Compositions and methods for modulating the immune system

Abstract

Compositions and methods for modulating the immune system generally include immunomodulatory exosomes derived from platelet-rich plasma that has been subjected to multiple freeze-thaw cycles and lyophilization. In one or more embodiments, the methods include administering the composition to a subject in an amount effective to modulate the immune system of the subject. In other embodiments, the methods include culturing hematopoietic progenitor cells or cells differentiated from hematopoietic progenitor cells with immunomodulatory exosomes to produce treated cells. The treated cells are then administered to the subject in an amount effective to modulate the immune system of the subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 313,579, filed February 24, 2022, the entirety of which is incorporated herein by reference. Summary of the Invention

[0002] The present disclosure describes compositions and methods for modulating the immune system of a subject.

[0003] In one aspect, the disclosure describes methods that generally include culturing hematopoietic progenitor cells or cells differentiated from hematopoietic progenitor cells with immunomodulatory exosomes to produce treated cells. The immunomodulatory exosomes (e.g., purified exosome product "PEP") are derived from platelet-rich plasma that has been subjected to multiple freeze-thaw cycles and lyophilization.

[0004] In one aspect, the present disclosure describes that the hematopoietic progenitor cells are myeloid progenitor cells. In one or more embodiments, the hematopoietic progenitor cells are lymphoid progenitor cells. In one or more other embodiments, the hematopoietic progenitor cells are peripheral blood mononuclear cells (PBMCs).

[0005] In one or more embodiments, the treated cells may be monocytes or macrophages.

[0006] In another aspect, the disclosure describes a method of treatment in which treated cells are administered to a subject. In one or more embodiments, the subject is suffering from an inflammation-associated disease state.

[0007] In one or more embodiments, the treated cells administered to the subject are M2 macrophages. In one or more of these embodiments, the M2 macrophages express CD163.

[0008] In one or more embodiments, the treated cells express CD14 or CD206, or both.

[0009] In one or more embodiments, the treated cells express IL-10, TGF-β, IL-1ra, or Arginase 1, or a combination thereof.

[0010] In another aspect, the disclosure describes a composition for use in treating an inflammation-associated disease state in a subject. Generally, the composition comprises immunomodulatory exosomes derived from platelet-rich plasma that has been subjected to multiple freeze-thaw cycles and lyophilization.

[0011] In one or more embodiments, the composition is formulated for intravenous administration. In one or more embodiments, the composition is formulated for intramuscular administration. In one or more embodiments, the composition is formulated for intraperitoneal administration.

[0012] In another aspect, the disclosure describes a method of treating an inflammation-associated disease state in a subject. In general, the method comprises administering to the subject a composition comprising immunomodulatory exosomes derived from platelet-rich plasma that has been subjected to multiple freeze-thaw cycles and lyophilization.

[0013] In one or more embodiments, the composition is administered intravenously. In one or more embodiments, the composition is administered intramuscularly. In one or more embodiments, the composition is administered intraperitoneally.

[0014] In one or more embodiments, administering the composition to a subject promotes the expansion of Treg cells. In some of these embodiments, the Treg cells are CD3 + CD4 + CD25 hi FoxP3 + In some of these embodiments, the Treg cells have a phenotype comprising CD127 lo The phenotype further comprises:

[0015] In one or more embodiments, administering the composition to a subject promotes the proliferation of M2 macrophages. In some of these embodiments, the M2 macrophages express CD163. In some of these embodiments, the M2 macrophages do not express detectable levels of CD80. In one or more embodiments, the M2 macrophages express CD14, CD206, or both.

[0016] In one or more embodiments, the method further comprises co-administering an additional therapeutic agent. In one or more of these embodiments, the additional therapeutic agent comprises an immunomodulatory monoclonal antibody or an immunomodulatory small molecule.

[0017] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly illustrates illustrative embodiments. In several places in this application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. [Brief description of the drawings]

[0018] [Figure 1] Overview of the use of purified exosome products (PEP) in the context of immune modulation. In vitro or in vivo exposure to PEP modulates the differentiation potential and / or phenotype of cell types derived from hematopoietic progenitor cells, including but not limited to monocytes, macrophages (Mφ), microglia, and T cells.

[0019] [Diagram 2] Scanning electron microscopy (SEM) images of PEP binding to immune cells: (A) T cells, (B) T cells (high magnification), (C) monocytes.

[0020] [Figure 3A]Schematic of the in vitro culture system (DEVERRA THERAPEUTICS, Seattle, Washington). Immobilized engineered Delta-like canonical Notch ligand 1 (Delta1ext-IgG) is used to proliferate and differentiate hematopoietic stem / progenitor cells (HSPCs). (B) Schematic showing immobilized Delta1ext-IgG construct interacting with the Notch receptor on hematopoietic stem / progenitor cells to promote proliferation of differentiated cell types. [Figure 3B] Schematic of the in vitro culture system (DEVERRA THERAPEUTICS, Seattle, Washington). Schematic showing immobilized Delta1ext-IgG constructs interacting with the Notch receptor on hematopoietic stem / progenitor cells to promote proliferation of differentiated cell types.

[0021] [Figure 4] Monocytes from mice were cultured in vitro with medium alone (control), medium + PEP (PEP), medium + lipopolysaccharide (LPS), and medium + lipopolysaccharide and PEP (LPS + PEP). PEP reduces the expression of proinflammatory cytokines typically secreted by M1 macrophages in response to LPS exposure.

[0022] [Diagram 5] Monocytes from mice were cultured in vitro with medium alone (control), medium + PEP (PEP), medium + lipopolysaccharide (LPS), and medium + lipopolysaccharide and PEP (LPS + PEP). PEP increases the expression of markers associated with M2 macrophages.

[0023] [Figure 6] Monocytes from mice were cultured in vitro with medium alone (control) or medium plus PEP (PEP). Monocyte proliferation, as measured by Ki-67 expression, was stimulated with PEP.

[0024] [Figure 7]Mouse-derived macrophages were cultured with medium alone (control), medium + PEP (PEP), medium + lipopolysaccharide (LPS), and medium + lipopolysaccharide and PEP (LPS + PEP) and assessed by flow cytometry. LPS induces IL-1β, a pro-inflammatory M1-associated cytokine. LPS-induced expression of IL-1β is reduced in the presence of PEP (top). LPS alone reduces expression of CD206, an M2-associated marker. In the presence of PEP, CD206 expression is increased in LPS-treated macrophages (bottom).

[0025] [Figure 8] Confocal microscopy analysis of mouse-derived macrophages in Figure 7. (A) IL-1β expression in LPS-stimulated macrophages. (B) Co-culture of LPS-treated macrophages with PEP increases the expression of CD206.

[0026] [Figure 9] Acute myocardial infarction (MI) was induced in a porcine model. MI was treated with stent placement alone (infarction) or stent placement plus PEP administration (MI+PEP). mRNA expression analysis was performed on tissue from porcine heart tissue in both groups.

