Regulatory T cell (Treg) extracellular vesicle compositions and methods

Abstract

The present disclosure provides anti-inflammatory extracellular vesicles (EVs) derived from ex vivo expanded human suppressive immune cells, such as regulatory T cells (Tregs). Such EVs are useful in the treatment of diseases such as amyotrophic lateral sclerosis (AES), Alzheimer's disease, and other neurological diseases, as well as inflammatory and autoimmune diseases or disorders.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 208,395, filed June 8, 2021, and U.S. Provisional Application No. 63 / 154,449, filed February 26, 2021, each of which is incorporated by reference in its entirety herein.

[0002] (1. Field) The present disclosure provides anti-inflammatory and reparative extracellular vesicles (EVs) derived from ex vivo expanded human suppressive immune cells, such as regulatory T cells (Tregs), and that are useful in the treatment of diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and other neurological diseases, as well as inflammatory, metabolic, and autoimmune diseases or dysfunctions. [Background technology]

[0003] (2.Background) Inflammation and neuroinflammatory mechanisms contribute to a wide variety of devastating diseases, including neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease, and multiple sclerosis, which result in significant health and economic burdens that only worsen over time.

[0004] Currently, no disease-modifying treatments are available for such diseases. Anti-inflammatory treatments have been available for decades in an attempt to ameliorate a number of neurodegenerative diseases. However, single drug / target approaches have made little progress.

[0005] Increasing studies point to the involvement of the immune system in the pathogenesis of such diseases and to immune cell dysfunction as a key mediator of disease pathogenesis. The complex signaling mechanisms and inherent redundancy of the immune system and its components may help explain the ineffectiveness of such single-drug / single-target anti-inflammatory approaches.

[0006] Recently, cell therapy with regulatory T cells (Tregs) has shown great promise, which may represent a more comprehensive approach to suppress immune system dysfunction that contributes to disease. For example, clinical trials involving the administration of expanded autologous Tregs to ALS patients have reported that Treg therapy slowed the rate of early and late disease progression, and that Treg suppressive function correlated with delayed disease progression (Thonhoff, JR et al., 2018, Neurology-Neuroimmunology Neuroinflammation 5(4)).

[0007] Nonetheless, there remains a need for the development of additional therapies that can suppress inflammation and / or promote anti-inflammatory immune system components, and that can do so in the pro-inflammatory and toxic disease state microenvironment. Summary of the Invention

[0008] (3. Overview) Presented herein are extracellular vesicles (EVs) that exhibit significant anti-inflammatory activity both in vitro and in vivo. The EVs presented herein are derived from ex vivo expanded human suppressive immune cells, e.g., regulatory T cells (Tregs). As shown herein, the EVs of the present disclosure retain the immunosuppressive activity of the cells from which they are derived. Furthermore, since EVs are not cells themselves, they avoid potential cell-based issues such as immune rejection and possible polarization into pro-inflammatory cell types. Thus, the anti-inflammatory EVs presented herein are particularly useful for the treatment of various diseases, such as, for example, neurodegenerative disorders, e.g., amyotrophic lateral sclerosis (ALS).

[0009] Results presented herein show that the disclosed EVs can potently suppress T-responder cell proliferation and pro-inflammatory myeloid, e.g., macrophage, activity in vitro, and exert potent anti-inflammatory effects in vivo, either via intravenous or intranasal administration. For example, in vivo results presented herein using the disclosed anti-inflammatory Treg EV composition show anti-inflammatory effects in models of inflammation and motor neuron degenerative disease modeling ALS. For example, results presented herein show that EVs can suppress brain and peripheral inflammation in an in vivo model of neuroinflammation, and can suppress inflammation and prolong survival in an in vivo model of amyotrophic lateral sclerosis (ALS). Results presented herein also show that Treg EVs have a greater inhibitory effect on pro-inflammatory immune cells than EVs derived from mesenchymal stem cells (MSCs).

[0010] Moreover, the anti-inflammatory EVs presented herein exhibit remarkable batch-to-batch consistency in size, stability, and activity, and display unique structural features as characterized, for example, by Treg EV surface markers and RNA profiles. Moreover, as shown herein, the methods presented herein yield potent anti-inflammatory EVs that exhibit similar structural and suppressive activity properties whether the original Treg starting material is obtained from healthy subjects or ALS patients.

[0011] In one aspect, provided herein is an isolated cell-free population of anti-inflammatory extracellular vesicles (EVs), wherein the anti-inflammatory EVs are derived from ex vivo expanded human suppressive immune cells, wherein: i) the population exhibits a size diameter distribution of about 50 nm to about 150 nm; ii) the population comprises EV surface CD2, CD25, and HLA-DRDPDQ; iii) the population comprises hsa-miR-1290, hsa-miR-146a-5p, and hsa-miR-155-5p micro-RNAs (miRNAs); and iv) the population exhibits the ability to suppress myeloid cells, e.g., macrophages, as measured by the ability to reduce pro-inflammatory cytokine production by myeloid cells (e.g., exhibiting the ability to reduce the expression of IL-6, IL-8, IL1β, or interferon-γ in myeloid cells) and the ability to increase the expression of one or more anti-inflammatory markers in myeloid cells (e.g., the ability to increase the expression of IL-10, Arg1, and / or CD206 in myeloid cells), or as measured by the ability to suppress the proliferation of responder T cells; and wherein the human suppressive immune cells are regulatory T cells (Tregs). In one embodiment, the human Tregs are derived from a healthy human subject. In one embodiment, the human Tregs are derived from a human subject diagnosed with or suspected of having amyotrophic lateral sclerosis (ALS).

[0012] In certain embodiments, the population of anti-inflammatory EVs further comprises EV surface CD44, CD29, CD4, and CD45. In certain embodiments, the population of anti-inflammatory EVs further comprises EV surface CD44, CD29, CD4, and CD45. In certain embodiments, the population of anti-inflammatory EVs further comprises EV surface CD9, CD63, and CD81. In certain embodiments, the population of anti-inflammatory EVs substantially lack EV surface CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP, CD146, CD86, CD326, CD133, CD142, CD31, and CD14. In certain embodiments, the population of anti-inflammatory EVs further comprises EV surface CD44, CD29, CD4, and CD45. In certain embodiments, the population of anti-inflammatory EVs further comprises EV surface CD44, CD29, CD4, and CD45, CD9, CD63, and CD81, and ii) substantially lacks EV surface CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP, CD146, CD86, CD326, CD133, CD142, CD31, and CD14.

[0013] In certain embodiments, the ratio of hsa-miR-146a-5p to hsa-miR-155-5p present in the population of anti-inflammatory EVs is about 2 to about 3. In certain embodiments, the abundance of hsa-miR-1290 in the population of anti-inflammatory EVs is at least twice the abundance of hsa-mir-155-5p. In specific embodiments, the ratio of hsa-miR-146a-5p to hsa-miR-155-5p present in the population of anti-inflammatory EVs is about 2 to about 3, and the abundance of hsa-miR-1290 in the population of anti-inflammatory EVs is at least twice the abundance of hsa-mir-155-5p.

[0014] In certain embodiments, at least about 90% of the EVs in the anti-inflammatory population exhibit a size diameter of about 50 nm to about 150 nm. In certain embodiments, the population of anti-inflammatory EVs exhibits a mean size diameter of about 80 nm to about 110 nm. In certain embodiments, the population of anti-inflammatory EVs exhibits a median size diameter of about 70 nm to about 110 nm. In certain embodiments, the population of anti-inflammatory EVs exhibits a mode size diameter of about 65 nm to about 95 nm. In specific embodiments, at least about 90% of the EVs in the population of anti-inflammatory EVs exhibit a size diameter of about 50 to about 150 nm, the population exhibiting a mean size diameter of about 80 nm to about 110 nm, a median size diameter of about 70 nm to about 110 nm, and a mode size diameter of about 65 nm to about 95 nm.

[0015] Provided herein are isolated cell-free populations of anti-inflammatory EVs, where the anti-inflammatory EVs are derived from ex vivo expanded human suppressive immune cells, e.g., regulatory T cells (Tregs). Also provided herein are pharmaceutical and cryopreserved compositions comprising the isolated cell-free populations of anti-inflammatory EVs described herein, methods of producing the EV populations, and methods of using EVs to treat diseases, such as neurodegenerative diseases, e.g., ALS.

[0016] In one aspect, provided herein is an isolated cell-free population of anti-inflammatory extracellular vesicles (EVs), wherein the anti-inflammatory EVs are derived from human suppressive immune cells that are expanded ex vivo.In some embodiments, the human suppressive immune cells are regulatory T cells (Tregs).In some embodiments, the Tregs are derived from healthy human subjects.

[0017] In some embodiments, the Tregs are from a human subject diagnosed with or suspected of having a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is Alzheimer's disease. In some embodiments, the neurodegenerative disorder is amyotrophic lateral sclerosis (ALS). In some embodiments, the neurodegenerative disorder is multiple sclerosis (MS). In some embodiments, the neurodegenerative disorder is Parkinson's disease.

[0018] In some embodiments, the Tregs are from a human subject who has been diagnosed with or is suspected of having had a stroke.

[0019] In some embodiments, the Tregs are from an elderly human subject.

[0020] In some embodiments, the Tregs are from multiple human subjects. In some embodiments, the Tregs are from multiple unrelated human subjects.

[0021] In some embodiments, anti-inflammatory EVs exhibit the ability to increase the expression of one or more anti-inflammatory markers in inflammatory cells.In some embodiments, inflammatory cells are myeloid cells.In some embodiments, anti-inflammatory EVs exhibit the ability to increase the expression of IL-10, Arg1, and / or CD206 in inflammatory cells.

[0022] In some embodiments, the anti-inflammatory EVs exhibit the ability to suppress inflammatory cells as measured by pro-inflammatory cytokine production by inflammatory cells. In some embodiments, the inflammatory cells are myeloid cells. In some embodiments, the myeloid cells are monocytes, macrophages, or microglia. In some embodiments, the macrophages are M1 macrophages. In some embodiments, the M1 macrophages are induced pluripotent stem cell (iPSC)-derived M1 macrophages.

[0023] In some embodiments, the ability to suppress inflammatory cells is measured by IL-6, IL-8, TNFα, IL1β, and / or interferon-γ production by inflammatory cells.

[0024] In some embodiments, anti-inflammatory EVs exhibit the ability to increase IL-1-, Arg1, and / or CD206 expression in inflammatory cells, e.g., myeloid cells, e.g., macrophages, as well as the ability to suppress IL-6, IL-8, TNFα, IL1β, and / or interferon-γ production.

[0025] In some embodiments, the anti-inflammatory EVs exhibit suppressive function as determined by the inhibition of proliferation of responder T cells. In some embodiments, proliferation of responder T cells is determined by flow cytometry or thymidine incorporation.

[0026] In some embodiments, the population is a population of anti-inflammatory EVs containing saline. In some embodiments, the population is a population of anti-inflammatory EVs containing saline. In some embodiments, the population is a population of anti-inflammatory EVs containing phosphate buffered saline.

[0027] In some embodiments, the population of anti-inflammatory EVs comprises exosomes and microvesicles. In some embodiments, the majority of the EVs are exosomes. In some embodiments, at least about 80%, about 90%, or about 95% of the EVs are exosomes. In some embodiments, the majority of the EVs are microvesicles. In some embodiments, at least about 80%, about 90%, or about 95% of the EVs are microvesicles.

[0028] In some embodiments, the population of anti-inflammatory EVs comprises at least about 50% exosomes. In some embodiments, at least about 60% of the EVs are exosomes. In some embodiments, at least about 70% of the EVs are exosomes.

[0029] In some embodiments, the population of anti-inflammatory EVs comprises at least about 50% microvesicles. In some embodiments, at least about 60% of the EVs are microvesicles. In some embodiments, at least about 70% of the EVs are microvesicles.

[0030] In some embodiments, the majority of EVs in the population of anti-inflammatory EVs provided herein have a diameter of about 30 nm to about 1000 nm. In some embodiments, the majority of EVs have a diameter of about 30 nm to about 100 nm, about 30 nm to about 150 nm, about 30 to about 200 nm, about 40 to about 100 nm, about 80 to about 100 nm, about 80 to about 110 nm, about 80 to about 125 nm, or about 100 to about 120 nm. In some embodiments, the majority of EVs have a diameter of about 60 nm to about 1000 nm, about 70 nm to about 1000 nm, about 80 nm to about 1000 nm, 100 to about 1000 nm, about 200 to about 1000 nm, or about 300 to about 1000 nm. In some embodiments, the majority of EVs have a diameter of about 20 nm to about 300 nm, about 20 nm to about 275 nm, about 20 to about 250 nm, about 20 to about 200 nm, or about 20 nm to about 175 nm.

[0031] In another aspect, provided herein is a pharmaceutical composition comprising an isolated cell-free population of anti-inflammatory EVs provided herein. In certain embodiments, a pharmaceutical composition comprising an isolated cell-free population of anti-inflammatory EVs provided herein in saline. In some embodiments, the population of anti-inflammatory EVs is about 1×10 6 ~Approx. 1×10 14 EV, approx. 1×10 8 ~Approx. 1×10 14 EV, approx. 1×10 8 ~Approx. 1×10 12 EV, approx. 1×10 8 ~Approx. 1×10 10 EV, approx. 1×10 10 ~Approx. 1×10 14 EV, or approximately 1 x 10 10 ~Approx. 1×10 12In some embodiments, the population of anti-inflammatory EVs is about 1×10 9 EV, about 5×10 9 EV, approx. 1×10 10 EV, about 5×10 10 EV, approx. 1×10 11 EV, about 5×10 11 EV, or approximately 1 x 10 12 In some embodiments, the population of anti-inflammatory EVs is about 1×10 6 ~Approx. 1×10 14 EV / ml, approximately 1 × 10 8 ~Approx. 1×10 14 EV / ml, approximately 1 × 10 8 ~Approx. 1×10 12 EV / ml, approximately 1 × 10 8 ~Approx. 1×10 10 EV / ml, approximately 1 × 10 10 ~Approx. 1×10 14 EV / ml, or approximately 1 x 10 10 ~Approx. 1×10 12 In some embodiments, the population of anti-inflammatory EVs is about 5×10 8 EV / ml, approximately 1 × 10 9 EV / ml, approximately 2.5 × 10 9 EV / ml, approximately 5 × 10 9 EV / ml, approximately 1 × 10 10 EV / ml, approximately 2.5 × 10 10 EV / ml, approximately 5 × 10 10 EV / ml, approximately 1 × 10 11 EV / ml, approximately 2.5 × 10 11 EV / ml, approximately 5 × 10 11 EV / ml, or approximately 1 x 10 12 Contains EVs / ml.

[0032] In some embodiments, the population of anti-inflammatory EVs contained in the pharmaceutical compositions provided herein comprises about 1 μg to about 200 mg EVs. In some embodiments, the population of anti-inflammatory EVs comprises about 1 μg to about 15 mg EVs. In some embodiments, the population of anti-inflammatory EVs comprises about 1 μg to about 15 mg EVs / ml.

[0033] In some embodiments, the pharmaceutical composition is a cryopreserved pharmaceutical composition. In some embodiments, the pharmaceutical composition has been previously cryopreserved.

[0034] In another aspect, provided herein is a cryopreserved composition comprising an isolated cell-free population of anti-inflammatory EVs provided herein.

[0035] In another aspect, provided herein is a method for producing an isolated cell-free population of anti-inflammatory extracellular vesicles (EVs), comprising: (a) ex vivo expanding a population of human suppressive immune cells in a culture medium to produce a culture comprising the cells, the culture medium, and anti-inflammatory EVs; and (b) isolating the anti-inflammatory EVs from the culture. In some embodiments, the human suppressive immune cell population is a population of regulatory T cells (Tregs).

[0036] In some embodiments, step b) comprises removing cells from the culture followed by polyethylene glycol precipitation of the culture. In some embodiments, step (b) comprises: (i) removing cells from the culture to produce a cell-free anti-inflammatory EV-containing solution; and (ii) isolating anti-inflammatory EVs from the cell-free anti-inflammatory EV-containing solution of (i).

[0037] In some embodiments, step (i) comprises passing the culture through a filter such that the cells are retained by the filter and thereby removed from the culture. In some embodiments, step (i) comprises microfiltration.

[0038] In some embodiments, step (ii) comprises step (ii-a): passing the cell-free anti-inflammatory EV-containing solution through the filter such that the anti-inflammatory EVs are retained by the filter. In some embodiments, the filter has a molecular weight cut-off (MWCO) of about 200 kilodaltons (kDa) to about 600 kDa. In some embodiments, the filter has a MWCO of about 500 kDa.

[0039] In some embodiments, step (ii) comprises ultrafiltration. In some embodiments, step (ii) further comprises step (ii-b): performing buffer exchange, such that the isolated cell-free population of anti-inflammatory EVs produced is an isolated cell-free population of anti-inflammatory EVs containing a buffer. In some embodiments, the buffer is a saline-containing buffer. In some embodiments, the saline-containing buffer is physiological saline. In some embodiments, the saline-containing buffer is PBS.

[0040] In some embodiments, step (ii-b) comprises diafiltration.

[0041] In some embodiments, steps (ii-a) and (ii-b) are performed simultaneously.

[0042] In some embodiments, step (b) comprises tangential flow filtration.

[0043] In some embodiments, the culture medium in step (a) is serum-free. In some embodiments, the culture medium in step (a) contains serum. In some embodiments, the serum is human AB serum. In some embodiments, the serum is depleted of serum-derived EVs.

[0044] In some embodiments, the method for producing an isolated cell-free population of anti-inflammatory EVs further comprises, prior to step (a), enriching Tregs from a cell sample suspected of containing Tregs to produce a baseline Treg cell population, which is a population of Tregs that are subsequently expanded in step (a). In some embodiments, the cell sample is a leukapheresis cell sample. In some embodiments, the method further comprises obtaining a cell sample from a donor by leukapheresis. In some embodiments, the cell sample is not stored overnight or frozen before performing the enrichment step. In some embodiments, the cell sample is obtained within 30 minutes before the start of the enrichment step. In some embodiments, the enrichment step comprises removing CD8+ / CD19+ cells and then enriching for CD25+ cells. In some embodiments, step (a) is performed within 30 minutes of the enrichment step.

[0045] In some embodiments, step (a) of the method for producing an isolated cell-free population of anti-inflammatory EVs comprises culturing Tregs in a culture medium containing beads coated with anti-CD3 and anti-CD28 antibodies. In some embodiments, the beads are first added to the culture medium within about 24 hours from the start of the culture. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are added to the culture medium about 14 days after the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture medium.

[0046] In some embodiments, step (a) further comprises adding IL-2 to the culture medium within about 6 days of initiation of the culture. In some embodiments, step (a) further comprises supplementing the culture medium with IL-2 about every 2-3 days after IL-2 is initially added to the culture medium.

[0047] In some embodiments, step (a) further comprises adding rapamycin to the culture medium within about 24 hours of initiation of culturing. In some embodiments, step a) further comprises supplementing the culture medium with rapamycin about every 2-3 days after rapamycin is initially added to the culture medium.

[0048] In some embodiments, step (a) is automated. In some embodiments, step (a) is performed in a bioreactor.

[0049] In some embodiments, step (b) of the method of producing an isolated cell-free population of anti-inflammatory EVs may begin at any time during step a).

[0050] In some embodiments, the Tregs enriched in step (a) are from a healthy human subject. In some embodiments, the Tregs are from a human subject diagnosed with or suspected of having a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), or Parkinson's disease. In some embodiments, the Tregs are from a human subject diagnosed with or suspected of having a stroke. In some embodiments, the Tregs are from an elderly human subject. In some embodiments, the Tregs are from multiple human subjects.

[0051] In some embodiments, the human suppressive immune cell population expanded in step (a) is a genetically engineered human suppressive immune cell population.

[0052] In some embodiments, the population of Tregs expanded in step (a) is a population of genetically engineered Tregs.

[0053] In another aspect, provided herein is a pharmaceutical composition comprising an isolated cell-free population of anti-inflammatory EVs, wherein the population is produced by any one of the methods described herein.

[0054] In some embodiments, the method of producing an isolated cell-free population of anti-inflammatory EVs further comprises (c) cryopreserving the isolated cell-free population of anti-inflammatory EVs, thereby producing a cryopreserved isolated cell-free population of anti-inflammatory EVs. Also provided herein is a cryopreserved composition comprising an isolated cell-free population of anti-inflammatory EVs, wherein the cryopreserved composition is produced using such a method.

[0055] In some embodiments, the method further comprises thawing the cryopreserved isolated cell-free population of anti-inflammatory EVs after cryopreservation for about 1 week, 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, or about 24 months.Also provided herein are compositions, e.g., pharmaceutical compositions, comprising the isolated cell-free population of anti-inflammatory EVs, wherein the compositions, e.g., pharmaceutical compositions, are made using such methods.

[0056] In another aspect, provided herein is an isolated cell-free population of anti-inflammatory EVs, wherein the anti-inflammatory EVs are derived from an ex vivo expanded Treg cell population that exhibits the ability to suppress inflammatory cells as measured by pro-inflammatory cytokine production by the inflammatory cells, wherein the inflammatory cells are macrophages or monocytes from a human donor or generated from induced pluripotent stem cells, wherein the ex vivo expanded Treg cell population has been expanded from baseline Tregs, and wherein within the ex vivo expanded Treg cell population: (a) expression of one or more dysfunctional baseline signature gene products listed in Table 3 and / or Table 4 is decreased relative to expression of one or more gene products in baseline Tregs; (b) expression of one or more dysfunctional baseline signature gene products listed in Table 5 is decreased relative to expression of one or more gene products in baseline Tregs; (c) expression of one or more Treg-associated signature gene products listed in Table 6 is decreased compared to expression of said one or more gene products in baseline Tregs; (d) expression of one or more mitochondrial signature gene products listed in Table 7 is increased compared to expression of said one or more gene products in baseline Tregs; (e) expression of one or more cell proliferation signature gene products listed in Table 8 is increased compared to expression of said one or more gene products in baseline Tregs; or (f) expression of one or more maximum protein expression signature gene products listed in Table 9 is increased compared to expression of said one or more gene products in baseline Tregs. In some embodiments, provided herein is a pharmaceutical composition comprising an isolated cell-free population of anti-inflammatory EVs.

[0057] In another aspect, provided herein is a method of treating a disorder associated with impaired Treg function, comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein.

[0058] In another aspect, provided herein is a method of treating a disorder associated with a Treg deficiency, comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein.

[0059] In another aspect, provided herein is a method of treating a disorder associated with overactivation of the immune system, comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein.

[0060] In another aspect, provided herein is a method of treating an inflammatory condition driven by a T cell response, comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein.

[0061] In another aspect, provided herein is a method of treating an inflammatory condition driven by a myeloid cell response, comprising administering to a subject in need of such treatment a pharmaceutical composition provided herein. In some embodiments, the myeloid cell is a monocyte, macrophage, or microglia.

[0062] In another aspect, provided herein is a method for treating a neurodegenerative disorder in a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition provided herein. In some embodiments, the neurodegenerative disease is ALS, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, or Huntington's disease.

[0063] In some embodiments, the neurodegenerative disease is ALS, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, multiple sclerosis, or Huntington's disease.

[0064] In another aspect, provided herein is a method for treating an autoimmune disorder in a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition provided herein.In some embodiments, the autoimmune disorder is polymyositis, ulcerative colitis, inflammatory bowel disease, Crohn's disease, celiac disease, systemic sclerosis (scleroderma), multiple sclerosis (MS), rheumatoid arthritis (RA), type I diabetes, psoriasis, dermatomyositis, lupus, such as systemic lupus erythematosus or cutaneous lupus, myasthenia gravis, autoimmune nephropathy, autoimmune hemolytic anemia, autoimmune cytopenia, autoimmune encephalitis, autoimmune hepatitis, autoimmune uveitis, alopecia, thyroiditis, or pemphigus.

[0065] In another aspect, provided herein is a method of treating graft-versus-host disease in a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition provided herein. In some embodiments, the subject has undergone a bone marrow transplant, a kidney transplant, or a liver transplant.

[0066] In another aspect, provided herein is a method of improving pancreatic islet graft survival in a subject in need thereof, comprising administering to a subject in need of said treatment a pharmaceutical composition provided herein.

[0067] In another aspect, provided herein is a method of treating cardiac inflammation in a subject in need thereof, comprising administering to a subject in need thereof a pharmaceutical composition provided herein. In some embodiments, the cardiac inflammation is associated with atherosclerosis, myocardial infarction, ischemic cardiomyopathy, or heart failure.

[0068] In another aspect, provided herein is a method of treating neuroinflammation in a subject in need thereof, comprising administering to a subject in need thereof a pharmaceutical composition provided herein. In some embodiments, the neuroinflammation is associated with stroke, acute disseminated encephalomyelitis, acute optic neuritis, acute inflammatory demyelinating polyradiculoneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, Guillain-Barré syndrome, transverse myelitis, neuromyelitis optica, epilepsy, traumatic brain injury, spinal cord injury, encephalitis, central nervous system vasculitis, neurosarcoidosis, autoimmune or post-infectious encephalitis, or chronic meningitis.

