JP2019530470A - Ex vivo generation of γδ Foxp3 + regulatory T cells and their therapeutic use - Google Patents

Abstract

The present invention relates to methods for generating and expanding γδFoxp3 + regulatory T cells ex vivo, and therapeutic uses thereof. We performed ex vivo induction of Foxp3 + expression in human inducible tumor antigen-specific CD4 + TCRγδ non-restricted T cells and induction of autologous CD8-mediated T cell responses to tumor antigen-specific FOXP3-expressing CD4 + TCRγδ non-restricted T cells did. We have developed a method for ex vivo generated and expanded antigen-specific Foxp3 expressing CD3 + TCRγδ + unrestricted T cells committed to exert regulatory activity exclusively under any stimulation culture conditions Developed. In particular, the present invention relates to a method for generating ex vivo γδ Foxp3 + regulatory T cells having the following phenotype: CD3 + TCRγδ + Foxp3 +.

Description

The present invention relates to ex vivo methods for generating and expanding γδ Foxp3 ⁺ regulatory T cells and their therapeutic use.

  Background of the Invention

  γδ T cells make up about 1-5% of circulating T cells and act at the interface between innate and adaptive immunity. These cells combine the properties of innate and adaptive immune cells, making them attractive for use in therapy, particularly cancer immunotherapy. Indeed, γδ T cells produce inflammatory cytokines and can directly lyse infected or malignant cells and establish a memory response to the attacking pathogen upon re-exposure. Unlike classical αβ T cells, Vγ9 Vδ2 T cells (a major subset of the circulating γδ T cell pool) recognize untreated antigens such as phosphomonoesters. This recognition is mediated by TCR and is not limited by MHC molecules.

It has been shown in the art that regulatory γδT cells expressing Foxp3 can be induced under appropriate antigenic stimulation and cytokines (Casetti et al. JI 2009, 183: 3574-3577). These Foxp3 ⁺ regulatory γδ T cells are capable of inhibitory activity.

As of today, there is no suggestion in the art how to grow these Foxp3 ⁺ regulatory γδ T cells ex vivo for their therapeutic use.

The present invention thus provides a method for generating and expanding Foxp3 ⁺ γδ regulatory T cells ex vivo, and the therapeutic use of said Foxp3 ⁺ γδ regulatory T cells.
wrap up

The present invention relates to a method for generating ex vivo γδFoxp3 ⁺ regulatory T cells having the following phenotype: CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ , comprising: -γδT cell activator and the following agents: i) cAMP (Cyclic adenosine monophosphate) activator, ii) TGFβ (transforming growth factor beta) pathway activator, iii) mTOR inhibitor, optionally iv) IL-2, IL-7, IL-15, and TSLP At least one cytokine selected in the group, and optionally v) at least one TET enzyme activator (preferably selected from vitamin C and NaHS hydrogen sulfide releasing agents) and / or at least one DNMT inhibitor ( for example, RG108, DAC, or in the presence of 5AC etc.), at least 5 days CD3 ⁺ Culturing the CRγδ ⁺ T cells.

  In one embodiment, the γδ T cell activator is a polyclonal γδ T cell activator, preferably an anti-TCRγδ antibody or a non-peptide phosphoantigen.

  In another embodiment, the γδ T cell activator is an antigen-specific γδ T cell activator, preferably a tolerogenic dendritic cell (DC), using at least one bisphosphonate, preferably at least one aminobiphosphonate. Pulsed.

  In one embodiment of the invention, the cAMP activator is selected from the group comprising a prostaglandin E2 (PGE2), an EP2 or EP4 agonist, a membrane adenine cyclase activator, or a metabotropic glutamate receptor agonist.

  In one embodiment, the TGFβ pathway activator is selected from the group comprising TGFβ, bone morphogenetic protein (BMP), growth and differentiation factor (GDF), anti-Muellerian hormone (AMH), activin, and nodar.

  In one embodiment, the mTOR inhibitor is rapamycin, rapamycin analog, wortmannin; theophylline; caffeine; epigallocatechin gallate (EGCG), curcumin, resveratrol; genistein, 3,3-diindolylmethane (DIM) ), LY294002 (2- (4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one), PP242, PP30, Torin1, Ku-0063794, WAY-600, WYE-687, WYE-354, GNE477 , NVP-BEZ235, PI-103, XL765, and WJD008.

In one embodiment, the method of the invention further comprises a proliferation step, wherein the γδ Foxp3 ⁺ regulatory T cells obtained by the production method described above are transformed into a γδ T cell activator and the following agents: i) cAMP (Cyclic adenosine monophosphate) activator, ii) TGFβ (transforming growth factor beta) pathway activator, iii) mTOR inhibitor, optionally iv) IL-2, IL-7, IL-15, and TSLP At least one cytokine selected in the group, and optionally v) at least one TET enzyme activator (preferably selected from vitamin C and NaHS hydrogen sulfide releasing agent) and / or at least one DNMT inhibitor ( In the presence of RG108, DAC, or 5AC, for example, the cells are cultured for at least 5 days.

Another object of the present invention is an ex vivo generated γδ Foxp3 ⁺ regulatory T cell population obtainable by the methods described hereinabove.

A further object of the present invention relates to an ex vivo generated and expanded γδ Foxp3 ⁺ regulatory T cell population obtainable by a method according to the present invention.

The present invention also relates to an ex vivo generated γδ Foxp3 ⁺ regulatory T cell population that remains functionally stable in inflammatory conditions.

The invention further relates to immunogenic products comprising immunogenic dendritic cells loaded with inactivated γδ Foxp3 ⁺ regulatory T cells or γδ Foxp3 ⁺ regulatory T cell blebs, or γδ Foxp3 ⁺ regulatory T cell blebs.

The present invention also provides an immunogen loaded with inactivated γδ Foxp3 ⁺ regulatory T cells and at least one pharmaceutically acceptable excipient or γδ Foxp3 ⁺ regulatory T cell bleb, or γδ Foxp3 ⁺ regulatory T cell bleb. A pharmaceutical composition comprising a dendritic cell is provided.

Another object of the present invention is to provide immunogenic dendritic cells loaded with inactivated γδ Foxp3 ⁺ regulatory T cells and at least one adjuvant or γδ Foxp3 ⁺ regulatory T cell blebs, or γδ Foxp3 ⁺ regulatory T cell blebs. A vaccine composition comprising.

  A further object of the present invention relates to an immunogenic product, pharmaceutical composition or vaccine composition according to the present invention for use in the treatment of cancer.

The present invention also relates to a pharmaceutical composition comprising γδ Foxp3 ⁺ regulatory T cells and at least one pharmaceutically acceptable excipient.

  The present invention further relates to a pharmaceutical composition as described herein above for use in cell therapy.

  A further object of the present invention is as described herein above for use in treating inflammatory or autoimmune diseases or for preventing transplant rejection or graft-versus-host disease (GVHD). The pharmaceutical composition described.

  Definition

As used herein, “regulatory T cells” or “Tregs” inhibit inhibitory activity (ie, proliferation of conventional T cells) by either cell-cell contact or MLR inhibition (mixed lymphocyte reaction). Refers to a cell capable of inhibiting). These cells contain different subpopulations (MHCII restriction ^CD4 + Foxp3 ⁺ regulatory T cells, GanmaderutaFoxp3 ⁺ regulatory T cells, and including invariant Foxp3 ⁺ regulatory T cells, but not limited to).

As used herein, “invariant Foxp3 ⁺ regulatory T cells” refers to cells having the following phenotype: CD3 ⁺ Vα24 ⁺ Foxp3 ⁺ . These cells recognize non-peptide lipid antigens under CD1 restriction.

As used herein, “γδFoxp3 ⁺ regulatory T cells” refers to cells having the following phenotype: γδTCR + Foxp3 ⁺ . These cells recognize non-peptide phosphoantigens without MHC (major histocompatibility complex) restriction.

As used herein, “MHCII restricted CD4 ⁺ Foxp3 ⁺ regulatory T cells” refers to cells having the following phenotype: CD4 ⁺ CD25 ⁺ Foxp3 ⁺ . These cells recognize peptides (including foreign or self-peptides) identified by their αβTCR (T cell receptor) and presented by restricted MHC class II (major histocompatibility complex class II) molecules.

As used herein, the term “treatment” refers to therapeutic treatment and prophylactic and preventative measures, wherein the purpose is to prevent or slow down the targeted pathological disorder or condition ( Reduction, reduction). Persons in need of treatment include those already with the disorder as well as those who are susceptible to the disorder or who should be prevented from the disorder. After the subject or mammal has received a therapeutic amount of γδFoxp3 ⁺ regulatory T cells or a therapeutic amount of inactivated γδFoxp3 ⁺ regulatory T cells according to the present invention, the patient is observable in one or more of the following: Or has been successfully treated for a disease if it shows a measurable decrease or absence: a decrease in the number of pathogenic cells; a decrease in the percentage of total cells that are pathogenic; and / or a particular disease or condition Some relief of one or more related symptoms; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease can be readily measured by routine procedures familiar to physicians.

As used herein, a “therapeutically effective amount” is a γδ Foxp3 ⁺ modulatory property intended to induce a therapeutic response without causing significant negative or adverse side effects to the target. Refers to the number of T cells or inactivated γδ Foxp3 ⁺ regulatory T cells. A therapeutically effective amount may be administered prior to the onset of the disease being treated, for a prophylactic or preventative effect. Alternatively or in addition, a therapeutically effective amount may be administered after the onset of the disease being treated for therapeutic effect.

As used herein, "therapeutic response" refers to therapeutic benefit induced by GanmaderutaFoxp3 ⁺ regulatory T cell therapy or GanmaderutaFoxp3 ⁺ regulatory T cell vaccination in a subject. The therapeutic response (1) delays or prevents the onset of the disease being treated; (2) slows or stops the progression, exacerbation, or worsening of one or more symptoms of the disease being treated; (3) treated May result in remission of the symptoms of the disease being treated; (4) reducing the severity or incidence of the disease being treated; or (5) the fact of curing the disease being treated.

  As used herein, “about” before a number means more or less than 10% of the value of that number.

  As used herein, “subject or patient” refers to a mammal, preferably a human. In the present invention, the terms “subject” and “subject” may be used interchangeably. Examples of non-human mammals are pets (eg, dogs, cats, domestic pigs, rabbits, ferrets, hamsters, mice, rats, etc.); primates (eg, chimpanzees, monkeys, etc.); Pigs, rabbits, horses, sheep, goats, etc.). In one embodiment, the subject is waiting to receive medical treatment, or is / becomes / can be the subject of a medical procedure, or is monitored for the onset of disease. In one embodiment, the subject is an adult (eg, more than 18 subjects). In another embodiment, the subject is a child (eg, a subject under the age of 18). In one embodiment, the subject is male. In another embodiment, the subject is a female.

  As used herein, “allogeneic cell” refers to a cell that has been isolated from one subject (donor) and injected into another subject (recipient or host).

  As used herein, “autologous cell” refers to a cell that is isolated and injected into the same subject (recipient or host).

  Detailed description

The present invention relates to a method for generating γδ Foxp3 ⁺ regulatory T cells ex vivo.

In one embodiment, a method for generating γδFoxp3 ⁺ regulatory T cells ex vivo comprises:
Γδ T cell activators and the following agents: i) cAMP (cyclic adenosine monophosphate) activator, ii) TGFβ (transforming growth factor beta) pathway activator, iii) mTOR inhibitor, optionally iv) At least one cytokine selected in the group of IL-2, IL-7, IL-15, and TSLP (thymic stromal lymphopoietin), and optionally v) at least one TET enzyme activator and / or Culturing CD3 ⁺ TCRγδ ⁺ T cells, preferably CD3 ⁺ TCRγδ ⁺ CD45RA ⁺ T cells in the presence of at least one DNMT inhibitor for at least 5 days;
-Thereby obtaining an ex vivo generated population of γδ Foxp3 ⁺ regulatory T cells, preferably from γδ naive (CD45RA ⁺ ) T cells.

In one embodiment, CD3 ⁺ TCRγδ ⁺ T cells, preferably CD3 ⁺ TCRγδ ⁺ CD45RA ⁺ T cells, are obtained from a blood sample by any technique well known in the art. In one embodiment, CD3 ⁺ TCRγδ ⁺ T cells, preferably CD3 ⁺ TCRγδ ⁺ CD45RA ⁺ T cells are isolated from PBMC (peripheral blood mononuclear cells) by flow cytometry. In one embodiment, CD3 ⁺ TCRγδ ⁺ T cells, preferably CD3 ⁺ TCRγδ ⁺ CD45RA ⁺ T cells, may be isolated from frozen PBMC.

In one embodiment, obtaining isolated CD3 ⁺ TCRγδ ⁺ T cells, preferably CD3 ⁺ TCRγδ ⁺ CD45RA ⁺ T cells, can be improved by any initial purification step. CD3 ⁺ TCRγδ ⁺ T cells, preferably CD3 ⁺ TCRγδ ⁺ CD45RA ⁺ T cells, are stimulated with antigen pulse tolerogenic DCs (eg, ovalbumin pulse tolerogenic DCs) in the presence of soluble anti-CD28 and anti-CD40 antibodies. To do. In one embodiment, the duration of stimulation ranges between 1 hour and 24 hours, preferably between 10 hours and 20 hours, more preferably between about 16 hours. After stimulation, the cells are washed, for example with PBS, and stained with anti-CD154 and anti-CD4 antibodies for sorting. Purified CD3 ⁺ TCRγδ ⁺ CD154 ⁺ T cells may be enriched and used for the following activation steps.

In one embodiment, CD3 ⁺ TCR γδ ⁺ T cells are activated in the presence of a γδ T cell activator. The γδ T cell activator can be a polyclonal γδ T cell activator or an antigen-specific γδ T cell activator.

  In the present invention, the polyclonal γδ T cell activator is a TCR γδ activator. Examples of TCRγδ activators include anti-TCRγδ antibodies, such as purified mouse anti-human TCRγδ clone B1 (Ref. 555715, BD Biosciences), anti-human TCRγδ antibody (Ref. 331209, Biolegend), monoclonal TCRγδ antibody (Ref. NBP2-222489 or NBP2- 22510, Novus Biologicals), anti-mouse γδ TCR antibody (reference 12-5711-81, eBioscience), TCR γδ antibody (reference MAB7297, R & D Systems), anti-T cell receptor γδ antibody (reference ABIN 2372990, antibody online), anti-TCR gamma + TCR Delta antibody (reference ab25663, Abcam), anti-γδTCR antibody clone IMMU510 (Beckman Coulter); non-peptide phosphoantigen (also called phosphorylated non-peptide antigen), isoprenyl pyrophosphate (IPP), (E) -4-hydroxy -3-Methyl-but-2-enyl diphosphate (HMB-PP) and their analogs such as bromohydrin diphosphate (BrHPP) and 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP) F1-ATPase; Apolipoprotein A-1; Mycobacterium tuberculosis; UL16 binding protein 4 (ULBP4); CD1c; CD1d tetramer loaded with sulfatide; Endothelial protein C receptor (EPCR) , Lipoexapeptides; phycoerythrin, histidyl tRNA synthase, butyrophyllin 3A1, and the like.

  In another embodiment, the polyclonal γδ T cell activator is MHC class I associated A (MICA).

  In another embodiment, the polyclonal γδ T cell activator is an immunogenic apoptotic body from a cancer cell or a bleb from a cancer cell or from a tissue lysate.

  Cancer cells can be derived from tumor biopsies or from the proliferation of circulating cancer cells.

  Immunogenic apoptotic bodies from cancer cells are obtained using, for example, anthracyclines (including doxorubicin, daunorubicin, idarubicin, and mitoxantrone); oxaliplatin, UVC or gamma-treated cancer cells that release apoptotic bodies Or can be isolated directly from anthracyclines (including doxorubicin, daunorubicin, idarubicin, and mitoxantrone); oxaliplatin; UVC or gamma treated cancer.

  The bleb constitutes an important immunogenic particle. They are heterogeneous vesicles formed on the surface of apoptotic cells. In one embodiment, the size of the bleb is in the range of 0.05-5 μm, preferably 0.1-1 μm. Various immunogenic cell death (ICD) inducers can induce the release of blebs from apoptotic cells or autophagic cells, such as irradiation at 5000 rads, as well as some antineoplastic agents (doxorubicin) , Oxaliplatin, and cisplatin). Immunogenic cancer cell blebs may in particular be obtained from apoptotic cancer cells or from cancer cell autophagy after treatment with chemical or physical inducers.

  In one embodiment, the polyclonal γδ T cell activator is an anti-TCRγδ antibody or a non-peptide phosphoantigen, such as isoprenyl pyrophosphate (IPP).

  In one embodiment, the polyclonal γδ T cell activator, preferably the anti-TCRγδ antibody, is soluble in the culture medium. In another embodiment, a polyclonal γδ T cell activator is coated on the culture plate.

  In one embodiment, a polyclonal γδ T cell activator, preferably an anti-TCRγδ antibody, is used in the presence of feeder cells, preferably autologous feeder cells.

  Feeder cells are: ΔCD3 cells (T cell depleted accessory cells), irradiated PBMC, irradiated DC, artificial APC (antigen presenting cells), Sf9 cells, insect cells, pools of PBMC or B cells from different subjects, KCD40L cells , EBV transformed B cell lines, and EBV transformed lymphoblast cells (LCL).