[0027] [Figure 10A] Assessment of the ratio of M2 macrophage phenotype (CD163) to mature macrophage population (25F9) following acute myocardial infarction. Immunohistochemistry and confocal microscopy. [Figure 10B] Assessment of the ratio of M2 macrophage phenotype (CD163) to mature macrophage population (25F9) following acute myocardial infarction. Quantification via ImageJ analysis.

[0028] [Figure 11]Immunohistochemistry of porcine urethral tissue for general macrophages (Mφ), CD163 M2-specific macrophages, and DAPI. Merge overlay highlights that PEP samples contained colocalized staining of macrophages and CD163+ tissue. (n=12, 15, 20) Scale=20 μm.

[0029] [Figure 12] ImageJ blinded quantification of immunohistochemical staining in Figure 11. M2:M1 specific macrophages (M1:M2) ratios were determined from comparison of M2:(Mφ-M2) image areas. Haldane correction was used to account for division by zero in M2:M1 ratios. This correction adds 0.5 to all values ​​in the list to ensure the calculation is error-free. Graphs represent raw ratios of M2:M1 macrophages. Red = statistical outliers. The dotted line at y=1 indicates the value where the ratios are equal. Logarithmic base 2 scale indicates that for values ​​Log2>0, M2:M1>1 and for values ​​Log2<0, M2:M1<1. ***=p<0.05. Red = statistical outliers. The dotted line at y=0 indicates the value where the ratios are equal. (n=12, 15, 20)

[0030] [Figure 13] Chemotaxis of polarized microglia pretreated with LPS, IL-4, or untreated showed no migration into the lower reservoir without PEP.

[0031] [Figure 14] Chemotaxis of polarized microglia pretreated with LPS for 24 h, resulting in an M1 phenotype, showed negligible chemotaxis towards the bottom reservoir of PEP-supplemented medium.

[0032] [Figure 15] Chemotaxis of polarized microglia pretreated with IL-4 to induce the M2 phenotype showed dose-dependent chemotaxis towards PEP.

[0033] [Figure 16]Chemotaxis of untreated naive microglia (M0) demonstrated a strong dose-dependent chemotaxis to PEP.

[0034] [Figure 17] Proliferative effect of PEP on polarized microglia. Microglia were polarized into three categories: M1 (LPS pretreated), M2 (IL-4 pretreated), and M0 (untreated). After polarization, microglia were treated with 10% PEP in serum-free medium. M2 and M0 showed increased proliferation with PEP treatment compared to M1 microglia.

[0035] [Figure 18] Flow cytometry results of T cell proliferation assay. (A) In the absence of T cell stimulation, PEP did not affect the number of T cells. (B) Upon CD3 / CD28 stimulation of T cells, CD4 T cell proliferation was inhibited by the presence of PEP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The present disclosure describes compositions and methods for modulating the immune system of a subject. The methods generally utilize the immunomodulatory properties of immunomodulatory exosomes derived from platelet-rich plasma and subjected to multiple freeze-thaw cycles and lyophilization. In one or more methods, a composition comprising immunomodulatory exosomes can be administered directly to a subject in an amount effective to modulate the immune system of the subject. In one or more other methods, hematopoietic progenitor cells or cells differentiated from hematopoietic progenitor cells are cultured with immunomodulatory exosomes to produce treated cells. The treated cells can be administered to a subject in an amount effective to modulate the immune system of the subject.

[0037] In brief, the present disclosure demonstrates the ability of immunomodulatory exosomes to bind to and regulate immune cells. In particular, immunomodulatory exosomes support the polarization of macrophages to an M2 anti-inflammatory phenotype and the reduction of an M1 pro-inflammatory profile both in vitro and in vivo. This is evidenced by changes in cytokine production and gene expression. In addition, immunomodulatory exosomes (e.g., PEP) recruit and support the proliferation of M0 and M2 polarized microglia. Furthermore, immunomodulatory exosomes inhibit the proliferation of activated CD4+ T cells. Thus, immunomodulatory exosomes can be used to support the reduction of inflammation in clinical diseases.

[0038] Immunomodulatory Exosomes In one or more aspects, the present disclosure describes compositions comprising immunomodulatory exosomes, and methods of using the immunomodulatory exosomes. In one or more embodiments, the immunomodulatory exosomes are derived from platelet-rich plasma that has been subjected to multiple freeze-thaw cycles and lyophilization. In one or more embodiments, the immunomodulatory exosomes can be prepared as described in International Patent Application No. PCT / US2018 / 065627 (published as International Publication No. WO2019 / 118817), U.S. Patent Publication No. 2021 / 0169812(A1), U.S. Patent No. 10,596,123, or International Patent Application No. PCT / US2021 / 054547 (published as International Publication No. WO2022 / 081557(A1)).

[0039] In one or more embodiments, the immunomodulatory exosomes may be a purified exosome product, referred to herein as PEP. PEP is a purified exosome product prepared using a freeze-drying process that produces a product with a structure different from exosomes prepared using conventional methods. The production of purified exosome product (PEP) involves separating plasma from blood and isolating a solution of exosomes from the separated plasma by filtration and centrifugation. PEP is fully characterized and methods of preparation of PEP are described in International Patent Application No. PCT / US2018 / 065627 (published as WO 2019 / 118817), U.S. Patent Application Publication No. 2021 / 0169812(A1), and U.S. Patent No. 10,596,123, which are incorporated herein by reference in their entireties. Briefly, PEP is a purified exosome product prepared using a freeze-drying process that produces a product with a structure different from exosomes prepared using conventional methods. For example, PEP typically has a spherical or spheroidal structure and an intact lipid bilayer, rather than the crystalline structure resulting from lipid reaggregation of the exosome lipid bilayer after exosomes are disrupted during conventional exosome preparation methods. The spherical or spheroidal exosome structures generally have a diameter of 300 nm or less. Typically, PEP preparations contain spherical or spheroidal exosome structures with a relatively narrow size distribution. In some preparations, PEP contains spherical or spheroidal exosome structures with a mean diameter of about 110 nm ± 90 nm, with the majority of the exosome structures having a mean diameter of 110 nm ± 50 nm, such as 110 nm ± 30 nm.

[0040] Unmodified PEP preparations, e.g., PEP preparations whose characteristics have not been altered by selection or separation of the exosome population in the preparation, may be characterized by CD63 + Exosomes and CD63 - Naturally contains a mixture with exosomes. CD63 - Exosomes can inhibit unlimited cell proliferation, and thus CD63 + Exosomes and CD63 -Unmodified PEP preparations that naturally contain exosomes can stimulate cell proliferation for wound repair and / or tissue regeneration or limit uncontrolled cell proliferation.

[0041] In addition, CD63 + Exosome sorting revealed that CD63 was expressed in naturally isolated PEP preparations. + Extract the exosomes and then elucidate the desired amount of CD63 + By returning exosomes, CD63 in the PEP product + Exosomal CD63 - The ratio of PEP to exosomes can be controlled. In one or more embodiments, the PEP preparation comprises CD63 - It may only have exosomes.