[0069] In another aspect, provided herein is a method for treating a regulatory T cell disorder (Tregopathy) in a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition provided herein. In some embodiments, the regulatory T cell disorder is caused by a loss-of-function mutation in FOXP3, CD25, cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), LPS-responsive beige-like anchor protein (LRBA), or BTB domain and CNC homolog 2 (BACH2) genes, or a gain-of-function mutation in signal transducer and activator of transcription 3 (STAT3).

[0070] In some embodiments, the anti-inflammatory EVs administered to the subject are derived from Tregs that are autologous to the subject. In some embodiments, the anti-inflammatory EVs are derived from Tregs that are allogeneic to the subject.

[0071] In some embodiments, the pharmaceutical composition is administered via intranasal administration. In some embodiments, the intranasal administration is by aerosol inhalation or nasal drops. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered by local injection.

[0072] In some embodiments, the therapeutic method provided herein further comprises administering to the subject a pharmaceutical composition comprising a therapeutic population of Tregs, wherein the Tregs have been expanded ex vivo and cryopreserved, and wherein the Tregs are not further expanded prior to administration. In some embodiments, the therapeutic population of Tregs is autologous to the subject. In some embodiments, the therapeutic population of Tregs is allogeneic to the subject. In some embodiments, the pharmaceutical composition comprising a therapeutic population of Tregs is administered intravenously. In some embodiments, the pharmaceutical composition comprising anti-inflammatory EVs and the pharmaceutical composition comprising a therapeutic population of Tregs are administered to the patient on the same day.

[0073] In some embodiments, the therapeutic method provided herein comprises administering to a subject in need of treatment a pharmaceutical composition comprising an isolated cell-free population of anti-inflammatory EVs, wherein the EVs have been cryopreserved and thawed before being administered to the subject.In some embodiments, the therapeutic method provided herein comprises administering to a subject in need of treatment a pharmaceutical composition comprising an isolated cell-free population of anti-inflammatory EVs, wherein the EVs have been stored at 4°C before being administered to the subject, for example, stored overnight at 4°C.In certain embodiments, the therapeutic method provided herein comprises administering to a subject in need of treatment a pharmaceutical composition comprising an isolated cell-free population of anti-inflammatory EVs, wherein the EVs have been cryopreserved, thawed and stored at 4°C before being administered to the subject, for example, stored overnight at 4°C.

[0074] In certain embodiments, the methods of treatment provided herein comprise administering to a subject in need of treatment a pharmaceutical composition comprising an isolated cell-free population of anti-inflammatory EVs, wherein the EVs have undergone at least two freeze / thaw cycles prior to administration to the subject, e.g., about 2 to about 20 freeze / thaw cycles prior to administration to the subject.

[0075] Further exemplary embodiments are as follows: 1. An isolated cell-free population of anti-inflammatory extracellular vesicles (EVs), the anti-inflammatory EVs being derived from ex vivo expanded human suppressive immune cells; where: i) the population exhibits a size diameter distribution of about 50 nm to about 150 nm; ii) the population comprises EV surface CD2, CD25, and HLA-DRDPDQ; iii) the population comprises hsa-miR-1290, hsa-miR-146a-5p, and hsa-miR-155-5p micro-RNAs (miRNAs); iv) the population exhibits the ability to suppress myeloid cells, as measured by the ability to reduce pro-inflammatory cytokine production by myeloid cells and increase expression of one or more anti-inflammatory markers in myeloid cells, or by the ability to suppress proliferation of responder T cells; and wherein the human suppressive immune cells are regulatory T cells (Tregs); The isolated cell-free population. 2. The population of anti-inflammatory EVs of embodiment 1, wherein at least about 90% of the EVs in said population exhibit a size diameter of about 50 nm to about 150 nm. 3. A population of anti-inflammatory EVs according to embodiment 1 or 2, wherein said population exhibits an average size diameter of between about 80 nm and about 110 nm. 4. A population of anti-inflammatory EVs according to any one of embodiments 1 to 3, wherein said population exhibits a median size diameter of about 70 nm to about 110 nm. 5. A population of anti-inflammatory EVs according to any one of embodiments 1 to 4, wherein said population exhibits a modal size diameter of between about 65 nm and about 95 nm. 6. A population of anti-inflammatory EVs according to embodiment 1, wherein at least about 90% of the EVs in said population exhibit a size diameter of about 50 to about 150 nm, and wherein said population exhibits a mean size diameter of about 80 nm to about 110 nm, a median size diameter of about 70 nm to about 110 nm, and a mode size diameter of about 65 nm to about 95 nm. 7. The population of anti-inflammatory EVs of any one of embodiments 1 to 6, wherein said population further comprises EV surface CD44, CD29, CD4, and CD45. 8. The population of anti-inflammatory EVs of any one of embodiments 1 to 7, wherein said population further comprises EV surface CD9, CD63, and CD81. 9. The population comprises EV surface CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP, CD146, CD86, CD326, CD133, CD142, CD31 9. The population of anti-inflammatory EVs according to any one of embodiments 1 to 8, wherein the population of anti-inflammatory EVs is substantially devoid of CD14. 10. The population of anti-inflammatory EVs of embodiment 1 or 6, wherein the population further comprises EV surface CD44, CD29, CD4, CD45, CD9, CD63, and CD81, and wherein the population is substantially devoid of EV surface CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP, CD146, CD86, CD326, CD133, CD142, CD31, and CD14. 11. A population of anti-inflammatory EVs according to any one of embodiments 1 to 10, wherein the ratio of hsa-miR-146a-5p to hsa-miR-155-5p in said population is from about 2 to about 3. 12. A population of anti-inflammatory EVs according to any one of embodiments 1 to 11, wherein the abundance of hsa-miR-1290 is at least twice the abundance of hsa-mir-155-5p. 13. The population of anti-inflammatory EVs according to any one of embodiments 1 to 12, wherein said Tregs are derived from a healthy human subject. 14. The population of anti-inflammatory EVs according to any one of embodiments 1 to 12, wherein said Tregs are derived from a human subject diagnosed with or suspected of having a neurodegenerative disorder. 15. The population of anti-inflammatory EVs according to any one of embodiments 1 to 14, wherein said anti-inflammatory EVs exhibit the ability to increase expression of IL-10, Arg1, and / or CD206 in myeloid cells. 16. The population of anti-inflammatory EVs according to any one of embodiments 1 to 15, wherein said anti-inflammatory EVs exhibit an ability to reduce expression of IL-6, IL-8, IL1β, or interferon-γ in myeloid cells. 17. The population of anti-inflammatory EVs according to embodiment 1, wherein proliferation of responder T cells is determined by flow cytometry or thymidine incorporation. 18. The population of anti-inflammatory EVs according to any one of embodiments 1 to 17, wherein said population is a population of anti-inflammatory EVs containing saline. 19. An isolated cell-free population of anti-inflammatory extracellular vesicles (EVs), wherein said anti-inflammatory EVs are derived from ex vivo expanded human suppressive immune cells. 20. The population of anti-inflammatory EVs of embodiment 19, wherein the human inhibitory immune cells are regulatory T cells (Tregs). 21. The population of anti-inflammatory EVs according to embodiment 20, wherein said Tregs are derived from a healthy human subject. 22. The population of anti-inflammatory EVs of embodiment 21, wherein the Tregs are derived from a human subject diagnosed with or suspected of having a neurodegenerative disorder. 23. The population of anti-inflammatory EVs of embodiment 22, wherein the neurodegenerative disorder is Alzheimer's disease. 24. The population of anti-inflammatory EVs of embodiment 22, wherein the neurodegenerative disorder is amyotrophic lateral sclerosis (ALS). 25. The population of anti-inflammatory EVs of embodiment 22, wherein the neurodegenerative disease is multiple sclerosis (MS). 26. The population of anti-inflammatory EVs of embodiment 22, wherein the neurodegenerative disease is Parkinson's disease. 27. The population of anti-inflammatory EVs according to embodiment 20, wherein said Tregs are derived from a human subject diagnosed with or suspected of having had a stroke. 28. The population of anti-inflammatory EVs of embodiment 20, wherein the Tregs are derived from an elderly human subject. 29. The population of anti-inflammatory EVs according to any one of embodiments 20 to 28, wherein the Tregs are from a plurality of human subjects. 30. The population of anti-inflammatory EVs of embodiment 29, wherein the Tregs are from a plurality of unrelated human subjects. 31. The population of anti-inflammatory EVs according to any one of embodiments 19 to 30, wherein said anti-inflammatory EVs exhibit the ability to increase the expression of one or more anti-inflammatory markers in inflammatory cells. 32. The population of anti-inflammatory EVs of embodiment 31, wherein the inflammatory cells are myeloid cells. 33. The population of anti-inflammatory EVs of embodiment 31 or 32, wherein the anti-inflammatory EVs exhibit the ability to increase expression of IL-10, Arg1, and / or CD206 in inflammatory cells. 34. The population of anti-inflammatory EVs according to any one of embodiments 19 to 33, wherein said anti-inflammatory EVs exhibit the ability to suppress inflammatory cells as measured by pro-inflammatory cytokine production by the inflammatory cells. 35. The method of embodiment 34, wherein the inflammatory cells are myeloid cells. 36. The population of anti-inflammatory EVs of embodiment 35, wherein the myeloid cells are monocytes, macrophages, or microglia. 37. The population of anti-inflammatory EVs of embodiment 36, wherein the macrophages are M1 macrophages. 38. The population of anti-inflammatory EVs of embodiment 37, wherein the M1 macrophages are induced pluripotent stem cell (iPSC)-derived M1 macrophages. 39. The population of anti-inflammatory EVs according to any one of embodiments 31 to 38, wherein the ability to suppress inflammatory cells is measured by IL-6, IL-8, TNFα, IL1β, and / or interferon-γ production by the inflammatory cells. 40. The population of anti-inflammatory EVs according to any one of embodiments 19 to 39, wherein said anti-inflammatory EVs exhibit suppressive function as measured by suppression of proliferation of responder T cells. 41. The population of anti-inflammatory EVs according to embodiment 40, wherein the proliferation of responder T cells is determined by flow cytometry or thymidine incorporation. 42. The population of anti-inflammatory EVs of any one of embodiments 19-41, wherein said population is a population of anti-inflammatory EVs containing saline. 43. The population of anti-inflammatory EVs according to any one of embodiments 19 to 41, wherein said population is a population of anti-inflammatory EVs containing saline. 44. The population of anti-inflammatory EVs of any one of embodiments 19-41, wherein said population is a population of anti-inflammatory EVs containing phosphate buffered saline. 45. The population of anti-inflammatory EVs according to any one of embodiments 19 to 44, wherein the population of anti-inflammatory EVs comprises exosomes and microvesicles. 46. ​​The population of anti-inflammatory EVs described in embodiment 45, wherein the majority of the EVs are exosomes. 47. The population of anti-inflammatory EVs of embodiment 46, wherein at least about 80%, about 90%, or about 95% of the EVs are exosomes. 48. The population of anti-inflammatory EVs according to embodiment 47, wherein the majority of the EVs are microvesicles. 49. The population of anti-inflammatory EVs of embodiment 48, wherein at least about 80%, about 90%, or about 95% of the EVs are microvesicles. 50. The population of anti-inflammatory EVs described in embodiment 45, wherein the majority of the EVs have a diameter of about 30 nm to about 1000 nm. 51. A population of anti-inflammatory EVs according to embodiment 45, wherein the majority of the EVs have a diameter of about 30 nm to about 100 nm, about 30 nm to about 150 nm, about 30 to about 200 nm, about 40 to about 100 nm, about 80 to about 100 nm, about 80 to about 110 nm, about 80 to about 125 nm, or about 100 to about 120 nm. 52. The population of anti-inflammatory EVs according to embodiment 25, wherein the majority of the EVs have a diameter of about 60 nm to about 1000 nm, about 70 nm to about 1000 nm, about 80 nm to about 1000 nm, 100 to about 1000 nm, about 200 to about 1000 nm, or about 300 to about 1000 nm. 53. A pharmaceutical composition comprising an isolated cell-free population of anti-inflammatory EVs according to any one of embodiments 1 to 52. 54. The population of anti-inflammatory EVs is about 1 × 10 6 ~Approx. 1×10 14 EV, approx. 1×10 8 ~Approx. 1×10 14 EV, approx. 1×10 8~Approx. 1×10 12 EV, approx. 1×10 8 ~Approx. 1×10 10 EV, approx. 1×10 10 ~Approx. 1×10 14 EV, or approximately 1 x 10 10 ~Approx. 1×10 12 54. The pharmaceutical composition of embodiment 53, comprising an EV. 55. The population of anti-inflammatory EVs is about 1 × 10 6 ~Approx. 1×10 14 EV / ml, approximately 1 × 10 8 ~Approx. 1×10 14 EV / ml, approximately 1 × 10 8 ~Approx. 1×10 12 EV / ml, approximately 1 × 10 8 ~Approx. 1×10 10 EV / ml, approximately 1 × 10 10 ~Approx. 1×10 14 EV / ml, or approximately 1 x 10 10 ~Approx. 1×10 12 54. The pharmaceutical composition of embodiment 53, comprising EV / ml. 56. The pharmaceutical composition of embodiment 53, wherein the population of anti-inflammatory EVs comprises from about 1 μg to about 200 mg EVs. 57. The pharmaceutical composition of embodiment 53, wherein the population of anti-inflammatory EVs comprises from about 1 μg to about 15 mg EVs. 58. The pharmaceutical composition of embodiment 53, wherein the population of anti-inflammatory EVs comprises from about 1 μg to about 15 mg EV / ml. 59. The pharmaceutical composition according to any one of embodiments 53 to 58, wherein said pharmaceutical composition is a cryopreserved pharmaceutical composition. 60. The pharmaceutical composition according to any one of embodiments 53 to 58, wherein the pharmaceutical composition has been previously cryopreserved. 61. A cryopreserved composition comprising an isolated cell-free population of anti-inflammatory EVs according to any one of embodiments 1 to 53. 62. A method for producing an isolated cell-free population of anti-inflammatory extracellular vesicles (EVs), comprising: a. expanding a population of human suppressive immune cells ex vivo in culture medium to produce a culture comprising the cells, culture medium, and anti-inflammatory EVs; and b. Isolating the anti-inflammatory EVs from the culture. The method comprising: 63. The method of embodiment 62, wherein the human suppressive immune cell population is a population of regulatory T cells (Tregs). 64. The method of embodiment 62 or 63, wherein step b) comprises removing cells from the culture and then polyethylene glycol precipitation of the culture. 65. Step b): i) removing the cells from the culture to produce a cell-free, anti-inflammatory EV-containing solution; and ii) isolating anti-inflammatory EVs from the cell-free anti-inflammatory EV-containing solution of i). 64. The method of embodiment 62 or 63, comprising: 66. The method of embodiment 65, wherein step i) comprises passing the culture through a filter such that the cells are retained by the filter and thereby removed from the culture. 67. The method of embodiment 65 or 66, wherein step i) comprises microfiltration. 68. The method of any one of embodiments 65 to 67, wherein step ii) comprises step ii-a): passing the acellular anti-inflammatory EV-containing solution through a filter such that the anti-inflammatory EVs are retained by the filter. 69. The method of embodiment 68, wherein the filter has a molecular weight cut-off (MWCO) of about 200 kilodaltons (kDa) to about 600 kDa. 70. The method of embodiment 69, wherein the filter has a MWCO of about 500 kDa. 71. The method of any one of embodiments 65 to 70, wherein step ii) comprises ultrafiltration. 72. The method according to any one of embodiments 68 to 71, wherein step ii) further comprises: step ii-b): performing a buffer exchange such that the produced isolated cell-free population of anti-inflammatory EVs is an isolated cell-free population of anti-inflammatory EVs containing a buffer. 73. The method of embodiment 72, wherein the buffer is a saline-containing buffer. 74. The method of embodiment 73, wherein the saline-containing buffer is physiological saline. 75. The method of embodiment 74, wherein the saline-containing buffer is PBS. 76. The method of any one of embodiments 73 to 75, wherein step ii-b) comprises diafiltration. 77. The method according to any one of embodiments 73 to 76, wherein steps ii-a) and ii-b) are carried out simultaneously. 78. The method of any one of embodiments 62-77, wherein step b) comprises tangential flow filtration. 79. The method according to any one of embodiments 62 to 78, wherein the culture medium in step a) is serum-free. 80. The method according to any one of embodiments 62 to 79, wherein the culture medium of step a) contains serum. 81. The method of embodiment 80, wherein the serum is human AB serum. 82. The method of embodiment 80 or 81, wherein the serum is depleted of serum-derived EVs. 83. The method of any one of embodiments 62 to 82, further comprising, prior to step a), enriching Tregs from a cell sample suspected of containing Tregs to produce a baseline Treg cell population, which is a population of Tregs that are subsequently expanded in a). 84. The method of embodiment 83, wherein the cell sample is a leukapheresis cell sample. 85. The method of embodiment 83 or 84, further comprising obtaining the cell sample from a donor by leukapheresis. 86. The method of any one of embodiments 83 to 85, wherein the cell sample is not stored overnight or frozen before performing the concentration step. 87. The method of any one of embodiments 83 to 86, wherein the cell sample is obtained no more than 30 minutes prior to the start of the enrichment step. 88. The method of any one of embodiments 82 to 87, wherein the enrichment step comprises removing CD8+ / CD19+ cells followed by enrichment for CD25+ cells. 89. The method of any one of embodiments 62-88, wherein step a) is carried out within 30 minutes of the concentrating step. 90. The method according to any one of embodiments 62 to 89, wherein step a) comprises culturing Tregs in a culture medium comprising beads coated with anti-CD3 and anti-CD28 antibodies. 91. The method of embodiment 90, wherein the beads are first added to the culture medium within about 24 hours of initiation of the culture. 92. The method of embodiment 90 or 91, wherein beads coated with anti-CD3 and anti-CD28 antibodies are added to the culture medium about 14 days after beads coated with anti-CD3 and anti-CD28 antibodies are added to the culture medium for the first time. 93. The method according to any one of embodiments 90 to 92, wherein step a) further comprises adding IL-2 to the culture medium within about 6 days from the initiation of the culture. 94. The method of embodiment 93, wherein step a) further comprises supplementing the culture medium with IL-2 about every 2-3 days after IL-2 is initially added to the culture medium. 95. The method of any one of embodiments 90 to 94, wherein step a) further comprises adding rapamycin to the culture medium within about 24 hours from the initiation of the culture. 96. The method of embodiment 95, wherein step a) further comprises supplementing the culture medium with rapamycin about every 2-3 days after rapamycin is initially added to the culture medium. 97. The method of any one of embodiments 62 to 96, wherein step a) is automated. 98. The method of any one of embodiments 62 to 97, wherein step a) is carried out in a bioreactor. 99. The method of any one of embodiments 62-98, wherein step b) can begin at any time during step a). 100. The method of any one of embodiments 63 to 99, wherein the Tregs are from a healthy human subject. 101. The method of any one of embodiments 63-99, wherein the Tregs are from a human subject diagnosed with or suspected of having a neurodegenerative disorder. 102. The method of embodiment 101, wherein the neurodegenerative disorder is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), or Parkinson's disease. 103. The method of any one of embodiments 63-102, wherein said Tregs are from a human subject diagnosed with or suspected of having had a stroke. 104. The method of any one of embodiments 63 to 102, wherein said Tregs are from an elderly human subject. 105. The method of any one of embodiments 63 to 104, wherein the Tregs are from multiple human subjects. 106. The method of embodiment 62, wherein the human suppressive immune cell population is a genetically engineered human suppressive immune cell population. 107. The method of any one of embodiments 63 to 106, wherein the population of Tregs is a population of genetically engineered Tregs. 108. A pharmaceutical composition comprising an isolated cell-free population of anti-inflammatory EVs, wherein the population is produced by any one of the methods of embodiments 62 to 107. 109. The method of any one of embodiments 62 to 107, further comprising: c) cryopreserving the isolated cell-free population of anti-inflammatory EVs, thereby producing an isolated cell-free population of cryopreserved anti-inflammatory EVs. 110. The method of embodiment 109, further comprising thawing the cryopreserved, isolated cell-free population of anti-inflammatory EVs after cryopreservation for about 1 week, 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, or about 24 months. 111. A pharmaceutical composition comprising an isolated cell-free population of anti-inflammatory EVs according to embodiment 110. 112. An isolated cell-free population of anti-inflammatory EVs, the anti-inflammatory EVs derived from an ex vivo expanded Treg cell population that exhibits the ability to suppress inflammatory cells as measured by pro-inflammatory cytokine production by the inflammatory cells, wherein the inflammatory cells are macrophages or monocytes from a human donor or generated from induced pluripotent stem cells, wherein the ex vivo expanded Treg cell population has been expanded from baseline Tregs, and wherein within the ex vivo expanded Treg cell population: a) expression of one or more dysfunctional baseline signature gene products listed in Table 3 and / or Table 4 is decreased relative to expression of said one or more gene products in baseline Tregs; b) expression of one or more dysfunctional baseline signature gene products listed in Table 5 is decreased relative to expression of said one or more gene products in baseline Tregs; c) increased expression of one or more Treg-associated signature gene products listed in Table 6 compared to expression of said one or more gene products in baseline Tregs; d) expression of one or more mitochondrial signature gene products listed in Table 7 is increased relative to expression of said one or more gene products in baseline Tregs; e) increased expression of one or more cell proliferation signature gene products listed in Table 8 compared to expression of said one or more gene products in baseline Tregs; or f) expression of one or more of the highest protein expression signature gene products listed in Table 9 is increased compared to expression of said one or more gene products in baseline Tregs; 2. An isolated cell-free population of anti-inflammatory EVs. 113. A pharmaceutical composition comprising an isolated cell-free population of anti-inflammatory EVs according to embodiment 112. 114. A method for treating a disorder associated with impaired Treg function, comprising administering to a subject in need of said treatment a composition described in any one of embodiments 53-60, 108, 111, or 113. 115. A method for treating a disorder associated with a Treg deficiency, comprising administering to a subject in need of said treatment a pharmaceutical composition described in any one of embodiments 53-60, 108, 111, or 113. 116. A method for treating a disorder associated with overactivation of the immune system, comprising administering to a subject in need of such treatment a pharmaceutical composition according to any one of embodiments 53-60, 108, 111, or 113. 117. A method for treating an inflammatory condition driven by a T cell response, comprising administering to a subject in need of said treatment a pharmaceutical composition described in any one of embodiments 53 to 60, 108, 111, or 113. 118. A method for treating an inflammatory condition driven by a myeloid cell response, comprising administering to a subject in need of said treatment a pharmaceutical composition according to any one of embodiments 53-60, 108, 111, or 113. 119. The method of embodiment 118, wherein the myeloid cells are monocytes, macrophages, or microglia. 120. A method for treating a neurodegenerative disorder in a subject in need thereof, comprising administering to a subject in need thereof a pharmaceutical composition according to any one of embodiments 53-60, 108, 111, or 113. 121. The method of embodiment 120, wherein the neurodegenerative disease is ALS, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, or Huntington's disease. 122. A method for treating an autoimmune disorder in a subject in need thereof, comprising administering to a subject in need thereof a pharmaceutical composition according to any one of embodiments 53-60, 108, 111, or 113. 123. The method of embodiment 122, wherein the autoimmune disorder is polymyositis, ulcerative colitis, inflammatory bowel disease, Crohn's disease, celiac disease, systemic sclerosis (scleroderma), multiple sclerosis (MS), rheumatoid arthritis (RA), type I diabetes, psoriasis, dermatomyositis, systemic lupus erythematosus, cutaneous lupus, myasthenia gravis, autoimmune nephropathy, autoimmune hemolytic anemia, autoimmune cytopenia, autoimmune encephalitis, autoimmune hepatitis, autoimmune uveitis, alopecia, thyroiditis, or pemphigus. 124. A method for treating graft-versus-host disease in a subject in need thereof, comprising administering to a subject in need thereof a pharmaceutical composition according to any one of embodiments 53-60, 108, 111, or 113. The method according to embodiment 106, wherein the subject has undergone a bone marrow, kidney, or liver transplant. 125. A method for improving survival of a pancreatic islet graft in a subject in need thereof, comprising administering to a subject in need of said treatment a pharmaceutical composition according to any one of embodiments 53-60, 108, 111, or 113. 126. A method for treating cardiac inflammation in a subject in need thereof, comprising administering to a subject in need thereof a pharmaceutical composition according to any one of embodiments 53-60, 108, 111, or 113. 127. The method of embodiment 126, wherein the cardiac inflammation is associated with atherosclerosis, myocardial infarction, ischemic cardiomyopathy, or heart failure. 128. A method for treating neuroinflammation in a subject in need thereof, comprising administering to a subject in need thereof a pharmaceutical composition according to any one of embodiments 53-60, 108, 111, or 113. 129. The method of embodiment 128, wherein the neuroinflammation is associated with stroke, acute disseminated encephalomyelitis, acute optic neuritis, acute inflammatory demyelinating polyradiculoneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, Guillain-Barré syndrome, transverse myelitis, neuromyelitis optica, epilepsy, traumatic brain injury, spinal cord injury, encephalitis, central nervous system vasculitis, neurosarcoidosis, autoimmune or post-infectious encephalitis, or chronic meningitis. 130. A method for treating a regulatory T cell disorder in a subject in need thereof, comprising administering to a subject in need of said treatment a pharmaceutical composition according to any one of embodiments 53-60, 108, 111, or 113. 131. The method of embodiment 130, wherein the regulatory T cell disorder is caused by a loss-of-function mutation in the genes FOXP3, CD25, cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), LPS-responsive beige-like anchor protein (LRBA), or BTB domain, and CNC homolog 2 (BACH2), or a gain-of-function mutation in signal transducer and activator of transcription 3 (STAT3). 132. The method of any one of embodiments 114 to 131, wherein the anti-inflammatory EVs are derived from Tregs that are autologous to the subject. 133. The method of any one of embodiments 114-131, wherein the anti-inflammatory EVs are derived from Tregs that are allogeneic to the subject. 134. The method of any one of embodiments 114-133, wherein the pharmaceutical composition is administered via intranasal administration. 135. The method of embodiment 134, wherein said intranasal administration is by aerosol inhalation or nasal drops. 136. The method of any one of embodiments 114-135, wherein the pharmaceutical composition is administered intravenously. 137. The method of any one of embodiments 114 to 135, wherein the pharmaceutical composition is administered by local injection. 138. The method of any one of embodiments 114-137, further comprising administering to the subject a pharmaceutical composition comprising a therapeutic population of Tregs, wherein the Tregs have been expanded ex vivo and cryopreserved, and wherein the Tregs have not been further expanded prior to administration. 139. The method of embodiment 138, wherein the therapeutic population of Tregs is autologous to the subject. 140. The method of embodiment 138, wherein the therapeutic population of Tregs is allogeneic to the subject. 141. The method of any one of embodiments 138-140, wherein the pharmaceutical composition comprising the therapeutic population of Tregs is administered intravenously. 142. The method according to any one of embodiments 138 to 141, wherein the pharmaceutical composition comprising anti-inflammatory EVs and the pharmaceutical composition comprising the therapeutic population of Tregs are administered to the patient on the same day. 143. The method of any one of embodiments 114 to 140, wherein the isolated cell-free population of anti-inflammatory EVs has been cryopreserved and thawed prior to administration to the subject. 144. The method of any one of embodiments 114 to 140, wherein the isolated cell-free population of anti-inflammatory EVs is stored overnight at 4°C before being administered to the subject. 145. The method of embodiment 144, wherein the isolated cell-free population of anti-inflammatory EVs was cryopreserved prior to administration to the subject, and then thawed and stored at 4°C overnight. 146. The method of any one of embodiments 114 to 140, wherein the isolated cell-free population of anti-inflammatory EVs has undergone at least two freeze / thaw cycles before being administered to the subject. 147. The method of embodiment 146, wherein the isolated cell-free population of anti-inflammatory EVs has undergone about 2 to about 20 freeze / thaw cycles before being administered to the subject. [Brief description of the drawings]

[0076] (4. BRIEF DESCRIPTION OF THE DRAWINGS) [Figure 1] Figure 1. Process flow diagram of an exemplary process for Treg isolation, enrichment, and ex vivo expansion.