  Preferably, the feeder cells used in the present invention are ΔCD3 cells isolated by negative selection from PBMC by incubation with anti-CD3 coated beads and then irradiated at 3000 rad.

  In one embodiment, the ratio of T cells / supporting cells ranges from about 1: 100 to about 1: 10000, preferably 1: 1000 to 1: 5000. Within the scope of the present invention, the expression “1: 100 to 1: 10000” is 1: 100, 1: 200, 1: 300, 1: 400, 1: 500, 1: 600, 1: 700, 1: 800. 1: 900, 1: 1000, 1: 1250, 1: 1500, 1: 1750, 1: 2000, 1: 2250, 1: 2500, 1: 2750, 1: 3000, 1: 3250, 1: 3500, 1, : 3750, 1: 4000, 1: 4250, 1: 4500, 1: 4750, 1: 5000, 1: 5250, 1: 5500, 1: 5750, 1: 6000, 1: 6250, 1: 6500, 1: 6750 1: 7000, 1: 7250, 1: 7500, 1: 7750, 1: 8000, 1: 8250, 1: 8500, 1: 8750, 1: 9000, 1: 9250, 1: 9500, 1:97 0, and 1: including the 10000, but are not limited to these.

  In the present invention, the antigen-specific γδ T cell activator is a tolerogenic dendritic cell (DC).

  As used herein, “tolerogenic DC” refers to a DC capable of inducing tolerance. In one embodiment, the tolerogenic DC may secrete more inhibitory cytokines (such as IL-10 and TGFβ) than pro-inflammatory cytokines (such as IL-12, IL-23, or TNFα). Is possible. In one embodiment, DCs are defined as tolerogenic if they secrete IL-10 and IL-12 in a ratio of IL-10: IL-12> 1.

  In one embodiment, tolerogenic DCs express major histocompatibility (MHC) class Ia and / or MHC class Ib on their surface. Presentation of MHC class Ia refers to “classical” presentation through HLA-A, HLA-B and / or HLA-C molecules, whereas presentation of MHC class Ib includes HLA-E, HLA-F, HLA -Refers to "non-classical" antigen presentation through G and / or HLA-H molecules.

  In one embodiment, tolerogenic DCs express 50% MHC class Ia molecules and 50% MHC class Ib molecules on their surface. In one embodiment, tolerogenic DCs express 45% MHC class Ia molecules and 55% MHC class Ib molecules on their surface. In one embodiment, tolerogenic DCs express 40% MHC class Ia molecules and 60% MHC class Ib molecules on their surface. In one embodiment, tolerogenic DCs express 35% MHC class Ia molecules and 65% MHC class Ib molecules on their surface. In one embodiment, tolerogenic DCs express 30% MHC class Ia molecules and 70% MHC class Ib molecules on their surface. In one embodiment, tolerogenic DCs express 25% MHC class Ia molecules and 75% MHC class Ib molecules on their surface. In one embodiment, tolerogenic DCs express 20% MHC class Ia molecules and 80% MHC class Ib molecules on their surface. In one embodiment, tolerogenic DCs express 15% MHC class Ia molecules and 85% MHC class Ib molecules on their surface. In one embodiment, tolerogenic DCs express 10% MHC class Ia molecules and 90% MHC class Ib molecules on their surface. In one embodiment, tolerogenic DCs express 5% MHC class Ia molecules and 95% MHC class Ib molecules on their surface. In one embodiment, tolerogenic DCs express only MHC class Ib molecules on their surface.

  In one embodiment, tolerogenic DCs express 50% HLA-A, HLA-B, and / or HLA-C molecules and 50% HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 45% HLA-A, HLA-B, and / or HLA-C molecules and 55% HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 40% HLA-A, HLA-B, and / or HLA-C molecules and 60% HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 35% HLA-A, HLA-B, and / or HLA-C molecules and 65% HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 30% HLA-A, HLA-B, and / or HLA-C molecules and 70% HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 25% HLA-A, HLA-B, and / or HLA-C molecules and 75% HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 20% HLA-A, HLA-B, and / or HLA-C molecules and 80% HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 15% HLA-A, HLA-B, and / or HLA-C molecules and 85% HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 10% HLA-A, HLA-B, and / or HLA-C molecules and 90% HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express 5% HLA-A, HLA-B, and / or HLA-C molecules and 95% HLA-E molecules on their surface. In one embodiment, tolerogenic DCs express only HLA-E molecules on their surface.

Methods for obtaining tolerogenic DC are well known in the art. An exemplary method is the generation of immunotolerogenic DC from CD14 ⁺ monocytes. For example, CD14 ⁺ monocytes are cultured in the presence of GM-CSF and IL-4 or in the presence of GM-CSF and IFNα for the generation of immature DC.

  Methods for inhibiting MHC class Ia molecule expression on the surface of tolerogenic DCs or inducing expression of HLA-E molecules are well known.

  Inhibition of the TAP transporter (transporter associated with antigen processing) leads to decreased expression of MHC class Ia molecules, which promotes HLA-E molecule expression on the surface of tolerogenic DCs.

  Exemplary methods for inhibiting the TAP transporter in the endoplasmic reticulum include CRISPR-CAS-9 technology, silencing RNA, transfected DC using UL-10 viral protein from CMV (cytomegalovirus), or virus Including but not limited to the use of proteins.

  Examples of viral proteins that can inhibit the TAP transporter include, but are not limited to, HSV-11CP47 protein, varicella virus UL49.5 protein, cytomegalovirus US6 protein, or gammaherpesvirus EBV BNLF2a protein.

  Another method is the use of chemical products to inhibit the expression of MHC class Ia molecules without altering HLA-E expression on the surface of tolerogenic DCs. Examples of chemical products include but are not limited to 5'-methyl-5'-thioadenosine or leptomycin B.

  Tolerogenic DCs are pulsed for about 24 hours in the presence of at least one bisphosphonate, preferably aminobiphosphonate. Examples of biphosphonates include zoledronic acid (or zoledronic acid), pamidronic acid, alendronic acid, risedronic acid, ibandronic acid, incadronic acid, etidronic acid, tiludronic acid, combinations thereof, their salts, and their hydration Including, but not limited to. Preferably, the bisphosphonate is zoledronic acid or zoledronate.

  In one embodiment, bisphosphonates, in particular zoledronic acid, are used at a concentration of 10 nM to 50 μM. Within the scope of the present invention, the expression “10 nM to 50 μM” includes but is not limited to 50 nM, 100 nM, 250 nM, 500 nM, 750 nM, 1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM.

  In one embodiment, a cAMP activator added to the culture allows activation of the cAMP pathway. Examples of cAMP activators include, but are not limited to, PGE2 (prostaglandin E2), EP2 or EP4 agonists, membrane adenine cyclase activators (such as forskolin), or metabotropic glutamate receptor agonists. Examples of PGE2 include reference P5640 or P0409 PGE2 (Sigma-Aldrich), reference 2296 PGE2 (R & D Systems), reference 2268 PGE2 (BioVision), reference 72192 PGE2 (Stemcell), reference ab144539 PGE2 (Abcam), And reference 14010 PGE2 (Cayman Chemical), but is not limited thereto.

  In one embodiment, a cAMP activator, preferably PGE2, is used at a concentration ranging from 0.01 μM to 10 μM. Within the scope of the present invention, the expression “0.01 μM to 10 μM” means 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0 1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3 Including, but not limited to, 5 μM, 4 μM, 4.5 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM. In certain embodiments, PGE2 is at a concentration in the range of 0.03 μM to 1.5 μM.

  In one embodiment, the TGFβ pathway activator added in the culture allows activation of the TGFβ pathway. Examples of TGFβ pathway activators include the TGFβ family (TGFβ1, TGFβ2, TGFβ3), bone morphogenetic protein (BMP), growth and differentiation factor (GDF), anti-Muellerian hormone (AMH), activin, and nodal, It is not limited to these. Examples of TGFβ include TGFβ1 (Sigma-Aldrich) of reference T7039, TGFβ2 of reference T2815 (Sigma-Aldrich), TGFβ3 of reference T5425 (Sigma-Aldrich), human TGFβ1 of reference P01137 (R & D systems), human TGFβ1 of reference 580702 (Biolegend), including, but not limited to, TGFβ1 (HumanZyme) of reference HZ-1011, TGFβ1 (Affymetrix eBioscience) of reference 14-8348-62.

  In one embodiment, pathway activators are used at concentrations ranging from 1 ng / ml to 20 ng / ml. Within the scope of the present invention, the expression “1 ng / ml to 20 ng / ml” means 2 ng / ml, 2.5 ng / ml, 3 ng / ml, 3.5 ng / ml, 4 ng / ml, 4.5 ng / ml, 5 ng / ml, 5.5 ng / ml, 6 ng / ml, 6.5 ng / ml, 7 ng / ml, 7.5 ng / ml, 8 ng / ml, 8.5 ng / ml, 9 ng / ml, 9.5 ng / ml, 10 ng including, but not limited to, / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml. In certain embodiments, TGFβ is at a concentration in the range of 2.5 ng / ml to 7.5 ng / ml.

  In one embodiment, an mTOR inhibitor added to the culture allows inhibition of the mTOR pathway. Examples of mTOR inhibitors are rapamycin (also called sirolimus) and its analogs (also called rapalog); wortmannin; theophylline; caffeine; epigallocatechin gallate (EGCG); curcumin; resveratrol; 3-diindolylmethane (DIM); LY294002 (2- (4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one); PP242; PP30; Torin1; Ku-0063794; WAY-600; 687; WYE-354; and mTOR and PI3K bispecific inhibitors (such as, but not limited to, GNE477, NVP-BEZ235, PI-103, XL765, and WJD008). Examples of rapamycin include rapamycin (Sigma-Aldrich) with reference R0395, Rapamycin (Selleckchem) with reference S1039, rapamycin (Tocris) with reference 1292, rapamycin with reference R-5000 (LC Laboratories), rapamycin with reference Tlrl-rap (InvivoGen) ), Reference ab120224 rapamycin (Abcam), reference R0395 rapamycin (Sigma-Aldrich).

  Examples of compounds of the same chemical class as rapamycin used clinically include everolimus (code name RAD001), temsirolimus (code name CCI-779, NSC 683864), zotarolimus (code name ABT-578). Not limited.

  In one embodiment, an mTOR inhibitor, preferably rapamycin, is used at a concentration ranging from 0.1 nM to 50 nM. Within the scope of the present invention, the expression “0.1 nM to 50 nM” means 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM. 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM 27 nM, 28 nM, 29 nM, 30 nM, 31 nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41 nM, 42 nM, 43 nM, 44 nM, 45 nM, 46 nM, 47 nM, 48 nM, 49 nM It is not limited to these.

  In one embodiment, at least one cytokine selected from IL-2, IL-7, IL-15, and TSLP can be added to the culture.

  In one embodiment, IL-2 is used at a concentration ranging from 10 IU / ml to 1000 IU / ml. Within the scope of the present invention, the expression “10 IU / ml to 1000 IU / ml” means 15 IU / ml, 20 IU / ml, 25 IU / ml, 30 IU / ml, 35 IU / ml, 40 IU / ml, 45 IU / ml, 50 IU / ml. 55 IU / ml, 60 IU / ml, 65 IU / ml, 70 IU / ml, 75 IU / ml, 80 IU / ml, 85 IU / ml, 90 IU / ml, 95 IU / ml, 100 IU / ml, 150 IU / ml, 200 IU / ml, 250 IU / ml, 300 IU / ml, 350 IU / ml, 400 IU / ml, 450 IU / ml, 500 IU / ml, 550 IU / ml, 600 IU / ml, 650 IU / ml, 700 IU / ml, 750 IU / ml, 800 IU / ml, 850 IU / ml , 900 IU / ml, 950 IU / ml, but not limited thereto. In certain embodiments, IL-2 is used at concentrations ranging from 50 IU / ml to 250 IU / ml.

  In one embodiment, IL-7 is used at a concentration ranging from 1 ng / ml to 100 ng / ml. Within the scope of the present invention, the expression “1 ng / ml to 100 ng / ml” means 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml. 40 ng / ml, 45 ng / ml, 50 ng / ml, 55 ng / ml, 60 ng / ml, 65 ng / ml, 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, 90 ng / ml, 95 ng / ml, 100 ng Including / ml, but not limited to.

  In one embodiment, IL-15 is used at a concentration ranging from 1 ng / ml to 50 ng / ml. Within the scope of the present invention, the expression “1 ng / ml to 50 ng / ml” means 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml. Including, but not limited to, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 45 ng / ml. In certain embodiments, IL-15 is used at concentrations ranging from 10 ng / ml to 30 ng / ml.

  In one embodiment, TSLP is used at a concentration ranging from 1 ng / ml to 100 ng / ml. Within the scope of the present invention, the expression “1 ng / ml to 100 ng / ml” means 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml. 40 ng / ml, 45 ng / ml, 50 ng / ml, 55 ng / ml, 60 ng / ml, 65 ng / ml, 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, 90 ng / ml, 95 ng / ml, 100 ng Including / ml, but not limited to.

  Examples of TET activators include, but are not limited to, vitamin C and NaHS hydrogen sulfide release agents (also known as H2S donors).

  In one embodiment, vitamin C is used at a concentration ranging from 1 to 100 μg / ml.

  In one embodiment, NaHS hydrogen sulfide releasing agent is used at a concentration in the range of 0.25 to 8 mM.

  Examples of DNMT inhibitors are 2- (1,3-dioxo-1,2-dihydro-2H-isoindol-2-yl) -3- (1H-indol-3-yl) propanoic acid (RG108), 5 -Including but not limited to aza-2'-deoxycytidine (decitabine or DAC) and 5-azacytidine (5AC).

  In one embodiment, RG108 is used at a concentration in the range of 20 to 500 μM.

  In one embodiment, the DAC is used at a concentration in the range of 0.1 to 2 μM.

  In one embodiment, 5AC is used at a concentration in the range of 0.1 to 10 μM.

  In one embodiment, neutralizing antibodies can be added to the culture to prevent the generation of other populations of regulatory T cells.

  Examples of neutralizing antibodies include, but are not limited to, anti-IFNγ antibodies, anti-IL-4 antibodies, and / or anti-IL12 antibodies.

  Examples of anti-IFNγ antibodies include, but are not limited to, Affymetrix eBioscience (reference 14-7318), R & D systems (reference MAB285), Novus Biologicals (reference AF-485-NA).

  Examples of anti-IL-4 antibodies include, but are not limited to, R & D Systems (reference MAB304, MAB204, or MAB204), Affymetrix eBioscience (reference 14-7048), GeneTex (reference GTX10755).

  Examples of anti-IL-12 antibodies include, but are not limited to, Affymetrix eBioscience (reference 16-7129 or 16-8126), Biolegend (reference 508803), R & D Systems (reference MAB219, AF-219, or AB-219). do not do.

  In one embodiment, the culture medium used in the cultures of the invention comprises (i) one or more pH buffer systems; (ii) inorganic salts; (iii) trace elements; (iv) free amino acids; Vitamins; (vi) hormones; (vii) carbon / energy sources.

  Examples of inorganic salts are calcium bromide, calcium chloride, calcium phosphate, calcium nitrate, calcium nitrite, calcium sulfate, magnesium bromide, magnesium chloride, magnesium sulfate, potassium bicarbonate, potassium bromide, potassium chloride, phosphorus dihydride Potassium acid, potassium disulfate, dipotassium hydrogen phosphate, potassium nitrate, potassium nitrite, potassium sulfite, potassium sulfate, sodium hydrogen carbonate, sodium bromide, sodium chloride, sodium disulfate, sodium hydrogen carbonate, sodium dihydrogen phosphate, phosphorus Including but not limited to disodium oxyhydrogen, sodium sulfate and mixtures thereof.

  Examples of trace elements are cobalt (Co), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), molybdenum (Mo), nickel (Ni), selenium (Se), zinc (Zn) And salts thereof, but are not limited thereto.

  Examples of free amino acids are L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L Including leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, taurine, L-threonine, L-tryptophan, L-tyrosine, L-valine and mixtures thereof. Not limited.

  Examples of vitamins are biotin (vitamin H); calcium D-pantothenate; choline chloride; folic acid (vitamin B9); myo-inositol; nicotinamide; pyridoxal (vitamin B6); riboflavin (vitamin B2); thiamine (vitamin B1); Including but not limited to cobalamin (vitamin B12); ascorbic acid; α-tocopherol (vitamin E) and mixtures thereof.

  Examples of carbon / energy sources include, but are not limited to, D-glucose; pyruvic acid; lactic acid; ATP; creatine; creatine phosphate and mixtures thereof.

  In one embodiment, the culture medium is a commercially available cell culture medium, particularly selected in the group comprising RPMI 1640 medium from IMDM (Iscove's Modified Dulbecco's Medium) from GIBCO® or GIBCO®. Is done.

  In other embodiments, the culture medium is a serum-free culture medium, such as AIM-V medium from GIBCO®, X-VIVO 10, 15, and 20 medium from LONZA.

  In another embodiment, the culture medium comprises an additional compound selected in the group comprising in particular fetal calf serum, pooled human AB serum, cytokines, and growth factors; in particular the group comprising penicillin, streptomycin, and mixtures thereof Further antibiotics selected in can be added.

  In one embodiment, the culture medium is IMDM.