[0042] In one or more embodiments, the PEP preparation comprises CD63 + Exosomes and CD63 - Exosomes may contain both CD63 and CD63. + Exosomal CD63 - The ratio of CD63 to exosomes can vary, at least in part, depending on the amount of cell expansion desired in a particular application. + / CD63 - Exosome ratio is CD63 + Desired cell proliferation induced by exosomes and CD63 achieved through cell contact inhibition - In certain scenarios, such as tissues with non-adherent cells (e.g., blood-derived components), this ratio can be adjusted to provide the appropriate balance of cell proliferation or cell inhibition for the tissue being treated. For example, in tissues with non-adherent cells, cell-to-cell contact is not a trigger, so CD63 + The ratio of exosomes can be reduced to avoid unlimited cell proliferation. Conversely, if one wishes to expand a clonal population of cells, such as in allogeneic cell-based therapies or immunotherapy, one can use CD63 to ensure that one can obtain a large cell population from a very small source. +The ratio of exosomes can be increased.

[0043] Thus, in one or more embodiments, CD63 in the PEP preparation + Exosomal CD63 - The ratio of CD63 to exosomes can be at least 1:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 11:1, at least 12:1, at least 13:1, at least 14:1, at least 15:1, or at least 16:1. + Exosomal CD63 - The ratio of CD63 to exosomes can be at most 15:1, at most 16:1, at most 17:1, at most 18:1, at most 19:1, at most 20:1, at most 25:1, or at most 30:1. + Exosomes vs. CD63 - The ratio of exosomes can be 1:1 to 30:1, 2:1 to 20:1, 4:1 to 15:1, or 8:1 to 10:1. In one or more specific embodiments, the PEP product contains a 9:1 ratio of CD63 + Exosomes vs. CD63 - In one or more particular embodiments, the exosomes are formulated to contain a natural PEP, such as CD63. + Exosomal CD63 - An unmodified ratio of PEP to exosomes may also be used.

[0044] In one or more embodiments, the PEP may be modified to include one or more exogenous active agents. As used herein, the term "exogenous" refers to a material that is not naturally present in the PEP exosome. Because PEP may be prepared from a variety of starting materials, an active agent may be "exogenous" to a PEP prepared from one source material, even though it may be endogenous (i.e., naturally occurring) in a PEP exosome prepared from another source. Thus, the assessment of whether an active agent is exogenous depends on the source material used to prepare the PEP. Exemplary exogenous active agents include, but are not limited to, nucleic acids or polypeptides. Methods for transforming extracellular vesicles and exemplary exogenous active agents are described in detail in International Patent Application No. PCT / US2019 / 043172, published as U.S. Patent Application Publication No. 2021 / 0259969(A1) and International Publication No. WO2020 / 023594.

[0045] Monocytes, macrophages, microglia, and T cells Monocytes are circulating myeloid cells that give rise to tissue macrophages and dendritic cells. Regulatory monocytes regulate other regulatory cells (e.g., regulatory T cells (T cells)) through the production of soluble regulatory factors (e.g., IL-10, TGF-β, indoleamine 2,3 deoxygenase (IDO), arginase, nitric oxide (NO), etc.). reg ) and / or by enhancing regulatory feedback loops, via the expression of inhibitory or regulatory cell surface molecules (including, for example, PD-L1, PD-L2) (VanGundy et al. BMC Immunol. 2014;15:8.).

[0046] Macrophages are large mononuclear phagocytes differentiated from monocytes. Macrophages can adopt a range of phenotypes from a proinflammatory or "M1" phenotype to a less inflammatory or "M2" phenotype that may be associated with the resolution of inflammation (e.g., Spiller et al. Adv. Drug Del. Rev. 2017;122:74-83). The role of M1 or M2 macrophages has been suggested in therapeutic strategies ranging from regenerative medicine to cancer immunotherapy.

[0047] Microglia are macrophage cells present in the central nervous system (CNS). Similar to peripheral macrophages, microglia can polarize into "M1" proinflammatory and "M2" anti-inflammatory phenotypes (Guo et al., Front Aging Neurosci. 2022 Feb 16;14:815347. doi:10.3389 / fnagi.2022.815347. PMID:35250543; PMCID:PMC8888930).

[0048] As used herein, a macrophage or microglial cell exhibiting an "M1-like" phenotype may express or produce one or more of CD14, CD68, CD80, CD86, iNos, CCL2, TNFα, IFNβ, IFNγ, IL-1β, IL-6, IL-12, IL-17, or IL-23. As used herein, a macrophage or microglial cell exhibiting an "M2-like" phenotype expresses or produces one or more of CD14, CD68, CD163, CD206, IL-4, IL-6, IL-10, IL-13, TGF-β, IL-1RA, RETN, or arginase 1.

[0049] Conventional T cells are important lymphocytes in the context of adaptive immune responses. Upon stimulation via their T cell receptor (TCR), in combination with additional costimulatory signals, T cells are activated and undergo rapid cell division. Helper T cells express CD3 and CD4 on their surface, and these cells can differentiate into several subsets to defend against various types of pathogens. However, prolonged activation of T cells can lead to chronic inflammation and undesirable immunopathology (e.g., in cases of autoimmunity). Regulatory T cells (Tregs) are a subpopulation of T cells that regulate the immune system and maintain tolerance to self-antigens. In one or more embodiments, Tregs ... + CD4 + CD25 hi FoxP3 + In one or more embodiments, Tregs have the CD127 lo It may further have a phenotype.

[0050] Methods for Producing Macrophages and Monocytes In another aspect, the present disclosure describes a method of generating M2-like macrophages, microglial cells, and / or regulatory monocytes. The present disclosure may further describe a method of expanding macrophages, microglial cells, or monocytes.

[0051] In one or more embodiments, the methods include co-culturing hematopoietic progenitor cells or cells differentiated from hematopoietic cells with immunomodulatory exosomes. Figure 1. Typically, hematopoietic progenitor cells or cells differentiated from hematopoietic cells can be co-cultured with immunomodulatory exosomes for 24 hours to 30 days, although methods of generating M2-like macrophages, microglial cells, or regulatory monocytes can include co-culturing the cells with immunomodulatory exosomes for periods outside this range.

[0052] The hematopoietic progenitor cells or cells differentiated from hematopoietic cells can be any cells that can differentiate into M2-like macrophages, microglia, or regulatory monocytes. Thus, the hematopoietic progenitor cells can be myeloid progenitor cells or lymphoid progenitor cells. Thus, in one or more embodiments, the method includes deriving macrophages, microglial cells, or monocytes, or all three, from a hematopoietic stem cell source. The hematopoietic stem cell source can include any suitable source of hematopoietic stem cells. Exemplary sources include umbilical cord blood, bone marrow, peripheral blood, and the like.

[0053] In one or more embodiments, the method of inducing macrophages, microglial cells, monocytes, or all three from a hematopoietic stem cell source may include co-culturing the hematopoietic stem cells with a Delta-1 Notch ligand. Figure 3A In one or more embodiments, the Delta-1 Notch ligand may include dilanubicel (also known as NLA101) from Deverra Therapeutics, Inc. (Seattle, Washington).

