Regulatory t cells exposed to microgravity and uses thereof

Abstract

Methods of producing immunosuppressive Treg cells by exposing Treg cells to microgravity are disclosed. The Treg cells may be obtained directly from a sample from a subject, or CD4+ T cells may be induced to become Tregs. Compositions comprising the Treg cells exposed to microgravity are disclosed. The compositions may comprise other cell types and / or other components, such as cytokines.

Description

REGULATORY T CELLS EXPOSED TO MICROGRAVITY AND USES THEREOFBACKGROUND OF INVENTION

[0001] This application claims priority under 35 U.S.C. § 199(e) to U.S. Provisional Application, U.S.S.N. 63 / 738129, filed December, 23, 2024 which is incorporated herein by reference.BACKGROUND OF INVENTION

[0002] Adoptive regulatory T (Treg) cell therapies have been proposed to treat a variety of human diseases. Methods for Treg cell manufacturing have undergone robust preclinical examination, however, not without various challenges. Of these challenges, a key barrier to successful production of Treg-based therapies is the difficulty in maintaining a stable and suppressive Treg cell phenotype following ex vivo cellular expansion. These suboptimal cellular phenotypes then likely necessitate the large loading doses required for therapeutic efficacy, increasing both the time and cost to manufacture these cellular therapies.Advancements in the field to maintain this suppressive phenotype before treatment are necessary before the true potential of these therapies can be realized.SUMMARY OF INVENTION

[0003] The present disclosure generally relates to immunosuppressive regulatory T (Treg) cells that have been exposed to microgravity. Aspects of the disclosure relate to producing immunosuppressive Treg cells by exposing naturally occurring Treg (nTregs) cells and / or induced Treg (iTreg) cells to microgravity. The nTreg cells may be obtained directly from a blood sample from a subject (e.g., a human subject). Additionally, or alternatively, CD4+ T cells obtained from a peripheral blood sample or a bone marrow sample may be induced to become Tregs (iTregs). Other aspects of the disclosure relate to compositions comprising the Treg cells that have been exposed to microgravity and the uses of such cells and compositions. The compositions may include other cell types and / or other components, such as cytokines. In some cases, exposure of the cells or composition to microgravity (or stimulated microgravity) increases the immunosuppressive activity of the Treg cells within the compositions. Additional aspects of the disclosure relate to methods of producing immunosuppressive Tregs by exposing said Tregs to microgravity. The disclosure further provides methods of treating an autoimmune disease, inflammatory disease, neurological 1 / 191#14753190vldisease, or a genetic disorder using Treg cells exposed to microgravity and / or compositions and kits thereof disclosed herein.

[0004] Aspects of the disclosure relate to Treg cells exposed to microgravity. In some embodiments, Treg cells are exposed to microgravity for at least 0.1 hours. In some embodiments, Treg cells are exposed to microgravity for less than 30 days. In some embodiments, Treg cells are exposed to microgravity for between 0.1 hours and 30 days. In some embodiments, Treg cells are exposed to microgravity for between 2 to 216 hours (or about 10 days).

[0005] Other aspects of the disclosure relate to compositions comprising a microgravity cell culture (MCC) medium comprising a plurality of Treg cells. In some embodiments, the composition has been exposed to microgravity for between 2 to 216 hours.

[0006] Additional aspects of the disclosure relate to Treg cells exposed to microgravity (e.g., for between 2 to 216 hours) and / or compositions comprising a microgravity cell culture (MCC) medium comprising a plurality of Treg cells, wherein the composition has been exposed to microgravity (e.g., for between 2 to 216 hours) for use as a medicament. The disclosure, in some aspects, further relates to Treg cells exposed to microgravity for between 2 to 216 hours and / or compositions comprising a microgravity cell culture (MCC) medium comprising a plurality of Treg cells, wherein the composition has been exposed to microgravity for between 2 to 216 hours for use in treating an autoimmune disease, inflammatory disease, a neurological disease, or a genetic disorder.

