtained and enriched for tregs therefrom prior to ex vivo expansion. Exemplary methods of generating, obtaining, enriching, and expanding Treg populations ex vivo are described in international patent application No. pct/US2020/63378, which is incorporated herein by reference in its entirety. In some embodiments, tregs from which EVs are obtained are expanded according to the disclosure described in section 5.2.2 below. In some embodiments, tregs from which EVs are obtained are expanded according to the disclosure described in section 6.12 below.

In certain embodiments, the enrichment step is automated. In certain embodiments, the enrichment step is performed in a closed system. In certain embodiments, the enrichment step is automated and performed in a closed system. In particular embodiments, in CliniMACSAn enrichment step is performed in the system. In a specific embodiment, at +.>The enrichment step was performed in the Plus system. In certain embodiments, the amplification step is automated. In certain embodiments, the amplification step is performed in a closed system. In certain embodiments, the amplification step is automated and performed in a closed system. In a specific embodiment, in a bioreactor (e.g., terumo BCT +. >Cell expansion system) is used for the expansion step. In some embodiments, the enrichment step and the amplification step are performed in different systems (e.g., in CliniMACS +.>Enrichment step is performed in the system and in Terumo BCT +.>The amplification step is carried out in a cell amplification system, or in +.>Enrichment step was performed in Plus system and in Terumo BCT +.>The amplification step is performed in a cell amplification system). In a specific embodiment, the enriched cell population produced by the enrichment step is transferred thereto in a closed stateIn a system in which the amplification step is performed in the step. In other embodiments, the enrichment step and the amplification step are performed in the same system. In a specific embodiment, the same system is a closed system.

In some embodiments, the population of tregs is obtained from a serum sample suspected of containing tregs. In some embodiments, the population of tregs is derived from a cell sample suspected of containing tregs, from a donor via a leukopenia method or from a donor via a blood sample. In some embodiments, the population of tregs is obtained from a biological sample suspected of containing tregs.

In some embodiments, the population of tregs is enriched from a biological sample from a human donor subject. In some embodiments, the donor of the biological sample is a patient subject to be treated by an anti-inflammatory EV population derived from a population of tregs. In other embodiments, the donor of the biological sample is different from the patient subject to be treated by the anti-inflammatory EV population derived from the Treg population. The biological sample may be any sample suspected of containing tregs, possibly containing tregs or known to contain tregs. These biological samples may be collected directly from the subject, or may be samples resulting from one or more processing steps, such as separation, e.g., selection or enrichment, centrifugation, washing, and/or incubation. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebral spinal fluid, joint fluid, tissue and organ samples, including treated samples obtained thereby.

In some aspects, the sample is a blood or blood-derived sample, or is derived from a apheresis product or a leukocyte removal product. Exemplary samples include whole blood, peripheral Blood Mononuclear Cells (PBMCs), white blood cells, bone marrow, and thymus.

In some embodiments, the biological sample is a blood-derived sample, e.g., a sample derived from whole blood, serum, or plasma. In some embodiments, the biological sample is or includes peripheral blood mononuclear cells. In some embodiments, the biological sample is a peripheral blood or serum sample. In some embodiments, the biological sample is a lymph node sample.

Methods for obtaining cell populations suspected of containing, possibly containing, or known to contain tregs from these biological donor samples are known in the art. For example, lymphocytes may be obtained from a surrounding blood sample by a leukapheresis procedure. In some embodiments, tregs are enriched from a lymphocyte population. In some embodiments, duplicate peripheral blood samples are obtained from a donor for Treg generation. In some embodiments, two or more peripheral blood samples are obtained from a donor. In some embodiments, the donor sample undergoes a volume reduction during the enrichment process.

In some embodiments, biological samples (e.g., leukocyte-removed samples or blood samples) from more than one donor are mixed to produce an allogeneic population of tregs prior to the enrichment process. In some embodiments, biological samples (e.g., leukocyte-removed samples or blood samples) from more than one unrelated donor are mixed to produce an allogeneic population of tregs prior to the enrichment process. In some embodiments, biological samples (e.g., leukocyte-depleted samples or blood samples from 2, 3, 4, 5, 10, 20, 50 or more donors) are mixed.

Tregs may be enriched from a biological sample by any method known in the art. In some embodiments, magnetic bead separation (e.g., cliniMACS Tubing Set LS (162-01),Plus apparatus or CliniMACSInstrument), fluorescent cell sorting and/or disposable closed column based cell sorter to enrich tregs from the sample.

Enrichment includes enrichment of cells that express one or more markers and may represent an increase in the number or percentage of these cells in a population of cells, but does not necessarily result in the complete absence of cells that do not express the markers. Depletion of cells expressing one or more markers refers to a reduction in the number or percentage of such cells in a population of cells, but does not necessarily result in complete elimination of all cells expressing one or more such markers.

In some embodiments, the enrichment comprises an affinity or immunoaffinity based separation step of cells expressing one or more markers (e.g., treg cell surface markers). These isolation steps may be based on positive selection of cells in which one or more markers are retained, and/or based on negative selection (depletion) of cells in which one or more markers are not retained.

The isolation may be based on expression (e.g., positive or negative expression) or expression level (e.g., high or low expression) of one or more markers (e.g., treg cell surface markers). In this regard, "high expression" and "low expression" are generally relative to the whole cell population. In some embodiments, cell separation may be based on CD8 expression. In some embodiments, cell isolation may be based on CD19 expression. In some embodiments, cell separation may be based on high CD25 expression.

In some embodiments, cell separation may be based on high CD9 expression. In some embodiments, cell isolation may be based on high CD63 expression. In some embodiments, cell separation may be based on high CD81 expression. In some embodiments, cell separation may be based on high CD44 expression. In some embodiments, cell isolation may be based on high CD29 expression. In some embodiments, cell separation may be based on high CD45 expression.

Thus, in some embodiments, enrichment of tregs may include incubation with antibodies or binding partners that specifically bind to markers (e.g., treg cell surface markers), followed by a washing step and separation of cells that bind to the antibodies or binding partners from those that do not.

In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a sphere or bead, e.g., a nanoparticle, microbead, nanobead, including agarose, magnetic bead, or paramagnetic bead. In some embodiments, the ball or bead may be packed into a column to achieve immunoaffinity chromatography. In some embodiments of the present invention, in some embodiments,the antibody or binding partner is detectably labeled. In some embodiments, the antibody or binding partner is attached to a small, magnetically reactive particle or microparticle, such as a nanoparticle or paramagnetic bead. These beads are known and commercially available (e.g.,(Life Technologies,Carlsbad,CA)、/>beads (Miltenyi Biotec, san Diego, calif.) or +.>Bead reagent (IBA, germany)). These particles or microparticles may be incubated with the cell population to be enriched and then placed in a magnetic field. This results in cells attached to the particles or microparticles via the antibody or binding partner being attracted to the magnet and separated from unbound cells. This method allows either retention of cells attached to the magnet (positive selection) or removal of cells attracted to the magnet (negative selection).

In some embodiments, the methods of generating Treg populations provided herein comprise both positive and negative selections during the enrichment step.

In some embodiments, the biological sample is obtained within about 25-35min, about 35-45min, about 45-60min, about 60-75min, about 75-90min, about 90-120min, about 120-150min, about 150-180min, about 2-3h, about 3-4h, about 4-5h, or about 5-6h of the beginning of the enrichment step. In some embodiments, the sample is obtained within about 30 minutes of the start of the enrichment step. In some embodiments, the biological sample is not stored (e.g., at 4 ℃) overnight.

In some embodiments, enrichment of tregs from a human sample comprises depletion of a cd8+ cell sample. In some embodiments, enrichment of tregs from a human sample comprises depletion of a cd19+ cell sample. In some embodiments, enrichment of tregs from a biological sample includes depletion of cd8+ cells and cd19+ cell samples. In some embodiments, tregs are derived from a biological sampleEnrichment of a cell population for CD25 ^{High height} Enrichment of cells. In some embodiments, enrichment of tregs from a biological sample includes enrichment of a cell population for cd25+ cells. In some embodiments, enrichment of tregs from a biological sample includes depletion of cd8+ cells and cd19+ cells from the sample and cell populations for CD25 ^{High height} Enrichment of cells. In some embodiments, enrichment of tregs from a biological sample includes depletion of cd8+/cd19+ cells and enrichment for cd25+ cells.

In some embodiments, the population of cells enriched for tregs comprises, as determined by flow cytometry, relative to cd4+cd25 in tregs prior to enrichment ^{High height} Proportion of Treg, cd4+cd25 ^{High height} The proportion of Treg increases. In a specific embodiment, CD4+CD25 ^{High height} The proportion of Treg increases by about 2-fold to about 4-fold, about 4-fold to about 6-fold, about 6-fold to about 8-fold, about 8-fold to about 10-fold, about 10-fold to about 15-fold, about 15-fold to about 20-fold, about 20-fold to about 25-fold, about 25-fold to about 30-fold, about 30-fold to about 35-fold, about 35-fold to about 40-fold, about 40-fold to about 45-fold, about 45-fold to about 50-fold.

In some embodiments, the population of cells enriched for tregs comprises, as determined by flow cytometry, relative to cd4+cd25 in tregs prior to enrichment ^{High height} CD127 ^{Low and low} Proportion of Treg, cd4+cd25 ^{High height} CD127 ^{Low and low} The proportion of Treg increases. In a specific embodiment, CD4+CD25 ^{High height} CD127 ^{Low and low} The proportion of Treg increases by about 2-fold to about 4-fold, about 4-fold to about 6-fold, about 6-fold to about 8-fold, about 8-fold to about 10-fold, about 10-fold to about 15-fold, about 15-fold to about 20-fold, about 20-fold to about 25-fold, about 25-fold to about 30-fold, about 30-fold to about 35-fold, about 35-fold to about 40-fold, about 40-fold to about 45-fold, about 45-fold to about 50-fold.

In some embodiments, the population of cells enriched for tregs comprises cd25+ tregs, wherein expression of CD25 in the Treg is elevated relative to expression of CD25 in the Treg prior to enrichment, as determined by flow cytometry. In specific embodiments, the expression of CD25 is increased by at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold.

In some embodiments, the population of cells enriched for tregs comprises cd127+ tregs, wherein expression of CD127 in the Treg is elevated relative to expression of CD127 in the Treg prior to enrichment, as determined by flow cytometry. In specific embodiments, the expression of CD127 is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, or at least about 3-fold.

In some embodiments, the particle size of tregs in the enriched population of tregs is increased relative to the particle size of tregs prior to enrichment, as determined by flow cytometry. In specific embodiments, the granularity of tregs is increased at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, or at least about 3-fold.

In some embodiments, the tregs in the Treg enriched population are increased in size relative to the size of the tregs prior to enrichment, as determined by flow cytometry. In specific embodiments, the Treg increases in size by at least about 1.2-fold, at least about 1.5-fold, or at least about 2-fold.

In another aspect, a population of tregs for isolating an anti-inflammatory EV provided herein is enriched from a biological sample and further amplified.

In some embodiments, the amplification step is performed within about 4-5 days after the enrichment step is complete. In some embodiments, the amplification step is performed within about 3-4 days after the enrichment step is complete. In some embodiments, the amplification step is performed within about 2-3 days after the enrichment step is complete. In some embodiments, the amplification step is performed within about 1-2 days after the enrichment step is complete. In some embodiments, the amplification step is performed within about 24 hours after the enrichment step is complete. In some embodiments, the amplification step is performed within about 12 hours after the enrichment step is complete. In some embodiments, the amplification step is performed within about 6 hours after the enrichment step is complete. In some embodiments, the amplification step is performed within about 3 hours after the enrichment step is complete. In some embodiments, the amplification step is performed within about 2 hours after the enrichment step is complete. In some embodiments, the amplification step is performed within about 1 hour after the enrichment step is complete. In some embodiments, the amplification step is performed within about 30 minutes after the enrichment step is complete.

Such expansion of the Treg population may include culturing the cells that have been enriched from the biological sample in a medium, for example, serum-free medium (e.g., texMACS medium), serum-depleted medium, or serum-containing medium.

In certain embodiments, the expanding step comprises culturing tregs in a medium comprising human serum (e.g., a TexMACS GMP medium supplemented with human serum). In a specific embodiment, the medium comprises 5% or less human serum. In a specific embodiment, the medium comprises 4% or less human serum. In a specific embodiment, the medium comprises 3% or less human serum. In a specific embodiment, the medium comprises 2% or less human serum. In a specific embodiment, the medium comprises 1% or less human serum. In a specific embodiment, the medium comprises 0.5% or less human serum. In a specific embodiment, the medium comprises less than 5% human serum. In a specific embodiment, the medium comprises less than 4% human serum. In a specific embodiment, the medium comprises less than 3% human serum. In a specific embodiment, the medium comprises less than 2% human serum. In a specific embodiment, the medium comprises less than 1% human serum. In a specific embodiment, the medium comprises less than 0.5% human serum. In a specific embodiment, the medium comprises 0-0.5% human serum. In a specific embodiment, the medium comprises 0.5-1% human serum. In a specific embodiment, the medium comprises 1-2% human serum. In a specific embodiment, the medium comprises 2-3% human serum. In a specific embodiment, the medium comprises 3-4% human serum. In a specific embodiment, the medium comprises 4-5% human serum. In a specific embodiment, the medium comprises about 0.5% human serum. In another specific embodiment, the medium comprises about 1% human serum. In another specific embodiment, the medium comprises about 2% human serum. In another specific embodiment, the medium comprises about 3% human serum. In another specific embodiment, the medium comprises about 4% human serum. In another specific embodiment, the medium comprises about 5% human serum.

In certain embodiments, the expansion step comprises culturing tregs in a medium comprising human AB serum (e.g., texMACS GMP medium supplemented with human AB serum). In a specific embodiment, the medium comprises 5% or less human AB serum. In a specific embodiment, the medium comprises 4% or less human AB serum. In a specific embodiment, the medium comprises 3% or less human AB serum. In a specific embodiment, the medium comprises 2% or less human AB serum. In a specific embodiment, the medium comprises 1% or less human AB serum. In a specific embodiment, the medium comprises 0.5% or less human AB serum. In a specific embodiment, the medium comprises less than 5% human AB serum. In a specific embodiment, the medium comprises less than 4% human AB serum. In a specific embodiment, the medium comprises less than 3% human AB serum. In a specific embodiment, the medium comprises less than 2% human AB serum. In a specific embodiment, the medium comprises less than 1% human AB serum. In a specific embodiment, the medium comprises less than 0.5% human AB serum. In a specific embodiment, the medium comprises 0-0.5% human AB serum. In a specific embodiment, the medium comprises 0.5-1% human AB serum. In a specific embodiment, the medium comprises 1-2% human AB serum. In a specific embodiment, the medium comprises 2-3% human AB serum. In a specific embodiment, the medium comprises 3-4% human AB serum. In a specific embodiment, the medium comprises 4-5% human AB serum. In a specific embodiment, the medium comprises about 0.5% human AB serum. In another specific embodiment, the medium comprises about 1% human AB serum. In another specific embodiment, the medium comprises about 2% human AB serum. In another specific embodiment, the medium comprises about 3% human AB serum. In another specific embodiment, the medium comprises about 4% human AB serum. In another specific embodiment, the medium comprises about 5% human AB serum.

In some embodimentsAt about 37℃and about 5% CO ₂ Culturing cells enriched from the biological sample. In some embodiments, the cells enriched from the biological sample are cultured under Good Manufacturing Practice (GMP) conditions. In some embodiments, the cells enriched from the biological sample are cultured in a closed system.

In some embodiments, the cells enriched from the biological sample are cultured in an automated system. In some embodiments, the cells enriched from the biological sample are cultured in a closed and automated system. In some embodiments, in Terumo BCTCells enriched from the biological sample are cultured in a cell expansion system.

In some embodiments, expansion of the population of tregs begins within 25-35min, 20-40min, 15-45min, or 10-50min of enrichment from the biological sample. In some embodiments, expansion of the population of tregs begins within about 30 minutes of enrichment from the biological sample.

As described above, once amplification begins, EV separation may be performed at any point during the amplification process or at the completion of the amplification process.

Tregs may be expanded ex vivo by culturing cells in the presence of one or more expansion agents. In some embodiments, the amplification agent is IL-2. The appropriate concentration of IL-2 in the medium can be determined by one skilled in the art. In some embodiments, the concentration of IL-2 in the cell culture medium is about 5-10IU/mL, about 10-20IU/mL, about 20-30IU/mL, about 30-40IU/mL, about 40-50IU/mL, about 50-100IU/mL, about 100-200IU/mL, about 200-300IU/mL, about 300-400IU/mL, about 400-500IU/mL, about 500-600IU/mL, about 600-700IU/mL, about 700-800IU/mL, about 800-900IU/mL, about 900-1000IU/mL, about 1000-1500IU/mL, about 1500-2000IU/mL, about 2000-2500IU/mL, about 2500-3000IU/mL, about 3000-3500IU/mL, about 3500-4000IU/mL, about 4000-4500IU/mL, about 4500-5000IU/mL, about 5000-6000IU/mL, about 6000-7000/mL, about 9007000 IU/mL, about 8000-8000 IU/mL, about 8000 IU-8000, or about 10,000 IU-0. In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 100IU/mL. In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 150IU/mL. In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 200IU/mL. In a specific embodiment, the concentration of IL-2 in cell culture medium is about 250IU/mL. In a specific embodiment, the concentration of IL-2 in cell culture medium is about 300IU/mL. In a specific embodiment, the concentration of IL-2 in cell culture medium is about 400IU/mL. In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 500IU/mL. In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 600IU/mL. In a specific embodiment, the concentration of IL-2 in cell culture medium is about 700IU/mL. In a specific embodiment, the concentration of IL-2 in cell culture medium is about 800IU/mL. In certain embodiments, the amplifying step comprises adjusting the concentration of IL-2 based on the number of cells. Cell number means the number of all cells in culture, including enriched Treg cells, which account for the majority of cells in culture and in particular embodiments, for greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 99% or 100% of cells in culture. In a specific embodiment, the expansion step comprises culturing tregs in a medium containing about 200IU/mL IL-2 until the number of cells reaches 600 x 10 ⁶ Treg are then cultured in medium containing about 250IU/mL IL-2.

In some embodiments, IL-2 is first added to the culture within about 4-5 days of the initial culture. In some embodiments, IL-2 is first added to the culture within about 3-4 days of the initial culture. In some embodiments, IL-2 is first added to the culture within about 2-3 days of the initial culture. In some embodiments, IL-2 is first added to the culture within about 1-2 days of the initial culture. In some embodiments, IL-2 is first added to the culture within about 24 hours of initiating the culture. In some embodiments, IL-2 is first added to the culture within about 12 hours of initiating the culture. In some embodiments, IL-2 is first added to the culture within about 6 hours of initiating the culture. In some embodiments, IL-2 is first added to the culture within about 3 hours of initiating the culture. In some embodiments, IL-2 is first added to the culture within about 2 hours of initiating the culture. In some embodiments, IL-2 is first added to the culture within about 1 hour of initiating the culture. In some embodiments, IL-2 is first added to the culture within about 30 minutes of initiating the culture. In some embodiments, IL-2 is first added to the culture within about 4-5 days after the enrichment step is complete. In some embodiments, IL-2 is first added to the culture within about 3-4 days after the enrichment step is complete. In some embodiments, IL-2 is first added to the culture within about 2-3 days after the enrichment step is complete. In some embodiments, IL-2 is first added to the culture within about 1-2 days after the enrichment step is complete. In some embodiments, IL-2 is first added to the culture within about 24 hours after the enrichment step is complete. In some embodiments, IL-2 is first added to the culture within about 12 hours after the enrichment step is complete. In some embodiments, IL-2 is first added to the culture within about 6 hours after the enrichment step is completed. In some embodiments, IL-2 is first added to the culture within about 3 hours after the enrichment step is complete. In some embodiments, IL-2 is first added to the culture within about 2 hours after the enrichment step is complete. In some embodiments, IL-2 is first added to the culture within about 1 hour after the enrichment step is complete. In some embodiments, IL-2 is first added to the culture within about 30 minutes after the enrichment step is complete. In some embodiments, IL-2 is supplemented about every 1, 2, 3, 4, or 5 days. In some embodiments, IL-2 is supplemented about every 1-2 days. In some embodiments, IL-2 is supplemented about every 2-3 days. In some embodiments, IL-2 is supplemented about every 3-4 days. In some embodiments, IL-2 is supplemented about every 4-5 days.

In some embodiments, the amplification agent activates CD3, e.g., the amplification agent is an anti-CD 3 antibody. In some embodiments, the amplification agent activates CD28, e.g., the amplification agent is an anti-CD 28 antibody.

In some embodiments, the amplification agent is a soluble anti-CD 3 antibody. In a specific embodiment, the anti-CD 3 antibody is OKT3. In some embodiments, the concentration of the soluble anti-CD 3 antibody in the medium is about 0.1-0.2ng/mL, about 0.2-0.3ng/mL, about 0.3-0.4ng/mL, about 0.4-0.5ng/mL, about 0.5-1ng/mL, about 1-5ng/mL, about 5-10ng/mL, about 10-15ng/mL, about 15-20ng/mL, about 20-25ng/mL, about 25-30ng/mL, about 30-35ng/mL, about 35-40ng/mL, about 40-45ng/mL, about 45-50ng/mL, about 50-60ng/mL, about 60-70ng/mL, about 70-80ng/mL, about 80-90ng/mL, or about 90-100ng/mL.

In some embodiments, the amplification agent is a soluble anti-CD 28 antibody. Non-limiting examples of anti-CD 28 antibodies include NA/LE (e.g., BD Pharmingen), IM1376 (e.g., beckman Coulter), or 15×10 ⁸ (e.g., miltenyi Biotec). In some embodiments, the concentration of the soluble anti-CD 28 antibody in the medium is about 1-2ng/mL, about 2-3ng/mL, about 3-4ng/mL, about 4-5ng/mL, about 5-10ng/mL, about 10-15ng/mL, about 15-20ng/mL, about 20-25ng/mL, about 25-30ng/mL, about 30-35ng/mL, about 35-40ng/mL, about 40-45ng/mL, about 45-50ng/mL, about 50-60ng/mL, about 60-70ng/mL, about 70-80ng/mL, about 80-90ng/mL, about 90-100ng/mL, about 100-200ng/mL, about 200-300ng/mL, about 300-400ng/mL, about 400-500ng/mL, 500-600ng/mL, 600-700ng/mL, about 700-800ng/mL, about 800-1000 ng/mL, or about 800-900 ng/mL.