[0054] In other embodiments, the method includes co-culturing cells differentiated from hematopoietic progenitor cells with immunomodulatory exosomes. The cells differentiated from hematopoietic progenitor cells can be peripheral blood mononuclear cells (PBMCs) or non-peripheral blood mononuclear cells. Exemplary PBMCs include, but are not limited to, B cells, T cells (e.g., CD4 + T cells, CD8 + cells, including Tregs, natural killer (NK) cells, macrophages, monocytes, dendritic cells, or myeloid-derived suppressor cells (MDSCs).

[0055] In one or more embodiments, PEP can promote the proliferation of macrophages. In particular, administration of PEP can promote the proliferation of M2-like macrophages.

[0056] How to use In another aspect, the disclosure describes a method that includes modulating the immune system using immunomodulatory exosomes. The method may include contacting the immunomodulatory exosomes with hematopoietic cells (or cells differentiated from hematopoietic cells) in vivo or ex vivo. Thus, in one or more embodiments, the method may include administering to a subject cells differentiated from hematopoietic cells (i.e., "treated cells," such as, for example, monocytes, M2-like macrophages, or other peripheral blood mononuclear cells) by contact with the immunomodulatory exosomes in an amount effective to modulate the immune system of the subject. In other embodiments, the method may include directly administering to a subject immunomodulatory exosomes in an amount effective to modulate the immune system of the subject.

[0057] The subject can be a human or a non-human animal, such as a livestock animal, a zoo animal, or a companion animal. Exemplary non-human animal subjects include, but are not limited to, members of the family Hominidae (including, for example, chimpanzees, gorillas, or orangutans), the genus Bovidae (including, for example, cattle), the genus Capricorn (including, for example, goats), the genus Ovis (including, for example, sheep), the genus Porcine (including, for example, pigs), the family Equidae (including, for example, horses), members of the family Cervidae (including, for example, deer, elk, moose, caribou, reindeer), members of the family Bisonidae (including, for example, bison), the family Felidae (including, for example, tigers, lions, and domestic cats), the family Canidae (including, for example, wolves and domestic dogs), birds (including, for example, turkeys, chickens, ducks, and geese), rodents (including, for example, mice or rats), members of the family Leporidae (including, for example, rabbits or hares), members of the family Mustelidae (including, for example, ferrets), or members of the order Chiroptera (including, for example, bats).

[0058] Treatment of a disease may be preventive or may be initiated after a subject shows one or more symptoms or clinical signs of the disease. A treatment that is preventive, e.g., that is initiated before a subject shows symptoms or clinical signs of the disease, is referred to herein as treatment of a subject "at risk" of having the disease. As used herein, the term "at risk" refers to a subject who may or may not actually have the described risk. Thus, for example, a subject "at risk" of an infectious condition is one who is present in an area where other individuals have been identified as having an infectious condition, and / or one who may be exposed to an infectious agent, even if the subject has not yet developed any detectable indicators of infection by the infectious agent, and whether or not the subject may carry subclinical amounts of the infectious agent. As another example, a subject "at risk" of a non-infectious condition is one who has one or more risk factors associated with the condition, such as, for example, genetic predisposition, ancestry, age, sex, geographic location, lifestyle, or medical history. Treatment can also be continued after symptoms have disappeared, e.g., to prevent or delay their recurrence.

[0059] Thus, treated cells and / or immunomodulatory exosomes produced as described herein can be administered before, during, or after a subject first exhibits symptoms or clinical signs of a condition, or in the case of an infectious condition, before, during, or after the subject first comes into contact with an infectious agent. Treatment initiated before a subject first exhibits symptoms or clinical signs associated with a disease may result in a reduced likelihood that the subject will experience clinical evidence of the disease, a reduced severity of symptoms and / or clinical signs of the disease, and / or a complete recovery from the disease, compared to a subject to whom the composition is not administered. Treatment initiated after a subject first exhibits symptoms or clinical signs associated with a disease may result in a reduced severity of symptoms and / or clinical signs of the disease, and / or a complete recovery from the disease, compared to a subject to whom the composition is not administered.

[0060] Thus, in one or more embodiments, the methods include administering to a subject having or at risk of having a particular condition an effective amount of treated cells. In other embodiments, the methods include administering to a subject having or at risk of having a particular condition an effective amount of immunomodulatory exosomes. In either case, an "effective amount" is an amount effective to reduce, limit the progression of, ameliorate, or reverse to any extent a symptom or clinical sign associated with the condition.

[0061] In some cases, the subject may suffer from an inflammation-associated disease state. Exemplary inflammation-associated disease states include, but are not limited to, cardiovascular disease (e.g., chronic pulmonary respiratory disease, acute respiratory disease, chronic obstructive pulmonary disease (COPD), etc.), neurological disease including neuroinflammatory disease (e.g., amyotrophic lateral sclerosis (ALS), Alzheimer's disease, multiple sclerosis, transverse myelitis, neuritis, neurosarcoidosis, Parkinson's disease, etc.), gastrointestinal disease (e.g., Crohn's disease, ulcerative colitis, irritable bowel syndrome, inflammatory bowel disease, etc.), skin disease (e.g., eczema, dermatitis, psoriasis, etc.), autoimmune disease (e.g., systemic lupus erythematosus, rheumatoid arthritis, psoriasis, etc.), inflammatory joint disease, myocarditis, atherosclerosis, diabetes, or osteoporosis. In one or more embodiments, the subject may have a viral infection, such as a SARS-CoV-2 infection. The subject may have a known inflammation-associated disease. Alternatively, the subject may be suspected of having an inflammation-related disease.

[0062] In embodiments in which treated cells are administered to a subject, the treated cells generated as described herein may be formulated with a pharma- ceutically acceptable carrier. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating agent, diluent, antibacterial and / or antifungal agent, isotonicity agent, absorption delaying agent, buffer, carrier solution, suspension, hydrogel, scaffold, colloid, and the like. The use of such media and / or agents for pharma- ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the treated cells, its use in the therapeutic composition is contemplated. Supplementary active ingredients may also be incorporated into the formulation with the therapeutic cells. As used herein, "pharmaceutical acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., the substance may be administered to an individual together with the treated cells without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is included.

[0063] Thus, the treated cells generated as described herein may be formulated into a pharmaceutical composition. The pharmaceutical composition may be formulated in a variety of forms adapted to the preferred route of administration. Thus, the composition may be administered via known routes, including, for example, orally, parenterally (e.g., intradermally, transdermally, subcutaneously, intramuscularly, intravenously, intraperitoneally, etc.), or topically (e.g., intranasally, intrapulmonary, intramammary, intravaginally, intradermally, transdermally, intrarectally, etc.). The pharmaceutical composition may be administered to a mucosal surface, such as, for example, by administration to the nasal or respiratory mucosa (e.g., by spray or aerosol). The composition may also be administered via sustained or delayed release.

[0064] Thus, the treated cells can be provided in any suitable form, including, but not limited to, in the form of a solution, suspension, emulsion, spray, aerosol, or any mixture. The composition can be delivered in a formulation that includes any pharma- ceutically acceptable excipient, carrier, or vehicle. For example, the formulation can be provided in a conventional topical dosage form, such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like. The formulation can further include one or more additives, including, for example, an adjuvant, a skin penetration enhancer, a colorant, an aroma, a flavoring, a moisturizer, a thickener, and the like.