[0007] In some aspects, the disclosure further relates to Treg cells exposed to microgravity (e.g., for between 2 to 216 hours) and / or compositions comprising a microgravity cell culture (MCC) medium comprising a plurality of Treg cells, wherein the composition has been exposed to microgravity (e.g., for between 2 to 216 hours) for use during and / or after surgery.

[0008] In some aspects, the disclosure relates to methods for producing immunosuppressive Treg cells by exposing a plurality of Treg cells to microgravity, for example by placing iTregs and / or nTregs into a cell culture media and exposing them to stimulated microgravity.

[0009] In some aspects, the disclosure relates to methods for treating an autoimmune disease, inflammatory disease, neurological disease, or a genetic disorder. In some embodiments, the method comprises administering Treg cells exposed to microgravity (e.g., for between 2 to 216 hours) and / or of compositions comprising a microgravity cell culture (MCC) medium comprising a plurality of Treg cells, wherein the composition has been exposed to microgravity (e.g., for between 2 to 216 hours).2 / 191#14753190vl

[0010] Other methods disclosed herein relate to methods of treating a subject during and / or after surgery. In some embodiments, the methods comprise administering Treg cells exposed to microgravity (e.g., for between 2 to 216 hours) and / or of compositions comprising a microgravity cell culture (MCC) medium comprising a plurality of Treg cells, wherein the composition has been exposed to microgravity (e.g., for between 2 to 216 hours).

[0011] The disclosure also provides for kits comprising Treg cells exposed to microgravity (e.g,. for between 2 to 216 hours) and / or of compositions comprising a microgravity cell culture (MCC) medium comprising a plurality of Treg cells, wherein the composition has been exposed to microgravity (e.g., for between 2 to 216 hours).

[0012] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure.DEFINITIONSRegulatory T (Treg) cells

[0013] The term “Regulatory T cells” or “Treg cells” refers to a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Treg cells are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. As used herein, the term Treg refers to any cell that expresses CD4 and FoxP3. Optional biomarkers may include CD3+, CD25+, CD5+, CD14-, CD19-, CD39+, CD103+, CTLA4+, Folate receptor 4 (FR4)+, GITR+, CD223+, LAP+, LRRC32 / GARP+, BDCA-4+, Ox40 / CD134+, CD62L+.

[0014] As used herein, Treg cells includes the terms “Natural Treg cells” or “nTreg cells” which refer to Treg cells isolated directly from a peripheral blood sample and the terms “Induced Treg cells” or “ iTreg cells”, which refers to Treg cells that have been obtained via ex vivo differentiation and expansion of CD4+ T cells isolated from a peripheral blood sample, an organ (e.g., thymus), peripheral tissues (e.g., gut-associated lymphoid tissue, skin, lung, liver, etc.), bone marrow, secondary lymph organs (e.g., spleen and lymph nodes), an non-lymphoid tissues (e.g., adipose tissue, skin, muscle, lung, intestine, tumor tissue).Cytokines

[0015] The term “cytokines” refers to a broad category of proteins that mediate various aspects of cell signaling. It is known in the art that cytokines exert their function by interacting with a specific cytokine receptor on the target cell surface. As used herein, the 3 / 191#14753190vlterm cytokines may include chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, hormones or growth factors. Exemplary cytokines include, but are not limited to IL-2, IL-7, IL-10, IL-35, IL15, IL-12, IFN-gamma, and / or TGF-beta.T responder cells

[0016] The term “T responder cells” as used herein, refers to CD4 T helper cells that are defined as being CD4+ CD25- (to ensure that they are not Treg-like).Adjuvant

