EP4731233A1 - Extracellular vesicles from regulatory b cells - Google Patents

Abstract

The present invention relates to extracellular vesicles derived from regulatory B cells, and the use thereof in therapy.

Description

EXTRACELLULAR VESICLES FROM REGULATORY B CELLS FIELD OF INVENTION [0001] The present invention relates to extracellular vesicles derived from regulatory B cells, and the use thereof in therapy. BACKGROUND OF INVENTION [0002] Regulatory B cells constitute a subset of B cells capable of suppressing immune responses through the secretion of cytokines, such as IL-10, IL-35 and TGF-β, and other soluble factors, such as granzyme B, as well as through the expression of cell surface molecules such as CD1d, TIM-1, FasL and PD-L1 or PD-1. Breg cells have been shown to modulate allergy, transplantation, cancer, infections and chronic metabolic diseases. [0003] In particular, Breg cells have been hypothesized to contribute to the maintenance of long-term graft function, after identification of a higher absolute number of granzyme B positive B cells in patients with operationally tolerant kidney grafts (i.e., tolerant patients without immunosuppressive treatment). In these patients, Breg cells have been shown to inhibit T cell proliferation and induce T cell apoptosis partially through granzyme B, two mechanisms which may contribute to induce a tolerogenic environment. [0004] These results suggest a potential therapeutic role of Breg cells for inducing and/or maintaining a pro-tolerogenic environment. However, use of Breg cells in cell therapy is prevented because of high risks of immunogenicity and embolism and because of the limited amount of biological material, complex production and storage. [0005] Extracellular vesicles are sub-cellular membrane-delimited particles secreted by almost all cell types into the extracellular space. [0006] As the gradual understanding of extracellular vesicles evolved over decades and different fields, they were first identified following categories based on their biogenesis or function. As such, vesicles of endosomal origin released by the fusion between multivesicular bodies (MVB) and the plasma membrane were named “exosomes”. Vesicles directly shed from membrane budding were described as “microvesicles”, and vesicles generated upon apoptosis were designated as “apoptotic bodies”. However, even though intracellular origin may modulate vesicles’ identity and activity, there is no way yet to distinguish and purify these specific subtypes, motivating the use of the broader term “extracellular vesicles” (EVs). It has now become consensual that the term “extracellular vesicles” (EVs) describes all heterogeneous populations of membrane- bound particles, with diameters ranging from 30 nm to a few micrometers, that can be secreted by any cell in a constitutive manner or in response to stimuli. [0007] In practice, EVs have been separated in subpopulations based on their size, differential ultracentrifugation being the more widespread method of isolation. Thus, features reported in the literature are usually attributed to a specific size range: medium/large EVs are pelleted between 2000 g and 10 000 g, while small EVs are collected by ultracentrifugation between 10000 g and 100000 g. [0008] Thus, the three main subtypes of extracellular vesicles are microvesicles, exosomes and apoptotic bodies, discriminated based on their size, content and function. [0009] Extracellular vesicles are found in almost all types of body fluids, such as blood, urine, breast milk, saliva and semen. [0010] Extracellular vesicles display active biomolecules at their surface, but also contain proteins, lipids, metabolites and nucleic acids in their core. [0011] EVs have attracted increasing interest in the last few decades since the first evidence of their role in intracellular communication. Indeed, due to their unique combination of features, comprising active biomolecules on their surface as well as genetic material, soluble proteins and other metabolites sheltered in their core, EVs have the ability to regulate a wide range of biological processes in a pleiotropic manner involving surface receptors or delivery of content. [0012] Extracellular vesicles have been recognized as mediators of intercellular communication through the delivery of effector molecules to targeted cells, tissues and organs. They also have been shown to possess the ability of regulating a wide range of biological processes, such as coagulation, immunity, tissue regeneration and bone calcification. [0013] EVs may be used as biomarkers for the diagnosis of many diseases, including cancer, coronary artery diseases, liver and kidney diseases or neurodegenerative diseases. Remarkably, the access to EVs through non-invasive body fluid collection, as well as their unique composition reflective of cell origin makes them very good candidates for study in liquid biopsies. [0014] In addition to their potential for diagnosis, EVs have been increasingly considered for therapy, as evidence accumulated of their beneficial effect in variety of applications including tissue repair, vaccination, treatment of lysosomal storage disorders or diabetes. [0015] Use of extracellular vesicles in therapy has been contemplated both in the context of drug delivery and regenerative medicine. Indeed, due to their capacity of functioning as endogenous intercellular cargo transfer systems, extracellular vesicles have been studied for use as potential selective vehicles for drug delivery while avoiding off-target effects. Indeed, EVs can be modified by loading specific cargo endogenously or exogenously or by engineering active surface molecules for targeting. Additionally, due to their decreased immunogenicity as compared to their parental cells, likely due to a lower number of MHC molecules at their surface, extracellular vesicles represent an advantageous therapeutic tool. Also, unlike cells, extracellular vesicles exhibit a long storage life, and can be further modified by engineering active surface molecules prolonging their half-life, which represent an advantage as well. [0016] Thus, EVs bring the advantages of a cell free, affordable, “off the shelf” treatment with no risk of uncontrolled replication and the possibility to access privileged areas of the organism even by systemic administration because of their ability to cross biological barriers. [0017] Overall, EVs show great promises as biomarkers and treatment for a wide range of pathologies. [0018] In this context, the Inventors surprisingly demonstrated that regulatory B cells expressing granzyme B are able to secrete extracellular vesicles. Additionally, the Inventors demonstrated that regulatory B cell-derived extracellular vesicles have the ability to inhibit T cell proliferation and to induce cell death. Therefore, the regulatory B- cell derived extracellular vesicles of the present invention represent a valuable therapeutic tool for inducing immune tolerance and in the prevention or treatment of various disease or condition including transplant rejection, graft-versus-host disease (GvHD), or autoimmune disease for example. SUMMARY [0019] The invention relates to an isolated regulatory B cell (Breg)-derived extracellular vesicle wherein said extracellular vesicle comprises Galectin-3. [0020] The invention also pertains to an isolated regulatory B cell (Breg)-derived extracellular vesicle obtained or obtainable by a method comprising: a) culturing B cells in a culture medium with a stimulation cocktail until Breg cells are induced and Breg- derived extracellular vesicles are produced in the culture medium, and b) collecting said Breg-derived extracellular vesicle in the culture medium, wherein said stimulation cocktail comprises soluble human CD40L, class B CpG oligodeoxynucleotide, anti- human IgG/IgM/IgA polyclonal antibodies, human IL-21 and human IL-2. [0021] The invention further relates to an isolated regulatory B cell (Breg)-derived extracellular vesicle obtained or obtainable by using a fluidic system comprising at least one container, a culture medium contained by the container, Breg cells, a culture medium agitator, means for controlling the speed of the agitator adapted for the growth of the Breg cells, wherein the means for controlling the speed of the agitator, the agitator and the shape and dimensions of the container are adapted to the generation of a turbulent flow of the culture medium in the container to exert shear stresses on the Breg cells in order to achieve the production of extracellular vesicles, the Kolmogorov length of the flow being less than or equal to 50 µm. [0022] The invention also pertains to an isolated regulatory B cell (Breg)-derived extracellular vesicle obtained or obtainable by a method comprising: a) culturing Breg cells in a culture medium, b) applying a turbulent flow in the culture medium until Breg- derived extracellular vesicles are produced in the culture medium, wherein the Kolmogorov length of the flow is less than or equal to 50 µm, and c) collecting said Breg- derived extracellular vesicle in the liquid medium. [0023] In some embodiments, the extracellular vesicle comprises Galectin-3. [0024] In some embodiments, the extracellular vesicle comprises Programmed cell Death protein 1 (PD-1). In some embodiments, the extracellular vesicle comprises Programmed cell Death Ligand 1 (PD-L1). [0025] In some embodiments, the extracellular vesicle comprises Tumor Necrosis Factor Receptor 2 (TNFR2). In some embodiments, the extracellular vesicle comprises Tumor Necrosis Factor (TNF). [0026] In some embodiments, the extracellular vesicle comprises at least one marker selected from CD9, CD63, CD81, and Tsg101. [0027] In some embodiments, the extracellular vesicle comprises at least one marker selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, HLA-DQ, CD19, CD20, CD24, CD29, CD40, CD44, and CD45. [0028] In some embodiments, the extracellular vesicle has a diameter ranging from 40 nm to 300 nm. [0029] In some embodiments, the extracellular vesicle is capable of inhibiting T cell proliferation, increasing T cell apoptosis, and/or increasing proinflammatory cytokine secretion by T cells. [0030] The invention also relates to a composition or pharmaceutical composition comprising the isolated Breg-derived extracellular vesicle of the invention, and optionally at least one pharmaceutically acceptable excipient. [0031] In some embodiments, the isolated Breg-derived extracellular vesicle or the composition of the invention is for use as a medicament. [0032] In some embodiments, the isolated Breg-derived extracellular vesicle or the composition of the invention is for use in inducing immune tolerance in a subject in need thereof. [0033] In some embodiments, the isolated Breg-derived extracellular vesicle or the composition of the invention is for use in the prevention, reduction and/or treatment of transplant rejection, graft-versus-host disease (GvHD), autoimmune disease, or abnormal or excessive immune response. [0034] In some embodiments, the isolated Breg-derived extracellular vesicle or the composition of the invention is for use in inhibiting T cell proliferation, increasing T cell apoptosis, and/or increasing proinflammatory cytokine secretion by T cells in a subject in need thereof. [0035] The invention further relates to a kit comprising the isolated Breg-derived extracellular vesicle of the invention, and instructions for use. In some embodiments, the isolated Breg-derived extracellular vesicle of the invention has a suppressive activity. In some embodiments, the isolated Breg-derived extracellular vesicle of the invention has an immuno-suppressive activity. In some embodiments, the isolated Breg-derived extracellular vesicle of the invention induces immune tolerance. DEFINITIONS [0036] In the present invention, the following terms have the following meanings. [0037] The terms “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. [0038] “About” preceding a figure encompasses plus or minus 10%, or less, of the value of said figure. It is to be understood that the value to which the term “about” refers is itself also specifically, and preferably, disclosed. [0039] “Extracellular vesicle” or “EV” refers to heterogenous vesicles formed by budding of the plasma membrane of eukaryotic cells, to the exterior of the cell. Extracellular vesicles are produced by all cells in a constitutive way or in response to stimuli. Extracellular vesicles are heterogeneous in size with diameters ranging from about 10 nm to about 5000 nm, but also in biogenesis pathway or cellular source. Examples of extracellular vesicles include, but are not limited to, microvesicles, microvesicle-like particles, prostasomes, exosomes, dexosomes, texosomes, ectosomes, oncosomes, microparticles, apoptotic bodies. [0040] As used herein, the terms “prevent”, “preventing” and “prevention” refer to prophylactic and preventative measures, wherein the object is to reduce the chances that a subject develop the pathologic condition or disorder over a given period of time. Such a reduction may be reflected, e.g., in a delayed onset of at least one symptom of the pathologic condition or disorder in the subject. [0041] “Subject” is intended to include living organisms in which an immune response can be elicited. Preferably, the term “subject” refers to a warm-blooded animal, more preferably a mammal. The term “mammal” refers here to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a primate, more preferably a human. In some embodiments, a subject may be a “patient”, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of the targeted disease or condition, such as, for example, transplant rejection, graft-versus-host disease (GvHD), autoimmune disease, abnormal or excessive immune response. In some embodiments, the subject is an adult (for example a subject above the age of 18). In some embodiments, the subject is a child (for example a subject below the age of 18). In some embodiments, the subject is a male. In some embodiments, the subject is a female. In some embodiments, the subject is affected, preferably is diagnosed, with transplant rejection, graft-versus- host disease (GvHD), autoimmune disease, abnormal or excessive immune response. In some embodiments, the subject is at risk of developing transplant rejection, graft-versus- host disease (GvHD), autoimmune disease, abnormal or excessive immune response. Examples of risks factor include, but are not limited to, genetic predisposition, or familial history of transplant rejection, graft-versus-host disease (GvHD), autoimmune disease, abnormal or excessive immune response. [0042] “Therapeutically effective amount” refers to the level or amount of Breg- derived extracellular vesicles, as described herein that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of a disease, disorder, or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease, disorder, or condition; (3) bringing about ameliorations of the symptoms of the disease, disorder, or condition; (4) reducing the severity or incidence of the disease, disorder, or condition; or (5) curing the disease, disorder, or condition. A therapeutically effective amount may be administered prior to the onset of the disease, disorder, or condition, for a prophylactic or preventive action. Alternatively, or additionally, the therapeutically effective amount may be administered after initiation of the disease, disorder, or condition, for a therapeutic action. [0043] “Treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. In some embodiments, a subject is successfully "treated" for a disease or disorder if, after receiving a therapeutic amount of Breg-derived extracellular vesicles, according to the present invention, the subject shows at least one of the following: relief to some extent of one or more of the symptoms associated with the disease or disorder to be treated; reduced morbidity and mortality; and improvement in quality-of-life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician. DETAILED DESCRIPTION [0044] The present invention first relates to an isolated regulatory B cell (Breg)-derived extracellular vesicle. [0045] As used herein, the term “extracellular vesicle” or “EV” refers to heterogenous vesicles of cellular origin. Typically, extracellular vesicles are formed by budding of the plasma membrane of eukaryotic cells, to the exterior of the cell. [0046] In some embodiments, the extracellular vesicle refers to a subcellular product. [0047] Such subcellular product typically has a membrane structure, is non-dividing, mobile and migratory. [0048] Extracellular vesicles are typically made up of a membrane of cellular origin which form an intraluminal compartment or core. The extracellular vesicles of the invention may comprise or contain various biological components or biomolecules, either bound to their membrane or GPI anchored (for instance displayed at their surface), or present within their intraluminal compartment. [0049] In some embodiments, the extracellular vesicle comprises lipids, proteins, nucleic acids and/or metabolites. [0050] As used herein, the term “comprises” within the expression “the extracellular vesicle comprises a component” means that said component is either associated to the membrane of the EV (e.g. said component is a transmembrane component or it is anchored or bound to the membrane), for instance displayed at its surface, or is contained within the intraluminal or internal compartment of the EV. [0051] The lipids comprised or associated to the membrane of the extracellular vesicles of the invention for instance include cholesterol, sphingomyelins, phostophatidyl- cholines, phosphatidylserines, phosphatidylethanolamines, phosphatidylinositols, ceramide… [0052] The lipid composition of the membrane of the extracellular vesicles of the invention is typically of 35-60% cholesterol, 8-20% sphingomyelins, 10-30% phostophatidylcholines, 5-15% phosphatidylserines, 1-15% phosphatidylethanolamines, 0.1-5 % phosphatidylinositols. Other lipids, such as e.g. ceramide, may be present in smaller percentages. [0053] The proteins comprised in the extracellular vesicles of the invention for instance include tetraspanins (CD9, CD81, CD63, …), adhesion and targeting proteins (integrins, P-selectin…), proteoglycans, antigen presenting molecule MHC class I, lysosomal- associated membrane proteins (LAMP1/2), heat shock proteins (HSC70, HSP84), cytoskeletal proteins (actin, tubulin), ESCRT components (ALIX, TSG101), signal transduction proteins (syntenin, EGFR…), membrane fusion proteins (Annexin, Rab GTPases), enzymes, or cytokines. [0054] In some embodiments, the extracellular vesicles of the invention comprise tetraspanins (CD9, CD81, CD63, …), adhesion and targeting proteins (integrins, P- selectin…), proteoglycans, antigen presenting molecule MHC class I, and/or lysosomal- associated membrane proteins (LAMP1/2) as transmembrane proteins. [0055] In some embodiments, the extracellular vesicles of the invention comprise heat shock proteins (HSC70, HSP84), cytoskeletal proteins (actin, tubulin), ESCRT components (ALIX, TSG101), signal transduction proteins (syntenin, EGFR…), membrane fusion proteins (Annexin, Rab GTPases), enzymes, and/or cytokines in the lumen of the EVs. [0056] The extracellular vesicles of the invention may also comprise DNA and/or RNA, such as e.g. messenger RNA (mRNAs), microRNAs (miRNAs), long non-coding RNAs (lncRNAs), small nuclear/nucleolar RNAs (snRNA, snoRNA), vault RNA or piwi interacting RNA (piRNA), small fragments of ribosomal RNA and/or transfer RNA. [0057] The extracellular vesicles of the invention may also comprise metabolites, such as e.g. fatty acid esters, amides, alcohols, nucleotides and derivatives, organic acids, sugars and/or carnitines. [0058] According to the guidelines released in 2018 by the International Society for Extracellular Vesicles for the study of extracellular vesicles, “extracellular vesicle” is to be used as the generic term for particles naturally released from the cell that are delimited by a lipid bilayer and cannot replicate. Extracellular vesicles can then be subdivided based on measurable characteristics such as cell of origin, molecular markers, size, density, function, etc. The three main subtypes of extracellular vesicles are microvesicles, exosomes and apoptotic bodies. [0059] “Exosomes”, also referred to as intraluminal vesicles (ILVs), correspond to a subtype of extracellular vesicles enclosed within a single outer membrane, formed through the fusion of multivesicular bodies with the plasma membrane. They are secreted by all cell types and their size typically ranges from 30 to 150 nm in diameter. Exosomes have been found in plasma, urine, semen, saliva, bronchial fluid, cerebral spinal fluid (CSF), breast milk, serum, amniotic fluid, synovial fluid, tears, lymph, bile, and gastric acid. [0060] “Microvesicles” or “MV”, also referred to as “ectosomes” or “microparticles” are extracellular vesicles that are released into the extracellular environment by the outward budding, or pinching, and fission of the cell’s plasma membrane. Their size typically ranges from 30 nm to 1000 nm in diameter. They play a role in intercellular communication and can transport molecules such as mRNA, miRNA, and proteins between cells. [0061] “Apoptotic bodies” refer to a type of extracellular vesicles that are released by dying cells undergoing apoptosis into the extracellular space. Their size typically ranges from 50 nm to 5000 nm in diameter. Apoptotic bodies comprise intact organelles, chromatin, and small amounts of glycosylated proteins. [0062] The extracellular vesicles of the invention can be of any shape. In some embodiments, the extracellular vesicles of the invention have a spherical shape. [0063] In some embodiments, the extracellular vesicle has a diameter ranging from about 10 nm to about 5000 nm, preferably from about 10 nm to about 3000 nm, preferably from about 10 nm to about 2000 nm, preferably from about 20 nm to about 1000 nm, preferably from about 30 nm to about 500 nm, preferably from about 40 nm to about 300 nm, preferably from about 50 nm to about 200 nm, more preferably from about 60 nm to about 180 nm. [0064] In some embodiments, the extracellular vesicle has a diameter of about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nm. [0065] As used herein, the term “regulatory B cell (Breg)-derived extracellular vesicle” means that the extracellular vesicle originates from, or has been produced by a regulatory B cell. [0066] “B cell” or “B lymphocyte” refers to a type of white blood cell of the lymphocyte subtype which mature in the bone marrow. B cells are responsible for humoral immunity which is part of the adaptive immune system. B cells produce antigen-specific immunoglobulins which may either be secreted or inserted into the plasma membrane where they serve as B-cell receptor (BCR). When a naïve or memory B cell is activated by an antigen, it proliferates and differentiates into an antibody-secreting effector cell, also known as a plasmablast or a plasma cell. Additionally, B cells present antigens (and are therefore designated as professional antigen-presenting cells (APCs)) and secrete cytokines. [0067] “Regulatory B cell” or “Breg cell” or “Breg” refers to a subset of B cells which regulate inflammation and immune responses. In particular, regulatory B cells may suppress an abnormal or excessive immune response and play a role in immune tolerance. Breg cells may exert their immunoregulatory functions for instance through cytokine secretion, in particular interleukin (IL)-10, IL-35 and transforming growth factor (TGF)- β, and/or intercellular contacts, including ligand-receptor interactions, such as CTLA- 4/CD86, CD40/CD40L and Fas/FasL. Breg cells have the ability to dampen T-cell driven immune responses. Some Bregs are Granzyme+ regulatory B cells. [0068] In some embodiments, the extracellular vesicle is produced by a regulatory B cell in a constitutive way. In some embodiments, the extracellular vesicle is produced by a regulatory B cell in response to a stimulus. [0069] An “isolated” Breg-derived extracellular vesicle, as used herein, is intended to refer to an extracellular vesicle that is modified or removed from its natural state. In particular, an isolated Breg-derived extracellular vesicle may