[0065] The preparation may be provided in a convenient unit dosage form and can be prepared by a method well known in the field of pharmacy.The method of preparing the composition containing pharmaceutically acceptable carrier comprises the step of associating treated cells with carrier, which may contain one or more accessory ingredients.In general, the preparation can be prepared by uniformly and / or intimately combining active compound with liquid carrier, finely divided solid carrier, or both, and then, if necessary, shaping the product into desired preparation.

[0066] The amount of treated cells administered may vary depending on a variety of factors, including but not limited to the specific treated cells administered, the subject's weight, health status, and / or age, and the administration site and / or administration route. Thus, the absolute weight of treated cells contained in a given unit dosage form may vary widely and depends on factors such as the subject's species, age, weight, and health status, and / or administration method. Therefore, it is not practical to generally state what constitutes an amount of treated cells that is effective for all possible applications. However, those skilled in the art can easily determine the appropriate amount with due consideration of such factors.

[0067] Thus, in one or more embodiments, the method includes, for example, measuring about 1×10 5 ~Approx. 1×10 10This may include administering sufficient treated cells to provide a dose of cells to the subject, although in one or more embodiments the method may be practiced by administering treated cells at a dose outside this range. The dose may be determined as a total number of cells or as a ratio of cells per body weight (cells / kg). The absolute minimum dose is 1×10 3 The absolute maximum dose should be approximately 1 x 10 15 Typically, it should be 1×10 5 ~Approx. 1×10 10 Doses of cells can be administered up to three times daily.

[0068] A single dose may be administered all at once, continuously over a period of time, or in multiple separate doses. When multiple doses are used, the dosages of each dose may be the same or different. For example, 2×10 10 The dose is 2 x 10 10 may be administered as a single dose of 1×10 10 or 1.5×10 10 followed by a first dose of 0.5 × 10 10 When multiple administrations are used to deliver a single dose, the intervals between administrations may be the same or different.

[0069] In one or more embodiments, the treated cells may be administered, for example, once to multiple doses per week, although in one or more embodiments, the method may include a course of treatment that includes administering doses of treated cells at a frequency outside this range. When a course of treatment includes administering multiple doses within a particular period of time, each dose may be the same or different. For example, a course of treatment may include a loading dose of an initial dose, followed by a maintenance dose that is lower than the loading dose. Also, when multiple doses are administered within a particular period of time, the interval between doses may be the same or different.

[0070] In certain embodiments, the treated cells may be administered to a subject about once a month to about five times a week.

[0071] In one or more embodiments, the treated cells are M2-like macrophages. In some cases, the M2-like macrophages express CD163 and / or CD206. In some cases, the M2-like macrophages further express IL-4, IL-10, TGF-β, IL-1RA, RETN, or arginase 1, or a combination of two or more thereof.

[0072] In embodiments in which the immunomodulatory exosomes are administered to a subject, the immunomodulatory exosomes may be formulated with a pharma- ceutically acceptable carrier. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating agent, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption retardant, buffer, carrier solution, suspension, hydrogel, scaffold, colloid, and the like. The use of such media and / or agents for pharma- ceutically active substances is well known in the art. Except where any conventional media or agent is incompatible with the immunomodulatory exosomes, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the formulation with the immunomodulatory exosomes. As used herein, "pharma- ceutically acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., the substance may be administered to an individual together with the immunomodulatory exosomes without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is included.

[0073] Thus, the immunomodulatory exosomes can be formulated into a pharmaceutical composition. The pharmaceutical composition can be formulated in various forms adapted to the preferred route of administration. Thus, the composition can be administered via known routes, including, for example, orally, parenterally (e.g., intradermally, transdermally, subcutaneously, intramuscularly, intravenously, intraperitoneally, etc.), or topically (e.g., intranasally, intrapulmonary, intramammary, intravaginally, intradermally, transdermally, intrarectally, etc.). The pharmaceutical composition can be administered to a mucosal surface, such as, for example, by administration to the nasal or respiratory mucosa (e.g., by spray or aerosol). The composition can also be administered via sustained or delayed release.

[0074] Thus, the immunomodulatory exosomes may be provided in any suitable form, including, but not limited to, in the form of a solution, suspension, emulsion, spray, aerosol, or any mixture. The composition may be delivered in a formulation that includes any pharma- ceutically acceptable excipient, carrier, or vehicle. For example, the formulation may be delivered in a conventional topical dosage form, such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like. The formulation may further include one or more additives, including, for example, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, and the like.

[0075] The formulations may be conveniently provided in unit dosage form and can be prepared by methods known in the art of pharmacy. The method of preparing the composition with a pharmaceutically acceptable carrier includes associating the immunomodulatory exosomes with a carrier that may contain one or more accessory ingredients. In general, the formulations can be prepared by uniformly and / or intimately combining the active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

[0076] The amount of immunomodulatory exosomes administered may vary depending on a variety of factors, including, but not limited to, the specific immunomodulatory exosomes administered, the weight, health status, and / or age of the subject, and the site and / or route of administration. Thus, the absolute weight of immunomodulatory exosomes contained in a given unit dosage form may vary widely and depends on factors such as the species, age, weight, and health status of the subject, and / or the method of administration. Thus, it is not practical to generally describe the amount that constitutes an effective amount of immunomodulatory exosomes for all possible applications. However, those skilled in the art can easily determine the appropriate amount with due consideration of such factors.

[0077] In one or more embodiments, the method comprises, for example, about 1×10 5 pieces~approx. 1×10 16 This may include administering sufficient immunomodulatory exosomes to provide a dose of 1×10 to the subject, although in one or more embodiments the method may be practiced by administering immunomodulatory exosomes at doses outside this range. The dose may be determined as a total number of immunomodulatory exosomes or as a ratio of immunomodulatory exosomes per body weight (exosomes / kg). In one or more embodiments, PEP may be administered on a per weight basis, either as a total mg dose or mg per body weight (mg / kg). In one or more embodiments the minimum dose delivered is 1×10 3 In one or more embodiments, the maximum dose delivered is 1×10 20 immunomodulatory exosomes. Typically, 1×10 5 Immunomodulatory exosomes ~ approx. 1 x 10 16 The dose of immunomodulatory exosomes can be administered up to three times daily.

[0078] A single dose may be administered all at once, continuously over a period of time, or in multiple separate doses. When multiple doses are used, the dosages of each dose may be the same or different. For example, 2×10 10 The dose is 2 x 1010 may be administered as a single dose or continuously over a 24 hour period, and may be administered in a dose of 1×10 10 may be administered as two doses of 1.5×10 immunomodulatory exosomes or 10 A first dose of 0.5 × 10 immunomodulatory exosomes followed by 0.5 × 10 10 The immunomodulatory exosomes may be administered as a second administration of the individual immunomodulatory exosomes. When multiple administrations are used to deliver a single dose, the intervals between administrations may be the same or different.