[0017] The term “adjuvant,” as used herein, refers to a substance or combination of substances that increases the effectiveness of another substance or drug. In some embodiments, administration of one or more of the following adjuvants with any one of the immunosuppressive Treg cells or compositions disclosed herein further enhances the therapeutic effect of the Treg cells and / or compositions: intravenous immunoglobulin (IVIG), disease modifying antirheumatic drugs (DMARDS) (e.g., chloroquine, hydroxychloroquine, leflunomide, methotrexate, sulfasalazine, mesalamine); immunosuppressants (e.g., azathioprine, 6-mercaptopurine, cyclophosphamide, mycophenolate mofetil); calcineurin inhibitors (e.g., cyclosporine, pimecrolimus, tacrolimus); mTOR inhibitors (e.g., everolimus, sirolimus); biologies (e.g., abatacept, adalimumab, anakinra, anifrolumab, axatilimab, belimumab, benralizumab, brodalumab, canakinumab, certolizumab pegol, dupilumab, eculizumab, efgartigimod, emapalumab, etanercept, golimumab, guselkumab, infliximab, ixekizumab, mepolizumab, omalizumab, reslizumab, rilonacept, risankizumab, rituximab, sarilumab, secukinumab, tezepelumab, tildrakizumab, tocilizumab, ustekinumab, vedolizumab); JAK inhibitors (e.g., baricitinib, filgotinib, tofacitinib, upadacitinib); integrin inhibitors (natalizumab, vedolizumab); SIP receptor modulators (e.g., fingolimod, ozanimod, ponesimod, siponimod); complement inhibitors (e.g., eculizumab, ravulizumab); phosphodiesterase (PDE) inhibitors (e.g., apremilast, roflumilast); corticosteroids (e.g., dexamethasone, hydrocortisone, methylprednisolone, prednisone, triamcinolone, budesonide, fluticasone); antihistamines (e.g., cetirizine, diphenhydramine, famotidine, fexofenadine, loratadine, ranitidine); non-steroidal anti-inflamatory drugs (NSAIDs) (e.g., aspirin, celecoxib, diclofenac, ibuprofen, naproxen, indomethacin); antifibrotics (e.g., nintedabib, pirfenidone); leukotriene modifiers (e.g., montelukast, zafirlukast); immunomodulatory antibiotics (e.g., clarithromycin, dapsone, metronidazole, minocycline); probiotic s / prebiotics (e.g., bifidobacterium spp., lactobacillus spp.); antimetabolites (e.g., 6-mercaptopurine,4 / 191#14753190vlthioguanine); anti-protease (e.g., alpha- 1 antitrypsin); cholinesterase inhibitors (e.g., donepezil, rivastigmine, galantamine); NMDA receptor antagonist e.g., memantine);Alzheimer’s disease modifying therapies (e.g., lecanemab, aducanumab, donanemab, dopaminergic therapies, levodopa / carbidopa, pramipexole, ropinirole, selegiline, rasagiline); ALS disease modifying therapies (e.g., riluzole, edaravone); MS disease modifying therapies (e.g., interferon beta-la, glatiramer acetate, fingolimod, dimethyl fumarate, natalizumab, ocrelizumab); and kinase inhibitors (e.g., belumosudil, ibrutinib, ruxolitinib).

[0018] In some embodiments, the adjuvant comprises one or more metabolic supplements. In some embodiments, the adjuvant comprises one or more stimulators. In some embodiments, the adjuvant comprises one or more chemical additives.Metabolic supplement

[0019] The term “metabolic supplement” as used herein refers to any substance or combination of compounds added to the culture medium to support and optimize cellular metabolism. Exemplary metabolic supplements include amino acids (e.g. arginine, glutamine, serine, glycine); glucose; human serum; vitamins (e.g. riboflavin, biotin, folic acid); lipids (e.g. palmitic acid, oleic acid, linoleic acid); minerals; and nucleotides (e.g. adenosine, guanosine, uridine, cytidine. In some embodiments, any media disclosed herein comprises one or more metabolic supplements.Chemical Additive