be substantially free of other cellular material and/or chemicals, in particular those that would interfere with therapeutic uses of the extracellular vesicle, including without limitation, cells, cell membranes, enzymes, hormones, and other proteinaceous or non-proteinaceous components. For example, a Breg-derived extracellular vesicle naturally present in a living animal is not "isolated" but the same Breg-derived extracellular vesicle partially or completely separated from the coexisting materials of its natural state is “isolated”. Typically, a preparation of isolated Breg-derived extracellular vesicle contains the Breg- derived extracellular vesicle at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, greater than about 96% pure, greater than about 97% pure, greater than about 98% pure, or greater than about 99% pure. [0070] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses Galectin-3 as a marker or biomarker. [0071] Galectin-3 typically refers to the protein referenced as NP_002297.2 in the NCBI database, as updated on April 3, 2023. Alternative names for Galectin-3 include “LGALS3”, “Galectin 3”, “GALIG”, “Advanced Glycation End-Product Receptor 3”, “Lectin, Galactoside-Binding, Soluble, 3”, “Carbohydrate-Binding Protein 3”, “Galactose-Specific Lectin 3”, “Laminin-Binding Protein”, “IgE-Binding Protein”, “35 KDa Lectin”, “Lectin L-29”, “MAC-2”, “GALBP”, “MAC2”, “Epididymis Secretory Sperm Binding Protein”, “Galactoside-Binding Protein”, “MAC-2 Antigen”, “Mac-2 Antigen”, “CBP 35”, “CBP35”, “Gal-3”, “GAL3”, “L-31”, and “L31”, as non- limiting examples. [0072] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human Galectin-3 gene sequence corresponds to NCBI Gene ID: 3958, as updated on April 3, 2023. The human Galectin-3 gene consists of 6 exons on chromosome 14q22.3. The major transcript encompasses 958 nucleotides and encodes a 250 amino acid protein. [0073] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses PD-1 as a marker or biomarker. In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses PD-1. [0074] Programmed cell Death protein 1 (PD-1) [Homo sapiens] typically refers to the protein referenced as NP_005009.2 in the NCBI database, as updated on June 12, 2024. Alternative names for PD-1 include PDCD1 (programmed cell death 1), CD279, hPD-1, SLEB2, hSLE1, as non-limiting examples. [0075] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human PD- 1 gene sequence corresponds to NCBI Gene ID: 5133, as updated on June 6, 2024. The human PD-1 gene consists of 6 exons on chromosome 2. The major transcript encompasses 2097 nucleotides and encodes a 288 amino acid protein. [0076] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses PD-L1 as a marker or biomarker. In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses PD-L1. [0077] Programmed cell Death Ligand 1 (PD-L1) [Homo sapiens] typically refers to the protein referenced as NP_054862.1 in the NCBI database, as updated on May 13, 2024. Alternative names for PD-L1 include CD274, B7-H, B7H1, PDCD1L1, PDCD1LG1, PDL1, hPD-L1, as non-limiting examples. [0078] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human PD- L1 gene sequence corresponds to NCBI Gene ID: 29126, as updated on June 9, 2024. The human PD-L1 gene consists of 7 exons on chromosome 9. The major transcript encompasses 3634 nucleotides and encodes a 290 amino acid protein. [0079] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses TNFR2 as a marker or biomarker. In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses TNFR2. [0080] Tumor necrosis factor receptor 2 (TNFR2) [Homo sapiens] typically refers to the protein referenced as NP_001057.1 in the NCBI database, as updated on June 12, 2024. Alternative names for TNFR2 include TNF receptor superfamily member 1B (TNFRSF1B), p75, TBPII, TNFBR, CD120b, TNFR1B, TNFR80, TNF-R75, p75TNFR, TNF-R-II, as non-limiting examples. [0081] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference TNFR2 gene sequence corresponds to NCBI Gene ID: 7133, as updated on June 6, 2024. The human TNFR2 gene consists of 13 exons on chromosome 1. The major transcript encompasses 3687 nucleotides and encodes a 461 amino acid protein. [0082] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses TNF as a marker or biomarker. In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses TNF. [0083] Tumor necrosis factor (TNF) [Homo sapiens] typically refers to the protein referenced as NP_000585 in the NCBI database, as updated on April 7, 2024. Alternative names for TNF include DIF, TNFA, TNFSF2, TNLG1F, TNF-alpha, as non-limiting examples. [0084] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference TNF gene sequence corresponds to NCBI Gene ID: 7124, as updated on May 5, 2024. The human TNF gene consists of 4 exons on chromosome 6. The major transcript encompasses 1678 nucleotides and encodes a 233 amino acid protein. [0085] “Marker” or “biomarker” as used herein refers to a variable, such as a protein or a lipid, which can be detected on or within extracellular vesicles. [0086] In some embodiments, the marker is displayed at the surface of the Breg-derived extracellular vesicle. In some embodiments, the marker is integrated into the lipid membrane of the Breg-derived extracellular vesicle. In some embodiments, the marker is inside (i.e., in the lumen) the Breg-derived extracellular vesicle. [0087] Methods for detecting markers expressed by extracellular vesicles are well known in the art and include for example Western blot analysis, bicinchoninic acid (BCA) assay, and flow cytometry. [0088] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least one marker selected from CD9, CD63, CD81, and Tsg101. [0089] CD9 typically refers to the protein referenced as NP_001760.1 in the NCBI database, as updated on December 24, 2022. Alternative names for CD9 include “TSPAN29”, “MRP-1”, “Tetraspanin-29”, “MIC3”, “P24”, “Cell Growth-Inhibiting Gene 2 Protein”, “Motility Related Protein-1”, “Leukocyte Antigen MIC3”, “CD9 Antigen (P24)”, “CD9 Antigen”, “5H9 Antigen”, “BA2”, “Motility-Related Protein”, “BA-2/P24 Antigen”, “Antigen CD9”, “TSPAN-29”, “Tspan-29”, “DRAP-27”, and “BTCC-1”, as non-limiting examples. [0090] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human CD9 gene sequence corresponds to NCBI Gene ID: 928, as updated on March 29, 2023. The human CD9 gene consists of 13 exons on chromosome 12p13.31. The major transcript encompasses 1225 nucleotides and encodes a 228 amino acid protein comprising four transmembrane domains. [0091] CD63 typically refers to the protein referenced as NP_001771.1 in the NCBI database, as updated on December 29, 2022. Alternative names for CD63 include “TSPAN30”, “ME491”, “MLA1”, “Lysosomal-Associated Membrane Protein 3”, “Lysosome Integral Membrane Protein 1”, “Ocular Melanoma-Associated Antigen”, “CD63 Antigen (Melanoma 1 Antigen)”, “Melanoma-Associated Antigen ME491”, “Tetraspanin-30”, “Granulophysin”, “CD63 Antigen”, “Tspan-30”, “LAMP-3”, “OMA81H”, “Limp1”, “Lysosome-Associated Membrane Glycoprotein 3”, and “Melanoma-Associated Antigen MLA1”, as non-limiting examples. [0092] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human CD63 gene sequence corresponds to NCBI Gene ID: 967, as updated on March 29, 2023. The human CD63 gene consists of 14 exons on chromosome 12q13.2. The major transcript encompasses 1023 nucleotides and encodes a 238 amino acid protein comprising four transmembrane domains. [0093] CD81 typically refers to the protein referenced as NP_004347.1 in the NCBI database, as updated on December 27, 2022. Alternative names for CD81 include “TSPAN28”, “Tetraspanin-28”, “TAPA1”, “CD81 Antigen (Target Of Antiproliferative Antibody 1)”, “26 KDa Cell Surface Protein TAPA-1”, “CD81 Antigen”, “Tspan-28”, “TAPA-1”, “S5.7”, “Target Of The Antiproliferative Antibody 1”, and “CVID6”, as non- limiting examples. [0094] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human CD81 gene sequence corresponds to NCBI Gene ID: 975, as updated on March 29, 2023. The human CD81 gene consists of 13 exons on chromosome 11p15.5. The major transcript encompasses 1482 nucleotides and encodes a 236 amino acid protein comprising four transmembrane domains. [0095] Tsg101 typically refers to the protein referenced as NP_006283.1 in the NCBI database, as updated on February 26, 2023. Alternatives names for Tsg101 include “Tumor Susceptibility 101”, “VPS23”, “Tumor Susceptibility Gene 101 Protein”, “ESCRT-I Complex Subunit TSG101”, “Tumor Susceptibility Gene 101”, “Tumor Susceptibility Gene 10”, “TSG10”, and “Tumor Susceptibility Protein”, as non-limiting examples. [0096] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human Tsg101 gene sequence corresponds to NCBI Gene ID: 7251, as updated on March 29, 2023. The human Tsg101 gene consists of 11 exons on chromosome 11p15.1. The major transcript encompasses 1534 nucleotides and encodes a 390 amino acid protein comprising a coiled-coil domain. [0097] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least 2, or 3 markers selected from CD9, CD63, CD81, and Tsg101. In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses 2, 3 or 4 markers selected from CD9, CD63, CD81, and Tsg101. [0098] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least one marker selected from HLA-A, HLA-B, HLA-C, HLA- DR, HLA-DP, HLA-DQ, CD19, CD20, CD24, CD29, CD40, CD44, and CD45. [0099] Typically, the markers HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, HLA-DQ, CD19, CD20, CD24, CD29, CD40, CD44, and CD45 are membrane markers that may be displayed at the surface of the Breg-derived extracellular vesicle. [0100] HLA-A typically refers to the protein referenced as NP_006283.1 in the NCBI database, as updated on February 26, 2023. Alternatives names for HLA-A include “Major Histocompatibility Complex, Class I, A”, “HLA Class I Histocompatibility Antigen, A Alpha Chain”, “HLAA”, “HLA Class I Histocompatibility Antigen, A-1 Alpha Chain”, “MHC Class I Antigen HLA-A Heavy Chain”, “Leukocyte Antigen Class I-A”, and “Human Leukocyte Antigen A”, as non-limiting examples. [0101] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human HLA-A gene sequence corresponds to NCBI Gene ID: 3105, as updated on March 29, 2023. The human HLA-A gene consists of 8 exons on chromosome 6p22.1. The major transcript encompasses 1535 nucleotides and encodes a 365 amino acid protein. [0102] HLA-B typically refers to the protein referenced as NP_005505 in the NCBI database, as updated on March 25, 2023. Alternatives names for HLA-B include “Major Histocompatibility Complex, Class I, B”, “HLA Class I Histocompatibility Antigen, B Alpha Chain”, “HLAB”, “AS”, “MHC Class I Antigen HLA-B Alpha Chain”, “MHC Class I Antigen HLA-B Heavy Chain”, MHC HLA-B Transmembrane Glycoprotein”, “MHC HLA-B Cell Surface Glycoprotein”, “Leukocyte Antigen Class I-B”, “HLA Class I Antigen HLA-B”, “MHC Class I Antigen SHCHA”, “Human Leukocyte Antigen B”, “Ankylosing Spondylitis”, “MHC Class I Molecule”, “MHC Class 1 Antigen”, and “B- 4901”, as non-limiting examples. [0103] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human HLA-B gene sequence corresponds to NCBI Gene ID: 3106, as updated on March 29, 2023. The human HLA-B gene consists of 8 exons on chromosome 6p21.33. The major transcript encompasses 1536 nucleotides and encodes a 362 amino acid protein. [0104] HLA-C typically refers to the protein referenced as NP_002108.4 in the NCBI database, as updated on March 18, 2023. Alternatives names for HLA-C include “Major Histocompatibility Complex, Class I, C”, “HLA Class I Histocompatibility Antigen, C Alpha Chain”, “HLA-JY3”, “D6S204”, “PSORS1”, “HLAC”, “Major Histocompatibility Antigen HLA-C”, “MHC Class I Antigen Heavy Chain HLA-C”, “Human Leukocyte Antigen-C Alpha Chain”, “Psoriasis Susceptibility 1”, “Human Leukocyte Antigen C”, “HLA-C Antigen”, “HLA-Cw”, “HLC-C”, and “MHC”, as non-limiting examples. [0105] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human HLA-C gene sequence corresponds to NCBI Gene ID: 3107, as updated on March 29, 2023. The human HLA-C gene consists of 8 exons on chromosome 6p21.33. The major transcript encompasses 1542 nucleotides and encodes a 366 amino acid protein. [0106] HLA-DR is a heterodimer consisting of an alpha chain (HLA-DRA) and a beta chain (HLA-DRB). [0107] HLA-DRA typically refers to the HLA-DRA1 protein referenced as NP_061984.2 in the NCBI database, as updated on December 24, 2022. Alternatives names for HLA-DRA1 include “Major Histocompatibility Complex, Class II, DR Alpha”, “HLA-DRA1”, “HLA Class II Histocompatibility Antigen, DR Alpha Chain”, “MHC Class II Antigen DRA”, and “Histocompatibility Antigen HLA-DR Alpha”, as non-limiting examples. [0108] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human HLA-DRA1 gene sequence corresponds to NCBI Gene ID: 3122, as updated on March 29, 2023. The human HLA-DRA1 gene consists of 5 exons on chromosome 6p21.32. The major transcript encompasses 1235 nucleotides and encodes a 254 amino acid protein. [0109] HLA-DRB typically refers to the HLA-DRB1 protein referenced as NP_002115.2 in the NCBI database, as updated on March 19, 2023. Alternatives names for HLA-DRB1 include “HLA-DRB1”, “Major Histocompatibility Complex, Class II, DR Beta 1”, “HLA-DR1B”, “Major Histocompatibility Complex, Class II, DR Beta 1 Precursor”, “HLA Class II Histocompatibility Antigen, DR-1 Beta Chain”, “HLA Class II Histocompatibility Antigen, DRB1 Beta Chain”, “MHC Class II HLA-DR Beta 1 Chain”, “Human Leucocyte Antigen DRB1”, “Human Leukocyte Antigen DRB1”, “Lymphocyte Antigen DRB1”, “DRB1”, and “SS1”, as non-limiting examples. [0110] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human HLA-DRB1 gene sequence corresponds to NCBI Gene ID: 3123, as updated on March 29, 2023. The human HLA-DRB1 gene consists of 6 exons on chromosome 6p21.32. The major transcript encompasses 1223 nucleotides and encodes a 266 amino acid protein. [0111] Variants of HLA-DRB include HLA-DRB3 corresponding to the NCBI Gene ID: 3125, as updated on March 29, 2023; HLA-DRB4 corresponding to the NCBI Gene ID: 3126, as updated on March 29, 2023; and HLA-DRB5 corresponding to the NCBI Gene ID: 3127, as updated on March 29, 2023. [0112] HLA-DP is a heterodimer consisting of an alpha chain (HLA-DPA) and a beta chain (HLA-DPB). [0113] HLA-DPA typically refers to the HLA-DPA1 protein referenced as NP_001229454.1 in the NCBI database, as updated on February 19, 2023. Alternatives names for HLA-DPA1 include “Major Histocompatibility Complex, Class II, DP Alpha”, “HLA-DP1A”, “HLA Class II Histocompatibility Antigen, DP Alpha 1 Chain”, “MHC Class II DP3-Alpha”, “HLA-SB Alpha Chain”, “DP(W3)”, “DP(W4)”, “HLASB”, “MHC Class II Antigen DP Alpha Chain”, “MHC Class II HLA-DPA1 Antigen”, “MHC Class II DP Alpha Chain”, “MHC Class II Antigen”, “MHC Class II Protein”, “MHC Class II DPA1”, “HLA DPA1”, “HLA-DPA1”, “HLADP”, “DPA1” and “PLT1”, as non- limiting examples. [0114] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human HLA-DPA1 gene sequence corresponds to NCBI Gene ID: 3113, as updated on March 29, 2023. The human HLA-DPA1 gene consists of 6 exons on chromosome 6p21.32. The major transcript encompasses 1666 nucleotides and encodes a 260 amino acid protein. [0115] HLA-DPB typically refers to the HLA-DPB1 protein referenced as NP_002112.3 in the NCBI database, as updated on March 25, 2023. Alternatives names for HLA-DPB1 include “Major Histocompatibility Complex, Class II, DP Beta 1”, “HLA-DP1B”, “HLA Class II Histocompatibility Antigen, DP(W4) Beta Chain”, “HLA Class II Histocompatibility Antigen, DP Beta 1 Chain”, “MHC Class II Antigen DPB1”, “HLA-DP Histocompatibility Type, Beta-1 Subunit”, “MHC Class II HLA-DP-Beta-1”, “MHC HLA DPB1”, “HLA-DPB1”, “HLA-DP” and “DPB1”, as non-limiting examples. [0116] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human HLA-DPB1 gene sequence corresponds to NCBI Gene ID: 3115, as updated on March 29, 2023. The human HLA-DPB1 gene consists of 6 exons on chromosome 6p21.32. The major transcript encompasses 3991 nucleotides and encodes a 258 amino acid protein. [0117] HLA-DQ is a heterodimer consisting of an alpha chain (HLA-DQA) and a beta chain (HLA-DQB). [0118] HLA-DQA typically refers to the HLA-DQA1 protein referenced as NP_002113.2 in the NCBI database, as updated on March 17, 2023. Alternatives names for HLA-DQA1 include “Major Histocompatibility Complex, Class II, DQ Alpha 1”, “CELIAC1”, “HLA Class II Histocompatibility Antigen, DQ Alpha 1 Chain”, “MHC Class II DQA1”, “DC-1 Alpha Chain”, “DC-Alpha”, “HLA-DQA1”, “HLA-DCA”, “HLA Class II Histocompatibility Antigen DQ Alpha Chain”, “MHC Class II DQ Alpha Chain”, “MHC Class II HLA-DQ-Alpha-1”, “MHC Class II Antigen DQA1”, “MHC Class II Protein”, “MHC HLA-DQ Alpha”, “HLA-DQA1*”, “HLA-DQB1”, “DQ-A1”, and “DQA1”, as non-limiting examples. [0119] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human HLA-DQA1 gene sequence corresponds to NCBI Gene ID: 3117, as updated on March 29, 2023. The human HLA-DQA1 gene consists of 6 exons on chromosome 6p21.32. The major transcript encompasses 1574 nucleotides and encodes a 255 amino acid protein. [0120] HLA-DQB typically refers to the HLA-DQB1 protein referenced as NP_002114.3 on the NCBI database as updated on March 18, 2023. Alternatives names for HLA-DQB1 include “Major Histocompatibility Complex, Class II, DQ Beta 1”, “CELIAC1”, “HLA-DQB1”, “IDDM1”, “HLA Class II Histocompatibility Antigen, DQ Beta 1 Chain”, “MHC Class II Antigen DQB1”, “MHC Class II HLA-DQ Beta Glycoprotein”, “MHC Class II Antigen HLA-DQ-Beta-1”, and “MHC Class II DQ Beta Chain”, as non-limiting examples. [0121] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human HLA-DQB1 gene sequence corresponds to NCBI Gene ID: 3119, as updated on March 29, 2023. The human HLA-DQB1 gene consists of 6 exons on chromosome 6p21.32. The major transcript encompasses 1605 nucleotides and encodes a 261 amino acid protein. [0122] CD19 typically refers to the protein referenced as NP_001761.3 in the NCBI database, as updated on January 22, 2023. Alternatives names for CD19 include “CD19 Molecule”, “B-Lymphocyte Surface Antigen B4”, “T-Cell Surface Antigen Leu-12”, “Differentiation Antigen CD19”, “B-Lymphocyte Antigen CD19”, “CD19 Antigen”, “CVID3”, and “B4”, as non-limiting examples. [0123] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human CD19 gene sequence corresponds to NCBI Gene ID: 930, as updated on March 29, 2023. The human CD19 gene consists of 14 exons on chromosome 16p11.2. The major transcript encompasses 1918 nucleotides and encodes a 556 amino acid protein. [0124] CD20 typically refers to the protein referenced as NP_690605.1 in the NCBI database, as updated on December 27, 2022. Alternatives names for CD20 include “MS4A1”, “Membrane Spanning 4-Domains A1”, “Bp35”, “FMC7”, “B1”, “Membrane- Spanning 4-Domains, Subfamily A, Member 1”, “Leukocyte Surface Antigen Leu-16”, “B-Lymphocyte Antigen CD20”, “CD20 Antigen”, “Membrane-Spanning 4-Domains Subfamily A Member 1”, “B-Lymphocyte Cell-Surface Antigen B1”, “B-Lymphocyte Surface Antigen B1”, “CD20 Receptor”, “LEU-16”, “CVID5”, and “S7”, as non-limiting examples. [0125] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human CD20 gene sequence corresponds to NCBI Gene ID: 931, as updated on March 29, 2023. The human CD20 gene consists of 8 exons on chromosome 11q12.2. The major transcript encompasses 3556 nucleotides and encodes a 297 amino acid protein. [0126] CD24 typically refers to the protein referenced as NP_001346013.1 in the NCBI database, as updated on February 12, 2023. Alternatives names for CD24 include “CD24A”, “CD24 Antigen (Small Cell Lung Carcinoma Cluster 4 Antigen)”, “Signal Transducer CD24”, “Small Cell Lung Carcinoma Cluster 4 Antigen", and “CD24 Antigen”, as non-limiting examples. [0127] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human CD24 gene sequence corresponds to NCBI Gene ID: 100133941, as updated on March 29, 2023. The human CD24 gene consists of 5 exons on chromosome 6q21. The major transcript encompasses 2156 nucleotides and encodes a 80 amino acid protein. [0128] CD29 typically refers to the protein referenced as NP_002202.2 in the NCBI database, as updated on February 19, 2023. Alternatives names for CD29 include “ITGB1”, “Integrin Subunit Beta 1”, “GPIIA”, “MSK12”, “FNRB”, “MDF2”, “Integrin, Beta 1 (Fibronectin Receptor, Beta Polypeptide, Antigen CD29 Includes MDF2, MSK12)”, “Glycoprotein IIa”, “Integrin Beta-1”, “Very Late Activation Protein, Beta Polypeptide”, “Fibronectin Receptor Subunit Beta”, “Integrin VLA-4 Beta Subunit”, “VLA-4 Subunit Beta”, “Integrin Beta 1”, “CD29 Antigen”, “VLA-BETA”, and “VLAB”, as non-limiting examples. [0129] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human CD29 gene sequence corresponds to NCBI Gene ID: 3688, as updated on March 29, 2023. The human CD29 gene consists of 18 exons on chromosome 10p11.22. The major transcript encompasses 3735 nucleotides and encodes a 798 amino acid protein. [0130] CD40 typically refers to the protein referenced as NP_001241.1 in the NCBI database, as updated on January 1, 2023. Alternatives names for CD40 include “CD40 Molecule”, “Bp50”, “TNFRSF5”, “P50”, “Tumor Necrosis Factor Receptor Superfamily Member 5”, “CD40 Molecule, TNF Receptor Superfamily Member 5”, “CD40L Receptor”, “Tumor Necrosis Factor Receptor Superfamily, Member 5”, “B Cell Surface Antigen CD40”, “B-Cell Surface Antigen CD40”, “B Cell-Associated Molecule”, “CD40 Antigen”, “CDW40”, and “CDw40”, as non-limiting examples. [0131] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human CD40 gene sequence corresponds to NCBI Gene ID: 958, as updated on March 29, 2023. The human CD40 gene consists of 9 exons on chromosome 20q13.12. The major transcript encompasses 1682 nucleotides and encodes a 277 amino acid protein. [0132] CD44 typically refers to the protein referenced as NP_000601.3 in the NCBI database, as updated on March 26, 2023. Alternatives names for CD44 include “HUTCH- I”, “HCELL”, “CSPG8”, “MC56”, “Pgp1”, “MDU2”, “MDU3”, “MIC4”, “IN”, “Hematopoietic Cell E- And L-Selectin Ligand”, “GP90 Lymphocyte Homing/Adhesion Receptor”, “Chondroitin Sulfate Proteoglycan 8”, “Extracellular Matrix Receptor III”, “Homing Cell Adhesion Molecule”, “Heparan Sulfate Proteoglycan”, “Phagocytic Glycoprotein 1”, “Phagocyte Glycoprotein 1”, “Hyaluronate Receptor”, “In(Lu) Related- P80”, “Hermes Antigen”, “CD44 Antigen”, “Hermes-1”, “ECMR-III”, “HUTCH-1”, “ECM-III”, “Epican”, “PGP-1”, “CD44R”, “CDw44”, “H-CAM”, “LHR”, “CD44 Antigen (Homing Function And Indian Blood Group System)”, “Homing Function And Indian Blood Group System”, “Extracellular Matrix Receptor-III”, “Cell Surface Glycoprotein CD44”, “Indian Blood Group Antigen”, “Phagocytic Glycoprotein I”, “Soluble CD44”, “CDW44”, and “PGP-I”, as non-limiting examples. [0133] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human CD44 gene sequence corresponds to NCBI Gene ID: 960, as updated on March 29, 2023. The human CD44 gene consists of 20 exons on chromosome 11p13. The major transcript encompasses 5431 nucleotides and encodes a 742 amino acid protein. [0134] CD45 typically refers to the protein referenced as NP_002829.3 in the NCBI database, as updated on March 15, 2023. Alternatives names for CD45 include “PTPRC”, “Protein Tyrosine Phosphatase Receptor Type C”, “T200”, “GP180”, “LCA”, “Receptor- Type Tyrosine-Protein Phosphatase C”, “CD45 Antigen”, “L-CA”, “Protein Tyrosine Phosphatase, Receptor Type, C Polypeptide”, “T200 Leukocyte Common Antigen”, “Leukocyte Common Antigen”, “T200 Glycoprotein”, “EC 3.1.3.48”, “IMD105”, “CD45R”, “B220”, and “LY5”, as non-limiting examples. [0135] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human CD45 gene sequence corresponds to NCBI Gene ID: 5788, as updated on March 29, 2023. The human CD45 gene consists of 37 exons on chromosome 1q31.3-q32.1. The major transcript encompasses 5357 nucleotides and encodes a 1306 amino acid protein. [0136] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 markers selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, HLA-DQ, CD19, CD20, CD24, CD29, CD40, CD44, and CD45. In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 markers selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, HLA-DQ, CD19, CD20, CD24, CD29, CD40, CD44, and CD45. [0137] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least one