In some embodiments, both the anti-CD 3 antibody and the anti-CD 28 antibody are present in the cell culture medium. In some embodiments, the anti-CD 3 antibody and the anti-CD 28 antibody are attached to a solid surface. In some embodiments, anti-CD 3 antibodies and anti-CD 28 antibodies are attached to the beads. In some embodiments, the beads loaded with CD28 antibody, anti-biotin antibody, and CD 3-biotin (e.g., 3.5 μm particles) are present in the cell culture medium. These beads are commercially available (e.g., MACS GMP ExpAct Treg kit,M-450CD3/CD28T cell expanding agent). In a specific embodiment, the ratio of anti-CD 3 antibody to anti-CD 28 antibody on the bead is about 100: 1. 90: 1. 80: 1. 70: 1. 60: 1. 50: 1. 40: 1. 30: 1. 20: 1. 10: 1.9: 1. 8: 1. 7: 1. 6: 1. 5: 1. 4: 1. 3: 1. 2: 1. 1: 1. 1: 2. 1: 3. 1: 4. 1: 5. 1: 6. 1: 7. 1: 8. 1: 9. 1: 10. 1: 20. 1: 30. 1: 40. 1: 50. 1: 60. 1: 70. 1: 80. 1:90 or 1:100. in some embodiments, the population of tregs is cultured in the presence of both IL-2 and beads loaded with CD28 antibodies, anti-biotin antibodies, and CD 3-biotin. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 4-5 days of initial culture. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 3-4 days of initial culture. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 2-3 days of initial culture. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 1-2 days of initial culture. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 24 hours of initial culture. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 12 hours of initial culture. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 6 hours of initial culture. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 3 hours of initial culture. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 2 hours of initial culture. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 1 hour of initial culture. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 30 minutes of initial culture. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 4-5 days after the enrichment step is complete. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are removed within about 3-4 days after the enrichment step is complete First to the culture. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 2-3 days after the enrichment step is complete. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 1-2 days after the enrichment step is complete. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 24 hours after the enrichment step is complete. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 12 hours after the enrichment step is complete. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 6 hours after the enrichment step is completed. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 3 hours after the enrichment step is complete. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 2 hours after the enrichment step is complete. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 1 hour after the enrichment step is complete. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are first added to the culture within about 30 minutes after the enrichment step is complete. In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are added to the medium again about 14 days after the first addition of the anti-CD 3 and anti-CD 28 antibody coated beads to the medium (e.g., if the cell number has not reached the target cell number at that time). In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are added to the medium again about 13 days after the first addition of the anti-CD 3 and anti-CD 28 antibody coated beads to the medium (e.g., if the cell number has not reached the target cell number at that time). In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are added to the medium again about 12 days after the first addition of the anti-CD 3 and anti-CD 28 antibody coated beads to the medium (e.g., if the cell number has not reached the target cell number at that time). In some embodiments, the coating is to be applied The anti-CD 3 and anti-CD 28 antibody coated beads are re-added to the medium about 11 days after the first addition of the anti-CD 3 and anti-CD 28 antibody beads to the medium (e.g., if the cell number has not reached the target cell number at that time). In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are added to the medium again about 10 days after the first addition of the anti-CD 3 and anti-CD 28 antibody coated beads to the medium (e.g., if the cell number has not reached the target cell number at that time). In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are added to the medium again about 9 days after the first addition of the anti-CD 3 and anti-CD 28 antibody coated beads to the medium (e.g., if the cell number has not reached the target cell number at that time). In some embodiments, the anti-CD 3 and anti-CD 28 antibody coated beads are added to the medium again about 8 days after the first addition of the anti-CD 3 and anti-CD 28 antibody coated beads to the medium (e.g., if the cell number has not reached the target cell number at that time). Cell number means the number of all cells in culture, including enriched Treg cells, which account for the majority of cells in culture and in particular embodiments, for greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 99% or 100% of cells in culture. In certain embodiments, the number of target cells is 1×10 ⁸ Up to 1X 10 ¹⁰ Individual cells. In certain embodiments, the number of target cells is 1×10 ⁹ Up to 5X 10 ⁹ Individual cells. In certain embodiments, the number of target cells is 2×10 ⁹ Up to 5X 10 ⁹ Individual cells. In certain embodiments, the number of target cells is 2×10 ⁹ Up to 2.5X10 ⁹ Individual cells. In a specific embodiment, the number of target cells is 1X 10 ⁹ Individual cells. In another specific embodiment, the number of target cells is 1.5X10 ⁹ Individual cells. In another specific embodiment, the number of target cells is 2X 10 ⁹ Individual cells. In another specific embodiment, the target cell number is 2.5X10 ⁹ Individual cells. In another specific embodiment, the number of target cells is 3X 10 ⁹ Each thinAnd (5) cells. In another specific embodiment, the number of target cells is 3.5X10 ⁹ Individual cells. In another specific embodiment, the number of target cells is 4X 10 ⁹ Individual cells. In another specific embodiment, the number of target cells is 4.5X10 ⁹ Individual cells. In another specific embodiment, the number of target cells is 5X 10 ⁹ Individual cells. In a specific embodiment, the ratio of beads to cells in the culture is 10: 1. 9: 1. 8: 1. 7: 1. 6: 1. 5: 1. 4: 1. 3: 1. 2:1 or 1:1.

One or more amplificants may be added to the medium every 1, 2, 3, 4 or 5 days. In a specific embodiment, the amplification agent is added to the medium every 1-2 days. In a specific embodiment, the amplificant is added to the medium every 2-3 days. In a specific embodiment, the amplificant is added to the medium every 3-4 days. In a specific embodiment, the amplificant is added to the medium every 4-5 days. In other specific embodiments, the amplification agent is added to the culture medium on days 6, 8, and 11, wherein day 0 is the day the biological sample is obtained from the subject. In some specific embodiments, the amplification agent is not added to the culture medium on day 13, wherein day 0 is the day the biological sample is obtained from the subject.

In some embodiments, the one or more amplicons are added to the culture for a first time within about 30 minutes to 1 hour, 1 to 2 hours, 2 to 4 hours, 4 to 6 hours, 6 to 8 hours, 8 to 10 hours, 10 to 12 hours, 12 to 14 hours, 14 to 16 hours, 16 to 18 hours, 18 to 24 hours, 24 to 36 hours, 36 to 48 hours, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 6 hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days of the initial culture. In some embodiments, the one or more amplificants are added to the culture for the first time within about 4-5 days of the initial culture. In some embodiments, the one or more amplificants are added to the culture for the first time within about 3-4 days of the initial culture. In some embodiments, the one or more amplificants are added to the culture for the first time within about 2-3 days of the initial culture. In some embodiments, one or more amplificants are added to the culture for the first time within about 1-2 days of the initial culture. In some embodiments, the one or more amplificants are added to the culture for the first time within about 24 hours of the initial culture. In some embodiments, the one or more amplificants are added to the culture for the first time within about 12 hours of the initial culture. In some embodiments, the one or more amplificants are added to the culture for the first time within about 6 hours of the initial culture. In some embodiments, the one or more amplificants are added to the culture for the first time within about 3 hours of the initial culture. In some embodiments, the one or more amplificants are added to the culture for the first time within about 2 hours of the initial culture. In some embodiments, the one or more amplificants are added to the culture for the first time within about 1 hour of initiating the culture. In some embodiments, the one or more amplificants are added to the culture for the first time within about 30 minutes of the initial incubation. In some embodiments, the one or more amplicons are first added to the culture within about 30 minutes to 1 hour, 1 to 2 hours, 2 to 4 hours, 4 to 6 hours, 6 to 8 hours, 8 to 10 hours, 10 to 12 hours, 12 to 14 hours, 14 to 16 hours, 16 to 18 hours, 18 to 24 hours, 24 to 36 hours, 36 to 48 hours, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 6 hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after completion of the enrichment step. In some embodiments, the one or more amplificants are added to the culture for a first time within about 4-5 days after the enrichment step is completed. In some embodiments, the one or more amplificants are added to the culture for a first time within about 3-4 days after the enrichment step is completed. In some embodiments, the one or more amplificants are added to the culture for a first time within about 2-3 days after the enrichment step is completed. In some embodiments, the one or more amplificants are added to the culture for a first time within about 1-2 days after the enrichment step is completed. In some embodiments, the one or more amplificants are added to the culture for a first time within about 24 hours after completion of the enrichment step. In some embodiments, the one or more amplificants are added to the culture for a first time within about 12 hours after completion of the enrichment step. In some embodiments, the one or more amplificants are added to the culture for a first time within about 6 hours after completion of the enrichment step. In some embodiments, the one or more amplificants are added to the culture for a first time within about 3 hours after completion of the enrichment step. In some embodiments, the one or more amplificants are added to the culture for a first time within about 2 hours after completion of the enrichment step. In some embodiments, the one or more amplificants are added to the culture for a first time within about 1 hour after the enrichment step is completed. In some embodiments, the one or more amplificants are added to the culture for a first time within about 30 minutes after the enrichment step is complete. In some embodiments, the one or more amplification agents are added to the medium again about 14 days after the first addition of the amplification agents to the medium. In some embodiments, the one or more amplification agents are added to the medium again about 13 days after the first addition of the amplification agents to the medium. In some embodiments, the one or more amplification agents are added to the medium again about 12 days after the first addition of the amplification agents to the medium. In some embodiments, the one or more amplification agents are added to the medium again about 11 days after the first addition of the amplification agents to the medium. In some embodiments, the one or more amplification agents are added to the medium again about 10 days after the first addition of the amplification agents to the medium. In some embodiments, the one or more amplificants are added to the medium again about 9 days after the first addition of the amplificants to the medium. In some embodiments, the one or more amplification agents are added to the medium again about 8 days after the first addition of the amplification agents to the medium.

If no amplification agent is added to the culture on a given day, that day is considered to be the "resting day". In some embodiments, no expansion agent is administered during the day prior to the day of harvesting the Treg population. In some embodiments, no amplification agent is administered during 2, 3, 4, 5, or 6 days prior to the day of harvesting the Treg population.

In some embodiments, the population of tregs may be expanded ex vivo by culturing the cells in the presence of one or more agents that inhibit the mammalian target of rapamycin (mTor). In some embodiments, the mTor inhibitor is rapamycin. In some embodiments, the mTor inhibitor is an analog of rapamycin ("rapamycin analog"), e.g., temsirolimus, everolimus, or diphensirolimus. In some embodiments, the mTor inhibitor is ICSN3250, OSU-53, or AZD8055. In some embodiments, the concentration of rapamycin in the cell culture medium is about 1-20nmol/L, about 20-30nmol/L, about 30-40nmol/L, about 40-50nmol/L, about 50-60nmol/L, about 60-70nmol/L, about 70-80nmol/L, about 80-90nmol/L, about 90-100nmol/L, about 100-150nmol/L, about 150-200nmol/L, about 200-250nmol/L, about 250-300nmol/L, about 300-350nmol/L, about 350-400nmol/L, about 400-450nmol/L, about 450-500nmol/L, about 500-600nmol/L, about 600-700nmol/L, about 700-800nmol/L, about 800-900 nmol/L, or about 900-1000nmol/L. In some embodiments, the concentration of rapamycin in the cell culture is about 100nmol/L.

In some embodiments, the mTor inhibitor is added to the culture for a first time within about 30 minutes to 1 hour, 1 to 2 hours, 2 to 4 hours, 4 to 6 hours, 6 to 8 hours, 8 to 10 hours, 10 to 12 hours, 12 to 14 hours, 14 to 16 hours, 16 to 18 hours, 18 to 24 hours, 24 to 36 hours, 36 to 48 hours, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days of the initial culture. In some embodiments, the mTor inhibitor is added to the culture medium about every 1, 2, 3, 4, or 5 days. In some embodiments, the mTor inhibitor is added to the culture medium about every 4-5 days. In some embodiments, the mTor inhibitor is added to the culture medium about every 3-4 days. In some embodiments, the mTor inhibitor is added to the culture medium about every 2-3 days. In some embodiments, the mTor inhibitor is added to the culture medium about every 1-2 days.

In certain embodiments, the amplification step is performed in a bioreactor that includes an extra-capillary space. In some embodiments, the flow rate of the Extracapillary (EC) medium of the bioreactor may be maintained at about 0-1mL/min, about 0-0.8mL/min, about 0-0.6mL/min, about 0-0.4mL/min, about 0-0.2mL/min, about 0.2-1mL/min, about 0.2-0.8mL/min, about 0.2-0.6mL/min, about 0.2-0.4mL/min, about 0.4-1mL/min, about 0.4-0.8mL/min, about 0.4-0.6mL/min, about 0.6-1mL/min, about 0.6-0.8mL/min, or about 0.8-1mL/min. In some embodiments, the flow rate of the EC media of the bioreactor may be maintained at about 0mL/min, about 0.1mL/min, about 0.2mL/min, about 0.3mL/min, about 0.4mL/min, about 0.5mL/min, about 0.6mL/min, about 0.7mL/min, about 0.8mL/min, about 0.9mL/min, or about 1mL/min. In some embodiments, the amplifying step comprises adjusting the flow rate of the EC medium of the bioreactor based on the number of cells. Cell number means the number of all cells in culture, including enriched Treg cells, which account for the majority of cells in culture and in particular embodiments, for greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 99% or 100% of cells in culture. In a specific embodiment, the amplifying step comprises maintaining the flow rate of the EC medium at 0 until the number of cells reaches 500X 10 ⁶ The flow rate of the EC media was then increased to about 0.2mL/min and the EC media flow rate was maintained at about 0.2mL/min until the cell number reached 750X 10 ⁶ The flow rate of the EC media was then increased to about 0.4mL/min and the EC media was maintained at about 0.4mL/min until the cell number reached 1,000X10 ⁶ The flow rate of the EC media was then increased to about 0.6mL/min and the EC media was maintained at about 0.6mL/min until the cell number reached 1,500X10 ⁶ And then the flow rate of the EC media was increased to about 0.8mL/min and the flow rate of the EC media was maintained at about 0.8mL/min. In certain embodiments, the extracapillary medium comprises rapamycin.

The population of tregs may be expanded by culturing them for an appropriate duration to produce a population of fully expanded tregs. For example, a population of tregs may be expanded for a time sufficient to obtain a desired number or amount of anti-inflammatory EVs. In another example, the population of tregs may be expanded for a time sufficient to achieve one or more characteristics. In certain embodiments, once the Treg population achieves one or more characteristics, an anti-inflammatory EV may be isolated.

For example, the proportion of cd4+cd25+ cells present in the expanded Treg culture may be monitored, e.g., using flow cytometry. For example, in some embodiments, the population of fully expanded tregs is a population of cells containing greater than 70% cd4+cd25+ cells, as determined by flow cytometry.

The number of cd4+cd25+ cells may be determined daily or every 2, 3, 4 or 5 days. In certain embodiments, if the culture does not comprise a fully expanded population of tregs on day 15 (where day 0 is the day the biological sample was obtained from the subject), the cells may be reactivated with one or more expansion agents. In certain embodiments, if the culture does not comprise a sufficiently expanded population of therapeutic tregs on day 14 (where day 0 is the day the biological sample was obtained from the subject), the cells may be reactivated with one or more expansion agents. In certain embodiments, if the culture does not comprise a sufficiently expanded population of therapeutic tregs on day 13 (where day 0 is the day the biological sample was obtained from the subject), the cells may be reactivated with one or more expansion agents. In certain embodiments, if the culture does not comprise a sufficiently expanded population of therapeutic tregs on day 12 (where day 0 is the day the biological sample was obtained from the subject), the cells may be reactivated with one or more expansion agents. In certain embodiments, if the culture does not comprise a sufficiently expanded population of therapeutic tregs at day 11 (where day 0 is the day the biological sample was obtained from the subject), the cells may be reactivated with one or more expansion agents. In certain embodiments, if the culture does not comprise a sufficiently expanded population of therapeutic tregs on day 10 (where day 0 is the day the biological sample was obtained from the subject), the cells may be reactivated with one or more expansion agents. In certain embodiments, if the culture does not comprise a sufficiently expanded population of therapeutic tregs at day 9 (where day 0 is the day the biological sample was obtained from the subject), the cells may be reactivated with one or more expansion agents. In certain embodiments, if the culture does not comprise a sufficiently expanded population of therapeutic tregs on day 8 (where day 0 is the day the biological sample was obtained from the subject), the cells may be reactivated with one or more expansion agents.

In some embodiments, the population of tregs is expanded by culturing for about 6-30 days, about 10-30 days, about 15-25 days, or about 18-22 days. In some embodiments, the population of tregs is expanded by culturing for about 15, 16, 18, 19, 20, 21, 22, 23, 24, or 25 days. In certain embodiments, for example, embodiments comprising an automated, semi-automated, or at least one automated step, the Treg population is expanded by culturing for about 6-15 days, about 8-15, about 8-12 days, or about 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days.

Viability of the cells expanded in culture may be determined using any method known in the art. For example, trypan blue exclusion may be used to determine viability of the expanded cells in culture. Trypan blue is dye that is dye-repellent to cells with intact membranes (living cells), but is absorbed by cells with impaired membrane integrity (non-living cells). Thus, living cells appear transparent under an optical microscope, while non-living cells appear blue. Equal amounts of trypan blue and cell suspension were mixed and counted. Viability was expressed as a percentage of trypan blue dye-repellent cells. In some embodiments, the population of tregs comprises about 60%, 65% or 70% viable cells, as determined by trypan blue exclusion. In some embodiments, the population of tregs comprises greater than about 70% viable cells, as determined by trypan blue exclusion. For example, in certain embodiments, the population of tregs comprises about 75%, 80%, 85%, 90%, 95% or greater than 95% viable cells, as determined by trypan blue exclusion. In some embodiments, viability of the cells expanded in culture is determined every 2-3 days. In some embodiments, viability of the cells expanded in culture is determined daily or every 2, 3, 4, or 5 days.

In some embodiments, the cells are washed one or more times during culturing to remove reagents present during culturing or culturing and/or to supplement the culture medium with one or more other reagents. In some embodiments, the cells are washed during incubation or culture to reduce or remove the amplification agent. The medium may be replaced about every 2, 3, 4, 5, 6 or 7 days, for example, every 2-3 days or every 3-4 days. In some embodiments, only a portion of the medium (e.g., about 50% of the medium) is replaced. In other embodiments, the entire culture medium is replaced. In some embodiments, the cell culture is not centrifuged during medium exchange. In certain embodiments, anti-inflammatory EVs may be isolated and the medium removed during any or each of these points during the amplification process.

To avoid having EV in the serum, the culture from which the EV was isolated may comprise cells cultured in a medium containing EV-depleted serum, e.g., serum free of EV. For example, cells can be cultured in a medium containing EV-depleted or EV-free Fetal Bovine Serum (FBS). In another example, cells may be cultured in a medium containing EV-depleted or EV-free human serum, e.g., human AB serum. In specific examples, cells can be cultured in a medium containing exosome-depleted serum, e.g., exosome-free serum, e.g., exosome-depleted or exosome-free FBS, or exosome-depleted or exosome-free human serum, e.g., human AB serum.

In a specific non-limiting example, isolating the culture of the EV therefrom can include culturing the cells in a medium containing EV-depleted serum, e.g., serum free of EV, for a period of 16 hours, 24 hours, or 48 hours prior to isolation. For example, cells may be cultured in a medium containing EV-depleted or EV-free Fetal Bovine Serum (FBS) for a period of 16 hours, 24 hours, or 48 hours prior to isolation. In another example, cells may be cultured in a medium containing EV-depleted or EV-free human serum, e.g., human AB serum, for a period of 16 hours, 24 hours, or 48 hours prior to isolation. In specific examples, cells may be cultured in a medium containing exosome-depleted serum, e.g., exosome-free serum, e.g., exosome-depleted or exosome-free FBS, or exosome-depleted or exosome-free human serum, e.g., human AB serum, for a period of 16 hours, 24 hours, or 48 hours prior to isolation.

5.2.2 exemplary procedure for isolation and expansion of regulatory T cells from leukopenia or blood sample products

In some embodiments, the EV may be isolated from tregs expanded using the following protocol. The procedure may be applied to the isolation and expansion of leukocyte removal products or blood sample products from, for example, ALS patients, alzheimer's patients, or patients exhibiting different disorders, e.g., different neurodegenerative disorders, or from healthy subjects.

5.2.2.1 step 1: patient leukocyte removal/blood sample product processing

The leukocyte removal or blood sample product should be treated within 24 hours.

The total volume of the leukocyte removal product should be between 100mL and 840mL relative to the leukocyte removal. If the leukocyte removal product is less than 100mL, an equal volume of CliniMACS buffer with 1% human serum albumin (HAS) should be added. The volume reduction of the leukocyte removal product can be performed by the PeriCell protocol and CS490.1 kit (PeriCell) using GE Healthcare/Biosafe Sepax 2 RM.

The leukocyte removal product or blood product was purified using the GE Healthcare-Biosafe Sepax 2RM Neatcell protocol and CS900.2 kit.

5.2.2.2 step 2: treg enrichment

Cd8+ and cd19+ cells can be depleted using the clinic macs kit according to the manufacturer's instructions.This includes Cd8+ and cd19+ microbeads label cells to be depleted, then use The Plus apparatus was combined with CliniMACS PBS/EDTA buffer in 1% HSA, cliniMACS Tubing Set LS and software series DEPLETION 2.1 for automated cell separation.

Subsequently, the method can enableCd25+ Treg enriched populations were positive selected with clinimmacs.This includes enrichment with CD 25-labeled cells of the CD25 microbeads are then used The Plus apparatus was combined with CliniMACS PBS/EDTA buffer in 1% HSA, cliniMACS Tubing Set LS and software series ENRICHMENT 3.2.3.2 for automated cell separation.

5.2.2.3 step 3: treg expansion

On day 0 Treg expansion was initiated from cd25+ enriched white blood cell removal/blood sample product.

The cd25+ enriched leukocyte removal product was centrifuged, the pellet was washed in TexMACS medium with 5% human AB serum, centrifuged again and at 0.8-1.0×10 ⁶ The resulting pellet was resuspended in TexMACS medium with 5% human AB serum at a density of individual cells/mL. Cells were transferred to flasks and incubated at 37℃in 95% air and 5% CO ₂ Is incubated for 16-18 hours in the wet mixture of (C).

After each medium change, the cell concentration should be maintained at 0.5X10 ⁶ Individual cells/mL to 1.2X10 ⁶ Between individual cells/mL. EV may be isolated from the medium removed from the Treg culture at one or more medium exchanges. The medium may be frozen prior to isolation of the EV.

For removal of cell culture medium, flasks were left undisturbed for at least 20 minutes before 50% of the total culture medium volume was removed.

Viability was assessed by trypan blue. If the cell viability is greater than 90%, the cells are expanded by exchanging cell culture media to obtain 0.5X10 ⁶ Individual cells/mL to 1.2X10 ⁶ Individual cells/mL.

On day 1, cells were stimulated with CD3/CD28 beads using the MACS GMP ExpAct Treg kit. The kit contains 3.5 μm particles preloaded with CD28 antibody, anti-biotin antibody and CD 3-biotin. Each vial contained 1X 10 ⁹ Individual ExpAct Treg beads (2×10 ⁵ /μl). MACS for initial stimulationGMP extract Treg beads and Treg cells should be at 4:1 bead: proportion of cells. For activation, the cell concentration should be about 0.5-0.7X10 for the MACS GMP ExpAct Treg kit (CD 3/CD28 beads) ⁶ Individual cells/mL. Activation was performed on day 1 and again on day 15.

Cells were expanded in TexMACS medium with 5% human AB serum supplemented with 100nmol/L rapamycin and 500IU/ml IL-2.

The medium was changed and supplemented with rapamycin on day 4, 6, 8, 11, 13, 15, 18, 20 and 22. IL-2 was supplemented on days 6, 8, 11, 15, 18 and 20. EV may be isolated from the medium removed from the Treg culture at one or more medium exchanges. The medium may be frozen prior to isolation of the EV.

5.2.2.4 step 4: treg harvesting

In certain embodiments, tregs may be harvested. For example, in a particular embodiment, tregs may be harvested on day 25. MACS GMP activating beads can be removed, for example, using CliniMACSDepletion Tubing Set LS (168-01) and software design 2.1 according to manufacturer's instructions or standard procedures. In certain embodiments, the expanded Treg cell product can meet the criteria as shown in table 1. In certain embodiments, the final harvested Treg cell product can meet the criteria as shown in table 1.

The EV (whether Treg harvesting is performed or not) can be isolated from the culture medium on the day Treg will be harvested. For example, EV may be isolated from the medium removed from the Treg culture at the time of harvesting or at the time of harvesting the Treg. The medium may be frozen prior to isolation of the EV.

TABLE 1 expanded Treg/Treg criteria

5.3Composition and method for producing the same

In certain aspects, compositions comprising an isolated, cell-free, anti-inflammatory EV population as described herein are provided. For example, provided herein are compositions comprising an isolated, cell-free, anti-inflammatory EV population suitable for administration to a subject, e.g., a human subject.

In certain aspects, pharmaceutical compositions comprising the isolated, cell-free anti-inflammatory EV populations described herein are provided. In certain embodiments, provided herein are pharmaceutical compositions comprising an isolated, cell-free, anti-inflammatory EV population and a buffer, e.g., a sterile buffer, e.g., a buffer comprising saline. In a specific embodiment, the pharmaceutical composition comprises an isolated, cell-free, anti-inflammatory EV population and physiological saline. In a specific embodiment, the pharmaceutical composition comprises an isolated, cell-free, anti-inflammatory EV population and physiological saline. In a specific embodiment, the pharmaceutical composition comprises an isolated, cell-free, anti-inflammatory EV population and 0.9% saline. In particular embodiments, the pharmaceutical composition comprises an isolated, cell-free, anti-inflammatory EV population and phosphate buffered saline.

In some embodiments, the compositions provided herein are pharmaceutical compositions comprising an anti-inflammatory EV population provided herein and a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the compositions provided herein are pharmaceutical compositions comprising an effective amount of an anti-inflammatory EV population provided herein and a carrier, excipient, or diluent, i.e., an amount of an anti-inflammatory EV population provided herein sufficient to result in a desired result.