[0079] In one or more embodiments, the immunomodulatory exosomes may be administered, for example, once to multiple times per week, although in one or more embodiments, the method may include a course of treatment that includes administering a dose of the immunomodulatory exosome at a frequency outside this range. When a course of treatment includes administering multiple doses within a particular time period, each dose may be the same or different. For example, a course of treatment may include a loading dose of an initial dose, followed by a maintenance dose that is lower than the loading dose. Also, when multiple doses are administered within a particular time period, the interval between doses may be the same or different.

[0080] In certain embodiments, the immunomodulatory exosomes may be administered to a subject about once a month to about five times a week.

[0081] In one or more embodiments, the method further comprises administering one or more additional therapeutic agents. The one or more additional therapeutic agents may be administered before, after, and / or simultaneously with the administration of the immunomodulatory exosomes or treated cells. The additional therapeutic agent(s) may be co-administered with the immunomodulatory exosomes or treated cells. As used herein, "co-administered" refers to two or more components of a combination administered such that the therapeutic or prophylactic effect of the combination may be greater than the therapeutic or prophylactic effect of either component administered alone. The two components may be co-administered simultaneously or sequentially. Components co-administered simultaneously may be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes when the components are administered such that each component may be present at the treatment site at the same time. Alternatively, sequential co-administration of two components may include when at least one component has been removed from the treatment site, but at least one cellular effect of administering the component (e.g., cytokine production, activation of a specific cell population, etc.) persists at the treatment site until one or more additional components are administered to the treatment site. Thus, co-administered combinations may, in certain circumstances, include components that are not in a chemical or physical mixture with each other. In other embodiments, the immunomodulatory exosomes or treated cells and additional therapeutic agents may be administered as part of a mixture or cocktail. In some aspects, administration of immunomodulatory exosomes or treated cells may allow for the efficacy of lower doses of other therapeutic modalities when compared to administration of the other therapeutic agent(s) alone, thereby reducing the likelihood, severity, and / or extent of toxicity observed when higher doses of the other therapeutic agent(s) are administered.

[0082] Exemplary additional therapeutic agents include, but are not limited to, monoclonal antibodies, antibody fragments, multispecific antibody-drug conjugates, nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, antibiotics, antivirals (e.g., remdesivir or oseltamivir), or convalescent plasma. Exemplary therapeutic monoclonal antibodies include, but are not limited to, remdesivir, baricitinib, adalimumab, sotrovimab, benralizumab, raxibacumab, or guselkumab.

[0083] In several places in the above description, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0084] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "one or more embodiments," or "some embodiments" or the like mean that the particular features, configurations, compositions, or characteristics described in connection with the embodiment are included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification do not necessarily refer to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, and characteristics may be combined in any suitable manner in one or more embodiments.

[0085] In the foregoing description, the words "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0086] The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

[0087] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0088] Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (eg, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0089] In any method disclosed herein that includes separate steps, the steps may be performed in any practicable order, and, if desired, any combination of two or more steps may be performed simultaneously.

[0090] Exemplary embodiments In embodiment 1, Culturing hematopoietic progenitor cells or cells differentiated from hematopoietic progenitor cells with immunomodulatory exosomes to produce treated cells; A method, wherein the immunomodulatory exosomes are derived from platelet-rich plasma that has been subjected to multiple freeze-thaw cycles and lyophilization.

[0091] Embodiment 2 is the method of embodiment 1, wherein the hematopoietic progenitor cells comprise bone marrow progenitor cells.

[0092] Embodiment 3 is the method of embodiment 1, wherein the hematopoietic progenitor cells comprise lymphoid progenitor cells.

[0093] Embodiment 4 is the method of embodiment 1, wherein the cells differentiated from hematopoietic progenitor cells are peripheral blood mononuclear cells (PBMCs).

[0094] Embodiment 5 is the method of embodiment 4, wherein the PBMCs comprise bone marrow cells.

[0095] Embodiment 6 is the method of embodiment 5, wherein the bone marrow cells comprise monocytes or macrophages.

[0096] Embodiment 7 is the method of embodiment 4, wherein the PBMCs comprise lymphocytes.

[0097] Embodiment 8 is the method of embodiment 7, wherein the lymphocytes comprise B cells, T cells, or NK cells.

[0098] Embodiment 9 is the method of embodiment 1, wherein the treated cells comprise monocytes or macrophages.

[0099] Embodiment 10 is the method of any one of embodiments 1 to 9, further comprising administering treated cells to a subject.

[0100] Embodiment 11 is the method of embodiment 10, wherein the subject is suffering from an inflammation-related disease state.

[0101] Embodiment 12 is the method of embodiment 11, wherein the inflammation-related disease state comprises a chronic pulmonary respiratory disease, an acute respiratory disease, an inflammatory joint disease, atherosclerosis, diabetes, a cardiovascular disease, an inflammatory bowel disease, a neuroinflammatory disease, or osteoporosis.

[0102] Embodiment 13 is the method of embodiment 11, wherein the inflammation-associated disease state comprises infection with SARS-CoV-2.

[0103] Embodiment 14 is the method of any one of embodiments 1 to 13, wherein the treated cells comprise M2 macrophages.

[0104] Embodiment 15 is the method of embodiment 14, wherein the M2 macrophages express CD163.

[0105] Embodiment 16 is the method of any one of embodiments 1 to 15, wherein the treated cells express CD14 or CD206, or both.

[0106] Embodiment 17 is a method according to any one of embodiments 1 to 16, wherein the treated cells express IL-10, TGF-β, IL-1ra, or arginase 1, or a combination thereof.

[0107] Embodiment 18 is a composition comprising immunomodulatory exosomes for use in treating an inflammation-related disease condition in a subject, comprising: A composition in which immunomodulatory exosomes are derived from platelet-rich plasma that has been subjected to multiple freeze-thaw cycles and lyophilization.

[0108] Embodiment 19 is the composition of embodiment 18, wherein the inflammation-related disease state comprises a chronic pulmonary respiratory disease, an acute respiratory disease, an inflammatory joint disease, atherosclerosis, diabetes, a cardiovascular disease, an inflammatory bowel disease, a neuroinflammatory disease, or osteoporosis.

[0109] Embodiment 20 is the composition of embodiment 19, wherein the inflammation-associated disease state comprises infection with SARS-CoV-2.

[0110] Embodiment 21 is the composition of embodiment 19, wherein the inflammation-related disease state comprises chronic obstructive pulmonary disease (COPD).

[0111] Embodiment 22 is the composition of embodiment 19, wherein the inflammation-related disease state comprises myocarditis.

[0112] Embodiment 23 is the composition of any one of embodiments 18-22, wherein the composition is formulated for intravenous administration.

[0113] Embodiment 24 is the composition of any one of embodiments 18-22, wherein the composition is formulated for intramuscular administration.

[0114] Embodiment 25 is the composition of any one of embodiments 18-22, wherein the composition is formulated for intraperitoneal administration.

[0115] Embodiment 26 is the composition of any of embodiments 18-25, wherein the composition further comprises one or more additional active agents.

[0116] Embodiment 27 is a composition according to any of embodiments 18-26 for use in treating domestic animals, domesticated animals, zoo animals, or humans.