[0020] The term “chemical additives” as used herein refers to any chemically defined substance that is intentionally added to the culture medium to support or modify the growth, survival, differentiation, or function of cells in vitro. Exemplary chemical additives include antibiotic s / antimyc otic s (e.g. penicillin- streptomycin, amphotericin B); small molecule inhibitors / activators (e.g. kinase inhibitors, STAT5 activators); recombinant albumin; all-trans retinoic acid; taurine; antioxidants (e.g. N-acetylcysteine). In some embodiments, any media disclosed herein comprises one or more chemical additives.Stimulators

[0021] The term “stimulators” as used herein refers to substances or compounds added to promote specific cellular activities such as proliferation, activation, differentiation, or the secretion of desired molecules. Exemplary stimulators include mitogenic stimulators (e.g. phytohemagglutinin (PHA), concanavalin A (ConA)); chemical stimulators [e.g. ionomycin,5 / 191#14753190vlphorbol 12-myristate 13-acetate (PMA)]; antigen- specific stimulators (e.g. anti-CD3 / CD28 antibodies, antigen-peptide complexes); and differentiation stimulators (e.g. all-trans retinoic acid (ATRA)). In some embodiments, any media disclosed herein comprises one or more stimulators.Peripheral blood mononuclear cells (PBMCs)

[0022] The term “Peripheral blood mononuclear cells” or “PBMCs,” as used herein, refers to any peripheral blood cell having a round nucleus. These cells include lymphocytes (T cells, B cells, NK cells) and monocytes. They do not include erythrocytes and platelets nor do they include granulocytes (e.g., neutrophils, basophils, and eosinophils). These cells can be extracted from whole blood using methods known in the art, for example, via ficoll, a hydrophilic polysaccharide that separates layers of blood, and gradient centrifugation, which will separate the blood into a top layer of plasma, followed by a layer of PBMCs (buffy coat) and a bottom fraction of polymorphonuclear cells (such as neutrophils and eosinophils) and erythrocytes.CD4+ T cells

[0023] The term “CD4+ T cells,” as used herein includes the terms “CD4+ helper T cells” or “helper T cells” and refers to Thl, Th2, Thl7, T regulatory (Treg), and follicular T helper cells (Tfh). Thl cells, defined by the expression of lineage cytokine interferon (IFN)-y and the master transcription factor T-bet, participate in type 1 immune responses to intracellular pathogens, such as mycobacterial species and viruses; Th2 cells, defined by the expression of lineage cytokines interleukin (IL)-4 / IL-5 / IL-13 and the master transcription factor GATA3, participate in type 2 immune responses to larger extracellular pathogens, such as helminths; Thl7 cells, defined by the expression of lineage cytokines IL-17 / IL-22 and the master transcription factor RORyt, participate in type 3 immune responses to extracellular pathogens, including some bacteria and fungi; Tfh cells, by producing IL-21 and expressing Bcl6, help B cells produce corresponding antibodies; whereas Foxp3-expressing Treg cells, unlike Thl / Th2 / Thl7 / Tfh exerting their effector functions, regulate immune responses to maintain immune cell homeostasis, and prevent immunopathology.Differentiation cell culture (DCC) medium

[0024] The term “differentiation” medium as used herein refers to a cell culture medium specifically tailored to drive differentiation of CD4+ T cells into induced Treg cells. In some 6 / 191#14753190vlembodiments, the differentiation medium comprises a basal media (e.g., a serum free basal media such as TexMACS™ GMP). In some embodiments, the basal media comprises Penicillin-Streptomycin Solution e.g., 1%), Human AB serum (or patient derived serum at 10%), GMP ActiveMax® Human T cell Activation / Expansion CD3 / CD28 Beads (e.g., 1,000,000 beads for every 106cells), GMP Human IL-2 Protein (e.g., 100 lU / mL), GMP Recombinant Human TGF-beta 1 (e.g., 1 ng / mL), and / or retinoic acid (Powder USP) (e.g., 100 nM). The differentiation media may comprise other compounds in other embodiments.Stimulation cell culture (SCC) Media