[0077] [Diagram 2]Figures 2A-2K. Figure 2A: Diagram showing the two EV populations generated (mixed Treg-derived EVs and enriched or pure Treg EVs), as well as references indicating which populations were utilized in the experiments shown in Figures 2B-2K. The mixed EV population obtained from Treg cultures was produced using the improved Treg ex vivo expansion protocol described in Example 1. Tregs were obtained from ALS patients and the culture medium utilized in this expansion process contained 5% human AB serum. Thus, the anti-inflammatory EVs isolated from this culture are present in the EV medium along with serum. The Treg-derived anti-inflammatory EV population is estimated to be approximately 20-30% of the total EV population. The second Treg population was harvested from healthy patient samples and expanded using the improved Treg ex vivo expansion protocol described in Example 1, but with culture medium containing exosome-depleted fetal bovine serum (FBS) instead of human AB serum. Thus, the anti-inflammatory EV population produced from this culture constitutes a pure batch of EVs derived from ex vivo expanded human Tregs. Figures 2B-2F and 2I-2K: These experiments utilized EV populations isolated using PEG. Figures 2G-2H: These experiments utilized EV populations isolated using tangential flow filtration (TFF). Figure 2B: Treg mixed EVs reduce iPSC-derived M1 IL-6 protein by ~70% following co-culture of 1 x 108 Treg EVs per 50,000 M1 cells stimulated overnight with LPS / IFNγ. Figure 2C: Mixed Treg EVs are able to suppress Tresp proliferation upon incremental dosing. Figure 2D: Pure Treg EV batches demonstrate the ability to suppress IL-6 transcripts. Figure 2E: Pure Treg EVs suppress M1 Il-6 protein after overnight stimulation. Figure 2F: Pure Treg EVs suppress Tresp proliferation upon incremental dosing. FIG. 2G: Mixed Treg EVs were shown to be able to suppress M1 IL-6 protein production, regardless of whether isolation was performed via PEG precipitation or TFF (n=3; PEG and TFF isolation protocols were performed on expanded Tregs from the same three patients from a clinical trial).2H: Mixed Treg EVs were shown to be able to suppress Treg proliferation, regardless of whether isolation was performed via PEG precipitation or TFF (n=3; PEG and TFF isolation protocols were performed on expanded Tregs from the same three patients). Figure 2I: Exemplary size profile of Treg EVs produced as described in this example, showing a single peak distribution within 20-200 nm. Figure 2J: Graph showing Miltenyi MACSPlex Exosome Kit (Miltenyi Biotec) analysis of Treg (mixed) EVs and media EVs. Figure 2K: Graph showing Miltenyi MACSPlex Exosome Kit (Miltenyi Biotec) analysis of Treg (mixed) EVs and media EVs. ALS Treg EVs n=7, media EVs n=3. Values ​​are shown as mean + / - SEM, analyzed by one-way ANOVA with Tukey's post-hoc test. ** indicates p-value less than 0.01; *** indicates p-value less than 0.001.

[0078] [Diagram 3] Figures 3A-3D. The anti-inflammatory effect of Treg EVs was evaluated in an LPS-induced neuroinflammation model. Briefly, 2 mg / kg LPS was injected intraperitoneally. Two hours after injection, pure Treg EVs were administered intranasally. Twelve hours after intranasal administration, CD11b+ myeloid cells in brain regions and spleen were isolated. To evaluate the anti-inflammatory effect, pro-inflammatory transcripts were analyzed. Figure 3A: Diagram illustrating the LPS-induced neuroinflammation model and Treg EV treatment paradigm. Figure 3B: Intranasal Treg EVs reduce IL-6 and IL-1β transcripts in the hippocampus. Figure 3C: Reduced IL-6 transcripts after intranasal Treg EV treatment in the cerebral cortex of mice. Figure 3D: Reduced peripheral myeloid cell activation after intranasal treatment with Treg EVs; showing reduced IL-6 and TNF transcripts in CD11b+ myeloid cells from the spleen. p-values ​​are *p<0.05 and **p<0.01.

[0079] [Figure 4]Figures 4A-4F. Treg EVs were administered intranasally to SOD1 mice every 2 weeks starting on day 90 (approximately 20 days after symptoms began to appear in this model) to evaluate the clinical benefit of multiple doses of intranasal Treg EVs to mice. After mice were sacrificed, inflammatory markers in the lumbar region of the inflamed spinal cord were evaluated by RNA analysis. Figure 4A: Diagram showing the intranasal Treg EV treatment paradigm for the SOD1 mouse model of ALS. Figure 4B: Intranasal Treg EV treatment increases the odds of survival compared to intranasal PBS treatment. Figure 4C: Treg EV treatment delayed disease progression as defined by a modified scoring system used to assess the progression of mouse ALS; the effect was more pronounced when mice were going through the acute phase of disease progression. Figure 4D: Treatment significantly extended disease duration. Figure 4E: Average life span is extended in animals that received Treg EV treatment. Figure 4F: Lumbar spinal cord was dissected after animals reached their ethical endpoint. RNA analysis was performed using lumbar spinal cord tissue to examine inflammatory markers. Decreased levels of inflammatory markers were observed in the spinal cord, while increased signals of Treg (FOXP3) and anti-inflammatory M2 macrophages (CD206) were observed in treated animals. Values ​​are presented as mean + / - SEM using one-way ANOVA with Tukey's post-hoc test (PBS n=3, LPS+PBS n=4, LPS+Treg EV n=4, Trev EV only n=3. * indicates p-value less than 0.05; *** indicates p-value less than 0.001.

[0080] [Diagram 5]Figures 5A-5C. Figure 5A: Treg EVs were able to suppress M1 pro-inflammatory IL-6 protein by 46% at a dose of 1x108 EVs and 30.6% at a dose of 1x107 EVs, compared to MSC EVs, which suppressed by 13.7% and 3.3%, respectively. Figure 5B: Treg EVs suppressed M1 pro-inflammatory IL-8 protein by 60% at a dose of 1x108 EVs and 50% at a dose of 1x107 EVs, compared to MSC EVs, which showed 20% suppression at a dose of 1x108. "Control Exo" in Figures 5A and 5B: EVs derived from non-exosome-depleted medium without cell culture. Figure 5C: Treg EVs suppressed T cell proliferation to a greater extent than MSC EVs in a comparative study.

[0081] [Figure 6] Figures 6A-6C. Treg EV stability and function were assessed after 1-20 freeze / thaw cycles and after storage at -20°C for 3, 6, or 12 months. Figure 6A: No loss in Treg EV particle number was observed after multiple (up to 20) freeze / thaw cycles. Figure 6B: No significant deviation in Treg EV particle size was observed during the same freeze / thaw cycles. Figure 6C: Treg EV suppression of T cell proliferation did not decrease over time during cryopreservation at -20°C.

[0082] [Figure 7] Figures 7A-7B. Figure 7A: Treg EV concentration (EV particles / ml medium) after isolation of EVs from growth medium. EV concentration from medium only ("Medium") is also shown. Figure 7B: Fold increase in EVs from ex vivo expanded Treg cell population compared to EVs from medium only.

[0083] [Figure 8] Figure 8. Exemplary size profile of Treg EVs isolated using the TFF protocol. The profile shows an EV mean value of 92.1 nm ± 4.2 nm and a mode value of 73.3 nm ± 6.1 nm.

[0084] [Figure 9]Figures 9A-9B. Figure 9A: Treg EVs derived from ex vivo expanded ALS patient Tregs induce M1 cells to increase Arg1 mRNA expression in an EV concentration-dependent manner. Figure 9B: Treg EVs derived from ex vivo expanded ALS patient Tregs induce M1 cells to increase CD206 mRNA expression in an EV concentration-dependent manner.

[0085] [Figure 10] 10A-10C. FIG. 10A: Treg EVs were shown to be significantly more potent than MSC EVs in suppressing M1 pro-inflammatory IL-6 protein production (n=3 for each group; *** indicates p-value less than 0.001 compared to corresponding MSC EVs). FIG. 10B: Treg EVs were shown to be significantly more potent than MSC EVs in suppressing T cell proliferation (n=3 for each group; *** indicates p-value less than 0.001 compared to corresponding MSC EVs). FIG. 10C: Treg EVs were shown to be significantly more potent than MSC EVs in suppressing M1 pro-inflammatory IL-8 protein production (n=3 for each group; *** indicates p-value less than 0.001 compared to corresponding MSC EVs). Treg EVs utilized in these experiments were pure Treg EVs, and MSC EVs utilized in these experiments were pure MSC EVs.

[0086] [Figure 11] Figures 11A-11B. Figure 11A: Mean particle size of EVs isolated via TFF. Figure 11B: Mode particle size of EVs isolated via TFF.

[0087] [Figure 12] Figure 12. Recovery rate of EVs isolated via TFF (n=6, results reported as mean ± SD).

[0088] [Figure 13] FIG 13. Flow chart of the process for generating Treg populations in a bioreactor.

[0089] [Figure 14] Figures 14A-14F. Figure 14A: Diagram illustrating an LPS-induced acute inflammation model in which WT mice were administered LPS via intraperitoneal injection and then treated with a single tail vein (IV) injection of various doses of Treg EVs. After overnight treatment, mice were sacrificed and peripheral immune cells were isolated from the mouse spleens for subsequent inflammatory transcript analysis. Figure 14B: Diagram illustrating an LPS-induced acute inflammation model in which WT mice were administered LPS via intraperitoneal injection and then treated with a single tail vein (IV) injection of various doses of Treg EVs. After overnight treatment, mice were sacrificed and brain tissues (hippocampus and cerebral cortex) were isolated and transcript analysis of neuroinflammatory markers was performed. Figure 14C: Graph showing fold change in pro-inflammatory transcripts of IL6 and iNOS in spleen-derived CD11b+ myeloid cells after IV treatment of Treg EVs. Figure 14D: Graph showing fold change of IL1b and IFNγ inflammatory transcripts in spleen-derived CD11b+ myeloid cells after IV Treg EV treatment. Figure 14E: Graph showing fold change of CD206 (MRC1) and CD163 anti-inflammatory related transcripts in CD11b+ myeloid cells after Treg EV treatment. Figure 14F: Graph showing fold change of FOXP3 and IL2RA (CD25) in fresh spleen isolated CD4+CD25+ Tregs after Treg EV treatment. Data are presented as mean ± SEM and statistical analysis was performed by one-way ANOVA with Tukey's post-hoc test (PBS n=5, LPS n=5, LPS+Treg EV 1x109 n=5 (peripheral tissue), 4-5 (brain tissue), LPS+Treg EV 1x1010 n=5 (peripheral tissue), 4-5 (brain tissue), LPS+Treg EV 1x1011 n=5 (peripheral tissue), 4-5 (brain tissue)). p-values ​​are *p<0.05, **p<0.01, and ***p<0.001.

[0090] [Figure 15]Figures 15A-15B. Figure 15A: Graphs show fold change in IL-6, IL1b, and TNF RNA in the hippocampus after IV Treg EV treatment. Figure 15B: Graphs show fold change in IL-6, IL1b, and TNF in the cerebral cortex after Treg EV treatment. Data are presented as mean ± SEM using one-way ANOVA with Tukey's post-hoc test (PBS n=5, LPS n=5, LPS+Treg EV 1x109 n=4-5, LPS+Treg EV 1x1010 n=4-5, LPS+Treg EV 1x1011 n=5).

[0091] [Figure 16] Figures 16A-16B. Figure 16A: Size distribution of TFF-isolated Treg EVs generated by nanoparticle tracking analysis. Figure 16B: Particle size data of TFF-isolated Treg EVs generated by nanoparticle tracking analysis.

[0092] [Figure 17] Figure 17. Treg EV suppression of T cell proliferation in vitro (n=6).

[0093] [Figure 18] Figure 18. Quantification of Treg functional proteins in Treg EVs using ELISA.

[0094] [Figure 19] 19A-19B. Quantification of residual IL2 and albumin concentrations as a percentage of the original total amount in concentrated batches after TFF and in final exemplary dose formulations.

[0095] [Figure 20]Figures 20A-20E. Figure 20A: Stability of Treg EVs at room temperature and 4°C. Presented are combined results with Treg EVs from bioreactor runs BioR4-6 up to the 8 hour time point as well as BioR5 and BioR6 for all time points. Figure 20B: Stability of Treg EVs at room temperature and 4°C showing a breakdown of the individual sample results combined in the results shown in Figure 20A. Figure 20C: Stability of particle size distribution of Treg EVs at room temperature and 4°C. As in Figure 20A, presented are combined results with Treg EVs from bioreactor runs BioR4-6 up to the 8 hour time point as well as BioR5 and BioR6 for all time points. Figure 20D: Stability of Treg EVs after long-term storage at -20°C and -80°C. n=3 (Treg EVs from BioR4-6) for each time point. Figure 20E: Particle size distribution of Treg EVs after long-term storage at -20° C. and -80° C. For each time point, n=3 (Treg EVs from BioR4-6).

[0096] [Figure 21] Figure 21. Ability of Treg EVs to suppress activated pro-inflammatory M1 cells; ** indicates p-value less than 0.01; *** indicates p-value less than 0.001.

[0097] [Figure 22] Figure 22. EV surface protein signature. Top panel: bioreactor TFF Treg EVs. Bottom panel: media EVs.

[0098] [Diagram 23] Figure 23. ALS patient Treg EV surface protein signature.

[0099] [Figure 24] Figures 24A-24B. Graphs showing levels of exosomal markers associated with ALS expanded Treg EVs and control expanded Treg EVs (n=3 for ALS expanded Treg EVs, n=6 for control expanded Treg EVs). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0100] (5. Detailed Description) Described herein are anti-inflammatory EV populations derived from ex vivo expanded human suppressive immune cells, e.g., regulatory T cells (Tregs). The EVs presented herein exhibit significant anti-inflammatory activity both in vitro and in vivo. For example, results presented herein show that the EVs of the present disclosure can potently suppress T-responder cell proliferation and pro-inflammatory myeloid, e.g., macrophage, activity in vitro, and exert potent anti-inflammatory effects in vivo. Briefly, results presented herein show that EVs can suppress brain and peripheral inflammation in an in vivo model of neuroinflammation, and can suppress inflammation, prolong survival, and delay late disease progression in an in vivo model of amyotrophic lateral sclerosis (ALS). The anti-inflammatory EVs exhibit suppressive effects on pro-inflammatory responses via either intravenous or intranasal administration. The results presented herein demonstrate that Treg EVs have a stronger suppressive effect on pro-inflammatory immune cells than EVs derived from mesenchymal stem cells (MSCs).

[0101] Moreover, the anti-inflammatory EVs presented herein exhibit remarkable batch-to-batch consistency in size, stability, and activity, and display unique structural features as characterized, for example, by Treg EV surface markers and RNA profiles. Moreover, as shown herein, the methods presented herein yield potent anti-inflammatory EVs that exhibit similar structural and suppressive activity properties whether the original Treg starting material is obtained from healthy subjects or ALS patients.

[0102] Without wishing to be bound by theory or mechanism, the EVs of the present disclosure appear to retain the immunosuppressive activity of the cells from which they are derived. Furthermore, because EVs are not cells themselves, they avoid potential cell-based issues such as immune rejection and possible polarization into pro-inflammatory cell types. Thus, the anti-inflammatory EVs presented herein are particularly useful for the treatment of various diseases, such as, for example, neurodegenerative disorders, e.g., amyotrophic lateral sclerosis (ALS).

[0103] Provided herein are isolated cell-free populations of anti-inflammatory EVs, where the anti-inflammatory EVs are derived from ex vivo expanded human suppressive immune cells, e.g., regulatory T cells (Tregs). Also provided herein are pharmaceutical and cryopreserved compositions comprising the isolated cell-free populations of anti-inflammatory EVs described herein, methods of producing the EV populations, and methods of using EVs to treat diseases, such as neurodegenerative diseases, e.g., ALS.

[0104] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each individual value falling within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually recited herein.

[0105] Unless specifically stated otherwise or apparent from the context, as used herein, the terms "a," "an," and "the" are understood to be in the singular or plural and refer to "one or more."

[0106] Unless specifically indicated otherwise, the terms "include," "such as," and similar terms are intended to convey inclusion, but not limitation.

[0107] The terms "or" and "and" can be used interchangeably and can be understood to mean "and / or."

[0108] Description herein of any aspect or embodiment of the invention using terms such as "comprising," "having," "including," or "containing" with reference to an element or elements is intended to provide support for similar aspects or embodiments of the invention that "consist," "consist essentially of," or "substantially comprise" that particular element or elements, unless otherwise specified or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood to also describe a composition consisting of that element, unless otherwise specified or clearly contradicted by context).

[0109] The terms "about" and "approximately" used herein are interchangeable and should be understood to generally refer to a numerical range around a given numerical value, as well as all numerical values ​​within a recited numerical range (e.g., unless otherwise specified, "about 5 to 15" means "about 5 to about 15"). Furthermore, all numerical ranges herein should be understood to include each and every integer within the range. Unless otherwise specified, these terms mean within plus or minus 10% of the given value or range. When an integer is required, these terms mean within plus or minus 10% of the given value or range, either rounded up or down to the nearest integer.

[0110] (5.1 Anti-inflammatory Extracellular Vesicles (EVs)) Provided herein is an isolated cell-free population of anti-inflammatory extracellular vesicles (EVs), wherein the anti-inflammatory EVs are derived from human suppressive immune cells expanded ex vivo.In some embodiments, the anti-inflammatory EVs are derived from human regulatory T cells (Tregs).

[0111] In certain embodiments, the isolated cell-free population of anti-inflammatory EVs is produced by the methods described herein, e.g., in Section 5.2, below.

[0112] For ease of reference, unless otherwise stated, the terms "extracellular vesicles," "EVs," "extracellular vesicle particles," and "EV particles" are used interchangeably herein. Furthermore, as will be self-evident, as used herein, references to "anti-inflammatory extracellular vesicles," "anti-inflammatory EVs," "anti-inflammatory exosomes," and the like, should be understood to include Treg EVs and Treg exosomes, as described in detail herein. For ease of explanation, not all embodiments presented herein are reproduced to recite, for example, both "anti-inflammatory EVs" and "Treg EVs," but each embodiment reciting such an "anti-inflammatory" embodiment should be understood to include and be replaced by the corresponding "Treg EVs" and "Treg exosomes," as these are described herein.

[0113] EVs are membrane-bound particles released by cells. Typically, EVs are composed of one or more components from the cell from which they are released, such as one or more DNA, RNA (e.g., coding RNA and / or non-coding RNA, e.g., mRNA, microRNA, and / or long non-coding RNA), proteins (e.g., signaling proteins, receptors, other surface proteins, glycoproteins, and / or enzymes), or non-protein, e.g., lipid components. The size of EVs usually ranges from about 30 nm to about 1000 nm in diameter. Larger EVs are sometimes called "microvesicles". Roughly speaking, microvesicles have a diameter larger than about 200 nm. Smaller EVs are sometimes called "exosomes". Roughly speaking, exosomes have a size diameter ranging from about 30-40 nm to about 150-200 nm. Methods for determining the particle size and concentration of EVs are well known to those skilled in the art.

[0114] Methods for determining particle size, concentration, and purity of EVs are well known, including determination using dynamic light scattering or single particle tracking analysis, or utilizing techniques such as flow cytometry, ELISA, or electron microscopy.See, for example, Balaj et al. (2015) Sci Rep 5, 10266, Nakai et al. (2016) Sci Rep 6, 33935, and Carnino et al. (2019) Respiratory Research 20:240.In certain embodiments, routine determination can be performed using a nanoparticle analyzer, such as a NanoSight (Malvern Panalytical) nanoparticle analyzer.

[0115] In some embodiments, EVs can be analyzed for the presence of exosome markers and / or Treg markers (e.g., CD25) by Western blot, ELISA, and other protein-related assays, or by protein analysis using commercially available arrays such as Exo-Check™ Exosome Antibody Array (System Biosciences), and / or MACSPlex Exosome Kit (Miltenyi Biotec). In certain embodiments, the EV population can be analyzed for the presence of serum-associated proteins. In certain embodiments, the EV population described herein is substantially free of serum-associated proteins.

[0116] In some embodiments, the anti-inflammatory EVs described herein exhibit the ability to increase the expression of one or more anti-inflammatory markers in inflammatory cells.For example, in certain embodiments, the anti-inflammatory EVs described herein exhibit the ability to increase the transcription and / or mRNA expression level of one or more genes encoding anti-inflammatory proteins in inflammatory cells.In another example, in certain embodiments, the anti-inflammatory EVs described herein exhibit the ability to increase the translation, processing, secretion, and / or activation of one or more anti-inflammatory proteins produced by inflammatory cells.

[0117] In a specific embodiment, the anti-inflammatory marker is IL-10, Arg1, and / or CD206. In a specific example, the inflammatory cell is a myeloid cell, e.g., a monocyte, macrophage, or microglia, e.g., a human inflammatory cell, e.g., a human monocyte, macrophage, or microglia.

[0118] In some embodiments, the anti-inflammatory EVs described herein exhibit the ability to suppress inflammatory cells. For example, in some embodiments, the anti-inflammatory EVs described herein exhibit the ability to suppress inflammatory cells as measured by the production of pro-inflammatory cytokines by inflammatory cells.