  In some specific embodiments, the culture medium is IMDM cell culture medium; 1% (w / w) to 5% (w / w) fetal calf serum; 10 IU / ml to 200 IU / ml penicillin; 10 IU / ml. From 100 mM to 200 IU / ml streptomycin; 0.1 mM to 10 mM mixture of amino acids selected in the group comprising non-essential amino acids, in particular alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine; Contains 0.5 mM to 10 mM glutamine, 10 mM to 25 mM HEPES (pH 7.6-7.8).

  In one embodiment, the medium is nTreg polarized medium. We have identified “nTreg Polarized Medium” as at least one cAMP activator as described hereinabove, at least one TGFβ pathway activator as described hereinabove, and Defined as a medium such as RPMI medium containing at least one mTor inhibitor as described above. In a preferred embodiment, “nTreg polarization medium” refers to RPMI medium containing TGFβ, rapamycin, and PGE2.

  In other embodiments, the medium is an inflammatory medium. We have defined “inflammatory media” as inflammatory cytokines such as IL-1β (10 ng / ml), IL-6 (30 ng / ml), IL-21 (50 ng / ml), IL-23 (30 ng / ml), IL-2 (100 UI / ml) and the like (for example, IMDM).

In one embodiment, the culture to generate γδ Foxp3 ⁺ regulatory T cells of the invention is performed for at least 5 days, at least 6 days, at least 7 days, at least 8 days. Within the scope of the present invention, the expression “at least 5 days” includes 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days. However, it is not limited to these.

In one embodiment, a portion of the culture medium is discarded once, twice, three times, four times, or five times during the time course of the production medium and replaced with the same volume of fresh culture medium. Within the scope of the present invention, the term “portion” means at least 20% (v / v), at least 25% (v / v), at least 30% (v / v), at least 35% of the volume of the culture medium ( v / v), at least 40% (v / v), at least 45% (v / v), at least 50% (v / v), at least 55% (v / v), at least 60% (v / v) at least It is intended to mean 65% (v / v), at least 70% (v / v), at least 75% (v / v). In certain embodiments, 40% (v / v) to 60% (v / v) of the volume of culture medium from step a) is discarded. In certain embodiments, the discarded volume is replaced with the same volume of fresh culture medium. Within the scope of the present invention, the expression “fresh culture medium” refers to a culture medium that is not in contact with any CD3 ⁺ T cells.

In one embodiment, a method for generating γδFoxp3 ⁺ regulatory T cells ex vivo comprises:
-CD3 ⁺ TCRγδ ⁺ T cells, preferably CD3 ⁺ TCRγδ ⁺ CD45RA ⁺ T cells, in the presence of autologous ΔCD3 feeder cells and coated anti-TCRγδ antibodies, and the following agents: i) PGE2, ii) TGFβ, iii) Culturing for at least 5 days in the presence of rapamycin, optionally iv) at least one cytokine selected in the group of IL-2 and IL-15, and optionally v) vitamin C;
Thereby obtaining an ex vivo generated population of γδ Foxp3 ⁺ regulatory T cells, preferably from γδ naive (CD45RA ⁺ ) T cells.

In one embodiment, a method for generating γδFoxp3 ⁺ regulatory T cells ex vivo comprises:
In the presence of tolerogenic DC pulsed with zoledronate for about 24 hours and in the presence of ΔCD3 feeder cells and the following agents: i) PGE2, ii) TGFβ, iii) rapamycin, optionally iv) IL-2 and At least one cytokine selected in the group of IL-15, and optionally v) culturing CD3 ⁺ TCRγδ ⁺ T cells, preferably CD3 ⁺ TCRγδ ⁺ CD45RA ⁺ T cells in the presence of vitamin C for at least 5 days;
-Thereby obtaining an ex vivo generated population of γδ Foxp3 ⁺ regulatory T cells, preferably from γδ naive (CD45RA ⁺ ) T cells.

The present invention also relates to an ex vivo method of generating and expanding γδ Foxp3 ⁺ regulatory T cells, including:
-Generating γδ Foxp3 ⁺ regulatory T cells as described herein above,
The presence of γδ T cell activators (preferably autologous ΔCD3 feeder cells and tolerogenic DC pulsed with coated anti-TCRγδ antibody or zoledronate for about 24 hours, and ΔCD3 feeder cells. And) the following agents: i) cAMP (cyclic adenosine monophosphate) activator (preferably PGE2), ii) TGFβ (transforming growth factor beta) pathway activator (preferably TGFβ), iii) mTOR inhibition An agent (preferably rapamycin), optionally iv) at least one cytokine selected in the group of IL-2, IL-7, IL-15, and TSLP (preferably IL-2 and / or IL-15), and Optionally v) at least one TET enzyme activator (preferably vitamins C and N) HS is selected from hydrogen sulfide release agent) and / or at least one DNMT inhibitors (e.g. RG108, DAC, or GanmaderutaFoxp3 ⁺ regulatory T cells generated by contacting in the presence of such) 5AC, at least 5 days To proliferate.
Thereby obtaining a proliferating population of γδ Foxp3 ⁺ regulatory T cells.

In one embodiment, an ex vivo generated γδ Foxp3 ⁺ regulatory T cell population is isolated by flow cytometry based on the following phenotype: CD3 ⁺ TCR γδ ⁺ CD45RO ⁺ Foxp3 ⁺ .

In one embodiment, the isolated γδ Foxp3 ⁺ regulatory T cell population thus obtained is expanded ex vivo by culturing these cells in the presence of a polyclonal γδ T cell activator. Examples of polyclonal γδ T cell activators are listed herein above. Alternatively, other examples of polyclonal T cell activators that can be used during proliferation include, but are not limited to, mitogens (eg PMA / ionomycin etc.), superantigens, anti-CD3 antibodies. Preferably, an anti-CD3 monoclonal antibody is coated. In one embodiment, a polyclonal γδ T cell activator can be used in the presence of feeder cells as described herein above.

In another embodiment, the isolated γδ Foxp3 ⁺ regulatory T cell population thus obtained is then transferred to these cells in the presence of an antigen-specific γδ T cell activator as described herein above. Is grown ex vivo by culturing. In one embodiment, an antigen-specific γδ T cell activator can be used in the presence of feeder cells as described herein above.

In one embodiment, culturing to expand γδ Foxp3 ⁺ regulatory T cells generated ex vivo of the invention is performed for at least 5 days, at least 6 days, at least 7 days, at least 8 days. Within the scope of the present invention, the expression “at least 5 days” means 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days. 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th or more, including these It is not limited to.

In one embodiment, a portion of the culture medium is discarded once, twice, three times, four times, or five times during the time course of the production medium and replaced with the same volume of fresh culture medium. Within the scope of the present invention, the term “portion” means at least 20% (v / v), at least 25% (v / v), at least 30% (v / v), at least 35% of the volume of the culture medium ( v / v), at least 40% (v / v), at least 45% (v / v), at least 50% (v / v), at least 55% (v / v), at least 60% (v / v) at least It is intended to mean 65% (v / v), at least 70% (v / v), at least 75% (v / v). In certain embodiments, 40% (v / v) to 60% (v / v) of the volume of culture medium from step a) is discarded. In certain embodiments, the discarded volume is replaced with the same volume of fresh culture medium. Within the scope of the present invention, the expression “fresh culture medium” refers to a culture medium that is not in contact with any CD3 ⁺ T cells.

In one embodiment, γδ Foxp3 ⁺ regulatory T cells are autologous ΔCD3 feeders in the presence of PGE2 (1 μM), TGFβ (5 ng / ml), rapamycin (10 nM), and IL-2 (100 UI / ml) in IMDM-5. cells (125 ¹⁰⁵ cells / ml) and coated anti TCRganmaderuta in the presence of the antibody (2 [mu] g / ml), were obtained by flow cytometry from PBMC ^{CD3 +} TCRγδ + CD45RA + T cells (5.10 ¹⁰³ cells / ml ) Is cultured ex vivo. On day 1, IL-2 (100 UI / ml) and IL-15 (10 ng / ml) are added to the culture. Every 3 days, half of the medium volume is discarded and fresh medium containing PGE2 (50 nM), TGFβ (5 ng / ml), rapamycin (1 nM), IL-2 (100 μl / ml), and IL-15 (10 ng / ml). Replace with When cells begin to grow, they are split every 2-3 days and every 9 days in medium containing PGE2 (1 μM), TGFβ (5 ng / ml), rapamycin (10 nM), and IL-2 (100 UI / ml). Can be cultured in the presence of ΔCD3 feeder cells and coated anti-TCRγδ antibody.

In another embodiment, γδ Foxp3 ⁺ regulatory T cells are in the presence of tolerogenic DC pulsed with zoledronate for about 24 hours, as well as ΔCD3 feeder cells (125 10 ⁵ cells / ml) in IMDM-5, PGE2 (1μM), TGFβ (5ng / ml), in the presence of rapamycin (10 nM), and IL-2 (100UI / ml) , obtained from PBMC by flow cytometry (5.10 ¹⁰³ cells / ml) CD3 ⁺ generating ex vivo by culturing TCRγδ ⁺ CD45RA ⁺ T cells (5.10 ¹⁰³ cells / ml). On day 1, IL-2 (100 UI / ml), IL-15 (10 ng / ml), and TGFβ (5 ng / ml) are added to the culture. Every 3 days, half of the medium volume is discarded and fresh medium containing PGE2 (50 nM), TGFβ (5 ng / ml), rapamycin (1 nM), IL-2 (100 UI / ml), and IL-15 (10 ng / ml) Replace with When cells began to grow, they were split every 2 or 3 days in the presence of ΔCD3 feeder cells and PGE2 (1 μM), TGFβ (5 ng / ml), rapamycin (10 nM), and IL-2 (100 UI / ml). Can be restimulated every 9 days with tolerogenic DCs.

In this embodiment, tolerogenic DCs were obtained by culturing CD14 ⁺ monocytes isolated from PBMC in the presence of AIMV supplemented with GMCSF (100 ng / ml) and IL-4 (10 ng / ml). . On days 3 and 6, the medium is discarded and replaced with fresh medium containing GM-CSF and IL-4. On day 6, tolerogenic DCs are pulsed for 24 hours in the presence of zoledronate (100 nM).

The present invention also relates to γδ Foxp3 ⁺ regulatory T cells obtainable by the ex vivo generation method described herein above.

The invention also relates to γδ Foxp3 ⁺ regulatory T cells obtainable by the ex vivo production and expansion methods described herein above.

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells obtained by the production and expansion methods of the present invention comprises at least 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ cells.

In one embodiment, the population of GanmaderutaFoxp3 ⁺ regulatory T cells obtained by the method for generating and proliferation of the present invention the following ^phenotypes: with a CD3 ⁺ TCRγδ + Foxp3 +.

In one embodiment, the γδFoxp3 ⁺ regulatory T cells obtainable or obtained by the method of the invention express the Vδ2 isotype. In one embodiment, the γδ Foxp3 ⁺ regulatory T cells of the invention do not express the Vδ2 isotype.

In another embodiment, the γδ Foxp3 ⁺ regulatory T cells obtainable or obtained by the methods of the present invention express the Vγ9 isotype. In one embodiment, the γδ Foxp3 ⁺ regulatory T cells of the invention do not express the Vγ9 isotype.

In one embodiment, the γδFoxp3 ⁺ regulatory T cells obtainable or obtained by the method of the present invention express the Vγ9Vδ2 isotype. In one embodiment, the γδFoxp3 ⁺ regulatory T cells of the invention do not express the Vγ9Vδ2 isotype.

In one embodiment, the γδFoxp3 ⁺ regulatory T cells of the invention express the Vδ3 isotype. In one embodiment, the γδ Foxp3 ⁺ regulatory T cells of the invention express the Vδ4 isotype. In one embodiment, the γδ Foxp3 ⁺ regulatory T cells of the invention express the Vδ5 isotype. In one embodiment, the γδ Foxp3 ⁺ regulatory T cells of the invention express the Vδ6 isotype.

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD45RO ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD127 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-1R1 ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-6R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-23R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-33R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CD45RO ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CD127 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ CD45RO ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ CD127 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD45RO ⁺ CD127 ⁺ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-1R1 ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-6R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-23R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-33R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD45RO ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD127 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CD45RO ⁺ CD127 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-1R1 ⁻ CTLA4 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-6R ⁻ CTLA4 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-23R ⁻ CTLA4 ⁺ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-33R ⁻ CTLA4 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-1R1 ⁻ CD127 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-6R ⁻ CD127 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-23R ⁻ CD127 ⁺ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-33R ⁻ CD127 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-1R1 ⁻ CD45RO ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-6R ⁻ CD45RO ⁺ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-23R ⁻ CD45RO ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-33R ⁻ CD45RO ⁺ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-1R1 ⁻ IL-6R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-1R1 ⁻ IL-23R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-1R1 ⁻ IL-33R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-6R ⁻ IL-23R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-6R ⁻ IL-33R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ IL-23R ⁻ IL-33R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-1R1 ⁻ CTLA4 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-6R ⁻ CTLA4 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-23R ⁻ CTLA4 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-33R ⁻ CTLA4 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-1R1 ⁻ CD127 ⁺ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-6R ⁻ CD127 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-23R ⁻ CD127 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-33R ⁻ CD127 ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-1R1 ⁻ CD45RO ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-6R ⁻ CD45RO ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-23R ⁻ CD45RO ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-33R ⁻ CD45RO ⁺ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-1R1 ^- IL-6R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-1R1 ⁻ IL-23R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-1R1 ^- IL-33R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-6R ⁻ IL-23R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-6R ⁻ IL-33R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ IL-23R ⁻ IL-33R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-1R1 ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-6R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-23R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-33R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-1R1 ⁻ IL-6R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-1R1 ⁻ IL-23R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-1R1 ^- IL-33R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-6R ⁻ IL-23R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-6R ⁻ IL-33R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-23R ⁻ IL-33R ⁻ .

In one embodiment, said population of GanmaderutaFoxp3 ⁺ regulatory T cells have the following ^{phenotypes: CD4 + Foxp3 + TCRγδ + CTLA4} + CD45RO + CD127 + IL-6R - IL-23R - IL-33R -.

In one embodiment, said population of GanmaderutaFoxp3 ⁺ regulatory T cells have the following ^{phenotypes: CD4 + Foxp3 + TCRγδ + CTLA4} + CD45RO + CD127 + IL-1R1 - IL-23R - IL-33R -.

In one embodiment, said population of GanmaderutaFoxp3 ⁺ regulatory T cells have the following ^{phenotypes: CD4 + Foxp3 + TCRγδ + CTLA4} + CD45RO + CD127 + IL-1R1 - IL-6R - IL-33R -.

In one embodiment, said population of GanmaderutaFoxp3 ⁺ regulatory T cells have the following ^{phenotypes: CD4 + Foxp3 + TCRγδ + CTLA4} + CD45RO + CD127 + IL-1R1 - IL-6R - IL-23R -.

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-1R1 ^- IL-6R ^- IL-23R ^- IL-33R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-1R1 ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-6R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-23R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-33R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-1R1 ⁻ IL-6R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-1R1 ⁻ IL-23R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-1R1 ⁻ IL-33R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-6R ⁻ IL-23R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-6R ⁻ IL-33R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD125RO ⁺ CD127 ⁺ IL-23R ⁻ IL-33R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD125RO ⁺ CD127 ⁺ IL-6R ⁻ IL-23R ⁻ IL-33R ⁻ .

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD125RO ⁺ CD127 ⁺ IL-1R1 ⁻ IL-23R ⁻ IL-33R ⁻ .

In one embodiment, said population of GanmaderutaFoxp3 ⁺ regulatory T cells have the following ^{phenotypes: CD4 + Foxp3 + TCRγδ + CD25} + CTLA4 + CD45RO + CD127 + IL-1R1 - IL-6R - IL-33R -.

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD45RO ⁺ CD127 ⁺ IL-1R1 ⁻ IL-6R ⁻ IL-23R ⁻ .

In one embodiment, said population of γδ Foxp3 ⁺ regulatory T cells has the following phenotype: CD4 ⁺ Foxp3 ⁺ TCR γδ ⁺ CD25 ⁺ CTLA4 ⁺ CD125R ⁺ CD127 ⁺ IL-1R1 ⁻ IL-6R ⁻ IL-23R ⁻ IL -33R ^-.

In one embodiment, the γδ Foxp3 ⁺ regulatory T cells of the present invention do not present a regulatory T cell specific demethylation region (TSDR) of the gene Foxp3. In another embodiment, the γδ Foxp3 ⁺ regulatory T cells of the present invention present a regulatory T cell specific demethylation region (TSDR) of the gene Foxp3. In one embodiment, γδ Foxp3 ⁺ regulatory T cells present a percentage of TSDR demethylation of gene FOXP3 that is at least 30%, 40%, greater than 50%. A protocol for measuring the percentage of promoter demethylation is given in the materials and methods part of the examples.

In another embodiment, γδ Foxp3 ⁺ regulatory T cells present an enriched percentage of acetylated histone in the Foxp3 promoter region that is at least greater than 10%, 20%, 30%, 40%, or 50%. A protocol for measuring the concentration of acetylated histones in percentage is given in the materials and methods part of the examples.

An example of the phenotypic characteristics of a population of γδ Foxp3 ⁺ regulatory T cells of the present invention is shown in FIG.