As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the united states pharmacopeia, european pharmacopeia, or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The carrier, excipient or diluent may be any pharmaceutically acceptable carrier, excipient or diluent known in the art. Examples of pharmaceutically acceptable carriers include non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials, formulation aids or carriers. Pharmaceutically acceptable carriers can include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solution, dextrose solution, and 5% human serum. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used.

Excipients may include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, coating agents, colorants, disintegrants, emulsifiers, extenders, fillers, flavoring agents, diluents, lubricants, flavorants, preservatives, propellants, mold release agents, sterilizing agents, sweeteners, solubilizing agents, wetting agents and mixtures thereof. The term "excipient" may itself represent a carrier or diluent.

In some embodiments, the pharmaceutical composition comprises an anti-inflammatory EV population provided herein suspended in a sterile buffer. In some embodiments, the pharmaceutical compositions provided herein comprise a population of anti-inflammatory EVs in a buffer suitable for administration to a human subject. Examples of buffers suitable for administration to a human subject include saline-containing buffers such as phosphate buffered saline, physiological saline (physiological saline), physiological saline (normal saline), or 0.9% saline.

The pharmaceutical compositions may be formulated to be compatible with the intended route of administration. For example, pharmaceutical compositions may generally be formulated to be suitable for administration by routes including intranasal, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, intraarterial, intraventricular, intrathecal, intraurethral, intrasternal and intrasynovial), intradermal, oral (e.g., swallowed, sublingual), inhalation, nasal, e.g., nasal drops, intracavity, intracranial, ophthalmic, e.g., intraocular and transdermal (topical).

In certain embodiments, for example, a pharmaceutical composition provided herein comprising an isolated, cell-free, anti-inflammatory EV population as described herein has been formulated to be suitable for intranasal administration to a subject, e.g., a human subject.

In certain embodiments, the pharmaceutical compositions provided herein comprising an isolated, cell-free, anti-inflammatory EV population as described herein have been formulated to be suitable for injection, infusion, or implantation into a subject, e.g., a human subject.

In particular embodiments, the pharmaceutical compositions provided herein comprising an isolated, cell-free, anti-inflammatory EV population as described herein have been formulated to be suitable for intravenous administration to a subject, e.g., a human subject.

In another example, in a specific embodiment, a pharmaceutical composition provided herein comprising an isolated, cell-free, anti-inflammatory EV population as described herein has been formulated to be suitable for subcutaneous administration to a subject, e.g., a human subject.

In another example, in a specific embodiment, a pharmaceutical composition provided herein comprising an isolated, cell-free, anti-inflammatory EV population as described herein has been formulated to be suitable for intramuscular administration to a subject, e.g., a human subject.

In certain embodiments, the compositions have been formulated in the form of solutions, suspensions, emulsions, micelles, liposomes, microspheres, or nanosystems, e.g., the pharmaceutical compositions provided herein comprising an isolated, cell-free, anti-inflammatory EV population as described herein.

In certain embodiments, the compositions may be stored frozen, e.g., the compositions may be stored at-20 ℃ or-80 ℃, e.g., the pharmaceutical compositions provided herein comprising an isolated, cell-free, anti-inflammatory EV population as described herein. For example, in particular embodiments, the composition, e.g., pharmaceutical composition, may be stored frozen, e.g., at-20 ℃ or-80 ℃ for about 1 week, 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, or about 24 months. In particular embodiments, the composition, e.g., a pharmaceutical composition, may then be thawed and administered to a patient.

In certain embodiments, the compositions may be stored frozen, e.g., the compositions may be stored at-20 ℃ or-80 ℃, e.g., the pharmaceutical compositions provided herein comprising an isolated, cell-free, anti-inflammatory EV population as described herein, thawed, and then refrozen. In particular embodiments, the composition, e.g., pharmaceutical composition, may be thawed and then refrozen 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times. In particular embodiments, the composition, e.g., a pharmaceutical composition, may then be thawed and administered to a patient.

In certain embodiments, the compositions can be stored at about 2 ℃ to about 8 ℃ (e.g., at about 4 ℃), e.g., a pharmaceutical composition provided herein comprising an isolated, cell-free, anti-inflammatory EV population as described herein. For example, in particular embodiments, the composition, e.g., a pharmaceutical composition, can be stored at about 2 ℃ to about 8 ℃ (e.g., at about 4 ℃) for less than about 2 weeks, less than about 1 week, less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days, less than about 7 days, less than about 6 days, less than about 5 days, less than about 4 days, less than about 3 days, less than about 2 days, less than about 1 day, or about overnight. In particular embodiments, the composition, e.g., a pharmaceutical composition, may be stored at 4 ℃ prior to administration to a subject, e.g., a human subject, e.g., may be thawed after freezing, and then stored at 4 ℃ prior to administration to a subject, e.g., a human subject.

In certain embodiments, provided herein are cryopreserved compositions, e.g., pharmaceutical compositions, comprising an isolated, cell-free anti-inflammatory EV population as described herein. In particular embodiments, a cryopreserved, isolated cell-free anti-inflammatory EV population may be cryopreserved for about 1 week, 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, or about 24 months, and then may be thawed and administered to a patient after cryopreservation.

In certain embodiments, provided herein are compositions comprising an isolated, cell-free, anti-inflammatory EV population as described herein, wherein the population comprises about 1 x 10 ⁶ Up to about 1X 10 ¹⁶ EV, about 1×10 ⁷ Up to about 1X 10 ¹⁶ EV, 1×10 ⁸ Up to about 1X 10 ¹⁶ EV, about 1×10 ⁹ Up to about 1X 10 ¹⁶ EV, 1×10 ¹⁰ Up to about 1X 10 ¹⁶ EV, about 1×10 ¹¹ Up to about 1X 10 ¹⁶ EV, 1×10 ¹² Up to about 1X 10 ¹⁶ EV, about 1×10 ¹³ Up to about 1X 10 ¹⁶ EV, 1×10 ⁶ Up to about 1X 10 ¹⁵ EV, about 1×10 ⁷ Up to about 1X 10 ¹⁵ EV, 1×10 ⁸ Up to about 1X 10 ¹⁵ EV, about 1×10 ⁹ Up to about 1X 10 ¹⁵ EV, 1×10 ¹⁰ Up to about 1X 10 ¹⁵ EV, about 1×10 ¹¹ Up to about 1X 10 ¹⁵ EV, 1×10 ¹² Up to about 1X 10 ¹⁵ EV, about 1×10 ¹³ Up to about 1X 10 ¹⁵ EV, 1×10 ⁶ Up to about 1X 10 ¹⁴ EV, about 1×10 ⁷ Up to about 1X 10 ¹⁴ EV, 1×10 ⁸ Up to about 1X 10 ¹⁴ EV, about 1×10 ⁹ Up to about 1X 10 ¹⁴ EV, 1×10 ¹⁰ Up to about 1X 10 ¹⁴ EV, about 1×10 ¹¹ Up to about 1X 10 ¹⁴ EV, 1×10 ¹² Up to about 1X 10 ¹⁴ EV, about 1×10 ¹³ Up to about 1X 10 ¹⁴ EV, 1×10 ⁶ Up to about 1X 10 ¹³ EV, about 1×10 ⁷ Up to about 1X 10 ¹³ EV, 1×10 ⁸ Up to about 1X 10 ¹³ EV, about 1×10 ⁹ Up to about 1X 10 ¹³ EV, 1×10 ¹⁰ Up to about 1X 10 ¹³ EV, about 1×10 ¹¹ Up to about 1X 10 ¹³ EV, 1×10 ¹² Up to about 1X 10 ¹³ EV, 1×10 ⁶ Up to about 1X 10 ¹² EV, about 1×10 ⁷ Up to about 1X 10 ¹² EV, 1×10 ⁸ Up to about 1X 10 ¹² EV, about 1×10 ⁹ Up to about 1X 10 ¹² EV, 1×10 ¹⁰ Up to about 1X 10 ¹² EV, about 1×10 ¹¹ Up to about 1X 10 ¹² EV, about 1×10 ⁶ Up to about 1X 10 ¹¹ EV, about 1×10 ⁷ Up to about 1X 10 ¹¹ EV, 1×10 ⁸ Up to about 1X 10 ¹¹ EV, about 1×10 ⁹ Up to about 1X 10 ¹¹ EV, 1×10 ¹⁰ To about1×10 ¹¹ EV, about 1×10 ⁶ Up to about 1X 10 ¹⁰ EV, about 1×10 ⁷ Up to about 1X 10 ¹⁰ EV, 1×10 ⁸ Up to about 1X 10 ¹⁰ EV, about 1×10 ⁶ EV, about 1×10 ⁷ EV, 1×10 ⁸ Up to about 1X 10 ⁹ EV, about 1×10 ¹⁰ Up to about 1X 10 ¹¹ EV, 1×10 ¹² Up to about 1X 10 ¹³ EV, about 1×10 ⁶ EV, about 1×10 ⁷ EV, about 1×10 ⁸ EV, about 2×10 ⁸ EV, about 3×10 ⁸ EV, about 4×10 ⁸ EV, about 5×10 ⁸ EV, about 6×10 ⁸ EV, about 7×10 ⁸ EV, about 8×10 ⁸ EV, about 9×10 ⁸ EV, about 1×10 ⁹ EV, about 5×10 ⁹ EV, about 1×10 ¹⁰ EV, about 1×10 ¹¹ EV, about 1×10 ¹² EV, about 1×10 ¹³ EV, about 1×10 ¹⁴ EV, about 1×10 ¹⁵ EV or about 1×10 ¹⁶ And (5) EV.

In certain embodiments, provided herein are compositions comprising an isolated, cell-free, anti-inflammatory EV population as described herein, wherein the population comprises 1 x 10 ⁶ Up to about 1X 10 ¹⁶ EV/ml, about 1X 10 ⁷ Up to about 1X 10 ¹⁶ EV/ml, 1X 10 ⁸ Up to about 1X 10 ¹⁶ EV/ml, about 1X 10 ⁹ Up to about 1X 10 ¹⁶ EV/ml, 1X 10 ¹⁰ Up to about 1X 10 ¹⁶ EV/ml, about 1X 10 ¹¹ Up to about 1X 10 ¹⁶ EV/ml, 1X 10 ¹² Up to about 1X 10 ¹⁶ EV/ml, about 1X 10 ¹³ Up to about 1X 10 ¹⁶ EV/ml, 1X 10 ⁶ Up to about 1X 10 ¹⁵ EV/ml, about 1X 10 ⁷ Up to about 1X 10 ¹⁵ EV/ml, 1X 10 ⁸ Up to about 1X 10 ¹⁵ EV/ml, about 1X 10 ⁹ Up to about 1X 10 ¹⁵ EV/ml, 1X 10 ¹⁰ Up to about 1X 10 ¹⁵ EV/ml, about 1X 10 ¹¹ Up to about 1X 10 ¹⁵ EV/ml, 1X 10 ¹² Up to about 1X 10 ¹⁵ EV/ml, about 1X 10 ¹³ Up to about 1X 10 ¹⁵ EV/ml, about 1X 10 ⁶ Up to about 1X 10 ¹⁴ EV/ml of eachAbout 1X 10 ⁷ Up to about 1X 10 ¹⁴ EV/ml, 1X 10 ⁸ Up to about 1X 10 ¹⁴ EV/ml, about 1X 10 ⁹ Up to about 1X 10 ¹⁴ EV/ml, 1X 10 ¹⁰ Up to about 1X 10 ¹⁴ EV/ml, about 1X 10 ¹¹ Up to about 1X 10 ¹⁴ EV/ml, 1X 10 ¹² Up to about 1X 10 ¹⁴ EV/ml, about 1X 10 ¹³ Up to about 1X 10 ¹⁴ EV/ml, 1X 10 ⁶ Up to about 1X 10 ¹³ EV/ml, about 1X 10 ⁷ Up to about 1X 10 ¹³ EV/ml, 1X 10 ⁸ Up to about 1X 10 ¹³ EV/ml, about 1X 10 ⁹ Up to about 1X 10 ¹³ EV/ml, 1X 10 ¹⁰ Up to about 1X 10 ¹³ EV/ml, about 1X 10 ¹¹ Up to about 1X 10 ¹³ EV/ml, 1X 10 ¹² Up to about 1X 10 ¹³ EV/ml, 1X 10 ⁶ Up to about 1X 10 ¹² EV/ml, about 1X 10 ⁷ Up to about 1X 10 ¹² EV/ml, 1X 10 ⁸ Up to about 1X 10 ¹² EV/ml, about 1X 10 ⁹ Up to about 1X 10 ¹² EV/ml, 1X 10 ¹⁰ Up to about 1X 10 ¹² EV/ml, about 1X 10 ¹¹ Up to about 1X 10 ¹² EV/ml, about 1X 10 ⁶ Up to about 1X 10 ¹¹ EV/ml, about 1X 10 ⁷ Up to about 1X 10 ¹¹ EV/ml, 1X 10 ⁸ Up to about 1X 10 ¹¹ EV/ml, about 1X 10 ⁹ Up to about 1X 10 ¹¹ EV/ml, 1X 10 ¹⁰ Up to about 1X 10 ¹¹ EV/ml, about 1X 10 ⁶ Up to about 1X 10 ¹⁰ EV/ml, about 1X 10 ⁷ Up to about 1X 10 ¹⁰ EV/ml, 1X 10 ⁸ Up to about 1X 10 ¹⁰ EV/ml, about 1X 10 ⁶ EV/ml, about 1X 10 ⁷ EV/ml, 1X 10 ⁸ Up to about 1X 10 ⁹ EV/ml, about 1X 10 ¹⁰ Up to about 1X 10 ¹¹ EV/ml, 1X 10 ¹² Up to about 1X 10 ¹³ EV/ml, about 1X 10 ⁶ About 1×107 EVs/ml, about 1×108 EVs/ml, about 2×108 EVs/ml, about 3×108 EVs/ml, about 4×108 EVs/ml, about 5×10 EV/ml ⁸ EV/ml, about 6X 10 ⁸ EV/ml, about 7X 10 ⁸ EV/ml, about 8×108 EV/ml, about 9×10 ⁸ EV/ml, about 1X 10 ⁹ EV/ml, about 5X10 ⁹ EV/ml, about 1X 10 ¹⁰ EV/ml, about 1X 10 ¹¹ EV/ml, about 1X 10 ¹² EV/ml, about 1X 10 ¹³ EV/ml, about 1X 10 ¹⁴ EV/ml, about 1X 10 ¹⁵ EV/ml or about 1X 10 ¹⁶ EV/mls of each

In certain embodiments, provided herein are compositions comprising an isolated, cell-free, anti-inflammatory EV population as described herein, wherein the population comprises from about 1 μg to about 200mg EV, from about 1 μg to about 150mg EV, from about 1 μg to about 100mg EV, from about 1 μg to about 75mg EV, from about 1 μg to about 50mg EV, from about 1 μg to about 25mg EV, from about 1 μg to about 20mg EV, from about 1 μg to about 15mg EV, from about 1 μg to about 10mg EV, from about 1 μg to about 5mg EV, from about 1 μg to about 1mg EV, from about 1 μg to about 500 μg EV, from about 1 μg to about 250 μg EV, from about 1 μg to about 125 μg EV, from about 1 μg to about 100 μg, from about 1 μg to about 50 μg EV, from about 1 μg to about 25 μg EV, from about 1 μg to about 20 μg EV, from about 1 μg to about 10 μg EV, from about 1 μg to about 5 μg EV, from about 10 μg to about gEV, from about 10 μg to about 5 μg EV, from about 1 μg to about 1mg EV, from about 1 μg to about 500 μg EV, from about 1 μg to about 250 μg EV, from about 1 μg to about 125 μg EV, from about 1 μg to about 10 μg to about 125 μg EV, from about 10 μg to about 100 μg EV, from about 10 μg to about 10 μg EV, from about 10 μg to about 100 μg EV.

In certain embodiments, provided herein are compositions comprising an isolated, cell-free, anti-inflammatory EV population as described herein, wherein the population comprises from about 1 μg to about 200mg EV/ml, from about 1 μg to about 150mg EV/ml, from about 1 μg to about 100mg EV/ml, from about 1 μg to about 75mg EV/ml, from about 1 μg to about 50mg EV/ml, from about 1 μg to about 25mg EV/ml, from about 1 μg to about 20mg EV/ml, from about 1 μg to about 15mg EV/ml, from about 1 μg to about 10mg EV/ml, from about 1 μg to about 5mg EV/ml, from about 1 μg to about 1mg EV/ml, from about 1 μg to about 500 μg gEV/ml, from about 1 μg to about 250 μg EV/ml, from about 1 μg to about 125 μg EV/ml, from about 1 μg to about 100 μg gEV/ml about 1 μg to about 50 μg EV/ml, about 1 μg to about 25 μg EV/ml, about 1 μg to about 20 μg EV/ml, about 1 μg to about 10 μg EV/ml, about 1 μg to about 5 μg EV/ml, about 10 μg to about 500 μg EV/ml, about 10 μg to about 250 μg EV/ml, about 10 μg to about 125 μg EV/ml, about 10 μg to about 100 μg EV/ml, about 10 μg to about 50 μg EV/ml, about 10 μg to about 25 μg EV/ml, about 10 μg to about 20 μg EV/ml, about 100 μg to about 500 μg EV/ml, about 100 μg to about 250 μg EV/ml, or about 100 μg to about 125 μg EV/ml.

In some embodiments, a composition comprising an anti-inflammatory EV population provided herein does not comprise a contaminant. In some embodiments, a composition comprising an anti-inflammatory EV population provided herein comprises a sufficiently low level of contaminants to be suitable for administration, e.g., therapeutic administration, to a subject, e.g., a human subject. Contaminants include, for example, bacteria, fungi, mycoplasma, endotoxins, or residual beads from Treg expansion cultures. In some embodiments, a composition comprising an anti-inflammatory EV population provided herein comprises less than about 5EU/kg endotoxin. In some embodiments, a composition comprising an anti-inflammatory EV population provided herein comprises about or less than about 100 beads/3 x 10 ⁶ Individual cells.

In some embodiments, the composition comprising the anti-inflammatory EV population provided herein is substantially free of components used during the Treg cell expansion process and/or the EV isolation process. For example, in some embodiments, a composition comprising an anti-inflammatory EV population provided herein is substantially free of IL2. In specific embodiments, for example, a composition comprising an anti-inflammatory EV population provided herein comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or less IL2 (as a percentage of the original amount of IL2 in Treg cell culture).

As discussed herein, in certain embodiments, treg cells from which Treg EVs are obtained may be cultured in an albumin-containing medium. In certain embodiments, a composition comprising an anti-inflammatory EV population provided herein is substantially free of albumin. In specific embodiments, for example, a composition comprising an anti-inflammatory EV population provided herein comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or less albumin (as a percentage of the original amount of albumin in Treg cell culture).

In some embodiments, the compositions comprising the anti-inflammatory EV population provided herein are sterile. In some embodiments, the separation or enrichment of cells is performed in a closed or sterile environment, e.g., to minimize errors, user handling, and/or contamination. In some embodiments, sterility can be readily achieved, for example, by filtration through a sterile filtration membrane.

In certain embodiments, provided herein are pharmaceutical compositions comprising a population of anti-inflammatory EVs described herein, wherein the pharmaceutical compositions comprise any amount or concentration of an anti-inflammatory EV described herein, e.g., a Treg EV. For example, in certain embodiments, provided herein are compositions comprising about 1 x 10 ⁹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 formulated in unit dosage form ⁹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 in unit dosage formulations comprising in saline, e.g., sterile saline, such as sterile saline for injection ⁹ Pharmaceutical composition of individual Treg EV. In another embodiment, provided herein are compositions comprising about 1 x 10 in unit dose formulated in 1mL, 2mL, 3mL, 4mL, or 5mL saline, e.g., sterile saline, such as sterile saline for injection ⁹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are methods for treating a subject with a composition comprising 2mL of saline, e.g., sterile saline, such as sterile saline for injectionUnit dose formulations comprising about 1 x 10 ⁹ Pharmaceutical composition of individual Treg EV. The pharmaceutical composition may be present as a single unit dose or as multiple unit doses, for example, in vials, such as sterile vials. For example, in particular embodiments, provided herein are compositions comprising about 1 x 10 in a vial in 1mL, 2mL, 3mL, 4mL, or 5mL sterile saline as a unit dose ⁹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 in 2mL sterile saline as a unit dose in a vial ⁹ Pharmaceutical composition of individual Treg EV.

In certain embodiments, provided herein are pharmaceutical compositions comprising a population of anti-inflammatory EVs described herein, wherein the pharmaceutical compositions comprise any amount or concentration of an anti-inflammatory EV described herein, e.g., a Treg EV. For example, in certain embodiments, provided herein are compositions comprising about 5 x 10 ⁹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 5 x 10 formulated in unit dosage form ⁹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 5 x 10 in unit dosage formulations comprising in saline, e.g., sterile saline, such as sterile saline for injection ⁹ Pharmaceutical composition of individual Treg EV. In another embodiment, provided herein are compositions comprising about 5 x 10 in unit dosage formulated in 1mL, 2mL, 3mL, 4mL, or 5mL saline, e.g., sterile saline, such as sterile saline for injection ⁹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 5 x 10 in unit dosage formulated in 2mL of saline, e.g., sterile saline, such as sterile saline for injection ⁹ Pharmaceutical composition of individual Treg EV. The pharmaceutical composition may be present as a single unit dose or as multiple unit doses, for example, in vials, such as sterile vials. For example, in particular embodiments, provided herein are compositions comprising about 5 x 10 in a vial in 1mL, 2mL, 3mL, 4mL, or 5mL sterile saline as a unit dose ⁹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are 2mL absence in a vialThe unit dose of the bacteria saline contains about 5×10 ⁹ Pharmaceutical composition of individual Treg EV.

In certain embodiments, provided herein are pharmaceutical compositions comprising a population of anti-inflammatory EVs described herein, wherein the pharmaceutical compositions comprise any amount or concentration of an anti-inflammatory EV described herein, e.g., a Treg EV. For example, in certain embodiments, provided herein are compositions comprising about 1 x 10 ¹⁰ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 formulated in unit dosage form ¹⁰ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 in unit dosage formulations comprising in saline, e.g., sterile saline, such as sterile saline for injection ¹⁰ Pharmaceutical composition of individual Treg EV. In another embodiment, provided herein are compositions comprising about 1 x 10 in unit dose formulated in 1mL, 2mL, 3mL, 4mL, or 5mL saline, e.g., sterile saline, such as sterile saline for injection ¹⁰ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 in unit dose formulations in 2mL of saline, e.g., sterile saline, such as sterile saline for injection ¹⁰ Pharmaceutical composition of individual Treg EV. The pharmaceutical composition may be present as a single unit dose or as multiple unit doses, for example, in vials, such as sterile vials. For example, in particular embodiments, provided herein are compositions comprising about 1 x 10 in a vial in 1mL, 2mL, 3mL, 4mL, or 5mL sterile saline as a unit dose ¹⁰ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 in 2mL sterile saline as a unit dose in a vial ¹⁰ Pharmaceutical composition of individual Treg EV.

In certain embodiments, provided herein are pharmaceutical compositions comprising a population of anti-inflammatory EVs described herein, wherein the pharmaceutical compositions comprise any amount or concentration of an anti-inflammatory EV described herein, e.g., a Treg EV. For example, in certain embodiments, provided herein are compositions comprising about 5 x 10 ¹⁰ Pharmaceutical composition of individual Treg EV. In particular embodimentsWherein the formulation is provided herein in unit dosage form comprising about 5X 10 ¹⁰ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 5 x 10 in unit dosage formulations comprising in saline, e.g., sterile saline, such as sterile saline for injection ¹⁰ Pharmaceutical composition of individual Treg EV. In another embodiment, provided herein are compositions comprising about 5 x 10 in unit dosage formulated in 1mL, 2mL, 3mL, 4mL, or 5mL saline, e.g., sterile saline, such as sterile saline for injection ¹⁰ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 5 x 10 in unit dosage formulated in 2mL of saline, e.g., sterile saline, such as sterile saline for injection ¹⁰ Pharmaceutical composition of individual Treg EV. The pharmaceutical composition may be present as a single unit dose or as multiple unit doses, for example, in vials, such as sterile vials. For example, in particular embodiments, provided herein are compositions comprising about 5 x 10 in a vial in 1mL, 2mL, 3mL, 4mL, or 5mL sterile saline as a unit dose ¹⁰ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 5 x 10 in 2mL sterile saline as a unit dose in a vial ¹⁰ Pharmaceutical composition of individual Treg EV.