marker selected from CD9, CD63, CD81, Tsg101, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, HLA-DQ, CD19, CD20, CD24, CD29, CD40, CD44, and CD45. In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 markers selected from CD9, CD63, CD81, Tsg101, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, HLA-DQ, CD19, CD20, CD24, CD29, CD40, CD44, and CD45. In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 markers selected from CD9, CD63, CD81, Tsg101, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, HLA-DQ, CD19, CD20, CD24, CD29, CD40, CD44, and CD45. [0138] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least one marker selected from Galectin-3, PD-1, PD-L1, TNFR2, TNF, CD9, CD63, CD81, Tsg101, HLA-A, HLA-B, HLA-C, HLA-DR, HLA- DP, HLA-DQ, CD19, CD20, CD24, CD29, CD40, CD44, and CD45. In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 markers selected from Galectin-3, PD-1, PD-L1, TNFR2, TNF, CD9, CD63, CD81, Tsg101, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, HLA-DQ, CD19, CD20, CD24, CD29, CD40, CD44, and CD45. In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 markers selected from Galectin-3, PD-1, PD-L1, TNFR2, TNF, CD9, CD63, CD81, Tsg101, HLA-A, HLA-B, HLA-C, HLA-DR, HLA- DP, HLA-DQ, CD19, CD20, CD24, CD29, CD40, CD44, and CD45. [0139] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses Galectin-3, PD-1, PD-L1, TNFR2, TNF, CD9, CD63, CD81, Tsg101, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, HLA-DQ, CD19, CD20, CD24, CD29, CD40, CD44, CD45 or any combination thereof. [0140] Preferably, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least three markers selected from the group consisting of Galectin-3, PD- 1, PD-L1, TNFR2, TNF, CD9, CD63, CD81, Tsg101, HLA-A, HLA-B, HLA-C, HLA- DR, HLA-DP, HLA-DQ, CD19, CD20, CD24, CD29, CD40, CD44, and CD45, wherein said at least three markers comprise at least one transmembrane protein and at least one intraluminal protein. [0141] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 markers selected from the group consisting of the proteins encoded by the genes identified on Figure 10B. [0142] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 markers selected from the group consisting of the proteins indicated on Figure 10C. [0143] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 markers selected from the group consisting of the proteins listed in Table A below. Table A: Proteins expressed by Breg-derived extracellular vesicles Gene Protein Protein Description NKRF NKRF NF-kappa-B-repressing factor TNF TNFA Tumor necrosis factor IL2RA IL2RA Interleukin-2 receptor subunit alpha ICAM1 ICAM1 Intercellular adhesion molecule 1 FES FES Tyrosine-protein kinase Fes/Fps UCHL1 UCHL1 Ubiquitin carboxyl-terminal hydrolase isozyme L1 CCL3;CCL3L1 CCL3;CL3L1 C-C motif chemokine 3 PRF1 PERF Perforin-1 IL2RB IL2RB Interleukin-2 receptor subunit beta LGALS3 LEG3 Galectin-3 TNFRSF1B TNR1B; Tumor necrosis factor receptor superfamily TNFR2 member 1B ITGAX ITAX Integrin alpha-X TNFAIP3 TNAP3 Tumor necrosis factor alpha-induced protein 3 ITGA3 ITA3 Integrin alpha-3 CCR10 CCR10 C-C chemokine receptor type 10 ITGA1 ITA1 Integrin alpha-1 TRAF2 TRAF2 TNF receptor-associated factor 2 TRAF1 TRAF1 TNF receptor-associated factor 1 SLC39A6 S39A6 Zinc transporter ZIP6 MELK MELK Maternal embryonic leucine zipper kinase PDCD1 PDCD1; PD- Programmed cell death protein 1 1 BATF BATF Basic leucine zipper transcriptional factor ATF- like THEMIS2 THMS2 Protein THEMIS2 TRAF7 TRAF7 E3 ubiquitin-protein ligase TRAF7 NLRC5 NLRC5 Protein NLRC5 DTX1 DTX1 E3 ubiquitin-protein ligase DTX1 NCSTN NICA Nicastrin GBP5 GBP5 Guanylate-binding protein 5 FCRL5 FCRL5 Fc receptor-like protein 5 IL4I1 OXLA L-amino-acid oxidase CARD9 CARD9 Caspase recruitment domain-containing protein 9 SEMA4A SEM4A Semaphorin-4A IL21 IL21 Interleukin-21 IL21R IL21R Interleukin-21 receptor SLAMF7 SLAF7 SLAM family member 7 CD274 PD-L1 Programmed cell death 1 ligand 1 [0144] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 markers selected from the group consisting of the proteins listed in Table B. [0145] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 markers selected from the group consisting of NKRF, TNFA, IL2RA, CCL3/CL3L1, PERF, LEG3, TNR1B/TNFR2, PDCD1/PD-1, OXLA, SLAF7, or PD-L1. [0146] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, or 221 markers selected from the group consisting of the proteins encoded by the genes listed in Table 3. [0147] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, or 221 markers selected from the group consisting of the proteins listed in Table 3. [0148] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, or 185 markers selected from the group consisting of the proteins encoded by the genes listed in Table 4. [0149] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, or 185 markers selected from the group consisting of the proteins listed in Table 4. [0150] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses LYAR. In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses CASP8. In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses RIPK3. [0151] In some embodiments, the isolated Breg-derived extracellular vesicle comprises, contains, or expresses Granzyme B. [0152] Granzyme B typically refers to the protein referenced as NP_004122.2 in the NCBI database, as updated on March 14, 2023. Alternative names for Granzyme B include “GZMB”, “CTSGL1”, “CGL1”, “SECT”, “HLP”, “T-Cell Serine Protease 1- 3E”, “Cathepsin G-Like 1”, “CGL-1”, “CSP-B”, “CTLA1”, “CCPI”, “CSPB”, “Granzyme B (Granzyme 2, Cytotoxic T-Lymphocyte-Associated Serine Esterase 1)”, “Cytotoxic T-Lymphocyte Proteinase 2”, “Cytotoxic Serine Protease B”, “Human Lymphocyte Protein”, “Fragmentin 2”, “EC 3.4.21.79”, “C11”, “Cytotoxic T- Lymphocyte-Associated Serine Esterase 1”, “Lymphocyte Protease”, “Fragmentin-2”, “Granzyme 2”, “Granzyme-2”, “EC 3.4.21”, “CTLA-1”, and “GRB”, as non-limiting examples. [0153] In the NCBI databases (https://www.ncbi.nlm.nih.gov), the reference human Granzyme B gene sequence corresponds to NCBI Gene ID: 3002, as updated on March 29, 2023. The human Granzyme B gene consists of exons on chromosome 14q12. The major transcript encompasses 891 nucleotides and encodes a 247 amino acid protein. [0154] In some embodiments, the extracellular vesicle has a high concentration of the at least one marker it comprises. In some embodiments, the concentration of said at least one marker in the extracellular vesicle is higher than that in Breg cells from which the extracellular vesicle is derived, i.e. the extracellular vesicle is enriched in said at least one marker compared to the Breg cells from which it is derived. In some embodiments, the concentration of said at least one marker is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5 or 10-fold of that in the Breg cells from which the extracellular vesicle is derived. [0155] In some embodiments, the extracellular vesicle does not express one or more marker(s) selected from the group consisting of CD3, CD4, CD8, CD56, CD105, CD2, CD1c, CD25, CD49e, ROR1, CD209, CD63, CD11c, MCSP, CD146, CD326, CD86, CD133.1, CD69, CD142, CD31 and CD14. [0156] In some embodiments, the extracellular vesicle may be targeted to a desired cell type or tissue. This targeting can be achieved by expressing on the surface of the extracellular vesicle a targeting moiety which binds to a cell surface moiety expressed on the surface of the cell to be targeted. Typically, the targeting moiety may be a peptide within a disclosed vesicle targeted fusion protein. However, it can also be independently expressed as a fusion protein with a vesicle transmembrane moiety. [0157] Techniques that may be used for characterizing Breg-derived extracellular vesicles are well-known and include e.g. Nanoparticle Tracking Analysis – NTA (coupled to antibody fluorescence detection), Tunable Resistive Pulse Sensor - TRPS, (Immuno)- Electron microscopy - EM/IEM, Atomic Force Microscopy - AFM, Super resolution microscopy, Exoview, Conventional flow cytometry, Small particles flow cytometry - Imagestream, Nano flow cytometry, Bead assay cytometry, Raman spectroscopy, Dynamic Light Scattering - DLS, Western Blot, ELISA, Asymmetric Flow field Flow fractionation coupled to DLS - A4F/DLS (optionally combined with UV absorbance, refractometer, and/or fluorescence), Mass spectrometry, Micro BCA/BCA, Bradford, Sulfophosphovanilin assay, Capillary electrophoresis instruments, Nucleic acid sequencing, etc. [0158] Various techniques may be combined for characterizing Breg-derived extracellular vesicles. For instance, Breg-derived extracellular vesicles of the invention may be characterized by using at least two techniques selected from the group consisting of Nanoparticle Tracking Analysis – NTA (coupled to antibody fluorescence detection), Tunable Resistive Pulse Sensor - TRPS, (Immuno)-Electron microscopy - EM/IEM, Atomic Force Microscopy - AFM, Super resolution microscopy, Exoview, Conventional flow cytometry, Small particles flow cytometry - Imagestream, Nano flow cytometry, Bead assay cytometry, Raman spectroscopy, Dynamic Light Scattering - DLS, Western Blot, ELISA, Asymmetric Flow field Flow fractionation coupled to DLS - A4F/DLS (optionally combined with UV absorbance, refractometer, and/or fluorescence), Mass spectrometry, Micro BCA/BCA, Bradford, Sulfophosphovanilin assay, Capillary electrophoresis instruments, and Nucleic acid sequencing. [0159] Preferably, the Breg-derived extracellular vesicles of the invention may be characterized by using at least two complementary techniques, such as e.g.: - at least one imaging technique (including electron microscopy, atomic force microscopy, or super resolution microscopy…), - at least one non-imaging single-particle technique (including resistive pulse sensing, nanoparticle tracking analysis, flow cytometry, or Raman spectroscopy…). [0160] In addition to differences in marker expression, the isolated Breg-derived extracellular vesicle of the invention also exhibits functional or therapeutic effects that are distinct from extracellular vesicles derived from other cell types. [0161] In some embodiments, the isolated Breg-derived extracellular vesicle is capable of inhibiting T cell proliferation, increasing T cell apoptosis, and/or increasing proinflammatory cytokine secretion by T cells. [0162] In some embodiments, the isolated Breg-derived extracellular vesicle is capable of inhibiting T cell proliferation. [0163] In some embodiments, the isolated Breg-derived extracellular vesicle is capable of increasing T cell apoptosis. [0164] In some embodiments, the isolated Breg-derived extracellular vesicle is capable of increasing proinflammatory cytokine secretion by T cells. [0165] In some embodiments, the isolated Breg-derived extracellular vesicle is obtained or obtainable by a method comprising: a) culturing B cells in a culture medium with a stimulation cocktail until Breg cells are induced and Breg-derived extracellular vesicles are produced in the culture medium, and b) collecting said Breg-derived extracellular vesicle in the culture medium. [0166] Therefore, another object of the present invention is an isolated Breg-derived extracellular vesicle obtained or obtainable by a method comprising: a) culturing B cells in a culture medium with a stimulation cocktail until Breg cells are induced and Breg-derived extracellular vesicles are produced in the culture medium, and b) collecting said Breg-derived extracellular vesicle in the culture medium. [0167] In some embodiments, B cells are cultured in an extracellular vesicle-free culture medium. In some embodiments, the extracellular vesicle-free culture medium is TexMACS™ medium (said medium is commercially available, for example from Miltenyi Biotec – reference 130-097-196). In some embodiments, the extracellular vesicle-free culture medium is RPMI (Roswell Park Memorial Institute) medium supplemented with 10% fetal bovine serum, depleted in bovine extracellular vesicles by ultracentrifugation at 100,000g for 18 hours. In some embodiments, the vesicle-free culture medium is supplemented with L-glutamine and/or penicillin/streptomycin. [0168] In some embodiments, the stimulation cocktail used to induce Breg cells comprises or consists of soluble human CD40-Ligand (CD40L), and/or class B CpG oligodeoxynucleotide, and/or anti-human IgG/IgM/IgA polyclonal antibodies, and/or human IL-21, and/or human IL-2. [0169] In some embodiments, the stimulation cocktail comprises from about 10 ng/mL to about 100 ng/mL of soluble human CD40L, preferably about 50 ng/mL of soluble human CD40L (said human CD40L is commercially available, for example from Miltenyi Biotec - reference 130-096-711). [0170] In some embodiments, the stimulation cocktail comprises from about 0.1 µg/mL to about 10 µg/mL class B CpG oligodeoxynucleotide, preferably about 1 µg/mL class B CpG oligodeoxynucleotide (said class B CpG oligodeoxynucleotide is commercially available, for example from InvivoGen – reference tlrl-2006). [0171] In some embodiments, the stimulation cocktail comprises from about 0.5 µg/mL to 50 µg/mL anti-human IgG/IgM/IgA polyclonal antibodies, preferably 5 µg/mL anti- human IgG/IgM/IgA polyclonal antibodies. In some embodiments, anti-human IgG/IgM/IgA polyclonal antibodies are F(ab')₂ fragment Goat Anti-Human IgA + IgG + IgM (H+L) polyclonal antibodies (said F(ab')₂ fragment Goat Anti-Human IgA + IgG + IgM (H+L) polyclonal antibodies are commercially available, for example from Jackson ImmunoResearch Laboratories – reference 109-006-064). [0172] In some embodiments, the stimulation cocktail comprises from about 1 ng/mL to about 100 ng/mL human IL-21, preferably about 10 ng/mL human IL-21 (said human IL- 21 is commercially available, for example from R&D Systems – reference 8879-IL- 010/CF). [0173] In some embodiments, the stimulation cocktail comprises from about 5 IU/mL to about 500 IU/mL of human IL-2, preferably 50 IU/mL human IL-2 (said human IL-2 is commercially available, such as for example Proleukin (Aldesleukine) 18 millions UI from Novartis). [0174] B cells may be cultured under any suitable culture conditions, for example with regards to temperature, humidity, CO₂. Suitable culture conditions are well-known to one skilled in the art and may be selected, for example, depending on the cells. In some embodiments, B cells are cultured in culture conditions suitable for human cells. In some embodiments, B cells are cultured at 37°C and 5% CO₂. [0175] In some embodiments, B cells are culture for at least 12, 24, 36, 48, 60, 72, 84, 96, 108, or 120 hours. In some embodiments, B cells are cultured for at least 72 hours. [0176] In some embodiments, B cells are cultured at a concentration of at least about 1x10⁴, 5x10⁴, 1x10⁵, 5x10⁵, 1x10⁶, 5x10⁶, 1x10⁷, or 5x10⁷ cells per milliliter of culture medium, preferably at a concentration of at least about 1x10⁶ cells/mL of culture medium. In some embodiments, B cells are cultured at a concentration ranging from about 1x10⁴ to about 5x10⁷ cells/mL of culture medium, preferably from about 1x10⁵ to about 1x10⁷ cells/mL of culture medium, more preferably from about 5x10⁵ to about 5x10⁶ cells/mL of culture medium. In some embodiments, B cells are cultured at a concentration of about 1x10⁴, 5x10⁴, 1x10⁵, 5x10⁵, 1x10⁶, 5x10⁶, 1x10⁷, or 5x10⁷ cells/mL of culture medium, preferably at a concentration of 1x10⁶ cells/mL of culture medium. [0177] In some embodiments, B cells are cultured in a multiple-well cell culture plate. Examples of multiple-well cell culture plates include 4-well, 6-well, 8-well, 12-well, 24-well, 48-well, and 96-well cell culture plates. In some embodiments, B cells are cultured in a 6-well cell culture plate. [0178] In some embodiments, the isolated Breg-derived extracellular vesicle is obtained or obtainable by using a fluidic system comprising at least one container, a culture medium contained by the container, Breg cells, a culture medium agitator, means for controlling the speed of the agitator adapted for the growth of the Breg cells, wherein the means for controlling the speed of the agitator, the agitator and the shape and dimensions of the container are adapted to the generation of a turbulent flow of the culture medium in the container to exert shear stresses on the Breg cells in order to achieve the production of extracellular vesicles, the Kolmogorov length of the flow being less than or equal to 50 µm. [0179] Therefore, another object of the present invention is an isolated Breg-derived extracellular vesicle obtained or obtainable by using a fluidic system comprising at least one container, a culture medium contained by the container, Breg cells, a culture medium agitator, means for controlling the speed of the agitator adapted for the growth of the Breg cells, wherein the means for controlling the speed of the agitator, the agitator and the shape and dimensions of the container are adapted to the generation of a turbulent flow of the culture medium in the container to exert shear stresses on the Breg cells in order to achieve the production of extracellular vesicles, the Kolmogorov length of the flow being less than or equal to 50 µm. [0180] In some embodiments, the isolated Breg-derived extracellular vesicle is obtained or obtainable by a method comprising: a) culturing Breg cells in a culture medium, b) applying a turbulent flow in the culture medium until Breg-derived extracellular vesicles are produced in the culture medium, wherein the Kolmogorov length of the flow is less than or equal to 50 µm, and c) collecting said Breg-derived extracellular vesicle in the culture medium. [0181] Thus, another object of the present invention is an isolated Breg-derived extracellular vesicle obtained or obtainable by a method comprising: a) culturing Breg cells in a culture medium, b) applying a turbulent flow in the culture medium until Breg-derived extracellular vesicles are produced in the culture medium, wherein the Kolmogorov length of the flow is less than or equal to 50 µm, and c) collecting said Breg-derived extracellular vesicle in the culture medium. [0182] “Agitator” as used herein refers to a means or a combination of means allowing by action on the culture medium to generate at least one flow, to promote mixing of the culture medium or to generate turbulence in the culture medium. [0183] In some embodiments, the agitator of the fluidic system consists of at least one blade. In some embodiments, the agitator of the fluidic system consists of 2, 3, 4, 5, 6, 7, 8 or more blades. [0184] In some embodiments, the at least one blade of the agitator is a vertical blade. [0185] In some embodiments, the agitator of the fluidic system is an agitator of the propeller type, such as marine or propeller with profiled blades, or a turbine, such as a Rushton turbine, or an agitation anchor, or a barrier agitator, or a helical ribbon propeller, or a bladed wheel, or a toother wheel, or a magnetic agitator or a combination of these agitators. [0186] In some embodiments, the agitator is a rotary or orbital agitator, the shape and size of which are adapted, with the shape and dimensions of the container, to the generation of a turbulent flow of the culture medium in the container. [0187] In some embodiments, the container may comprise static structures such as baffles, or structures forming partial barriers to liquid movement, such as those used in a static mixer. [0188] In some embodiments, the container of the fluidic system is a spinner flask with a capacity of 100 mL, comprising a blade with a diameter of 3.8 cm and a working volume of less than 100 mL. [0189] In some embodiments, the container of the fluidic system is a spinner flask with a capacity of 500 mL, comprising a blade with a diameter of 7.6 cm and a working volume of 200 mL to 500 mL. [0190] In some embodiments, the container of the fluidic system is a spinner flask with a capacity of 1000 mL, comprising a blade with a diameter of 10.8 cm and a working volume greater than or equal to 300 mL and less than 1 L. [0191] In some embodiments, the container of the fluidic system is a spinner flask whose structural features (i.e., capacity, diameter of the blade and working volume) are all increased or decreased proportionally to those mentioned above for the spinner flask with a capacity of 100 mL, or for the spinner flask with a capacity of 500 mL, or for the spinner flask with a capacity of 1000 mL. In some embodiments, the container of the fluidic system is a spinner flask whose structural features (i.e., capacity, diameter of the blade and working volume) are all increased or decreased in a non-proportional manner with respect to those mentioned above for the spinner flask with a capacity of 100 mL, or for the spinner flask with a capacity of 500 mL, or for the spinner flask with a capacity of 1000 mL, in particular during a change in scale. [0192] In some embodiments, the container of the fluidic system is a bioreactor, the working volume of which is from 400 mL to 1000 mL and the diameter of the blade of which is 6 cm. [0193] In some embodiments, the container of the fluidic system is a bioreactor whose working volume and diameter of the blade are increased or decreased proportionally to the respective values of 400 mL and 6 cm. In some embodiments, the container of the fluidic system is a bioreactor whose working volume and diameter of the blade are increased or decreased in a non-proportional manner with respect to the respective values of 400 mL and 6 cm, in particular during a change in scale. [0194] In some embodiments, the Kolmogorov length of the flow is less than or equal to 50 µm, preferably less than or equal to 40 µm, more preferably less than or equal to 35 µm. In some embodiments, the Kolmogorov length of the flow is from 5 to 50 µm, preferably from 5 to 41 µm, preferably from 10 to 41 µm, more preferably from 5 to 35 µm, even more preferably from 10 to 35 µm. In some embodiments, the Kolmogorov length of the flow is equal to 25 µm. [0195] In some embodiments, the duration of the turbulent agitation at a Kolmogorov length less than or equal to 50 µm, for example from 17 to 35 µm, is greater than or equal to 15 minutes, preferably from about 20 minutes to about 10 hours, more preferably from about 20 minutes to about 8 hours, even more preferably from about 1 hour to about 6 hours, even more preferably between about 2 hours and about 3 hours, even more preferably about 2 hours or alternatively about 3 hours or alternatively about 4 hours. In some embodiments, the duration of the turbulent agitation at a Kolmogorov length equal to 25 µm is equal to about 6 hours. [0196] Breg cells described herein, may be cultured in any suitable culture medium. Suitable culture media are well-known to one skilled in the art and may be selected, for example, depending on the cells. Example of suitable media include RPMI (Roswell Park Memorial Institute) 1640 medium. The culture medium may be supplemented with additional substances such as serum and serum components, vitamins, reducing agents, and/or buffering agents. [0197] In some embodiments, Breg cells are culture in RPMI 1640 culture medium containing penicillin and streptomycin. [0198] Breg cells may be cultured under any suitable culture conditions, for example with regards to temperature, humidity, CO₂. Suitable culture conditions are well-known to one skilled in the art and may be selected, for example, depending on the cells. In some embodiments, Breg cells are cultured in culture conditions suitable for human cells. In some embodiments, Breg cells are cultured at 37°C and 5% CO2. [0199] In some embodiments, Breg cells are culture for at least 6 hours. In some embodiments, Breg cells are cultured for at least 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, or 24 hours. [0200] In some embodiments, Breg cells are cultured at a concentration of at least about 1x10⁴, 5x10⁴, 1x10⁵, 5x10⁵, 1x10⁶, 5x10⁶, 1x10⁷, or 5x10⁷ cells per milliliter of culture medium, preferably at a concentration of at least about 3.5x10⁵ cells/mL of culture medium. In some embodiments, Breg cells are cultured at a concentration ranging from about 1x10⁴ to about 5x10⁷ cells/mL of culture medium, preferably from about 1x10⁵ to about 1x10⁷ cells/mL of culture medium, more preferably from about 5x10⁵ to about 5x10⁶ cells/mL of culture medium. In some embodiments, Breg cells are cultured at a concentration of about 1x10⁴, 5x10⁴, 1x10⁵, 5x10⁵, 1x10⁶, 5x10⁶, 1x10⁷, or 5x10⁷ cells/mL of culture medium, preferably at a concentration of 3.5x10⁵ cells/mL of culture medium. [0201] Another object of the present invention is a method for isolating the Breg-derived extracellular vesicle as described herein. [0202] In some embodiments, the method comprises: - collecting culture medium from Breg cells, - centrifuging the culture medium, e.g. for 10 min at 400g, until B cells fall in the pellet, - collecting supernatant from the first centrifugation, - centrifuging the culture medium, e.g. for 10 min at 2,000g, until dead cells fall in the pellet, - collecting supernatant from the second centrifugation, - applying a second centrifugation to the supernatant, e.g. for 30 min at 10,000g, until subcellular debris and larger vesicles fall in the pellet, - collecting supernatant from the third centrifugation, - applying a fourth centrifugation to the supernatant, e.g. for 1h30 at 150,000g until extracellular vesicles are pelleted, - recovering Breg-derived extracellular vesicle from the pellet. [0203] Another object of the present invention is a kit comprising at least one isolated Breg-derived extracellular vesicle as described herein and instructions for use. [0204] By “kit” is intended any manufacture (e.g., a package or a container) comprising at least one isolated Breg-derived extracellular vesicle according to the present invention. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. [0205] In some embodiments, the kit is suitable for delivery (e.g., local injection) of the isolated Breg-derived extracellular vesicles to a subject. [0206] The kit can comprise one or more containers selected from the group consisting of a bottle, a vial, an ampoule, a blister pack, and a syringe. [0207] The present invention also provides packaging and kits comprising compositions for use in the methods of the present invention. [0208] The kit can further include one or more of instructions for use in treating and/or preventing a disease, condition or disorder of the present invention, one or more syringes, one or more applicators, or a sterile solution suitable for reconstituting a pharmaceutical composition of the present invention. [0209] Another object of the present invention is a composition comprising, consisting essentially of, or consisting of at least one isolated Breg-derived extracellular vesicle as described herein. [0210] As used herein, “consisting essentially of”, with reference to a composition, means that the at least one isolated Breg-derived extracellular vesicle is the only active agent, therapeutic agent, or agent with a biologic activity within said composition. [0211] Another object of the present invention is a pharmaceutical composition comprising, consisting essentially of, or consisting of at least one isolated Breg-derived extracellular vesicle as described herein, and at least one pharmaceutically acceptable excipient. [0212] The term “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. Said excipient does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a mammal, more preferably a human. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA. [0213] Pharmaceutically acceptable excipients that may be used in the pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat. [0214] In one embodiment, the pharmaceutical composition comprises vehicles which are pharmaceutically acceptable for a formulation capable of being injected to a subject. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. [0215] Another object of the present invention is a medicament comprising, consisting essentially of, or consisting of at least one isolated Breg-derived extracellular vesicle as described herein. [0216] In some embodiments, the extracellular vesicle, the composition, pharmaceutical composition, or medicament as described herein are formulated for administration to a subject. [0217] The extracellular vesicle, composition, pharmaceutical composition, or medicament as described herein may be administered or formulated to be administered systemically, orally, parenterally, by injection, topically, by inhalation spray, rectally, nasally, or via an implanted reservoir. The term administration used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the extracellular vesicle composition, pharmaceutical composition, or medicament as described herein are to be administered locally (e.g. topically) or systemically. [0218] In some embodiments, the extracellular vesicle, the composition, pharmaceutical composition, or medicament as described herein are formulated for administration by injection, such as for example subcutaneous injection, or by infusion, such as for example intravenous infusion. Examples of forms adapted for injection include, but are not limited to, solutions, such as, for example, sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to use, such as, for example, powder, liposomal forms and the like. [0219] In one embodiment, the Breg-derived extracellular vesicle or the composition described above is to be administered to the subject in need thereof in a therapeutically effective amount. [0220] The term “therapeutically effective amount”, as used herein, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired preventive and/or therapeutic result. [0221] It will be however understood that the total daily usage of the Breg-derived extracellular vesicle or the composition described above will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disease being treated and the severity of the disease; activity of the Breg- derived extracellular vesicle or the composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the Breg-derived extracellular vesicle or composition employed; the duration of the treatment; drugs used in combination or coincidental with the Breg- derived extracellular vesicle or composition employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. The total dose required for each treatment may be administered by multiple doses or in a single dose. [0222] The effective amount for prevention or treatment of a disease or condition may be from about 0.01 ng to about 10,000 nM of the composition, pharmaceutical composition, or medicament. The composition may comprise a concentration of about, at least about, or at most about 0.01, 1.0, 10.0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 nM, of Breg-derived extracellular vesicles, or any range derivable therein. [0223] The above numerical values may also be the dosage that is to be administered to the patient based on the patient’s weight, expressed as ng/kg, mg/kg, or g/kg, and any range derivable from those values. The composition may have a concentration of Breg- derived extracellular vesicles of 0.01, 1.0, 10.0, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ng/ml, or any range derivable therein. The effective amount may be from about 0.01 ng/mL to about 10,000 ng/mL of the composition. [0224] In one embodiment, a therapeutically effective amount of the Breg-derived extracellular vesicle or of the composition is to be administered once a day, twice a day, three times a day or more. [0225] In one embodiment, a therapeutically effective amount of the Breg-derived extracellular vesicle or of the composition is to be administered every day, every two days, every three days, every four days, every five days, every six days. [0226] In one embodiment, a therapeutically effective amount of the Breg-derived extracellular vesicle or of the composition is to be administered every week, every two weeks, every three weeks. [0227] In one embodiment, a therapeutically effective amount of the Breg-derived extracellular vesicle or of the composition is to be administered every month, every two months, every three months, every four months, every five months, every six months. [0228] In a preferred embodiment, a therapeutically effective amount of the Breg- derived extracellular vesicle or of the composition is to be administered every 12 hours, every 24 hours, every 36 hours, every 48 hours, every 60 hours, every 72 hours, every 96 hours. [0229] In a preferred embodiment, a therapeutically effective amount of the Breg- derived extracellular vesicle or of the composition is to be administered every 60 hours. [0230] In one embodiment, a therapeutically effective amount of the Breg-derived extracellular vesicle or of the composition is to be administered for about 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, 1 month, 2 months, 3 months, 6 months, 1 year or more. [0231] In one embodiment, a therapeutically effective amount of the Breg-derived extracellular vesicle or of the composition is to be administered for a period of time ranging from about one week to about eight weeks, from about two weeks to about seven weeks, from about two weeks to about six weeks, from about two weeks to about five weeks. [0232] In a preferred embodiment, a therapeutically effective amount of the Breg- derived extracellular vesicle or of the composition is to be administered for a period of time ranging from about 10 days to about 40 days, from about 15 days to about 35 days, from about 20 days to about 30 days. [0233] In some embodiments, the composition, pharmaceutical composition, or medicament is to be administered in an amount of 0.001 to 1000 mg/day. In some embodiments, the composition, pharmaceutical composition, or medicament is to be administered in a range from about 0.001 mg/kg to about 1000 mg/kg, about 0.01 mg/kg to about 100 mg/kg, about 10 mg/kg to about 250 mg/kg, about 0.1 mg/kg to about 15 mg/kg; or any range in which the low end of the range is any amount between 0.001 mg/kg and 900 mg/kg and the upper end of the range is any amount between 0.1 mg/kg and 1000 mg/kg (e.g., 0.005 mg/kg and 200 mg/kg, 0.5 mg/kg and 20 mg/kg). Effective doses will also vary, as recognized by those skilled in the art, depending on the diseases treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments such as use of other agents. [0234] In one embodiment, the Breg-derived extracellular vesicle or the composition described above is to be administered before, concomitantly with or after a therapeutic drug. [0235] Examples of suitable therapeutic drugs include for instance chemotherapeutic agents, targeted therapy agents, cytotoxic agents (or cytotoxins), cell cycle-synchronizing agents, ligands for cellular receptor(s), immunomodulatory agents, pro-apoptotic agents, lytic peptides, anti-angiogenic agents, cytokines, growth factors, and hormones. [0236] Suitable examples of immunosuppressor agents include those described under subgroup L04 of the Anatomical Therapeutic Chemical Classification System. [0237] Suitable examples of immunosuppressor agents include, but are not limited to: - antimetabolites, such as, e.g.: ^ antifolates, including aminopterin, methotrexate, pemetrexed, pralatrexate, pteropterin, raltitrexed, denopterin, trimetrexate, pemetrexed, and the like; ^ purine analogues, including pentostatin, cladribine, clofarabine, fludarabine, nelarabine, tioguanine, mercaptopurine, and the like; ^ pyrimidine analogues, including fluorouracil, capecitabine, doxifluridine, tegafur, tegafur/gimeracil/oteracil, carmofur, floxuridine, cytarabine, gemcitabine, azacytidine, decitabine, and the like; and ^ hydroxycarbamide); - macrolides, such as, e.g., tacrolimus, ciclosporin, pimecrolimus, abetimus, gusperimus, and the like; - immunomodulatory imide drugs, such as, e.g., lenalidomide, pomalidomide, thalidomide, apremilast, and the like; - IL-1 receptor antagonists, such as, e.g., anakinra, and the like); - mTOR inhibitors, such as, e.g., sirolimus, everolimus, ridaforolimus, temsirolimus, umirolimus, zotarolimus, and the like); - serum-targeting antibodies, such as, e.g., eculizumab, adalimumab, afelimomab, certolizumab pegol, golimumab, infliximab, nerelimomab, mepolizumab, omalizumab, faralimomab, elsilimomab, lebrikizumab, ustekinumab, secukinumab, and the like; - cell-targeting antibodies, such as, e.g., muromonab-CD3, otelixizumab, teplizumab, visilizumab, clenoliximab, keliximab, zanolimumab, efalizumab, erlizumab, obinutuzumab, rituximab, ocrelizumab, pascolizumab, gomiliximab, lumiliximab, teneliximab, toralizumab, aselizumab, galiximab, gavilimomab, ruplizumab, belimumab, blisibimod, ipilimumab, tremelimumab, bertilimumab, lerdelimumab, metelimumab, natalizumab, tocilizumab, odulimomab, basiliximab, daclizumab, inolimomab, zolimomab aritox, atorolimumab, cedelizumab, fontolizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, siplizumab, talizumab, telimomab aritox, vapaliximab, vepalimomab, and the like; - fusion antibodies, such as, e.g., abatacept, belatacept, etanercept, pegsunercept, aflibercept, alefacept, rilonacept and the like. [0238] As used herein, the term “pro-apoptotic agent” refers to any molecule able to induce apoptosis or programmed cell death in a cell upon administration. [0239] Suitable examples of pro-apoptotic agents include, but are not limited to, histone deacetylase inhibitors (such as, e.g., sodium butyrate, depsipeptide and the like), bortezomib, deguelin, favopiridol, fenretinide, fludarabine, kaempferol, miltefosine, narciclasine, obatoclax, oblimersen, and oncrasin. [0240] Suitable examples of cytokines include, but are not limited to, chemokines, tumor necrosis factors, interleukins, and colony-stimulating factors. [0241] Suitable examples of chemokines include, but are not limited to, chemokine C-C motif ligand (CCL) 1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, chemokine C-X-C motif ligand (CXCL) 1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, fractalkine, chemokine C motif ligand (XCL) 1, and XCL2. [0242] Suitable examples of tumor necrosis factors include, but are not limited to, tumor necrosis factor (TNF) α, lymphotoxin, OX40L, CD40LG, Fas ligand, CD70, CD153, 4- 1BB ligand, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor κ-Β ligand (RANKL), a proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF), and ectodysplasin A (EDA). [0243] Suitable examples of interleukins include, but are not limited to, interleukin (IL) 1α, IL-1β, IL-1Ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL- 26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36α, IL-36β, IL- 36γ, IL-36Ra, IL-37, IL-38, interferon (IFN) α, IFNβ, IFNκ, and IFNω. [0244] Suitable examples of colony-stimulating factors include, but are not limited to, granulocyte-macrophage colony-stimulating factor (GM-CSF) (including granulocyte- colony stimulating factor (G-CSF) and macrophage colony-stimulating factor (M-CSF)), haematopoietin, and thrombopoietin. [0245] It will also be understood by the skilled artisan that the particular therapeutic drug for co-administration will depend on the disease or condition to be prevented and/or treated. [0246] In an exemplary embodiment, where the Breg-derived extracellular vesicle or the composition described above is for inducing immune tolerance in a subject in need thereof, or preventing and/or reducing transplant rejection, it may be desirable that an immunosuppressant agent be co-administered. [0247] Another object of the present invention is an isolated Breg-derived extracellular vesicle as described herein, for use as a medicament. [0248] In some embodiments, the extracellular vesicle is used as a drug carrier. [0249] Another object of the present invention is an isolated Breg-derived extracellular vesicle as described herein, for use in inhibiting T cell proliferation, increasing T cell apoptosis, and/or increasing proinflammatory cytokine secretion by T cells in a subject in need thereof. [0250] The present invention also concerns a pharmaceutical composition for use in inhibiting T cell proliferation, increasing T cell apoptosis, and/or increasing proinflammatory cytokine secretion by T cells in a subject in need thereof, wherein said pharmaceutical composition comprises at least one isolated Breg-derived extracellular vesicle as described herein. [0251] The present invention also relates to a method of inhibiting T cell proliferation, increasing T cell apoptosis, and/or increasing proinflammatory cytokine secretion by T cells in a subject in need thereof, said method comprising administering to the subject at least one isolated Breg-derived extracellular vesicle or a pharmaceutical composition as described herein. [0252] Another object of the present invention is an isolated Breg-derived extracellular vesicle or a pharmaceutical composition for inhibiting T cell proliferation, increasing T cell apoptosis, and/or increasing proinflammatory cytokine secretion by T cells in a subject in need thereof, wherein said pharmaceutical composition comprises at least one isolated Breg-derived extracellular vesicle as described herein. [0253] Another object of the present invention is the use of at least one isolated Breg- derived extracellular vesicle or a pharmaceutical composition as described herein in the manufacture of a medicament for inhibiting T cell proliferation, increasing T cell apoptosis, and/or increasing proinflammatory cytokine secretion by T cells in a subject in need thereof. [0254] Another object of the present invention is an isolated Breg-derived extracellular vesicle as described herein, for use in inducing immune tolerance in a subject in need thereof. [0255] The present invention also concerns a pharmaceutical composition for use in inducing immune tolerance in a subject in need thereof, wherein said pharmaceutical composition comprises at least one isolated Breg-derived extracellular vesicle as described herein. [0256] The present invention also relates to a method of inducing immune tolerance in a subject in need thereof, said method comprising administering to the subject at least one isolated Breg-derived extracellular vesicle or a pharmaceutical composition as described herein. [0257] Another object of the present invention is an isolated Breg-derived extracellular vesicle or a pharmaceutical composition for inducing immune tolerance in a subject in need thereof, wherein said pharmaceutical composition comprises at least one isolated Breg-derived extracellular vesicle as described herein. [0258] Another object of the present invention is the use of at least one isolated Breg- derived extracellular vesicle or a pharmaceutical composition as described herein in the manufacture of a medicament for inducing immune tolerance in a subject in need thereof. [0259] “Immune tolerance”, as used herein, relates to a state of unresponsiveness of the immune system to specific substances or tissues that have the capacity to elicit an immune response while preserving immune response against other substances or tissues. [0260] “Immune response”, as used herein, includes T cell-mediated and/or B cell- mediated immune responses. Exemplary immune responses include, but are not limited to, T cell responses (e.g., cytokine production and cellular cytotoxicity), but also immune responses that are indirectly affected by T cell activation (e.g., macrophages). Immune cells involved in the immune response include lymphocytes (such as B cells and T cells, including CD4⁺, CD8⁺, T_h1 and T_h2 cells), antigen presenting cells (e.g., professional antigen presenting cells such as dendritic cells), natural killer cells, myeloid cells (such as macrophages, eosinophils, mast cells, basophils, and granulocytes). [0261] Another object of the present invention is an isolated Breg-derived extracellular vesicle as described herein, for use in the prevention, reduction and/or treatment of transplant rejection, graft-versus-host disease (GvHD), autoimmune disease, or abnormal or excessive immune response in a subject in need thereof. [0262] The present invention also concerns a pharmaceutical composition for use in the prevention, reduction and/or treatment of transplant rejection, graft-versus-host disease (GvHD), autoimmune disease, or abnormal or excessive immune response in a subject in need thereof, wherein said pharmaceutical composition comprises at least one isolated Breg-derived extracellular vesicle as described herein. [0263] The present invention also relates to a method of preventing, reducing and/or treating transplant rejection, graft-versus-host disease (GvHD), autoimmune disease, or abnormal or excessive immune response in a subject in need thereof, said method comprising administering to the subject at least one isolated Breg-derived extracellular vesicle or a pharmaceutical composition as described herein. [0264] Another object of the present invention is an isolated Breg-derived extracellular vesicle or a pharmaceutical composition for preventing, reducing and/or treating transplant rejection, graft-versus-host disease (GvHD), autoimmune disease, or abnormal or excessive immune response in a subject in need thereof, wherein said pharmaceutical composition comprises at least one isolated Breg-derived extracellular vesicle as described herein. [0265] Another object of the present invention is the use of at least one isolated Breg- derived extracellular vesicle or a pharmaceutical composition as described herein in the manufacture of a medicament for preventing, reducing and/or treating transplant rejection, graft-versus-host disease (GvHD), autoimmune disease, or abnormal or excessive immune response in a subject in need thereof. [0266] “Preventing transplant rejection” or “prevention of transplant rejection” and “reducing transplant rejection” or “reduction of transplant rejection” are meant to encompass prevention or inhibition of immune transplant rejection, as well as delaying the onset or the progression of immune transplant rejection. The terms are also meant to encompass prolonging survival of a transplant in a subject, or reversing failure of a transplant in a subject. Further, the terms are meant to encompass ameliorating a symptom of an immune transplant rejection, including, for example, ameliorating an immunological complication associated with immune rejection, such as, e.g., interstitial fibrosis, chronic graft arteriosclerosis, or vasculitis. [0267] The term “transplantation”, “transplant” and variations thereof refer to the insertion of a transplant (also called graft) into a recipient, whether the transplantation is syngeneic (where the donor and recipient are genetically identical), allogeneic (where the donor and recipient are of different genetic origins but of the same species), or xenogeneic (where the donor and recipient are from different species). Thus, in a typical scenario, the host is human and the graft is an isograft, derived from a human of the same or different genetic origins. In another scenario, the graft is derived from a species different from that into which it is transplanted, including animals from phylogenetically widely separated species, for example, a baboon heart being transplanted into a human host. [0268] The term “transplant rejection”, as used herein, encompasses both acute and chronic transplant rejection. [0269] “Acute rejection” is the rejection by the immune system of a tissue transplant- recipient when the transplanted tissue is immunologically foreign. Acute rejection is characterized by infiltration of the transplant tissue by immune cells of the recipient, which carry out their effector function and destroy the transplant tissue. The onset of acute rejection is rapid and generally occurs in humans within a few weeks after transplant surgery. Generally, acute rejection can be inhibited or suppressed with immunosuppressive drugs such as rapamycin, cyclosporin, anti-CD40L monoclonal antibody and the like. [0270] “Chronic rejection” generally occurs in humans within several months to years after engraftment, even in the presence of successful immunosuppression of acute rejection. Fibrosis is a common factor in chronic rejection of all types of organ transplants. [0271] In some embodiments, the transplant rejection is an allogeneic transplant rejection. Accordingly, in one embodiment, the donor of the transplant is a human. The donor of the transplant can be a living donor or a deceased donor, namely a cadaveric donor. [0272] In some embodiments, the transplant is an organ, a tissue or cells. [0273] As used herein, the term “organ” refers to a solid vascularized organ that performs a specific function