In one embodiment, the population of γδ Foxp3 ⁺ regulatory T cells express Foxp3 with a median fluorescence intensity (MFI) at least equivalent to Foxp3 MFI measured in naive regulatory T cells. As used herein, “naïve regulatory T cells” refers to T cells having the phenotype Foxp3 ⁺ CD45RA ⁺ CD4 ⁺ CD25 ⁺ CD127 ⁻ .

In one embodiment, γδ Foxp3 ⁺ regulatory T cells express Foxp3 with a median fluorescence intensity (MFI) of at least 2000.

In one embodiment, γδ Foxp3 ⁺ regulatory T cells express Foxp3 with a median fluorescence intensity (MFI) of at least 2 or 3 times the Foxp3 MFI measured in naive regulatory T cells.

  In one embodiment, γδ regulatory T cells express Foxp3 with a median fluorescence intensity (MFI) of at least 2000, 3000, 4000, 5000, 10000, 20000, 30000, 40000, 50000, 60000, 70000.

In one embodiment, the γδ Foxp3 ⁺ regulatory T cell population comprises CD3 ⁺ CD4 ⁺ cells that express at least 65% Foxp3. The expression “at least 65%” means 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 752%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 82%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, and 100%.

  As used herein, the term “expression” can alternatively refer to transcription of a molecule (ie, expression of mRNA) or translation of a molecule (ie, expression of a protein). In one embodiment, detecting expression may correspond to intracellular detection. In another embodiment, detecting expression may correspond to surface detection, ie, detection of molecules expressed on the cell surface. In another embodiment, detecting expression may correspond to extracellular detection, ie, detection of secretion. In another embodiment, detecting expression may correspond to intracellular detection, surface detection, and / or extracellular detection. Methods for determining expression levels are well known to those of skill in the art, and include cellular transcriptomes (in embodiments where expression is related to transcription of the molecule) or proteome (expression is related to translation of the cytotoxic molecule). But not limited to).

  In one embodiment of the invention, the expression of the molecule is assessed at the mRNA level. Methods for assessing the transcription level of a molecule are well known in the prior art. Examples of such methods are RT-PCR, RT-qPCR, Northern blots, hybridization techniques (such as using microarrays), and combinations thereof (hybridization, sequencing of amplicons obtained by RT-PCR). Examples include, but are not limited to, next generation DNA sequencing (NGS) or RNA-seq (also known as “all transcriptome shotgun sequencing”). In another embodiment of the invention, the expression of the molecule is assessed at the protein level. Methods for determining protein levels in a sample are well known in the art. Examples of such methods are immunohistochemistry, multiplex method (Luminex), Western blot, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, fluorescence-linked immunosorbent assay (FLISA), enzyme immunoassay ( EIA), radioimmunoassay (RIA), flow cytometry (FACS) and the like.

  In another embodiment, determining the expression level of at least one molecule corresponds to detecting and / or quantifying the binding of the ligand to the molecule. In one embodiment, the ligand is an antibody specific for the molecule, and the method of the invention comprises detecting and / or quantifying a complex formed between the antibody and the molecule. Complexes can be detected when the ligand is covalently bound, for example, to a detectable molecule, such as an antibody constant fragment (Fc) or a fluorescent compound (eg, a cyanine dye, Alexa dye, Quantum dye, etc.). . Complexes can also be detected when the ligand is tagged by different means well known to those skilled in the art. For example, tags used with the present invention include hemagglutinin tag, polyarginine tag, polyhistidine tag, Myc tag, Strep tag, S tag, HAT tag, 3 × flag tag, calmodulin-binding peptide tag, SBP tag, chitin binding domain tag , GST tag, maltose binding protein tag, fluorescent protein tag, T7 tag, V5 tag, and Xpress tag, or a tag selected from the group consisting of them. The use of ligands thus allows on the one hand the identification and detection of molecules depending on the ligand used and on the other hand the quantification of the complexes formed.

  In one embodiment, determining the expression level of the molecule is performed by flow cytometry, immunofluorescence, or image analysis, such as high content analysis. Preferably, the expression level of the molecule is determined by flow cytometry. In one embodiment, cells are fixed and permeabilized prior to performing flow cytometric analysis, thereby allowing detection of intracellular proteins.

  In one embodiment, determining the expression level of a molecule in a cell population includes determining the percentage of cells in the cell population that express the molecule (ie, cell “+” for the molecule). Preferably, the percentage of cells expressing the molecule is measured by FACS.

  The terms “expressed (or +)” and “not expressed (or −)” are well known in the art and refer to the expression level of a cell marker of interest, corresponding to “+” Cell marker expression levels are high or moderate, also referred to as “+/−”. A cell marker corresponding to “-” refers to the null expression level of the cell marker or less than 10% of the cell population expressing the cell marker.

  The expression level of the cell marker of interest is the median fluorescence intensity (MFI) of cells from a cell population stained with a fluorescently labeled antibody specific for this marker, but with the irrelevant specificity but the same isotype, It is determined by comparing the fluorescence intensity (FI) of cells from the same cell population with the same fluorescent probe and stained with a fluorescently labeled antibody from the same species (referred to as isotype control). Cells from a population that is stained with a fluorescent-labeled antibody specific for this marker and that shows an equivalent or lower MFI than cells stained with an isotype control do not express this marker and thus Specify (-) or negative. Cells from a population that are specific for this marker and show MFI values that exceed those stained with the isotype control are expressing this marker and are therefore designated (+) or positive.

In one embodiment, the γδ Foxp3 ⁺ regulatory T cells are capable of inhibitory activity similar to that of naive CD4 ⁺ CD25 ⁺ CD45RA ⁺ CD127 ⁻ regulatory T cells. Determination of the suppressive activity of a cell population is well known in the art and can be performed by conventional assays such as standard polyclonal cell-cell contact Treg suppression assays or autologous MLR suppression assays as described in the Examples. it can.

Another object of the present invention is a population of γδ Foxp3 ⁺ regulatory T cells that remain functionally stable when placed in an inflammatory condition.

In one embodiment, a population of γδ Foxp3 ⁺ regulatory T cells obtainable or obtained by the ex vivo generation and expansion methods of the present invention remains functionally stable when placed in an inflammatory condition. Has characteristics.

  As used herein, “functionally stable” is non-secreted or hyposecreted of IL-17, ie, less than 200 ng / ml, 100 ng / ml, 50 ng / ml, and still has the ability to suppress, ie, perform. It refers to being able to inhibit the proliferation of conventional T cells as shown in the examples.

  As used herein, an “inflammatory condition” is an inflammatory cytokine such as IL-1β (10 ng / ml), IL-6 (30 ng / ml), IL-21 (50 ng / ml), IL-23. (30 ng / ml), IL-2 (100 UI / ml) and the like, a medium rich in aromatic acids, preferably tryptophan, such as IMDM. A method for determining whether a population of regulatory T cells remains functionally stable in an inflammatory condition is preferably coated anti-CD3 (4 μg / ml), preferably soluble anti-CD28. In the presence of (4 μg / ml), culturing regulatory T cells in inflammatory medium as described herein above. After incubation for 36-72 hours, the presence of IL-17 in the culture supernatant is measured. Recognition of IL-17 in the culture supernatant can be performed by a conventional method known in the art, for example, a sandwich ELISA anti-IL-17. Briefly, after the plate is coated with capture anti-IL-17 antibody, the culture supernatant is added to each well with a dilution series. After incubation, detection anti-IL-17 antibody is added to each well. The ELISA may be performed by any colorimetric means known in the art, such as using a detection antibody labeled with biotin, a polystreptavidin HRP amplification system, and o-phenylenediamine dihydrochloride base solution. Develop. IL-17 levels inferior to 200 ng / ml, 100 ng / ml, 50 ng / ml correspond to no secretion or low secretion of IL-17.

Without wishing to be bound by theory, the present inventors found that the stroma of malignant tumor cells is highly abundant in regulatory T cells, particularly immunosuppressive activity against NK cells (immunity of local cancers). It includes TIL (tumor infiltrating lymphocytes) that exert a high probability of explaining escape. Inactivated γδ Foxp3 ⁺ regulatory T cells represent an antigenic target that induces an immune response against γδ Foxp3 ⁺ regulatory T cells present in TIL, thereby preventing their immunosuppressive activity and against tumor cells Enables cytotoxic activity of effector cells (eg, NK cells). The inventors thus suggest the use of a vaccine composition comprising inactivated γδFoxp3 ⁺ regulatory T cells as an active ingredient.

One object of the present invention is an immunogenic product comprising, consisting essentially of or consisting of inactivated γδ Foxp3 ⁺ regulatory T cells, as described herein above.

In one embodiment, the immunogenic product comprises essentially inactivated γδFoxp3 ⁺ regulatory T cells having the following phenotype CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ as described hereinabove ^: Or consist of.

As used herein, the term “consisting essentially of” refers to an immunogenic product, pharmaceutical composition, vaccine, or medicament wherein at least one γδFoxp3 ⁺ regulatory T cell population or antibody of the invention is Means the only therapeutic agent or drug with biological activity within said immunogenic product, pharmaceutical composition, vaccine or medicament.

In one embodiment, an immunogenic product is generated as described herein above and optionally ex vivo propagated, inactivated γδFoxp3 ⁺ regulatory with the following phenotype CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ Contain, consist essentially of, or consist of T cells.

One object of the present invention is an immunogenic product comprising blebs from γδ Foxp3 ⁺ regulatory T cells as described herein above.

One object of the present invention is an immunogenic product comprising immunogenic dendritic cells (immunogenic DC) loaded with blebs from γδ Foxp3 ⁺ regulatory T cells as described hereinabove. .

As used herein, “immunogenic DC” refers to a DC capable of inducing an immunogenic response. In one embodiment, the immunogenic DC has the following phenotype: CD80 ^high CD83 ^high CD86 ^high HLA class I ^high and HLA class II ^high , with IL-10 at a ratio of IL-12 / IL-10> 1 Secretes IL-12.

Methods for obtaining immunogenic DC are well known in the art. An exemplary method is the generation of immunogenic DC from CD14 ⁺ monocytes. For example, CD14 ⁺ monocytes are cultured in the presence of GM-CSF (about 20 ng / mL) and IFNα (about 10 ng / mL) to obtain MoDC. MoDC maturation can be induced by a cytokine cocktail consisting of IL-1β (about 10 ng / ml), IL-6 (about 10 ng / ml) TNFα (about 200 U / ml), and PGE2 (about 10 ng / ml).

  Another object of the present invention is a pharmaceutical composition comprising, consisting essentially of, or consisting of an immunogenic product and at least one pharmaceutically acceptable excipient as described herein above. It is.

Another object of the invention consists essentially of, comprising inactivated γδ Foxp3 ⁺ regulatory T cells with the following phenotype CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ and at least one pharmaceutically acceptable excipient, Or a pharmaceutical composition comprising the same.

Another object of the present invention is to provide inactivated γδ Foxp3 ⁺ regulatory T cells and at least one of the following phenotypes CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ generated and expanded ex vivo by the methods described herein above: A pharmaceutical composition comprising, consisting essentially of, or consisting of a pharmaceutically acceptable excipient.

Another object of the present invention is to bleb and at least one pharmaceutically acceptable shaping of γδFoxp3 ⁺ regulatory T cells having the following phenotype CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ as described herein above: A pharmaceutical composition comprising, consisting essentially of, or consisting of an agent.

Another object of the present invention is to provide an immunogenic DC loaded with a bleb of γδ Foxp3 ⁺ regulatory T cells and at least one medicament having the following phenotype CD3 ⁺ TCR γδ ⁺ Foxp3 ⁺ as described hereinabove ^: A pharmaceutical composition comprising, consisting essentially of, or consisting of a pharmaceutically acceptable excipient.

  As used herein, the term “excipient” refers to any and all solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying. It refers to agents. For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by regulatory authorities (eg, FDA Office or EMA) .

  By “pharmaceutically acceptable” is meant that the components of the pharmaceutical composition are compatible with each other and not deleterious to the subject to whom it is administered. Examples of pharmaceutically acceptable excipients include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, or combinations thereof.

  Another object of the present invention is a vaccine composition comprising, consisting essentially of or consisting of the immunogenic products described herein above.

Another object of the present invention is a vaccine composition comprising, consisting essentially of or consisting of inactivated γδ Foxp3 ⁺ regulatory T cells having the following phenotype CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ .

Another object of the invention includes inactivated γδFoxp3 ⁺ regulatory T cells generated and expanded ex vivo by the methods described hereinabove and having the following phenotype CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ A vaccine composition consisting essentially of or consisting of.

Another object of the invention includes, consists essentially of or consists of a bleb of γδFoxp3 ⁺ regulatory T cells having the following phenotype CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ as described herein above It is a vaccine composition.

Another object of the invention includes immunogenic DC loaded with blebs of γδ Foxp3 ⁺ regulatory T cells having the following phenotype CD3 ⁺ TCR γδ ⁺ Foxp3 ⁺ as described hereinabove, and then essentially A vaccine composition consisting of or consisting of.

As used herein, “inactivated” T cells refer to T cells that are viable but have reduced or no effector function, ie, have lost their pathogenic potential. Examples of cell surface markers for inactivated T cells include, but are not limited to, 7-aminoactinomycin D (7-AAD), calreticulin and heat shock protein 90 (HSP-90). Thus, inactivated T cells express 7-AAD and / or calreticulin and / or HSP-90. Inactivated γδFoxp3 ⁺ regulatory T cells of the present invention have lost their suppressive activity but are still immunogenic. An example of a T cell effector function assay is, but is not limited to, a T cell proliferation assay. T cell proliferation may be assessed in fixed versus non-fixed T cells. Briefly, the T cell proliferation assay is aimed at determining the percentage of viable proliferating cells in fixed versus non-fixed T cells by flow cytometry. After staining T cells with CFSE, anti-CD3 antibody, and 7-AAD, live proliferating cells are defined as the CFSE low fraction in gated CD3 ⁺ 7-AAD- cells.

In one embodiment, the γδ Foxp3 ⁺ regulatory T cells are inactivated by any method known in the art. Examples of methods for inactivating cells preferably include irradiation with about 2500-3000 rads and / or chemical inactivation, such as exposure to cisplatin, carboplatin, oxaliplatin, mitomycin C, or anthracyclines. However, it is not limited to these.

  In one embodiment, the vaccine composition of the present invention further comprises at least one adjuvant. Examples of adjuvants that can be used in vaccine compositions are ISA51; emulsions such as CFA, MF59, montanide, AS03, and AF03; inorganic salts such as alum, calcium phosphate, iron salt, zirconium salt, and AS04; TLR ligands such as TLR2 ligands (such as outer surface protein A or OspA), TLR3 ligands (such as poly I: C), TLR4 ligands (such as MPL and GLA), TLR5 ligands, TLR7 / 8 ligands (such as imiquimod) Polysaccharides such as chitin, chitosan, α-glucan, β-glucan, fructan, mannan, dextran, lentinan, inulin based adjuvants (eg gamma inulin), TLR9 ligands (eg CpG ODN) ) Such as; TLR9 and STING ligands, for example including K3 CpG and CGAMP, without limitation. As used herein, “adjuvant” refers to an agent that enhances an immune response to an antigen and / or modulates it toward a desired immune response.

In one embodiment, the inactivated γδ Foxp3 ⁺ regulatory T cells are specific for at least one non-peptide phosphoantigen as described herein above.

In another embodiment, the inactivated γδFoxp3 ⁺ regulatory T cells are specific for at least one non-peptide phosphoantigen present on the apoptotic body of the cancer cell.

In another embodiment, the inactivated γδFoxp3 ⁺ regulatory T cells are specific for at least one non-peptide phosphoantigen present on the bleb of the cancer cell.

In one embodiment, the inactivated γδFoxp3 ⁺ regulatory T cells present in the immunogenic product, pharmaceutical composition, or vaccine composition of the invention are human γδFoxp3 ⁺ regulatory T cells.

In one embodiment, the inactivated γδFoxp3 ⁺ regulatory T cells present in the immunogenic product, pharmaceutical composition or vaccine composition of the invention are autologous γδFoxp3 ⁺ regulatory T cells.

In one embodiment, the inactivated γδFoxp3 ⁺ regulatory T cells present in the immunogenic product, pharmaceutical composition, or vaccine composition of the invention are allogeneic γδFoxp3 ⁺ regulatory T cells.

In another embodiment, the immunogenic product, pharmaceutical composition, or vaccine composition of the invention may be individualized for the patient. As used herein, an “individualized” immunogenic product or vaccine composition consists of γδ Foxp3 ⁺ regulatory T cells generated and expanded ex vivo using at least one patient-specific epitope. Refers to use. In this embodiment, γδ Foxp3 ⁺ regulatory T cells used as immunogenic products or in vaccine compositions are ex vivo in the presence of apoptotic bodies of cancer cells obtained from patients or in the presence of blebs of cancer cells. Generated and propagated thereby providing at least one patient-specific epitope.

In one embodiment, an immunogenic product, pharmaceutical composition, or vaccine composition of the invention comprises, consists essentially of or consists of inactivated γδ Foxp3 ⁺ regulatory T cells as an active ingredient.

In one embodiment, the immunogenic product, pharmaceutical composition or vaccine composition of the present invention has at least 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ non-active ingredients as active ingredients. Contains, consists essentially of or consists of activated γδ Foxp3 ⁺ regulatory T cells.