In certain embodiments, provided herein are pharmaceutical compositions comprising a population of anti-inflammatory EVs described herein, wherein the pharmaceutical compositions comprise any amount or concentration of an anti-inflammatory EV described herein, e.g., a Treg EV. For example, in certain embodiments, provided herein are compositions comprising about 1 x 10 ¹¹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 formulated in unit dosage form ¹¹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 in unit dosage formulations comprising in saline, e.g., sterile saline, such as sterile saline for injection ¹¹ Pharmaceutical composition of individual Treg EV. In another embodiment, provided herein is a saline solution at 1mL, 2mL, 3mL, 4mL, or 5mL, e.g., sterile saline, such as sterile saline for injectionComprising about 1X 10 in unit dosage formulation ¹¹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 in unit dose formulations in 2mL of saline, e.g., sterile saline, such as sterile saline for injection ¹¹ Pharmaceutical composition of individual Treg EV. The pharmaceutical composition may be present as a single unit dose or as multiple unit doses, for example, in vials, such as sterile vials. For example, in particular embodiments, provided herein are compositions comprising about 1 x 10 in a vial in 1mL, 2mL, 3mL, 4mL, or 5mL sterile saline as a unit dose ¹¹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 in 2mL sterile saline as a unit dose in a vial ¹¹ Pharmaceutical composition of individual Treg EV.

In certain embodiments, provided herein are pharmaceutical compositions comprising a population of anti-inflammatory EVs described herein, wherein the pharmaceutical compositions comprise any amount or concentration of an anti-inflammatory EV described herein, e.g., a Treg EV. For example, in certain embodiments, provided herein are compositions comprising about 5 x 10 ¹¹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 5 x 10 formulated in unit dosage form ¹¹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 5 x 10 in unit dosage formulations comprising in saline, e.g., sterile saline, such as sterile saline for injection ¹¹ Pharmaceutical composition of individual Treg EV. In another embodiment, provided herein are compositions comprising about 5 x 10 in unit dosage formulated in 1mL, 2mL, 3mL, 4mL, or 5mL saline, e.g., sterile saline, such as sterile saline for injection ¹¹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 5 x 10 in unit dosage formulated in 2mL of saline, e.g., sterile saline, such as sterile saline for injection ¹¹ Pharmaceutical composition of individual Treg EV. The pharmaceutical composition may be present as a single unit dose or as multiple unit doses, for example, in vials, such as sterile vials. For example, in a specific embodiment, the presentProvided herein are compositions comprising about 5 x 10 in vials as unit doses in 1mL, 2mL, 3mL, 4mL or 5mL sterile saline ¹¹ Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 5 x 10 in 2mL sterile saline as a unit dose in a vial ¹¹ Pharmaceutical composition of individual Treg EV.

In certain embodiments, provided herein are pharmaceutical compositions comprising a population of anti-inflammatory EVs described herein, wherein the pharmaceutical compositions comprise any amount or concentration of an anti-inflammatory EV described herein, e.g., a Treg EV. For example, in certain embodiments, provided herein are compositions comprising about 1 x 10 ¹² Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 formulated in unit dosage form ¹² Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 in unit dosage formulations comprising in saline, e.g., sterile saline, such as sterile saline for injection ¹² Pharmaceutical composition of individual Treg EV. In another embodiment, provided herein are compositions comprising about 1 x 10 in unit dose formulated in 1mL, 2mL, 3mL, 4mL, or 5mL saline, e.g., sterile saline, such as sterile saline for injection ¹² Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 in unit dose formulations in 2mL of saline, e.g., sterile saline, such as sterile saline for injection ¹² Pharmaceutical composition of individual Treg EV. The pharmaceutical composition may be present as a single unit dose or as multiple unit doses, for example, in vials, such as sterile vials. For example, in particular embodiments, provided herein are compositions comprising about 1 x 10 in a vial in 1mL, 2mL, 3mL, 4mL, or 5mL sterile saline as a unit dose ¹² Pharmaceutical composition of individual Treg EV. In particular embodiments, provided herein are compositions comprising about 1 x 10 in 2mL sterile saline as a unit dose in a vial ¹² Pharmaceutical composition of individual Treg EV.

5.4 methods of treatment

Provided herein are methods of treatment comprising administering to a subject in need thereof an effective amount of an anti-inflammatory EV population as described herein.

In some embodiments, the subject is diagnosed with or suspected of having a condition associated with Treg dysfunction. In some embodiments, the subject is diagnosed with or suspected of having a disorder associated with Treg deficiency. In some embodiments, the subject is diagnosed with or suspected of having a condition driven by a T cell response (e.g., an inflammatory condition). In some embodiments, the subject is diagnosed with or suspected of having a condition driven by a bone marrow cell response (e.g., an inflammatory condition). In some embodiments, the subject is diagnosed with or suspected of having a condition for which a bone marrow cell response is helpful (e.g., caused by or exacerbated by a bone marrow cell response). In certain embodiments, the condition is an inflammatory, autoimmune or neurodegenerative disorder. In specific embodiments, the bone marrow cells are monocytes, macrophages or microglia. In certain embodiments, the bone marrow cells comprise microglia in the brain. In certain embodiments, the bone marrow cells comprise monocytes or macrophages in the peripheral, external, central nervous system.

In some embodiments, the subject is diagnosed with or suspected of having a neurodegenerative disease. In some embodiments, the subject is diagnosed with or suspected of having alzheimer's disease, amyotrophic lateral sclerosis, huntington's disease, parkinson's disease, or frontotemporal dementia.

In some embodiments, the subject is diagnosed with or suspected of having a condition that would benefit from down-regulation of the immune system.

In some embodiments, the subject is diagnosed with or suspected of having an autoimmune disease. The autoimmune disease may be, for example, systemic sclerosis (scleroderma), polymyositis, ulcerative colitis, inflammatory bowel disease, crohn's disease, celiac disease, multiple Sclerosis (MS), rheumatoid Arthritis (RA), type I diabetes, psoriasis, dermatomyositis, lupus, for example, systemic lupus erythematosus or cutaneous lupus, myasthenia gravis, autoimmune kidney disease, autoimmune hemolytic anemia, autoimmune cytopenia, autoimmune encephalitis, autoimmune hepatitis, autoimmune uveitis, alopecia, thyroiditis, or pemphigus.

In some embodiments, the subject is diagnosed with or suspected of having heart failure or ischemic cardiomyopathy.

In some embodiments, the subject is diagnosed with or suspected of having graft versus host disease, e.g., after undergoing an organ transplant (e.g., a kidney transplant or a liver transplant), or after undergoing a stem cell transplant (e.g., a hematopoietic stem cell transplant, including a bone marrow transplant).

In some embodiments, the subject is diagnosed with or suspected of having neurogenic inflammation. The neurogenic inflammation may be associated with, for example, stroke, acute Disseminated Encephalomyelitis (ADEM), acute optic neuritis, acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, gillin-barre syndrome, transverse myelitis, neuromyelitis optica (NMO), epilepsy, traumatic brain injury, spinal cord injury, encephalitis Central Nervous System (CNS) vasculitis, nervous system sarcoidosis, post-autoimmune or post-infection encephalitis, or chronic meningitis.

In some embodiments, the subject is diagnosed with or suspected of having a liver disorder. The liver condition may be, for example, fatty liver, e.g., non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), primary cholangitis, autoimmune hepatitis, liver cancer, hepatitis a, hepatitis b, and hepatitis c. In certain embodiments, the liver disorder is NAFLD. In certain embodiments, the liver disorder is NASH.

In some embodiments, the subject is diagnosed with or suspected of having Alcoholic Hepatitis (AH) or Alcoholic Steatohepatitis (ASH).

In some embodiments, the subject is diagnosed with or suspected of having a metabolic disorder. The metabolic disorder may be, but is not limited to, fibrosis, metabolic syndrome, NAFLD, and NASH.

In some embodiments, the subject is in need of improved islet transplantation survival, and the method comprises administering to the subject an effective amount of an anti-inflammatory EV population as described herein or a pharmaceutical composition as described herein, in combination with islet transplantation.

In some embodiments, the subject is diagnosed with or suspected of having cardiac inflammation, e.g., associated with atherosclerosis, myocardial infarction, ischemic cardiomyopathy, cardiac inflammation associated with heart failure.

In some embodiments, the subject is diagnosed with or suspected of having Chronic Inflammatory Demyelinating Polyneuropathy (CIDP). In some embodiments, the subject is diagnosed with or suspected of having Acute Inflammatory Demyelinating Polyneuropathy (AIDP). In some embodiments, the subject is diagnosed with or suspected of having a gehlrabi-gilbert syndrome (GBS).

In some embodiments, the subject has a stroke.

In some embodiments, the subject is diagnosed with or suspected of having cancer, e.g., a blood cancer.

In some embodiments, the subject is diagnosed with or suspected of having asthma.

In some embodiments, the subject is diagnosed with or suspected of having eczema.

In some embodiments, the subject is diagnosed with or suspected of having a disorder associated with excessive activation of the immune system.

In some embodiments, the subject is diagnosed with or suspected of having Treg lesions (tregolopathy). The Treg lesions may be caused by FOXP3, CD25, cytotoxic T lymphocyte-associated antigen 4 (CTLA 4), LPS-reactive beige anchor-like protein (LRBA) or BTB domain or CNC homologous gene 2 (BACH 2) gene function deletion mutations or signal transduction and activator of transcription 3 (STAT 3) function gain mutations.

In some embodiments, the donor subject is an elderly subject, e.g., a subject at least 65, at least 70, at least 75, at least 80, at least 85, or at least 90 years old.

In some embodiments, about 1×10 is administered ⁸ Up to about 1X 10 ¹⁴ EV, about 1×10 ⁸ Up to about 1X 10 ¹² EV, about 1×10 ⁸ Up to about 1X 10 ¹⁰ EV, about 1×10 ¹⁰ Up to about 1X 10 ¹⁴ EV, about 1×10 ¹⁰ Up to about 1X 10 ¹² EV, about 1×10 ⁶ Up to about 1X 10 ⁷ EV, about 1×10 ⁷ Up to about 1X 10 ⁸ EV, about 1×10 ⁸ Up to about 1X 10 ⁹ EV, about 1×10 ⁹ Up to about 1X 10 ¹⁰ EV, about 1×10 ¹⁰ Up to about 1X 10 ¹¹ EV, about 1×10 ¹² Up to about 1X 10 ¹³ EV, about 1×10 ¹³ Up to about 1X 10 ¹⁴ EV or about 1×10 ¹⁴ Up to about 1X 10 ¹⁵ And (5) EV.

In some embodiments, about 1 x 10 per dose is administered, for example ⁸ EV, about 2×10 ⁸ EV, about 3×10 ⁸ EV, about 4×10 ⁸ EV, about 5×10 ⁸ EV, about 6×10 ⁸ EV, about 7×10 ⁸ EV, about 8×10 ⁸ EV, about 9×10 ⁸ EV, about 1×10 ⁹ EV, about 2×10 ⁹ EV, about 3×10 ⁹ EV, about 4×10 ⁹ EV, about 5×10 ⁹ EV, about 6×10 ⁹ EV, about 7×10 ⁹ EV, about 8×10 ⁹ EV, about 9×10 ⁹ EV or about 1×10 ¹⁰ And (5) EV.

In some embodiments, about 1×10 is administered ⁸ Up to about 1X 10 ¹⁴ EV/ml, about 1X 10 ⁸ Up to about 1X 10 ¹² EV/ml, about 1X 10 ⁸ Up to about 1X 10 ¹⁰ EV/ml, about 1X 10 ¹⁰ Up to about 1X 10 ¹⁴ EV/ml, about 1X 10 ¹⁰ Up to about 1X 10 ¹² EV/ml, about 1X 10 ⁶ Up to about 1X 10 ⁷ EV/ml, about 1X 10 ⁷ Up to about 1X 10 ⁸ EV/ml, about 1X 10 ⁸ Up to about 1X 10 ⁹ EV/ml, about 1X 10 ⁹ Up to about 1X 10 ¹⁰ EV/ml, about 1X 10 ¹⁰ Up to about 1X 10 ¹¹ EV/ml, about 1X 10 ¹² Up to about 1X 10 ¹³ EV/ml, about 1X 10 ¹³ Up to about 1X 10 ¹⁴ EV/mL or about 1X 10 ¹⁴ Up to about 1X 10 ¹⁵ EV/mL.

In some embodiments, about 1 x 10 per dose is administered, for example ⁸ EV/ml, about 2X 10 ⁸ EV/ml, about 3X 10 ⁸ EV/ml, about 4X 10 ⁸ EV/ml, about 5X 10 ⁸ EV/ml, about 6X 10 ⁸ EV/ml, about 7X 10 ⁸ EV/ml, about 8X 10 ⁸ EV/ml, about 9X 10 ⁸ EV/ml, about 1X 10 ⁹ EV/ml, about 2X 10 ⁹ EV/ml, about 3X 10 ⁹ EV/ml, about 4X 10 ⁹ EV/ml, about 5X 10 ⁹ EV/ml, about 6X 10 ⁹ EV/ml, about 7X 10 ⁹ EV/ml, about 8X 10 ⁹ EV/ml, about 9X 10 ⁹ EV/ml or about 1X 10 ¹⁰ EV/ml.

In some embodiments, about 1 μg to about 100 μg EV, about 100 μg to about 200 μg EV, about 200 μg to about 300 μg EV, about 300 μg to about 400 μg EV, about 400 μg to about 500 μg EV, about 500 μg to about 600 μg EV, about 600 μg to about 700 μg EV, about 700 μg to about 800 μg EV, about 800 μg to about 900 μg EV, about 900 μg to about 1mg EV, about 1mg to about 10mg EV, about 10mg to about 20mg EV, about 20mg to about 30mg EV, about 30mg to about 40mg EV, about 40mg to about 50mg EV, about 50mg to about 60mg EV, about 60mg to about 70mg EV, about 70mg to about 80mg EV, about 80mg to about 90mg EV, about 90mg to about 100mg EV, about 100mg to about 110mg, about 110mg to about 120mg, about 120mg to about 130mg, about 130mg to about 170mg EV, about 180mg to about 170mg or about 180mg to about 190mg EV is administered.

In some embodiments, about 1 μg to about 100 μg EV/mL, about 100 μg to about 200 μg EV/mL, about 200 μg to about 300 μg EV/mL, about 300 μg to about 400 μg EV/mL, about 400 μg to about 500 μg gEV/mL, about 500 μg to about 600 μg EV/mL, about 600 μg to about 700 μg EV/mL, about 700 μg to about 800 μg EV/mL, about 800 μg to about 900 μg EV/mL, about 900 μg to about 1mg EV/mL, about 1mg to about 10mg EV/mL, about 10mg to about 20mg EV/mL, about 20mg to about 30mg EV/mL, about 30mg to about 40mg EV/mL, about 40mg to about 50mg EV/mL, about 50mg to about 60mg EV/mL, about 60mg to about 70mg, about 70mg to about 80mg, about 80mg to about 90mg, about 90mg to about 90mg EV/mL, about 180mg to about 170mg to about 120mg, about 180mg to about 130mg, about 180mg to about 120mg, about 180mg to about 130mg, about 180mg to about 120mg, about 100mg to about 130mg EV/mL.

The anti-inflammatory EV population may be administered to the subject by any suitable route. For example, an anti-inflammatory EV population can be administered to the subject by routes including intranasal, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, intraarterial, intraventricular, intrathecal, intraurethral, intrasternal, and intrasynovial), intradermal, oral (e.g., swallowed, sublingual), inhalation, nasal, e.g., nasal drops, intracavity, intracranial, ocular, e.g., intraocular, and transdermal (topical). In particular embodiments, for example, an anti-inflammatory EV population can be administered to the subject in a pharmaceutical composition formulated for administration by a route including intranasal, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, intraarterial, intraventricular, intrathecal, intraurethral, intrasternal, and intrasynovial), intradermal, oral (e.g., swallowed, sublingual), inhalation, nasal, e.g., nasal drops, intracavity, intracranial, ocular, e.g., intraocular, and transdermal (topical).

In certain embodiments, for example, the methods of treatment provided herein comprise administering to a subject in need of treatment a pharmaceutical composition comprising an effective amount of an isolated, cell-free, anti-inflammatory EV population as described herein and formulated for intranasal administration to a subject, e.g., a human subject.

In certain embodiments, for example, the methods of treatment provided herein comprise administering to a subject in need of treatment a pharmaceutical composition comprising an effective amount of an isolated, cell-free, anti-inflammatory EV population as described herein and formulated to be suitable for injection, infusion, or transplantation into a subject, e.g., a human subject.

In certain embodiments, for example, the methods of treatment provided herein comprise administering to a subject in need of treatment a pharmaceutical composition comprising an effective amount of an isolated, cell-free, anti-inflammatory EV population as described herein and formulated for intravenous administration to a subject, e.g., a human subject.

In certain embodiments, for example, the methods of treatment provided herein comprise administering to a subject in need of treatment a pharmaceutical composition comprising an effective amount of an isolated, cell-free, anti-inflammatory EV population as described herein and formulated for subcutaneous administration to a subject, e.g., a human subject.

In certain embodiments, for example, the methods of treatment provided herein comprise administering to a subject in need of treatment a pharmaceutical composition comprising an effective amount of an isolated, cell-free, anti-inflammatory EV population as described herein and formulated for intramuscular administration to a subject, e.g., a human subject.

More than one anti-inflammatory EV population may be administered to the subject. For example, the anti-inflammatory EV population may be administered to the subject weekly, every other week, every three weeks, once a month, every other month, every 3 months, every 6 months, every 12 months, every 18 months, annually, every other year, every 3 years, or every 5 years.

The anti-inflammatory EV population administered to the subject can be autologous to the subject. The anti-inflammatory EV population administered to the subject can be allogeneic to the subject. The anti-inflammatory EV population administered to the subject may be derived from human inhibitory immune cells, e.g., tregs, from more than one individual. For example, the anti-inflammatory EV population administered to the subject can be a mixed anti-inflammatory EV population, wherein some or all of the anti-inflammatory EV population is allogeneic to the subject.

In certain embodiments, more than one anti-inflammatory EV population may be administered to the subject. For example, the anti-inflammatory EV population may be administered to the subject weekly, every other week, every three weeks, once a month, every other month, every 3 months, every 6 months, every 12 months, every 18 months, annually, every other year, every 3 years, or every 5 years.

5.4.1 other therapies

In some embodiments, a subject treated according to the methods of treatment described herein also receives one or more additional therapies.

In some embodiments, an effective amount of an ex vivo expanded population of tregs is additionally administered to the subject. In some embodiments, the population of tregs has been cryopreserved. In some embodiments, the cryopreserved population of tregs has been thawed and administered to the subject without further expansion.

In some embodiments, the population of tregs or the cryopreserved population of tregs may be one of the population of tregs from which the EV administered to the subject has been isolated. In some embodiments, the population of tregs or cryopreserved population of tregs is the population of tregs described in international patent application No. pct/US2020/63378, or is produced by the method described in international patent application No. pct/US2020/63378, the entire contents of which are incorporated herein by reference.

In some embodiments, about 1×10 is applied ⁶ Up to about 2X 10 ⁶ About 2X 10 ⁶ Up to about 3X 10 ⁶ About 3X 10 ⁶ Up to about 4X 10 ⁶ About 4X 10 ⁶ Up to about 5X 10 ⁶ About 5X 10 ⁶ Up to about 6X 10 ⁶ About 6X 10 ⁶ Up to about 7X 10 ⁶ About 7X 10 ⁶ Up to about 8X 10 ⁶ About 8X 10 ⁶ Up to about 9X 10 ⁶ About 9X 10 ⁶ Up to about 1X 10 ⁷ About 1X 10 ⁷ Up to about 2X 10 ⁷ About 2X 10 ⁷ Up to about 3X 10 ⁷ About 3X 10 ⁷ Up to about 4X 10 ⁷ About 4X 10 ⁷ Up to about 5X 10 ⁷ About 5X 10 ⁷ Up to about 6X 10 ⁷ About 6X 10 ⁷ Up to about 7X 10 ⁷ About 7X 10 ⁷ Up to about 8X 10 ⁷ About 8X 10 ⁷ Up to about 9X 10 ⁷ About 9X 10 ⁷ Up to about 1X 10 ⁸ About 1X 10 ⁸ Up to about 2X 10 ⁸ About 2X 10 ⁸ Up to about 3X 10 ⁸ About 3X 10 ⁸ Up to about 4X 10 ⁸ About 4X 10 ⁸ Up to about 5X 10 ⁸ About 5X 10 ⁸ Up to about 6X 10 ⁸ About 6X 10 ⁸ Up to about 7X 10 ⁸ About 7X 10 ⁸ Up to about 8X 10 ⁸ About 8X 10 ⁸ Up to about 9X 10 ⁸ About 9X 10 ⁸ Up to about 1X 10 ⁹ Cd4+cd25+ cells per kg body weight of the subject. In some embodiments, 1X 10 is applied ⁶ The number of tregs, e.g., cd4+cd25+ cells (+/-10%) per kg subject body weight.

In some embodiments, about 1 x 10 is administered to the patient ⁶ Up to about 2X 10 ⁶ About 2X 10 ⁶ Up to about 3X 10 ⁶ About 3X 10 ⁶ Up to about 4X 10 ⁶ About 4X 10 ⁶ Up to about 5X 10 ⁶ About 5X 10 ⁶ Up to about 6X 10 ⁶ About 6X 10 ⁶ Up to about 7X 10 ⁶ About 7X 10 ⁶ To about 8X 106, about 8X 106 to about 9X 106, about 9X 10 ⁶ To about 1X 107, about 1X 10 ⁷ Up to about 2X 10 ⁷ About 2X 10 ⁷ Up to about 3X 10 ⁷ About 3X 10 ⁷ Up to about 4X 10 ⁷ About 4X 10 ⁷ Up to about 5X 10 ⁷ About 5X 10 ⁷ Up to about 6X 10 ⁷ About 6X 10 ⁷ Up to about 7X 10 ⁷ About 7X 10 ⁷ Up to about 8X 10 ⁷ About 8X 10 ⁷ Up to about 9X 10 ⁷ About 9X 10 ⁷ Up to about 1X 10 ⁸ About 1X 10 ⁸ Up to about 2X 10 ⁸ About 2X 10 ⁸ Up to about 3X 10 ⁸ About 3X 10 ⁸ Up to about 4X 10 ⁸ About 4X 10 ⁸ Up to about 5X 10 ⁸ About 5X 10 ⁸ Up to about 6X 10 ⁸ About 6X 10 ⁸ Up to about 7X 10 ⁸ About 7X 10 ⁸ Up to about 8X 10 ⁸ About 8X 10 ⁸ Up to about 9X 10 ⁸ About 9X 10 ⁸ Up to about 1X 10 ⁹ Individual tregs, e.g. CD4 ⁺ CD25 ⁺ And (3) cells.

In some embodiments, about 1 x 10 is administered to the patient in one infusion ⁶ Up to about 2X 10 ⁶ About 2X 10 ⁶ Up to about 3X 10 ⁶ About 3X 10 ⁶ Up to about 4X 10 ⁶ About 4X 10 ⁶ Up to about 5X 10 ⁶ About 5X 10 ⁶ Up to about 6X 10 ⁶ About 6X 10 ⁶ Up to about 7X 10 ⁶ About 7X 10 ⁶ Up to about 8X 10 ⁶ About 8X 10 ⁶ Up to about 9X 10 ⁶ About 9X 10 ⁶ Up to about 1X 10 ⁷ About 1X 10 ⁷ Up to about 2X 10 ⁷ About 2X 10 ⁷ Up to about 3X 10 ⁷ About 3X 10 ⁷ Up to about 4X 10 ⁷ About 4X 10 ⁷ Up to about 5X 10 ⁷ About 5X 10 ⁷ Up to about 6X 10 ⁷ About 6X 10 ⁷ Up to about 7X 10 ⁷ About 7X 10 ⁷ Up to about 8X 10 ⁷ About 8X 10 ⁷ Up to about 9X 10 ⁷ About 9X 10 ⁷ Up to about 1X 10 ⁸ About 1X 10 ⁸ Up to about 2X 10 ⁸ About 2X 10 ⁸ Up to about 3X 10 ⁸ About 3X 10 ⁸ Up to about 4X 10 ⁸ About 4X 10 ⁸ Up to about 5X 10 ⁸ About 5X 10 ⁸ Up to about 6X 10 ⁸ About 6X 10 ⁸ Up to about 7X 10 ⁸ About 7X 10 ⁸ Up to about 8X 10 ⁸ About 8X 10 ⁸ Up to about 9X 10 ⁸ About 9X 10 ⁸ Up to about 1X 10 ⁹ Individual tregs, e.g. CD4 ⁺ CD25 ⁺ And (3) cells.