or group of functions within an organism. The term organ includes, but is not limited to, heart, lung, kidney, liver, pancreas, skin, uterus, bone, cartilage, small or large bowel, bladder, brain, breast, blood vessels, esophagus, fallopian tube, gallbladder, ovaries, pancreas, prostate, placenta, spinal cord, limb including upper and lower, spleen, stomach, testes, thymus, thyroid, trachea, ureter, urethra, uterus. [0274] As used herein, the term “tissue” refers to any type of tissue in human or animals, and includes, but is not limited to, vascular tissue, skin tissue, hepatic tissue, pancreatic tissue, neural tissue, urogenital tissue, gastrointestinal tissue, skeletal tissue including bone and cartilage, adipose tissue, connective tissue including tendons and ligaments, amniotic tissue, chorionic tissue, dura, pericardia, muscle tissue, glandular tissue, facial tissue, ophthalmic tissue. [0275] The term “cells” refers here to a composition enriched for cells of interest, preferably a composition comprising at least 30%, preferably at least 50%, even more preferably at least 65 % of said cells. In some embodiments, the cells are selected from the group comprising or consisting of multipotent hematopoietic stem cells derived from bone marrow, peripheral blood, or umbilical cord blood; or pluripotent (i.e., embryonic stem cells [ES] or induced pluripotent stem cells [iPS]) or multipotent stem cell-derived differentiated cells of different cell lineages, including, but not limited to, cardiomyocytes, β-pancreatic cells, hepatocytes, neurons and the like. [0276] In some embodiments where the transplantation is an allogeneic hematopoietic stem cell transplantation (HSCT), the cells are selected from the group comprising or consisting of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. [0277] “HSCT” or “hematopoietic stem cell transplantation” is a transplantation therapy which can be curative for patients affected with leukemia and lymphomas (including, without limitation, acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), myelodysplasia syndrome (MDS), myeloproliferative syndrome, Hodgkin lymphomas, non-Hodgkin lymphomas, chronic lymphatic leukemia (CLL) and multiple myeloma). However, an important limitation of allogeneic HSCT is the development of graft-versus-host-disease (GvHD), which occurs in a severe form in about 30-50% of humans who receive this therapy. [0278] “Graft-versus-host-disease” or “GvHD” as used herein, refers to a systemic disorder that occurs when the graft’s immune cells recognize the host’s cells as foreign and attack them. GvHD can occur after allogeneic bone marrow transplant or hematopoietic stem cell transplant, or following transplantation of solid organs with are rich in lymphoid cells, such as liver, or following transfusion of un-irradiated blood. [0279] As used herein, the term “autoimmune disease” refers to a disease in which the immune system produces an immune response (e.g., a B cell or a T cell response) against an antigen that is part of the normal host (that is an auto-antigen), with consequent injury to tissues. In an autoimmune disease, the immune system of the host fails to recognize a particular antigen as “self” and an immune reaction is mounted against the host’s tissues expressing the antigen. [0280] Exemplary autoimmune diseases contemplated in the present invention include, but are not limited to, rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjogren’s syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (including Crohn’s disease and ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, alopecia areata, type 1 diabetes, non-obese diabetes, myasthenia gravis, Grave’s disease, Hashimoto’s thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosis, autoimmune thrombocytopenia purpura, Goodpasture’s syndrome, Addison’s disease, systemic sclerosis, polymyositis, dermatomyositis, acquired hemophilia, thrombotic thrombocytopenic purpura, and uveitis. [0281] As used herein, the term “abnormal or excessive immune response” refers to any immune reaction that may be detrimental or harmful for the subject. It may refer to any undesired immune reaction, preferably any undesired immune reaction directed to (i) proteins expressed in the course of gene therapy, (ii) vectors (such as, e.g., viral vectors) used in the course of gene therapy and/or (iii) therapeutic proteins. Such proteins include, e.g., factor VIII (hemophilia A) and other coagulation factors, enzyme replacement therapies, monoclonal antibodies, polyclonal antibodies, enzymes or cytokines. [0282] The isolated Breg-derived extracellular vesicle or the pharmaceutical composition as described herein may be administered to a subject in order to suppress an immune response, especially to prevent immune reactions to specific proteins when their expression is restored by gene therapy in those subjects with corresponding genetic deficiencies. Thus, the isolated Breg-derived extracellular vesicle or the pharmaceutical composition as described herein may be used to prevent immune reactivity towards proteins normally absent in the subject due to mutations, while their reconstitution is achieved by gene therapy. Moreover, protein therapy is an area of medical innovation that is becoming more widespread, and involves the application of proteins, such as enzymes, antibodies or cytokines, directly to subjects as therapeutic products. One of the major hurdles in delivery of such medicaments involves the immune responses directed against the therapeutic protein themselves. Administration of protein-based therapeutics is often accompanied by administration of immune suppressants, which are used in order to facilitate a longer lifetime of the protein and therefore increased uptake of the protein into the cells and tissues of the organism. General immune suppressants can however be disadvantageous due to the unspecific nature of the immune suppression that is carried out, resulting in unwanted side effects in the patient. Therefore, this approach can be applied to suppress an immune response against therapeutic proteins and peptides, such as therapeutic antibodies, cytokines, enzymes or any other protein administered to a subject. [0283] In some embodiments, the isolated Breg-derived extracellular vesicle or the pharmaceutical composition as described herein may be administered to a subject in order to suppress an immune response, especially to prevent immune reactions to vectors used in gene therapy, in particular viral vectors used in gene therapy. [0284] Another object of the present invention is the use of the isolated Breg-derived extracellular vesicle as described herein for inhibiting T cell proliferation, increasing T cell apoptosis, and/or increasing proinflammatory cytokine secretion by T cells in a sample. [0285] Still another object of the present invention is an in vitro method for inhibiting T cell proliferation, increasing T cell apoptosis, and/or increasing proinflammatory cytokine secretion by T cells in a sample, said method comprising a step of contacting said sample with the isolated Breg-derived extracellular vesicle as described herein. [0286] As used herein, a “sample” refers to any biological material obtained via suitable methods known to the person skilled in the art from a subject. The sample may be collected in a clinically acceptable manner, e.g., in a way that cells, nucleic acids (such as DNA and RNA), proteins and/or extracellular vesicles are preserved. A “sample” may be a body tissue and/or a bodily fluid, preferably a bodily fluid. Examples of bodily fluids include, but are not limited to, blood, plasma, serum, lymph, ascetic fluid, cystic fluid, urine, bile, nipple exudate, vomitus, breast milk, tears, wound drainage, feces, vaginal secretions, synovial fluid, bronchoalveolar lavage fluid, sputum, amniotic fluid, peritoneal fluid, cerebrospinal fluid, pleural fluid, pericardial fluid, semen, saliva, sweat and alveolar macrophages, tissue lysates, biopsies and extracts prepared from diseased tissues. [0287] In some embodiments, the sample is previously taken or retrieved from a subject, i.e., the methods as described herein do not comprise an active step of recovering a sample from the subject. Consequently, according to some embodiments, the methods as described herein are non-invasive methods, i.e., the methods of the invention are in vitro methods. [0288] Assays suitable for measuring inhibition of T cell proliferation by the isolated Breg-derived extracellular vesicle as described herein are well-known in the art, and include staining the T cells with a proliferation dye and analyzing their proliferation by flow cytometry. BRIEF DESCRIPTION OF THE DRAWINGS [0289] Figures 1A-B are a combination of graphs showing the number of extracellular vesicles per cell (Figure 1A) and the size of extracellular vesicles (Figure 1B-C) produced by unstimulated B cells and granzyme B+ Breg cells. B cells from healthy donors were expanded with or without a stimulation cocktail in regulatory B cells expressing Granzyme B during 72 h and were submitted to a mechanical stimulation during 6 h. EVs produced spontaneously and under mechanical stimulation were isolated from the culture supernatant by differential centrifugation. Extracellular vesicles were then characterized in quantity (Figure 1A) and size (Figure 1B) with Nanotracking Analysis (NS300, Malvern) or by cryo-electron microscopy (Figure 1C). Data were analyzed with Prism v8 software (GraphPad). [0290] Figures 2A-D are a combination of photographs from cryo-electron microscopy showing extracellular vesicles produced spontaneously (Figures 2A-B) or by mechanical stimulation (Figures 2C-D) by unstimulated B cells (Figures 2A and C) or granzyme B+ Breg cells (Figures 2B and D). B cells from healthy donors were expanded with or without a stimulation cocktail in regulatory B cells expressing Granzyme B during 72 h and were submitted to a mechanical stimulation during 6 h. EVs produced spontaneously (Figures 2A-B) and under mechanical stimulation (Figures 2C-D) were isolated from the culture supernatant by differential centrifugation. Extracellular vesicles were then characterized by cryo-electron microscopy (CRYO-EM). The CRYO-TEM images were analyzed with the ImageJ software. [0291] Figure 3 is a graph showing the density of extracellular vesicles produced by unstimulated B cells and granzyme B+ Breg cells. B cells from healthy donors were expanded with or without a stimulation cocktail in regulatory B cells expressing Granzyme B during 72 h and were submitted to a mechanical stimulation during 6 h. EVs produced spontaneously and under mechanical stimulation were isolated from the culture supernatant by differential centrifugation. Extracellular vesicles were then characterized by cryo-electron microscopy. The density of EVs to electrons was assessed by measuring gray averages. Data were analyzed with Prism v8 software (GraphPad). Results represent the difference of density compared to the density of extracellular vesicles spontaneously produced by unstimulated B cells. [0292] Figures 4A-C are a combination of graphs showing the analysis of unstimulated B cells and granzyme B+ Breg cells by single-cell RNA sequencing. B cells from healthy donors were expanded with or without a stimulation cocktail in regulatory B cells expressing Granzyme B during 72 h. Their transcriptome (Figure 4A) and expression of Galectin-3 (Figures 4B-C) were analyzed by single-cell RNA sequencing. [0293] Figure 5 is a combination of photographs of Western-blot showing the detection of CD9, CD63, CD81, Tsg101, Galectin-3 and Calnexin by western blot. B cells from healthy donors were expanded with or without a stimulation cocktail in regulatory B cells expressing Granzyme B during 72 h. EVs produced spontaneously were isolated from the culture supernatant by differential centrifugation. Spontaneously produced extracellular- vesicles were then lysed and characterized by western-blot. Expression of CD9, CD63, CD81, Tsg101, Galectin-3 and Calnexin (Figure 5) was analyzed in unstimulated B cells and granzyme B+ Breg cells and in extracellular vesicles produced by unstimulated B cells and extracellular vesicles produced by granzyme B+ Breg cells. Images were analyzed with ImageJ software. [0294] Figure 6 is a graph showing the detection of 37 markers on extracellular vesicles produced by unstimulated B cells and granzyme B+ Breg cells. B cells from healthy donors were expanded with or without a cocktail of stimulation in regulatory B cells expressing Granzyme B during 72 h. EVs produced spontaneously were isolated from the culture supernatant by differential centrifugation. Spontaneously produced extracellular- vesicles were then characterized by flow cytometry (MACSPlex Exosome Kit, Miltenyi). Expression of CD3, CD4, CD19, CD8, HLA-DR/DP/DQ, CD56, CD105, CD2, CD1c, CD25, CD49e, ROR1, CD209, CD9, SSEA-4, HLA-A/B/C, CD63, CD40, CD62P, CD11c, CD81, MCSP, CD146, CD41b, CD42a, CD24, CD326, CD44, CD86, CD133.1, CD29, CD69, CD142, CD45, CD31, CD20 and CD14 was analyzed. Data were analyzed with Prism v8 software (GraphPad). [0295] Figures 7A-H are a combination of flow cytometry plots showing the inhibition of effector T cell proliferation by extracellular vesicles secreted spontaneously (Figures 7C and 7D) or after mechanical stimulation (Figures 7G and 7H) by unstimulated B cells (Figures 7C and 7G) or by granzyme B+ Breg cells (Figures 7D and 7H). B cells from healthy donors were expanded with or without a stimulation cocktail in regulatory B cells expressing Granzyme B during 72 h and were submitted to a mechanical stimulation during 6 h. EVs produced spontaneously or under mechanical stimulation were isolated from the culture supernatant by differential centrifugation and co-cultured with CD4+CD25- T cells after labelling with a cell proliferation dye and stimulation of their proliferation with anti-CD3/CD28 beads. Proliferation was then analyzed by flow cytometry. Unstimulated CD4+CD25- T cells were used as negative control (Figures 7A and 7E), while CD4+CD25- T cells activated with anti-CD3/CD28 beads were used as positive control (Figures 7B and 7F). Dead cells were excluded of the analysis. Analysis was performed with FlowJo v10 software. [0296] Figures 8A-D are a combination of graphs showing CD4+ T cell suppressive functions of Breg-derived EVs. To determine the regulatory effect of EVs, increasing amounts of EVs were cultured with CD4+CD25- T lymphocytes for 3 days after activation with CD3/CD28 beads. Proliferation (Fig. 8A) and viability (Fig. 8C) were then analyzed by flow cytometry. Validation of the biological effect on proliferation (Fig. 8B) and viability (Fig. 8D) was achieved by producing EVs from 30 to 50 million producing B cells (n=10). Dead cells were excluded from the proliferation analysis. Statistical significance was tested using linear regression and Kruskal-Wallis test where *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns, non-significant. [0297] Figure 9 is a graph showing an impoverishment of miRNAs involved in inflammatory processes in Breg-derived EVs. The miRNA content of Breg-derived EVs (n=6) and (n=5) EVs from unstimulated B cells, and their cells of origin, were characterized by in situ hybridization on a panel of 827 human miRNAs (Nanostring Technologies). After background subtraction, differential miRNA expression analysis was performed by t-test. The miRNAs were considered differentially expressed between EVs produced by unstimulated B cells versus Breg-derived EVs with a p-value <0.05 and a fold change ≥ or ≤1.5. [0298] Figures 10A-C are a combination of graphs showing an enrichment of proteins linked to the regulation of T cell proliferation and cell death in EVs produced by GzmB+ Breg. The protein content of Breg-derived EVs and EVs produced by unstimulated B cells (n=4) was characterized by mass spectrometry allowing the detection of 6055 proteins in all the samples. Unsupervised clustering analysis of the gene ontology was performed on the diffentially expressed proteins. The analysis associate Breg-derived EVs up-regulated proteins to 18 biological processes, and in particular two clusters of proteins associated with the regulation of T-cell proliferation and the up-regulation of T-cell apoptosis. Proteins associated with these processes showing a FoldChange >1.5 and an adjusted p- value <0.05 enabled the identification of 36 proteins of interest (Fig.10A-C), including 11 proteins of biological interest in the mechanism of action of Breg (bold boxes on Fig. 10B-C). Fig.10B shows the 36 identified genes of interest, and Fig.10C shows the 36 identified proteins of interest. [0299] Figures 11A-D are a combination of graphs showing that Breg-derived EVs exhibit suppressive activity partially dependent on PD-1 or PD-L1, TNFR2 or TNF, and Galectin-3 (Gal-3). To characterize the involvement of protein targets on Breg suppressive function, Breg were expanded for 24h in presence or absence of anti-TNFR2 (Fig. 11A), and then co-cultured with CD4+ CD25- T lymphocytes. To assess the involvement of proteins of interest on the ability of Breg to differentiate and produce EVs, Breg were expanded for 72h in presence of an inhibitor of Galectin-3 (GB1107) (Fig. 11B). To test the involvement of these protein targets in CD4+ T Cell inhibition mediated by Breg EVs, EVs produced by 20.10⁶ Breg were pre-incubated with anti-PD1 antibodies (10 µg/ml) and then cultured with 2.5.10⁴ CD4+ CD25- T cells for 3 days after activation with CD3/CD28 beads (Fig. 11C-D) (n=2). Proliferation was analyzed by Cell proliferation dye (CPD) and KI67 expression by flow cytometry. Dead cells were excluded from the analysis. [0300] Figure legend: in all Figures disclosed herein, “EVs B NS” designates EVs produced by unstimulated B cells; “EVs Breg GzmB+” or “EVs Breg” designates EVs produced by GzmB+ Breg cells; and “stim” means stimulation or stimulated. EXAMPLES [0301] The present invention is further illustrated by the following examples. Example 1: Breg-derived extracellular vesicles characterization Materials and Methods B cell Isolation [0302] This study was conducted using blood samples from healthy donors obtained from the French Blood Establishment (EFS, Nantes, France) with written informed consent. Fresh human PBMCs were isolated from buffy coat of healthy donors by Ficoll gradient centrifugation using SepMate PBMC Isolation Tubes (Stemcell Technologies). B cells were isolated by negative selection of human PBMCs using a human B cell Isolation kit II (Miltenyi Biotec). Granzyme B+ regulatory B cell Expansion [0303] B cells were cultured at a concentration of 1 x 10⁶ cells/mL in TexMACS™ medium (Miltenyi Biotec), an EV-free culture medium, supplemented with L-glutamine and penicillin/streptomycin (Life Technologies) for 3 days at 37°C in a 5% CO₂ in the presence of a stimulation cocktail containing 50 ng/ml soluble CD40L (Miltenyi Biotec), 1µg/ml class B CpG oligodeoxynucleotide (ODN) 2006 (InvivoGen), 5 µg/ml F(ab’)₂ goat anti-human IgG/IgM/IgA (H + L) polyclonal Abs (anti-BCR) (Jackson ImmunoResearch Laboratories), 10 ng/ml of IL-21 (R&D Systems) and 50 IU/ml human IL-2 (Novartis). Unstimulated B cells were used as negative control. B cells cultured 72 h with or without stimulation cocktail were centrifuged and submitted to mechanical stimulation. Their supernatant were used for spontaneous extracellular vesicles isolation. Cell Multiplexing and single-cell RNA Sequencing [0304] scRNAseq using the CITE-seq method (Stoeckius et al., 2018, Genome Biol.) was essentially carried out as described in Abidi et al. (2020, Front. Immunol). For each experiment, cells were marked with viability dye (Fixable Viability Dye eFluor 450, 1/1000 in PBS without azide or protein, Invitrogen) for 25 min. Living cells were sorted on an ARIA III (BD Biosciences) and marked with conjugated DNA sequences (HashTag Oligonucleotide, HTO, Chromium Single Cell 3' Feature Barcode Kit, PN-1000079) specific of the donor and the experimental condition following CITE-seq protocols (Stoeckius et al., 2018, Genome Biol.). Cells were then pooled with similar amounts and 20000 total cells were loaded onto a Chromium controller (10X genomics) for libraries preparation (Chromium Next GEM Single Cell 3' Kit v3.1, ref PN-1000121; Chromium Next GEM Chip G Single Cell Kit, ref PN-1000120) and sequenced on a Nova-Seq 6000 (Illumina) at the GenoBird platform (IRS-UN, CHU Nantes). Raw reads were then aligned to the reference genome GRCh38 using the aligner STAR. Single-cell RNA sequencing analysis [0305] Analysis was performed using R (version 4.0.4) and Rstudio (version 1.3.1056). Data was further analyzed using the Seurat package (Hao et al., 2021, Cell) (v4.0.2) for the demultiplexing and pre-processing steps. Briefly, cells with less than 200 genes and more than 4000 genes or 25% of mitochondrial genes were excluded. Dimensional reductions (PCA and UMAP) and differential gene expression were performed with Seurat (Hao et al., 2021, Cell) v4.0.2 and MAST (Finak et al., 2015, Genome Biol.) v1.16.0. Production of extracellular vesicles by mechanical stimulation [0306] B cells cultured 72 h with or without stimulation cocktail were stimulated for 6 h in RPMI 1640 culture medium supplemented with penicillin/streptomycin (Gibco) in spinner flask of 100 mL or 1 L at a Kolmogorov length of 25 µm. Production conditions are detailed in Table 1 below. B cells were centrifuged and the supernatant was harvested for the isolation of mechanical stimulation extracellular vesicles. Table 1: Summary table of production conditions Production Cell Agitation Cells Conditions Volume Time volume concentration Speed Unstimulated Mechanical 100 mL 3.7E5 cells/mL 50 mL 300 RPM 6 h B cells stimulation 100 mL 3.5E5 cells/mL 50 mL 300 RPM 6 h Granzyme B+ Mechanical 2.02E5 1 L 500 mL 180 RPM 6 h Breg cells stimulation cells/mL Unstimulated Spontaneous 6-well plate 1E6 cells/mL 45 mL - 72 h B cells Granzyme B+ Spontaneous 6-well plate 1E6 cells/mL 45 mL - 72 h Breg cells Isolation of extracellular vesicles [0307] The EVs produced spontaneously or after mechanical stimulation were isolated from the supernatant by differential centrifugation. Briefly, the supernatant was submitted to a first centrifugation step at 2,000 g for 10 min to remove dead cells. Then, subcellular debris and larger vesicles were removed by centrifugation at 10,000 g for 30 min. Finally, extracellular vesicles were isolated by ultracentrifugation at 150,000 g for 1h30 (Ultracentrifuge Optima XE 90, Beckman Coulter). The EV pellet was then resuspended in sterile PBS for further characterization or in an EV-free complete RPMI medium for T cell suppression assay. The EV-free complete RPMI medium consisted in RPMI 1640 culture medium (Gibco) supplemented with 10% fetal bovine serum (Lonza), L- Glutamine and penicillin/streptomycin (Life Technologies) previously depleted of EVs by ultracentrifugation during 18 h at 10,000 g. EVs were stored at -80°C before being characterized. Quantification of extracellular vesicles [0308] Size and concentration of EVs were characterized using NTA (Nanoparticles Tracking Analysis, Nanosight NS300, laser 405 nm, Malvern) from the supernatant and from the isolated EVs. Samples were diluted in sterile PBS to obtain between 7E7 and 7E8 particles/mL in 1 mL. Five 60-second videos were recorded under constant flow, with a Camera Level set to 16 (acquisition rate: 25 frames/s). Data analysis was performed with the NTA NanoSight 3.3 software by choosing a detection threshold of 5. Western-blot [0309] B cells and their EVs produced spontaneously were lysed in RIPA Buffer (Cell Signaling Technology) supplemented with protease inhibitors (ThermoFisher Scientific) and the amount of protein was determined by BCA Assay (Interchim).10 µg of protein were separated on a 4-15% Mini-Protean TGX Precast protein gel and were transferred onto a nitrocellulose membrane (Biorad). After blocking for 2 h in PBS-0.1% Tween-20 containing 5% milk, membranes were incubated overnight at 4◦C with primary antibodies, washed, and incubated for 1 h with specific secondary peroxidase-coupled antibodies (Table 2). Proteins of interest were finally detected with the SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific) using a ChemiDoc Imaging System (Biorad).