In one embodiment, the immunogenic product, pharmaceutical composition or vaccine composition of the present invention has about 10 ⁴ , 5 × 10 ⁴ , 10 ⁵ , 5 × 10 ⁵ , 10 ⁶ , 5 × 10 ⁶ as the active ingredient. , including ^{10 7, 5 × 10 7,} 10 8, 5 × 10 8, 10 9, 5 × 10 9, 10 10 or inactivated GanmaderutaFoxp3 ⁺ regulatory T cells, it consists essentially of or consisting .

In one embodiment, the γδ Foxp3 ⁺ regulatory T cells, inactivated γδ Foxp3 ⁺ regulatory T cells, immunogenic products, pharmaceutical compositions, or vaccine compositions of the invention are frozen.

  In one embodiment, the immunogenic product, pharmaceutical composition, or vaccine composition of the invention may be administered by subcutaneous injection, intramuscular injection, intraperitoneal injection, or intravenous injection, or directly into the tumor. .

  In one embodiment, an immunogenic product, pharmaceutical composition, or vaccine composition of the invention may be administered to a subject at least once, twice, three times, four times, five times per year. Examples of dosing schedules include administration of immunogenic products or vaccine compositions on day 0, 4 weeks after 0 day, 8 weeks after 0 day, 12 weeks after 0 day, and 24 weeks after 0 day. Including but not limited to.

Another object of the invention is to administer to a subject a therapeutically effective amount of inactivated γδ Foxp3 ⁺ regulatory T cells, or an immunogenic product, pharmaceutical composition or vaccine composition of the invention. A method for treating cancer in a subject in need thereof.

Another object of the present invention is a method for eliciting an immune response against γδ Foxp3 ⁺ regulatory T cells present in the TIL of a subject suffering from cancer, comprising a therapeutically effective amount Administration of an inactivated γδ Foxp3 ⁺ regulatory T cell or an immunogenic product, pharmaceutical composition, or vaccine composition of the invention as described herein above to a subject.

  Examples of cancers that can be treated using the immunogenic products, pharmaceutical compositions, or vaccine compositions of the present invention include adrenal cortex cancer, anal cancer, bladder cancer, ependymoma, medulloblastoma, supratent primordial Neuroectodermal, pineal tumor, hypothalamic glioma, breast cancer, carcinoid tumor, carcinoma, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing familial tumor (pnet), Extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germ cell tumor, extragonadal tumor, gestational choriocarcinoma, head and neck cancer, hypopharyngeal cancer, islet cell cancer, laryngeal cancer, leukemia, Acute lymphoblastic leukemia, oral cancer, liver cancer, lung cancer, small cell lymphoma, AIDS-related, lymphoma, central nervous system (primary) lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, non-Hodgkin's disease, malignant mesothelioma, Melanoma, Merkel cell carcinoma, metastatic squamous cell carcinoma, Multiple myeloma, plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disease, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, Low-grade ovarian tumor, pancreatic cancer, exocrine, pancreatic cancer, paranasal and nasal cavity cancer, parathyroid cancer, pheochromocytoma cancer, pituitary cancer, plasma cell neoplasm, rhabdomyosarcoma, rectal cancer, renal cell Cancer, salivary gland cancer, Sezary syndrome, Kaposi sarcoma, small intestine cancer, soft tissue sarcoma, thymoma, malignant thyroid cancer, urethral cancer, uterine cancer, sarcoma, childhood abnormal cancer, vaginal cancer, vulvar cancer or Wilms tumor, chemotherapy This includes, but is not limited to, benign conditions associated with treatment, such as lupus, rheumatoid arthritis, and skin diseases.

  In one embodiment, cancers that can be treated using the immunogenic products, pharmaceutical compositions, or vaccine compositions of the present invention include breast cancer, prostate cancer, ovarian cancer, and glioblastoma, It is not limited to.

Another object of the present invention is a method for preparing the immunogenic product of the present invention comprising:
Providing a biological sample from the subject to be treated, preferably a blood sample, and optionally a tumor sample from the subject to be treated;
Generation of CD3 ⁺ TCRγδ ⁺ T cells isolated from biological samples, preferably γδFoxp3 ⁺ regulatory T cells from CD3 ⁺ TCRγδ ⁺ CD45RA ⁺ T cells as described herein above and Growing,
Inactivating the γδ Foxp3 ⁺ regulatory T cells obtained in the previous step,
Thereby obtaining an immunogenic product of the invention.

  In a preferred embodiment, the generation and expansion steps are performed in the presence of tolerogenic dendritic cells (DCs) and pulsed with an apoptotic tumor body or bleb obtained from a subject tumor sample.

  Another object of the invention is a method for treating cancer in a subject in need thereof, comprising administering to the subject an immunogenic product, pharmaceutical composition or vaccine composition of the invention. including.

Another object of the invention is a method for treating cancer in a subject in need thereof, comprising:
-Preparing an immunogenic product as described herein above,
-Optionally preparing a pharmaceutical or vaccine composition comprising an immunogenic product;
-Optionally subjecting the subject to plasma exchange therapy;
-Administering the immunogenic product, pharmaceutical composition or vaccine composition of the invention to a subject.

Without wishing to be bound by theory, the inventors have committed that the γδFoxp3 ⁺ regulatory T cells of the present invention exert an immunosuppressive function and are self-reactive pathogenic immune effector cells ( CD4 ⁺ cells, CD8 ⁺ cells, including B cells or natural NK cells) may be possible, but they then suggest that they are unable to exert cytotoxic properties against autologous cells.

One object of the present invention consists essentially of a γδFoxp3 ⁺ regulatory T cell or γδFoxp3 ⁺ regulatory T cell population as described herein above and comprising at least one pharmaceutically acceptable excipient. Or a pharmaceutical composition consisting of them.

Another object of the present invention comprises, consists essentially of γδFoxp3 ⁺ regulatory T cells with the following phenotype CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ and at least one pharmaceutically acceptable excipient, or It is a pharmaceutical composition comprising them.

Another object of the present invention is to provide γδFoxp3 ⁺ regulatory T cells having the following phenotype CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ produced and expanded ex vivo by the methods described hereinabove and at least one pharmaceutically A pharmaceutical composition comprising, consisting essentially of or consisting of acceptable excipients.

One object of the present invention is a γδ Foxp3 ⁺ regulatory T cell or γδ Foxp3 ⁺ regulatory T cell population or pharmaceutical composition as described herein above for use in adoptive therapy.

Another object of the present invention is to provide a γδ Foxp3 ⁺ regulatory T cell or γδ Foxp3 ⁺ regulatory T cell population or medicament as described herein above for use in treating inflammatory or autoimmune diseases. It is a composition.

  Examples of inflammatory or autoimmune diseases are acute disseminated encephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-antibody TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic neuropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, Autoimmune immunodeficiency, autoimmune inner ear disease, autoimmune myocarditis, autoimmune ovitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura, autoimmune thyroid disease, Autoimmune urticaria, axon and neuropathy, Baro's disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating multiple Neuritis, Chronic recurrent multifocal osteomyelitis, Churg-Strauss syndrome, Scarous pemphigoid / benign mucocele pemphigoid, Crohn's disease, Kogan syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST Disease, essential mixed cryoglobulinemia, demyelinating neuropathy, herpetic dermatitis, dermatomyositis, Devic disease, discoid lupus, Dosler syndrome, endometriosis, eosinophilic esophagitis, eosinophilic Fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibromyalgia, giant cell arteritis, giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, multiple occurrences Granulomatosis with vasculitis (Wegener syndrome), Graves' disease, Guillain-Barre syndrome, Hashimoto encephalitis, Hashimoto thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, pregnancy Pes, hypogammaglobulinemia, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, IgG4-related sclerosis, immunomodulating lipoprotein, inclusion body myositis, interstitial cystitis, juvenile arthritis , Juvenile diabetes (type 1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert Eaton syndrome, leukoclastic vasculitis, lichen planus, sclerosis lichen, woody conjunctivitis, linear IgA disease, lupus, chronic Lyme disease, Meniere Disease, microscopic polyangiitis, mixed connective tissue disease, Mohren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, optic neuromyelitis, neutropenia, ocular scarring Pemphigoid, optic neuritis, recurrent rheumatism, childhood autoimmune neuropsychiatric disorders related to streptococci, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Lomberg syndrome, pa -Sonage-Turner syndrome, flatitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, and type III polyglandular Autoimmune syndrome, rheumatic polymyalgia, polymyositis, post-myocardial infarction syndrome, epicardial detachment syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, Pyoderma gangrenosum, erythroblastosis, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter syndrome, relapsing polychondritis, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis , Sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis, Zac syndrome, sympathetic ophthalmitis, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura, Tolosa Hunt syndrome, transverse myelitis, type 1 diabetes, ulcerative colon Including, but not limited to, inflammation, undifferentiated connective tissue disease, uveitis, vasculitis, vesicular skin disease, and leukoplakia.

  Examples of inflammatory or autoimmune diseases include, but are not limited to, rheumatoid arthritis, type 1 diabetes, and multiple sclerosis.

Another object of the present invention, transplant rejection, or graft-versus-host disease for use in in preventing (GVHD), described herein above GanmaderutaFoxp3 ⁺ regulatory T cells or GanmaderutaFoxp3 ⁺ regulatory A T cell population or a pharmaceutical composition.

In one embodiment, the γδ Foxp3 ⁺ regulatory T cells are specific for at least one non-peptide phosphoantigen described hereinabove.

In another embodiment, the γδ Foxp3 ⁺ regulatory T cells are specific for at least one non-peptide phosphoantigen that was present in the tissue lysate.

In one embodiment, the pharmaceutical composition of the invention comprises at least 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ γδ Foxp3 ⁺ regulatory T cells as active ingredients, then essentially Become or consist of.

In one embodiment, the pharmaceutical composition of the present invention comprises about 10 ⁴ , 5 × 10 ⁴ , 10 ⁵ , 5 × 10 ⁵ , 10 ⁶ , 5 × 10 ⁶ , 10 ⁷ , 5 × 10 ⁷ , 10 as the active ingredient. ⁸ , 5 × 10 ⁸ , 10 ⁹ , 5 × 10 ⁹ , 10 ¹⁰ γδFoxp3 ⁺ regulatory T cells comprising, consisting essentially of or consisting of.

In one embodiment, a γδ Foxp3 ⁺ regulatory T cell, γδ regulatory T cell population, or medicament of the invention is frozen.

In one embodiment, the γδ Foxp3 ⁺ regulatory T cells present in the pharmaceutical composition of the invention are human γδ regulatory T cells.

In one embodiment, the γδFoxp3 ⁺ regulatory T cells present in the pharmaceutical composition of the invention are autologous γδFoxp3 ⁺ regulatory T cells.

In one embodiment, the γδFoxp3 ⁺ regulatory T cells present in the pharmaceutical composition of the invention are allogeneic γδFoxp3 ⁺ regulatory T cells.

  In one embodiment, the pharmaceutical composition of the invention may be administered to a subject by subcutaneous injection, intramuscular injection, intraperitoneal injection, or intravenous injection.

  In one embodiment, the pharmaceutical composition of the invention may be administered to a subject at least once, twice, three times, four times, five times a week.

  In another embodiment, the pharmaceutical composition of the invention may be administered to a subject at least once, twice, three times, four times, five times a month.

  In another embodiment, the pharmaceutical composition of the invention may be administered to a subject at least once, twice, three times, four times, five times over a three month period.

Another object of the present invention is a method for treating an inflammatory or autoimmune disease in a subject in need thereof, comprising a therapeutically effective amount as described herein above. administering to a subject a γδFoxp3 ⁺ regulatory T cell or γδFoxp3 ⁺ regulatory T cell population or a pharmaceutical composition thereof.

  T-cell vaccination induces regulatory networks that specifically suppress immunogenic T cells by activating specific T cells for clonal type specific determinants (anti-idiotypic response). Shown in the field. Also, anti-ergotypic responses directed at activation markers (corresponding to ergotopes) may also partially explain the suppression of targeted regulatory T cell populations.

Another object of the present invention is an antibody that recognizes the TCR (T cell receptor) of γδ Foxp3 ⁺ regulatory T cells of the present invention.

In one embodiment, an antibody that recognizes a TCR of a γδ Foxp3 ⁺ regulatory T cell of the invention recognizes at least one of CDR1, CDR2, and CDR3 (complementarity determining regions 1, 2, and 3) of the TCR.

In another embodiment, an antibody that recognizes a TCR of a γδ Foxp3 ⁺ regulatory T cell of the invention recognizes CDR3 of the TCR.

  Another object of the present invention is a pharmaceutical composition comprising, consisting essentially of or consisting of said antibody and at least one pharmaceutically acceptable excipient.

  Another object of the present invention is the use of said antibody for treating cancer in a subject in need thereof.

In one embodiment, antibodies against γδ Foxp3 ⁺ regulatory T cells of the invention are derived from antibodies produced after immunization of mammals (including humans) with the immunogenic compositions described herein above. Become.

  In another embodiment, the antibodies may also be obtained by cloning relevant DNA material encoding them starting from B cells obtained, for example, from said mammal (including from said human).

In another embodiment, the antibodies also sequence the amino acid sequences of antibodies collected from said mammal (including from said human) to produce related recombinant antibodies against γδ Foxp3 ⁺ regulatory T cells of the invention. May be obtained by synthesizing and then synthesizing the antibody-encoding DNA molecule or a portion thereof including its CDRs.

Preparation of antibodies to γδ Foxp3 ⁺ regulatory T cells of the present invention by immunization with an immunogenic composition of the present invention is simple for those skilled in the art using general technical knowledge from the art. Can be implemented.

Alternatively, antibodies against γδ Foxp3 ⁺ regulatory T cells of the present invention may be obtained after immortalization of the human B lymphocytes that produce them; their cDNAs can also be cloned, and their or their It can be further used to produce derivatives.

As used herein, the term “antibody” is used to refer to molecules having useful antigen binding specificity. One skilled in the art will readily appreciate that the term is a fragment or derivative of an antibody, but may also cover polypeptides that may exhibit the same or closely similar functionality. Such antibody fragments or derivatives are intended to be encompassed by the term “antibody” as used herein. By “antibody” or “antibody molecule” for the purpose of passive immunotherapy, not only whole immunoglobulin molecules but also fragments thereof, such as Fab, F (ab ′) 2, Fv, and γδFoxp3 ⁺ regulatory, are used herein. Those other fragments that retain the ability to bind and inactivate T cells are also contemplated. Similarly, the term “antibody” includes genetically engineered derivatives of antibodies, such as single chain Fv molecules (scFv) and domain antibodies (dAbs).

In some embodiments, the antibody against γδ Foxp3 ⁺ regulatory T cells of the present invention comprises a polyclonal antibody.

In some embodiments, the antibodies against γδ Foxp3 ⁺ regulatory T cells of the present invention comprise monoclonal antibodies.

  The term “monoclonal antibody” as used herein includes any isolated Ab, such as a conventional monoclonal antibody hybridoma, but isolated monospecific antibody produced by any cell, such as a mammal. Also included are samples of the same human immunoglobulin expressed in cell lines.

  The variable heavy chain (V H) and variable light chain (V L) domains of antibodies are included in antigen recognition, a fact that was first recognized by early protease digestion experiments. Further confirmation was found by “humanization” of rodent antibodies. A rodent-derived variable domain may be fused to a human-derived constant domain such that the resulting antibody retains the antigen specificity of the rodent parent antibody (Morrison et al. (1984) Proc Natl. Acad. Sci. USA 81, 6851-6855). It is known from experiments involving bacterial expression of antibody fragments (all containing one or more variable domains) that antigen specificity is conferred by variable domains and is independent of constant domains. These molecules are Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); sub. H and V. sub. Single-chain Fv (ScFv) molecules in which L partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci. USA 85, 5879) and single domain antibodies (dabs) containing isolated V domains (Ward et al (1989) Nature 341, 544). A general review of the techniques involved in the synthesis of antibody fragments that retain their specific binding sites is found in Winter & Milstein (1991, Nature 349, 293-299).

  The term “ScFv molecule” encompasses a molecule in which the V H and V L partner domains are linked via a flexible oligopeptide. Engineered antibodies (eg, ScFv antibodies) are described in J. Huston et al, (1988) "Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single chain Fv analogue produced in E. coli", Proc Natl. Acad. Sci. USA, 85, pp. 5879-5883, and A. Pluckthun, (1991) "Antibody engineering; Advances from use of E. coli expression systems", Bio / technology 9 (6): 545. -51 (incorporated herein by reference) can be used to make.

  Suitable monoclonal antibodies that are reactive as described herein are known in the art, eg, “Monoclonal Antibodies; A manual of techniques”, H Zola (CRC Press, 1988), and “Monoclonal Hybridoma Antibodies: Techniques and Applications ", SGR Hurrell (CRC Press, 1982).

  Further embodiments include humanized antibodies in which complementarity determining regions (CDRs), regions of mouse antibodies that have been contacted with antigens, have been transferred to a human antibody framework. Such antibodies are almost completely human and rarely cause adverse antibody responses when administered to patients. Several chimeric or humanized antibodies have been registered as therapeutic drugs and are now widely used in various indications (Borrebaeck & Carlsson, 2001, Curr. Opin. Pharmacol. 1: 404-408) .