[0119] In some embodiments, the ability to suppress inflammatory cells is measured by IL-6, TNFα, IL1β, IL8, and / or interferon-γ production by inflammatory cells. In some embodiments, the ability to suppress inflammatory cells is measured by IL-6 production by inflammatory cells.

[0120] In certain embodiments, inflammatory cells are myeloid cells, such as monocytes, macrophages, or microglia, for example, human inflammatory cells, such as human myeloid cells, such as human monocytes, macrophages, or microglia.In specific examples, myeloid cells, such as monocytes, macrophages, or microglia, are derived from human donors or are produced from induced pluripotent stem cells.In some embodiments, macrophages are M1 macrophages, for example, induced pluripotent stem cell (iPSC)-derived M1 macrophages.

[0121] In some embodiments, the anti-inflammatory EVs described herein exhibit the ability to suppress proinflammatory M1 cells. In some embodiments, the ability to suppress proinflammatory M1 cells is measured by the IL-6 production by proinflammatory M1 cells.

[0122] In certain embodiments, the anti-inflammatory EVs described herein exhibit the ability to suppress IL-6 protein production in pro-inflammatory M1 cells by about 20% to about 70%, about 20% to about 50%, about 25% to about 50%, about 30% to about 50%, or about 25% to about 45%.

[0123] In certain embodiments, the anti-inflammatory EVs described herein exhibit the ability to suppress inflammatory cells as determined by suppression of responder T cell proliferation. In certain embodiments, responder T cell proliferation is determined by flow cytometry or thymidine incorporation, e.g., tritiated thymidine incorporation.

[0124] In certain embodiments, the anti-inflammatory EVs described herein exhibit the ability to suppress inflammatory cells (e.g., as measured by pro-inflammatory cytokine production and / or responder T cell proliferation), as well as the ability to increase the expression of one or more inflammatory markers in inflammatory cells.

[0125] In some embodiments, the population of anti-inflammatory EVs described herein comprises exosomes. In other embodiments, the population of anti-inflammatory EVs described herein comprises microvesicles. In yet other embodiments, the population of anti-inflammatory EVs described herein comprises exosomes and microvesicles.

[0126] In some embodiments, the majority of the EVs in the population of anti-inflammatory EVs described herein are exosomes.For example, in some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more of the EVs in the population of anti-inflammatory exosomes described herein are exosomes.

[0127] In some embodiments, the majority of the EVs in the anti-inflammatory EV population described herein are microvesicles.For example, in some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more of the EVs in the anti-inflammatory exosome population described herein are microvesicles.

[0128] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a size diameter of about 5 nm to about 1000 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a size diameter of about 10 nm to about 1000 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a size diameter of about 15 nm to about 1000 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a size diameter of about 20 nm to about 1000 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a size diameter of about 30 nm to about 1000 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a size diameter of about 20 nm to about 300 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a size diameter of about 20 nm to about 275 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a size diameter of about 20 nm to about 250 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a size diameter of about 20 nm to about 200 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a size diameter of about 20 nm to about 175 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a size diameter of about 50 nm to about 200 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a size diameter of about 50 nm to about 175 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a size diameter of about 50 nm to about 150 nm.

[0129] In certain embodiments, a majority of the EVs (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more) of the EVs of a population of anti-inflammatory EVs described herein have a size diameter of about 5 nm to about 1000 nm, about 10 nm to about 1000 nm, about 15 nm to about 1000 nm, about 20 nm to about 1000 nm, or about 30 nm to about 1000 nm.

[0130] In certain embodiments, a majority of the EVs (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more) of the EVs of a population of anti-inflammatory EVs described herein have a size diameter of about 20 nm to about 300 nm, about 20 nm to about 275 nm, about 20 nm to about 250 nm, about 20 nm to about 200 nm, or about 20 nm to about 175 nm.

[0131] In certain embodiments, a majority of the EVs (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more) of the EVs in a population of anti-inflammatory EVs described herein have a size diameter of about 20 nm to about 200 nm.

[0132] In certain embodiments, a majority of the EVs (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more) of the EVs of a population of anti-inflammatory EVs described herein have a size diameter of about 50 nm to about 200 nm, about 50 nm to about 175 nm, or about 50 nm to about 150 nm.

[0133] In certain embodiments, a majority of the EVs (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more) of the EVs in a population of anti-inflammatory EVs described herein have a size diameter of about 50 nm to about 150 nm.

[0134] In certain embodiments, the majority of the EVs in the population of anti-inflammatory EVs described herein have a size diameter of less than about 300 nm, less than about 200 nm, less than about 150 nm, or less than about 100 nm. For example, in certain embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more of the EVs in the population of anti-inflammatory exosomes described herein have a size diameter of less than about 300 nm, less than about 200 nm, less than about 150 nm, or less than about 100 nm.

[0135] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein are from about 30 nm to about 300 nm, from about 30 nm to about 250 nm, from about 30 nm to about 200 nm, from about 30 nm to about 160 nm, from about 30 nm to about 150 nm, from about 30 nm to about 100 nm, from about 40 nm to about 300 nm, from about 40 nm to about 200 nm, from about 40 nm to about 160 nm, from about 40 nm to about 150 nm, from about 40 nm to about 100 nm, from about 60 nm to about 300 nm, from about 60 nm to about 200 nm, from about 60 nm to about 160 nm, from about 60 nm to about 150 nm, from about 60 nm to about 125 nm, from about The size diameter is from 60 nm to about 110 nm, from about 60 nm to about 100 nm, from about 60 nm to about 80 nm, from about 70 nm to about 300 nm, from about 70 nm to about 200 nm, from about 70 nm to about 160 nm, from about 70 nm to about 150 nm, from about 70 nm to about 125 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 300 nm, from about 80 nm to about 200 nm, from about 80 nm to about 160 nm, from about 80 nm to about 150 nm, from about 80 nm to about 125 nm, from about 80 nm to about 110 nm, from about 80 nm to about 100 nm, or from about 110 nm to about 120 nm.

[0136] In certain embodiments, a majority of the EVs (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more) of the EVs in a population of anti-inflammatory EVs described herein are between about 30 nm and about 300 nm, about 30 nm and about 250 nm, about 30 nm and about 200 nm, about 30 nm and about 160 nm, about 30 nm and about 150 nm, about 30 nm and about 100 nm, about 40 nm and about 300 nm, about 40 nm and about 200 nm, about 40 nm and about 160 nm, about 40 nm and about 150 nm, about 40 nm and about 100 nm, or more. nm, about 60nm to about 300nm, about 60nm to about 200nm, about 60nm to about 160nm, about 60nm to about 150nm, about 60nm to about 125nm, about 60nm to about 110 nm, about 60nm to about 100nm, about 60nm to about 80nm, about 70nm to about 300nm, about 70nm to about 200nm, about 70nm to about 160nm, about 70nm to about 150nm , about 70 nm to about 125 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 300 nm, about 80 nm to about 200 nm, about 80 nm to about 160 nm, about 80 nm to about 150 nm, about 80 nm to about 125 nm, about 80 nm to about 110 nm, about 80 nm to about 100 nm, or about 110 nm to about 120 nm.

[0137] In certain embodiments, a majority of the EVs (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more) of the EVs of a population of anti-inflammatory EVs described herein have a size diameter of about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 110 nm to about 120 nm, about 150 nm, about 175 nm, about 200 nm, about 250 nm, or about 300 nm.

[0138] In certain embodiments, a majority of the EVs (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more) of the EVs of a population of anti-inflammatory EVs described herein have a size diameter of greater than about 300 nm, greater than about 400 nm, greater than about 500 nm, greater than about 500 nm, greater than about 700 nm, or greater than about 800 nm.

[0139] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have size diameters of about 200 nm to about 1000 nm, about 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 500 nm to about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, about 200 nm to about 800 nm, about 300 nm to about 800 nm, about 400 nm to about 800 nm, about 500 nm to about 800 nm, about 600 nm to about 800 nm, about 200 nm to about 600 nm, about 300 nm to about 600 nm, about 400 nm to about 600 nm, about 200 nm to about 500 nm, or about 300 nm to about 500 nm.

[0140] In certain embodiments, a majority of the EVs (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more) of the EVs in a population of anti-inflammatory EVs described herein are between about 200 nm and about 1000 nm, between about 300 nm and about 1000 nm, between about 400 nm and about 1000 nm, or between about 500 nm and about 1000 nm. nm, about 500 nm to about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, about 200 nm to about 800 nm, about 300 nm to about 800 nm, about 400 nm to about 800 nm, about 500 nm to about 800 nm, about 600 nm to about 800 nm, about 200 nm to about 600 nm, about 300 nm to about 600 nm, about 400 nm to about 600 nm, about 200 nm to about 500 nm, or about 300 nm to about 500 nm.

[0141] In certain embodiments, a majority of the EVs (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more) of the EVs of a population of anti-inflammatory EVs described herein have a size diameter of about 400 nm, about 450 nm, about 500 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, 800 nm, about 850 nm, about 900 nm, about 950 nm, or about 1000 nm.

[0142] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have an average size diameter of about 30 nm to about 1000 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have an average size diameter of less than about 300 nm, less than about 200 nm, less than about 150 nm, or less than about 100 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein are from about 30 nm to about 300 nm, from about 30 nm to about 250 nm, from about 30 nm to about 200 nm, from about 30 nm to about 160 nm, from about 30 nm to about 150 nm, from about 30 nm to about 100 nm, from about 40 nm to about 300 nm, from about 40 nm to about 200 nm, from about 40 nm to about 160 nm, from about 40 nm to about 150 nm, from about 40 nm to about 100 nm, from about 60 nm to about 300 nm, from about 60 nm to about 200 nm, from about 60 nm to about 160 nm, from about 60 nm to about 150 nm, from about 60 nm to about 125 nm, from about 60 nm to about 250 nm, from about 60 nm to about 300 nm, from about 60 nm to about 25 ...300 nm, from about 60 nm to about 250 nm, from about 60 nm to about 160 nm, from about 60 nm to about 150 nm, from about 60 nm to about 125 nm, from about 60 nm to about 100 nm, from about 60 nm to about 300 nm, from about 60 nm to about 250 nm, from about 60 nm to about 100 nm, from about 60 nm to about 300 nm, The average size diameter is from about 0 nm to about 110 nm, from about 60 nm to about 100 nm, from about 60 nm to about 80 nm, from about 70 nm to about 300 nm, from about 70 nm to about 200 nm, from about 70 nm to about 160 nm, from about 70 nm to about 150 nm, from about 70 nm to about 125 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 300 nm, from about 80 nm to about 200 nm, from about 80 nm to about 160 nm, from about 80 nm to about 150 nm, from about 80 nm to about 125 nm, from about 80 nm to about 110 nm, from about 80 nm to about 100 nm, or from about 110 nm to about 120 nm.

[0143] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have an average size diameter of about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 110 nm to about 120 nm, about 150 nm, about 175 nm, about 200 nm, about 250 nm, or about 300 nm.

[0144] In certain embodiments, the EVs in the population of anti-inflammatory EVs described herein have an average size diameter of about 80 nm to about 110 nm, about 80 nm to about 100 nm, about 80 to about 95 nm, about 80-90 nm, about 85 nm to about 110 nm, about 85 nm to about 100 nm, about 85 to about 95 nm, about 90 nm to about 110 nm, about 90 nm to about 100 nm, or about 90 to about 95 nm.

[0145] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have an average size diameter of greater than about 300 nm, greater than about 400 nm, greater than about 500 nm, greater than about 500 nm, greater than about 700 nm, or greater than about 800 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have an average size diameter of about 200 nm to about 1000 nm, about 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 500 nm to about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, about 200 nm to about 800 nm, about 300 nm to about 800 nm, about 400 nm to about 800 nm, about 500 nm to about 800 nm, about 600 nm to about 800 nm, about 200 nm to about 600 nm, about 300 nm to about 600 nm, about 400 nm to about 600 nm, about 200 nm to about 500 nm, or about 300 nm to about 500 nm.

[0146] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have an average size diameter of about 400 nm, about 450 nm, about 500 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, 800 nm, about 850 nm, about 900 nm, about 950 nm, or about 1000 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have an average size diameter of about 80 nm to about 110 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have an average size diameter of about 85 nm to about 100 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have an average size diameter of about 80 nm to about 100 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have an average size diameter of about 85 nm to about 95 nm.

[0147] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a median size diameter of about 30 nm to about 1000 nm, hi certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a median size diameter of less than about 300 nm, less than about 200 nm, less than about 150 nm, or less than about 100 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein are from about 30 nm to about 300 nm, from about 30 nm to about 250 nm, from about 30 nm to about 200 nm, from about 30 nm to about 160 nm, from about 30 nm to about 150 nm, from about 30 nm to about 100 nm, from about 40 nm to about 300 nm, from about 40 nm to about 200 nm, from about 40 nm to about 160 nm, from about 40 nm to about 150 nm, from about 40 nm to about 100 nm, from about 60 nm to about 300 nm, from about 60 nm to about 200 nm, from about 60 nm to about 160 nm, from about 60 nm to about 150 nm, from about 60 nm to about 125 nm, from about 60 nm to about 110 nm, from about 60 nm to about 100 nm, from about 60 nm to about 80 nm, from about 70 nm to about 100 nm, from about 70 nm to about 125 nm, from about 70 nm to about 130 nm, from about 70 nm to about 140 nm, from about 70 nm to about 150 nm, from about 70 nm to about 160 nm, from about 70 nm to about 150 nm, from about 70 nm to about 10 ... In some embodiments, the median size diameter is about 0 nm to about 300 nm, about 70 nm to about 200 nm, about 70 nm to about 160 nm, about 70 nm to about 150 nm, about 70 nm to about 125 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 75 nm to about 100 nm, about 75 nm to about 195 nm, about 75 nm to about 90 nm, about 75 nm to about 85 nm, about 80 nm to about 300 nm, about 80 nm to about 200 nm, about 80 nm to about 160 nm, about 80 nm to about 150 nm, about 80 nm to about 125 nm, about 80 nm to about 110 nm, about 80 nm to about 100 nm, about 80 nm to about 95 nm, about 85 nm to about 95 nm, or about 110 nm to about 120 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a median size diameter of about 30 nm to about 1000 nm.

[0148] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a median size diameter of about 70 nm to about 100 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a median size diameter of about 75 nm to about 100 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a median size diameter of about 75 nm to about 95 nm.

[0149] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a median size diameter of about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 110 nm to about 120 nm, about 150 nm, about 175 nm, about 200 nm, about 250 nm, or about 300 nm.

[0150] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a median size diameter of greater than about 300 nm, greater than about 400 nm, greater than about 500 nm, greater than about 500 nm, greater than about 700 nm, or greater than about 800 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a median size diameter of about 200 nm to about 1000 nm, about 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 500 nm to about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, about 200 nm to about 800 nm, about 300 nm to about 800 nm, about 400 nm to about 800 nm, about 500 nm to about 800 nm, about 600 nm to about 800 nm, about 200 nm to about 600 nm, about 300 nm to about 600 nm, about 400 nm to about 600 nm, about 200 nm to about 500 nm, or about 300 nm to about 500 nm.

[0151] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a median size diameter of about 400 nm, about 450 nm, about 500 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, 800 nm, about 850 nm, about 900 nm, about 950 nm, or about 1000 nm.

[0152] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a modal size diameter of about 30 nm to about 1000 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a modal size diameter of less than about 300 nm, less than about 200 nm, less than about 150 nm, or less than about 100 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein are from about 30 nm to about 300 nm, from about 30 nm to about 250 nm, from about 30 nm to about 200 nm, from about 30 nm to about 160 nm, from about 30 nm to about 150 nm, from about 30 nm to about 100 nm, from about 40 nm to about 300 nm, from about 40 nm to about 200 nm, from about 40 nm to about 160 nm, from about 40 nm to about 150 nm, from about 40 nm to about 100 nm, from about 60 nm to about 300 nm, from about 60 nm to about 200 nm, from about 60 nm to about 160 nm, from about 60 nm to about 150 nm, from about 60 nm to about 125 nm, from about 60 nm to about 150 ... The mode size diameter is about 60 nm to about 100 nm, about 60 nm to about 80 nm, about 70 nm to about 300 nm, about 70 nm to about 200 nm, about 70 nm to about 160 nm, about 70 nm to about 150 nm, about 70 nm to about 125 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 300 nm, about 80 nm to about 200 nm, about 80 nm to about 160 nm, about 80 nm to about 150 nm, about 80 nm to about 125 nm, about 80 nm to about 110 nm, about 80 nm to about 100 nm, or about 110 nm to about 120 nm. In certain embodiments, the EVs in the population of anti-inflammatory EVs described herein have a modal size diameter of about 65 nm to about 85 nm, about 65 nm to about 80 nm, about 65 nm to about 75 nm, about 70 nm to about 85 nm, about 70 nm to about 80 nm, or about 70 nm to about 75 nm.

[0153] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a modal size diameter of about 65 nm to about 95 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a modal size diameter of about 75 nm to about 85 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a modal size diameter of about 75 nm to about 85 nm.

[0154] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a modal size diameter of about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 110 nm to about 120 nm, about 150 nm, about 175 nm, about 200 nm, about 250 nm, or about 300 nm.

[0155] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a modal size diameter of greater than about 300 nm, greater than about 400 nm, greater than about 500 nm, greater than about 500 nm, greater than about 700 nm, or greater than about 800 nm. In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a mode size diameter of about 200 nm to about 1000 nm, about 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 500 nm to about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, about 200 nm to about 800 nm, about 300 nm to about 800 nm, about 400 nm to about 800 nm, about 500 nm to about 800 nm, about 600 nm to about 800 nm, about 200 nm to about 600 nm, about 300 nm to about 600 nm, about 400 nm to about 600 nm, about 200 nm to about 500 nm, or about 300 nm to about 500 nm.

[0156] In certain embodiments, the EVs of the population of anti-inflammatory EVs described herein have a modal size diameter of about 400 nm, about 450 nm, about 500 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, 800 nm, about 850 nm, about 900 nm, about 950 nm, or about 1000 nm.

[0157] In some embodiments, the population of anti-inflammatory EVs described herein is a population of anti-inflammatory EVs containing a buffer. The anti-inflammatory EVs described herein are derived from ex vivo expanded human suppressive immune cells, e.g., Tregs. As described in detail below, such a population of anti-inflammatory EVs can be isolated from a culture comprising ex vivo expanded human suppressive immune cells, e.g., Tregs, and culture medium. In some cases, as part of the process of isolating EVs from a culture, the culture medium can be replaced with a buffer, e.g., a sterile buffer, e.g., a buffer suitable for administration to humans, e.g., suitable for administration to humans for therapeutic use. In such cases, the resulting isolated cell-free population of anti-inflammatory EVs can be referred to as a population of anti-inflammatory EVs containing a buffer.

[0158] Similarly, in some embodiments, the population of anti-inflammatory EVs described herein is a population of anti-inflammatory EVs that contains saline.In certain embodiments, the population of anti-inflammatory EVs described herein is a population of anti-inflammatory EVs that contains normal saline.In certain embodiments, the population of anti-inflammatory EVs described herein is a population of anti-inflammatory EVs that contains 0.9% saline.In certain embodiments, the population of anti-inflammatory EVs described herein is a population of anti-inflammatory EVs that contains phosphate buffer saline.

[0159] The isolated cell-free populations of anti-inflammatory EVs described herein are substantially free of cellular material, particulates, or other contaminants (e.g., organelles, lipids, cholesterol) from the cell or tissue source from which the EVs are derived, e.g., from the human suppressive immune cells, e.g., Tregs, from which the EVs are derived. For example, the isolated cell-free populations of anti-inflammatory EVs described herein typically contain or are free of less than about 5 weight percent, less than about 1 weight percent, less than about 0.5 weight percent, less than about 0.1 weight percent, or less than about 0.01 weight percent of cellular material, particulates, or other contaminants (e.g., organelles, lipids, cholesterol) from the cell or tissue source from which the EVs are derived, e.g., from the human suppressive immune cells, e.g., Tregs, from which the EVs are derived.

[0160] In certain embodiments, the isolated cell-free population of anti-inflammatory EVs described herein is present in a composition that is substantially free of other EVs. For example, in certain embodiments, the isolated cell-free population of anti-inflammatory EVs described herein is present in a composition that contains less than about 20%, less than about 10%, less than about 5%, or less than about 1% of other EVs.

[0161] In certain embodiments, the isolated cell-free population of anti-inflammatory EVs described herein is present in a composition comprising other EVs, wherein the isolated cell-free population of anti-inflammatory EVs comprises about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more than about 95% of the EVs in the composition. In a specific embodiment, the other EVs are serum EVs, such as bovine serum EVs or human serum EVs.

[0162] In certain embodiments, the cell-free population of anti-inflammatory EVs is comprised of anti-inflammatory EVs that contain one or more cargoes, e.g., drugs, detectable labels, proteins, nucleic acids, e.g., RNAs such as mRNAs and / or miRNAs, that are heterologous to the EVs. The cargo(s) of the resulting anti-inflammatory EVs may be present within the EVs and / or on the surface of the EVs.

[0163] In some embodiments, the cell-free population of anti-inflammatory EVs is produced from genetically engineered Treg cells, e.g., engineered to express a cargo that is loaded into the EVs when the EVs are produced. Alternatively, one or more cargoes may be introduced into the culture medium, e.g., during the EV production process, and loaded into the EVs when the EVs are produced. In yet another alternative, one or more cargoes may be introduced into the EVs via techniques such as electroporation or sonication. The cargo of the resulting anti-inflammatory EVs may be present within the EVs and / or on the EV surface.

[0164] 5.1.1. Gene Product Expression Profiles The population of anti-inflammatory EVs provided herein can be characterized by its gene product expression profile. For example, when the population of anti-inflammatory EVs is derived from a population of Tregs cultured in serum-containing medium containing serum EVs (also called "medium EVs"), the gene product expression profile of the population of anti-inflammatory EVs can be compared with medium EVs. In this context of EV gene product expression discussed herein, two values ​​can be considered to be substantially the same if the difference between them is not statistically significant (e.g., p≧0.1, ≧0.05, ≧0.01, or ≧0.001) and / or if the fold difference between the two values ​​is less than about 1.5-fold or less than about 2-fold (increase or decrease).

[0165] A change in gene product expression can be expressed as a "fold increase" or a "fold decrease," or as the binary logarithm (or "log2") of the fold change. In some embodiments, gene product expression is increased at least about 4-fold. In some embodiments, gene product expression is increased about 5-10 fold, about 10-15 fold, about 15-20 fold, about 20-25 fold, about 25-30 fold, about 30-35 fold, about 35-40 fold, about 40-45 fold, about 45-50 fold, about 50-60 fold, about 60-70 fold, about 70-80 fold, about 80-90 fold, about 90-100 fold, or at least about 100 fold. In some embodiments, gene product expression is decreased at least about 4-fold. In some embodiments, gene product expression is increased by about 5-10 fold, about 10-15 fold, about 15-20 fold, about 20-25 fold, about 25-30 fold, about 30-35 fold, about 35-40 fold, about 40-45 fold, about 45-50 fold, about 50-60 fold, about 60-70 fold, about 70-80 fold, about 80-90 fold, about 90-100 fold, or at least about 100 fold.

[0166] In some embodiments, the population of anti-inflammatory EVs provided herein is characterized by its gene product expression profile as shown in Table 10. In some embodiments, the population of anti-inflammatory EVs provided herein is characterized by detectable expression of at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or all of the 191 gene products listed in Table 10. In certain embodiments, such gene products include at least one of MIF, LGALS3, and S100A4. In other specific embodiments, such gene products include at least MIF, LGALS3, and S100A4.

[0167] In some embodiments, the population of anti-inflammatory EVs provided herein exhibits an increased expression level of at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 310, at least 320, at least 330, at least 340, at least 350, at least 360, at least 370, at least 380, at least 390, at least 400, at least 410, at least 420, at least 430, at least 440, at least 450, at least 460, at least 470, at least 480, at least 490, at least 500, at least 500, at least 510, at least 520, at least 530, at least 540, at least 550, at least 560, at least 570, at least 580, at least 590, at least 600, at least 610, at least 620, at least 630, at least 640, at least 650, at least 660, at least 670, at least 680, at least 690, at least The gene products are characterized by 0 or all increased expression (e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold expression, at least 0.5, at least 1, at least 2, at least 3, at least 5, at least 7, at least 8, at least 9, or at least 10 log2 fold expression, and / or statistically significant increased expression (e.g., p<0.1, <0.05, <0.01, or <0.001)). In certain embodiments, such gene products include at least one of MIF, LGALS3, and S100A4. In other specific embodiments, such gene products include at least MIF, LGALS3, and S100A4.

[0168] Gene product expression can be determined by any method known in the art, for example, quantitative real-time PCR, Fluidigm™ chip assay, RNA sequencing, or proteomics analysis (e.g., single-shot proteomics).In a specific embodiment, gene product expression is determined by proteomics analysis (e.g., single-shot proteomics).In a specific embodiment, proteomics analysis is performed by mass spectrometry.