Cryo-electron microscopy (CRYO-EM) [0310] CryoEM was performed using R2/2 quantifoil grids (Quantifoil). 3 μl of the sample were deposited on airglow-discharged quantifoil. The sample excess was blotted with a filter paper, and the grid plunged into liquid‑nitrogen-cooled ethane. The grid was rapidly transferred and kept under liquid nitrogen. For observation, the grids were mounted in a 626 Gatan holder using its cryo-transfer device. The observations were made in a Tecnai 200kV equipped with a field-emission gun (ThermoFisher Scientific). Images of the sample were recorded using a direct electron detection camera, K2 Summit (Gatan/Ametek). The images were aligned and summed as recommended by manufacturer. They were recorded at 15,000 magnification (pixel size 2.5 Å) using a total dose of about 20 electrons/Å2. Multiplex EV surface marker analysis [0311] Analysis of surface protein expression on B cells EVs produced spontaneously was performed using the MACSPlex Exosome kit human (Miltenyi Biotec) following the manufacturers instruction. Briefly, the exosome concentration was indirectly determined by quantifying the protein concentration by BCA assay as describe above. Extracellular vesicles from regulatory or unstimulated B cells produced spontaneously (20 µg) were incubated 1h at room temperature protected from light with exosome capture beads directed against 37 markers (CD1c, CD2, CD3, CD4, CD8, CD9, CD11c, CD14, CD19, CD20, CD24, CD25, CD29, CD31, CD40, CD41b, CD42a, CD44, CD45, CD49e, CD56, CD62p, CD63, CD69, CD81, CD86, CD105, CD133.1, CD142, CD146, CD209, CD326, HLA- ABC, HLA-DR DP DQ, MCSP, ROR1 and SSEA-4) and two isotype controls (mIgG1 and REA control). After washing, a detection antibody cocktail directed against CD9, CD63 and CD81 was added to the beads and incubated for 15 minutes at room temperature. Samples were washed and analyzed by flow cytometry (Canto II, BD Bioscience). A control was performed by incubating exosome capture beads with the buffer only. Background value from the buffer and the isotype control were subtracted from the median fluorescence intensity value (MFI) of each bead population. The MFI from the detection antibody cocktail was then calculated for each bead population and normalyzed by dividing with the mean of the detection antibody cocktail MFI according to the manufacturer’s instruction. Acquisition and analysis [0312] Flow cytometry data acquisition was performed with the CANTO II flow cytometer (BD Bioscience). Flow cytometry analysis were performed with FlowJo v10. Quantitative analysis was performed using Excel software. Western-Blot and CRYO-EM images were analyzed using ImageJ Imaging software (FIJI). Figures were generated with Prism v8 software. Results [0313] In order to characterize the secretion of EVs by Granzyme B+ Breg cells, the inventors developed two production models. B cells were stimulated for 72 h with the Granzyme B+ Breg cell stimulation cocktail described above in the “Granzyme B+ regulatory B cell Expansion” section. The supernatant was kept while the Granzyme B+ Breg cells were subjected to mechanical stimulation for 6 h at 300 rpm. EVs produced spontaneously (i.e., during the 72 h-culture with the stimulation cocktail) and after mechanical stimulation were isolated by differential centrifugation and characterized with NTA (NS300, Malvern). Quantification of EVs produced by each cell type showed that Granzyme B+ Breg cells spontaneously secreted 3 to 4 times more EVs than unstimulated B cells, with 78 EVs/cell in unstimulated B cells versus 305 EVs/cell in Granzyme B+ Breg cells (as shown on Figure 1A). Moreover, mechanical stimulation increased EV secretion of Granzyme B+ Breg cells by nearly 2-fold, with 305 EVs/cell in spontaneous production versus 554 EVs/cell in mechanical stimulation (as shown on Figure 1A). [0314] The size of EVs produced spontaneously and by mechanical stimulation was characterized by NTA (NS300, Malvern) and by cryo-TEM. The data showed a decrease in the size of EVs produced by mechanical stimulation compared to EVs produced spontaneously by unstimulated B cells (177.8 nm for spontaneously produced EVs versus 67.3 nm for mechanical stimulation EVs, as shown on Figures 1B-C) and by Granzyme B+ Breg cells (165.2 nm for spontaneously produced EVs versus 135.5 nm for mechanical stimulation EVs, as shown on Figures 1B-C). In addition, Cryo-TEM data showed the presence of a protein ring on the surface of spontaneously produced EVs (Figures 2A-D), and a difference in the content of spontaneously produced Granzyme B+ Breg cells EVs, with a higher electron density of spontaneously produced Granzyme B+ Breg cells EVs as compared to mechanical stimulation Granzyme B+ Breg cells EVs (Figure 3). [0315] To explore the transcriptomic impact of the in vitro stimulation of B cells toward the induction of Granzyme B+ Bregs, single cell RNA-seq was performed on both B cell populations. When represented in UMAP (Uniform Manfold proximation and Projection) the data show a highly dichotomic profile of Granzyme B+ Bregs and unstimulated B cells highlighting important variations in their transcripts (Figure 4A). Galectin-3 (LGALS3 gene) was one of the most differentially expressed genes, over-expressed in in vitro induced Granzyme B+ Breg cells compared to unstimulated cells, and was homogeneously expressed among Granzyme B+ Breg cells (Figures 4B and 4C). [0316] To determine the purity and type of EVs isolated, the expression of CD9, CD63, CD81, Tsg101, Galectin-3, and Calnexin was analyzed by western-blot in lysed unstimulated B cells and Granzyme B+ Breg cells, as well as in spontaneously produced EVs isolated from unstimulated B cells and Granzyme B+ Breg cells (Figure 5). The absence of Calnexin in the EV fractions both from unstimulated B cells and Granzyme B+ Breg cells indicates the absence of cellular contaminants (Figure 5). An enrichment of the exosomal markers CD9, CD63, CD81, and Tsg101 (Figure 5) could be detected in EVs from Granzyme B+ Breg cells, as compared to EVs from unstimulated B cells. Detection of Galectin-3 in both Granzyme B+ Breg cells and EVs isolated from Granzyme B+ Breg cells, and not in unstimulated B cells and EVs from unstimulated B cells (Figure 5), confirmed the results of the single-cell RNA sequencing analysis. [0317] Finally, a characterization of the membrane protein profile of EVs spontaneously produced by unstimulated B cells and Granzyme B+ Breg cells was performed by MACSPlex technology (Miltenyi) by flow cytometry. The analysis revealed the presence of all tested B cell markers: CD19, HLA-DP/DR/DQ, HLA-A/B/C, CD40, CD24, CD44, CD29, CD20, at the surface of spontaneously produced EVs from both unstimulated B cells and Granzyme B+ Breg cells (Figure 6). Weak expression of CD9, SSEA-4, CD62P, CD81, CD41b, CD42a and CD45 could also be detected in spontaneously produced EVs from Granzyme B+ Breg cells (Figure 6). Example 2: Impact of Breg-derived extracellular vesicles on T cell proliferation Materials and Methods B cell Isolation [0318] PBMCs were isolated from buffy coat of healthy donors, and B cells were isolated from PBMCs as described above in Example 1. T cell Isolation [0319] PBMCs were isolated from buffy coat of healthy donors as described above in Example 1. CD4+CD25- T Cells were isolated by a double negative selection of human PBMC using respectively Human CD4+ T Cell Isolation kit and Human CD25 MicroBeads II (Miltenyi Biotec). All the isolation were performed using the AutoMACS Pro Separator according to the manufacturer’s instructions (Miltenyi Biotec). Granzyme B+ regulatory B cell Expansion [0320] Granzyme B+ regulatory B cells were induced as described above in Example 1. Production of extracellular vesicles by mechanical stimulation [0321] Unstimulated B cells and Granzyme B+ regulatory B cells were stimulated mechanically as described above in Example 1 in order to induce the production of extracellular vesicles. Isolation of extracellular vesicles [0322] Extracellular vesicles were isolated from supernatants as described above in Example 1. T Cell suppression assay [0323] 5 x 10⁴ Cell Proliferation Dye (CPD eFluor670, eBioscience) labeled allogenic CD4+CD25- T cells were activated with anti-CD3/CD28 beads (at a 1:1 of beads:CD4+CD25- T cells ratio) (Gibco, ThermoFisher Scientific) and were co-cultured with extracellular vesicles produced spontaneously or by mechanical stimulation by 40 x 10⁶ Granzyme B+ regulatory B cells or unstimulated B cells during 72 h at 37°C and 5% CO₂ in EV-free complete RPMI and 96 U-bottom culture plate. A culture condition of T cells alone, with and without proliferation stimulation was performed as a control. After 72 h, cells were resuspended and then transferred to a FACS 96-well plate, and dead cells were labeled with the Fixable Viability Dye eFluor450 (FVD) dye following the manufacturer's instructions (Invitrogen). Cells were then labeled with anti-CD3-PerCP Cy5.5 (1/25) and anti-CD4-PeCy7 (1/25) antibodies (BD Bioscience). Cell proliferation and viability were assessed by flow cytometry (Canto II, BD Bioscience). Dead cells and debris were excluded from the proliferation analysis on FlowJo v10 software. Results [0324] To test their regulatory properties, EVs produced spontaneously or by mechanical stimulation were cultured with CD4+CD25- T cells stimulated with anti-CD3/CD28 beads for 72 h. The proliferation and viability of these cells were analyzed by flow cytometry. Unstimulated CD4+CD25- T cells were used as a negative control, and as shown on Figures 7A and 7E no proliferation could be detected. CD4+CD25- T cells activated with anti-CD3/CD28 beads were used as a positive control, and as shown on Figures 7B and 7F, 82% of the cells proliferated. On the other hand, as shown on Figures 7C and 7G, EVs produced by unstimulated B cells, both by spontaneous production (Figure 7C) and mechanical stimulation (Figure 7C), were capable of inhibiting T cell proliferation. Finally, as shown on Figures 7D and 7H, EVs secreted by Granzyme B+ regulatory B cells exhibited a superior inhibitory effect, with spontaneously produced EVs reducing CD4+CD25- T cells proliferation by 48% (Figure 7D) compared to a 38% reduction for EVs produced by mechanical stimulation (Figure 7H). Example 3: Breg-derived extracellular vesicles characterization Materials and Methods T Cell suppression assay [0325] 5.10⁴ Cell Proliferation Dye (CPD eFluor670, EBioscience) labeled allogenic CD4+ CD25- T cells were activated with anti-CD3/CD28 beads (at a 1:1 of beads:CD4+CD25- T cells ratio) (Gibco, ThermoFisher Scientific) and were co-cultured with EVs produced spontaneously by 5.10⁶ to 70.10⁶ of regulatory or unstimulated B cells (B NS) during 72h at 37°C and 5% CO₂ in EVs-free complete RPMI and 96 U-bottom culture plate. A culture condition of T cells alone, with and without proliferation stimulation was performed as a control. After 72h, cells are resuspended and then transferred to a FACS 96-well plate, and dead cells are labeled with the Fixable Viability Dye eFluor450 (FVD) dye following the manufacturer's instructions (Invitrogen). Cells were then labeled with anti-CD3-PerCP Cy5.5 (1/25) and anti-CD4-PeCy7 (1/25) antibodies (BD Bioscience). Cell proliferation and viability were assessed by flow cytometry (Canto II, BD Bioscience). Dead cells and debris were excluded from the proliferation analysis on FlowJo v10 software. Statistical significance was tested using linear regression and Kruskal-Wallis test where *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns, non-significant. miRNA extraction and profiling [0326] Total RNA from EVs produced by Breg cells and EVs produced by unstimulated B cells, including microRNA, were extracted using miRNeasy Micro Kit (Qiagen) according to the manufacturer’s protocol. Total RNA quantification was performed with the NanoDrop spectrophotometer. MicroRNA profiling was performed with 100 ng of total EVs RNA using the nCounter v3 miRNA panel, allowing the detection of 827 human microRNA, six positive controls, eight negative controls, six ligation controls, and five mRNA reference controls (ACTB, B2M, GAPDH, RPL19 and RPLP0), according to the manufacturer’s instructions (Nanostring Technologies). Normalization was performed using positive controls and the mean expression of the 50 top expressed miRNA in all the samples. miRNA with a mean raw count >50 in at least 2 samples by conditions were analyzed. miRNA were considered as differentially expressed with a FoldChange (FC) > 1.5 and a p-value from a Student t-test < 0.1. Mass spectrometry Protein digestion [0327] S-Trap^TM micro spin column (Protifi, Hutington, USA) digestion was performed on 20 µg of EVs from human B cells according to manufacturer’s instructions. Briefly, samples were supplemented with 20% SDS to a final concentration of 5%, reduced with 20 mM TCEP (Tris(2-carboxyethyl) phosphine hydrochloride) and alkylated with 50 mM CAA (chloracetamide) for 5min at 95^oC. Aqueous phosphoric acid was then added to a final concentration of 2.5% following by the addition of S-Trap binding buffer (90% aqueous methanol, 100 mM TEAB, pH 7.1). Mixtures were then loaded on S-Trap columns. Six washes were performed for thorough SDS elimination. Samples were digested with 1 µg of trypsin (Promega) at 47°C for 1h30. After elution, peptides were vacuum dried and resuspended in 50 µL TEAB 50 mM and HiPPR^TM Detergent Removal (ThermoFisher, Waltham, USA) was performed according to manufacturer’s instructions, using 100 µL of resin to remove any residual detergent. After which, the peptides were dried again and resuspended in 50 µL of TFA 0.1% in order to carry out a peptide clean- up on homemade stage tip using C18 Empore^TM disks (CDS Analytical, Oxford, USA). Two C18 disks were assembled for each sample. The disks were washed twice with 50 µL ACN 100% at 1500 g for 2 min and then with 50 µL of TFA 0.1% twice 2 min at 1500 g. The samples have been added onto the disks and centrifuged 2 min at 1500 g. The flow through has been passed through the disks a second time in order to maximize peptide retention. Peptides have been washed twice with 50 µL of TFA 0.1% and, finally, eluted with a mix of ACN 70% formic acid 0.1%. The eluted peptides were resuspended in 2% ACN, 0.1% formic acid in HPLC-grade water prior to MS analysis. NanoLC-MS/MS protein identification and quantification [0328] The tryptic peptides were resuspended in 25 µL and a quantity of 200 ng was injected on a nanoelute (Bruker Daltonics, Germany) HPLC (high-performance liquid chromatography) system coupled to a timsTOF Pro (Bruker Daltonics, Germany) mass spectrometer. HPLC separation (Solvent A: 0.1% formic acid in water, 2% acetonitrile; Solvent B: 0.1% formic acid in acetonitrile) was carried out at 250 nL/min using a packed emitter column (C18, 25 cm×75μm 1.6μm) (Ion Optics, Australia) using a 40 min gradient elution (2 to 11% solvent B during 19 min; 11 to 16% during 7 min; 16% to 25% during 4 min; 25% to 80% for 3 min and finally 80% for 7 min to wash the column). Mass-spectrometric data were acquired using the parallel accumulation serial fragmentation (PASEF) acquisition method in DIA mode. The measurements were carried out over the m/z range from 100 to 1700 Th. The range of ion mobilities values from 0.85 to 1.27 V s/cm2 (1/k0). The total cycle time was set to 0.95 s. Mass spectrometry data analysis [0329] Data analysis was performed using DIA-NN software (version 1.8.1). A search against the human UniProtKB/Swiss-Prot Homo sapiens database (downloaded the 23th of January, 2023, 26619 entries) was performed using library free workflow. For this purpose, “FASTA digest for library free search/library generation” and “Deep learning spectra, RTs and IMs prediction” options were checked for precursor ion generation. A maximum of 1 trypsin missed cleavages was allowed and the maximum variable modification was set to 2. Carbamidomethylation (Cys) was set as the fixed modification, whereas protein N-terminal methionine excision, methionine oxidation and N-terminal acetylation were set as variable modifications. The peptide length range was set to 7–30 amino acids, precursor charge range 2–4, precursor m/z range 300–1300, and fragment ion m/z range 300–1300. To search the parent mass and fragment ions, accuracy was set to 10 ppm manually. The false discovery rates (FDRs) at the protein and peptide level were set to 1%. Match between runs was allowed. For the quantification strategy, Robust LC (high precision) was used as advised in the software documentation, whereas default settings were kept for the other algorithm parameters. [0330] Statistical and bioinformatic analysis were performed with Perseus software (version 1.6.15.0) freely available at www.perseus-framework.org. All protein intensities were log2 transformed to perform statistics. For statistical comparison, four groups were set, each containing up to 4 biological replicates. The columns were then reordered in order to separate the turbulence and spontaneous conditions. The data were filtered from the spontaneous conditions to keep only proteins with at least 50% valid values in at least one group. Next, the data were imputed to fill missing data points by creating a Gaussian distribution of random numbers with a standard deviation of 33% relative to the standard deviation of the measured values and 1.8 standard deviation downshift of the mean to simulate the distribution of low signal values. t-test FDR<0.05, S0=0.1 was performed. Proteins with a P-value adjusted (P.adj) < 0.05 and a FoldChange (FC) > 1.5 were considered as enriched in EVs derived from GzmB+ Breg. Protein involvement in Biological Process analysis was performed on R by Gene ontology (ClusterProfiler R Package). Redundant terms were reduced using the rrvgo R package. Proteins contained in ontologies “Regulation of T cell proliferation” and “Lymphocyte Apoptotic Process” were considered as Target Proteins and were classified by their P.Adj and Log2FC. The present mass spectrometry work was performed by the Proteomics Platform Necker, Université Paris Cité - Structure Fédérative de Recherche Necker, INSERM US24/CNRS UAR3633, Paris 75015, France. Proteic target inhibition [0331] In order to identify the involvement of target proteins in the mechanism of action and activity of EVs secreted by Breg, the involvement of the proteins of interest was tested at different levels: during the expansion and production of EVs by Breg, during Breg-T CD4+ coculture, and during Breg-derived EVs / T CD4+ coculture. Proteic target inhibition on Breg expansion [0332] B cells were labelled for proliferation with eFluor450 according to the manufacturer instruction (Invitrogen) and were then expanded in the presence or absence of the stimulation cocktail described above for 72h 37°C 5% CO2 at 1.10⁶ cells/ml in flat- bottom plate. Implication of proteic target on Breg expansion was assessed by simultaneously adding during the expansion Galectin-3 inhibitor (GB1107, MCE) at incremental concentrations (1 to 30 µM). After 72h, cells are resuspended and then transferred to a FACS 96-well plate, and dead cells are labeled with the LIVE/DEAD