In some embodiments, the cryopreserved composition comprising the population of tregs is administered within about 30 minutes, about 1 hour, about 2-3 hours, about 3-4 hours, about 4-5 hours, about 5-6, about 6-7 hours, about 7-8 hours, about 8-9 hours, or about 9-10 hours of thawing the cryopreserved composition comprising the population of tregs. Between thawing and administration, the cryopreserved composition comprising the population of tregs may be stored at about 2 ℃ to about 8 ℃ (e.g., at about 4 °).

In some embodiments, a subject is administered one dose of a population of tregs or a composition comprising a population of tregs. In some embodiments, more than one population of tregs or a composition comprising a population of tregs is administered. In some embodiments, the population of tregs or the composition comprising the population of tregs is administered twice or more. In some embodiments, the Treg population or the composition comprising the Treg population is administered every 1-2 weeks, 2-3 weeks, 3-4 weeks, 4-5 weeks, 5-6 weeks, 6-7 weeks, 7-8 weeks, 8-9 weeks, 9-10 weeks, 10-11 weeks, 11-12 weeks, every 1-2 months, 2-3 months, 3-4 months, 4-5 months, 5-6 months, 6-7 months, 7-8 months, 8-9 months, 9-10 months, 10-11 months, 11-12 months, 13-14 months, 14-15 months, 15-16 months, 16-17 months, 17-18 months, 18-19 months, 19-20 months, 20-21 months, 21-22 months, 22-23 months, 23-24 months, every 1-2 years, 2-3 years, 3-4 years, or 4-5 years.

In some embodiments, about 1 x 10 is administered in the first administration ⁶ Each Treg per kg of subject body weight, and the number of tregs administered is increased in the second, third and subsequent administrations. In some embodiments, about 1 x 10 is administered in the first two administrations ⁶ Each Treg is per kg of subject body weight, and the number of tregs administered is increased every second thereafter (e.g., 4 th, 6 th, 8 th, and 10 th administrations). Thus, for example, for the first and second months, about 1X 10 may be administered per month ⁶ Each Treg is administered per kg of subject body weight, and for the third and fourth months, about 2 x 10 can be administered per month ⁶ Each Treg is administered about 3 x 10 per kg of subject body weight, and/or for the fifth and sixth months ⁶ Individual cells per kg body weight of the subject.

In some embodiments, the methods of treatment provided herein comprise administering to the subject a population of autologous tregs or a composition comprising a population of autologous tregs. In other embodiments, a method of treating a neurodegenerative disorder in a subject comprises administering to the subject a population of allogeneic tregs or a composition comprising a population of allogeneic tregs.

In some embodiments, the subject is additionally administered IL-2. The dose of IL-2 may be about 0.5-1X 10 ⁵ IU/m ² About 1 to 1.5X10 ⁵ IU/m ² About 1.5 to 2X 10 ⁵ IU/m ² About 2 to 2.5X10 ⁵ IU/m ² About 2.5 to 3X 10 ⁵ IU/m ² About 3 to 3.5X10 ⁵ IU/m ² About 3.5 to 4X 10 ⁵ IU/m ² About 4 to 4.5X10 ⁵ IU/m ² About 4.5 to 5X 10 ⁵ IU/m ² About 5 to about 6×10 ⁵ IU/m ² About 6 to 7X 10 ⁵ IU/m ² About 7 to 8X 10 ⁵ IU/m ² About 8 to about 9X 10 ⁵ IU/m ² About 9 to 10X 10 ⁵ IU/m ² About 10 to 15X 10 ⁵ IU/m ² About 15 to 20X 10 ⁵ IU/m ² About 20 to 25X 10 ⁵ IU/m ² About 25 to 30X 10 ⁵ IU/m ² About 30 to 35X 10 ⁵ IU/m ² About 35 to 40X 10 ⁵ IU/m ² About 40 to 45X 10 ⁵ IU/m ² About 45 to 50X 10 ⁵ IU/m ² About 50 to 60X 10 ⁵ IU/m ² About 60 to 70X 10 ⁵ IU/m ² About 70 to 80X 10 ⁵ IU/m ² About 80 to 90X 10 ⁵ IU/m ² Or about 90 to 100X 10 ⁵ IU/m ² . In a specific embodiment, 2 x 10 is administered to the subject ⁵ IU/m ² Is a IL-2 of (C).

IL-2 may be administered once, twice or more per month. In some embodiments, IL-2 is administered three times per month. In some embodiments, IL-2 is administered subcutaneously. IL-2 may be administered at least 2 weeks, at least 3 weeks, or at least 4 weeks prior to the anti-inflammatory EV population.

In some embodiments, a subject treated according to the methods described herein receives one or more additional therapies for treating alzheimer's disease. Other therapies for treating alzheimer's disease may include acetylcholinesterase inhibitors (e.g., donepezilGalanthamine->Or rivastigmine->) Or NMDA receptor antagonists (e.g., memantine ++ > And). Other therapies may also include anti-inflammatory agents (e.g., non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, indomethacin, and sulindac sulfide)), neuronal death-related protein kinase (DAPK) inhibitors, such as derivatives of 3-aminopyridazine, cyclooxygenase (COX-1 and-2) inhibitors, or antioxidants, such as vitamins C and E.

In some embodiments, a subject treated according to the methods described herein receives one or more additional therapies for treating ALS. Other therapies for treating ALS may include riluzoleOr riluzole

5.4.2 methods for determining the efficacy of treatment

The effect of the methods of treatment provided herein can be assessed by monitoring the clinical signs and symptoms of the disease to be treated.

The therapeutic methods described herein of efficacy may be evaluated about 20 weeks, about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, about 56 weeks, about 60 weeks, about 64 weeks, about 68 weeks, about 72 weeks, about 76 weeks, about 80 weeks, about 84 weeks, about 88 weeks, about 92 weeks, about 96 weeks, about 100 weeks, about 2-3 months, 3-4 months, 4-5 months, 5-6 months, 6-7 months, 7-8 months, 8-9 months, about 9-10 months, about 10-11 months, about 11-12 months, about 12-18 months, about 18-24 months, about 1-2 years, about 2-3 years, about 3-4 years, about 4-5 years, about 5-6 years, about 6-7 years, about 7-8 years, about 8-9 or about 9-10 years after initiation of treatment according to the methods described herein.

In some embodiments, the methods of treatment provided herein result in a change in the Appel ALS score from baseline. In the context of evaluating the effect of a therapeutic method, the term "baseline" refers to a measured pretreatment. The Appel ALS score measures the overall development of disability or altered function. In some embodiments, an Appel ALS score is reduced in a subject treated according to the methods provided herein, as compared to baseline, which is indicative of an improvement in symptoms. In other embodiments, the Appel ALS score remains unchanged in subjects treated according to the methods provided herein as compared to baseline.

In some embodiments, the methods of treatment provided herein result in a change in amyotrophic lateral sclerosis function rating scale-revision (ALSFRS-R) score as compared to baseline. ALSFRS-R scores assess the progression of disability or altered function. In some embodiments, an increased ALSFRS-R score in a subject treated according to the methods provided herein as compared to baseline, which is indicative of an improvement in symptoms. In other embodiments, the Appel ALSFRS-R score remains unchanged in subjects treated according to the methods provided herein as compared to baseline.

In some embodiments, the treatment methods provided herein result in a change in forced vital capacity (FVC; strength of muscle used for exhalation) from baseline, where the maximum value is the strongest measurement. In some embodiments, FVC is elevated in a subject treated according to the methods provided herein compared to baseline. In other embodiments, FVC remains unchanged in subjects treated according to the methods provided herein compared to baseline.

In some embodiments, the methods of treatment provided herein result in a change in comparison to the maximum inspiratory pressure (MIP; the intensity of the muscle used for inspiration), where the maximum value is the strongest measurement. In some embodiments, MIP is increased in a subject treated according to the methods provided herein compared to baseline. In other embodiments, MIPs remain unchanged in subjects treated according to the methods provided herein compared to baseline.

In some embodiments, the methods of treatment provided herein result in a change in neuropsychiatric symptom questionnaire (NPI-Q) compared to baseline. NPI-Q provides a symptom severity and affliction rating for each reported symptom, as well as a total severity and affliction score that reflects the sum of individual domain scores. In some embodiments, the NPI-Q score is reduced in a subject treated according to the methods provided herein compared to baseline. In other embodiments, the NPI-Q score remains unchanged in subjects treated according to the methods provided herein compared to baseline.

In some embodiments, the methods of treatment provided herein result in a reduced frequency of GI symptoms, allergies, or seizures as compared to baseline.

In some embodiments, the methods of treatment provided herein result in altered CSF amyloid and/or CSF tau protein (CSF-tau) compared to baseline. In some embodiments, CSF amyloid and/or CSF tau levels are reduced in a subject treated according to the methods provided herein compared to baseline. In other embodiments, CSF amyloid and/or CSF tau levels remain unchanged in a subject treated according to the methods provided herein as compared to baseline.

In some embodiments, the methods of treatment provided herein result in an alteration in Clinical Dementia Rating (CDR) compared to baseline. CDRs rate memory, directionality, judgment and problem solving, social things, family and hobbies, and personal care, and then generate global ratings ranging from 0-non-damaging to 3-severe damage. In some embodiments, CDR reduction in a subject treated according to the methods provided herein is compared to baseline. In other embodiments, CDRs remain unchanged in subjects treated according to the methods provided herein as compared to baseline.

In some embodiments, the methods of treatment provided herein result in an alteration of the Alzheimer's Disease Assessment Scale (ADAS) -cog13 score compared to baseline. The ADAS-cog tested cognitive performance and had an upper limit of 85 (poorly performing) and a lower limit of zero (best performing). In some embodiments, the ADAS-cog13 score is reduced in a subject treated according to the methods provided herein compared to baseline. In other embodiments, the ADAS-cog13 score remains unchanged in subjects treated according to the methods provided herein.

In some embodiments, wherein the method of treatment comprises administration of an anti-inflammatory EV population and administration of a Treg population or a cryopreserved Treg population, the method results in an increase in Treg suppression function in blood relative to baseline. In some embodiments, the methods of treatment provided herein result in an increase in Treg inhibition function in blood to week 4, week 8, week 16, week 24, week 30, or week 36 relative to baseline. In some embodiments, the methods of treatment provided herein result in an increase in Treg inhibition function in the blood by week 24 relative to baseline. In some embodiments, the methods of treatment provided herein result in an increase in the number of tregs in the blood relative to baseline. In some embodiments, the methods of treatment provided herein result in an increase in Treg numbers in the blood to week 4, week 8, week 16, week 24, week 30, or week 36 relative to baseline. In some embodiments, the methods of treatment provided herein result in an increase in Treg numbers in the blood by week 24 relative to baseline.

5.5 kit

Provided herein are therapeutic compositions comprising the anti-inflammatory EV populations provided herein or kits comprising the compositions of the anti-inflammatory EV populations provided herein.

In some embodiments, kits provided herein include instructions for use, other reagents (e.g., sterile water or saline solution for dilution of the composition), or reagents for collecting a biological sample, treating a component of a biological sample, such as a tube, container, or syringe, and/or for quantifying the amount of one or more surface markers in a sample (e.g., a detection reagent, such as an antibody).

In some embodiments, the kit contains one or more containers containing an anti-inflammatory EV population provided herein or a composition comprising an anti-inflammatory EV population provided herein. The one or more containers containing the composition may be disposable vials or multiple use vials. In some embodiments, the article of manufacture or kit may further comprise a second container comprising a suitable diluent. In some embodiments, the kit contains instructions for using (e.g., diluting and/or administering) an anti-inflammatory EV population provided herein or a composition comprising a population of tregs provided herein.

In certain embodiments, the kits provided herein comprise one or more unit doses of an anti-inflammatory EV as described herein in one or more containers, e.g., one or more vials. Such a kit may, for example, contain a single unit dose per container, e.g., a single unit dose per vial, or may contain multiple unit doses per container, e.g., multiple unit doses per vial.

6. Examples

6.1 example 1: improved Treg production protocol

6.1.1 detailed information of improved Treg production procedure

The following is a list of representative steps showing an embodiment of an improved Treg ex vivo amplification protocol. This exemplary protocol may be used, for example, as part of a method of producing an isolated, cell-free, anti-inflammatory EV population as provided herein. A more detailed summary of this representative Treg production procedure is shown in fig. 1 and provided below in 6.1.2. As mentioned, in the description of fig. 1, the improved Treg production method may be used to expand tregs from ALS patients. It is to be understood that the Treg production methods provided herein can also be applied to expand tregs from other starting materials, including from a subject suffering from other disorders, e.g., other neurodegenerative disorders, or from a cell sample of a healthy donor subject. Exemplary methods of generating, obtaining, enriching, and expanding Treg populations ex vivo using improved methods, such as this, are also described in international patent application No. pct/US2020/63378, which is incorporated herein by reference in its entirety.

Donor cell isolation.

1) The product was removed (or blood sample) using fresh white blood cells immediately after separation on day 0. They were not stored overnight at 4 ℃.

Treg enrichment.

2) Cd25+ cells are obtained after leukocyte removal (or blood sample aspiration); enrichment involves volume reduction, purification, followed by cd8+/cd19+ depletion, followed by cd25+ enrichment.

And (5) amplifying.

3) Freshly isolated cd25+ cells were immediately (within about 30 minutes) placed in culture. They were not first cryopreserved.

4) IL-2 was administered every 2-3 days on days 6, 8 and 11. Cells are not discarded during expansion and thus the protocol is bigger.

5) During medium exchange, half of the medium was removed and supplemented on days 1, 4 and 6. Cells removed during these medium exchanges were not returned to the flask.

6) During medium exchange, the cells are typically not centrifuged.

The EV can be isolated at any point during the amplification process. For example, if desired, EV can be isolated from the medium removed in 5) above.

6.1.2 exemplary procedure for isolation and expansion of regulatory T cells (Tregs) from leukopenia or blood sample products

The procedure may be applied to the isolation and expansion of leukocyte removal products or blood sample products from, for example, ALS patients, alzheimer's patients, or patients exhibiting different disorders, e.g., different neurodegenerative disorders, or from healthy subjects.

6.1.2.1 step 1: patient leukocyte removal/blood sample product processing

The leukocyte removal product should be treated within 24 hours. The total volume of the leukocyte removal product should be between 100mL and 840 mL. If the leukocyte removal product is less than 100mL, an equal volume of CliniMACS buffer with 1% human serum albumin (HAS) should be added.

The volume reduction of the leukocyte removal product was performed by the PeriCell protocol and CS490.1 kit (PeriCell) using GE Healthcare/Biosafe Sepax 2 RM.

The leukocyte removal product was purified using the GE Healthcare-Biosafe Sepax 2RM Neatcell protocol and CS900.2 kit.

6.1.2.2 step 2: treg enrichment

The cd8+ and cd19+ cells were depleted using a clinic macs kit according to the manufacturer's instructions, which included labeling the cells to be depleted with cd8+ and cd19+ microbeads, followed by use ofThe Plus apparatus was combined with CliniMACS PBS/EDTA buffer in 1% HSA, cliniMACS Tubing Set LS and software series DEPLETION 2.1 for automated cell separation.

Subsequently, the population was enriched for cd25+ tregs by positive selection using clinic macs, which included labeling cells with CD25 enriched for CD25 microbeads, followed by use ofThe Plus apparatus was combined with CliniMACS PBS/EDTA buffer in 1% HSA, cliniMACS Tubing Set LS and software series ENRICHMENT 3.2.3.2 for automated cell separation.

6.1.2.3 step 3: treg expansion

Cell culture and harvest parameters:

slow-growing amplifications: cell culture for 25 days, cell number was estimated<2×10 ⁹ Individual cells-a second leukocyte removal/blood sample may be required.

Normal amplification: cell culture is carried out for 25 days, and the estimated cell number is more than or equal to 2 multiplied by 10 ⁹ Individual cells.

Rapid growth and amplification: in cell culture for less than or equal to 15 days, the estimated cell number is more than or equal to 2 multiplied by 10 ⁹ In individual cells.

All cell culture expansion should maintain a purity of 70% cd4+cd25+ cells and a viability of greater than 70% prior to aseptic filling into vials and cryopreservation.

The cd25+ enriched leukocyte removal/blood sample product was centrifuged, the pellet was washed in TexMACS medium with 5% human AB serum, centrifuged again and at 0.8-1.0x10 ⁶ The resulting pellet was resuspended in TexMACS medium with 5% human AB serum at a density of individual cells/mL. Cells were transferred to flasks and incubated at 37℃in 95% air and 5% CO ₂ Is incubated for 16-18 hours in the wet mixture of (C).

On day 1, cells were stimulated with CD3/CD28 beads using the MACS GMP ExpAct Treg kit. The kit contains 3.5 μm particles preloaded with CD28 antibody, anti-biotin antibody and CD 3-biotin. Each vial contained 1X 10 ⁹ Individual ExpAct Treg beads (2×10 ⁵ /μl). For initial stimulation, MACS GMP ExpAct Treg beads and Treg cells should be at 4:1 bead: proportion of cells. For activation, the cell concentration should be about 0.5-0.7X10 for the MACS GMP ExpAct Treg kit (CD 3/CD28 beads) ⁶ Individual cells/mL. Activation was performed on day 1 and again on day 15.

The medium was changed and supplemented with rapamycin on day 4, 6, 8, 11, 13, 15, 18, 20 and 22. IL-2 was supplemented on days 6, 8, 11, 15, 18 and 20. EV can be isolated from the medium removed from Treg culture at harvest. The medium may be frozen prior to isolation of the EV.

6.1.2Step 4: treg harvesting (optional if Treg culture is used for EV isolation)

Optionally, the expanded tregs may be harvested. For example, the expanded tregs may be harvested on day 25. MACS GMP-activating beads can be removed using CliniMACS Depletion Tubing Set LS (168-01) and software design 2.1 according to the manufacturer's instructions.

If the final harvested cell product is intended for therapeutic use, it should meet the release criteria as shown in Table 2.

Table 2 release criteria

6.2 example 2: identification of ex vivo expanded Treg cell populations by proteomics

The experiment provided in this example describes proteomic analysis of baseline tregs and tregs that have been expanded ex vivo using the improved expansion methods described in example 1 above and in section 5.2.1.1 above. These experimental results demonstrate that tregs produced via these methods constitute a unique, non-naturally occurring population of tregs. Likewise, thus, the anti-inflammatory EV population derived from these ex vivo expanded tregs also constitutes a unique, non-naturally occurring EV population. As discussed below, these features are significantly different from baseline Treg gene product features and indicate the health and efficacy of the expanded tregs from which the EV population described herein can be derived.

6.2.1 groups analyzed in the experiments

Baseline tregs—tregs derived from baseline levels of 2 ALS patients.

Expanded tregs-expanded as described herein, in particular, tregs of the same patient following the procedure described in section 5.2.1.1 and example 1 above.

6.2.2 proteomic profiling methods

Proteomic profiling via single-shot proteomic analysis was performed on Treg baseline and Treg expanded cells. The cell pellet was lysed by RIPA buffer. For each sample, 5 μg of protein supernatant was mixed with NuPAGE LDS sample buffer (Thermo, NP 0007) and boiled at 90 ℃ for 10 minutes. Proteins were isolated on a preformed NuPAGE Bis-Tris 10% protein gel (Invitrogen, NP0301 BOX). For staining, the gel was first fixed with Destatin I (40% MeOH,7% AcOH) for 15 min, stained with Coomassie dye (0.025% Brilliant blue R-250, 40% MeOH,7% AcOH) for 5 min, destained with Destatin I buffer for 30 min, 2 times, and kept in water overnight. 4 bands were excised for each sample. With Destatin II (40% MeOH,50mM NH) ₄ CO ₃ ) The bands were further completely decolorized, equilibrated in water, dehydrated with 75% ACN and incubated in 50mM ammonium bicarbonate solution for 1hr. Then, each strip was crushed and digested with 22. Mu.l trypsin solution (20. Mu.l 50mM ammonium bicarbonate and 2. Mu.l 100 ng/. Mu.l trypsin (GenDepot: T9600)) overnight at 37 ℃. The digests were then acidified by addition of 20 μl of 2% formic acid (Thermo, 85178). Peptides were extracted from the gel by adding 350 μl 100% ACN for 15min and collected by centrifugation at 21,000rpm for 5 min. The extracted peptides were dried in SpeedVac (Thermo Scientific SC 210). For MS runs, peptides from all 4 bands were resuspended in 20 μl of 5% methanol+0.1% FA solution, mixed together, and used with Thermo Scientific ^TM EASY-nLC ^TM On-line separation Exploris Orbitrap, 480 mass spectrometer (Thermo Fisher Scientific, san Jose, CA) of the 1200 liquid chromatography system. 1. Mu.g was isolated on-line using a packed column with sub-2. Mu. m C18 beads (Reprosil-Pur Basic C18, cat# r119.b9.0003, dr. Maisch GmbH) 20cm long with an inner diameter of 100. Mu.m. A linear reverse gradient of 2-30% B (100% ACN) was run for 90 minutes.

Raw mass spectral data were processed through Proteome Discoverer (PD 2.0.0.802 version; thermo Fisher Scientific). Spectrum and 350-10,000Da mass range and trypsin is used Peptides from the human RefSeq protein database (downloaded by RefProtDB at 2020-03-24) digested in the P computer and with up to 2 missing cleavages. The mass error was set to 20ppm for the precursor mass and 0.02Da for the fragment mass. The following dynamic modifications: acetyl (protein N-terminal), oxidized (M), urea methylated (C), deStreak (C) and deamidated (NQ). Verification of peptide-spectral match (PSM) by Percolator (v 2.05)2007,PMID 17952086) by et al. The target strict and relaxed FDR levels of Percolator were set to 0.01 and 0.05 (1% and 5%) respectively. Label-free quantification of PSM was performed using the area detector module (Area Detector Module) of Proteome Discoverer.

Protein inference and quantification was performed by gpGrouper (v1.0.040) using consensus peptide iBAQ area distribution (Saltzman et al 2018 PMID 30093420). The resulting protein values are the median values normalized and log transformed for downstream analysis. For statistical evaluation, the missing values were interpolated by sampling the normal distribution N (μ -1.8σ,0.8σ), where μ, σ are the mean and standard deviation of the quantified values. To evaluate the inter-group differences, a moderate t-test (ritche et al, 2015) was used as implemented in the R package limma. Multiple hypothesis testing corrections were performed by the Benjamini-Hochberg program (Benjamini and Hochberg, 1995). Pathway analysis for phenotype association was examined using the reactiome, kyoto genome encyclopedia (Kyoto Encyclopedia of Genes and Genome, KEGG) and gene set enrichment analysis (Gene Set Enrichment Analysis, GSEA).

6.2.3 proteomics research discoveries

In this example, gene products were identified from expanded Treg cell populations as well as from baseline tregs (freshly isolated, enriched tregs pre-expanded, herein from ALS patient cell samples) via unbiased single shot proteomic analysis of mass spectra. The following results were obtained:

1. baseline Treg gene product characteristics resolved after the Treg expansion process were identified. Such features indicate, for example, dysfunctional epigenetic/methylation mechanisms in baseline tregs. Methylation gene product characteristics were also identified within this baseline signature.

2. Unique proteomic gene product characteristics of expanded tregs were identified, which indicate that the population of Treg cells produced via the methods provided herein is novel and that the expanded population of Treg cells represents a robust, effective population. Importantly, this feature is quite conserved among the different ALS patient tregs undergoing the amplification process.

3. The enhanced Treg gene product profile after amplification can be further defined by at least the following unique gene product profile:

treg-related gene product characteristics.

b. Mitochondrial gene product characterization.

c. Cell proliferation gene product characteristics (cell division, cell cycle and DNA replication/repair).

d. The highest protein expression gene product characteristic after the amplification process.