[0117] Embodiment 28 is a method of treating an inflammation-associated disease state in a subject, comprising: The method includes administering to a subject immunomodulatory exosomes derived from platelet-rich plasma that has been subjected to multiple freeze-thaw cycles and lyophilization.

[0118] Embodiment 29 is the method of embodiment 28, wherein the inflammation-related disease state comprises a chronic pulmonary respiratory disease, an acute respiratory disease, an inflammatory joint disease, atherosclerosis, diabetes, a cardiovascular disease, an inflammatory bowel disease, a neuroinflammatory disease, or osteoporosis.

[0119] Embodiment 30 is the method of embodiment 29, wherein the inflammation-associated disease state comprises infection with SARS-CoV-2.

[0120] Embodiment 31 is the method of embodiment 29, wherein the inflammation-associated disease state comprises chronic obstructive pulmonary disease (COPD).

[0121] Embodiment 32 is the method of embodiment 29, wherein the inflammation-associated disease state comprises myocarditis.

[0122] Embodiment 33 is the method of any of embodiments 28-32, wherein the method comprises administering a composition comprising immunomodulatory exosomes, wherein the composition is formulated for intravenous administration.

[0123] Embodiment 34 is the method of any of embodiments 28-32, wherein the method comprises administering a composition comprising immunomodulatory exosomes, wherein the composition is formulated for intramuscular administration.

[0124] Embodiment 35 is the method of any of embodiments 28-32, wherein the method comprises administering a composition comprising immunomodulatory exosomes, wherein the composition is formulated for intraperitoneal administration.

[0125] Embodiment 36 is the method according to any one of embodiments 28 to 35, wherein administering immunomodulatory exosomes promotes the proliferation of Treg cells.

[0126] Embodiment 37 is a method for treating Treg cells comprising administering to a patient a therapeutically effective amount of Treg cells, comprising administering + CD4 + CD25 hi FoxP3 + 37. The method of embodiment 36, wherein the patient has a phenotype comprising:

[0127] Embodiment 38 is a method for treating Tregs comprising administering to a patient a therapeutically effective amount of Tregs comprising administering to lo 38. The method of embodiment 37, wherein the phenotype further comprises:

[0128] Embodiment 39 is the method of any one of embodiments 28 to 38, wherein administering the immunomodulatory exosomes promotes the proliferation of M2 macrophages.

[0129] Embodiment 40 is the method of embodiment 39, wherein the M2 macrophages express CD163.

[0130] Embodiment 41 is the method of embodiment 39 or embodiment 40, wherein the M2 macrophages do not express CD80.

[0131] Embodiment 42 is the method of any one of embodiments 39 to 41, wherein the M2 macrophages express CD14 or CD206, or both.

[0132] Embodiment 43 is the method of any of embodiments 28-42, further comprising co-administering an additional therapeutic agent.

[0133] Embodiment 44 is the method of embodiment 43, wherein the additional therapeutic agent comprises an immunomodulatory monoclonal antibody or an immunomodulatory small molecule. EXAMPLES

[0134] The present invention is illustrated in the following examples. The specific examples, materials, amounts, and treatments should be interpreted broadly in accordance with the scope and spirit of the invention described herein.

[0135] Scanning Electron Microscope (SEM) Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and resuspended in human plasma. One vial of PEP (75 mg) was reconstituted in 1 mL of sterile saline. PEP and PBMCs were then mixed and gently agitated for 30-60 min. After co-incubation, samples were washed using centrifugation and sterile PBS. The supernatant was removed and samples were resuspended in tramp fixative for electron microscopy.

[0136] qPCR Cells were purified with Trizol reagent and RNA was isolated using RNA isolation spin columns. After quantification and quality assessment using a spectrophotometer (NANODROP 2000, Thermo Fisher Scientific, Inc., Waltham, MA), RNA samples were reverse transcribed into cDNA. Gene expression was then quantified by real-time quantitative PCR using the SYBR GREEN RT-qPCR kit. Reactions were measured in a quantitative real-time thermal cycler. Transcript levels were monitored at 200 rpm for 1 h. ΔΔCt method and normalized to housekeeping genes.

[0137] Immunohistochemistry (IHC) Formalin-fixed samples were embedded in paraffin and cut into 10 μm sections on a microtome. Further sections were deparaffinized with successive xylene washes, rehydrated in decreasing amounts of ethanol baths, and finally washed with water. Antigen retrieval was performed by immersing sections in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) and boiling in a pressure cooker for 10 min. Sections were then permeabilized with blocking buffer (PBS + 5% normal donkey serum, 5% BSA, 0.2% Triton-X) for 1 h at room temperature. Primary antibodies against Mφ and M2 macrophages were diluted in blocking buffer overnight at 4°C. Secondary antibodies (ALEXA FLUOR, Thermo Fisher Scientific, Inc., Waltham, MA) were then diluted 1:500 in blocking buffer and incubated for 1 h at room temperature. After washing, PROLONG Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, Inc., Waltham, MA) was added to the sections, coverslips were applied, and images were taken on an inverted fluorescence microscope with a variable fluorescence objective (AXIO OBSERVER, Carl ZEISS AG, Oberkochen, Germany).

[0138] In vitro culture of mouse monocytes Peripheral blood mononuclear cells (PBMCs) were isolated from mouse blood. + Cells were treated with PEP and / or LPS and the treated cells were harvested for analysis.

[0139] In vitro culture of mouse macrophages Mice were sacrificed according to IACUC standards. Femurs were harvested from sacrificed mice and flushed with PBS to isolate bone marrow cells. Bone marrow cells were washed and cultured in medium containing macrophage colony stimulating factor to promote macrophage differentiation. Adherent macrophages were harvested, treated with PEP and / or LPS, and then harvested for analysis.

[0140] Flow cytometry of mouse monocytes or macrophages Treated mouse monocytes or macrophages were harvested and washed in PBS. For detection by flow cytometry, cells were stained with fluorescently conjugated antibodies (anti-Ki-67 for proliferation, anti-IL-1β for identification of M1 macrophages, and anti-CD206 for identification of M2 macrophages). After incubation with fluorescent antibodies, cells were washed and analyzed by flow cytometer.

[0141] Microglial chemotaxis HMC3 cells (ATCC: CRL-3304; American Type Culture Collection, Manassas, VA) were plated at 3 × 10 5 The wells were plated at a density of 100 cells / well and incubated at 37°C and 5% CO2 for 24 hours. After 24 hours, two wells were treated with 20 ng / mL recombinant human IL-4 (R&D Systems, Inc., Minneapolis, MN). After 24 hours, two more wells were treated with 10 ng / mL LPS (Sigma-Aldrich, St. Louis, MO). After another 24 hours, all wells were washed with PBS and treated with serum-free minimal essential medium (EMEM). Cells were then trypsinized and plated at 3 × 10 cells / well for chemotaxis on 96-well plates (INCUCYTE Clearview, Essen Bioscience, Inc., Ann Arbor, MI). 3 Plates were seeded at a density of 1000 cells / well. The bottom reservoir was filled with PBS and returned to the incubator to allow cells to attach. After 2 hours, the bottom reservoir of each group was replaced with serum-free EMEM or a range of PEP dilutions in serum-free EMEM (1.5%, 2.5%, 5%, and 10%). Plates were monitored for 6 days in the incubator (Sartorius AG, Göttingen, Germany) and analyzed using a live cell analysis system (Sartorius AG, Göttingen, Germany).