[0169] Similarly, the population of Tregs herein from which the anti-inflammatory EVs provided herein are derived can be characterized by their gene product expression profile. For example, the gene product expression profile of the population of Tregs provided herein can be compared to baseline Tregs. In this context, the term "baseline" or "baseline Treg cell population" refers to a population of Tregs enriched from a patient sample but not yet expanded. In this context of Treg gene product expression discussed herein, two values ​​are considered to be substantially the same if the difference between them is not statistically significant (e.g., ≧0.05) and / or if the binary logarithm of the fold difference between the two values ​​is less than about 2-fold (increase or decrease).

[0170] A change in gene product expression can be expressed as a "fold increase" or a "fold decrease," or as the binary logarithm (or "log2") of the fold change. In some embodiments, gene product expression is increased at least about 4-fold. In some embodiments, gene product expression is increased about 5-10 fold, about 10-15 fold, about 15-20 fold, about 20-25 fold, about 25-30 fold, about 30-35 fold, about 35-40 fold, about 40-45 fold, about 45-50 fold, about 50-60 fold, about 60-70 fold, about 70-80 fold, about 80-90 fold, about 90-100 fold, or at least about 100 fold. In some embodiments, gene product expression is decreased at least about 4-fold. In some embodiments, gene product expression is increased by about 5-10 fold, about 10-15 fold, about 15-20 fold, about 20-25 fold, about 25-30 fold, about 30-35 fold, about 35-40 fold, about 40-45 fold, about 45-50 fold, about 50-60 fold, about 60-70 fold, about 70-80 fold, about 80-90 fold, about 90-100 fold, or at least about 100 fold.

[0171] The level of gene product expression may be above or below the detection limit of the method used to measure gene product expression. In some embodiments, the expression level of a gene product listed in any of Tables 3-7 may be undetectable (i.e., below the detection level of the method used, such as single-shot proteomics) in the population of Tregs from which the anti-inflammatory EVs provided herein are derived. In some embodiments, the expression level of a gene product listed in any of Tables 3-7 may be detectable (i.e., above the detection level of the method used, such as single-shot proteomics) in the population of Tregs from which the anti-inflammatory EVs provided herein are derived. In some embodiments, the expression level of a gene product listed in any of Tables 3-7 may be detectable or undetectable in the population of Tregs from which the anti-inflammatory EVs provided herein are derived upon enrichment or expansion of the population of Tregs. Gene product expression may be determined by any method known in the art, such as quantitative real-time PCR, Fluidigm™ chip assay, RNA sequencing, or proteomic analysis (e.g., single-shot proteomics). In a specific embodiment, gene product expression is determined by proteomic analysis (e.g., single-shot proteomics). In a specific embodiment, the proteomic analysis is performed by mass spectrometry.

[0172] In some embodiments, expression of one or more of the gene products listed in Table 3 and / or Table 4 is decreased in Tregs from which the anti-inflammatory EVs provided herein are derived after expansion compared to baseline Tregs. In some embodiments, expression of one or more of the gene products listed in Table 3 is decreased in Tregs from which the anti-inflammatory EVs provided herein are derived after expansion compared to baseline Tregs. In some embodiments, expression of one or more of the gene products listed in Table 4 is decreased in Tregs from which the anti-inflammatory EVs provided herein are derived after expansion compared to baseline Tregs.

[0173] In some embodiments, expression of one or more of the gene products listed in Tables 5-9 is increased in Tregs from which the anti-inflammatory EVs provided herein are derived after expansion compared to baseline Tregs. In some embodiments, expression of one or more gene products (e.g., one or more of the gene products listed in Table 3) associated with a dysfunctional Treg phenotype is decreased in Tregs from which the anti-inflammatory EVs provided herein are derived after expansion compared to baseline Tregs. Dysfunctional Treg phenotypes include, for example, dysregulated calcium dynamics, loss of MECP2 binding ability to 5-hydroxymethylcytosine (5hmC)-DNA, dysregulated MECP2 expression or activity, and loss of MECP2 regulation, phosphorylation, or binding ability.

[0174] (5.1.2. Treg EV surface marker profile) The population of anti-inflammatory EVs provided herein can be characterized by its Treg EV surface marker profile.When the population of anti-inflammatory EVs is derived from a population of Tregs cultured in serum-containing medium containing serum EVs (also called "medium EVs"), the Treg EV surface marker profile of the population of anti-inflammatory EVs can be compared with the surface marker profile of medium EVs.For example, such comparison can be used to identify and optionally remove or subtract the aspect of the profile contributed by medium EVs.

[0175] In certain embodiments, EV surface marker profile can be generated using antibody-based method to detect epitopes on EV surface.For example, EV surface marker profile can be generated using well-known technique that utilizes a cocktail of various fluorescently labeled bead populations, each coated with specific antibody that binds to the surface epitope of interest, in combination with flow cytometry.For example, MACSPlex Exosome Kit (Miltenyi Biotec) utilizes such technique.In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by Treg surface marker profile generated using MACSPlex Exosome Kit (Miltenyi Biotec).

[0176] The EV surface marker profile can be presented using relative intensity units, e.g., median intensity units, e.g., median fluorescence intensity (MFI) units, such that the EV surface marker profile conveys the detectable presence or absence of the markers being assayed and further conveys the relative amounts of such markers.

[0177] In specific examples, the population of anti-inflammatory EVs described herein exhibit a Treg EV surface marker profile as shown in Example 21 below ("Treg EV surface marker signature"). In specific examples, the population of anti-inflammatory EVs described herein exhibit a Treg EV surface marker profile as shown in Example 21 below ("Treg EV surface marker signature"), generated using the techniques described in Example 21, e.g., using the MACSPlex Exosome Kit (Miltenyi Biotec).

[0178] Each of the surface markers (e.g., CD2, CD25, etc.) is well known, as are antibodies specific for epitopes present on such markers. Note that "HLA-DRDPDQ" refers to the HLA-class II molecules HLA-DR, HLA-DP, and HLA-DQ. Since the Treg EVs described herein are typically obtained from ex vivo expanded human Treg cells, in certain embodiments, such surface markers are human (e.g., human CD2, human HLA-DRDPDQ, CD25, etc.).

[0179] In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile comprising at least one, two, or all three of CD2, HLA-DRDPDQ, and / or CD25. In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile comprising CD2, HLA-DRDPDQ, and CD25. In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile comprising CD2, HLA-DRDPDQ, and CD25, wherein CD2 is present in at least 1-fold, 2-fold, or 5-fold greater abundance than HLA-DRDPDQ and / or CD25, as measured using an assay used to measure or identify the Treg EV surface marker profile, e.g., a MACSPlex Exosome Kit assay.

[0180] As used herein, when a population of anti-inflammatory EVs provided herein is described as being characterized by a Treg surface marker profile comprising one or more listed markers, this refers to a population of anti-inflammatory EVs comprising a detectable amount of the marker using an assay used to measure or identify the Treg EV surface marker profile. Such assays are well known in the art, for example, antibody-based detection assays, for example, antibody-based detection techniques including detection of directly or indirectly labeled, e.g., fluorescently labeled, antibodies specific for surface marker epitopes. Such assays include, for example, utilizing differentially labeled beads coated with antibodies specific for surface marker epitopes in combination with flow cytometry. In a specific embodiment, such assays can utilize, for example, the MACSPlex Exosome Kit assay as described in Example 21 ("Treg EV Surface Marker Signature") below.

[0181] In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that includes at least one, two, or all three of CD2, HLA-DRDPDQ, and / or CD25, and substantially lacks at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen ...

[0182] In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that includes CD2, HLA-DRDPDQ, and CD25, and substantially lacks at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nine, twenty, twenty-one, or all of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31, and / or CD14.

[0183] In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that includes CD2, HLA-DRDPDQ, and CD25, and is substantially devoid of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31, and CD14.

[0184] In certain embodiments, "substantially devoid" as used herein may refer to levels below the level of detection using an assay used to measure or identify a Treg EV surface marker profile, such as a MACSPlex exosome kit assay. In certain embodiments, "substantially devoid" as used herein may refer to levels at least 5-fold, 10-fold, 15-fold, or 20-fold lower than the levels of CD2, HLA-DRDPDQ, and / or CD25 (where such markers are part of a Treg EV surface marker profile) when measured using an assay used to measure or identify a Treg EV surface marker profile, such as a MACSPlex exosome kit assay. In certain embodiments, CD2 is part of a Treg EV surface marker profile, and "substantially devoid" as used herein refers to levels at least 5-fold, 10-fold, 15-fold, or 20-fold lower than the levels of CD2 when measured using an assay used to measure or identify a Treg EV surface marker profile, such as a MACSPlex exosome kit assay.

[0185] In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile that includes at least one, two, or all three of CD2, HLA-DRDPDQ, and / or CD25, and further includes at least one of CD44, CD29, CD4, and / or CD45. In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile that includes CD2, HLA-DRDPDQ, and CD25, and further includes at least one of CD44, CD29, CD4, and / or CD45. In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile that includes CD2, HLA-DRDPDQ, and CD25, and further includes CD44, CD29, CD4, and CD45. In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile comprising CD2, HLA-DRDPDQ, and CD25, and further comprising CD44, CD29, CD4, and CD45, where CD2 is present in at least 1-fold, 2-fold, or 5-fold greater abundance than HLA-DRDPDQ and / or CD25, as well as CD44, CD29, CD4, and CD45, as measured using an assay used to measure or identify the Treg EV surface marker profile, e.g., a MACSPlex Exosome Kit assay.

[0186] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such populations are further characterized by a Treg EV surface marker profile that is substantially devoid of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nine, twenty, twenty-one, or all of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CDl lc, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31, and / or CD14. In certain embodiments of any such population of anti-inflammatory EVs described immediately above herein, such populations are further characterized by a Treg EV surface marker profile that is substantially devoid of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31, and CD14.

[0187] In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that: i) comprises at least one, two, or all three of CD2, HLA-DRDPDQ, and / or CD25; ii) further comprises at least one of CD44, CD29, CD4, and / or CD45; and iii) further comprises at least one of HLA-ABC, CD24, CD69, CD41b, and / or CD42a. In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that includes: i) CD2, HLA-DRDPDQ, and CD25; ii) further includes at least one of CD44, CD29, CD4, and / or CD45; and iii) further includes at least one of HLA-ABC, CD24, CD69, CD41b, and / or CD42a, e.g., at least one of HLA-ABC, CD24, and / or CD69. In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile that includes: i) CD2, HLA-DRDPDQ, and CD25, ii) further includes CD44, CD29, CD4, and CD45, and iii) further includes at least one of HLA-ABC, CD24, CD69, CD41b, and / or CD42a, e.g., at least one of HLA-ABC, CD24, and / or CD69. In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile that includes: i) CD2, HLA-DRDPDQ, and CD25, ii) further includes CD44, CD29, CD4, and CD45, and iii) further includes HLA-ABC, CD24, and CD69, and optionally, CD41b and CD42a.In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ, and CD25; ii) further comprising CD44, CD29, CD4, and CD45; and iii) HLA-ABC, CD24, and CD69, and optionally, CD41b and CD42a, where CD2 is present in an amount at least 1-, 2-, or 5-fold higher than HLA-DRDPDQ and / or CD25, and CD44, CD29, CD4, CD45, HLA-ABC, CD24, CD69, and (if present), CD41b and CD42a, as measured using an assay used to measure or identify the Treg EV surface marker profile, e.g., a MACSPlex Exosome Kit assay.

[0188] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such populations are further characterized by a Treg EV surface marker profile that is substantially devoid of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nine, twenty, twenty-one, or all of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CDl lc, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31, and / or CD14. In certain embodiments of any such population of anti-inflammatory EVs described immediately above herein, such populations are further characterized by a Treg EV surface marker profile that is substantially devoid of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31, and CD14.

[0189] In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that: i) comprises at least one, two, or all three of CD2, HLA-DRDPDQ, and / or CD25; ii) further comprises at least one of CD44, CD29, CD4, and / or CD45; iii) further comprises at least one of HLA-ABC, CD24, CD69, CD41b, and / or CD42a, e.g., at least one of HLA-ABC, CD24, and / or CD69; and iv) further comprises at least one exosome or EV marker, e.g., at least one, two, or all three of CD81, CD63, and / or CD9. In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that: i) comprises CD2, HLA-DRDPDQ, and CD25; ii) further comprises at least one of CD44, CD29, CD4, and / or CD45; iii) further comprises at least one of HLA-ABC, CD24, CD69, CD41b, and / or CD42a, e.g., at least one of HLA-ABC, CD24, and / or CD69; and iv) further comprises at least one exosome or EV marker, e.g., at least one, two, or all three of CD81, CD63, and / or CD9. In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that: i) comprises CD2, HLA-DRDPDQ, and CD25; ii) further comprises CD44, CD29, CD4, and CD45; iii) further comprises at least one of HLA-ABC, CD24, CD69, CD41b, and / or CD42a, e.g., at least one of HLA-ABC, CD24, and / or CD69; and iv) further comprises at least one exosome or EV marker, e.g., at least one, two, or all three of CD81, CD63, and / or CD9.In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that: i) comprises CD2, HLA-DRDPDQ, and CD25; ii) further comprises CD44, CD29, CD4, and CD45; iii) further comprises at least one of HLA-ABC, CD24, CD69, and optionally, at least one of CD41b and CD42a; ​​and iv) further comprises at least one exosome or EV marker, e.g., at least one, two, or all three of CD81, CD63, and / or CD9. In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that includes: i) CD2, HLA-DRDPDQ, and CD25; ii) further includes CD44, CD29, CD4, and CD45; iii) further includes at least one of HLA-ABC, CD24, CD69, and optionally, CD41b and CD42a; ​​and iv) further includes at least one, two, or all three of CD81, CD63, and / or CD9. In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile that includes: i) CD2, HLA-DRDPDQ, and CD25; ii) CD44, CD29, CD4, and CD45; iii) HLA-ABC, CD24, CD69, and optionally, CD41b and CD42a; ​​and iv) at least one, two, or all three of CD81, CD63, and CD9. In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile that includes: i) CD2, HLA-DRDPDQ, and CD25; ii) CD44, CD29, CD4, and CD45; iii) HLA-ABC, CD24, CD69, and optionally, CD41b and CD42a; ​​and iv) CD81, CD63, and CD9.In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ, and CD25; ii) further comprising CD44, CD29, CD4, and CD45; iii) further comprising HLA-ABC, CD24, CD69, CD41b, and CD42a; ​​and iv) further comprising CD81, CD63, and CD9. In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile comprising: i) CD2, HLA-DRDPDQ, and CD25; ii) further comprising CD44, CD29, CD4, and CD45; iii) further comprising HLA-ABC, CD24, CD69, CD41b, and CD42a; ​​and iv) further comprising CD81, CD63, and CD9, where CD2 is present in at least 1-, 2-, or 5-fold greater abundance than HLA-DRDPDQ and / or CD25, and CD44, CD29, CD4, CD45, HLA-ABC, CD24, CD69, CD42a, CD81, CD63, and CD9, as measured using an assay used to measure or identify the Treg EV surface marker profile, e.g., a MACSPlex Exosome Kit assay.

[0190] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such populations are further characterized by a Treg EV surface marker profile that is substantially devoid of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nine, twenty, twenty-one, or all of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CDl lc, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31, and / or CD14. In certain embodiments of any such population of anti-inflammatory EVs described immediately above herein, such populations are further characterized by a Treg EV surface marker profile that is substantially devoid of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31, and CD14.

[0191] In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile: i) comprising CD2, HLA-DRDPDQ, and CD25; ii) further comprising CD44, CD29, CD4, and CD45; iii) further comprising HLA-ABC, CD24, CD69, CD41b, and CD42a; ​​iv) further comprising CD81, CD63, and CD9; and wherein the Treg EV surface marker profile is characterized by a substantial lack of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31, and CD14.

[0192] In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that includes: i) CD2, HLA-DRDPDQ, and CD25; ii) further includes CD44, CD29, CD4, and CD45; iii) further includes HLA-ABC, CD24, CD69, CD41b, and CD42a; ​​iv) further includes CD81, CD63 and CD9, where CD2 is present in at least 1-, 2-, or 5-fold higher abundance than HLA-DRDPDQ and / or CD25, and CD44, CD29, CD4, CD45, HLA-ABC, CD24, CD69, CD42a, CD81, CD63, and CD9, as measured using an assay used to measure or identify the Treg EV surface marker profile, e.g., a MACSPlex Exosome Kit assay; and The EV surface marker profile is characterized by a substantial lack of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31, and CD14.

[0193] In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile that: i) comprises at least one, two, or all three of CD2, HLA-DRDPDQ, and / or CD25, ii) further comprises at least one of CD44, CD29, CD4, and / or CD45, and iii) further comprises at least one of HLA-ABC, CD24, and / or CD69. In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile that: i) comprises CD2, HLA-DRDPDQ, and CD25, ii) further comprises at least one of CD44, CD29, CD4, and / or CD45, and iii) further comprises at least one of HLA-ABC, CD24, and / or CD69. In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that includes: i) CD2, HLA-DRDPDQ, and CD25, ii) further includes CD44, CD29, CD4, and CD45, and iii) further includes at least one of HLA-ABC, CD24, and / or CD69. In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that includes: i) CD2, HLA-DRDPDQ, and CD25, ii) further includes CD44, CD29, CD4, and CD45, and iii) further includes HLA-ABC, CD24, and CD69.

[0194] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such populations are further characterized by a Treg EV surface marker profile that is substantially devoid of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nine, twenty, twenty-one, or all of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CDl lc, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31, and / or CD14. In certain embodiments of any such population of anti-inflammatory EVs described immediately above herein, such populations are further characterized by a Treg EV surface marker profile that is substantially devoid of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31, and CD14.

[0195] In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that: i) comprises at least one, two, or all three of CD2, HLA-DRDPDQ, and / or CD25; ii) further comprises at least one of CD44, CD29, CD4, and / or CD45; iii) further comprises at least one of HLA-ABC, CD24, and / or CD69; and iv) further comprises at least one exosome or EV marker, e.g., at least one, two, or all three of CD81, CD63, and / or CD9. In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that: i) comprises CD2, HLA-DRDPDQ, and CD25; ii) further comprises at least one of CD44, CD29, CD4, and / or CD45; iii) further comprises at least one of HLA-ABC, CD24, and / or CD69; and iv) further comprises at least one exosome or EV marker, e.g., at least one, two, or all three of CD81, CD63, and / or CD9. In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that: i) comprises CD2, HLA-DRDPDQ, and CD25; ii) further comprises CD44, CD29, CD4, and CD45; iii) further comprises at least one of HLA-ABC, CD24, and / or CD69; and iv) further comprises at least one exosome or EV marker, e.g., at least one, two, or all three of CD81, CD63, and / or CD9.In certain embodiments, the population of anti-inflammatory EVs provided herein are characterized by a Treg EV surface marker profile that: i) comprises CD2, HLA-DRDPDQ, and CD25; ii) further comprises CD44, CD29, CD4, and CD45; iii) further comprises at least one of HLA-ABC, CD24, and CD69; and iv) further comprises at least one exosome or EV marker, e.g., at least one, two, or all three of CD81, CD63, and / or CD9. In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile that includes: i) CD2, HLA-DRDPDQ, and CD25, ii) further includes CD44, CD29, CD4, and CD45, iii) further includes at least one of HLA-ABC, CD24, and CD69; and iv) further includes at least one, two, or all three of CD81, CD63, and / or CD9. In certain embodiments, the population of anti-inflammatory EVs provided herein is characterized by a Treg EV surface marker profile that includes: i) CD2, HLA-DRDPDQ, and CD25, ii) further includes CD44, CD29, CD4, and CD45, iii) further includes at least one of HLA-ABC, CD24, and CD69; and iv) further includes CD81, CD63, and CD9.

[0196] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such populations are further characterized by a Treg EV surface marker profile that is substantially devoid of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nine, twenty, twenty-one, or all of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CDl lc, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31, and / or CD14. In certain embodiments of any such population of anti-inflammatory EVs described immediately above herein, such populations are further characterized by a Treg EV surface marker profile that is substantially devoid of CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP (mesenchymal stem cell-like protein), CD146, CD86, CD326, CD133, CD142, CD31, and CD14.

[0197] 5.1.3. Treg EV RNA Profile The population of anti-inflammatory EVs provided herein can be characterized by their Treg EV RNA profile, which characterizes the micro-RNA (miRNA) profile of the anti-inflammatory EV populations described herein.

[0198] Treg EV RNA profiles can be generated using well-known techniques, such as those described in Example 22 below ("Treg EV RNA Profile"). For example, RNA can be isolated from the EV population, enriched for small RNAs, e.g., RNAs about 15-200, e.g., 17-200 nucleotides in length, and sequenced, where sequences are counted when confirmed to correspond to sequences associated with known small miRNAs. Such "read counts" can be used to assess the abundance of a given miRNA within the EV population.

[0199] In one embodiment, the population of anti-inflammatory EVs provided herein are described as being characterized by a Treg RNA profile that includes three, four, or all five of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and / or hsa-miR-21-5p. In one embodiment, the population of anti-inflammatory EVs provided herein are described as being characterized by a Treg RNA profile that includes each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p.

[0200] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p at a hsa-miR-146a-5p / hsa-miR-155-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5. In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-1290 and hsa-miR-155-5p, and such population comprises hsa-miR-1290 at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher abundance than hsa-mir-155-5p.

[0201] In one embodiment, the population of anti-inflammatory EVs provided herein are described as being characterized by a Treg RNA profile that includes: i) three, four, or all five of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and / or hsa-miR-21-5p, and; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p. In certain embodiments, the population of anti-inflammatory EVs provided herein are described as being characterized by a Treg RNA profile comprising: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p, and ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p.

[0202] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p at a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5. In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-1290 and hsa-miR-155-5p, and such population comprises hsa-miR-1290 at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher abundance than hsa-mir-155-5p. In other embodiments of any such population of anti-inflammatory EVs described immediately above, such population comprises at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii). In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a hsa-miR-146a-5p / hsa-miR-155-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5; and such population comprises hsa-miR-1290 and hsa-miR-146a-5p in an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii).

[0203] In one embodiment, the population of anti-inflammatory EVs provided herein is selected from the group consisting of Tregs comprising: i) three, four, or all five of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and / or hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; and iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p. It has been described as being characterized by its RNA profile. In certain embodiments, the population of anti-inflammatory EVs provided herein are described as being characterized by a Treg RNA profile that includes: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) any one to five of the following: hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; and iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p.In certain embodiments, the population of anti-inflammatory EVs provided herein are described as being characterized by a Treg RNA profile comprising: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; and iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p.

[0204] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p at a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5. In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-1290 and hsa-miR-155-5p, and such population comprises hsa-miR-1290 at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher abundance than hsa-mir-155-5p. In other embodiments of any such population of anti-inflammatory EVs described immediately above herein, such populations comprise at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) and iii). In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5; and such population comprises hsa-miR-1290 and hsa-miR-146a-5p in an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) and iii).

[0205] In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) three, four, or all five of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and / or hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; and iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p. In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; and any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; and any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p. In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; and any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) are characterized by a Treg RNA profile comprising each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; and each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p.

[0206] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p at a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5. In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-1290 and hsa-miR-155-5p, and such population comprises hsa-miR-1290 at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher abundance than hsa-mir-155-5p. In other embodiments of any such population of anti-inflammatory EVs described immediately above herein, such populations comprise at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) and iii). In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5; and such population comprises hsa-miR-1290 and hsa-miR-146a-5p in an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) to iv).

[0207] In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) three, four, or all five of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and / or hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; and v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p.In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; and v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p.In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; and v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; and v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p. In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; and v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; and v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p.

[0208] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p at a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5. In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-1290 and hsa-miR-155-5p, and such population comprises hsa-miR-1290 at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher abundance than hsa-mir-155-5p. In other embodiments of any such population of anti-inflammatory EVs described immediately above herein, such populations comprise at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) and iii). In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5; and such population comprises hsa-miR-1290 and hsa-miR-146a-5p in an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) to v).

[0209] In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) three, four, or all five of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and / or hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; and vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p.In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; and vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p.In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; and vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; and vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; and vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; and vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; and vi) each of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p.

[0210] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p at a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5. In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-1290 and hsa-miR-155-5p, and such population comprises hsa-miR-1290 at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher abundance than hsa-mir-155-5p. In other embodiments of any such population of anti-inflammatory EVs described immediately above herein, such populations comprise at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) and iii). In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5; and such population comprises hsa-miR-1290 and hsa-miR-146a-5p in an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) to vi).

[0211] In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) three, four, or all five of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and / or hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; and vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p.In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; and vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p.In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; and vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; and vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; and vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; and vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) each of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p; and vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) each of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p; and vii) each of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and hsa-miR-103a-3p.