6.2.4 results

6.2.4.1 dysfunctional baseline tregs showed improved proteomic characteristics after expansion.

Single shot proteomic profiling identified peptide sequences of 82 gene products that were localized to a total of 3,709 gene products and found to be elevated in the baseline sample, but subsequently significantly reduced or lost during the amplification process (table 3; dysfunctional baseline gene production profile). The profiling includes gene products that have a p-value of p <0.05 after correction for error-finding rates and multiple hypothesis testing, while also having a fold change (log 2 FC > 2) of at least 4. Pathway analysis of these significant gene product groups indicated that dysfunctional Treg phenotypes included deregulated calcium dynamics (p=0.0278), loss of MECP2 binding capacity to 5hmC-DNA (p=6.96 e-6), deregulation of MECP2 expression and activity (p=0.0166) and MECP2 control (p=0.0303), phosphorylation (p=0.037) and loss of binding capacity (p= 0.0456). It has been previously shown that correct MECP2 expression and function is critical for Treg health and expression of the functional marker FOXP3 (PMID: 24958888).

In addition, multiple gene products in this feature are also targeted to histones and other proteins that are modified and play a role in controlling DNA melting to enable epigenetic changes, particularly DNA methylation that directly affect transcription. After expansion, expression of these dysfunctional epigenetic/methylation-related gene products is reduced in the Treg population relative to that observed for baseline tregs. See table 4 (methylation gene product profile).

Table 3: gene product that is elevated in baseline samples but subsequently significantly reduced or lost during the amplification process

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Table 4: dysfunctional epigenetic/methylation signatures in baseline tregs

6.2.4.2 enriched proteomic characterization of patient tregs post expansion observed between patient groups

Proteomic analysis of expanded tregs compared to baseline tregs identified peptide sequences that localized back to 391 unique gene products found in expanded tregs compared to baseline patient tregs among 3,709 gene products in total (table 5). These genes are a compilation of all significant gene products that have p values of p <0.05 after correction for false discovery rates and multiple hypothesis testing, while also having fold changes (log 2 FC > 2) of at least 4.

Table 5: gene product enriched in expanded tregs compared to baseline patient tregs

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6.2.4.3 enhanced Treg proteomics profile after amplification.

Phenotypic analysis shows that amplified Treg gene product characteristics include gene products from well-known pathways involved in functional processes, specifically enriched in Treg immune characteristics, mitochondrial activation and energetics, and cell proliferation, including cell division, cell cycle, and DNA replication/repair. Proteomic data are also partitioned to show the highest expression profile present in expanded patient tregs. Each of these gene product characteristics of the expanded Treg cell population is described below.

6.2.4.3.1 Treg-related gene product characteristics.

Proteomic analysis showed some gene products involved in the immunological pathways enriched in the expanded Treg cell population, as evidenced by their elevated expression relative to that observed in baseline tregs. These pathways include, for example, adaptive immune pathways (p= 0.00726), innate immune pathways (p=0.09), cytokine signaling in the immune system (p= 0.0338), MHC class II antigen presentation (p=9.33 e-13), PD-1 signaling (p=7.66 e-11), co-stimulation by the CD28 family (p=9.12 e-11), production of second messenger molecules (p=7.69 e-13), interferon signaling (p=1.31 e-7), downstream TCR signaling (p=1.31 e-7), and RUNX1 and FOXP3 control of regulatory T lymphocyte development (p=1.05 e-3). Table 6 (Treg-related gene product profile) lists gene products whose expression is elevated relative to baseline tregs, where these gene products are documented in the literature as important for proliferation, health, identification and/or mechanism of Treg cells.

As shown in table 6, treg-related gene product characteristics include, for example: ADAM10, AIMP1, AIMP2, ARG2, BCL2L1, BSG, CD2, CD28, CD38, CD74, CD84, CTLA4, FAS, FOXP3, GCLC, HAT1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HPGD, ICOS, IL1RN, IRF4, KPNA2, LGALS1, LGMN, PCNA, POFUT1, SATB1, SELPLG, STAT1, TFRC, and TNFRSF18.PMID: pubmed ID.

Table 6: the gene products in expanded tregs, which are important for proliferation, health, identification and/or mechanism of Treg cells, are recorded in the literature.

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6.2.4.3.2 mitochondrial Gene product characterization.

Mitochondria play an important role in Treg health and function. Proteomic studies showed large, enriched gene product profiles of mitochondrial-related genes in an elevated expression of ex vivo expanded Treg cell population relative to that observed in baseline tregs (p=2.96 e-29). See table 7. This document describes the importance of mitochondrial health and energetics in Treg function, which inevitably leads to Treg dysfunction (e.g. PMID: 30320604). Currently, targeting and restoration of mitochondrial function is examined as a means of restoring dysfunctional tregs (PMID: 30473188). For example, this mitochondrial gene product feature is highly enriched by pathways involved in mitochondrial replication (p=1.12 e-14) and mitochondrial energy metabolism (p=1.83 e-2). This gene product profile suggests that mitochondrial activation, function and recovery are important products of the Treg amplification process described herein.

As shown in table 7, mitochondrial gene product characteristics include, for example: ACAA2, ACADM, ACADVL, ACOT, ACSL1, ACSL4, ACSL5, AGK, AGMAT, AK, ARG2, ARL2, AUH, BCL2L1, BDH1, BNIP1, CDK1, CHDH, CIAPIN1, CISD2, COX17, CPOX, CPT1A, CPT2, CYB5B, DAP3, DHRS2, DNM1L, DUT, DYNLL1, ECI1, FDXR, FEN1, FKBP8, GK, GRSF1, HTRA2, L2HGDH, LACTB2, LRPPRC, MAIP1, MAOA, MPST, MRPL1, MRPL12, MRPL13, MRPL14, MRPL17, MRPL22 MRPL37, MRPL39, MRPL4, MRPL43, MRPL44, MRPL46, MRPL48, MRPS11, MRPS14, MRPS2, MRPS27, MRPS31, MRPS35, MRPS9, MTHFD2, MTX1, MYCBP, NDUFA8, NUDT1, OAT, PITRM1, PLSCR3, PMPCA, PPIF, PTRH2, PYCR2, REXO2, RMND1, SFXN2, SLC25A10, SLC25A19, SLC25A4, TIGAR, TIMM13, TIMM23, TMEM14C, TOMM, TOMM34, TOMM40 and TST.

Table 6: characterization of enriched mitochondrial-related gene products

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6.2.4.3.3 characteristics of the cell proliferation Gene product (cell division, cell cycle and DNA replication/repair)

Proteomic studies showed elevated expression of some gene products associated with the cell proliferation pathway relative to their expression in baseline tregs. See table 8. These enriched gene products include, for example, those associated with the cell cycle (p=0.0014), cell division (p= 0.0478), DNA replication (p=5.05 e-13), and DNA repair (p=0.0496) pathways.

As shown in table 8, cell proliferation gene product characteristics include, for example: ARL2, ARL3, BCCIP, CCDC124, CDK1, CDK2, CDK5, CDK6, CUL4B, DCTN3, FEN1, HELLS, LIG1, MAD2L1, MAEA, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, MCMBP, NUDC, PCNA, POLD1, POLD2, RALB, RBM38, RFC2, RFC3, RFC4, RFC5, RNASEH2A, RNASEH2B, SMC2.

Table 7: cell proliferation gene product characterization

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6.2.4.3.4 highest protein expression Gene product characterization (after amplification)

The gene products from the proteomic studies were then partitioned based on the highest protein expression observed in ex vivo expanded tregs produced by the methods provided herein, as quantified using absolute quantitative based Intensity (iBAQ) as a measure of protein abundance in the proteomic assay. The first 40 expressed gene products from this study are compiled in table 9. As mentioned in table 9, the expression of each of the members characteristic of this highest protein expression gene product was elevated relative to the expression observed in the baseline Treg.

Gene products that characterize the highest expressed protein include, for example: ACAA2, ACADM, ACADVL, ACOT, BSG, CACYBP, CD, CDK1, CPOX, DUT, ECI1, ENO3, FEN1, FKBP3, HIST1H2BJ, HLA-DQA1, HLA-DRA, HLA-DRB1, LGALS3, MCM5, MCM6, MCM7, MTHFD1, NAMPT, NME1, NQO1, PCNA, RAB1A, RALB, SLC A4, STAT1, STMN2, TUBA1B, TUBB4A, TUBB8, TXN, TXNRD1, and WARS.

Table 8: highest protein expression Gene product characterization

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6.3 example 3: extracellular vesicles derived from suppressive immune cells regulate inflammation in vitro and in vivo

6.3.1 methods

6.3.1.1 ex vivo Treg expansion

Tregs were enriched and expanded ex vivo following the procedure described in example 1 above. Tregs were cultured in human AB serum as described in example 1, or to isolate a pure Treg EV population, tregs were cultured using the same amplification protocol, but with exogenously depleted Fetal Bovine Serum (FBS) instead of serum.

Tregs are derived from healthy subjects or ALS patients as shown below.

6.3.1.2 extracellular vesicle isolation Using PEG

Extracellular Vesicles (EV) were isolated using polyethylene glycol Precipitation (PEG) and ExoQuick-TC reagent (System Biosciences, SBI) according to the manufacturer's protocol. Briefly, medium from Treg expansion culture was centrifuged at 3000×g for 15 min to remove cells and debris. PEG reagent was added to PEG: TC Medium 1:5 and the supernatant was centrifuged at a ratio sufficient to mix and refrigerated overnight at 4 ℃. The mixture was then centrifuged at 1500 Xg for 30 minutes, the supernatant was aspirated, again at 1500 Xg for 10 minutes, and the supernatant was aspirated again. The resulting EV pellet particles were resuspended in sterile PBS and diluted for Nanosight EV size/concentration analysis and ready for use. EV is stored at-20 ℃ while limiting freeze/thaw cycles.

6.3.1.3 extracellular vesicle isolation Using TFF

EV populations isolated using Tangential Flow Filtration (TFF) techniques are isolated using the protocols summarized herein.

Briefly, this isolation uses the Repligen KR2i TFF system, which enables isolation, concentration and diafiltration of Treg EV populations using buffers suitable for therapeutic use.

First, TFF and membrane area 85cm were used ² And a Midi 20cm 0.65 μm Spectrum mPES 0.75mm hollow fiber filter (D02-E65U-07-N) with fiber diameter 0.75mm circulated the medium from Treg expansion culture to filter out cells and debris. The process uses a flow rate of 100-200mL/min, which results in the production of about 2,000-5,000s ^-1 While maintaining a variable transmembrane pressure (TMP) driven by a holding hydraulic pressure of 5 psi.

The permeate from this step is then designed to concentrate and diafiltrate the EV population into the retentate by a continuous cycle. The process uses a TFF system and has a 115cm length ² Midi 20cm 500kD Spectrum mPES 0.5mm hollow fiber filter (D02-E500-05-N) with a membrane area and fiber diameter of 0.5mm to retain/concentrate all particles greater than about 60-80nm into the retention solution. The process uses 80-200mL/min flow rate, which results in the production of 2,000-7,500s ^-1 While maintaining and driving filtration at 10psi TMP. The final volume after concentration was targeted at about 20mL.

Sterile PBS was introduced into the circulation, resulting in 10 x diafiltration and replacement of the existing solution. 10 x diafiltration effectively results in a 10-fold change in the entire buffer volume and its implementation eliminates 99% + of the soluble material or impurities that will remain.

6.3.1.4 Nanosight EV size/concentration reading

EV readings were obtained using a Nanosight NS300 (Malvern Panalytical) particle analyzer. EV samples were diluted for reading and recorded at a rate of 50 μl per minute, at 3 times 60 seconds analysis, using continuous measurements for analysis, respectively, with the following parameters: camera level (12-15), temperature (22 ℃) and detection threshold (5). The concentration was recorded as particles/ml and the size statistic as mean and mode.

6.3.1.5 iPS-derived M1 spinal cord cultures

Bone marrow cells were generated using previously developed/described protocols (Thome et al, 2018,Molecular Neurodegeneration 13.1:1-11; zhao et al, 2020,Iscience 23.6:101192).

Briefly, bone marrow cells were generated using a 4-step culture method that allowed for the production of cd14+ cells from a control iPSC line. Cd14+ bone marrow cells were isolated using positive magnetic selection by Miltenyi Biotec CD beads, separation column and magnet device.

For M1 cells: CD14+ cells were cultured in complete RPMI medium (10% fetal bovine serum, 25mM HEPES, 1mM sodium pyruvate, 1 Xnonessential amino acids, 55. Mu.M 2-mercaptoethanol, 100 units/ml penicillin and 100. Mu.g/ml streptomycin) supplemented with 50ng/ml GMCSF (R & D systems) for 7 days to generate M0 cells for M1 use. Then, M0 cells were stimulated with 0.1ng/ml Lipopolysaccharide (LPS) (Sigma) and 0.2ng/ml IFNγ (Invitrogen) to differentiate bone marrow cells into pro-inflammatory M1 cells.

6.3.1.6MSC EV

Human Mesenchymal Stem Cells (MSCs) were obtained from bone marrow and cultured in T75 flasks using medium containing 10% FBS in minimal essential medium supplemented with antibiotic/antifungal solution. Cells were brought to 80% confluence in flasks, then medium was aspirated, washed with PBS and replaced with the same medium, but with 10% exosome-free FBS instead of normal FBS. T75 flasks were incubated for an additional 48 hours and EV was isolated from tissue culture medium using PEG isolation.

6.3.1.7 EV inhibition assay using bone marrow cells and Tresp proliferation assay

M0 (GM-CSF) cells were raised, pelleted and re-plated in 24 well plates at 50,000 cells/well using enzyme-free dissociation buffer.

M1 cells were stimulated with 0.1ng/ml LPS (Sigma) and 0.2ng/ml IFNγ (Invitrogen) for 1 hour to differentiate into M1 cells. Treg EV (1×10) was post M1 polarization overnight time point ⁸ Individual particles) were added to the culture, then M1 cells were collected for RNA analysis and medium was collected for protein analysis.

For response T cell (Tresp) proliferation assays, control Tresp was isolated using Miltenyi Biotec reagents and protocols to isolate cd4+cd25-T cells from surrounding blood. Tresp was plated in 96-well round bottom plates at 50,000 cells per well and stimulated with CD3/28 beads (Miltenyi Biotec).

Treg EV was added to the culture at progressively increasing doses and the whole experiment was maintained in Tresp culture. After 4 days of incubation, the Tresp was pulsed with tritium and proliferation was determined by examining tritium incorporation 18 hours after tritium pulsing.

6.3.1.8LPS-mouse model of induced neuroinflammation and SOD1 mouse model of ALS

For the acute LPS-induced neuroinflammation mouse model, C57Bl6 WT mice were intraperitoneally Injected (IP) with 2mg/kg LPS (Sigma; O111: B4) followed by intranasal administration of Treg EV (1X 10) 2 hours after LPS injection ⁹ Individual particles). Mice were then sacrificed 12 hours or 22 hours after intranasal administration and organs were harvested for RNA and protein analysis, specifically brain components (hippocampus and cortex) and spleen.Bone marrow cells were isolated from spleen by extracting single cells through a 40 μm cell strainer and using mouse CD11b beads and magnetic columns (Miltenyi Biotec).

Transgenic mice with SOD1-G93A mutations (see Gurney et al, 1994,Science 264.5166:1772-1775) were used as a model of motor neuron degeneration in ALS. Phenotypic analysis of SOD1 mice started on day 70 and Treg EV started on day 90 (1×10) ⁹ Particles) and continue every two weeks until the mice reach their ethical endpoint, thereby requiring their sacrifice. Mice phenotypes were assessed using a modified "BASH scoring system" in which SOD1 mice achieved a degenerative score of 0-6 as a result of the deterioration of the phenotype with disease progression. Phenotypes and increasing scores were evaluated as follows (but not necessarily in this order): +1 tremor, +1 gait abnormalities, +1 lower limb weakness/mild paralysis, +1 > 10% weight loss in adults, +1 lower limb rigidity, or both, +1 paralysis, which is end-stage, which results in mice being sacrificed and organs harvested for RNA and protein analysis.

6.3.1.9 RNA purification, RT-PCR analysis and protein ELISA

RNA was isolated from cells and tissues using Trizol reagent, followed by Direct-zol RNA MiniPrep Plus kit (Zymo Research) according to the manufacturer's recommendations. Quantitative RT-PCR was performed by SYBR Green (Bio-Rad) and the iQ5 Multicolor real-time PCR detection system (Bio-Rad) using a one-step RT-PCR kit. Primers for RT-PCR (IL-6, IL-1. Beta., TNF. Alpha., IL-10, arg-1, IFN-. Gamma., FOXP3 and CD 206) were obtained from Bio-Rad and performed according to the manufacturer's protocol. The relative expression level of each mRNA was assessed using the ΔΔct method and normalized to β -actin/control. Supernatants were collected from the co-culture paradigm and the amount of IL-6 protein was assessed using ELISA-based immunoassay (Invitrogen).

6.3.2 results

6.3.2.1 Treg-derived EV inhibited Tresp proliferation and pro-inflammatory iPSC-derived M1 cells.

Treg expansion medium was collected from expansion culture of ALS patient-derived tregs and EV was isolated using the PEG method described above. This resulted in a final EV mixture containing about 70% medium serum-derived EV and about 30% treg-derived EV (fig. 2A).

When 1X 10 ⁸ The Treg/medium EV mixture showed approximately 70% inhibition of M1 IL-6 protein production when administered at a dose of 50,000M 1 cells per particle. In contrast, medium EV alone showed only minimal effect (less than 20%). Fig. 2B.

When Treg EV mixtures were added to Tresp proliferation assays at progressively increasing doses, a dose-dependent inhibition of Tresp proliferation was observed, which was at 5×10 ⁷ The individual particles became apparent at a fixed dose of 50,000M 1 cells, reaching 65% inhibition, and at 1 x 10 ⁷ And 5X 10 ⁷ The individual particles were raised to 82% and 91% inhibition, respectively, per fixed dose of 50,000M 1 cells (fig. 2C).

To isolate a pure population of tregs EV, tregs were cultured using the same amplification protocol, but with exogenously depleted Fetal Bovine Serum (FBS) instead of serum. Since there will no longer be an EV from serum, this results in a population of Treg EVs with the same purity as the population of Treg from which the EV was derived. Pure Treg EVs were tested in the same manner as described above to examine their ability to inhibit proliferation of pro-inflammatory M1 cells and Tresp. Treg EV showed significant, dose-dependent inhibition capacity when examined for LPS-induced IL-6RNA production by M1 cells after 3 hours and 20 hours (fig. 2D). These data were confirmed by evaluating inhibition of LPS-induced IL-6 protein production by M1 cells after use of progressively higher doses of pure Treg EV; all of these displays are significantly valid (fig. 2E). The spectrum of inhibition of Tresp proliferation by pure Treg EVs was consistent with the spectrum of mixed populations with increasing doses of Treg EVs that conferred increased proliferation inhibition (fig. 2F).

When Treg-mixed EVs were isolated using TFF, the EV population retained the inhibitory activity observed with PEG isolation. In particular, treg-mixed EVs isolated using TFF reduced iPSC-derived M1 IL-6 protein in a dose-dependent manner similar to that observed when EVs were isolated using PEG (fig. 2G). In addition, mixed Treg EVs isolated using TFF were able to inhibit Tresp proliferation at progressively higher doses in a manner similar to that observed when EVs were isolated using PEG (fig. 2H).

Fig. 2I shows an exemplary size spectrum of an EV of Treg mix produced and PEG separated as described in this example, showing a single peak, indicating a diameter particle size distribution between about 50-150 nm. The size range was verified using Scanning Electron Microscopy (SEM). It should be noted that nanoparticle analysis of the multiple Treg EV populations obtained demonstrated a consistent particle size distribution between about 50nm and about 150nm (e.g., exhibiting average = 94.5nm, mode = 76.8nm, d10 = 56.6nm, d50 (median) = 86.4nm, d90 = 146.9 nm) as described herein.

Analysis using Miltenyi MACSPlex exosome kit (Miltenyi Biotec) found that the mixed EVs produced and isolated using this method expressed a combination of exosome markers including CD9, CD63 and CD81, however, in contrast, medium EV expressed only CD81 among these markers (fig. 2J). In addition, miltenyi MACSPlex exosome kit (Miltenyi Biotec) analysis also confirmed that Treg EVs were positive for CD2, CD4, CD25, CD44, CD29, CD45 and HLA-DRDPDQ, whereas in contrast, medium EVs did not express any of these markers (fig. 2K).

Overall, treg EVs derived from ex vivo expanded Treg cells demonstrate unique and Treg-conserved features and in vitro inhibitory functions similar to expanded Treg cells.

6.3.2.2 Treg EV inhibits inflammation in LPS-induced mouse neurogenic inflammation model

Mice were peripherally injected with LPS to induce neuroinflammatory mechanisms in the brain to test in vivo inhibition of Treg EV upon intranasal administration. Intranasal delivery of 1X 10 at 2 hours after LPS injection of 2mg/kg IP ⁹ Treg EV was used and mice were sacrificed after another 12 hours to examine neuroinflammatory parameters (fig. 3A). The Treg EVs used in these experiments were a cultured pure Treg EV population of ex vivo expanded tregs from healthy human subjects. Tregs were cultured in exosome-free FBS and EVs were isolated using the PEG method described above.

For neuroinflammatory analysis, hippocampus and cortex were isolated from sacrificed mice and RNA was extracted for RT-PCR analysis. Treg EV demonstrated the ability to significantly reduce hippocampal IL-6 and IL-1 β transcripts produced by LPS injection, whereas there was no significant change in hippocampal tnfα transcripts after treatment (fig. 3B).

When the cortex was examined, treg EV treatment-specific reduction in IL-6 transcripts was observed, while IL-1β and TNF transcripts remained elevated (fig. 3C). Peripheral inflammation was examined by analysis of inflammatory transcript changes in cd11b+ bone marrow cells isolated from spleen following LPS IP injection and Treg intranasal treatment.

Robust elevation of myelitis activation was observed in the spleen due to peripheral LPS injection (fig. 3D). Intranasal Treg EV treatment reduced peripheral bone marrow cell-derived IL-6 and TNF transcripts 14 hours after LPS injection.

Thus, in the LPS-induced in vivo neurogenic inflammation model, treg EV administration inhibits pro-inflammatory mechanisms centrally and peripherally.

6.3.2.3 in the SOD1 mouse model of ALS, treg EV inhibits inflammation, prolongs survival and slows down end-stage disease Development.

SOD1 motor neuron degeneration mouse model of ALS was used to examine the role of Treg EV in an inflammatory mechanism driven neurodegenerative model. When the animals have exhibited degenerative symptoms, 1X 10 use is started on day 90 ⁹ Intranasal treatment of individual Treg EV particles and these treatments were continued every 2 weeks until animals were sacrificed and tissues harvested for analysis (fig. 4A). The Treg EVs used in these experiments were a cultured pure Treg EV population of ex vivo expanded tregs from healthy human subjects. Tregs were cultured in exosome-free FBS and EVs were isolated using the PEG method described above.

The 2 week-interval treatment of Treg EV significantly improved the survival probability in the treated group compared to PBS-injected control (fig. 4B). In addition, treg EV treatment slowed disease progression later in motor neuron disease, as measured by an improved scoring system detailing the dyskinesia phenotype in mice (fig. 4C).

By statistics, the average disease duration from the first symptom increased from 85 days in Treg EV-treated animals to only 69 days in PBS-treated mice (fig. 4D). The mean life span in Treg EV-treated animals tended to increase compared to PBS control, 151.7 days (PBS control) vs.162.8 days (Treg EV treated animals), respectively (fig. 4E).

After the animals were sacrificed, RNA was extracted from the lumbar portion of the spinal cord to evaluate treatment-related inflammatory changes. Treg EVs have the ability to reduce TNF transcripts in SOD1 spinal cord and beneficial reduction of IL6, IL1 beta and ifnγ transcripts. In addition, the levels of anti-inflammatory Treg-related FOXP3 RNA increased with Treg EV treatment. Anti-inflammatory spinal cord-specific CD206 transcripts were also elevated in Treg EV treated SOD1 animals compared to controls (fig. 4F).