[0025] The term “stimulation” medium as used herein refers to a cell culture medium specifically tailored to activate and maintain the suppressive function of natural Treg cells. In some embodiments, the stimulation medium comprises a basal media (e.g., a serum free basal media such as TexMACS™ GMP). In some embodiments, the basal media comprises 1% Penicillin-Streptomycin Solution (e.g., 1%), Human AB serum (or patient derived serum at 10%), GMP ActiveMax® Human T cell Activation / Expansion CD3 / CD28 Beads (e.g. 1,000,000 beads for every 106cells), GMP Human IL-2 protein (e.g., 300 lU / mL), retinoic acid (Powder USP) (e.g. 100 nM), and / or MACS GMP Rapamycin (e.g., 100 nM). The stimulation media may comprise other compounds in other embodiments.Expansion cell culture (ECC) media

[0026] The term “expansion” medium as used herein refers to a cell culture medium specifically tailored to expand natural or induced Treg cells. In some embodiments, the expansion medium comprises a basal media (e.g., a serum free basal media such as TexMACS™ GMP). In some embodiments, the basal media is added in addition to differentiation and / or stimulation media. In some embodiments, the basal media comprises Penicillin-Streptomycin solution (e.g., 1%), Human AB serum (or patient derived serum at 10%), GMP Human IL-2 Protein (e.g., 300 lU / mL), MACS GMP Rapamycin (e.g., 50 nM), and / or Retinoic acid (Powder USP) (e.g., 50 nM). The expansion media may comprise other compounds in other embodiments.Microgravity cell culture (MCC) medium

[0027] The term “microgravity” medium as used herein refers to a cell culture medium specifically tailored for maintaining and / or promoting natural or induced Treg cell phenotypes during exposure to microgravity. In some embodiments, the microgravity 7 / 191#14753190vlmedium comprises a basal media (e.g., a serum free basal media such as TexMACS™ GMP). In some embodiments, the basal media comprises Penicillin-Streptomycin Solution (e.g. 1%), Human AB serum (or patient derived serum at 10%), cytokines [e.g., IL-2 (10 to 300 lU / mL), IL-7 (5 to 50 ng / mL), TGF-betal (1 to 100 ng / mL), IL-10 (1 to 100 ng / mL), IL-35 (5 to 100 ng / mL), IL-12 (0.1 to 100 ng / mL), IFN-gamma (0.1 to 100 ng / mL) and / or IL-15 (5 to 50 ng / mL)], nutritional / metabolic supplements (e.g. arginine (0.5 to 5 mM), glutamine (2 to 6 mM), serine (0.5 to 5 mM), glycine (0.5 to 10 mM)), chemical additives (e.g., taurine (0.1 to 5 mM), N-acetylcysteine (0.1 to 10 mM), all-trans retinoic acid (1 to 1000 nM)), stimulators (e.g., anti-CD3 / CD28 stimulatory beads at 1,000,000 beads for every 106cells), and / or other compounds (e.g. rapamycin (10 to 100 nM)). The differentiation media may comprise other compounds in other embodiments.Peripheral blood venipuncture

[0028] The term “peripheral blood venipuncture,” as used herein, refers to a minimally invasive procedure that involves extracting blood from a vein of a human subject from which PBMCs are isolated.Apheresis instrument

[0029] The terms “apheresis” or “apheresis instrument” refers to a medical technology in which the blood of a subject is passed through a device that separates out one or more particular blood components (e.g., white blood cells) and returns the remainder of blood components to the subject. When this isolated blood component is white blood cells, the process is referred to as leukapheresis.Subject