[0142] Microglial proliferation HMC3 cells (ATTC:CRL-3304; American Type Culture Collection, Manassas, VA) were plated at 3 × 10 5 Cells were plated at a density of 1000 cells / well and incubated for 24 hours at 37°C and 5% CO2. After 24 hours, two wells were treated with 20 ng / mL human recombinant IL-4 (R&D Systems, Inc., Minneapolis, MN). After 24 hours, two more wells were treated with 10 ng / mL LPS (Sigma-Aldrich, St. Louis, MO). After another 24 hours, cells were washed with PBS, and then half of the wells were treated with serum-free EMEM and the remaining wells with 10% PEP in serum-free medium. Plates were monitored for 6 days in an incubator (Sartorius AG, Gottingen, Germany) and analyzed using a live cell analysis system (Sartorius AG, Gottingen, Germany).

[0143] Flow cytometry in human T cells Peripheral blood mononuclear cells (PBMCs) were isolated from apheresis blood cones via a FICOLL (Cytiva, Marlborough, MA) gradient. Total PBMCs were plated in the absence (unstimulated) or presence (stimulated) of CD3 / CD28 stimulants, and in the absence or presence of PEP, for a total of 7 days. After 7 days, cells were harvested and stained with fluorescently conjugated antibodies for detection by flow cytometry. Anti-CD45 antibodies were used to detect leukocytes, anti-CD3 antibodies to detect T cells, and anti-CD4 antibodies to detect CD4+ helper T cells. Cells were washed and analyzed on a flow cytometer.

[0144] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, nucleotide sequence deposits in GenBank and RefSeq, and amino acid sequence deposits in SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference. In the event of any inconsistency between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application shall control. The foregoing detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be construed therefrom. The invention is not limited to the exact details shown and described, and modifications obvious to one skilled in the art are within the scope of the invention as defined by the claims.

[0145] Unless otherwise indicated, all numbers expressing amounts of ingredients, molecular weights, and the like used in the specification and claims should be understood in all instances to be modified by the term "approximately" or "about". Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending on the desired properties sought to be obtained by the present invention. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, all numerical values ​​inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of reported significant digits and by ordinary rounding techniques.

[0146] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

1. 1. A method comprising: Culturing hematopoietic progenitor cells or cells differentiated from hematopoietic progenitor cells with immunomodulatory exosomes to produce treated cells; The method, wherein the immunomodulatory exosomes are derived from platelet-rich plasma that has been subjected to multiple freeze-thaw cycles and lyophilization.

2. The method of claim 1 , wherein the hematopoietic progenitor cells comprise bone marrow progenitor cells.

3. The method of claim 1 , wherein the hematopoietic progenitor cells comprise lymphoid progenitor cells.

4. The method of claim 1 , wherein the cells differentiated from hematopoietic progenitor cells are peripheral blood mononuclear cells (PBMCs).

5. The method of claim 4 , wherein the PBMCs comprise bone marrow cells.

6. The method of claim 5 , wherein the bone marrow cells comprise monocytes or macrophages.

7. The method of claim 4 , wherein the PBMCs comprise lymphocytes.

8. 8. The method of claim 7, wherein the lymphocytes comprise B cells, T cells, or NK cells.

9. The method of claim 1 , wherein the treated cells comprise monocytes or macrophages.

10. A composition for use in treating a subject suffering from an inflammation-related disease state, comprising treated cells according to any one of claims 1 to 9, wherein the composition is administered to the subject in an amount effective to modulate the immune system of the subject.

11. 11. The composition of claim 10, wherein the inflammation-associated disease state comprises a chronic pulmonary respiratory disease, an acute respiratory disease, an inflammatory joint disease, atherosclerosis, diabetes, cardiovascular disease, inflammatory bowel disease, a neuroinflammatory disease, or osteoporosis.

12. 11. The composition of claim 10, wherein the inflammation-associated disease state comprises infection with SARS-CoV-2.

13. The composition of claim 10 , wherein the treated cells comprise M2 macrophages.

14. The composition of claim 13, wherein the M2 macrophages express CD163.

15. The composition of claim 10, wherein the treated cells express CD14 or CD206, or both.

16. 11. The composition of claim 10, wherein the treated cells express IL-10, TGF-β, IL-1ra, or arginase 1, or a combination thereof.

17. 1. A composition comprising immunomodulatory exosomes for use in treating an inflammation-associated disease state in a subject, comprising: The composition, wherein the immunomodulatory exosomes are derived from platelet-rich plasma that has been subjected to multiple freeze-thaw cycles and lyophilization.

18. 18. The composition of claim 17, wherein the inflammation-associated disease state comprises a chronic pulmonary respiratory disease, an acute respiratory disease, an inflammatory joint disease, atherosclerosis, diabetes, cardiovascular disease, inflammatory bowel disease, a neuroinflammatory disease, or osteoporosis.

19. 19. The composition of claim 18, wherein the inflammation-associated disease state comprises infection with SARS-CoV-2.

20. 19. The composition of claim 18, wherein the inflammation-associated disease state comprises chronic obstructive pulmonary disease (COPD).

21. 20. The composition of claim 18, wherein the inflammation-associated disease state comprises myocarditis.

22. The composition of any one of claims 17 to 21, wherein the composition is formulated for intravenous administration.

23. The composition of any one of claims 17 to 21, wherein the composition is formulated for intramuscular administration.

24. The composition of any one of claims 17 to 21, wherein the composition is formulated for intraperitoneal administration.

25. The composition of any one of claims 17 to 21, wherein the composition further comprises one or more additional active agents.

26. 22. The composition of any one of claims 17 to 21, wherein the composition is for use to treat farm animals, domesticated animals, zoo animals, or humans.

27. The composition of claim 25, wherein the composition is for use in treating livestock, domesticated animals, zoo animals, or humans.

28. The composition of claim 17, wherein administering the immunomodulatory exosomes to the subject promotes proliferation of Treg cells.

29. The Treg cells are CD3 ＋ CD4 ＋ CD25 ｈｉ FoxP3 ＋ 29. The composition of claim 28, having a phenotype comprising:

30. The Treg cells are CD127 ｌｏ 30. The composition of claim 29, having a phenotype further comprising:

31. The composition of claim 17, wherein administering the immunomodulatory exosome promotes proliferation of M2 macrophages.

32. The composition of claim 31 , wherein the M2 macrophages express CD163.

33. The composition of claim 31 , wherein the M2 macrophages do not express CD80.

34. 32. The composition of claim 31, wherein the M2 macrophages express CD14 or CD206, or both.

35. The composition described in any one of claims 17 to 21, wherein the composition is co-administered to the subject with an additional therapeutic agent.

36. 36. The composition of claim 35, wherein the additional therapeutic agent comprises an immunomodulatory monoclonal antibody or an immunomodulatory small molecule.

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