  The antibody is preferably a humanized antibody. Appropriately prepared non-human antibodies can be “humanized” by known methods, eg, by inserting the CDR regions of a murine antibody into the framework of a human antibody. Humanized antibodies use techniques and techniques described in Verhoeyen et al (1988) Science, 239, 1534-1536 and in Kettleborough et al, (1991) Protein Engineering, 14 (7), 773-783. Can be produced.

  In another embodiment, the antibody also encompasses a fully human antibody, which can be produced using recombinant techniques. Typically, large libraries containing billions of different antibodies are used. In contrast to past techniques, for example using mouse antibody chimerization or humanization, this technique does not rely on immunization of animals to produce specific antibodies. Instead, the recombinant library contains a vast number of pre-made antibody variants, in which the library is likely to have at least one antibody specific for any antigen.

  The frequency of administration may be determined clinically by following the decrease in antibody titer in the patient's serum over time, but in any event, 1 to 52 times a year, most preferably the year There can be a frequency of 1-12 times. The amount of antibody may vary according to the severity of the disease or the half-life of the antibody in the serum, but is preferably in the range of 1-10 mg / kg patient, preferably in the range of 1-5 mg / kg patient, Most preferred is a 1-2 mg / kg patient.

Different frequency and phenotypic characteristics between Foxp3 ⁺ and FOXP3 ^- CD3 ⁺ T cell populations, defined by their variable TCR recognition in TIL isolated from human peripheral blood (PBMC) and breast tumors. Analysis of Foxp3 ⁺ expression in human inducible tumor antigen-specific FOXP3 ex vivo expressing CD4 ⁺ TCRγδ ⁺ unrestricted T cells. NTreg composed of IL-2 (100 IU / ml) and TGFβ (5 ng / ml), PGE2 (1 μM), and Rapa (10 nM) using apoptotic tumor antigen (Ag) pulse tolerogenic DC (tDC) Specific pTregs were generated and propagated from naive CD4 ⁺ T cells in the presence of polarized medium. Unloaded tDC was used as a control. (A) Frequency and (B) expression level (assessed by MFI) of Foxp3 in CD4 ⁺ T cell culture. Phenotypic differences between Foxp3-expressing CD3 ⁺ CD4 ⁺ γδ T cell non-restricted T cells and currently described γδ T cell subtypes isolated from BC biopsies. Phenotypic identification of Foxp3-expressing CD3 ⁺ CD4 ⁺ γδT cells uses antibodies against CD3 (clone SK7), CD4 (clone SK3), CD8 (clone SK1), pan-γδT (clone IMMU510), and Foxp3 ⁺ (clone 259D) Was performed by flow cytometry. Use of TCRVδ among CD3 ⁺ CD4 ⁺ γδ T cells expressing Foxp3 ⁺ isolated from BC biopsy. Identification of TCRV γδ chain was performed using antibodies against CD3 (clone SK7), CD4 (clone SK3), CD8 (clone SK1), pan γδT (clone IMMU510), TCRVδ1 (REA173), TCRVδ2 (REA771), and Foxp3 ⁺ (clone 259D) Was performed by flow cytometry using Inhibitory ability of Foxp3-expressing CD3 ⁺ CD4 ⁺ γδ T cell non-restricted T cells isolated from BC biopsy. CFSE-labeled Tconv (TconvCFSE) was co-cultured with different ratios of sorted CD3 ⁺ CD4 ⁺ γδ T cells. The percent inhibition of TconvCFSE proliferation by CD4 ⁺ γδT cells was shown. Circulating fresh Treg from a healthy donor was used as a control. Generation of specific pathogenic CD4 ⁺ T cells, ie tumor antigen-specific FOXP3 expressing CD4 ⁺ TCRγδ ⁺ autologous CD8 ⁺ T cell lines that are functionally committed to lyse unrestricted T cells. The ability of CD8 + ^T cell clones to lyse the inductive pathogenic CD4 + ^T cells clones is evaluated using classical 7-AAD / CFSE cell-mediated cytotoxicity assay as described previously. Briefly, 4 days after stimulation, pathogenic CD4 ⁺ target cells or autolymphoblast cell lines are labeled with CFSE and placed in triplicate at 3 × 10 ⁴ per well in a 96 well U-bottom plate. did. CD8 ⁺ effector T cells (5: 1 E: T ratio) were added and incubation was carried out at 37 ° C. for 6 hours. At the end of the experiment, dead cells were labeled with 7-AAD to detect lysed cells. Cytolytic activity against target cells was analyzed using a FACSCalibur instrument based on areas showing double positive staining CFSE and 7-AAD. The cytolytic activity (%) of CD8 ⁺ T cell clones was calculated as positive cells / total CFSE positive cells for both CFSE and 7-AAD after subtracting spontaneous lysis (%) in the negative control. The percentage of cytolytic activity was then calculated using the following formula: cytolytic activity (%) [dead target cells (%) − natural death (%)] × 100 / [100−natural death (%) ]]. Analysis of Foxp3 ⁺ expression in lymphocytes present in TIL extracted from lumen A and lumen B breast subtypes. Tumor tissue from patients with lumen A and lumen B was minced with a scalpel and enzymatically digested by overnight incubation in collagenase type IV. Expression of the FOXP3 marker in lymphocytes present in isolated TIL was determined by flow cytometric analysis. Analysis of Foxp3 ⁺ expression in lymphocytes present in TIL extracted from three different breast cancer subgroups: patients with lumen A (n = 3), patients with lumen B (n = 3) and triple Tumor tissue from patients with negative breast cancer (TNBC) (n = 2) was minced with a scalpel and enzymatically digested by overnight incubation in collagenase type IV. Expression of the FOXP3 marker in lymphocytes present in isolated TIL was determined by flow cytometric analysis. Display of percentage of FOXP3 expression in CD3 ⁺ CD4 ⁺ TCRγδ ⁺ unrestricted T cells. Multiparametric flow cytometric analysis of lymphocytes present in TIL from lumen A and B breast subtypes. Lymphocytes present in TIL were stained on the cell surface using Ab against CD3, CD4, CD25, CD56, CD161. After fixation and permeabilization, Foxp3 and CTLA4 were stained intracellularly. It stimulated naive ^{CD3 + TCRγδ} + T cells from phenotypic and functional capability of suppressing the Ag-specific ^{CD3 + TCRγδ} + T cells generated ex vivo. Naive CD3 ⁺ TCRγδ ⁺ T cells were stimulated with zoledronic acid-treated auto-tDC in the presence of nTreg polarization medium and IL-2 (100 IU / ml) and IL-15 (10 ng / ml). (A) Overlay histogram exhibiting Foxp3 expression profile and (B) Inhibitory ability of Ag-specific CD3 ⁺ TCRγδ ⁺ T cells grown over 21 or 42 days.

  Example

  The invention is further illustrated by the following examples.

  Materials and methods

  Human blood sample. Blood samples from healthy individuals were derived from Etablissement Francais du Sang (EFS, Paris). Blood cells are collected using standard procedures.

  Human tumor sample. Tumor tissue samples were derived from patients with lumen A and lumen B breast cancer (Institut Jean Godinot, Lance).

  Cell purification and culture

  Peripheral blood mononuclear cells (PBMC) are isolated by density gradient centrifugation on Ficoll-Hypaque (Pharmacia). PBMC are used as fresh cells or stored frozen in liquid nitrogen. T cell subsets and T cell depleted accessory cells (ΔCD3 cells) are isolated from fresh or frozen PBMC. T cell depleted accessory cells (ΔCD3 cells) are isolated by negative selection from PBMC by incubation with anti-CD3 coated Dynabeads (Dynal Biotech) and irradiated with 3000 rads (referred to as ΔCD3 feeder).

CD4 ⁺ T cells are negatively selected using a CD4 ⁺ T cell isolation kit (Miltenyi Biotec, yielding a CD4 ⁺ T cell population of 96-99% purity). Subsequently, selected CD4 ⁺ T cells were analyzed using a FACSAria III Cell Sorter (Becton Dickinson) for CD4 ⁺ CD127 ^{− / lo} CD25 ^high (pTregs) and CD4 ⁺ CD127 ⁺ CD25 ^{neg / dim} [conventional helper CD4 T Cells (Tconv)] Prior to sorting into subpopulations, anti-CD4 (13B8.2) -FITC (Beckman Coulter), anti-CD25 (4E3) -APC (Miltenyi Biotec), and anti-CD127 (R34.34) -PE ( Beckman Coulter).

CD14 ⁺ monocytes are isolated from PBMC by positive selection using the MACS system.

CD3 ⁺ CD4 ⁺ CD127 ⁺ CD45RA ⁺ CD25 ⁻ TCRαβ ⁺ MHCII restriction (naïve conventional CD4 ⁺ T cells) is isolated from PBMC after magnetic enrichment (MACS system: CD4 microbeads) and FAC sorting. Prior to the sorting step, the enriched CD3 ⁺ CD4 ⁺ T cells were treated with anti-CD4 (13B8.2) -FITC (Beckman Coulter), anti-CD25 (4E3) -APC (Miltenyi Biotec), and anti-CD127 (R34.34). -Stain with PE (Beckman Coulter), anti-TCRαβ-BV421 (IP26) (Biolegend).

CD3 ⁺ CD45RA ⁺ invTCR Vα24 ⁺ CD1 restricted T cells are isolated from PBMC after magnetic enrichment (MACS system: anti-iNKT microbeads and FACS sorting). Prior to the sorting step, enriched CD3 ⁺ invTCR Vα24 ⁺ T cells were treated with anti-CD3 (UCHT-1) V450 anti-invariant TCR Vα24-JαQ (6B11) -PE (inv TCR) Vα24-JαQ (Becton Dickinson), and Stain with anti-CD45RA (T6D11) -FITC (Miltenyi Biotec).

CD3 ⁺ CD45RA ⁺ CD27 ⁺ TCRγδ ⁺ unrestricted T cells are isolated from PBMC after magnetic enrichment (MACS system: TCRγδ ⁺ T cell isolation kit) and FACS sorting. Prior to the sorting step, the enriched CD3 ⁺ TCRγδ ⁺ T cells were transformed into anti-CD3 (UCHT-1) V450, anti-TCR pan γδ ⁺ PE (IMMU510) (Beckman Coulter), anti-CD27-APC fluor 780 (O323) (ebioscience) ), And anti-CD45RA (T6D11) -FITC (Miltenyi Biotec).

  T cell subsets are cultured in 2% hypoxia in IMDM supplemented with 5% SVF, 100 IU / ml penicillin / streptomycin, 1 mM sodium pyruvate, 1 mM non-essential amino acids, glutamax, and 10 mM HEPES (IMDM-5 medium). To do.

  Breast cancer cell lines and cultures. Human breast cancer cell line MCF-7 was obtained from American Type Culture Collection (USA). Cells are maintained in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS). MCF-7 cells are treated with 5 μg / ml doxorubicin for 24 hours or by gamma irradiation (20 Gy). The degree of apoptosis is monitored by flow cytometric analysis (FACS). Cells are washed thoroughly before supplying DC.

  TIL isolation. The tumor tissue is minced with a scalpel in collagenase type IV (2 mg / mL, Roche Diagnostic GmbH) in DMEM high glucose medium supplemented with 2 mM glutamine (Gibco), 50 mg / mL gentamicin, and 0.25% human serum albumin. Enzymatic digestion by overnight incubation on a 37 ° C. rotary shaker.

  Generation of polyclonal, functionally committed FOXP3 expression regulatory T cells ex vivo.

Ex vivo generation of polyclonal functionally committed FOXP3-expressing CD3 ⁺ TCRαβ ⁺ MHCII restricted T cells: On day 0, T cells are seeded at 2.5 × 10 ⁵ cells / well in a 48-well plate, Stimulate with plate-bound anti-CD3 mAb (4 μg / ml) in the presence of ΔCD3 feeder (1M). Cells were supplemented with IMDM-5 medium (5% SVF, 100 IU / ml penicillin / streptomycin, 1 mM sodium pyruvate, 1 mM non-essential amino acids, glutamax, and 10 mM HEPES with 1 μM PGE2, 5 ng / ml TGFβ, and 10 nM Rapa. IMDM). On day 2, IL-2 (100 IU / ml) is added to the culture. Every 3 days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI / ml). On day 11, these CD4 ⁺ T cell lines were further expanded by restimulation with plate-bound anti-CD3 Ab (4 μg / ml). This restimulation was performed in the presence of ΔCD3 feeder, PGE2 1 μM, TGFβ 5 ng / ml, Rapa 10 nM, and IL-2 (100 UI / ml). Next, every 3 days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI / ml). On day 20, the phenotype of expanded CD4 ⁺ T cells was assessed by flow cytometry. 75% of stimulated naïve conventional T cells that became CD45RO ⁺ express Foxp3 ⁺ .

Ex vivo generation of polyclonal functionally committed FOXP3-expressing invariant T cells: On day 0, T cells were seeded at 1 × 10 ³ cells / well in a 96-well plate and the ΔCD3 feeder (2.5 Stimulate with plate-bound anti-inv TCR Vα24-JαQ (6B11) mAb (2 μg / ml) in the presence of × 10 ⁵ ). Cells are cultured in IMDM-5 media with 1 μM PGE2, 5 ng / ml TGFβ, 10 nM Rapa, IL-2 (100 UI / ml), and IL-15 (10 ng / ml). Every 3 days, IL-2 (100 IU / ml) and IL-15 (10 ng / ml) are added to the culture. On day 12, T cells were plated in anti-inv TCR plate-bound in the presence of ΔCD3 feeder, PGE2 1 μM, TGFβ 5 ng / ml, Rapa 10 nM, IL-2 (100 UI / ml), and IL-15 (10 ng / ml). Further growth was achieved by restimulation with Vα24-JαQ (6B11) mAb (2 μg / ml). Next, every 3 days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI / ml) and IL-15 (10 ng / ml). On day 21, the cells were analyzed by flow cytometry. 70% of stimulated CD3 ⁺ invTCR Vα24 ⁺ RA ⁺ T cells that became CD45RO ⁺ express Foxp3 ⁺ .

Ex vivo generation of polyclonal functionally committed FOXP3-expressing TCRγδ ⁺ T cells: On day 0, T cells were seeded at 1 × 10 ³ cells / well in a 96-well plate and the ΔCD3 feeder (2.5 Stimulate with plate-bound anti-TCRγδ mAb (2 μg / ml) in the presence of × 10 ⁵ ). Cells were treated with IMDM-5 medium (5% SVF, 100 IU / ml penicillin / streptomycin, with PGE2 1 μM, TGFβ 5 ng / ml, Rapa 10 nM, IL-2 (100 UI / ml), and IL-15 (10 ng / ml), IMDM supplemented with 1 mM sodium pyruvate, 1 mM non-essential amino acids, glutamax, and 10 mM HEPES). Every 3 days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI / ml) and IL-15 (10 ng / ml). On day 11, T cells were further expanded by restimulation with plate-bound anti-pan TCRγδ Ab (2 μg / ml). This restimulation was performed in the presence of ΔCD3 feeder, PGE2 1 μM, TGFβ 5 ng / ml, Rapa 10 nM, and IL-2 (100 UI / ml) and IL-15 (10 ng / ml). Next, every 3 days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI / ml) and IL-15 (10 ng / ml). On day 21, the cells were analyzed by flow cytometry. 65% of stimulated CD3 ⁺ CD45RA ⁺ CD27 ⁺ TCRγδ ⁺ T cells that became CD45RO ⁺ express Foxp3 ⁺ .

  Generation of antigen-specific functionally committed FOXP3-expressing T cells ex vivo.

Antigen production in ex vivo (ovalbumin) -specific manner functionally Foxp3 expression ^{CD3 + TCRαβ} + MHCII-restricted T cells that have been committed:
a) In vitro generation of ovalbumin-tolerant DC (referred to as tolerogenic monocyte-derived DC (Tol-Mo-DC)) from CD14 ⁺ monocytes: monocytes are used to generate immature DCs In 48 well flat bottom plates containing 0.5 ml AIMV per well supplemented with 100 ng / ml recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and 10 ng / ml human recombinant IL-4 Incubate. On the third day, 500 μl of medium containing cytokines was added. On day 6, Tol-Mo-DC is removed from 1) wells and IMDM-5 (5% SVF, 100 IU / ml penicillin / streptomycin, 1 mM sodium pyruvate, 1 mM non-essential amino acids, glutamax, and 10 mM HEPES is added. 2) and added to wells of a 48-well plate at a concentration of 3 × 10 ⁵ cells / ml in IMDM-5, 3) in IMDM-5 using specific Ag (OVA) Pulsed.
b) Ex vivo generation and proliferation of certain functionally committed FOXP3-expressing CD3 ⁺ TCRαβ ⁺ MHCII restricted T cells: On day 0, ovalbumin pulsed tDC was washed 1) twice with IMDM-5, 2) Add to wells of a 48-well plate in the presence of 2 × 10 ⁵ irradiated self-feeders, PGE2 1 μM, and Rapa 10 nM at a concentration of 3 × 10 ⁵ cells / ml in IMDM-5. Purified naïve conventional CD4 ⁺ T cells (isolated from PBMC previously frozen by FACS) are added to pulsed tDC. On day 1, IL-2 (100 IU / ml) and TGFβ (5 ng / ml) are added to the co-culture. Every 3 days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI / ml (T cell cloning medium)). On day 12, these T cells are further expanded by restimulation with ova-pulsed tDC in the presence of ΔCD3-feeder, PGE2 1 μM, TGFβ 5 ng / ml, Rapa 10 nM, IL-2 (100 UI / ml). Once T cells begin to grow, they can be split every 2-3 days using T cell cloning media and irradiation feeders. On day 21, cells are analyzed by flow cytometry. 85% of stimulated naive conventional CD4 ⁺ T cells that became CD45RO ⁺ express Foxp3 ⁺ . Ova-specific memory CD3 ⁺ TCRαβ ⁺ MHCII restricted T cells are committed to exert exclusively regulatory activity after 21 days of growth in nTreg polarized medium in any culture condition of stimulation To confirm the Ova-specific pTreg was obtained using nTreg polarization medium (containing a combination of IL-2, TGFβ, PGE2, and rapamycin) or TH-17 polarization medium (IL-2, IL-1, IL- 6 and IMDM medium containing IL-21 and IL-23 cytokine). Foxp3-expressing CD3 ⁺ CD4 ⁺ TCRαβ ⁺ MHCII restricted T cells grown for 21 days were stimulated every 3 days with plate-bound anti-CD3 mAb (4 μg / ml) in the presence of ΔCD3 feeder (1 M) in a 48-well plate Discard half of the clear volume and replace with fresh T cell cloning medium or TH-17 polarization medium for 21 days.