[0030] The term “subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cow, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development. A “subject in need thereof’ refers to an individual who has a disease, a sign 8 / 191#14753190vland / or symptom of a disease, or a predisposition toward a disease (e.g., a genetic predisposition), with the purpose to cure, heal, alleviate, relieve, alter, remedy, prevent, ameliorate, improve, or affect the disease, the symptom of the disease, or the predisposition toward the disease. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is human. In some embodiments, the mammal is a rodent. In some embodiments, the rodent is a mouse. In some embodiments, the rodent is a rat. In some embodiments, the mammal is a companion animal. A “companion animal” refers to pets and other domestic animals. Non-limiting examples of companion animals include dogs and cats; livestock, such as horses, cattle, pigs, sheep, goats, and chickens; and other animals, such as mice, rats, guinea pigs, and hamsters.Microgravity

[0031] The term “microgravity” refers to a measure of the degree to which an object is subjected to acceleration due to gravity. In general parlance, the term is used synonymously with zero gravity and weightlessness; however, microgravity refers to accelerations equivalent to one-millionth (IO-6) of the force of gravity at Earth’s surface. Thus, as used herein, the term microgravity is a general term encompassing any gravitational force an object can experience that is less than the gravitational force the object would experience if it were on the Earth’s surface. The acceleration due to gravity (g) on Earth is 9.8 m / s2or 32 ft / s2. Thus, in some embodiments, microgravity refers to the gravitational forces experienced by an object in low Earth orbit (LEO) or approximately 90% of Earth’s gravity. In some embodiments, microgravity refers to the gravitational forces experienced by an object on the moon, or about 16.5% of Earth’s gravity. In some embodiments, microgravity refers to the gravitational forces experienced by an object on Mars, or about 38% of Earth’s gravity. In some embodiments, the term “microgravity” refers to an object (e.g., a cell) in free fall (e.g., an object falling solely under the influence of gravity). Objects in free fall do not experience a change in gravitational force but instead are exposed to a constant gravitational force, and those gravitational forces are counteracted by orbital motion or other conditions.

[0032] Accordingly, in some embodiments, an object experiences accelerations that are greater than or equal to 10’9, greater than or equal to 10’8, greater than or equal to 10’7, greater than or equal to 10’6, greater than or equal to 10’5, greater than or equal to 10’4, greater than or equal to 10’3, greater than or equal to 10’2, greater than or equal to 101of the force of gravity at Earth’s surface. In some embodiments, the object experiences accelerations that are less than or equal to 101, less than or equal to 10’2, less than or equal to 10’3, less than or equal to 9 / 191#14753190vl10’4, less than or equal to 10’5, less than or equal to 10’6, less than or equal to 10’7, less than or equal to 10’8, or less than or equal to 10’9of the force of gravity at Earth’s surface.Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the object experiences accelerations that are greater than or equal to 10’9and less than or equal to 101of the force of gravity at Earth’s surface.Medicament

[0033] A “medicament” refers to a substance used for medical treatment. As used herein a medicament may refer to any Treg cell exposed to microgravity or composition thereof as disclosed herein. The medicament in some cases further comprises one or more adjuvants.Autoimmune disease

[0034] An “autoimmune disease” refers to a disease arising from an inappropriate immune response of the body of a subject against substances and tissues normally present in the body. In other words, the immune system mistakes some part of the body as a pathogen and attacks its own cells. This may be restricted to certain organs (e.g., in autoimmune thyroiditis) or involve a particular tissue in different places (e.g., Goodpasture’s disease which may affect the basement membrane in both the lung and kidney). The treatment of autoimmune diseases is typically with immunosuppression, e.g., medications which decrease the immune response. Exemplary autoimmune diseases include, but are not limited to, glomerulonephritis, Goodpasture’s syndrome, necrotizing vasculitis, lymphadenitis, polyarteritis nodosa, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis / polymyositis, antiphospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated vasculitis e.g., Wegener’s granulomatosis, microscopic polyangiitis), uveitis, Sjogren’s syndrome, Crohn’s disease, Reiter’s syndrome, ankylosing spondylitis, Lyme disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, and cardiomyopathy.