Fixable Blue Dead Cell Stain Kit following the manufacturer's instructions (Invitrogen). Cells were then labeled for membranous CD19-BV786 and then for Granzyme B-PeCy7 (Biolegend) and Ki67-BUV395 (BD Biosciences) after permeabilization with the FIX & PERM™ kit (Invitrogen). Flow cytometry was performed on Aurora (Cytek®) and analysis done using the OMIQ software from Dotmatics (www.omiq.ai, www.dotmatics.com). Proteic target inhibition on Breg suppressive function of CD4+ T cells [0333] B cells were expanded with the cocktail of stimulation described above in the presence or absence of TNFR2 blocking antibody (Biolegend) at 10 µg/mL for 24 hours 37°C 5% CO2 at 1.10⁶ cells/ml in flat-bottom plate.1.10⁵ Breg were then cocultured with 5.10⁴ CD4+ CD25- T cells labelled with CPD eFluor450 (Invitrogen) and activated with anti-CD3/CD28 beads (at a 1:1 of beads:CD4+CD25- T cells ratio) (Gibco, ThermoFisher Scientific) for 72h 37°C 5% CO₂ in complete RPMI and 96 U-bottom culture plate. After 72h, cells are resuspended and then transferred to a FACS 96-well plate, and dead cells are labeled with the LIVE/DEAD Fixable Blue Dead Cell Stain Kit following the manufacturer's instructions (Invitrogen). Cells were then labeled for membranous CD19- BV786, CD3-BUV615 (BD Biosciences) and TNFR2-PE (Biolegend) and then for Granzyme B-PeCy7 (Biolegend) and Ki67-BUV395 (BD Biosciences) after permeabilization with the FIX & PERM™ kit (Invitrogen). Flow cytometry was performed on Aurora (Cytek®) and analysis done using the OMIQ software from Dotmatics (www.omiq.ai, www.dotmatics.com). Proteic target inhibition on Breg-derived EV suppressive function of CD4+ T cells [0334] 2.5.10⁴ Cell Proliferation Dye (CPD eFluor450, EBioscience) labeled CD4+ CD25- T cells were activated with anti-CD3/CD28 beads (at a 1:1 of beads:CD4+CD25- T cells ratio) (Gibco, ThermoFisher Scientific) and were co-cultured with EVs produced spontaneously by 20.10⁶ of regulatory B cells (Breg) during 72h at 37°C and 5% CO2 in EVs-free complete RPMI and 96 U-bottom culture plate. Protein target blockade was achieved by pre-incubating EVs produced from Breg with an anti-PD1 blocking antibody (Bioxcell) for 15 min at 10 µg/mL. A culture condition of T cells alone, with and without proliferation stimulation was performed as a control. After 72h, cells are resuspended and then transferred to a FACS 96-well plate, and dead cells are labeled with the LIVE/DEAD Fixable Blue Dead Cell Stain Kit following the manufacturer's instructions (Invitrogen). Cells were then labeled for membranous CD19-BV786 and then for Granzyme B-PeCy7 (Biolegend) and Ki67-BUV395 (BD Biosciences) after permeabilization with the FIX & PERM™ kit (Invitrogen). Flow cytometry was performed on Aurora (Cytek®) and analysis done using the OMIQ software from Dotmatics (www.omiq.ai, www.dotmatics.com). Results Breg-derived EVs display suppressive properties in vitro [0335] To assess the regulatory potential of Breg-derived EVs, increasing amounts of EVs were cultured for 72h with CD4+ T cells activated by CD3/CD28 beads. CD4+ T cell proliferation and viability were then analyzed by flow cytometry. Breg-derived EVs inhibited CD4+ T cell proliferation in a dose-dependent manner, with a maximum effect of inhibition between 30 and 50 million producing cells (Figure 8A). At this level, Breg- derived EVs had a 40% inhibitory potential on proliferation, while addition of EVs from unstimulated B cells had no effect (Figure 8B). As regulatory cell suppressor mechanisms can also affect effector cell viability, the impact of Breg-derived EVs on effector T cell viability was also investigated. EVs produced by GzmB+ Breg also induced CD4+ T cell death in a dose-dependent manner (Figure 8C) with a 3-fold increase compared to EVs from 30 to 50 million of unstimulated B producing cells (Figure 8D). Breg-derived EVs display a reduction in pro-inflammatory miRNA [0336] Various studies have shown that EVs can be enriched in microRNAs with biological functions. miRNAs were isolated from EVs derived from GzmB+ Breg and from EVs derived from unstimulated B cells and analyzed by in situ hybridization using a panel allowing semi-quantitative detection of 827 human miRNAs. Analysis of the miRNAs differentially expressed between EVs produced by unstimulated B and EVs produced by GzmB+ Breg led to the identification of 11 miRNAs differentially expressed between EVs produced by unstimulated B and EVs produced by Breg. Only one miRNA showed enrichment in Breg-derived EVs, hsa-miR-4454/7975 (FC=1.9, Pval = 0.02). In contrast, a decrease was observed in the presence of 10 miRNAs described in the regulation of various inflammatory events in Breg-derived EVs compared to EVs from unstimulated B cells (FC≤1.5, Pval <0.05) (Figure 9). Breg-derived EVs display an enrichment of proteins involved in the regulation of effector T cell proliferation and cell death [0337] To understand the mechanism of action of EVs produced by GzmB+ Breg, their protein content was lysed and analyzed by mass spectrometry. This method enabled to identify 221 proteins differentially enriched in Breg-derived EVs compared to EVs from unstimulated B cells (Table 3). Table 3: List of proteins differentially expressed by Breg-derived EVs produced spontaneously Gene Protein Description Log Pval symbol symbol 2FC _adj CCL3 CCL3; C-C motif chemokine 3 6.16 0.01 CL3L1 NCSTN NICA Nicastrin 5,76 0,01 IL21 IL21 Interleukin-21 5,00 0,01 PDCD1 PDCD1; Programmed cell death protein 1 4,67 0,01 PD-1 TNFRSF1B TNR1B; Tumor necrosis factor receptor superfamily 4,56 0,01 TNFR2 member 1B WRAP73 WRP73 WD repeat-containing protein WRAP73 4,48 0,01 SLC52A2 S52A2 Solute carrier family 52. riboflavin 4,33 0,01 transporter. member 2 ITGA1 ITA1 Integrin alpha-1 4,27 0,00 IL2RA IL2RA Interleukin-2 receptor subunit alpha 4,26 0,01 NCAPD2 CND1 Condensin complex subunit 1 4,14 0,00 SCARB1 SCRB1 Scavenger receptor class B member 1 3,98 0,01 FCRL5 FCRL5 Fc receptor-like protein 5 3,96 0,03 FLT1 VGFR1 Vascular endothelial growth factor 3,90 0,01 receptor 1 CEP55 CEP55 Centrosomal protein of 55 kDa 3,84 0,00 TNF TNFA Tumor necrosis factor 3,72 0,01 EPB41L5 E41L5 Band 4.1-like protein 5 3,66 0,01 AICDA AICDA Single-stranded DNA cytosine deaminase 3,63 0,01 SLC31A1 COPT1 High affinity copper uptake protein 1 3,60 0,01 LGALS3 LEG3 Galectin-3 3,58 0,01 DTX1 DTX1 E3 ubiquitin-protein ligase DTX1 3,50 0,01 PPP1R16B PP16B Protein phosphatase 1 regulatory inhibitor 3,49 0,02 subunit 16B IL4I1 OXLA L-amino-acid oxidase 3,41 0,01 KIF4A KIF4A Chromosome-associated kinesin KIF4A 3,40 0,01 RACGAP1 RGAP1 Rac GTPase-activating protein 1 3,39 0,01 CARD9 CARD9 Caspase recruitment domain-containing 3,36 0,01 protein 9 GDPD4 GDPD4 Glycerophosphodiester phosphodiesterase 3,34 0,01 domain-containing protein 4 KIF23 KIF23 Kinesin-like protein KIF23 3,28 0,01 SHCBP1 SHCBP SHC SH2 domain-binding protein 1 3,17 0,01 ITGA3 ITA3 Integrin alpha-3 3,14 0,01 IQGAP3 IQGA3 Ras GTPase-activating-like protein 3,10 0,04 IQGAP3 SLC38A1 S38A1 Sodium-coupled neutral amino acid 3,07 0,01 symporter 1 KIF2C KIF2C Kinesin-like protein KIF2C 3,07 0,02 NCAPG CND3 Condensin complex subunit 3 3,07 0,01 CDC42SE1 C42S1 CDC42 small effector protein 1 3,03 0,02 CD70 CD70 CD70 antigen 3,03 0,03 INCENP INCE Inner centromere protein 3,02 0,05 SLC43A3 S43A3 Equilibrative nucleobase transporter 1 2,97 0,01 LDLR LDLR Low-density lipoprotein receptor 2,96 0,01 CD40LG CD40L CD40 ligand 2,91 0,02 FANCI FANCI Fanconi anemia group I protein 2,90 0,01 NCAPH CND2 Condensin complex subunit 2 2,89 0,01 CD226 CD226 CD226 antigen 2,88 0,04 CTH CGL Cystathionine gamma-lyase 2,84 0,01 NKRF NKRF NF-kappa-B-repressing factor 2,82 0,01 RMND5A RMD5A E3 ubiquitin-protein transferase RMND5A 2,81 0,01 CKS2 CKS2 Cyclin-dependent kinases regulatory 2,81 0,01 subunit 2 PRC1 PRC1 Protein regulator of cytokinesis 1 2,80 0,03 C2orf69 CB069 Mitochondrial protein C2orf69 2,80 0,01 KIF14 KIF14 Kinesin-like protein KIF14 2,78 0,01 SLCO4A1 SO4A1 Solute carrier organic anion transporter 2,76 0,01 family member 4A1 CIP2A CIP2A Protein CIP2A 2,74 0,02 RCN1 RCN1 Reticulocalbin-1 2,72 0,01 NIBAN2 NIBA2 Protein Niban 2 2,70 0,02 KCNN4 KCNN4 Intermediate conductance calcium- 2,70 0,01 activated potassium channel protein 4 RPLP1 RLA1 Large ribosomal subunit protein P1 2,69 0,04 RNF149 RN149 E3 ubiquitin-protein ligase RNF149 2,69 0,01 GINS3 PSF3 DNA replication complex GINS protein 2,66 0,04 PSF3 DHCR24 DHC24 Delta(24)-sterol reductase 2,65 0,01 UCHL1 UCHL1 Ubiquitin carboxyl-terminal hydrolase 2,64 0,04 isozyme L1 SLC39A6 S39A6 Zinc transporter ZIP6 2,63 0,03 CENPM CENPM Centromere protein M 2,63 0,00 NQO1 NQO1 NAD(P)H dehydrogenase [quinone] 1 2,62 0,01 APOBEC3B ABC3B DNA dC->dU-editing enzyme APOBEC- 2,60 0,01 3B SEMA4A SEM4A Semaphorin-4A 2,59 0,05 P2RY11 P2Y11 P2Y purinoceptor 11 2,59 0,01 CDC20 CDC20 Cell division cycle protein 20 homolog 2,58 0,01 TRIP13 PCH2 Pachytene checkpoint protein 2 homolog 2,57 0,03 MAP7D1 MA7D1 MAP7 domain-containing protein 1 2,56 0,05 CLSTN1 CSTN1 Calsyntenin-1 2,55 0,02 JOSD1 JOS1 Josephin-1 2,55 0,04 FGFBP2 FGFP2 Fibroblast growth factor-binding protein 2 2,53 0,02 OCC1 OCC1 Overexpressed in colon carcinoma 1 2,53 0,01 protein CKS1B CKS1 Cyclin-dependent kinases regulatory 2,49 0,01 subunit 1 REXO4 REXO4 RNA exonuclease 4 2,49 0,04 ZWILCH ZWILC Protein zwilch homolog 2,49 0,03 DNAAF5 DAAF5 Dynein axonemal assembly factor 5 2,48 0,04 MELK MELK Maternal embryonic leucine zipper kinase 2,44 0,01 CDCA5 CDCA5 Sororin 2,43 0,04 FLVCR1 FLVC1 Heme transporter FLVCR1 2,42 0,02 CD80 CD80 T-lymphocyte activation antigen CD80 2,41 0,05 ZCCHC4 ZCHC4 rRNA N6-adenosine-methyltransferase 2,41 0,03 ZCCHC4 CCDC61 CCD61 Centrosomal protein CCDC61 2,40 0,01 CHAC2 CHAC2 Glutathione-specific gamma- 2,39 0,02 glutamylcyclotransferase 2 HEG1 HEG1 Protein HEG homolog 1 2,38 0,01 RGS3 RGS3 Regulator of G-protein signaling 3 2,38 0,01 RRS1 RRS1 Ribosome biogenesis regulatory protein 2,37 0,01 homolog ACSL4 ACSL4 Long-chain-fatty-acid--CoA ligase 4 2,36 0,01 RECQL5 RECQ5 ATP-dependent DNA helicase Q5 2,36 0,01 SLC1A4 SATT Neutral amino acid transporter A 2,36 0,01 THEMIS2 THMS2 Protein THEMIS2 2,33 0,01 TRAF7 TRAF7 E3 ubiquitin-protein ligase TRAF7 2,33 0,01 PLK1 PLK1 Serine/threonine-protein kinase PLK1 2,32 0,01 NDC80 NDC80 Kinetochore protein NDC80 homolog 2,32 0,01 SMN1 SMN Survival motor neuron protein 2,31 0,03 GNA15 GNA15 Guanine nucleotide-binding protein 2,31 0,05 subunit alpha-15 CHEK1 CHK1 Serine/threonine-protein kinase Chk1 2,31 0,01 AURKB AURKB Aurora kinase B 2,29 0,02 USP36 UBP36 Ubiquitin carboxyl-terminal hydrolase 36 2,28 0,01 TRAF1 TRAF1 TNF receptor-associated factor 1 2,25 0,05 EMILIN2 EMIL2 EMILIN-2 2,25 0,01 ATP13A3 AT133 Polyamine-transporting ATPase 13A3 2,23 0,04 MTHFD2 MTDC Bifunctional methylenetetrahydrofolate 2,23 0,01 dehydrogenase/cyclohydrolase. mitochondrial ZDHHC5 ZDHC5 Palmitoyltransferase ZDHHC5 2,23 0,01 CDC42SE2 C42S2 CDC42 small effector protein 2 2,22 0,02 MED15 MED15 Mediator of RNA polymerase II 2,22 0,02 transcription subunit 15 SLC16A13 MOT13 Monocarboxylate transporter 13 2,22 0,01 TOP2A TOP2A DNA topoisomerase 2-alpha 2,20 0,01 GBP5 GBP5 Guanylate-binding protein 5 2,19 0,04 CDK1 CDK1 Cyclin-dependent kinase 1 2,18 0,02 ITGAX ITAX Integrin alpha-X 2,17 0,03 FLVCR2 FLVC2 Heme transporter FLVCR2 2,17 0,01 GPRIN1 GRIN1 G protein-regulated inducer of neurite 2,17 0,01 outgrowth 1 IL21R IL21R Interleukin-21 receptor 2,15 0,02 DCAF6 DCAF6 DDB1- and CUL4-associated factor 6 2,14 0,05 SLC29A2 S29A2 Equilibrative nucleoside transporter 2 2,13 0,02 RAB23 RAB23 Ras-related protein Rab-23 2,13 0,03 SMC4 SMC4 Structural maintenance of chromosomes 2,12 0,01 protein 4 FMNL3 FMNL3 Formin-like protein 3 2,12 0,02 SLC39A14 S39AE Metal cation symporter ZIP14 2,10 0,01 MTRR MTRR Methionine synthase reductase 2,10 0,03 SRGAP2 SRGP2 SLIT-ROBO Rho GTPase-activating 2,06 0,01 protein 2 NUDT9 NUDT9 ADP-ribose pyrophosphatase. 2,05 0,03 mitochondrial CCR10 CCR10 C-C chemokine receptor type 10 2,04 0,02 VEGFA VEGFA Vascular endothelial growth factor A. long 2,04 0,01 form KIF16B KI16B Kinesin-like protein KIF16B 2,04 0,05 NAMPT NAMPT Nicotinamide phosphoribosyltransferase 2,04 0,01 TAGLN3 TAGL3 Transgelin-3 2,04 0,01 SMC2 SMC2 Structural maintenance of chromosomes 2,02 0,01 protein 2 ICAM1 ICAM1 Intercellular adhesion molecule 1 2,02 0,01 KIF15 KIF15 Kinesin-like protein KIF15 2,02 0,01 IFITM2 IFM2 Interferon-induced transmembrane protein 2,01 0,01 2 UBE2T UBE2T Ubiquitin-conjugating enzyme E2 T 2,01 0,01 SLAMF7 SLAF7 SLAM family member 7 2,01 0,03 DLGAP5 DLGP5 Disks large-associated protein 5 2,01 0,01 CAMKK2 KKCC2 Calcium/calmodulin-dependent protein 2,00 0,03 kinase kinase 2 WDHD1 WDHD1 WD repeat and HMG-box DNA-binding 1,99 0,05 protein 1 CD274 PD-L1 Programmed cell death 1 ligand 1 1,98 0,01 FBXO6 FBX6 F-box only protein 6 1,98 0,02 SLC38A5 S38A5 Sodium-coupled neutral amino acid 1,97 0,01 transporter 5 TNFAIP3 TNAP3 Tumor necrosis factor alpha-induced 1,97 0,02 protein 3 SSTR3 SSR3 Somatostatin receptor type 3 1,95 0,04 FAM78A FA78A Protein FAM78A 1,94 0,01 CPM CBPM Carboxypeptidase M 1,94 0,04 SLC6A9 SC6A9 Sodium- and chloride-dependent glycine 1,94 0,02 transporter 1 TFRC TFR1 Transferrin receptor protein 1 1,93 0,01 NLRC5 NLRC5 Protein NLRC5 1,93 0,01 SEL1L SE1L1 Protein sel-1 homolog 1 1,92 0,04 ANGPTL6 ANGL6 Angiopoietin-related protein 6 1,92 0,03 CD19 CD19 B-lymphocyte antigen CD19 1,91 0,01 APOL2 APOL2 Apolipoprotein L2 1,91 0,03 EPHA4 EPHA4 Ephrin type-A receptor 4 1,88 0,02 SH3BP2 3BP2 SH3 domain-binding protein 2 1,86 0,01 DDX50 DDX50 ATP-dependent RNA helicase DDX50 1,86 0,03 CDKAL1 CDKAL Threonylcarbamoyladenosine tRNA 1,85 0,01 methylthiotransferase SPAG5 SPAG5 Sperm-associated antigen 5 1,85 0,03 SLC38A2 S38A2 Sodium-coupled neutral amino acid 1,85 0,03 symporter 2 MITD1 MITD1 MIT domain-containing protein 1 1,84 0,03 CLEC2D CLC2D C-type lectin domain family 2 member D 1,84 0,02 CACNA1A CAC1A Voltage-dependent P/Q-type calcium 1,84 0,05 channel subunit alpha-1A MCRIP1 MCRI1 Mapk-regulated corepressor-interacting 1,83 0,04 protein 1 ADRM1 ADRM1 Proteasomal ubiquitin receptor ADRM1 1,83 0,01 PRF1 PERF Perforin-1 1,83 0,03 PRIM1 PRI1 DNA primase small subunit 1,82 0,05 IL2RB IL2RB Interleukin-2 receptor subunit beta 1,82 0,01 KIAA0040 K0040 Uncharacterized protein KIAA0040 1,81 0,02 MRPS34 RT34 Small ribosomal subunit protein mS34 1,80 0,05 LYRM7 LYRM7 Complex III assembly factor LYRM7 1,80 0,05 SPTLC2 SPTC2 Serine palmitoyltransferase 2 1,78 0,01 JPT1 JUPI1 Jupiter microtubule associated homolog 1 1,78 0,02 BCAT1 BCAT1 Branched-chain-amino-acid 1,77 0,01 aminotransferase. cytosolic CYBRD1 CYBR1 Plasma membrane ascorbate-dependent 1,77 0,04 reductase CYBRD1 BATF BATF Basic leucine zipper transcriptional factor 1,76 0,04 ATF-like ADAM8 ADAM8 Disintegrin and metalloproteinase domain- 1,76 0,03 containing protein 8 RIOK1 RIOK1 Serine/threonine-protein kinase RIO1 1,75 0,02 ECD ECD Protein ecdysoneless homolog 1,75 0,05 MYOF MYOF Myoferlin 1,75 0,02 SDF4 CAB45 45 kDa calcium-binding protein 1,73 0,04 ERCC6L ERC6L DNA excision repair protein ERCC-6-like 1,72 0,01 SLC3A2 4F2 4F2 cell-surface antigen heavy chain 1,72 0,01 POLA2 MT1E;M DNA polymerase alpha subunit B 1,71 0,02 T1G;MT 1M;MT1 X;MT2 ZNF672 DPOA2 Zinc finger protein 672 1,71 0,04 MT1E ZN672 Metallothionein-2 1.71 0.03 NEU3 NEUR3 Sialidase-3 1,70 0,03 FES FES Tyrosine-protein kinase Fes/Fps 1,69 0,05 LAPTM5 LAPM5 Lysosomal-associated transmembrane 1,69 0,02 protein 5 TRAF2 TRAF2 TNF receptor-associated factor 2 1,69 0,04 PLOD3 PLOD3 Multifunctional procollagen lysine 1,69 0,03 hydroxylase and glycosyltransferase LH3 SLC16A1 MOT1 Monocarboxylate transporter 1 1,68 0,01 EZH2 EZH2 Histone-lysine N-methyltransferase EZH2 1,68 0,01 ARL3 ARL3 ADP-ribosylation factor-like protein 3 1,68 0,05 SLC39A10 S39AA Zinc transporter ZIP10 1,67 0,01 NIBAN1 NIBA1 Protein Niban 1 1,66 0,01 C7orf50 CG050 Uncharacterized protein C7orf50 1,66 0,02 TNFAIP8L1 TP8L1 Tumor necrosis factor alpha-induced 1,66 0,03 protein 8-like protein 1 UBTD2 UBTD2 Ubiquitin domain-containing protein 2 1,65 0,02 RPS19 RS19 Small ribosomal subunit protein eS19 1,64 0,04 MVP MVP Major vault protein 1,64 0,01 KIF20A KI20A Kinesin-like protein KIF20A 1,62 0,02 BASP1 BASP1 Brain acid soluble protein 1 1,60 0,01 HLA-DPA1 DPA1 HLA class II histocompatibility antigen. 