Generation of functionally committed FOXP3-expressing CD3 ⁺ TCRγδ ⁺ unrestricted T cells ex vivo:

In vitro generation of tumor-bearing tolerogenic DC (referred to as tolerogenic monocyte-derived DC (tDC)) from CD14 ⁺ monocytes: monocytes were transformed into 100 ng / ml recombinant human granulocyte-macrophage colony stimulating factor ( Incubate in 48 well flat bottom plates containing 0.5 ml AIMV per well supplemented with GM-CSF) and 10 ng / ml human recombinant IL-4. On day 3, 500 μl medium containing cytokines is added. On day 5, a portion of tDC was apoptotic MCF-7 over 24 hours at a DC / tumor cell ratio of 1: 2 in AIMV with GM-CSF (100 ng / mL), IL-4 (10 ng / mL). Co-culture with cells. Another portion of tDC is frozen at 2 × 10 ⁶ cells / vial in 90% FBS-10% DMSO.

Ex vivo generation and proliferation of tumor phosphoantigen specific functionally committed Foxp3 expressing CD3 ⁺ TCRγδ + non-restricted T cells.

On day 0, tumor antigen pulse tDC was washed 1 × with IMDM-5 twice, 2) 3 × in IMDM-5 in the presence of 2 × 10 ⁵ irradiated self-feeders, PGE2 1 μM, and Rapa 10 nM. Add to wells of a 48-well plate at a concentration of 10 ⁵ cells / ml. Purified CD3 ⁺ CD45RA ⁺ TCRγδ ⁺ non-restricted T cells (isolated from PBMC previously frozen by FACS) are added to pulsed tDC. On day 1, IL-2 (100 IU / ml) and TGFβ (5 ng / ml) are added to the co-culture. Every 3 days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 (100 UI / ml) with IL-2 (T cell cloning medium). On day 12, these T cells are further expanded by restimulation with tumor Ag pulse tDC in the presence of ΔCD3 feeder, PGE2 1 μM, TGFβ 5 ng / ml, Rapa 10 nM, and IL-2 (100 UI / ml). . Once T cells begin to grow, they can be split every 2-3 days with T cell cloning media and irradiation feeders. On day 21, cells are analyzed by flow cytometry. 75% of stimulated naive CD3 ⁺ CD45RA ⁺ TCRγδ ⁺ T cells that became CD45RO ⁺ express Foxp3 ⁺ .

Generation of tumor antigen-specific functionally committed FOXP3-expressing CD3 ⁺ invTCR Vα24 ⁺ CD1d restricted T cells ex vivo.
a) In vitro generation of tumor-bearing tolerogenic DC from CD14 ⁺ monocytes (referred to as tolerogenic monocyte-derived DC (tDC)): for the generation of immature DC expressing CD1d 48-well flat bottom plate containing 0.5 ml AIMV per well supplemented with 100 ng / ml recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and 10 ng / ml human recombinant IL-4 and AM580 (100 nM) Incubate in. On day 3, 500 μl medium containing cytokines is added. On day 5, a portion of tDC was transferred to apoptotic MCF- for 24 hours at a DC / tumor cell ratio of 1: 2 in AIMV with GM-CSF (100 ng / mL), IL-4 (10 ng / mL). Co-culture with 7 cells. Other portions of tDC are frozen in 90% FBS-10% DMSO at 2 × 10 6 / vial.
b) Ex vivo generation and proliferation of tumor antigen-specifically functionally committed Foxp3-expressing CD3 ⁺ invTCR Vα24 ⁺ CD1d restricted T cells on day 0: 1) Tumor antigen pulse tDC 2) with IMDM-5 Wash 2 times and add to wells of a 48-well plate at a concentration of 3 × 10 ⁵ cells / ml in IMDM-5 in the presence of 2 × 10 ⁵ irradiation self-feeders, PGE2 1 μM, and Rapa 10 nM. Purified CD3 ⁺ CD45RA ⁺ invTCR Vα24 ⁺ CD1 restricted T cells (isolated from PBMC previously frozen by FACS) are added to pulsed tDC. On day 1, IL-2 (100 IU / ml), IL-15 (10 ng / ml), and TGFβ (5 ng / ml) are added to the co-culture. Every 3 days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 (T cell cloning medium) with IL-2 (100 UI / ml) and IL-15 (10 ng / ml). On day 12, these T cells were transformed into tumor Ag pulse tDC in the presence of ΔCD3 feeder, PGE2 1 μM, TGFβ 5 ng / ml, Rapa 10 nM, IL-2 (100 UI / ml), and IL-15 (10 ng / ml). Further growth by restimulation with Once T cells begin to grow, they can be split every 2-3 days using T cell cloning media and irradiation feeders. On day 21, cells are analyzed by flow cytometry. 75% of stimulated CD3 ⁺ CD45RA ⁺ invTCR Vα24 ⁺ cells that became CD45RO ⁺ express Foxp3 ⁺ .

Ex vivo generation of phosphoantigen-specific functionally committed FOXP3-expressing CD3 ⁺ TCRγδ ⁺ unrestricted T cells.
a) In vitro generation of tolerogenic DC from CD14 ⁺ monocytes (referred to as tolerogenic monocyte-derived DC (Tol-Mo-DC)): monocytes are 100 ng / for generation of immature DC Culture in 48-well flat bottom plates containing 0.5 ml AIMV per well supplemented with ml recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and 10 ng / ml human recombinant IL-4. On the third day, 500 μl of medium containing cytokines was added. On day 6, the produced Tol-Mo-DC was removed from the well and IMDM-5 (5% SVF, 100 IU / ml penicillin / streptomycin, 1 mM sodium pyruvate, 1 mM non-essential amino acids, glutamax, and 10 mM HEPES added. Washed twice with IMDM) and frozen, or used for the generation and expansion of functionally committed FOXP3-expressing CD3 ⁺ TCRγδ ⁺ non-restricted T cells specific for phosphoantigen.
b) Phosphoantigen-specific functionally committed FOXP3-expressing CD3 ⁺ TCRγδ ⁺ ex vivo generation and proliferation: on day 0, 2 × 10 ⁵ irradiated self-feeders, PGE2 Add to wells of a 48-well plate at a concentration of 3 × 10 5 / ml in IMDM in the presence of 1 μM and Rapa 10 nM and zoledronic acid (100 nM). Purified CD3 ⁺ CD45RA ⁺ TCRγδ ⁺ unrestricted T cells (isolated from PBMC previously frozen by FACS) are added to pulsed tDC. On day 1, IL-2 (100 IU / ml), IL-15 (10 ng / ml), and TGFβ (5 ng / ml) are added to the co-culture. Every 3 days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 (T cell cloning medium) with IL-2 (100 UI / ml) and IL-15 (10 ng / ml). On day 12, these T cells were removed in the presence of ΔCD3 feeder, PGE2 1 μM, TGFβ 5 ng / ml, Rapa 10 nM, IL-2 (100 UI / ml), IL-15 (10 ng / ml), and zoledronic acid (100 nM). Further growth by restimulation with tDC. Once T cells begin to grow, they can be split every 2-3 days using T cell cloning media and irradiation feeders. On day 21, cells are analyzed by flow cytometry. 75% of stimulated CD3 ⁺ CD45RA ⁺ TCRγδ ⁺ T cells that became CD45RO ⁺ express Foxp3 ⁺ .

In vitro generation of stimulator cells for MLR assay: Monocytes were transformed into 20 ng / ml recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and 20 ng / ml for generation of immature DC (iDC). Incubate in 48 well flat bottom plates containing 0.5 ml RPMI-5 per well supplemented with human recombinant IL-4. On day 3, 500 μl medium containing cytokines is added. On day 5, a portion of iMDC was transferred to RPMI 1640 supplemented with GM-CSF (20 ng / mL), IL-4 (20 ng / mL), and 5% FBS for a DC / tumor cell ratio of 1: 2 Co-culture with apoptotic MCF-7 cells. Another portion of iDC is frozen at 2 × 10 ⁶ cells / vial (in 90% FBS-10% DMSO). When indicated, pulsed DCs are matured (mDC) for 2 days with tumor necrosis factor α (TNF-α; final 20 ng / mL) and PGE 2 (1 μM). In some experiments, TNF and PGE2 (at the same concentration), or lipopolysaccharide (LPS; 10-1000 ng / mL; Sigma) is added directly to the MLR. Irradiate the antigen-loaded DC stimulator at 30 Gy.

In vitro generation of TAP inhibitory stimulator cells for MLR assays: Mature DCs obtained as described above were used with 20 μg RNA synthesized from the pGem4Z vector containing the UL49.5 gene from BHV-1. Electroporation (reference: Lampen MH, Verweij MC, Querido B, van der Burg SH, Wiertz EJ, van Hall T). CD8 ⁺ T cell responses to TAP-inhibiting cells are readily detected in the human population (J Immunol. 2010 Dec 1; 185 (11): 6508-17.).

  Apoptotic T-cell-DC co-culture: immature DCs were cultured for 16 hours alone or with apoptotic cells (3 apoptotic cells: 1 iDC). DCs were then purified by immunomagnetic depletion of apoptotic T cells using anti-CD3 coated microbeads (Miltenyi Biotec) and electroporated with or without 20 μg synthetic RNA, 20 ng / ml GM- Incubated for 24 hours in RPMI-5 supplemented with CSF, 20 ng / ml human recombinant IL-4, and maturation cocktail (TNF-α 20 ng / ml and PGE2 1 μM).

  Flow cytometry analysis

mAb labeling. The following conjugated mAb is used. a) For CD3 ⁺ T cells: anti-CD4 (SK3) -PerCP-eFluor 710, anti-TCRαβ (IP26) -APC (ebioscience), anti-CD25 (B1.49.9) -PeCy55, anti-CD127 (R34.34)- APC-AF700 (Beckman Coulter), anti-CD3 (UCHT1) -BB515 anti-invariant TCR Vα24-JaQ (6B11) -PE, anti-Foxp3 (259D / C7) -PE-CF594 and anti-CD152 (BNI3) -BV421, anti-CD161 (DX12) BV605 and anti-CD56 (NCAM 16.2) BU395 (Becton Dickinson), anti-TCRαβ-BV421 (IP26) (Biolegend), anti-TCR pan-γδ ⁺ PE (IMMU510) (Beckman Coulter) and anti-CD27-APC fluor 780 (O323) (eb ioscience). Cells are stained for surface markers using a mixture of Ab diluted in PBS (FACS buffer) containing BSA / NaN ₃ (0.5% BSA, 0.01% NaN ₃ ) (in the dark, 30 minutes at 4 ° C). Intracellular staining of Foxp3 and CTLA-4 is performed using the FOXP3 staining kit obtained from ebioscience according to the manufacturer's instructions. An appropriate isotype control Ab is used for each staining combination. Samples are acquired on a BD LSR FORTESSA flow cytometer using BD FACSDIVA 8.0.1 software (Becton Dickinson). Results are expressed as percentage (%) or mean fluorescence intensity (MFI).
b) For inducible specific Treg: The presence of IL-1R1 in the inducible Treg is assessed using monoclonal anti-Foxp3 (259D / C7) -PE-CF594 Ab and polyclonal anti-IL1R1-PE (R & D Systems, FAB269P) did.

CFSE staining. Tconv is stained with 1 μM carboxy-fluorescein succinimidyl ester (CFSE) (CellTrace Cell Proliferation Kit; Molecular Probes / Invitrogen) in PBS for 8 minutes at a concentration of 1 × 10 ⁷ cells / mL at 37 ° C. . Labeling is stopped by washing the cells twice with RPMI 1640 culture medium containing 10% FBS. The cells are then resuspended at the desired concentration and then used for proliferation assays.

7-AAD (7-amino-actinomycin D) staining. Apoptosis of stimulated CFSE-labeled or unlabeled nTreg and Tconv was determined using a 7-AAD assay. Briefly, cultured cells are stained with 20 μg / mL nuclear dye 7-AAD (Sigma-Aldrich) for 30 minutes at 4 ° C. The FSC / 7-AAD dot plot, apoptosis ^{(FSC high / 7-AAD +} ) cells and apoptotic bodies ^{(FSC low / 7-AAD +} ) and strips ^{((FSC low / 7-AAD} - survive) (FSC ^high / 7-AAD ⁻ ), and live cells are identified as CD3 ⁺ 7-AAD ⁻ FSC ⁺ cells.

Phenotypic characteristics of Foxp3-expressing CD3 ⁺ CD4 ⁺ γδ T cell non-restricted T cells isolated from BC biopsy: Identification of TCR γδ T cell subsets was performed by flow cytometry. Panels show antibodies against CD3 (clone SK7), CD4 (clone SK3), CD8 (clone SK1), pan-γδT (clone IMMU510), TCR Vδ1 (REA173), TCR Vδ2 (REA771), and Foxp3 ⁺ (clone 259D) Was included.

  Functional assay

T cell proliferation. T cell proliferation is assessed in normoxia in RPMI supplemented with 5% FBS, 100 IU / ml penicillin / streptomycin, 1 mM sodium pyruvate, 1 mM non-essential amino acids, glutamax, and 10 mM HEPES (RPMI-5 medium). . At the completion of co-culture, stimulated CFSE-labeled Tconv is collected, co-stained with anti-CD3 mAb and 7-AAD, and percentage of viable proliferating cells in gated CD3 ⁺ 7-AAD− cells (defined as CFSE low fraction) Is determined by flow cytometry.

Induction of T cell apoptosis: Tumor antigen-specific functionally committed FOXP3-expressing CD3 ⁺ TCRγδ ⁺ unrestricted T cells are generated ex vivo as described above. Tumor antigen specific stimulated T cells were then irradiated at 254 nm (UV-C) (240 mJ / cm ² ) and cultured for 6 hours prior to co-culture with immature DCs. Apoptosis was confirmed by 7-AAD staining. On average, 75% of the cells are 7-AAD ⁺ .

Standard polyclonal cell-cell contact Treg suppression assay: CFSE-labeled Tconv (4 × 10 ⁴ cells / well) used as responder cells and a defined amount of Foxp3 T cells (blood Treg or ex vivo generated T cells) Incubate with ΔCD3-feeders (4 × 10 ⁴ cells / well) in the presence or absence for 4-5 days. Culturing is performed in round bottom plates coated with 0.2 μg / mL anti-CD3 mAb in 200 μL complete RPMI medium. Results are expressed as a percentage of proliferating CFSE low T cells or as a percentage of suppression calculated as follows: (100 × [(percent Tconv CFSE low cells−percent Tconv CFSE in co-culture with nTregs)) / Tconv CSFE low cell percentage].

Autologous MLR inhibition assay: CFSE-labeled Tconv CD4 ⁺ CD25 ⁻ T cells (5 × 10 ⁴ ) using 1 × 10 ⁴ pulsed iDC in RPMI-5 medium or IL-2 (20 IU / ml) 5 × 10 ³ in IMDM-5 medium supplemented with IL-1b (10 ng / ml), IL-6 (30 ng / ml), IL-21 (50 ng / ml), and IL-23 (30 ng / ml) Stimulation for 5-6 days in the presence or absence of a defined amount of Foxp3 T cells (blood Tregs or ex vivo generated T cells). Where indicated, cultures were IL-2 (20 IU / ml), IL-1β (10 ng / ml), IL-6 (30 ng / ml), IL-21 (50 ng / ml), and IL-23 (30 ng). / ml) in IMDM-5 medium. Results are expressed as a percentage of proliferating CFSE low T cells or as a percentage of suppression calculated as follows: (100 × [(percent Tconv CFSE low cells−percent Tconv CFSE in co-culture with nTregs) / Tconv CSFE low cell percentage).

Classical 7-AAD / CFSE cell-mediated cytotoxicity assay: Target cells were labeled with CFSE as described above and placed in triplicate at 3 × 10 ⁴ cells / well in a 96-well U-bottom plate. CD8 ⁺ effector T cells (5: 1 E: T ratio) were added and incubation was carried out at 37 ° C. for 6 hours. At the end of the experiment, dead cells were labeled with 7-AAD to detect lysed cells. Cytolytic activity against target cells was analyzed using a FACSCalibur instrument based on areas showing double positive staining CFSE and 7-AAD. The cytolytic activity (%) of CD8 ⁺ T cell clones was calculated as positive cells / total CFSE positive cells for both CFSE and 7-AAD after subtracting spontaneous lysis (%) in the negative control. The percentage of cytolytic activity was then calculated using the following formula: cytolytic activity (%) [dead target cells (%) − natural death (%)] × 100 / [100−natural death (%) ]].