[0035] In some embodiments, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, polymyositis, Sjogren syndrome, Hashimoto thyroiditis, Graves’ disease, type 1 insulin dependent diabetes, Addison disease, vitiligo, pernicious anemia, glomerulonephritis, myasthenia gravis, pulmonary fibrosis, psoriasis, psoriatic arthritis, Crohn’s disease, celiac disease, ulcerative colitis, autoimmune gastritis, multiple sclerosis, myasthenia gravis, and autoimmune encephalitis.10 / 191#14753190vlInflammatory disease

[0036] The terms “inflammatory disease” and “inflammatory condition” are used interchangeably herein, and refer to a disease or condition caused by, resulting from, or resulting in inflammation. Inflammatory diseases and conditions include those diseases, disorders or conditions that are characterized by signs of pain (dolor, from the generation of noxious substances and the stimulation of nerves), heat (calor, from vasodilatation), redness (rubor, from vasodilatation and increased blood flow), swelling (tumor, from excessive inflow or restricted outflow of fluid), and / or loss of function (functio laesa), which can be partial or complete, temporary or permanent. Inflammation takes on many forms and includes, but is not limited to, acute, adhesive, atrophic, catarrhal, chronic, cirrhotic, diffuse, disseminated, exudative, fibrinous, fibrosing, focal, granulomatous, hyperplastic, hypertrophic, interstitial, metastatic, necrotic, obliterative, parenchymatous, plastic, productive, proliferous, pseudomembranous, purulent, sclerosing, seroplastic, serous, simple, specific, subacute, suppurative, toxic, traumatic, and / or ulcerative inflammation. The term “inflammatory disease” may also refer to a dysregulated inflammatory reaction that causes an exaggerated response by macrophages, granulocytes, and / or T-lymphocytes leading to abnormal tissue damage and / or cell death. An inflammatory disease can be either an acute or chronic inflammatory condition and can result from infections or noninfectious causes.Inflammatory diseases include, without limitation, atherosclerosis, arteriosclerosis, autoimmune disorders, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatica (PMR), gouty arthritis, degenerative arthritis, tendonitis, bursitis, psoriasis, cystic fibrosis, arthrosteitis, rheumatoid arthritis, inflammatory arthritis, Sjogren’s syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, diabetes (e.g., Type I), myasthenia gravis, Hashimoto’s thyroiditis, Graves’ disease, Goodpasture’s disease, mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, pernicious anemia, inflammatory dermatoses, usual interstitial pneumonitis (UIP), asbestosis, silicosis, bronchiectasis, berylliosis, talcosis, pneumoconiosis, sarcoidosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, giant cell interstitial pneumonia, cellular interstitial pneumonia, extrinsic allergic alveolitis, Wegener’s granulomatosis and related forms of angiitis (temporal arteritis and polyarteritis nodosa), inflammatory dermatoses, hepatitis, delayed-type hypersensitivity reactions (e.g., poison ivy dermatitis), pneumonia, respiratory tract inflammation, Acute Respiratory Distress 11 / 191#14753190vlSyndrome (ARDS), encephalitis, immediate hypersensitivity reactions, asthma, hay fever, allergies, acute anaphylaxis, rheumatic fever, glomerulonephritis, pyelonephritis, cellulitis, cystitis, chronic cholecystitis, ischemia (ischemic injury), reperfusion injury, allograft rejection, host-versus-graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacryoadenitis, dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, pharyngitis, pleuritis, phlebitis, pneumonitis, proctitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, testitis, tonsillitis, urethritis, urocystitis, uveitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, angiitis, chronic bronchitis, osteomyelitis, optic neuritis, temporal arteritis, transverse myelitis, necrotizing fasciitis, and necrotizing enterocolitis..