1,60 0,01 DP alpha 1 chain TREX1 TREX1 Three-prime repair exonuclease 1 1,60 0,05 MKI67 KI67 Proliferation marker protein Ki-67 1,59 0,02 ADGRE5 AGRE5 Adhesion G protein-coupled receptor E5 1,59 0,02 HMMR HMMR Hyaluronan mediated motility receptor 1,59 0,02 SMARCAD SMRCD SWI/SNF-related matrix-associated actin- 1,58 0,05 1 dependent regulator of chromatin subfamily A containing DEAD/H box 1 OFD1 OFD1 Centriole and centriolar satellite protein 1,58 0,01 OFD1 RRM1 RIR1 Ribonucleoside-diphosphate reductase 1,58 0,01 large subunit POLR3H RPC8 DNA-directed RNA polymerase III subunit 1,57 0,05 RPC8 SDCBP2 SDCB2 Syntenin-2 1,57 0,05 GOSR2 GOSR2 Golgi SNAP receptor complex member 2 1,57 0,04 MTHFD1L C1TM Monofunctional C1-tetrahydrofolate 1,56 0,02 synthase. mitochondrial LIMA1 LIMA1 LIM domain and actin-binding protein 1 1,56 0,01 SLC1A5 AAAT Neutral amino acid transporter B(0) 1,56 0,03 PARP4 PARP4 Protein mono-ADP-ribosyltransferase 1,55 0,01 PARP4 STK38L ST38L Serine/threonine-protein kinase 38-like 1,54 0,01 SPART SPART Spartin 1,52 0,05 POLR2D RPB4 DNA-directed RNA polymerase II subunit 1,52 0,02 RPB4 POLA1 DPOLA DNA polymerase alpha catalytic subunit 1,52 0,03 NIP7 NIP7 60S ribosome subunit biogenesis protein 1,51 0,02 NIP7 homolog WARS1 SYWC Tryptophan--tRNA ligase. cytoplasmic 1,51 0,04 RIDA RIDA 2-iminobutanoate/2-iminopropanoate 1,51 0,03 deaminase [0338] To identify the biological processes in which the proteins are involved, an unsupervised clustering analysis of the gene ontology was performed. This analysis enabled to classify the proteins contained in EVs into 12 biological processes. Given the activation profile of Breg cells, the main biological processes were associated with cell proliferation and B-cell activation. However, two biological processes related to the function of Breg-derived EVs stand out. Indeed, two ontologies were associated with the regulation of T cell proliferation and the up-regulation of T cell apoptosis. Proteins associated with these processes with a FoldChange >1.5 and adjusted p-values <0.05 led to the identification of 36 proteins of interest (Figure 10A-C and Table A), including 11 markers of biological interest in the mechanism of action of Breg (Table B). Table A: Proteins expressed by Breg-derived extracellular vesicles Gene Protein Protein Description NKRF NKRF NF-kappa-B-repressing factor TNF TNFA Tumor necrosis factor IL2RA IL2RA Interleukin-2 receptor subunit alpha ICAM1 ICAM1 Intercellular adhesion molecule 1 FES FES Tyrosine-protein kinase Fes/Fps UCHL1 UCHL1 Ubiquitin carboxyl-terminal hydrolase isozyme L1 CCL3;CCL3L1 CCL3;CL3L1 C-C motif chemokine 3 PRF1 PERF Perforin-1 IL2RB IL2RB Interleukin-2 receptor subunit beta LGALS3 LEG3 Galectin-3 TNFRSF1B TNR1B; Tumor necrosis factor receptor superfamily TNFR2 member 1B ITGAX ITAX Integrin alpha-X TNFAIP3 TNAP3 Tumor necrosis factor alpha-induced protein 3 ITGA3 ITA3 Integrin alpha-3 CCR10 CCR10 C-C chemokine receptor type 10 ITGA1 ITA1 Integrin alpha-1 TRAF2 TRAF2 TNF receptor-associated factor 2 TRAF1 TRAF1 TNF receptor-associated factor 1 SLC39A6 S39A6 Zinc transporter ZIP6 MELK MELK Maternal embryonic leucine zipper kinase PDCD1 PDCD1; PD- Programmed cell death protein 1 1 BATF BATF Basic leucine zipper transcriptional factor ATF- like THEMIS2 THMS2 Protein THEMIS2 TRAF7 TRAF7 E3 ubiquitin-protein ligase TRAF7 NLRC5 NLRC5 Protein NLRC5 DTX1 DTX1 E3 ubiquitin-protein ligase DTX1 NCSTN NICA Nicastrin GBP5 GBP5 Guanylate-binding protein 5 FCRL5 FCRL5 Fc receptor-like protein 5 IL4I1 OXLA L-amino-acid oxidase CARD9 CARD9 Caspase recruitment domain-containing protein 9 SEMA4A SEM4A Semaphorin-4A IL21 IL21 Interleukin-21 IL21R IL21R Interleukin-21 receptor SLAMF7 SLAF7 SLAM family member 7 CD274 PD-L1 Programmed cell death 1 ligand 1 Table B: Proteins expressed by Breg-derived extracellular vesicles having biological interest in the mechanism of action of Breg Genes Protein Protein Description Names NKRF NKRF NF-kappa-B-repressing factor TNF TNFA Tumor necrosis factor IL2RA IL2RA Interleukin-2 receptor subunit alpha CCL3;CCL3L1 CCL3;CL3L1 C-C motif chemokine 3 PRF1 PERF Perforin-1 LGALS3 LEG3 Galectin-3 TNFRSF1B TNR1B; Tumor necrosis factor receptor superfamily TNFR2 member 1B PDCD1 PDCD1; PD-1 Programmed cell death protein 1 IL4I1 OXLA L-amino-acid oxidase SLAMF7 SLAF7 SLAM family member 7 CD274 PD-L1 Programmed cell death 1 ligand 1 [0339] Similarly, to understand the mechanism of action of EVs produced by GzmB+ Breg submitted to a mechanical stimulation (turbulent flow), their protein content was lysed and analyzed by mass spectrometry. This method enabled to identify 185 proteins differentially enriched in Breg-derived EVs produced under mechanical stimulation compared to EVs from unstimulated B cells produced under mechanical stimulation (Table 4). Table 4: List of proteins differentially expressed by Breg-derived EVs produced under mechanical stimulation G^{ene Symbol Protein} Description Log2fc Symbol BCHE CHLE Cholinesterase 3,05 PITPNM2 PITM2 Membrane-associated phosphatidylinositol 2,88 transfer protein 2 TCOF1 TCOF Treacle protein 2,69 SF3A3 SF3A3 Splicing factor 3A subunit 3 2,66 PNISR PNISR Arginine/serine-rich protein PNISR 2,62 ZRANB2 ZRAB2 Zinc finger Ran-binding domain-containing 2,6 protein 2 PDGFRB PGFRB Platelet-derived growth factor receptor beta 2,57 CCDC124 CC124 Coiled-coil domain-containing protein 124 2,51 IGHV3-35 HV335 Probable non-functional immunoglobulin 2,49 heavy variable 3-35 JMJD1C JHD2C Probable JmjC domain-containing histone 2,45 demethylation protein 2C RPL26L1 RL26L Ribosomal protein uL24-like 2,41 PM20D2 P20D2 Xaa-Arg dipeptidase 2,4 DYNLL1 DYL1 Dynein light chain 1, cytoplasmic 2,34 KRT15 K1C15 Keratin, type I cytoskeletal 15 2,33 PHIP PHIP PH-interacting protein 2,32 RPL21 RL21 Large ribosomal subunit protein eL21 2,31 GGACT GGACT Gamma-glutamylaminecyclotransferase 2,31 RBM8A RBM8A RNA-binding protein 8A 2,27 ORM2 A1AG2 Alpha-1-acid glycoprotein 2 2,26 XRCC1 XRCC1 DNA repair protein XRCC1 2,22 RNPS1 RNPS1 RNA-binding protein with serine-rich 2,21 domain 1 HSP90AB3P H90B3 Putative heat shock protein HSP 90-beta-3 2,2 CDC27 CDC27 Cell division cycle protein 27 homolog 2,2 JPT1 JUPI1 Jupiter microtubule associated homolog 1 2,19 SP140 SP140 Nuclear body protein SP140 2,19 PSIP1 PSIP1 PC4 and SFRS1-interacting protein 2,17 RIOX2 RIOX2 Ribosomal oxygenase 2 2,15 CPSF3 CPSF3 Cleavage and polyadenylation specificity 2,13 factor subunit 3 REPIN1 REPI1 Replication initiator 1 2,11 HLA-DQA1 DQA1 HLA class II histocompatibility antigen, DQ 2,09 alpha 1 chain SOWAHD SWAHD Ankyrin repeat domain-containing protein 2,08 SOWAHD HMGN3 HMGN3 High mobility group nucleosome-binding 2,08 domain-containing protein 3 LCN2 NGAL Neutrophil gelatinase-associated lipocalin 2,07 SRSF3 SRSF3 Serine/arginine-rich splicing factor 3 2,06 CUL4B CUL4B Cullin-4B 2,06 NEMF NEMF Ribosome quality control complex subunit 2,06 NEMF ELANE ELNE Neutrophil elastase 2,06 RPLP1 RLA1 Large ribosomal subunit protein P1 2,05 CALML3 CALL3 Calmodulin-like protein 3 2,04 SLTM SLTM SAFB-like transcription modulator 2,04 CLSTN1 CSTN1 Calsyntenin-1 2,04 ACTR1A ACTZ Alpha-centractin 2,04 HLA-DRB1 DRB1 HLA class II histocompatibility antigen, 2,04 DRB1 beta chain TRA2A TRA2A Transformer-2 protein homolog alpha 2,03 WDR5 WDR5 WD repeat-containing protein 5 2 GTF2B TF2B Transcription initiation factor IIB 2 CDH13 CAD13 Cadherin-13 1,97 INTS1 INT1 Integrator complex subunit 1 1,96 GSTM3 GSTM3 Glutathione S-transferase Mu 3 1,95 ZNF280C Z280C Zinc finger protein 280C 1,95 MEF2D MEF2D Myocyte-specific enhancer factor 2D 1,93 EXOSC1 EXOS1 Exosome complex component CSL4 1,92 RANBP9 RANB9 Ran-binding protein 9 1,92 DEK DEK Protein DEK 1,9 AZGP1 ZA2G Zinc-alpha-2-glycoprotein 1,89 RPL3 RL3 Large ribosomal subunit protein uL3 1,88 DHCR7 DHCR7 7-dehydrocholesterol reductase 1,88 SAFB2 SAFB2 Scaffold attachment factor B2 1,88 NDUFS2 NDUS2 NADH dehydrogenase [ubiquinone] iron- 1,87 sulfur protein 2, mitochondrial USP39 SNUT2 U4/U6,U5 tri-snRNP-associated protein 2 1,87 FAF2 FAF2 FAS-associated factor 2 1,85 ENGASE ENASE Cytosolic endo-beta-N- 1,84 acetylglucosaminidase ACP1 PPAC Low molecular weight phosphotyrosine 1,84 protein phosphatase SPAG5 SPAG5 Sperm-associated antigen 5 1,84 CTNNBL1 CTBL1 Beta-catenin-like protein 1 1,82 ZNF512 ZN512 Zinc finger protein 512 1,81 SBSN SBSN Suprabasin 1,81 CYB5B CYB5B Cytochrome b5 type B 1,81 TFCP2 TFCP2 Alpha-globin transcription factor CP2 1,8 ARL6IP1 AR6P1 ADP-ribosylation factor-like protein 6- 1,79 interacting protein 1 TUBG1 TBG1 Tubulin gamma-1 chain 1,78 COG2 COG2 Conserved oligomeric Golgi complex 1,78 subunit 2 MACROH2A1 H2AY Core histone macro-H2A,1 1,78 ARPC5 ARPC5 Actin-related protein 2/3 complex subunit 5 1,77 CMAS NEUA N-acylneuraminate cytidylyltransferase 1,77 CD79A CD79A B-cell antigen receptor complex-associated 1,76 protein alpha chain BAZ1B BAZ1B Tyrosine-protein kinase BAZ1B 1,76 SMC6 SMC6 Structural maintenance of chromosomes 1,76 protein 6 OSBPL3 OSBL3 Oxysterol-binding protein-related protein 3 1,75 NSA2 NSA2 Ribosome biogenesis protein NSA2 1,75 homolog CCDC6 CCDC6 Coiled-coil domain-containing protein 6 1,75 EXOSC10 EXOSX Exosome component 10 1,75 SMARCE1 SMCE1 SWI/SNF-related matrix-associated actin- 1,74 dependent regulator of chromatin subfamily E member 1 LUC7L2 LC7L2 Putative RNA-binding protein Luc7-like 2 1,74 TUBGCP2 GCP2 Gamma-tubulin complex component 2 1,74 NOL11 NOL11 Nucleolar protein 11 1,73 MTA1 MTA1 Metastasis-associated protein MTA1 1,73 ADA ADA Adenosine deaminase 1,73 H2BC3 H2B1B Histone H2B type 1-B 1,72 TOP1 TOP1 DNA topoisomerase 1 1,72 MAEA MAEA E3 ubiquitin-protein transferase MAEA 1,72 SBNO1 SBNO1 Protein strawberry notch homolog 1 1,72 C1orf198 CA198 Uncharacterized protein C1orf198 1,71 ENSA ENSA Alpha-endosulfine 1,71 VWA5A VMA5A von Willebrand factor A domain-containing 1,7 protein 5A RAD21 RAD21 Double-strand-break repair protein rad21 1,7 homolog LYAR LYAR Cell growth-regulating nucleolar protein 1,7 PWP1 PWP1 Periodic tryptophan protein 1 homolog 1,7 SELENBP1 SBP1 Methanethiol oxidase 1,7 U2SURP SR140 U2 snRNP-associated SURP motif- 1,7 containing protein WDR3 WDR3 WD repeat-containing protein 3 1,69 PAF1 PAF1 RNA polymerase II-associated factor 1 1,69 homolog PFKFB3 F263 6-phosphofructo-2-kinase/fructose-2,6- 1,69 bisphosphatase 3 HELZ2 HELZ2 Helicase with zinc finger domain 2 1,68 MRE11 MRE11 Double-strand break repair protein MRE11 1,68 SAFB SAFB1 Scaffold attachment factor B1 1,67 TRMT1L TRM1L TRMT1-like protein 1,67 DCTN5 DCTN5 Dynactin subunit 5 1,67 SURF6 SURF6 Surfeit locus protein 6 1,66 CHD4 CHD4 Chromodomain-helicase-DNA-binding 1,66 protein 4 PARP1 PARP1 Poly [ADP-ribose] polymerase 1 1,66 RCC1 RCC1 Regulator of chromosome condensation 1,65 C5orf52 CE052 Uncharacterized protein C5orf52 1,65 GTDC1 GTDC1 Glycosyltransferase-like domain-containing 1,65 protein 1 ZC3H4 ZC3H4 Zinc finger CCCH domain-containing 1,65 protein 4 GSTT1 GSTT1 Glutathione S-transferase theta-1 1,63 NPM1 NPM Nucleophosmin 1,63 GNL2 NOG2 Nucleolar GTP-binding protein 2 1,63 MDC1 MDC1 Mediator of DNA damage checkpoint 1,62 protein 1 METAP2 MAP2 Methionine aminopeptidase 2 1,62 FUCA2 FUCO2 Plasma alpha-L-fucosidase 1,62 PIH1D1 PIHD1 PIH1 domain-containing protein 1 1,62 FUCA1 FUCO Tissue alpha-L-fucosidase 1,62 OGA OGA Protein O-GlcNAcase 1,61 P2RY8 P2RY8 P2Y purinoceptor 8 1,61 LRWD1 LRWD1 Leucine-rich repeat and WD repeat- 1,61 containing protein 1 SF3B1 SF3B1 Splicing factor 3B subunit 1 1,61 CRTAC1 CRAC1 Cartilage acidic protein 1 1,61 CYB5R2 NB5R2 NADH-cytochrome b5 reductase 2 1,61 XRN2 XRN2 5'-3' exoribonuclease 2 1,6 SHMT1 GLYC Serine hydroxymethyltransferase, cytosolic 1,6 SNU13 NH2L1 NHP2-like protein 1 1,6 CCDC47 CCD47 PAT complex subunit CCDC47 1,6 CASP8 CASP8 Caspase-8 1,59 M6PR MPRD Cation-dependent mannose-6-phosphate 1,59 receptor SLC2A5 GTR5 Solute carrier family 2, facilitated glucose 1,59 transporter member 5 SLC16A13 MOT13 Monocarboxylate transporter 13 1,59 MBD1 MBD1 Methyl-CpG-binding domain protein 1 1,58 PIGR PIGR Polymeric immunoglobulin receptor 1,58 CDC16 CDC16 Cell division cycle protein 16 homolog 1,58 SMARCA5 SMCA5 SWI/SNF-related matrix-associated actin- 1,58 dependent regulator of chromatin subfamily A member 5 NOP58 NOP58 Nucleolar protein 58 1,58 POLR1B RPA2 DNA-directed RNA polymerase I subunit 1,58 RPA2 ILF3 ILF3 Interleukin enhancer-binding factor 3 1,58 MAF1 MAF1 Repressor of RNA polymerase III 1,58 transcription MAF1 homolog CMSS1 CMS1 Protein CMSS1 1,58 GP9 GPIX Platelet glycoprotein IX 1,58 SART3 SART3 Squamous cell carcinoma antigen 1,57 recognized by T-cells 3 HSPA13 HSP13 Heat shock 70 kDa protein 13 1,57 TRIP12 TRIPC E3 ubiquitin-protein ligase TRIP12 1,57 CNOT10 CNO10 CCR4-NOT transcription complex subunit 1,57 10 VAMP4 VAMP4 Vesicle-associated membrane protein 4 1,57 TRMT1 TRM1 tRNA (guanine(26)-N(2))- 1,56 dimethyltransferase FAM111B F111B Serine protease FAM111B 1,56 AKAP8L AKP8L A-kinase anchor protein 8-like 1,56 MAK16 MAK16 Protein MAK16 homolog 1,56 LARS2 SYLM Leucine--tRNA ligase, mitochondrial 1,55 PFKFB2 F262 6-phosphofructo-2-kinase/fructose-2,6- 1,55 bisphosphatase 2 PPAN SSF1 Suppressor of SWI41 homolog 1,55 HP1BP3 HP1B3 Heterochromatin protein 1-binding protein 3 1,55 ARRB2 ARRB2 Beta-arrestin-2 1,55 NOP2 NOP2 Probable 28S rRNA (cytosine(4447)-C(5))- 1,55 methyltransferase SNRPA1 RU2A U2 small nuclear ribonucleoprotein A' 1,55 MRPS35 RT35 Small ribosomal subunit protein mS35 1,54 CDC42SE2 C42S2 CDC42 small effector protein 2 1,54 QSOX2 QSOX2 Sulfhydryl oxidase 2 1,54 RIPK3 RIPK3 Receptor-interacting serine/threonine- 1,54 protein kinase 3 PPP1R14B PP14B Protein phosphatase 1 regulatory subunit 1,54 14B GPATCH11 GPT11 G patch domain-containing protein 11 1,53 EP400 EP400 E1A-binding protein p400 1,53 CDK5RAP3 CK5P3 CDK5 regulatory subunit-associated protein 1,53 3 GSPT2 ERF3B Eukaryotic peptide chain release factor 1,53 GTP-binding subunit ERF3B RANBP3 RANB3 Ran-binding protein 3 1,53 PPP2R5D 2A5D Serine/threonine-protein phosphatase 2A 56 1,52 kDa regulatory subunit delta isoform SPCS1 SPCS1 Signal peptidase complex subunit 1 1,52 EIF4G3 IF4G3 Eukaryotic translation initiation factor 4 1,52 gamma 3 NUP54 NUP54 Nucleoporin p54 1,52 SNRNP200 U520 U5 small nuclear ribonucleoprotein 200 kDa 1,52 helicase SRSF10 SRS10 Serine/arginine-rich splicing factor 10 1,51 NUTF2 NTF2 Nuclear transport factor 2 1,51 OAS1 OAS1 2'-5'-oligoadenylate synthase 1 1,51 EMILIN2 EMIL2 EMILIN-2 1,51 VAMP2 VAMP2 Vesicle-associated membrane protein 2 1,51 TMEM205 TM205 Transmembrane protein 205 1,5 SMARCA2 SMCA2 Probable global transcription activator 1,5 SNF2L2 [0340] Among the proteins differentially enriched in Breg-derived EVs produced under mechanical stimulation compared to EVs from unstimulated B cells produced under mechanical stimulation, some are associated with the regulation of T cell and of T cell apoptosis, such as e.g. LYAR, CASP8 and RIPK3, which represent markers of biological interest in the mechanism of action of Breg. The suppressive activity of Breg-derived EVs is at least partially dependent on PD-1, PD-L1, TNFR2, TNF and Galectin-3 [0341] Suppressive mechanisms of Breg-derived EVs are dependent of three main processes: (1) Breg expansion, (2) the ability of Breg to secrete EVs and (3) Breg-derived EVs suppressive function. To identify the involvement of target proteins in the suppressive activity of EVs secreted by Breg, the involvement of the proteins of interest was tested at different levels: during expansion, during production of EVs from Breg, during Breg / T CD4+ coculture, and during Breg-derived EVs / T CD4+ coculture. [0342] To identify the proteins involved in the suppressive activity directly mediated by Breg-derived EVs, EVs produced by 20 million Breg were pre-incubated or not with blocking antibodies and then cultured with CD4+ CD25- T cells activated with CD3/CD28 beads for 3 days. [0343] Firstly, Breg were incubated with a TNFR2 blocking antibody during their expansion and production of EVs, and then these cells were co-cultured with CD4+ CD25- T cells. Blocking the TNFR2 receptor led to an approximately 50% decrease in proliferation inhibition of CD4+ T cells, demonstrating that TNFR2 and/or TNF are indirectly involved in the suppressive activity of Breg-derived EVs by affecting their content and/or secretion (Figure 11A). [0344] Then, the inventors investigated whether blocking these proteins could block the EVs suppressive activity of Breg by blocking Breg expansion. Breg were incubated with a Galectin-3 inhibitor during their expansion for 72h. Inhibition of Galectin-3 reduced the ability of Breg to expand in vitro (Figure 11B) demonstrating that Galectin-3 also indirectly plays an important role in the suppressive activity of Breg-derived EVs by modulating the Breg expansion ability. [0345] Finally, blocking PD-1 in Breg-derived EVs reduced the proliferation-inhibitory effect of Breg-derived EVs by around 50% (Figure 11C-D). These results demonstrate that EVs produced by GzmB+ Breg can partially block CD4+ T cell proliferation via PD-1 and/or PD-L1 expression. Thus, PD-1 and/or PD-L1 expressed in Breg-derived EVs play an important role in the suppressive activity of Breg-derived EVs, said suppressive activity being at least partially dependent on PD-1 and/or PD-L1. In conclusion, the inventors showed that several protein markers are expressed in Breg- derived EVs, such as PD-1, TNFR2, TNF, which play a role in the suppressive activity of Breg and/or of Breg-derived EVs. To their knowledge, those proteins have never been described as markers associated with EVs.

Claims

CLAIMS 1. An isolated regulatory B cell (Breg)-derived extracellular vesicle wherein said extracellular vesicle comprises Galectin-3. 2. An isolated regulatory B cell (Breg)-derived extracellular vesicle obtained or obtainable by a method comprising: a) culturing B cells in a culture medium with a stimulation cocktail until Breg cells are induced and Breg-derived extracellular vesicles are produced in the culture medium, and b) collecting said Breg-derived extracellular vesicle in the culture medium, wherein said stimulation cocktail comprises soluble human CD40L, class B CpG oligodeoxynucleotide, anti-human IgG/IgM/IgA polyclonal antibodies, human IL-21 and human IL-2. 3. An isolated regulatory B cell (Breg)-derived extracellular vesicle obtained or obtainable by using a fluidic system comprising at least one container, a culture medium contained by the container, Breg cells, a culture medium agitator, means for controlling the speed of the agitator adapted for the growth of the Breg cells, wherein the means for controlling the speed of the agitator, the agitator and the shape and dimensions of the container are adapted to the generation of a turbulent flow of the culture medium in the container to exert shear stresses on the Breg cells in order to achieve the production of extracellular vesicles, the Kolmogorov length of the flow being less than or equal to 50 µm. 4. An isolated regulatory B cell (Breg)-derived extracellular vesicle obtained or obtainable by a method comprising: a) culturing Breg cells in a culture medium, b) applying a turbulent flow in the culture medium until Breg-derived extracellular vesicles are produced in the culture medium, wherein the Kolmogorov length of the flow is less than or equal to 50 µm, and c) collecting said Breg-derived extracellular vesicle in the liquid medium. 5. The isolated Breg-derived extracellular vesicle according to any one of claims 2 to 4, wherein said extracellular vesicle comprises Galectin-3. 6. The isolated Breg-derived isolated regulatory B cell (Breg)-derived extracellular vesicle according to any one of claims 1 to 5, wherein said extracellular vesicle comprises Programmed cell Death protein 1 (PD-1) and/or Programmed cell Death Ligand 1 (PD-L1). 7. The isolated Breg-derived isolated regulatory B cell (Breg)-derived extracellular vesicle according to any one of claims 1 to 6, wherein said extracellular vesicle comprises Tumor Necrosis Factor Receptor 2 (TNFR2) and/or Tumor Necrosis Factor (TNF). 8. The isolated Breg-derived isolated regulatory B cell (Breg)-derived extracellular vesicle according to any one of claims 1 to 7, wherein said extracellular vesicle comprises at least one marker selected from CD9, CD63, CD81, and Tsg101. 9. The isolated Breg-derived extracellular vesicle according to any one of claims 1 to 8, wherein said extracellular vesicle comprises at least one marker selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, HLA-DQ, CD19, CD20, CD24, CD29, CD40, CD44, and CD45. 10. The isolated Breg-derived extracellular vesicle according to any one of claims 1 to 9, wherein said extracellular vesicle is capable of inhibiting T cell proliferation, increasing T cell apoptosis, and/or increasing proinflammatory cytokine secretion by T cells. 11. A composition or pharmaceutical composition comprising the isolated Breg- derived extracellular vesicle according to any one of claims 1 to 10, and optionally at least one pharmaceutically acceptable excipient. 12. The isolated Breg-derived extracellular vesicle according to any one of claims 1 to 10, or the composition according to claim 11, for use as a medicament. 13. The isolated Breg-derived extracellular vesicle according to any one of claims 1 to 10, or the composition according to claim 11, for use in the prevention, reduction and/or treatment of transplant rejection, graft-versus-host disease (GvHD), autoimmune disease, or abnormal or excessive immune response. 14. The isolated Breg-derived extracellular vesicle according to any one of claims 1 to 10, or the composition according to claim 11, for use in inhibiting T cell proliferation, increasing T cell apoptosis, increasing proinflammatory cytokine secretion by T cells, and/or inducing immune tolerance in a subject in need thereof. 15. A kit comprising the isolated Breg-derived extracellular vesicle according to any one of claims 1 to 10, and instructions for use.