  DNA methylation measurement: Classically, a stable Treg gene signature is a highly demethylated CpG island within the conserved non-coding sequence 2 (CNS2) of the Treg-specific demethylation region (TSDR) Made up of. DNA methylation analysis of the TSDR region of gene FOXP3 was performed as described previously by Christopher Fuhrman (Fuhrman et al, Divergent Phenotypes of Human Regulatory T Cells Expressing the Receptors TIGIT and CD226, 2015, Journal of immunology). DNA was evaluated by quantitative PCR after bisulfite treatment. Briefly, nucleotides were isolated using the AllPrep DNA / RNA Mini Kit (Qiagen) or DNeasy tissue kit (Qiagen) as appropriate. Bisulfite treatment of genomic DNA was performed on 500 ng of DNA using EZ DNA Methylation Kit (Zymo Research). DNA standards are derived from unmethylated bisulfite converted human EpiTect control DNA (Qiagen) or universal methylated bisulfite converted human control DNA (Zymo Research). To obtain abundant standards, TSDR was PCR amplified using the following reaction: 25 μl of ZymoTaq PreMix buffer (Zymo Research) and 0.5 μM each of primer FOXP3_TSDRfwd (5′-ATATTTTTAGATAGGGATATGGAGATGATTTGTTTG-3′-sequence Reaction volume of 50 μM containing No. 1) and FOXP3_TSDRrev (5′-AATAAACATCACCTACCACATCCACCCAACAC-3′-SEQ ID No. 2). After incubation at 95 ° C for 10 minutes, amplification was performed as follows: 50 cycles of 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute. The amplified PCR product was purified using the QIAquick Gel Extraction Kit (Qiagen). The concentration of purified control TSDR DNA was determined using a GE NanoVue spectrophotometer (GE Healthcare Life Sciences). TSDR real-time PCR was performed with probes targeting methylated or demethylated target sequences. The reaction was performed in a 96 well white tray using the Roche LightCycler 480 system (Roche Diagnostics). Each reaction contained 10 μl of LightCycler 480 Probes Master Mix (Roche), 10 ng of bisulfite converted DNA sample or standard, 1 μM of each primer, and 150 nM of each probe, with a final reaction value of 20 μl. It was. The probes used for amplification were TSDR-forward 5'-GGTTTGTATTTGGGTTTTGTTTTATAGT-3 '(SEQ ID NO: 3) and TSDR-reverse 5'-CATAAAATAAAATATCTCACCCTCTTCCTTCCTCT-3' (SEQ ID NO: 4). The probe for detecting the target sequence was a FAM-labeled methylated probe, FAM-CGGTCGGATGCGTC-MGB-NFQ (SEQ ID NO: 5), or a VIC-labeled unmethylated probe, VIC-TGGTGGTTGGATGTGTTG-MGB-NFQ (SEQ ID NO: 6). . All samples were tested in triplicate. The protocol for real-time amplification is as follows: After initial denaturation at 95 ° C for 10 minutes, the samples were subjected to 50 cycles of 95 ° C for 15 seconds and 61 ° C for 1 minute. Fourteen different ratios of fully methylated and demethylated templates were used as real time standards. A 6th order polynomial was used to estimate the percentage of cells demethylated with TSDR for each sample.

  Measurement of histone acetylation: Histone acetylation analysis of four different sites of the FOXP3 gene was evaluated by the ChIP assay as previously described by Ling Lu (Ling Lu et al, PNAS 2014). Briefly, 50,000 cells of each treated nTreg cell sample were collected and cross-linked with 1% formaldehyde, then 120 μL lysis buffer [50 mM Tris.HCl, pH 8.0, 10 mM EDTA, 1% (Wt / vol) SDS, protease inhibitor mixture (1: 100 dilution; Sigma), 1 mM PMSF, 20 mM Na butyrate]. The chromatin in the lysate was sonicated to a 500-800 bp fragment and then 800 μL of RIPA ChIP buffer [10 mM Tris.HCl, pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% ( vol / 1) Triton X-100, 0.1% (wt / vol) SDS, 0.1% (wt / vol) Na-deoxycholic acid, protease inhibitor mixture (1: 100 dilution; Sigma), 1 mM PMSF And 20 mM Na butyrate]. Dynabeads Protein G (10 μL; Invitrogen) was incubated separately for 2 hours with 1 μg H3K4me3 (Abcam) or H3K9ac (Cell Signaling) or normal rabbit IgG negative control ChIP grade antibody. Next, 100 μL of sheared chromatin was immunoprecipitated separately from the pretreated antibody-bead complex and another 100 μL of fragmented chromatin for total input DNA extraction. The immunoprecipitated DNA was quantified by real-time PCR using the following primers: promoter, 5′-ACC GTA CAG CGT GGT TTT TC-3 ′ (SEQ ID NO: 7) and 5′-CTA CCT CCC TGC CATC CCT CT 3 '(SEQ ID NO: 8); CNS1, 5'-CCC AAG CCC TAT GTG TGATT-3' (SEQ ID NO: 9) and 5'-GTG TGT CAG GCC TTG TGC TA-3 '(SEQ ID NO: 10); CNS2, 5′-GTC CTC TCC ACAACC CAA GA-3 ′ (SEQ ID NO: 11) and 5′-GAC ACC ACG GAG GAA GAG AA-3 ′ (SEQ ID NO: 12); CNS3, 5′-AGG TGC CGA CCT TTA CTG TG- 3 ′ (SEQ ID NO: 13 ) And 5'-ACA ATA CGG CCT CCT CCT CT-3 '(SEQ ID NO: 14).

  result

a) Induction of Foxp3 ⁺ expression in ex vivo human-induced tumor antigen-specific CD4 ⁺ TCRγδ non-restricted T cells.

Optimal conditions are set up to induce tumor antigen-specific Foxp3 ⁺ expressing CD4 ⁺ TCRγδ non-restricted T cells, as previously described. FIG. 2 shows that apoptotic tumor antigen pulsed immunotolerogenic DC (“Tumor Ag-loaded tDC”) in the presence of IL-2 and nTreg polarized medium composed of TGFβ, PGE2, and rapamycin is antigen specific. High levels of Foxp3 ⁺ expression can be induced in non-stimulated naïve conventional CD4 ⁺ T cells (“naive Treg”) (frequency in FIG. 2A and MFI in FIG. 2B), unpulsed tDC (“unloaded tDC ")" Indicates that Foxp3 ⁺ expression cannot be induced in naive conventional CD4 ⁺ T cells in the presence of the same polarized medium.

b) Specific mobilization of pathogenic CD4 ⁺ γδ T cells expressing Foxp3 in human breast cancer.

  A novel subset of γδ T cells showing expression of CD4 and Foxp3 has been identified in TIL isolated from breast cancer (BC) biopsies. Indeed, although γδ T cells expressing Foxp3 are rare in PBMCs from normal individuals (<1%), they are strongly enriched in TIL purified from BC biopsies as shown in FIG. Yes (about 10 times).

In general, human γδ T cells are divided into two major structural subsets according to their TCRδ chain usage: Vδ1 and Vδ2 T cells (Vδ1 is the dominant tissue resident cell, whereas Vδ2 is the major subset in peripheral blood. The present inventors have examined the TCR Vδ chain in this new CD4 ⁺ Foxp3 ⁺ γδT subset by flow cytometry. FIG. 4 shows that the majority (> 85%) of pathogenic CD4 ⁺ γδT cells expressing Foxp3 are Vδ1 neg Vδ2 neg.

We next evaluated the ability of Foxp3-expressing CD3 ⁺ CD4 ⁺ γδ T cells unrestricted T cells isolated from BC biopsies to suppress function. FIG. 5 shows that, like fresh Tregs, these CD4 ⁺ γδ T cells expressing Foxp3 ⁺ exhibit inhibitory activity when using a standard polyclonal cell-cell contact Treg inhibition assay.

c) Induction of autologous CD8-mediated T cell response to tumor antigen-specific FOXP3-expressing CD4 ⁺ TCRγδ unrestricted T cells.

Pathogen used to load dendritic cells by establishing a culture system in which inflammatory DC loaded with apoptotic pathogenic CD4 ⁺ T cells co-cultured with autologous CD3 ⁺ naive T cells (inf DC) Generation of a CD8 ⁺ T cell line against sex CD4 ⁺ T cells can be induced. FIG. 6 shows their specific targets, their two targets by two CD8 ⁺ clones induced with apoptotic pathogenic CD4 ⁺ T cells loaded with -inf DC ("mDC") or -TAP-inhibited DC, respectively. FIG. 5 shows that inducible pathogenic CD4 ⁺ T cell clones can be lysed. However, when both CD8 ⁺ clones are tested against the autologous EBV cell line, they cannot lyse this target (FIG. 6).

d) Presence of Foxp3 ⁺ expressing T cells in tumor infiltrating lymphocytes (TIL) isolated from lumen B breast cancer.

Lumen A and B subtypes are both estrogen receptor positive (ER +) and low grade, lumen A tumors grow very slowly, and lumen B tumors grow more rapidly. Lumen A tumors have the best prognosis. Lumen B tumors are associated with poor clinical outcomes. The present inventors, the phenotype of lymphocytes in TIL isolated from both lumens subtypes of breast cancer was verified by flow ^cytometry, CD3 ⁺ CD4 ^{+ TCRαβ +} MHCII restriction and ^{CD3 + CD4} + ^TCRγδ + The presence of Foxp3 expression in non-restricted T cells was found. Foxp3 was not detected in TIL extracted from luminal A breast tumors (FIG. 7). Furthermore, a positive correlation is observed between a high percentage of Foxp3 expression in CD3 ⁺ CD4 ⁺ TCRγδ ⁺ non-restricted T cells and poor clinical outcome in breast cancer (FIG. 8).

Foxp3 expression ^{CD3 + CD4} + ^TCRαβ + MHCII-restricted T cells and Foxp3 expression ^{CD3 + TCRαβ} + unrestricted T cells, respectively, equivalent to approximately 20% and ^{CD3 + TCRγδ} + 23% of the ^{CD3 + TCRαβ} + T cells in the test sample To do. Foxp3 expression ^{CD3 + TCRγδ} + T ^cells, present the same phenotype profile and Foxp3 ⁺ CD3 ⁺ TCRαβ ⁺ T cells. These Foxp3 ⁺ TCRγδ ⁺ T cell ^population, Foxp3 ⁺ CD3 ^{+ TCRαβ +} T cell levels and similar levels Foxp3, CD25, and express CTLA4 (Figure 9).

d) Ex vivo production and expansion of specific CD3 ⁺ TCRγδ ⁺ expressing Foxp3 committed exclusively to exert regulatory activity.

Tests have shown that the ability of antigen-specific Tregs to suppress is much greater than that of polyclonal Tregs, and so we have exclusively regulated activity under any stimulus culture conditions. A method was set up for antigen-specific Foxp3 expressing CD3 ⁺ TCRγδ ⁺ non-restricted T cells committed and ex vivo.

FIG. 10 shows naive CD3 ⁺ TCRγδ ⁺ T cells (CD3 ⁺ CD45RA) stimulated with zoledronic acid-treated auto-tDC in the presence of nTreg polarized medium containing a combination of IL-15, IL-2, TGFβ, PGE2, and rapamycin. ⁽⁺ CD27 + TCRγδ ⁺ T cells) express Foxp3 after 21 days of growth and show significant functional inhibitory activity as assessed by standard polyclonal cell-cell contact Treg inhibition assay. Interestingly, FOXP3-expressing CD3 ⁺ TCRγδ ⁺ T cells grown for 21 days maintain their Foxp3 levels and their suppressive activity after an additional 21 days of culture in nTreg polarization medium.

Claims (18)

A method for ex vivo generation of γδFoxp3 ⁺ regulatory T cells having the following phenotype: CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ , comprising: γδT cell activator and the following agents: i) cAMP (cyclic) Adenosine monophosphate) activator, ii) TGFβ (transforming growth factor beta) pathway activator, iii) mTOR inhibitor, optionally iv) group of IL-2, IL-7, IL-15, and TSLP Culturing CD3 ⁺ TCRγδ ⁺ T cells for at least 5 days in the presence of at least one cytokine selected in and optionally v) at least one TET enzyme activator and / or at least one DNMT inhibitor.   The method according to claim 1, wherein the γδ T cell activator is a polyclonal γδ T cell activator, preferably an anti-TCRγδ antibody or a non-peptide phosphoantigen.   The γδ T cell activator is an antigen-specific γδ T cell activator, preferably a tolerogenic dendritic cell (DC), pulsed with at least one bisphosphonate, preferably at least one aminobiphosphonate. The method of claim 1.   The cAMP activator is selected from the group comprising a prostaglandin E2 (PGE2), an EP2 or EP4 agonist, a membrane adenine cyclase activator, or a metabotropic glutamate receptor agonist. The method described in the paragraph.   The TGFβ pathway activator is selected from the group comprising TGFβ, bone morphogenetic protein (BMP), growth and differentiation factor (GDF), anti-Muellerian hormone (AMH), activin, and nodar. The method according to any one of the above.   mTOR inhibitors are rapamycin, rapamycin analog, wortmannin; theophylline; caffeine; epigallocatechin gallate (EGCG), curcumin, resveratrol; genistein, 3,3-diindolylmethane (DIM), LY294002 (2 -(4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one), PP242, PP30, Torin1, Ku-0063794, WAY-600, WYE-687, WYE-354, GNE477, NVP-BEZ235, 6. The method of any one of claims 1-5, selected from the group comprising PI-103, XL765, and WJD008. The γδ Foxp3 ⁺ regulatory T cells obtained by the production method according to claims 1 to 6 are converted into γδ T cell activator and the following agents: i) cAMP (cyclic adenosine monophosphate) activator, ii) TGFβ (transformation) Growth factor beta) pathway activator, iii) mTOR inhibitor, optionally iv) at least one cytokine selected in the group of IL-2, IL-7, IL-15, and TSLP, and optionally v) at least 7. The method according to any one of claims 1 to 6, further comprising culturing for at least 5 days in the presence of one TET enzyme activator and / or at least one DNMT inhibitor. A gamma delta Foxp3 ⁺ regulatory T cell population generated ex vivo obtainable by the method of any one of claims 1-6. The γδ Foxp3 ⁺ regulatory T cell population generated and expanded ex vivo obtainable by the method of claim 7, wherein the γδ Foxp3 ⁺ regulatory T cells have the following phenotype: CD3 ⁺ TCR γδ ⁺ Foxp3 ⁺ . GanmaderutaFoxp3 ⁺ regulatory T cells, the following ^phenotype: CD3 ^{+ TCRγδ} + Foxp3 + having remains functionally stable in inflammation conditions, GanmaderutaFoxp3 ⁺ regulatory T cell population generated ex vivo. 11. The ex vivo generated γδ Foxp3 ⁺ regulatory T cell population of claim 10, wherein the regulatory T cell population has the following phenotype: CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ IL-1R1 ⁻ . The following ^phenotype: CD3 ^{+ TCRγδ} + Foxp3 + inactivated GanmaderutaFoxp3 ⁺ regulatory T cells with, or following ^phenotype: CD3 ^{+ TCRγδ} + Foxp3 has a ^{+ γδFoxp3 +} regulatory T cells of blebs, or following phenotype : An immunogenic product comprising an immunogenic dendritic cell loaded with a bleb of γδFoxp3 ⁺ regulatory T cells with CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ . The following ^phenotype: CD3 ^{+ TCRγδ} + Foxp3 + inactivated GanmaderutaFoxp3 ⁺ regulatory T cells with, or following ^phenotype: CD3 ^{+ TCRγδ} + Foxp3 has a ^{+ γδFoxp3 +} regulatory T cells of blebs, or following phenotype A pharmaceutical composition comprising an immunogenic dendritic cell loaded with a bleb of γδFoxp3 ⁺ regulatory T cells with CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ and at least a pharmaceutically acceptable excipient. The following ^phenotype: CD3 ^{+ TCRγδ} + Foxp3 + inactivated GanmaderutaFoxp3 ⁺ regulatory T cells with, or following ^phenotype: CD3 ^{+ TCRγδ} + Foxp3 has a ^{+ γδFoxp3 +} regulatory T cells of blebs, or following phenotype A vaccine composition comprising: an immunogenic dendritic cell loaded with a bleb of γδFoxp3 ⁺ regulatory T cells with CD3 ⁺ TCRγδ ⁺ Foxp3 ⁺ , and at least one adjuvant.   13. An immunogenic product, pharmaceutical composition, or vaccine composition according to any one of claims 10-12 for use in treating cancer. The following ^{phenotype: CD3 + TCRγδ + Foxp3 + γδFoxp3} + regulatory T cells with, and pharmaceutical compositions comprising at least one pharmaceutically acceptable excipient.   16. A pharmaceutical composition according to claim 15 for use in cell therapy.   16. A pharmaceutical composition according to claim 15 for use in treating inflammatory or autoimmune diseases or for preventing transplant rejection or graft-versus-host disease (GVHD).