[0212] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p at a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5. In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-1290 and hsa-miR-155-5p, and such population comprises hsa-miR-1290 at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher abundance than hsa-mir-155-5p. In other embodiments of any such population of anti-inflammatory EVs described immediately above herein, such populations comprise at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) and iii). In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5; and such population comprises hsa-miR-1290 and hsa-miR-146a-5p in an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) to vii).

[0213] In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) three, four, or all five of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and / or hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; and viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p.In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; and viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p.In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; and viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; and viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; and viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; and viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) each of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; and viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) each of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p; vii) each of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and hsa-miR-103a-3p; and viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) each of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p; vii) each of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and hsa-miR-103a-3p; and viii) each of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and hsa-miR-625-5p.

[0214] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p at a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5. In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-1290 and hsa-miR-155-5p, and such population comprises hsa-miR-1290 at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher abundance than hsa-mir-155-5p. In other embodiments of any such population of anti-inflammatory EVs described immediately above herein, such populations comprise at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) and iii). In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5; and such population comprises hsa-miR-1290 and hsa-miR-146a-5p in an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) to viii).

[0215] In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) three, four, or all five of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and / or hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p; and ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p.In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p; and ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p.In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p; and ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p; and ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p; and ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p; and ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) each of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p; and ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) each of one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p; vii) each of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p; and ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) each of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p; and ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hs. vii) each of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and hsa-miR-103a-3p; viii) each of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and hsa-miR-625-5p; and ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p. It has been described as being characterized by its RNA profile.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) each of one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p; vii) each of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and hsa-miR-103a-3p; viii) each of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and hsa-miR-625-5p; and ix) each of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and hsa-miR-98-5p.

[0216] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p at a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5. In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-1290 and hsa-miR-155-5p, and such population comprises hsa-miR-1290 at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher abundance than hsa-mir-155-5p. In other embodiments of any such population of anti-inflammatory EVs described immediately above herein, such populations comprise at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) and iii). In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5; and such population comprises hsa-miR-1290 and hsa-miR-146a-5p in an abundance that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) to ix).

[0217] In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) three, four, or all five of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and / or hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p; ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p;and x) a Treg RNA profile comprising any one to five of hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p, and / or hsa-miR-342-5p. In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) any one to five of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and / or hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p;ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p; and x) any one to five of hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p, and / or hsa-miR-342-5p. In one embodiment, the population of anti-inflammatory EVs provided herein comprises: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) any one to five of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and / or hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p;ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p; and x) any one to five of hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p, and / or hsa-miR-342-5p. In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) any one to five of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and / or hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p;ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p; and x) any one to five of hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p, and / or hsa-miR-342-5p. In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) any one to five of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and / or hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p;ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p; and x) any one to five of hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p, and / or hsa-miR-342-5p. In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) any one to five of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and / or hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and hsa-miR-625-5p;ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and hsa-miR-98-5p; and x) any one to five of hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p, and hsa-miR-342-5p. In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) each of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p; vii) any one to five of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and / or hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p;ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p; and x) any one to five of hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p, and / or hsa-miR-342-5p. In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) each of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p; vii) each of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and hsa-miR-103a-3p; viii) any one to five of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and / or hsa-miR-625-5p;ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p; and x) any one to five of hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p, and / or hsa-miR-342-5p. In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) each of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p; vii) hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and hsa-m; viii) each of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and hsa-miR-625-5p; ix) any one to five of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and / or hsa-miR-98-5p; and x) any one to five of hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p, and / or hsa-miR-342-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) each of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p; vii) each of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and hsa-miR-103a-3p; viii) each of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and hsa-miR-625-5p; ix) each of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and hsa-miR-98-5p; and x) any one to five of hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p, and / or hsa-miR-342-5p.In one embodiment, the population of anti-inflammatory EVs provided herein are selected from the group consisting of: i) each of hsa-miR-1290, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-let-7a-5p, and hsa-miR-21-5p; ii) each of hsa-miR-191-5p, hsa-miR-1246, hsa-let-7b-5p, hsa-miR-29a-3p, and hsa-miR-320a-3p; iii) each of hsa-miR-423-5p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-16-5p, and hsa-miR-26a-5p; iv) each of hsa-let-7g-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-26b-5p, and hsa-miR-342-3p; v) each of hsa-miR-146b-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-23a-3p, and hsa-miR-181a-5p; vi) each of hsa-miR-150-5p, hsa-miR-20a-5p, hsa-miR-27a-3p, hsa-let-7d-5p, and hsa-miR-17-5p; vii) each of hsa-miR-142-3p, hsa-miR-130b-3p, hsa-miR-25-3p, hsa-miR-142-5p, and hsa-miR-103a-3p; viii) each of hsa-miR-28-3p, hsa-let-7e-5p, hsa-miR-425-5p, hsa-miR-186-5p, and hsa-miR-625-5p; ix) each of hsa-miR-4516, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-486-5p, and hsa-miR-98-5p; and x) a Treg RNA profile comprising each of hsa-miR-181b-5p, hsa-miR-378a-3p, hsa-miR-30d-5p, hsa-miR-454-3p, and hsa-miR-342-5p.

[0218] In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p at a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5. In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-1290 and hsa-miR-155-5p, and such population comprises hsa-miR-1290 at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher abundance than hsa-mir-155-5p. In other embodiments of any such population of anti-inflammatory EVs described immediately above herein, such populations comprise at least one of hsa-miR-1290 or hsa-miR-146a-5p at an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) and iii). In certain embodiments of any such population of anti-inflammatory EVs described immediately herein above, such population comprises hsa-miR-146a-5p and hsa-miR-155-5p in a hsa-miR-146a-5p / hsa-miR-15-5p ratio of about 1 to 10, e.g., 1.5 to 6, e.g., about 2, about 3, about 2 to about 3, about 2 to about 4, about 4, or about 5; and such population comprises hsa-miR-1290 and hsa-miR-146a-5p in an abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the abundance of any of the miRNAs listed in ii) through x).

[0219] 5.2 Methods for Producing Anti-inflammatory EVs In one embodiment, provided herein is a method for producing an isolated cell-free population of anti-inflammatory EVs.

[0220] In certain embodiments, provided herein is a method of producing an isolated cell-free population of anti-inflammatory EVs, comprising: a) expanding ex vivo a population of human suppressive immune cells in culture medium to produce a culture comprising the human suppressive immune cell population, culture medium, and anti-inflammatory EVs (without wishing to be bound by theory or mechanism, the EVs are presumed to be released into the culture by the human suppressive immune cells during the ex vivo expansion period), and b) isolating the anti-inflammatory EVs from the culture.

[0221] In certain embodiments, provided herein is a method of producing an isolated cell-free population of anti-inflammatory EVs, comprising: a) expanding a human suppressive immune cell population ex vivo in culture medium, where the human suppressive immune cell population is a population of regulatory T cells (Tregs), to produce a culture comprising a Treg population, culture medium, and anti-inflammatory EVs, and b) isolating the anti-inflammatory EVs from the culture.

[0222] Methods for obtaining, optionally enriching, and expanding ex vivo human suppressive immune cells, such as Tregs, are well known. Exemplary methods are provided below.

[0223] For ease of explanation, a method for producing an isolated cell-free population of anti-inflammatory EVs may be presented herein as comprising: a) expanding a human suppressive immune cell population ex vivo in culture medium to produce a culture comprising the human suppressive immune cell population, culture medium, and anti-inflammatory EVs, and b) isolating the anti-inflammatory EVs from the culture. However, it should be understood that isolating the anti-inflammatory EVs from the culture may be performed at any time once ex vivo expansion has commenced, or may be performed repeatedly during the ex vivo expansion period.

[0224] For example, ex vivo expansion can include multiple rounds of expansion. In one non-limiting example, the culture medium containing EVs can be collected at the end of one or more rounds of expansion, and EVs can be isolated from the culture medium. In another non-limiting example, the culture medium containing EVs can be collected at the time of replenishing or replacing the culture medium of the culture during the expansion period. In another non-limiting example, EVs can be isolated at the end of the ex vivo expansion process. In yet another non-limiting example, EVs can be isolated at multiple time points during the ex vivo expansion process, for example, at one or more of the time points described above.

[0225] In some embodiments, the ex vivo expansion period lasts for about 24 hours, 48 ​​hours, or 72 hours. In some embodiments, the ex vivo expansion period lasts for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 10 days, 14 days, 2 weeks, 3 weeks, or longer.

[0226] In some embodiments, EVs can be isolated about 24 hours, 48 ​​hours, or 72 hours after growth. In some embodiments, EVs can be isolated about 24 hours, 48 ​​hours, or 72 hours after culture medium is replenished or replaced. In some embodiments, EVs are isolated every 2, 3, 4, or 5 days.

[0227] In certain embodiments, the culture medium containing EVs can be collected at one or more time points during the growth process, and the isolation of EVs is initiated within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, or overnight after the culture medium is collected.In certain embodiments, the culture medium containing EVs can be collected at one or more time points during the growth process and stored at 4°C before isolating EVs.In specific embodiments, for example, the culture medium containing EVs can be collected at one or more time points during the growth process and stored at 4°C for about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, or overnight before isolating EVs from the culture medium.

[0228] In certain embodiments, the culture medium containing the EVs can be harvested at one or more time points during the expansion process and stored, e.g., frozen, prior to isolating the EVs.

[0229] In certain embodiments, EVs can be isolated from cell culture by centrifugation, for example, differential centrifugation.In some embodiments, differential centrifugation can be used to isolate desired subpopulations of EVs.For example, differential centrifugation can be used to isolate subpopulations of EVs enriched for smaller particle diameter sizes (e.g., exosomes; EVs with particle sizes of less than about 300 nm, less than about 200 nm, less than about 160 nm, less than about 150 nm, less than about 130 nm, less than about 100 nm, or less than about 80 nm).In a particular non-limiting example, a centrifugation step at 2,000 g (3,000 rpm) for 20 minutes can be used to remove cell debris and dead cells, and a centrifugation step at 16,500 g (9,800 rpm) for 45 minutes or at 100,000 g (26,450 rpm) for 2 hours can be used to specifically isolate exosomes.

[0230] EVs can also be purified using density gradient centrifugation, which isolates EVs from cultures based on their buoyant density in solutions of either sucrose, iohexol, or iodixanol.

[0231] Further examples of methods used to isolate EVs include precipitation with organic solvents (e.g., polyethylene glycol, sodium acetate, or protamine), immunoprecipitation, separation using antibody-coated magnetic beads, microfluidic devices, and ultrafiltration, as described, for example, in Carnino et al., Respiratory Research (2019) 20:240 and Momen-Heravi et al., Biol. Chem. 2013; 394(10): 1253-1262. Further exemplary methods are isolation using heparin-conjugated agarose beads (see, for example, Balaj et al., (2015) Sci Rep 5, 10266) and purification using Tim4-affinity purification (see, for example, Nakai et al., (2016) Sci Rep 6, 33935).

[0232] Commercially available kits for EV isolation are also available, non-limiting examples include the exoEasy kit (Qiagen), the ExoQuick® kit (Systems Bioscience), and the EasySep™ Human Pan Extracellular Vesicle Positive Selection Kit (Stem Cell Technologies).

[0233] In some embodiments, the method of producing an isolated cell-free population of anti-inflammatory EVs provided herein includes (a) expanding a human suppressive immune cell population (e.g., a Treg cell population) ex vivo in culture medium to produce a culture comprising cells, culture medium, and anti-inflammatory EVs; and (b) isolating the anti-inflammatory EVs from the culture.

[0234] In one embodiment, the method of producing an isolated cell-free population of anti-inflammatory EVs and isolating the anti-inflammatory EVs from culture comprises polyethylene glycol (PEG) precipitation.In a specific embodiment, PEG is added to culture so that EVs are precipitated from culture.After removing the precipitate containing EVs from culture, EVs are washed to produce an isolated cell-free population of anti-inflammatory EVs.

[0235] An exemplary non-limiting protocol for isolation of EVs from cells (e.g., Tregs) using PEG precipitation can include: (i) centrifuging medium from a cell culture (e.g., ex vivo expanded human suppressive immune cells, e.g., Treg cell culture) at 3000×g for 15 minutes to remove cells and debris; (ii) adding PEG reagent to the supernatant, e.g., at a ratio of PEG:supernatant of 1:5; (iii) mixing thoroughly; (iv) refrigerating overnight at 4° C.; (v) centrifugation at 1500×g for 30 minutes; (vi) aspirating the supernatant; (vii) centrifugation again at 1500×g for 10 minutes; (viii) removing the supernatant, e.g., removing the supernatant by aspiration; and (ix) resuspending the resulting EV pellet in a sterile buffer, e.g., sterile PBS.

[0236] EVs can also be isolated from cell cultures using filtration, e.g., tangential flow filtration (TFF), which can be utilized to efficiently isolate and enrich EV populations in a scalable and reproducible manner, even when starting from large culture volumes.

[0237] In certain embodiments, for example, the isolating step (b) comprises removing cells from the culture to produce a cell-free population of anti-inflammatory EVs.

[0238] In certain embodiments, for example, the isolation step (b) comprises (i) removing cells from the culture to produce a cell-free anti-inflammatory EV-containing solution; and (ii) isolating the anti-inflammatory EVs from the cell-free anti-inflammatory EV-containing solution of (i). Steps (i) and (ii) may be performed separately, e.g., sequentially, as separate steps, or may be accomplished as a single step.

[0239] In some embodiments, step (b) comprises filtration, e.g., one or more filtration steps. In certain embodiments, the filtration comprises TFF. For example, some or all of the filtration can utilize TFF. For example, filtration can be utilized to remove cells and debris from the culture. Filtration can also be utilized to isolate and concentrate EVs, e.g., isolate and concentrate EVs of a particular size or size range. In certain embodiments, the removal of cells and debris and the isolation of EVs, e.g., isolate EVs of a particular size or size range, can be achieved using a single filtration step. In other embodiments, for example, a series (two or more) filtration steps can be utilized to remove cells and debris, isolate EVs, and isolate EVs of a particular size range. For example, one or more filtration steps can be utilized to first remove cells and debris to produce an EV-containing solution, and then isolate and concentrate an EV population from the solution, e.g., one or more filtration steps can be utilized to isolate and concentrate EVs of a particular size or size range from the solution.

[0240] In some embodiments, step (b), e.g., step (i), comprises filtration, e.g., microfiltration (e.g., microfiltration by TFF). For example, the culture can be passed through a filter, e.g., a 0.05 μm, 0.1 μm, 0.2 μm, 0.45 μm, 0.65 μm, or 0.8 μm filter, to remove cells and any debris from the culture and produce a cell-free anti-inflammatory EV-containing solution comprising an anti-inflammatory population. In a specific embodiment, the culture can be passed through a 0.65 μm filter to remove cells and any debris from the culture and produce a cell-free anti-inflammatory EV-containing solution comprising an anti-inflammatory population. In a specific embodiment, the culture can be circulated through a filter, e.g., a 0.05 μm, 0.1 μm, 0.2 μm, 0.45 μm, 0.65 μm, or 0.8 μm filter, using TFF, to remove cells and any debris from the culture and produce a cell-free anti-inflammatory EV-containing solution comprising an anti-inflammatory EV population. In certain embodiments, the culture can be circulated through a 0.65 μm filter using a TFF to remove cells and any debris from the culture and produce a cell-free anti-inflammatory EV-containing solution that contains the anti-inflammatory EV population. In a specific embodiment, the filter used in step (i) is an 85 cm 2 In a specific embodiment, the filter used in step (i) is a hollow fiber filter. In a specific embodiment, the filter used in step (i) is a hollow fiber filter with a fiber diameter of 0.75 mm. One or more filtrations can be utilized. One or more sizes of filters can be utilized. It should be understood that in addition to removing cells and debris, such filtration can also serve to isolate EVs of a particular size or size range.

[0241] In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is carried out at a flow rate of 20 to 1000 mL / min. In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is carried out at a flow rate of 50 to 500 mL / min. In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is carried out at a flow rate of 100 to 200 mL / min. In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is carried out at a flow rate of about 100 mL / min. In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is carried out at a flow rate of about 150 mL / min. In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is carried out at a flow rate of about 200 mL / min.

[0242] In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is performed for about 2,000 to 5,000 s -1 In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is performed using a hollow fiber filter having a shear rate of about 2,000 to 3,000 s -1 In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is performed using a hollow fiber filter having a shear rate of about 3,000 to 4,000 s -1 In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is performed using a hollow fiber filter having a shear rate of about 4,000 to 5,000 s -1 In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is performed using a hollow fiber filter having a shear rate of about 2,000 s -1 In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is performed using a hollow fiber filter having a shear rate of about 3,000 s -1In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is performed using a hollow fiber filter having a shear rate of about 4,000 s -1 In a specific embodiment, the microfiltration in step (i) (e.g., microfiltration by TFF) is performed using a hollow fiber filter having a shear rate of about 5,000 s -1 This is done using hollow fiber filters with a shear rate of 1000 s. Shear rate is a term used for hollow fiber membranes and is influenced by the flow rate and the radius of the fibers. Typical ranges of shear rate are 2000-12000 s. -1 However, preferably, the shear rate maintained in step (i) is about 2000 to 5000 s to avoid EV disruption and result in high EV recovery efficiency (e.g., greater than 90% or greater than 95% EV recovery). -1 (but not higher). In a specific embodiment, the shear rate maintained in step (i) is about 2,000 to 5,000 s -1 The flow rate is 100 to 200 mL / min, and a hollow fiber filter having a fiber diameter of 0.75 mm is used.

[0243] In certain embodiments, the retentate pressure in step (i) is maintained at about 5 psi. In certain embodiments, the shear rate maintained in step (i) is about 2,000 to 5,000 s -1 The flow rate is 100-200 mL / min, and a hollow fiber filter with a fiber diameter of 0.75 mm is used, resulting in a retentate pressure of approximately 5 psi.

[0244] In some embodiments, step (b) comprises step (ii), where step (ii) comprises filtration, e.g., ultrafiltration (e.g., ultrafiltration by TFF). In certain embodiments, step (ii) comprises passing the cell-free anti-inflammatory EV-containing solution through a filter, such that anti-inflammatory EVs, e.g., anti-inflammatory EVs of a particular size or size range, are retained by the filter. In certain embodiments, step (ii) comprises circulating the cell-free anti-inflammatory EV-containing solution through a filter using TFF, such that anti-inflammatory EVs are retained by the filter. One or more filtrations can be utilized. One or more sizes of filters can be utilized. Step (ii) can also serve to concentrate the EVs. In specific embodiments, the final volume of the EV-containing solution after concentration is about 5-200 mL. In specific embodiments, the final volume of the EV-containing solution after concentration is about 10-100 mL. In specific embodiments, the final volume of the EV-containing solution after concentration is about 10-50 mL. In specific embodiments, the final volume of the EV-containing solution after concentration is about 10 mL. In a specific embodiment, the final volume of the EV-containing solution after concentration is about 15mL.In a specific embodiment, the final volume of the EV-containing solution after concentration is about 20mL.In a specific embodiment, the final volume of the EV-containing solution after concentration is about 25mL.In a specific embodiment, the final volume of the EV-containing solution after concentration is about 30mL.

[0245] In some embodiments, at least one filter used in step (ii) has a molecular weight cut-off (MWCO) of about 50 kilodaltons (kDa) to about 750 kDa, about 100 kDa to about 750 kDa, about 300 kDa to about 750 kDa, or about 300 kDa to about 500 kDa. In some embodiments, the filter has a MWCO of about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 150 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa about 600 kDa, about 700 kDa, or about 750 kDa. In one embodiment, the filter has a MWCO of about 500 kDa. In some embodiments, the filter used in step (ii) has a pore size of about 0.3 μm, about 0.22 μm, about 0.2 μm, or about 0.1 μm. In specific embodiments, the filter used in step (ii) has a pore size of about 115 cm 2 In a specific embodiment, the filter used in step (ii) is a hollow fiber filter. In a specific embodiment, the filter used in step (ii) is a hollow fiber filter with a fiber diameter of 0.5 mm.

[0246] In certain embodiments, step (ii) is designed to retain EVs of a certain particle size or size range, for example, to retain EVs of about 50 nm to about 60 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 80 nm, about 80 nm, about 100 nm, about 150 nm, or greater than about 200 nm.

[0247] In some embodiments, step (ii) comprises the use of a filter designed to retain EVs greater than about 50 nm to about 60 nm and having a MWCO of about 300 kDa. In some embodiments, step (ii) comprises the use of a filter designed to retain EVs greater than about 50 nm and having a MWCO of about 300 kDa. In some embodiments, step (ii) comprises the use of a filter designed to retain EVs greater than about 70 nm to about 80 nm and having a MWCO of about 500 kDa. In some embodiments, step (ii) comprises the use of a filter designed to retain EVs greater than about 70 nm and having a MWCO of about 500 kDa. In some embodiments, step (ii) comprises the use of a filter designed to retain EVs greater than about 80 nm and having a MWCO of about 500 kDa. In some embodiments, step (ii) comprises the use of a filter designed to retain EVs greater than about 60 nm and having a MWCO of about 500 kDa.

[0248] In some embodiments, step (b), e.g., step (b)(ii), comprises performing a buffer exchange such that the produced isolated cell-free population of anti-inflammatory EVs is an isolated cell-free population of anti-inflammatory EVs containing a buffer. In certain embodiments, the buffer exchange comprises diafiltration. In a specific embodiment, the buffer exchange comprises TFF and diafiltration. In a specific embodiment, the diafiltration is performed at 2X to 100X. In a specific embodiment, the diafiltration is performed at 5X to 50X. In a specific embodiment, the diafiltration is performed at 5X to 20X. In a specific embodiment, the diafiltration is performed at 5X. In a specific embodiment, the diafiltration is performed at 10X. In a specific embodiment, the diafiltration is performed at 15X. In a specific embodiment, the diafiltration is performed at 20X.

[0249] For example, in some embodiments, step (b) comprises step (ii), which can comprise circulating the cell-free anti-inflammatory EV-containing solution through a filter using TFF, such that the anti-inflammatory EVs are retained by the filter, wherein the circulating comprises incorporating a suitable buffer into the solution, such that during the process, the buffer displaces the solution, thereby resulting in an isolated cell-free population of anti-inflammatory EVs containing the buffer.

[0250] In some embodiments, the buffer is a sterile buffer. In some embodiments, the buffer is a sterile buffer suitable for administration to humans, for example, suitable for administration to humans for therapeutic use. In a specific embodiment, the buffer is a saline-containing buffer. In one embodiment, the buffer is physiological saline. In one embodiment, the buffer is physiological saline. In one embodiment, the buffer is normal saline. In one embodiment, the buffer is 0.9% saline. In one embodiment, the buffer is phosphate buffered saline (PBS).

[0251] In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF, and optionally, diafiltration) is performed at a flow rate of 20 to 1000 mL / min. In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF, and optionally, diafiltration) is performed at a flow rate of 50 to 500 mL / min. In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF, and optionally, diafiltration) is performed at a flow rate of 80 to 200 mL / min. In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF, and optionally, diafiltration) is performed at a flow rate of 80 to 175 mL / min. In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF, and optionally, diafiltration) is carried out at a flow rate of about 80 mL / min. In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF, and optionally, diafiltration) is carried out at a flow rate of about 100 mL / min. In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF, and optionally, diafiltration) is carried out at a flow rate of about 125 mL / min. In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF, and optionally, diafiltration) is carried out at a flow rate of about 150 mL / min. In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF, and optionally, diafiltration) is carried out at a flow rate of about 175 mL / min. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (eg, ultrafiltration by TFF and, optionally, diafiltration) is performed at a flow rate of about 200 mL / min.

[0252] In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed for about 2,000 to 8,000 s. -1In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 2,000 to 7,500 s -1 In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 2,000 to 7,000 s -1 In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 2,000 to 3,000 s -1 In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 3,000 to 4,000 s -1 In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 4,000 to 5,000 s -1 In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 5,000 to 6,000 s -1 In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 6,000 to 7,000 s -1 In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 7,000 to 8,000 s -1In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 2,000 s -1 In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 3,000 s -1 In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 4,000 s -1 In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 5,000 s -1 In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 6,000 s -1 In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 7,000 s -1 In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 7,500 s -1 In a specific embodiment, the ultrafiltration (and optionally, diafiltration) in step (ii) (e.g., ultrafiltration by TFF and, optionally, diafiltration) is performed using a hollow fiber filter having a shear rate of about 8,000 s -1 This is done using hollow fiber filters with a shear rate of 1000 s. Shear rate is a term used for hollow fiber membranes and is influenced by the flow rate and the radius of the fibers. Typical ranges of shear rate are 2000-12000 s.-1 However, preferably, the shear rate maintained in step (ii) is about 2,000 to 8,000 s to avoid EV disruption and result in high EV recovery efficiency (e.g., greater than 90% or greater than 95% EV recovery). -1 , about 2,000~7,500s -1 , or about 2,000 to 7,000s -1 (but not higher). In a specific embodiment, the shear rate maintained in step (ii) is about 2,000 to 7,500 s -1 The flow rate is 80 to 200 mL / min, and a hollow fiber filter having a fiber diameter of 0.5 mm is used.