The results provided in this example show that intranasal administration of Treg EV populations, such as pure (medium-free EV) Treg EV populations, produces CNS benefits through the reduction of pro-inflammatory transcripts in multiple brain regions. Furthermore, an increase in anti-inflammatory transcripts in these same cells indicates Treg EV-induced repolarization of peripheral bone marrow cells.

6.4 example 4: treg EVs have greater inhibition of pro-inflammatory M1 cells than MSC EVs.

The ability of EVs isolated from tregs ("Treg EVs") and EVs isolated from mesenchymal stem cells ("MSC EVs") to suppress immune cells was tested. As demonstrated herein, treg EVs showed greater inhibition of pro-inflammatory M1 cells compared to MSC EVs. The Treg EVs used in these experiments were a cultured pure Treg EV population of ex vivo expanded tregs from healthy human subjects. Tregs were cultured in exosome-free FBS and EVs were isolated using the PEG method described above. The MSC EVs used in these experiments were isolated from cultures of human bone marrow MSCs using exosome-free FBS and isolated using the PEG method described above.

At 1X 10 ⁸ Dose sum of individual EV 1X 10 ⁷ At the dose of individual EVs, treg EVs were able to inhibit M1 pro-inflammatory IL-6 protein by 46% and 30.6%, respectively, as compared to 13.7% and 3.3% for MSC EVs, respectively (fig. 5A). At 1X 10 ⁸ The sum of the doses of (2) is 1×10 ⁷ Treg EV inhibited M1-derived pro-inflammatory IL-8 protein by 60% and 50%, respectivelyIn contrast, MSC EV was at 1X 10 ⁸ Shows 20% inhibition at the dose (figure 5B). In the comparative study, treg EV inhibited T cell proliferation more than MSC EV (fig. 5C).

These experiments are also provided in example 9 below, which includes, for example, a graph showing the statistical significance of these results.

6.5 example 5: stability and immune cell suppression of EV

Treg EV stability and function were evaluated after 1 to 20 freeze/thaw cycles and after-20 ° storage for 3, 6 or 12 months. After multiple freeze/thaw cycles, no loss of Treg EV particle number (fig. 6A) and no significant deviation in particle size (fig. 6B) were observed. Treg EV inhibition of T cell proliferation did not decrease with time in-20 ℃ frozen storage (fig. 6C). Treg EVs used in these experiments were derived from cultures of ex vivo expanded tregs obtained from ALS patients. Tregs were cultured in human serum (non-exosome depleted) and EVs were isolated using the PEG method described above.

6.6 example 6: EV yield of

ALS Treg EV concentration was measured after isolation from the amplification medium and from the medium EV alone (using PEG described above). FIG. 7A shows EV particle yield per ml media. The number of EVs in the amplification medium was increased 1.61-fold compared to the medium alone (fig. 7B). Thus, the increase in EV may result from an expanded population of tregs. It is estimated that about 30% of the total EV population from these samples is derived from expanded Treg cells.

6.7 example 7: particle size of EV isolated by TFF

Treg EV derived from ex vivo expanded ALS patient tregs cultured in human serum (non-exosome depletion) was isolated using Tangential Flow Filtration (TFF) techniques. Briefly, this isolation used the Repligen KR2i TFF system, which enabled the isolation, concentration and diafiltration of Treg EV populations using PBS buffer.

First, 85cm of hollow fibers were used with TFF and 0.65 μm Spectrum mPES ² The filter circulates the medium from the Treg expansion culture to filter out cells and debris.

The permeate from this step is then designed to concentrate and diafiltrate the EV population into the retentate by a continuous cycle. The process uses a TFF system and Spectrum mPES hollow fiber 115cm ² Filters (500 kD) to retain/concentrate all particles greater than about 60-80nm in the retentate.

Sterile PBS was introduced into the circulation, resulting in diafiltration and replacement of the existing solution. Treg EV isolated using TFF protocol showed a size spectrum with an average of 92.1nm±4.2nm and a mode of 73.3nm±6.1nm (fig. 8).

6.8 example 8: treg EV can induce transformation of pro-inflammatory M1 cells into anti-inflammatory M2 cells

Treg EV derived from ALS patient tregs (specifically ALS patient tregs expanded ex vivo in a medium containing exosome non-depleted serum) were added to M1 cell culture at various doses at time points overnight (18 hr). Treg EV was found to be able to induce Arg1 and CD206 mRNA (see fig. 9A and 9B, respectively), indicating the conversion of M1 cells to anti-inflammatory M2 cells.

Treg EVs used in these experiments were derived from cultures of ex vivo expanded tregs obtained from ALS patients. Tregs were cultured in human serum (non-exosome depleted) and EVs were isolated using the PEG method described above. As described above, proinflammatory M1 cells are polarized.

6.9 example 9: treg EVs have greater inhibition of pro-inflammatory M1 cells than MSC EVs.

As explained in example 4 above, EVs isolated from tregs ("Treg EVs") and EVs isolated from mesenchymal stem cells ("MSC EVs") were tested for their ability to suppress immune cells. As demonstrated herein, treg EVs showed greater inhibition of pro-inflammatory M1 cells compared to MSC EVs. The Treg EVs used in these experiments were a cultured pure Treg EV population of ex vivo expanded tregs from healthy human subjects. Tregs were cultured in exosome-free FBS and EVs were isolated using the PEG method described above. MSCs EV used in these experiments were isolated from cultures of human bone marrow MSCs and isolated using the PEG method described above.

Specifically, MSCs were obtained from the collaboration, where MSCs were obtained and grown from bone marrow and passaged 3-4 times, and then grown to 80% confluency in flasks in 1640 medium supplemented with 10% FBS. Then, the 1640 medium supplemented with 10% FBS was replaced with serum-free medium for 48 hours. After 48 hours in serum-free MSC medium, EVs were harvested from tissue culture medium.

Proinflammatory spinal cord studies and T cell proliferation studies were performed using iPSC-derived proinflammatory bone marrow cell procedures. For reproducibility, T cell proliferation assays used T cells isolated from the same control patients. Control EVs were isolated from medium containing 5% human AB serum that was never used in culture (i.e., effectively serum EVs). Control T cells were isolated from human blood. All assays were repeated three times.

The data indicate that Treg EVs were significantly more effective than MSC EVs in inhibiting M1 pro-inflammatory IL-6 protein production (fig. 10A), T cell proliferation (fig. 10B), and M1 pro-inflammatory IL-8 protein production (fig. 10C).

6.10 example 10: particle size of EV isolated by TFF

EV derived from the patient Treg expanded medium was isolated using Tangential Flow Filtration (TFF) techniques. TFF was performed using the two-step procedure described in example 3 above. The size spectrum of the separated EVs was measured and the data is shown in fig. 11A and 11B. Each column in the figure shows EV of Treg medium isolated from different patients from the expansion process.

Fig. 11A shows the average value of the particle diameter. Fig. 11B shows the mode value of the particle diameter. The data show that Treg EVs isolated using the TFF protocol showed a size spectrum with an average of 87.38nm (fig. 11A) and a mode of 71.58nm (fig. 11B).

For patients #1-4, treg expansion was performed using the flask production method as described in example 1. For patients #5 and #6, a bioreactor (Terumo BCT was usedCell expansion system) for Treg expansion. The data show that EV size is not significantly different between the two amplification methods.

6.11 example 11: TFF EV recovery

EV recovery after TFF isolation was calculated via Nanosight particle analysis as described in section 6.3.1.4 above. The particle/mL value was used and multiplied by the solution volumes of the original medium and total isolation product, respectively, to calculate the total EV number. FIG. 12 shows the recovery level of EV recovered from the original medium by TFF separation.

6.12 example 12: automated Treg expansion

In the isolation and enrichment (CD 25) ⁺ enrichment/CD8+CD19+ depletion, e.g. viaPlus or CliniMACS->) Thereafter, CD25 is used ⁺ The enriched cells were incubated in the Quantum cell expansion system (Terumo BCT).

Within 24 hours of the start of the isolated and enriched cell culture (preferably within 30 minutes after isolation and enrichment) (day 0) at 4:1 bead ratio cell ratio activation of CD25 with anti-CD 3/anti-CD 28 beads ⁺ And (3) cells. IL-2 and rapamycin are also added on day 0 (preferably within 30 minutes of isolation and enrichment) within 24 hours of the start of the isolated and enriched cell culture.

The medium was supplemented with IL-2 every 3-4 days and the IL-2 concentration was adjusted based on the cell number (i.e. the number of all cells in culture, including the enriched Treg cells). Specifically, the cells were cultured in a medium containing about 200IU/mL IL-2 until the number of cells reached 600X 10 ⁶ And then cultured in a medium containing about 250IU/mL IL-2. The medium also contains human AB serum (e.g., 1% or 0.5% human AB serum).

The flow rate of the Extracapillary (EC) medium was also adjusted based on the cell number (i.e., the number of all cells in culture, including the enriched Treg cells). Specifically, the flow rate of the EC medium was maintained at 0 until the number of cells reached 500X 10 ⁶ Then increase to about 0.2mL/min and maintained at about 0.2mL/min until the cell number reached 750X 10 ⁶ Then raised to about 0.4mL/min and maintained at about 0.4mL/min until the cell number reaches 1,000X10 ⁶ Then raised to about 0.6mL/min and maintained at about 0.6mL/min until the cell number reaches 1,500X10 ⁶ Then raised to about 0.8mL/min and maintained at about 0.8mL/min. The EC medium contained rapamycin. From day 1, cells were expanded in a Quantum bioreactor. Cell count and viability were determined daily. Glucose and lactate levels in the medium were also measured daily.

Before day 11 or on day 11, if the cell expansion yields the desired cell dose (. Gtoreq.2.5X10) ⁹ Individual cells), the cells are harvested and cryopreserved after removal of the beads. If the cell expansion process did not reach the dose by day 11, then 1:1 bead ratio cells were reactivated with anti-CD 3/anti-CD 28 beads. The amplification process may be continued in the bioreactor from day 12 to day 15, as desired. Cell count and viability were measured daily. Once the cell expansion process achieved the required cell dose (occurring on any of the days between 12 and 15), the cells were harvested immediately and cryopreserved after bead removal. See fig. 13 for a corresponding process flow diagram.

6.13 example 13: identification of TFF isolated EVs by proteomics

Proteomic analysis was performed on Treg EV samples isolated from three TFFs. These samples have a mixture of serum EV supplemented with 5% human AB medium used in GMP production. Two independent samples of medium supplemented with 5% human AB serum were identified to subtract serum EV background and obtain unique features of Treg EV. Serum EV (also referred to as "medium EV") was also isolated using TFF.

The proteomic profile of Treg EVs was compared to the culture medium serum EVs, effectively generating a proteomic profile of Treg EVs by subtracting the background. The data presented in table 10 below shows the highest gene product enriched in Treg EV compared to background medium EV, which reached a modified p-value cut-off of less than 0.1. The positive "log 2 of fold change in medium EV vs. Treg EV" values in the table represent enrichment of the corresponding gene product in Treg EV compared to background medium EV. Values are shown as log2 fold change. Also shown in the table are the adjusted p-values, as well as the intensity-based absolute quantification (iBAQ) values of the gene products from the mass spectrometry runs for the three Treg EV samples and the two medium EV samples.

Table 9: compared to medium EV, the gene product enriched in Treg EV

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The 191 gene products in table 10 were further analyzed using the reactiome pathway database. Table 11 provides the first 200 paths from the analysis. Table 12 provides the selected immune pathways.

Table 11: the first 200 reactiome pathways

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Table 12: selected immune pathways

6.14Example 14: in vivo immune cell action of whole body (IV) administered Treg EV in LPS-induced mouse neurogenic inflammation model.

The experimental design described in this example was designed to evaluate the effect of systemic (intravenous) administration of Treg EVs in a model of LPS-induced mouse neurogenic inflammation (as described above).

Tregs from healthy subjects cultured from the bioreactor produced Treg EV used and TFF isolated. See example 15 below (specifically, treg used in these experiments was from example 15 bioreactor run # 4). Mice were peripherally injected with LPS to induce neuroinflammatory mechanisms in the brain to test in vivo inhibition of Treg EV upon intravenous administration. After LPS injection of 2mg/kg IP for the inflammatory mouse model, the dose was varied (1X 10 ⁹ 、1×10 ¹⁰ And 1X 10 ¹¹ ) Treg EV was administered to mice via tail vein Injection (IV). After overnight treatment, mice were sacrificed. The spleen was dissected to isolate immune cells, including CD11b+ bone marrow cells, CD4+CD25+ Treg cells, and CD4+CD25-effector T cells. See, fig. 14A-14B.

IV-effect of administered Treg EV on peripheral immune cell characteristics.Transcript analysis of spleen-derived CD11b+ bone marrow cells showed induction of pro-inflammatory transcripts, such as IL-6, iNOS, IL-1b and IFNγ, following LPS inflammatory induction in mice (FIG. 14C). Increasing the dose of IV-administered Treg EV demonstrated a dose of 1X 10 ¹⁰ (61%) and 1X 10 ¹¹ At a dose of (75%) in the myelitis IL-6 transcript, and at these same doses (1X 10 ¹⁰ (64%) and 1X 10 ¹¹ (85%)) a corresponding significant decrease in iNOS transcripts in the same cells (fig. 14C). Reduced trend was observed in IL-1 beta and ifnγ transcripts, where knots were observed at higher doses of administrationThe result is best (fig. 14D). Anti-inflammatory transcripts from these bone marrow cells following LPS activation and subsequent IV Treg EV administration were then analyzed. At higher Treg EV doses, activated bone marrow cells showed increased anti-inflammatory transcripts like MRC1 (mannose receptor/CD 206) and CD163 (fig. 14E) compared to LPS-injected control animals alone. These results demonstrate that IV-administered Treg EV reduces pro-inflammatory transcripts in activated bone marrow cells in the LPS mouse inflammation model and increases anti-inflammatory transcripts in these same activated cells. These results indicate a shift to M2, or anti-inflammatory spinal phenotype.

Transcript analysis of spleen-derived CD4+CD25+ (Treg) and CD4+CD25+ (T effector; teff) cell populations was performed to evaluate the immune-modulating effect of Treg EV. Transcript analysis of the spleen-derived cd4+cd25+ Treg cell population showed reduced expression of the Treg health and functional marker FOXP3 following LPS-induced inflammation (fig. 14F). Treatment with Treg EV increased FOXP3 transcript levels in a dose-dependent manner, at 1 x 10 ¹¹ Expression was significantly elevated (fig. 14F). Furthermore, treatment with Treg EV increased Treg health and function marker IL2RA transcript (CD 25) in a dose-dependent manner, wherein at 1×10 ¹¹ Expression at this time was significantly elevated (fig. 14F). At the same time point, measurements of multiple inflammatory transcripts (TNF, IFNγ, IL-10 and IL-2) from spleen-derived CD4+CD25-T effector (Teff) cell populations indicated that LPS treatment did not result in significant Teff cell activation. No significant changes in these transcripts were also observed in the Teff cell population following Treg EV treatment alone, indicating that administration of human Treg EV alone was insufficient to drive T cell immune responses.

Effect of IV administered Treg EV on neuroinflammatory changes in the brain.The results presented above demonstrate that intravenous administration of Treg EV significantly modulates the characteristics of peripheral immune cells in the LPS-induced inflammation model. Then, the effect of IV-administered Teg EV on neuroinflammatory changes in the central nervous system (brain) was evaluated. Hippocampal and cortical brain tissue was isolated from the same treated animals whose peripheral tissues were evaluated above. Transcript analysis from these brain tissues was performed to evaluate peripheral (IV) -administered Tre g EV reduces the potential for neurogenic inflammation. In the hippocampus of LPS-treated mice, in 1X 10 ¹¹ IV delivered Treg EV produced a reduction in the proinflammatory transcripts of IL-6 and IL-1 beta at the highest dose of IL, with a modest reduction in IL-6 (fig. 15A). In the cortex, a dose-dependent increasing trend of inhibition of IL-6 and IL-1β was observed (FIG. 15B). Treatment with LPS alone did not significantly increase TNF transcript levels in the hippocampus or cortex relative to PBS control. Likewise, treg EV administered did not affect TNF transcript levels in these tissues.

6.15Example 15: particle size distribution of TFF-separated Treg EV produced by bioreactor

In a medium containing 1% human AB serum, a series of EV bioreactor production runs (BioR 1-BioR 6) from healthy patient Treg expansion were performed according to the protocol described herein (see, e.g., example 12), using the TFF techniques described (see, e.g., example 3 above) and isolating EVs using the specific parameters shown in table 13 below.

TABLE 13

Nanoparticle chase analysis of Treg EVs isolated using TFF of Nanosight NS300 (see section 6.3.1.4 above) demonstrated a significantly consistent population of-20-200 nm.

As shown in fig. 16A, the single peak generated in nanoparticle analysis describes a uniform EV population, as opposed to a heterogeneous population that would be represented by multiple strong peaks within the distribution.

In addition, population size parameters were significantly repeatable between 6 runs for both mean and mode particle sizes. Specifically, treg EV showed a size spectrum with mean 89.4nm, median 86.3nm and mode 73.2nm (fig. 16B).

6.16Example 16: treg EV inhibits T cell proliferation in vitro

A bioactivity assay was performed on Treg EVs from the bioreactor run described in example 15 to examine the inhibition of Treg EVs in a T cell proliferation assay. In vitro dose response studies have shown that once activated with CD3/CD28 beads, 1 x 10 in their ability to inhibit proliferation of T-responsive cells (Tresp) ⁷ Each Treg EV is equivalent to 50,000 Treg cells. Treg EV inhibition was observed to be 86.12% ± 5.17%, n=6 (fig. 17).

6.17Example 17: quantification of functional proteins in Treg EV.

Treg EVs from the bioreactor run described in example 15 above were further identified as described in this example.

An enzyme-linked immunoassay (ELISA) was used to quantify the Treg-conserved functional protein in Treg EV. Each of CD73, CTLA4 and CD25 was found to be present in Treg EV, but hardly present in medium EV alone (fig. 18). As also shown in fig. 18, each of these markers is also present at significant levels in Treg cells from which Treg EVs were obtained. Interestingly, CD73 and CTLA4 play an important role in Treg inhibition of functional mechanisms, and CD25 (IL 2 RA) is a known marker of Treg cell health and function.

6.18Example 18: quantification of residual IL2 and albumin

The impurity profile of the Treg EV produced was produced as described in example 15 above, by quantification of residual IL2 and albumin (as a percentage of the original amount in culture).

After TFF, it was determined that only an average of 6.38% of total IL2 (n=6) was retained in the concentrated retentate (fig. 19A). This corresponds to less than 50ng of total IL2. When diluted to 1X 10 in 2mL of sterile saline per vial ¹¹ At doses of individual Treg EVs, for example, this will be equal to less than 200pg IL2.

After TFF, determineOnly an average of 5.58% of total albumin remained after TFF treatment, which was equivalent to Treg EV product with a total concentration of less than 10 grams (fig. 19B). When diluted to 1X 10 in 2mL of sterile saline per vial ¹¹ At a dose of Treg EV, for example, this will be equal to less than 20mg albumin. It should be noted that both IL2 and albumin are commonly used as direct injection to patients or in cell therapy administration, and both are known to be well tolerated at much higher amounts than this.

6.19Example 19: stability and particle size distribution of Treg EV at room temperature, 4 ℃, -20 ℃ and-80 °

Treg EVs produced from bioreactor-cultured tregs of healthy patients (see example 15 above, in particular EVs from BioR 4-6) and isolated via TFF techniques described herein (see, e.g., example 3 above) were used for these stability experiments, diluted to 1×10 in 2ml of 0.9% sodium chloride solution (sterile saline solution for injection) ¹¹ Exemplary unit doses per EV per dose/vial. Treg EV particle concentration and particle size parameter checks were performed at both Room Temperature (RT) and 4 ℃ for a sustained period of time to evaluate the stability of Treg EV products. Treg EV was stable at exemplary unit doses up to the upper limit of the 48 hour examination, 48 hours (fig. 20A-20B). In addition, as assessed via nanoparticle analysis, the particle size distribution remained stable at 4 ℃ and room temperature storage at dose for mean, mode and median particle size (fig. 20C). Furthermore, both Treg stability and Treg particle size stability remained stable after long term storage (up to 3 months, the last test time point) at-20 ℃ and-80 ℃ (fig. 20D-20E).

6.20Example 20: stability and particle size distribution of Treg EV at room temperature, 4 ℃, -20 ℃ and-80 °

The Treg EV from the bioreactor run described in example 15 isolated by TFF carried out this example.

Treg EV was co-cultured overnight with iPSC-derived, proinflammatory spinal cord M1 cells that had been activated with GM-CSF and LPS/IFNy. Treg EVs inhibited the pro-inflammatory IL-6 cytokine production of M1 cells by about 40% (fig. 21), which is significantly greater than the inhibitory activity of Mesenchymal Stem Cell (MSC) EVs or the minimal inhibition mediated by serum EV control populations, thereby further demonstrating the potential of Treg EVs described herein as therapeutic agents for the treatment of autoimmune and inflammatory disorders.

6.21Example 21: treg EV surface marker profile

To further identify the Treg EV population described herein, EV surface marker analysis was performed on Treg EVs from the 6 bioreactor runs described in example 15 and TFF isolation (see, e.g., example 3). As mentioned therein, treg EVs are derived from tregs obtained from healthy subjects and expanded ex vivo, and TFF isolated Treg EVs. The resulting Treg EV surface marker profile is discussed herein.

Treg EV surface proteins were evaluated using Miltenyi MACSPlex exosome kit (Miltenyi Biotec) and analyzed on a macquant analyzer flow cytometer (Miltenyi Biotec) according to the manufacturer's instructions. Briefly, the EV population is incubated overnight with a mixture of multiple fluorescently labeled bead populations coated with specific antibodies that target different surface epitopes. EV detection reagents are used to form sandwich complexes on the beads, which are then analyzed based on their unique fluorescent characteristics. Then, different positive populations were measured using a macquant flow cytometer.

The Treg EV determined was from 6 bioreactor runs described in example 15. As mentioned therein, treg EVs are derived from tregs obtained from healthy subjects and expanded ex vivo, and TFF isolated Treg EVs. The Treg EV characteristics of these populations obtained are shown in the upper panel of fig. 22, which is the average data for the 6 Treg EV populations. The control "medium EV" was derived from bioreactor medium (cell-free culture) supplemented with 1% serum. This provided a control population for the experiment and also allowed differential identification of Treg EV from medium EV alone (fig. 22, lower panel). For example, figure 22, treg EV results shown in upper panels removed potential contributions from medium EV alone.

Using the TFF protocol described in example 3 above, ALS patient Treg EVs were prepared following Treg expansion according to the protocol described in example 1 above. Features of ALS patient-derived Treg EV are shown in figure 23. Since these Treg EVs were also cultured in serum, the medium EV plates shown in the lower panels in fig. 22 also allow differential identification of these Treg EVs.

Features of Treg EVs from healthy subjects (fig. 22, upper panel) are similar to those of ALS patients (fig. 23). For example, CD9, CD63, and CD81, which are commonly used as exosome markers, are positive in both Treg EVs isolated from ALS patients as well as healthy subjects (see fig. 22, upper panels, each of fig. 23, and 24A). Markers associated with Treg EV specificity (see also, e.g., fig. 2J and 2K) were found to be positively associated with tregs from both ALS patients and healthy subjects (see each of fig. 22, 23 and 24B) compared to medium EV. Note that: in fig. 24A-B, healthy subjects are also referred to as "controls". Interestingly, CD2 is present in particularly high abundance in tregs derived from both healthy and ALS starting materials. Surface markers such as HLA-DRPDQ, CD25, CD44, CD45, CD29, CD4 and CD125 were also observed at particularly significant levels. It should be noted that "HLA-DRDPDQ" refers to HLA class II molecules HLA-DR, HLA-DP and HLA-DQ.

To verify that the observed features are unique to Treg EVs, and for example, instead of measuring the consequences of artefacts, surface markers were also evaluated for EVs derived from different cell types (cd14+ cells) using a Miltenyi MACSPlex assay. As expected, the characteristics from these EVs are different from those from Treg EVs.

6.22Example 22: treg EV RNA profile

To further identify the population of Treg EVs described herein, RNA components, in particular microrna components, from Treg EVs operated by the 6 bioreactors described in example 15 above were analyzed. As a control, EV analysis of the medium alone was also performed. The Treg EV RNA profile obtained by the analysis is described herein.