[0253] In certain embodiments, the transmembrane pressure in step (ii) is maintained at about 10 psi. In certain embodiments, the shear rate maintained in step (ii) is about 2,000 to 7,500 s -1 The flow rate is 80-200 mL / min, and a hollow fiber filter with a fiber diameter of 0.5 mm is used, resulting in a transmembrane pressure of approximately 10 psi.

[0254] In certain embodiments, the isolation step (b) comprises: a step (i) comprising microfiltration as described above, and a step (ii) comprising ultrafiltration as described above and optionally diafiltration as described above.

[0255] In some embodiments, the above step (i) is performed using one or more pumps (e.g., one or more automated pumps), such as a main pump and a back-up pump. In some embodiments, the above step (ii) is performed using one or more pumps (e.g., one or more automated pumps), such as a main pump and a back-up pump. In some embodiments, the above steps (i) and (ii) are each performed using one or more pumps (e.g., one or more automated pumps), such as a main pump and a back-up pump.

[0256] In certain non-limiting examples, the Repligen KR2i TFF system can be used to isolate EVs from cell culture, concentrate, and diafilter into a buffer suitable for therapeutic use. For example, EV isolation using TFF can include: (i) combining TFF with an 85 cm 2 Circulate the culture through a Midi 20 cm 0.65 µm Spectrum mPES hollow fiber filter (D02-E65U-07-N) with a membrane area of ​​100 μm and a fiber diameter of 0.75 mm to filter out cells and debris (e.g., for approximately 2,000–5,000 s) while maintaining a variable transmembrane pressure (TMP) driven by a retentate pressure of 5 psi. -1 (ii) using a flow rate of 100-200 mL / min resulting in a shear rate of 100-200 mL / min; and (ii) concentrating and diafiltering the EV product using the permeate of the process. For example, the process can be performed using a TFF system with continuous circulation and a 115 cm 2 A 500 kD Spectrum mPES hollow fiber filter (D02-E500-05-N) with a membrane area of ​​0.5 mm and a fiber diameter of 0.5 mm was used (for example, 2,000 to 7,500 s while maintaining and driving the filtration at a TMP of 10 psi). -1 A flow rate of 80-200 mL / min is utilized, resulting in a shear rate of 100-200 nm, to retain / concentrate particles larger than about 60-80 nm into a retentate. The incorporation of a suitable buffer (e.g., sterile saline or sterile PBS) into the circulation can be used to diafilter the EVs into a sterile solution acceptable for therapeutic use, replacing the existing solution.

[0257] In certain embodiments, the isolated EVs can be stored at −20° C. In certain embodiments, the isolated EVs can be stored at −20° C. with limited freeze / thaw cycles.

[0258] In certain embodiments, the isolated EVs can be stored at about 2° C. to about 8° C. (e.g., at about 4° C.), for example, at about 2° C. to about 8° C. (e.g., at about 4° C.) for up to about 1 week, for example, at about 2° C. to about 8° C. (e.g., at about 4° C.) for about overnight, up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, or up to about 7 days. In certain embodiments, the isolated EVs are stored at 4° C. for less than about 2 weeks, for example, at about 2° C. to about 8° C. (e.g., at about 4° C.) for less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, or less than about 8 days.

[0259] In certain embodiments, the methods presented herein for producing an isolated cell-free population of anti-inflammatory EVs produce approximately 1×10 8 ~Approx. 1×10 10 In one embodiment, the method presented herein for producing an isolated cell-free population of anti-inflammatory EVs results in a yield of about 5×10 EVs / ml culture medium. 8 ~Approx. 1×10 10 In one embodiment, the method provided herein for producing an isolated cell-free population of anti-inflammatory EVs results in a yield of about 1×10 EVs / ml culture medium. 9 ~Approx. 1×10 10 In one embodiment, the method presented herein for producing an isolated cell-free population of anti-inflammatory EVs results in a yield of about 5×10 EVs / ml culture medium. 9 ~Approx. 1×10 10 In one embodiment, the method provided herein for producing an isolated cell-free population of anti-inflammatory EVs results in a yield of about 1×10 EVs / ml culture medium. 9 EV / ml, approximately 2 × 10 9 EV / ml, approximately 3 × 10 9 EV / ml, approximately 4 × 10 9 EV / ml, approximately 5 × 10 9 EV / ml, approximately 6 × 10 9 EV / ml, approximately 7 × 10 9 EV / ml, approximately 8 × 10 9 EV / ml, approximately 9 × 10 9EV / ml, or approximately 1 x 10 10 Resulting in yield of EVs / ml culture medium.

[0260] The anti-inflammatory EVs provided herein can be derived from ex vivo expanded human suppressive immune cells, such as Tregs. Exemplary methods for expanding Tregs are provided herein.

[0261] In some embodiments, the anti-inflammatory EVs provided herein are derived from ex vivo expanded human suppressive immune cells, such as Tregs. Exemplary methods for expanding Tregs are provided herein.

[0262] 5.2.1. Cultivation, Enrichment, and Expansion of Human Suppressive Immune Cells The isolated cell-free population of anti-inflammatory EVs presented herein is derived from ex vivo expanded human suppressive immune cells. In one embodiment, the isolated cell-free population of anti-inflammatory EVs presented herein is derived from ex vivo expanded human Tregs.

[0263] In some embodiments, the human suppressive immune cells, such as Tregs, are derived from healthy human subjects.In some embodiments, the human suppressive immune cells, such as Tregs, are derived from two or more healthy human subjects.In certain embodiments, for example, the human suppressive immune cells, such as Tregs, are derived from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or more healthy human subjects. In other specific embodiments, for example, the human suppressive immune cells, e.g., Tregs, are from 2-50, 2-5, 2-10, 5-10, 5-50, 5-25, 10-15, 10-50, 10-25, 15-25, 25-30, 30-35, 35-40, 40-45, or 45-50 subjects. In some embodiments, the subjects are related. In some embodiments, the subjects are not unrelated.

[0264] In certain embodiments in which the human suppressive immune cells, such as Tregs, are derived from multiple human subjects, the method of producing an isolated cell-free population of anti-inflammatory EVs can include pooling cells from multiple human subjects together before expanding the cells ex vivo.In other certain embodiments in which the human suppressive immune cells, such as Tregs, are derived from multiple human subjects, the method of producing an isolated cell-free population of anti-inflammatory EVs can include separately expanding cells from one or more human subjects ex vivo and pooling the resulting anti-inflammatory EVs from each culture.

[0265] In some embodiments, the human suppressive immune cell, such as Treg, is derived from a donor subject that has been diagnosed with or is suspected of having a disorder associated with Treg dysfunction.In some embodiments, the donor subject has been diagnosed with or is suspected of having a disorder associated with Treg deficiency.In some embodiments, the donor subject has been diagnosed with or is suspected of having a condition driven by T cell response.

[0266] In some embodiments, the human inhibitory immune cells, such as Tregs, are derived from a donor subject diagnosed with or suspected of having a neurodegenerative disease. In some embodiments, the donor subject is diagnosed with or suspected of having Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis (MS), Parkinson's disease, Huntington's disease, or frontotemporal dementia.

[0267] In some embodiments, the human suppressive immune cells, e.g., Tregs, are derived from a donor subject diagnosed with or suspected of having a disorder that would benefit from down-regulation of the immune system.

[0268] In some embodiments, the human suppressive immune cell, such as Treg, is derived from a donor subject who has been diagnosed with or is suspected of having an autoimmune disease.The autoimmune disease can be, for example, systemic sclerosis (scleroderma), polymyositis, ulcerative colitis, inflammatory bowel disease, Crohn's disease, celiac disease, multiple sclerosis (MS), rheumatoid arthritis (RA), type I diabetes, psoriasis, dermatomyositis, lupus, such as systemic lupus erythematosus or cutaneous lupus, myasthenia gravis, autoimmune nephropathy, autoimmune hemolytic anemia, autoimmune cytopenia, autoimmune hepatitis, autoimmune uveitis, alopecia, thyroiditis, or pemphigus.

[0269] In some embodiments, the human suppressive immune cells, e.g., Tregs, are derived from a donor subject diagnosed with or suspected of having heart failure or ischemic cardiomyopathy. In some embodiments, the donor subject is diagnosed with or suspected of having graft-versus-host disease, e.g., after receiving an organ transplant (e.g., kidney or liver transplant) or after receiving a stem cell transplant (e.g., hematopoietic stem cell transplant, including bone marrow transplant).

[0270] In some embodiments, the human suppressive immune cells, such as Tregs, are derived from a donor subject diagnosed with or suspected of having neuroinflammation.Neuroinflammation can be associated with, for example, stroke, acute disseminated encephalitis, acute optic neuritis, transverse myelitis, neuromyelitis optica, epilepsy, traumatic brain injury, spinal cord injury, encephalitis, central nervous system (CNS) vasculitis, neurosarcoidosis, autoimmune or post-infectious encephalitis, or chronic meningitis.

[0271] In some embodiments, the human inhibitory immune cells, e.g., Tregs, are from a donor subject diagnosed with or suspected of having chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In some embodiments, the donor subject is diagnosed with or suspected of having acute inflammatory demyelinating polyradiculoneuropathy (AIDP). In some embodiments, the donor subject is diagnosed with or suspected of having Guillain-Barre syndrome (GBS).

[0272] In some embodiments, the human inhibitory immune cells, e.g., Tregs, are derived from a donor subject diagnosed with or suspected of having cardiac inflammation, e.g., cardiac inflammation associated with myocardial infarction, ischemic cardiomyopathy, heart failure.

[0273] In some embodiments, the human suppressive immune cells, eg, Tregs, are derived from a donor subject who has had a stroke.

[0274] In some embodiments, the human suppressive immune cells, e.g., Tregs, are derived from a donor subject diagnosed with or suspected of having cancer, e.g., a hematological cancer.

[0275] In some embodiments, the human suppressive immune cells, eg, Tregs, are derived from a donor subject diagnosed with or suspected of having asthma.

[0276] In some embodiments, the human suppressive immune cells, eg, Tregs, are derived from a donor subject diagnosed with or suspected of having eczema.

[0277] In some embodiments, the human suppressive immune cells, such as Tregs, are derived from a donor subject diagnosed with or suspected of having a disorder associated with overactivation of the immune system.

[0278] In some embodiments, the human suppressive immune cells, such as Treg, are derived from a donor subject who has been diagnosed with or is suspected of having a regulatory T cell disease.Regulatory T cell disease can be caused by, for example, FOXP3, CD25, cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), LPS-responsive beige-like anchor protein (LRBA), or BTB domain and CNC homolog 2 (BACH2) gene loss-of-function mutations, or signal transducer and activator of transcription 3 (STAT3) gain-of-function mutations.

[0279] In some embodiments, the human suppressive immune cells, e.g., Tregs, are derived from one or more adult subjects, e.g., one or more healthy adult subjects. In certain embodiments, the one or more adult subjects are at least 18, 20, 25, 30, 35, 40, 45, 50, or 55 years old. In certain embodiments, for example, the human inhibitory immune cells, e.g., Tregs, are from one or more adult subjects, where the one or more healthy adult subjects are about 18-55 years old, about 18-50 years old, about 18-45 years old, about 18-40 years old, about 18-35 years old, about 18-30 years old, about 18-25 years old, about 20-55 years old, about 25-55 years old, about 30-55 years old, about 35-55 years old, about 40-55 years old, about 25-50 years old, about 30-50 years old, about 35-45 years old, about 25-45 years old, about 40-50 years old.

[0280] In some embodiments, the human suppressive immune cells, e.g., Tregs, are from an elderly subject, e.g., a healthy elderly subject, e.g., a subject that is at least 65 years old, at least 70 years old, at least 75 years old, at least 80 years old, at least 85 years old, or at least 90 years old.

[0281] In some embodiments, the anti-inflammatory EVs provided herein are derived from a population of genetically engineered human suppressive immune cells, e.g., Tregs.

[0282] 5.2.1.1 Improved Methods for Ex vivo Expansion of Tregs In some embodiments, the isolated cell-free population of anti-inflammatory EVs provided herein is derived from a population of human Tregs that are expanded ex vivo.The method of expanding Tregs is well known.Described in this section is an improved method of expanding Tregs ex vivo.

[0283] In some embodiments, a biological donor sample containing or suspected of containing Tregs, such as a peripheral blood sample or thymus tissue, can be obtained from which Tregs can be obtained and enriched prior to ex vivo expansion. Exemplary production methods for obtaining, enriching and expanding a population of Tregs ex vivo are described in International Patent Application No. PCT / US2020 / 63378, which is incorporated herein by reference in its entirety. In some embodiments, the Tregs from which the EVs are obtained are expanded according to the disclosure set forth in Section 5.2.2 below. In some embodiments, the Tregs from which the EVs are obtained are expanded according to the disclosure set forth in Section 6.12 below.

[0284] In certain embodiments, the enrichment step is automated. In certain embodiments, the enrichment step is performed in a closed system. In certain embodiments, the enrichment step is automated and performed in a closed system. In a specific embodiment, the enrichment step is performed in a CliniMACS Prodigy® system. In a specific embodiment, the enrichment step is performed in a CliniMACS® Plus system. In certain embodiments, the expansion step is automated. In certain embodiments, the expansion step is performed in a closed system. In certain embodiments, the expansion step is automated and performed in a closed system. In a specific embodiment, the expansion step is performed in a bioreactor (e.g., a Terumo BCT Quantum® Cell Expansion System). In some embodiments, the enrichment step and the expansion step are performed in different systems (e.g., the enrichment step is performed in a CliniMACS Prodigy® system and the expansion step is performed in a Terumo BCT Quantum® Cell Expansion System, or the enrichment step is performed in a CliniMACS® Plus system and the expansion step is performed in a Terumo BCT Quantum® Cell Expansion System). In a specific embodiment, the enriched cell population produced by the enrichment step is transferred to a system in which the expansion step is performed in a closed system. In other embodiments, the enrichment step and the expansion step are performed in the same system. In a specific embodiment, the same system is a closed system.

[0285] In some embodiments, the population of Tregs is obtained from a serum sample suspected of containing Tregs. In some embodiments, the population of Tregs is obtained from a cell sample suspected of containing Tregs obtained from a donor via leukapheresis or from a donor via a blood sample. In some embodiments, the population of Tregs is obtained from a biological sample suspected of containing Tregs.

[0286] In some embodiments, the population of Tregs is enriched from a biological sample from a human donor subject. In some embodiments, the donor of the biological sample is the subject who will be treated with the population of anti-inflammatory EVs derived from the population of Tregs. In other embodiments, the donor of the biological sample is different from the patient subject who will be treated with the population of anti-inflammatory EVs derived from the population of Tregs. The biological sample can be any sample suspected of containing Tregs, likely to contain Tregs, or known to contain Tregs. Such biological samples may be taken directly from a subject, or may be samples resulting from one or more processing steps, such as separation, e.g., selection or enrichment, centrifugation, washing, and / or incubation. Biological samples include, but are not limited to, bodily fluids, e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, tissue, and organ samples, including processed samples derived therefrom.

[0287] In some embodiments, the sample is a blood or blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, and thymus.

[0288] In some embodiments, the biological sample is a blood-derived sample, such as a sample derived from whole blood, serum, or plasma. In some embodiments, the biological sample is or comprises peripheral blood mononuclear cells. In some embodiments, the biological sample is a peripheral blood or serum sample. In some embodiments, the biological sample is a lymph node sample.

[0289] Methods for obtaining a population of cells suspected of containing Tregs, likely to contain Tregs, or known to contain Tregs from such biological donor samples are known in the art.For example, lymphocytes can be obtained from peripheral blood samples by leukapheresis.In some embodiments, Tregs are enriched from a population of lymphocytes.In some embodiments, repeated peripheral blood samples are obtained from the donor to produce Tregs.In some embodiments, two or more peripheral blood samples are obtained from the donor.In some embodiments, the donor sample undergoes volume reduction during the enrichment process.

[0290] In some embodiments, biological samples (e.g., leukapheresis samples or blood samples) from multiple donors are pooled prior to the enrichment process to generate an allogeneic Treg population. In some embodiments, biological samples (e.g., leukapheresis samples or blood samples) from multiple unrelated donors are pooled prior to the enrichment process to generate an allogeneic Treg population. In some embodiments, biological samples (e.g., leukapheresis or blood samples from 2, 3, 4, 5, 10, 20, 50, or more donors) are pooled.

[0291] Tregs can be enriched from biological samples by any method known in the art. In some embodiments, Tregs are enriched from samples using magnetic bead separation (e.g., CliniMACS tubing set LS (162-01), CliniMACS® Plus instrument or CliniMACS Prodigy® instrument), fluorescent cell sorting, and / or disposable closed cartridge-based cell sorter.

[0292] Enrichment includes enriching for cells expressing one or more markers and can refer to increasing the number or percentage of such cells in a population of cells, but does not necessarily result in the complete absence of cells that do not express the marker. Depletion of cells expressing one or more markers refers to decreasing the number or percentage of such cells in a population of cells, but does not necessarily result in the complete elimination of all cells expressing such marker(s).

[0293] In some embodiments, enrichment of cells expressing one or more markers (e.g., Treg cell surface markers) comprises an affinity or immunoaffinity based separation step. Such a separation step can be based on positive selection, where cells expressing one or more markers are retained, and / or on negative selection (removal), where cells not expressing one or more markers are retained.

[0294] Separation can be based on expression (e.g., positive or negative expression) or expression levels (e.g., high or low expression) of one or more markers (e.g., Treg cell surface markers). In this context, "high expression" and "low expression" are usually relative to the entire population of cells. In some embodiments, cell separation can be based on CD8 expression. In some embodiments, cell separation can be based on CD19 expression. In some embodiments, cell separation can be based on high CD25 expression.

[0295] In some embodiments, the separation of cells can be based on high CD9 expression. In some embodiments, the separation of cells can be based on high CD63 expression. In some embodiments, the separation of cells can be based on high CD81 expression. In some embodiments, the separation of cells can be based on high CD44 expression. In some embodiments, the separation of cells can be based on high CD29 expression. In some embodiments, the separation of cells can be based on high CD45 expression.

[0296] Thus, in some embodiments, enrichment for Tregs can involve incubation with an antibody or binding partner that specifically binds to a marker (e.g., a Treg cell surface marker), typically followed by a washing step and separation of cells bound to the antibody or binding partner from cells that are not bound to the antibody or binding partner.

[0297] In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as spheres or beads, such as nanoparticles, microbeads, nanobeads (including agarose), magnetic beads, or paramagnetic beads. In some embodiments, the spheres or beads can be packed into a column to achieve immunoaffinity chromatography. In some embodiments, the antibody or binding partner is detectably labeled. In some embodiments, the antibody or binding partner is attached to small magnetically responsive particles or microparticles, such as nanoparticles or paramagnetic beads. Such beads are known and commercially available (e.g., Dynabeads® (Life Technologies, Carlsbad, CA), MACS® beads (Miltenyi Biotec, San Diego, CA), or Streptamer® bead reagent (IBA, Germany)). Such particles or microparticles can be incubated with the population of cells to be enriched and then placed in a magnetic field. This results in cells attached to the particles or microparticles via the antibody or binding partner being attracted to the magnet and separated from unbound cells. This method allows for the retention of cells attached to the magnet (positive selection) or the removal of cells attracted to the magnet (negative selection).

[0298] In some embodiments, the methods of producing a population of Tregs provided herein include both positive and negative selection during the enrichment step.

[0299] In some embodiments, the biological sample is obtained within about 25-35 minutes, about 35-45 minutes, about 45-60 minutes, about 60-75 minutes, about 75-90 minutes, about 90-120 minutes, about 120-150 minutes, about 150-180 minutes, about 2-3 hours, about 3-4 hours, about 4-5 hours, or about 5-6 hours from the start of the concentration step. In some embodiments, the sample is obtained within about 30 minutes from the start of the concentration step. In some embodiments, the biological sample is not stored overnight (e.g., stored at 4°C).

[0300] In some embodiments, enriching for Tregs from a human sample comprises removing a sample of CD8+ cells. In some embodiments, enriching for Tregs from a human sample comprises removing a sample of CD19+ cells. In some embodiments, enriching for Tregs from a biological sample comprises removing a sample of CD8+ cells and CD19+ cells. In some embodiments, enriching for Tregs from a biological sample comprises enriching a cell population by isolating the cell population with CD25 high In some embodiments, enriching for Tregs from a biological sample comprises enriching the cell population for CD25+ cells. In some embodiments, enriching for Tregs from a biological sample comprises removing CD8+ cells and CD19+ cells from the sample and enriching the cell population for CD25+ cells. high In some embodiments, enriching for Tregs from a biological sample comprises depleting CD8+ / CD19+ cells and enriching for CD25+ cells.

[0301] In some embodiments, the population of cells enriched for Tregs is enriched for CD4+CD25+ Tregs prior to enrichment as determined by flow cytometry. high Increased proportion of CD4+CD25 relative to Treg proportion high In a specific embodiment, CD4+CD25 highThe proportion of Tregs is increased by about 2-fold to about 4-fold, about 4-fold to about 6-fold, about 6-fold to about 8-fold, about 8-fold to about 10-fold, about 10-fold to about 15-fold, about 15-fold to about 20-fold, about 20-fold to about 25-fold, about 25-fold to about 30-fold, about 30-fold to about 35-fold, about 35-fold to about 40-fold, about 40-fold to about 45-fold, and about 45-fold to about 50-fold.

[0302] In some embodiments, the population of cells enriched for Tregs is enriched for CD4+CD25+ Tregs prior to enrichment as determined by flow cytometry. high CD127 low Increased proportion of CD4+CD25 relative to Treg proportion high CD127 low In a specific embodiment, CD4+CD25 high CD127 low The proportion of Tregs is increased by about 2-fold to about 4-fold, about 4-fold to about 6-fold, about 6-fold to about 8-fold, about 8-fold to about 10-fold, about 10-fold to about 15-fold, about 15-fold to about 20-fold, about 20-fold to about 25-fold, about 25-fold to about 30-fold, about 30-fold to about 35-fold, about 35-fold to about 40-fold, about 40-fold to about 45-fold, and about 45-fold to about 50-fold.

[0303] In some embodiments, the population of cells enriched for Tregs comprises CD25+ Tregs, wherein the expression of CD25 in the Tregs is increased compared to the expression of CD25 in the Tregs before enrichment as determined by flow cytometry. In a specific embodiment, the expression of CD25 is increased by at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold.

[0304] In some embodiments, the population of cells enriched for Tregs comprises CD127+ Tregs, wherein the expression of CD127 on the Tregs is increased compared to the expression of CD127 on the Tregs before enrichment as determined by flow cytometry. In specific embodiments, the expression of CD127 is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, or at least about 3-fold.

[0305] In some embodiments, the granularity of Tregs in the enriched population of Tregs is increased compared to the granularity of Tregs prior to enrichment as determined by flow cytometry. In specific embodiments, the granularity of Tregs is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, or at least about 3-fold.

[0306] In some embodiments, the size of Tregs in the enriched population of Tregs is increased compared to the size of Tregs before enrichment as determined by flow cytometry. In specific embodiments, the size of Tregs is increased by at least about 1.2-fold, at least about 1.5-fold, or at least about 2-fold.

[0307] In another embodiment, the population of Tregs used to isolate the anti-inflammatory EVs provided herein are enriched from a biological sample and further expanded.

[0308] In some embodiments, the expansion step is performed within about 4-5 days from the end of the enrichment step. In some embodiments, the expansion step is performed within about 3-4 days from the end of the enrichment step. In some embodiments, the expansion step is performed within about 2-3 days from the end of the enrichment step. In some embodiments, the expansion step is performed within about 1-2 days from the end of the enrichment step. In some embodiments, the expansion step is performed within about 24 hours from the end of the enrichment step. In some embodiments, the expansion step is performed within about 12 hours from the end of the enrichment step. In some embodiments, the expansion step is performed within about 6 hours from the end of the enrichment step. In some embodiments, the expansion step is performed within about 3 hours from the end of the enrichment step. In some embodiments, the expansion step is performed within about 2 hours from the end of the enrichment step. In some embodiments, the expansion step is performed within about 1 hour from the end of the enrichment step. In some embodiments, the expansion step is performed within about 30 minutes from the end of the enrichment step.

[0309] Expanding this population of Tregs can include culturing cells enriched from the biological sample in a medium, for example in serum-free medium (e.g., TexMACS medium), in serum-depleted medium, or in serum-containing medium.