As mentioned in example 15, treg EVs were from tregs obtained from healthy subjects and expanded ex vivo, and TFF isolated Treg EVs.

And (5) RNA extraction.The EV was cleaved using 3ml TRI reagent added to 1ml EV sample. The samples were mixed and then centrifuged at 12,000Xg for 5min at 4 ℃. The clarified supernatant was transferred to a new tube and incubated for 5 minutes at room temperature. BAN phase separation reagent was added to the supernatant (0.05 ml reagent per 1ml supernatant), followed by vigorous shaking and incubation at room temperature for 5min. To separate the aqueous phase from the organic phase, the sample was centrifuged at 12,000Xg at 4℃for 15min, and the RNA-containing aqueous phase was transferred to a new tube. To clear RNA from the aqueous phase and enrich for small RNA species (those of about 17-200 nucleotides in length), RNA Clean from Zymo Research (catalogue # R1015) was used &Concentrator ^TM -5 a kit. The procedure was followed by the manufacturer, but modified to add 1.5 volumes of ethanol to the aqueous phase.

microRNA-Seq library preparation (Zymo Research):a microRNA-Seq library was constructed from 100ng RNA. Briefly, RNA is linked to miRNA linkers. The excess linker is blocked to prevent interference in subsequent steps. The miRNA-linker ligation product was circularized (circularized) and the blocked linker dimer was removed. The circularized miRNA-adaptor product is then reverse transcribed. The resulting cDNA was amplified using indexed PCR primers and an index sequence added to the Illumina-compatible adapter. The microRNA-Seq library is sequenced on an Illumina NovaSeq sequencer to a sequencing depth of at least 500-1000 ten thousand read pairs per sample.

Alignment of sequence data:first, use Trim Galore-! (v 0.6.6) correction of Illumina NovaSeq read from microrna-Seq data file, then quality was analyzed by short read alignment with reference genome of interest using conventional tubing integrating Bowtie (v 1.3.0), samtools (v 1.11) and FASTX (v 0.0.14). Further miRNA-specific computational analyses were performed using the software programs miR Trace (v1.0.1), mirtop (v0.4.23) and isopirs (v1.16.0). If the sequence corresponds to a known miRNA sequence, the sequencing read is considered to be a miRNA count. Table 14 below provides the total number of miRNA read counts (treg_ev_1-6), the average of 6 total numbers (Treg EV AVG) and the Standard Deviation (SD) from each Treg EV population tested. Since Treg EV is derived from culture in serum (1% AV serum) The cultured Treg cells then also obtained a culture-only EV population and sequencing mirnas from this population.

TABLE 14

Small RNA reading number in EV	miRNA
		Treg_EV_1	689554
Treg_EV_2	1646852
		Treg_EV_3	953235
Treg_EV_4	1171101
		Treg_EV_5	2337624
Treg_EV_6	1211914
		Treg EV AVG	1335046.7
Treg EV SD	533364.3
		Vehicle EV	9811

Table 15 below provides the top 50 most abundant mirnas present in the Treg EVs tested, as determined based on the number of miRNA read counts obtained for each sequence, and the average of the 6 Treg EVs tested (Treg EV AVG). The table also provides miRNA read counts (treg_ev_1-6) for each of the individual Treg populations. Sd=standard deviation. As shown in table 14 (medium EV), medium-only EVs contained very few mirnas and as such did not significantly contribute to the Treg EV miRNA results shown herein.

TABLE 15

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And identifying the miRNA with the greatest abundance as hsa-miR-1290. On average, the abundance of this miRNA was about 26% based on the miRNA read count.

Interestingly, based on tam2.0 miRbase, the most abundant mirnas in the Treg EV RNA profile are inflammatory or immune related mirnas (Kozomara and Griffiths-Jones (2011) nuc.39D152-D157). See table 16 below.

Table 16

Of these inflammatory mirnas, two of the most abundant mirnas present in the Treg EV population are hsa-miR-146-5p and hsa-miR-155-5p. Table 17 below provides the abundance of these 2 miRNAs in the tested Treg populations (as assessed by read counts), the hsa-miR-146-5p/hsa-miR-155-5p ratio in each of the 6 tested Treg EV populations, and the ratio range (1.93-5.31) in the 6 populations. Based on these results, the average hsa-miR-146-5p/hsa-miR-155-5p ratio of the 6 Treg EV populations tested was about 2.7. Table 18 below shows the proportion of total miRNA reads identified as hsa-miR-146-5p, hsa-miR-155-5p or hsa-miR-146-5p and hsa-miR-155-5p.

TABLE 17

TABLE 18

All patent publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual patent publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

The scope of the invention is not limited by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

wherein:

ii) the population comprises EV surfaces CD2, CD25 and HLA-DRDPDQ;

iii) The population comprises hsa-miR-1290, hsa-miR-146a-5p and hsa-miR-155-5p microRNAs (miRNAs); and

Wherein the human suppressive immune cells are regulatory T cells (tregs).

2. The anti-inflammatory EV population of claim 1, wherein at least about 90% of EVs in the population exhibit a dimensional diameter of about 50nm to about 150 nm.

3. The anti-inflammatory EV population of claim 1 or 2, wherein the population exhibits an average size diameter of about 80nm to about 110 nm.

4. The anti-inflammatory EV population of any one of claims 1-3 wherein the population exhibits a median size diameter of about 70nm to about 110 nm.

5. The anti-inflammatory EV population of any one of claims 1-4, wherein the population exhibits a mode size diameter of about 65nm to about 95 nm.

6. The anti-inflammatory EV population of claim 1, wherein at least about 90% of EVs in the population exhibit a size diameter of about 50 to about 150nm and the population exhibits an average size diameter of about 80nm to about 110nm, a median size diameter of about 70nm to about 110nm, and a mode size diameter of about 65nm to about 95 nm.

7. The anti-inflammatory EV population of any one of claims 1-6 wherein the population further comprises EV surfaces CD44, CD29, CD4, and CD45.

8. The anti-inflammatory EV population of any one of claims 1-7 wherein the population further comprises EV surfaces CD9, CD63, and CD81.

9. The anti-inflammatory EV population of any one of claims 1-8 wherein the population is significantly devoid of EV surfaces CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP, CD146, CD86, CD326, CD133, CD142, CD31, and CD14.

10. The anti-inflammatory EV population of claim 1 or 6, wherein the population further comprises EV surfaces CD44, CD29, CD4, CD45, CD9, CD63, and CD81, and wherein the population is substantially devoid of EV surfaces CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP, CD146, CD86, CD326, CD133, CD142, CD31, and CD14.

11. The anti-inflammatory EV population of any one of claims 1-10 wherein the ratio of hsa-miR-146a-5p to hsa-miR-155-5p in the population is from about 2 to about 3.

12. The anti-inflammatory EV population of any one of claims 1-11 wherein hsa-miR-1290 is at least 2-fold more abundant than hsa-miR-155-5 p.

14. The anti-inflammatory EV population of any one of claims 1-12 wherein the tregs are from a human subject diagnosed with or suspected of having Amyotrophic Lateral Sclerosis (ALS).

15. The population of anti-inflammatory EVs of any one of claims 1-14, wherein the anti-inflammatory EVs exhibit the ability to increase expression of IL-10, arg1, and/or CD206 in bone marrow cells.

16. The population of anti-inflammatory EVs of any one of claims 1-15, wherein the anti-inflammatory EVs exhibit the ability to reduce expression of IL-6, IL-8, IL1 β, or interferon- γ in bone marrow cells.

17. The anti-inflammatory EV population of claim 1, wherein proliferation of said responsive T cells is determined by flow cytometry or thymidine incorporation.

18. The anti-inflammatory EV population of any one of claims 1 to 17 wherein the population is an anti-inflammatory EV population containing saline.

21. The anti-inflammatory EV population of claim 20 wherein the tregs are from healthy human subjects.

23. The anti-inflammatory EV population of claim 22, wherein the neurodegenerative disorder is alzheimer's disease.

24. The anti-inflammatory EV population of claim 22, wherein the neurodegenerative disorder is Amyotrophic Lateral Sclerosis (ALS).

25. The anti-inflammatory EV population of claim 22, wherein the neurodegenerative disease is Multiple Sclerosis (MS).

26. The anti-inflammatory EV population of claim 22, wherein the neurodegenerative disease is parkinson's disease.

27. The anti-inflammatory EV population of claim 20 wherein the tregs are from a human subject diagnosed with or suspected of having a stroke.

28. The anti-inflammatory EV population of claim 20 wherein the tregs are from an elderly subject.

29. The anti-inflammatory EV population of any one of claims 20-28 wherein the Treg is from a plurality of human subjects.

31. The population of anti-inflammatory EVs of any one of claims 19-30, wherein the anti-inflammatory EVs exhibit the ability to increase expression of one or more anti-inflammatory markers in inflammatory cells.

32. The anti-inflammatory EV population of claim 31 wherein the inflammatory cells are bone marrow cells.

33. The population of anti-inflammatory EVs according to claim 31 or 32, wherein the anti-inflammatory EVs exhibit the ability to increase expression of IL-10, arg1 and/or CD206 in inflammatory cells.

34. The population of anti-inflammatory EVs of any one of claims 19-33, wherein the anti-inflammatory EVs exhibit the ability to inhibit inflammatory cells as measured by pro-inflammatory cytokine production by the inflammatory cells.

35. The method of claim 34, wherein the inflammatory cell is a bone marrow cell.

36. The anti-inflammatory EV population of claim 35 wherein the bone marrow cells are monocytes, macrophages or microglia.

37. The anti-inflammatory EV population of claim 36 wherein the macrophages are M1 macrophages.

38. The anti-inflammatory EV population of claim 37 wherein the M1 macrophages are Induced Pluripotent Stem Cell (iPSC) -derived M1 macrophages.

39. The anti-inflammatory EV population of any one of claims 31-38 wherein the ability to inhibit inflammatory cells is measured by IL-6, IL-8, tnfa, IL1 β, and/or interferon- γ production by the inflammatory cells.

40. The anti-inflammatory EV population of any one of claims 19 to 39 wherein the anti-inflammatory EVs exhibit inhibitory function as determined by inhibition of responsive T cell proliferation.

41. The anti-inflammatory EV population of claim 40 wherein proliferation of the responsive T cells is determined by flow cytometry or thymidine incorporation.

42. The anti-inflammatory EV population of any one of claims 19 to 41 wherein the population is an anti-inflammatory EV population that contains saline.

43. The anti-inflammatory EV population of any one of claims 19 to 41 wherein the population is an anti-inflammatory EV population containing physiological saline.

44. The anti-inflammatory EV population of any one of claims 19 to 41 wherein the population is an anti-inflammatory EV population containing phosphate buffered saline.

45. The anti-inflammatory EV population of any one of claims 19-44 wherein the anti-inflammatory EV population comprises exosomes and microbubbles.

46. The anti-inflammatory EV population of claim 45 wherein a majority of EVs are exosomes.

47. The anti-inflammatory EV population of claim 46 wherein at least about 80%, about 90%, or about 95% of the EVs are exosomes.

48. The anti-inflammatory EV population of claim 47 wherein a majority of EVs are microbubbles.

49. The anti-inflammatory EV population of claim 48 wherein at least about 80%, about 90%, or about 95% of the EVs are microbubbles.

50. The anti-inflammatory EV population of claim 45 wherein a majority of EVs have diameters from about 30nm to about 1000 nm.

51. The anti-inflammatory EV population of claim 45 wherein a majority of EVs have diameters of about 30nm to about 100nm, about 30nm to about 150nm, about 30 to about 200nm, about 40 to about 100nm, about 80 to about 110nm, about 80 to about 125nm, or about 100 to about 120 nm.

52. The anti-inflammatory EV population of claim 25, wherein a majority of EVs have diameters of about 60nm to about 1000nm, about 70nm to about 1000nm, about 80nm to about 1000nm, 100 to about 1000nm, about 200 to about 1000nm, or about 300 to about 1000 nm.

53. A pharmaceutical composition comprising the isolated, cell-free anti-inflammatory EV population of any one of claims 1-52.

54. The pharmaceutical composition of claim 53, wherein the anti-inflammatory EV population comprises about 1 x 10 ⁶ Up to about 1X 10 ¹⁴ EV, about 1×10 ⁸ Up to about 1X 10 ¹⁴ EV, about 1×10 ⁸ Up to about 1X 10 ¹² EV, about 1×10 ⁸ Up to about 1X 10 ¹⁰ EV, about 1×10 ¹⁰ Up to about 1X 10 ¹⁴ EV or about 1×10 ¹⁰ Up to about 1X 10 ¹² And (5) EV.

55. The pharmaceutical composition of claim 53, wherein the anti-inflammatory EV population comprises about 1 x 10 ⁶ Up to about 1X 10 ¹⁴ EV/ml, about 1X 10 ⁸ Up to about 1X 10 ¹⁴ EV/ml, about 1X 10 ⁸ Up to about 1X 10 ¹² EV/ml, about 1X 10 ⁸ Up to about 1X 10 ¹⁰ EV/ml, about 1X 10 ¹⁰ Up to about 1X 10 ¹⁴ EV/ml or about 1X 10 ¹⁰ Up to about 1X 10 ¹² EV/ml.

56. The pharmaceutical composition of claim 53, wherein the anti-inflammatory EV population comprises about 1 μg to about 200mg EV.

57. The pharmaceutical composition of claim 53, wherein the anti-inflammatory EV population comprises about 1 μg to about 15mg EV.

58. The pharmaceutical composition of claim 53, wherein the anti-inflammatory EV population comprises about 1 μg to about 15mg EV/ml.

59. The pharmaceutical composition of any one of claims 53-58, wherein the pharmaceutical composition is a cryopreserved pharmaceutical composition.

60. The pharmaceutical composition of any one of claims 53-58, wherein the pharmaceutical composition was previously cryopreserved.

61. A cryopreserved composition comprising the isolated, cell-free anti-inflammatory EV population of any one of claims 1-53.

b. isolating the anti-inflammatory EV from the culture.

63. The method of claim 62, wherein the population of human suppressive immune cells is a population of regulatory T cells (tregs).

64. The method of claim 62 or 63, wherein step b) comprises removing cells from the culture, and then precipitating the culture with polyethylene glycol.

65. The method of claim 62 or 63, wherein step b) comprises:

66. The method of claim 65, wherein step i) comprises flowing the culture through a filter, thereby retaining cells through the filter and thereby removing cells from the culture.

67. The method of claim 65 or 66, wherein step i) comprises microfiltration.

68. The method of any one of claims 65-67, wherein step ii) comprises step ii-a): the solution containing the cell-free, anti-inflammatory EV is flowed through a filter, thereby retaining the anti-inflammatory EV through the filter.

69. The method of claim 68, wherein the filter has a molecular weight cut-off (MWCO) of about 200 kilodaltons (kDa) to about 600 kDa.

70. The method of claim 69, wherein the filter has a MWCO of about 500 kDa.

71. The method of any one of claims 65-70, wherein step ii) comprises ultrafiltration.

72. The method of any one of claims 68-71, wherein step ii) further comprises step ii-b): buffer exchange is performed such that the resulting isolated, cell-free anti-inflammatory EV population is an isolated, cell-free anti-inflammatory EV population containing buffer.

73. The method of claim 72, wherein the buffer is a saline-containing buffer.

74. The method of claim 73, wherein the saline-containing buffer is physiological saline.

75. The method of claim 74, wherein the saline-containing buffer is PBS.

76. The method of any one of claims 73-75, wherein step ii-b) comprises diafiltration.

77. The method of any one of claims 73-76, wherein steps ii-a) and ii-b) are performed simultaneously.

78. The method of any one of claims 62-77, wherein step b) comprises tangential flow filtration.

79. The method of any one of claims 62-78, wherein the medium in step a) is serum-free.

80. The method of any one of claims 62-79, wherein the medium in step a) comprises serum.

81. The method of claim 80, wherein the serum is human AB serum.

82. The method of claim 80 or 81, wherein serum is consumed for serum-derived EVs.

83. The method of any one of claims 62-82, further comprising, prior to step a), the step of enriching tregs from a cell sample suspected of containing tregs to produce a baseline population of Treg cells that is the population of tregs subsequently expanded in a).

84. The method of claim 83, wherein the cell sample is a leukocyte-depleted cell sample.

85. The method of claim 83 or 84, wherein the method further comprises obtaining the cell sample from a donor by a leukapheresis procedure.

86. The method of any one of claims 83-85, wherein the cell sample is not stored overnight or frozen prior to performing the enriching step.

87. The method of any one of claims 83-86, wherein the cell sample is obtained within 30 minutes before the enrichment step begins.

88. The method of any one of claims 82-87, wherein the enriching step comprises depleting cd8+/cd19+ cells prior to enriching for cd25+ cells.

89. The method of any one of claims 62-88, wherein step a) is performed within 30 minutes of the enriching step.

90. The method of any one of claims 62-89, wherein step a) comprises culturing the Treg in a medium comprising anti-CD 3 antibody and anti-CD 28 antibody coated beads.

91. The method of claim 90, wherein the beads are first added to the culture medium within about 24 hours of the start of the culture.

92. The method of claim 90 or 91, wherein the anti-CD 3 antibody and anti-CD 28 antibody coated beads are added to the culture medium about 14 days after the first adding the anti-CD 3 antibody and anti-CD 28 antibody coated beads to the culture medium.

93. The method of any one of claims 90-92, wherein step a) further comprises adding IL-2 to the culture medium within about 6 days of the start of the culture.

94. The method of claim 93, wherein step a) further comprises supplementing the medium with IL-2 about every 2-3 days after first adding IL-2 to the medium.

95. The method of any one of claims 90-94, wherein step a) further comprises adding rapamycin to the medium within about 24 hours of the start of the culturing.

96. The method according to claim 95, wherein step a) further comprises supplementing the culture medium with rapamycin every 2-3 days after first adding rapamycin to the culture medium.

97. The method of any one of claims 62-96, wherein step a) is automated.

98. The method of any one of claims 62-97, wherein step a) is performed in a bioreactor.

99. The method of any one of claims 62-98, wherein step b) can begin at any point during step a).

101. The method of any one of claims 63-99, wherein the tregs are from a human subject diagnosed with or suspected of having a neurodegenerative disorder.

102. The method of claim 101, wherein the neurodegenerative disorder is alzheimer's disease, amyotrophic Lateral Sclerosis (ALS), multiple Sclerosis (MS), or parkinson's disease.

106. The method of claim 62, wherein the population of human suppressive immune cells is a genetically engineered population of human suppressive immune cells.

108. A pharmaceutical composition comprising an isolated, cell-free, anti-inflammatory EV population, wherein the population is prepared by any one of the methods of claims 62-107.

109. The method of any one of claims 62-107, further comprising c) cryopreserving the isolated, cell-free anti-inflammatory EV population, thereby producing a cryopreserved, isolated, cell-free anti-inflammatory EV population.

110. The method of claim 109, further comprising thawing the cryopreserved, isolated cell-free anti-inflammatory EV population after about 1 week, 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, or about 24 months of cryopreservation.

111. A pharmaceutical composition comprising the isolated, cell-free, anti-inflammatory EV population of claim 110.

113. A pharmaceutical composition comprising the isolated, cell-free, anti-inflammatory EV population of claim 112.

114. A method of treating a condition associated with Treg dysfunction, the method comprising administering to a subject in need of such treatment the composition of any one of claims 53-60, 108, 111 or 113.

115. A method of treating a condition associated with Treg deficiency, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of claims 53-60, 108, 111 or 113.

116. A method of treating a disorder associated with excessive activation of the immune system, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of claims 53-60, 108, 111, or 113.

117. A method of treating an inflammatory condition driven by a T cell response, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of claims 53-60, 108, 111, or 113.

118. A method of treating an inflammatory condition driven by a bone marrow cell response, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of claims 53-60, 108, 111, or 113.

119. The method of claim 118, wherein the bone marrow cells are monocytes, macrophages or microglia.

120. A method of treating a neurodegenerative disorder in a subject in need thereof, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of claims 53-60, 108, 111, or 113.

121. The method of claim 120, wherein the neurodegenerative disease is ALS, alzheimer's disease, parkinson's disease, frontotemporal dementia, or huntington's disease.

122. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of claims 53-60, 108, 111, or 113.

123. The method of claim 122, wherein the autoimmune disease is polymyositis, ulcerative colitis, inflammatory bowel disease, crohn's disease, celiac disease, systemic sclerosis (scleroderma), multiple Sclerosis (MS), rheumatoid Arthritis (RA), type I diabetes, psoriasis, dermatomyositis, systemic lupus erythematosus, cutaneous lupus, myasthenia gravis, autoimmune kidney disease, autoimmune hemolytic anemia, autoimmune cytopenia, autoimmune encephalitis, autoimmune hepatitis, autoimmune uveitis, alopecia, thyroiditis, or pemphigus.

124. A method of treating graft versus host disease in a subject in need thereof, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of claims 53-60, 108, 111, or 113. The method of claim 106, wherein the subject has received a bone marrow transplant, a kidney transplant, or a liver transplant.

125. A method of improving islet transplantation survival in a subject in need thereof, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of claims 53-60, 108, 111, or 113.

126. A method of treating cardiac inflammation in a subject in need thereof, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of claims 53-60, 108, 111, or 113.

127. The method of claim 126, wherein the cardiac inflammation is associated with atherosclerosis, myocardial infarction, ischemic cardiomyopathy, or heart failure.

128. A method of treating neurogenic inflammation in a subject in need thereof, the method comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of claims 53-60, 108, 111, or 113.

129. The method of claim 128, wherein the neurogenic inflammation is associated with stroke, acute disseminated encephalomyelitis, acute optic neuritis, acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, gill-barre syndrome, transverse myelitis, optic neuromyelitis, epilepsy, traumatic brain injury, spinal cord injury, encephalitis, central nervous system vasculitis, nervous system sarcoidosis, autoimmune or post-infection encephalitis, or chronic meningitis.

130. A method of treating Treg lesions (tregolopathia) in a subject in need thereof, comprising administering to a subject in need of such treatment the pharmaceutical composition of any one of claims 53-60, 108, 111 or 113.

131. The method of claim 130, wherein the Treg lesions are caused by FOXP3, CD25, cytotoxic T lymphocyte-associated antigen 4 (CTLA 4), LPS-reactive beige anchor-like protein (LRBA) or BTB domain and CNC homologous gene 2 (BACH 2) loss of function mutations or signal transduction and activator of transcription 3 (STAT 3) function gain mutations.

132. The method of any one of claims 114-131, wherein the anti-inflammatory EV is derived from tregs autologous to the subject.

133. The method of any one of claims 114-131, wherein the anti-inflammatory EV is derived from tregs allogeneic to the subject.

134. The method of any one of claims 114-133, wherein the pharmaceutical composition is administered via intranasal administration.

135. The method of claim 134, wherein the intranasal administration is via spray or nasal drip.

136. The method of any one of claims 114-135, wherein the pharmaceutical composition is administered intravenously.

137. The method of any one of claims 114-135, wherein the pharmaceutical composition is administered by local injection.

138. The method of any one of claims 114-137, wherein the method further comprises administering to the subject a pharmaceutical composition comprising a population of therapeutic tregs, wherein the tregs have been expanded ex vivo and cryopreserved, and wherein the tregs have not been further expanded prior to administration.

141. The method of any one of claims 138-140, wherein the pharmaceutical composition comprising the population of therapeutic tregs is administered intravenously.

142. The method of any one of claims 138-141, wherein the pharmaceutical composition comprising the anti-inflammatory EV and the pharmaceutical composition comprising the population of therapeutic tregs are administered to the patient on the same day.

143. The method of any one of claims 114-140, wherein the isolated, cell-free, anti-inflammatory EV population has been cryopreserved and thawed prior to administration to the subject.

144. The method of any one of claims 114-140, wherein the isolated, cell-free anti-inflammatory EV population is stored overnight at 4 ℃ prior to administration to the subject.

145. The method of claim 144, wherein the isolated, cell-free anti-inflammatory EV population has been cryopreserved prior to administration to the subject, then thawed and stored overnight at 4 ℃.

146. The method of any one of claims 114-140, wherein the isolated, cell-free, anti-inflammatory EV population has undergone at least 2 freeze/thaw cycles prior to administration to the subject.

147. The method of claim 146, wherein the isolated, cell-free, anti-inflammatory EV population has undergone from about 2 to about 20 freeze/thaw cycles prior to administration to the subject.