[0310] In certain embodiments, the expansion step comprises culturing Tregs in a culture medium comprising human serum (e.g., TexMACS GMP medium supplemented with human serum). In a specific embodiment, the culture medium comprises 5% or less human serum. In a specific embodiment, the culture medium comprises 4% or less human serum. In a specific embodiment, the culture medium comprises 3% or less human serum. In a specific embodiment, the culture medium comprises 2% or less human serum. In a specific embodiment, the culture medium comprises 1% or less human serum. In a specific embodiment, the culture medium comprises 0.5% or less human serum. In a specific embodiment, the culture medium comprises less than 5% human serum. In a specific embodiment, the culture medium comprises less than 4% human serum. In a specific embodiment, the culture medium comprises less than 3% human serum. In a specific embodiment, the culture medium comprises less than 2% human serum. In a specific embodiment, the culture medium comprises less than 1% human serum. In a specific embodiment, the culture medium comprises less than 0.5% human serum. In a specific embodiment, the culture medium comprises 0.5-1% human serum. In a specific embodiment, the culture medium comprises 1-2% human serum. In a specific embodiment, the culture medium comprises 2-3% human serum. In a specific embodiment, the culture medium comprises 3-4% human serum. In a specific embodiment, the culture medium comprises 4-5% human serum. In a specific embodiment, the culture medium comprises about 0.5% human serum. In another specific embodiment, the culture medium comprises about 1% human serum. In another specific embodiment, the culture medium comprises about 2% human serum. In another specific embodiment, the culture medium comprises about 3% human serum. In another specific embodiment, the culture medium comprises about 4% human serum. In another specific embodiment, the culture medium comprises about 5% human serum.

[0311] In certain embodiments, the expansion step comprises culturing Tregs in a culture medium comprising human AB serum (e.g., TexMACS GMP medium supplemented with human AB serum). In a specific embodiment, the culture medium comprises 5% or less human AB serum. In a specific embodiment, the culture medium comprises 4% or less human AB serum. In a specific embodiment, the culture medium comprises 3% or less human AB serum. In a specific embodiment, the culture medium comprises 2% or less human AB serum. In a specific embodiment, the culture medium comprises 1% or less human AB serum. In a specific embodiment, the culture medium comprises 0.5% or less human AB serum. In a specific embodiment, the culture medium comprises less than 5% human AB serum. In a specific embodiment, the culture medium comprises less than 4% human AB serum. In a specific embodiment, the culture medium comprises less than 3% human AB serum. In a specific embodiment, the culture medium comprises less than 2% human AB serum. In a specific embodiment, the culture medium comprises less than 1% human AB serum. In a specific embodiment, the culture medium comprises less than 0.5% human AB serum. In a specific embodiment, the culture medium comprises 0-0.5% human AB serum. In a specific embodiment, the culture medium comprises 0.5-1% human AB serum. In a specific embodiment, the culture medium comprises 1-2% human AB serum. In a specific embodiment, the culture medium comprises 2-3% human AB serum. In a specific embodiment, the culture medium comprises 3-4% human AB serum. In a specific embodiment, the culture medium comprises 4-5% human AB serum. In a specific embodiment, the culture medium comprises about 0.5% human AB serum. In another specific embodiment, the culture medium comprises about 1% human AB serum. In another specific embodiment, the culture medium comprises about 2% human AB serum. In another specific embodiment, the culture medium comprises about 3% human AB serum. In another specific embodiment, the culture medium comprises about 4% human AB serum. In another specific embodiment, the culture medium comprises about 5% human AB serum.

[0312] In some embodiments, the enriched cells from the biological sample are incubated at about 37° C. and about 5% CO 2In some embodiments, the cells enriched from the biological sample are cultured under Good Manufacturing Practice (GMP) conditions. In some embodiments, the cells enriched from the biological sample are cultured in a closed system.

[0313] In some embodiments, the cells enriched from the biological sample are cultured in an automated system. In some embodiments, the cells enriched from the biological sample are cultured in a closed, automated system. In some embodiments, the cells enriched from the biological sample are cultured in a Terumo BCT Quantum® Cell Expansion System.

[0314] In some embodiments, expansion of the population of Tregs is initiated within 25-35 minutes, 20-40 minutes, 15-45 minutes, or 10-50 minutes of enrichment from the biological sample, hi some embodiments, expansion of the population of Tregs is initiated within about 30 minutes of enrichment from the biological sample.

[0315] As mentioned above, once proliferation has commenced, isolation of EVs can be performed at any point during or at the end of the proliferation process.

[0316] Tregs can be expanded ex vivo by culturing the cells in the presence of one or more proliferation agents. In some embodiments, the proliferation agent is IL-2. The appropriate concentration of IL-2 in the culture medium can be determined by one of skill in the art. In some embodiments, the concentration of IL-2 in the cell culture medium is about 5-10 IU / mL, about 10-20 IU / mL, about 20-30 IU / mL, about 30-40 IU / mL, about 40-50 IU / mL, about 50-100 IU / mL, about 100-200 IU / mL, about 200-300 IU / mL, about 300-400 IU / mL, about 400-500 IU / mL, about 500-600 IU / mL, about 600-700 IU / mL, about 700-800 IU / mL, about 800-900 IU / mL, about 900-10 In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 100 IU / mL, about 1000-1500 IU / mL, about 1500-2000 IU / mL, about 2000-2500 IU / mL, about 2500-3000 IU / mL, about 3000-3500 IU / mL, about 3500-4000 IU / mL, about 4000-4500 IU / mL, about 4500-5000 IU / mL, about 5000-6000 IU / mL, about 6000-7000 IU / mL, about 7000-8000 IU / mL, about 8000-9000 IU / mL, or about 9000-10,000 IU / mL. In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 150 IU / mL. In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 200 IU / mL. In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 250 IU / mL. In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 300 IU / mL. In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 400 IU / mL. In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 500 IU / mL. In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 600 IU / mL. In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 700 IU / mL. In a specific embodiment, the concentration of IL-2 in the cell culture medium is about 800 IU / mL.In some embodiments, the expansion step includes adjusting the IL-2 concentration according to the cell number. By cell number, we mean the number of all cells in the culture, including enriched Treg cells, which represent the majority of the cells in the culture, and in specific embodiments, more than 70%, more than 80%, more than 90%, more than 95%, more than 99%, or 100% of the cells in the culture. In a specific embodiment, the expansion step includes adjusting the IL-2 concentration according to the cell number, which represents ... 6 The method includes culturing Tregs in culture medium containing about 200 IU / mL IL-2 until the Tregs reach a concentration of about 1000, and then culturing the Tregs in culture medium containing about 250 IU / mL IL-2.

[0317] In some embodiments, IL-2 is added to the culture for the first time within about 4 to 5 days after the initiation of the culture. In some embodiments, IL-2 is added to the culture for the first time within about 3 to 4 days after the initiation of the culture. In some embodiments, IL-2 is added to the culture for the first time within about 2 to 3 days after the initiation of the culture. In some embodiments, IL-2 is added to the culture for the first time within about 1 to 2 days after the initiation of the culture. In some embodiments, IL-2 is added to the culture for the first time within about 24 hours after the initiation of the culture. In some embodiments, IL-2 is added to the culture for the first time within about 12 hours after the initiation of the culture. In some embodiments, IL-2 is added to the culture for the first time within about 6 hours after the initiation of the culture. In some embodiments, IL-2 is added to the culture for the first time within about 3 hours after the initiation of the culture. In some embodiments, IL-2 is added to the culture for the first time within about 2 hours after the initiation of the culture. In some embodiments, IL-2 is added to the culture for the first time within about 1 hour after the initiation of the culture. In some embodiments, IL-2 is added to the culture for the first time within about 30 minutes after the initiation of the culture. In some embodiments, IL-2 is added to the culture for the first time within about 4-5 days after the end of the enrichment step. In some embodiments, IL-2 is added to the culture for the first time within about 3-4 days after the end of the enrichment step. In some embodiments, IL-2 is added to the culture for the first time within about 2-3 days after the end of the enrichment step. In some embodiments, IL-2 is added to the culture for the first time within about 1-2 days after the end of the enrichment step. In some embodiments, IL-2 is added to the culture for the first time within about 24 hours after the end of the enrichment step. In some embodiments, IL-2 is added to the culture for the first time within about 12 hours after the end of the enrichment step. In some embodiments, IL-2 is added to the culture for the first time within about 6 hours after the end of the enrichment step. In some embodiments, IL-2 is added to the culture for the first time within about 3 hours after the end of the enrichment step. In some embodiments, IL-2 is added to the culture for the first time within about 2 hours after the end of the enrichment step.In some embodiments, IL-2 is first added to the culture within about 1 hour of the end of the enrichment step. In some embodiments, IL-2 is first added to the culture within about 30 minutes of the end of the enrichment step. In some embodiments, IL-2 is replenished about every 1, 2, 3, 4, or 5 days. In some embodiments, IL-2 is replenished about every 1-2 days. In some embodiments, IL-2 is replenished about every 2-3 days. In some embodiments, IL-2 is replenished about every 3-4 days. In some embodiments, IL-2 is replenished about every 4-5 days.

[0318] In some embodiments, the proliferative agent activates CD3, e.g., the proliferative agent is an anti-CD3 antibody. In some embodiments, the proliferative agent activates CD28, e.g., the proliferative agent is an anti-CD28 antibody.

[0319] In some embodiments, the proliferative agent is a soluble anti-CD3 antibody. In certain embodiments, the anti-CD3 antibody is OKT3. In some embodiments, the concentration of the soluble anti-CD3 antibody in the culture medium is about 0.1 to 0.2 ng / mL, about 0.2 to 0.3 ng / mL, about 0.3 to 0.4 ng / mL, about 0.4 to 0.5 ng / mL, about 0.5 to 1 ng / mL, about 1 to 5 ng / mL, about 5 to 10 ng / mL, about 10 to 15 ng / mL, about 15 to 20 ng / mL, about 20 to 25 ng / mL, about 25 to 30 ng / mL, about 30 to 35 ng / mL, about 35 to 40 ng / mL, about 40 to 45 ng / mL, about 45 to 50 ng / mL, about 50 to 60 ng / mL, about 60 to 70 ng / mL, about 70 to 80 ng / mL, about 80 to 90 ng / mL, or about 90 to 100 ng / mL.

[0320] In some embodiments, the proliferation agent is a soluble anti-CD28 antibody. Non-limiting examples of anti-CD28 antibodies include NA / LE (e.g., BD Pharmingen), IM1376 (e.g., Beckman Coulter), or 15×10 8(e.g., Miltenyi Biotec). In some embodiments, the concentration of the soluble anti-CD28 antibody in the culture medium is about 1 to 2 ng / mL, about 2 to 3 ng / mL, about 3 to 4 ng / mL, about 4 to 5 ng / mL, about 5 to 10 ng / mL, about 10 to 15 ng / mL, about 15 to 20 ng / mL, about 20 to 25 ng / mL, about 25 to 30 ng / mL, about 30 to 35 ng / mL, about 35 to 40 ng / mL, about 40 to 45 ng / mL, about 45 to 50 ng / mL, about 50 to 60 ng / mL, about 65 to 70 ng / mL, about 70 to 80 ng / mL, about 75 to 85 ng / mL, about 85 to 90 ng / mL, about 90 to 100 ng / mL, about 95 to 100 ng / mL, about 100 to 150 ng / mL, about 15 to 20 ng / mL, about 20 to 25 ng / mL, about 25 to 30 ng / mL, about 30 to 35 ng / mL, about 35 to 40 ng / mL, about 40 to 45 ng / mL, about 45 to 50 ng / mL, about 50 to 150 ng / mL, about 100 to 250 ng / mL, about 15 to 20 ng / mL, about 20 to 25 ng / mL, about 25 to 30 ng / mL, about 30 to 35 ng / mL, about 35 to 40 ng / mL, about 40 to 45 ng / mL, about 45 to 50 ng / 60ng / mL, about 60 to 70ng / mL, about 70 to 80ng / mL, about 80 to 90ng / mL, about 90 to 100ng / mL, about 100 to 200ng / mL, about 200 to 300ng / mL, about 300 to 400ng / mL, about 400 to 500ng / mL, 500 to 600ng / mL, 600 to 700ng / mL, about 700 to 800ng / mL, about 800 to 900ng / mL, or about 900 to 1000ng / mL.

[0321] In some embodiments, both anti-CD3 and anti-CD28 antibodies are present in the cell culture medium. In some embodiments, both anti-CD3 and anti-CD28 antibodies are attached to a solid surface. In some embodiments, both anti-CD3 and anti-CD28 antibodies are attached to beads. In some embodiments, CD28 antibody, anti-biotin antibody, and beads (e.g., 3.5 μm particles) loaded with CD3-biotin are present in the cell culture medium. Such beads are commercially available (e.g., MACS GMP ExpAct Treg kit, DYNABEADS® M-450 CD3 / CD28 T Cell Expander). In specific embodiments, the ratio of anti-CD3 antibody to anti-CD28 antibody on the beads is about 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100. In some embodiments, the population of Tregs is cultured in the presence of both IL-2 and CD28 antibody, anti-biotin antibody, and CD3-biotin loaded beads. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 4-5 days after initiating the culture. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 3-4 days after initiating the culture. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 2-3 days after initiating the culture. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 1-2 days after initiating the culture. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 24 hours after initiating the culture.In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 12 hours after the initiation of the culture. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 6 hours after the initiation of the culture. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 3 hours after the initiation of the culture. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 2 hours after the initiation of the culture. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 1 hour after the initiation of the culture. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 30 minutes after the initiation of the culture. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 4 to 5 days after the end of the enrichment step. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 3-4 days after the end of the enrichment step. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 2-3 days after the end of the enrichment step. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 1-2 days after the end of the enrichment step. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 24 hours after the end of the enrichment step. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 12 hours after the end of the enrichment step. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 6 hours after the end of the enrichment step. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture within about 3 hours after the end of the enrichment step.In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are added to the culture for the first time within about 2 hours after the end of the enrichment step. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are added to the culture for the first time within about 1 hour after the end of the enrichment step. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are added to the culture for the first time within about 30 minutes after the end of the enrichment step. In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are added back to the culture medium about 14 days after the beads coated with anti-CD3 and anti-CD28 antibodies are added to the culture medium for the first time (e.g., if the cell number has not reached the target cell number by that time). In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are added to the culture medium for the first time (e.g., if the cell number has not reached the target cell number by that time). In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are added back to the culture medium about 12 days after the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture medium (e.g., if the cell number has not reached the target cell number by that time). In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are added back to the culture medium about 11 days after the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture medium (e.g., if the cell number has not reached the target cell number by that time). In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are added back to the culture medium about 10 days after the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture medium (e.g., if the cell number has not reached the target cell number by that time).In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are added back to the culture medium about 9 days after the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture medium (e.g., if the cell number has not reached the target cell number by that time). In some embodiments, the beads coated with anti-CD3 and anti-CD28 antibodies are added back to the culture medium about 8 days after the beads coated with anti-CD3 and anti-CD28 antibodies are first added to the culture medium (e.g., if the cell number has not reached the target cell number by that time). Cell number refers to the number of all cells in the culture, including enriched Treg cells, which represent the majority of the cells in the culture, and in specific embodiments, more than 70%, more than 80%, more than 90%, more than 95%, more than 99%, or 100% of the cells in the culture. In some embodiments, the target cell number is 1×10. 8 ~1×10 10 In one embodiment, the target cell number is 1×10 9 ~5×10 9 In one embodiment, the target cell number is 2×10 9 ~5×10 9 In one embodiment, the target cell number is 2×10 9 ~2.5×10 9 In a specific embodiment, the target cell number is 1×10 9 In another specific embodiment, the target cell number is 1.5×10 9 In another specific embodiment, the target cell number is 2×10 9 In another specific embodiment, the target cell number is 2.5×10 9 In another specific embodiment, the target cell number is 3×10 9 In another specific embodiment, the target cell number is 3.5×10 9 In another specific embodiment, the target cell number is 4×10 9 In another specific embodiment, the target cell number is 4.5×10 9In another specific embodiment, the target cell number is 5×10 9 In specific embodiments, the ratio of beads to cells in the culture is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

[0322] The proliferation agent(s) can be added to the culture medium every 1, 2, 3, 4, or 5 days. In a specific embodiment, the proliferation agent is added to the culture medium every 1-2 days. In a specific embodiment, the proliferation agent is added to the culture medium every 2-3 days. In a specific embodiment, the proliferation agent is added to the culture medium every 3-4 days. In a specific embodiment, the proliferation agent is added to the culture medium every 4-5 days. In other specific embodiments, the proliferation agent is added to the culture medium on days 6, 8, and 11, where day 0 is the day the biological sample is obtained from the subject. In some specific embodiments, the proliferation agent is not added to the culture medium on day 13, where day 0 is the day the biological sample is obtained from the subject.

[0323] In some embodiments, the one or more growth agents are first added to the culture within about 30 minutes to 1 hour, within 1 to 2 hours, within 2 to 4 hours, within 4 to 6 hours, within 6 to 8 hours, within 8 to 10 hours, within 10 to 12 hours, within 12 to 14 hours, within 14 to 16 hours, within 16 to 18 hours, within 18 to 24 hours, within 24 to 36 hours, within 36 to 48 hours, within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 6 hours, within...

Claims

1. An isolated cell-free population of anti-inflammatory extracellular vesicles (EVs), the anti-inflammatory EVs being derived from ex vivo expanded human suppressive immune cells; where: i) the population exhibits a size diameter distribution of about 50 nm to about 150 nm; ii) the population comprises EV surface CD2, CD25, and HLA-DRDPDQ; iii) the population comprises hsa-miR-1290, hsa-miR-146a-5p, and hsa-miR-155-5p micro-RNAs (miRNAs); and iv) the population exhibits the ability to suppress myeloid cells as measured by the ability to reduce pro-inflammatory cytokine production by myeloid cells and increase expression of one or more anti-inflammatory markers in myeloid cells or by the ability to suppress the proliferation of responder T cells; and wherein the human suppressive immune cells are regulatory T cells (Tregs); The isolated cell-free population.

2. (i) the population exhibits an average size diameter of about 80 nm to about 110 nm; (ii) the population exhibits a median size diameter of about 70 nm to about 110 nm; and / or (iii) The population of anti-inflammatory EVs of claim 1, wherein said population exhibits a mode size diameter of about 65 nm to about 95 nm.

3. The population, (i) EV surface CD44, CD29, CD4, and CD45; or (ii) further comprises EV surface CD9, CD63, and CD81; and / or 2. The population of anti-inflammatory EVs of claim 1, wherein the population is substantially devoid of EV surface CD3, CD19, CD8, CD56, CD105, CD1c, CD49e, ROR1, CD209, SSEA-4, CD40, CD62P, CD11c, CD40, MSCP, CD146, CD86, CD326, CD133, CD142, CD31, and CD14.

4. (i) the ratio of hsa-miR-146a-5p to hsa-miR-155-5p in the population is about 2 to about 3; (ii) the abundance of hsa-miR-1290 is at least twice that of hsa-mir-155-5p; (iii) the Tregs are from a healthy human subject or from a human subject diagnosed with or suspected of having amyotrophic lateral sclerosis (ALS); (iv) the anti-inflammatory EVs exhibit the ability to increase the expression of IL-10, Arg1, and / or CD206 in myeloid cells; (v) the anti-inflammatory EVs exhibit the ability to reduce expression of IL-6, IL-8, IL1β, or interferon-γ in myeloid cells; (vi) proliferation of responder T cells is determined by flow cytometry or thymidine incorporation; and / or (vii) The population of anti-inflammatory EVs of claim 1, wherein the population is a saline-containing population of anti-inflammatory EVs.

5. (i) the ratio of hsa-miR-146a-5p to hsa-miR-155-5p in the population is about 2 to about 3; (ii) the abundance of hsa-miR-1290 is at least twice that of hsa-mir-155-5p; (iii) the Tregs are from a healthy human subject or from a human subject diagnosed with or suspected of having amyotrophic lateral sclerosis (ALS); (iv) the anti-inflammatory EVs exhibit the ability to increase the expression of IL-10, Arg1, and / or CD206 in myeloid cells; (v) the anti-inflammatory EVs exhibit the ability to reduce expression of IL-6, IL-8, IL1β, or interferon-γ in myeloid cells; (vi) proliferation of responder T cells is determined by flow cytometry or thymidine incorporation; and / or (vii) The population of anti-inflammatory EVs of claim 2, wherein the population is a saline-containing population of anti-inflammatory EVs.

6. An isolated cell-free population of anti-inflammatory extracellular vesicles (EVs), wherein the anti-inflammatory EVs are derived from ex vivo expanded human suppressive immune cells.

7. The population of anti-inflammatory EVs of claim 6, wherein the human inhibitory immune cells are regulatory T cells (Tregs).

8. The Treg: (i) Healthy human subjects; (ii) a human subject diagnosed with or suspected of having a neurodegenerative disorder; (iii) a human subject diagnosed with or suspected of having had a stroke; (iv) elderly human subjects; (v) multiple human subjects; or (vi) The population of anti-inflammatory EVs of claim 7, which is derived from a plurality of unrelated human subjects.

9. 9. The population of anti-inflammatory EVs of claim 8, wherein the Tregs are derived from a human subject diagnosed with or suspected of having a neurodegenerative disorder, the neurodegenerative disorder being Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), or Parkinson's disease.

10. (i) the population is a saline-containing population of anti-inflammatory EVs; (ii) the population is a saline-containing population of anti-inflammatory EVs; (iii) the population is a population of anti-inflammatory EVs containing phosphate buffered saline; (iv) The population of anti-inflammatory EVs of claim 6, wherein the population of anti-inflammatory EVs comprises exosomes and microvesicles.

11. The population of anti-inflammatory EVs of claim 10, wherein the population of anti-inflammatory EVs has a diameter of about 30 nm to about 1000 nm.

12. A pharmaceutical composition comprising an isolated cell-free population of anti-inflammatory EVs according to any one of claims 1 to 11.

13. 13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is a frozen and stored pharmaceutical composition.

14. The pharmaceutical composition according to claim 12, which has been previously frozen and stored.

15. 1. A method for producing an isolated cell-free population of anti-inflammatory extracellular vesicles (EVs), comprising: (a) expanding a population of human suppressive immune cells ex vivo in a culture medium to produce a culture comprising the cells, the culture medium, and the anti-inflammatory EVs; and (b) isolating the anti-inflammatory EVs from the culture.

16. The method of claim 15, wherein the human suppressive immune cell population is a population of regulatory T cells (Tregs).

17. (i) the Tregs are derived from healthy human subjects; (ii) the Tregs are derived from a human subject diagnosed with or suspected of having a neurodegenerative disorder; (iii) the Tregs are derived from a human subject diagnosed with or suspected of having had a stroke; (iv) the Tregs are derived from an elderly human subject; or (v) The method of claim 15, wherein the Tregs are derived from multiple human subjects.

18. The method of claim 15, further comprising: (c) cryopreserving the isolated cell-free population of anti-inflammatory EVs, thereby producing an isolated cell-free population of cryopreserved anti-inflammatory EVs.

19. 19. A pharmaceutical composition comprising an isolated cell-free population of anti-inflammatory EVs, wherein said population is produced by the method of any one of claims 15 to 18.

20. An isolated cell-free population of anti-inflammatory EVs derived from an ex vivo expanded Treg cell population that exhibits the ability to suppress inflammatory cells as measured by pro-inflammatory cytokine production by the inflammatory cells, wherein the inflammatory cells are macrophages or monocytes from a human donor or generated from induced pluripotent stem cells, wherein the ex vivo expanded Treg cell population has been expanded from baseline Tregs, and wherein within the ex vivo expanded Treg cell population: a) expression of one or more dysfunctional baseline signature gene products listed in Table 3 and / or Table 4 is decreased relative to expression of said one or more gene products in baseline Tregs; b) expression of one or more dysfunctional baseline signature gene products listed in Table 5 is decreased relative to expression of said one or more gene products in baseline Tregs; c) increased expression of one or more Treg-associated signature gene products listed in Table 6 compared to expression of said one or more gene products in baseline Tregs; d) expression of one or more mitochondrial signature gene products listed in Table 7 is increased relative to expression of said one or more gene products in baseline Tregs; e) increased expression of one or more cell proliferation signature gene products listed in Table 8 relative to expression of said one or more gene products in baseline Tregs; or f) increased expression of one or more of the highest protein expression signature gene products listed in Table 9 relative to expression of said one or more gene products in baseline Tregs; 2. An isolated cell-free population of anti-inflammatory EVs.

21. 21. A pharmaceutical composition comprising the isolated cell-free population of anti-inflammatory EVs according to claim 20.

22. 22. A pharmaceutical composition according to any one of claims 12 to 14 or 19 to 21 for use in a method for treating a disorder in a subject in need thereof, the disorder being: (i) Disorders associated with Treg dysfunction; (ii) disorders associated with Treg deficiency; (iii) disorders associated with overactivation of the immune system; (iv) inflammatory states driven by T cell responses; (v) inflammatory conditions driven by myeloid cell responses; (vi) neurodegenerative disorders; (vii) autoimmune disorders; (viii) graft-versus-host disease; (ix) disorders requiring improved islet graft survival; (x) inflammation of the heart; (xi) neuroinflammation; or (xii) The pharmaceutical composition, which is a regulatory T cell disease.

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