[0037] Additional exemplary inflammatory conditions include, but are not limited to, inflammation associated with acne, anemia (e.g., aplastic anemia, hemolytic autoimmune anemia), asthma, arteritis (e.g., polyarteritis, temporal arteritis, periarteritis nodosa, Takayasu’s arteritis), arthritis (e.g., crystalline arthritis, osteoarthritis, psoriatic arthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis and Reiter’s arthritis), ankylosing spondylitis, amylosis, amyotrophic lateral sclerosis, autoimmune diseases, allergies or allergic reactions, atherosclerosis, bronchitis, bursitis, chronic prostatitis, conjunctivitis, Chagas disease, chronic obstructive pulmonary disease, cermatomyositis, diverticulitis, diabetes (e.g., type I diabetes mellitus, Type II diabetes mellitus), a skin condition (e.g., psoriasis, eczema, bums, dermatitis, pruritus (itch)), endometriosis, Guillain-Barre syndrome, infection, ischemic heart disease, Kawasaki disease, glomerulonephritis, gingivitis, hypersensitivity, headaches (e.g., migraine headaches, tension headaches), ileus (e.g., postoperative ileus and ileus during sepsis), idiopathic thrombocytopenic purpura, interstitial cystitis (painful bladder syndrome), gastrointestinal disorder (e.g., selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn’s disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet’s syndrome, indeterminate colitis) and inflammatory bowel syndrome (IBS)), lupus, multiple sclerosis, morphea, myasthenia gravis, myocardial ischemia, nephrotic syndrome, pemphigus 12 / 191#14753190vlvulgaris, pernicious anemia, peptic ulcers, polymyositis, primary biliary cirrhosis, neuroinflammation associated with brain disorders (e.g., Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease), prostatitis, chronic inflammation associated with cranial radiation injury, pelvic inflammatory disease, reperfusion injury, regional enteritis, rheumatic fever, systemic lupus erythematosus, scleroderma, sarcoidosis, spondyloarthropathies, Sjogren’s syndrome, thyroiditis, transplantation rejection, tendonitis, trauma or injury (e.g., frostbite, chemical irritants, toxins, scarring, burns, physical injury), vasculitis, vitiligo, and Wegener’s granulomatosis.

[0038] In certain embodiments, the inflammatory disorder is selected from the group consisting of rheumatoid arthritis, psoriasis, asthma, hepatitis, arthritis, cardiovascular disease, Kawasaki disease, chronic obstructive pulmonary disorder, ulcerative colitis, Crohn’s disease, diabetes, vasculitis, gout, sarcoidosis, systemic lupus erythematosus, Hashimoto’s thyroiditis, Grave’s disease, ankylosing spondylitis, antiphospholipid antibody syndrome, chronic recurrent multifocal osteomyelitis, Henoch- Schonlein Purpura, idiopathic thrombocytopenic purpura, juvenile dermatomyositis, juvenile idiopathic arthritis, juvenile lupus, juvenile scleroderma, juvenile vasculitis, mixed connective tissue disease, myositis, poststreptococcal inflammatory syndrome, psoriatic arthritis, reactive arthritis, scleroderma, Sjogren’s syndrome, uveitis, vasculitis, encephalitis, atherosclerosis, myocardial infarction, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, obesity, sepsis, chronic viral infection, aging, wound injury, hemophagocytic lymphohistiocytosis, atopic dermatitis, pemphigus vulgaris, Guillain-Barre syndrome, hemophilia, B cell autoimmunity, acute respiratory distress syndrome, osteoarthritis. The compounds disclosed herein may also be useful in treating inflammation associated with cancer.Genetic disease

[0039] The term “genetic disease” refers to a disease caused by one or more abnormalities in the genome of a subject, such as a disease that is present from birth of the subject. In some embodiments, the disease comprises an inflammatory and / or autoimmune component.Genetic diseases may be heritable and may be passed down from the parents’ genes. A genetic disease may also be caused by mutations or changes of the DNAs and / or RNAs of the subject. In such cases, the genetic disease will be heritable if it occurs in the germline.Exemplary genetic diseases include, but are not limited to, Aarskog-Scott syndrome, Aase syndrome, achondroplasia, acrodysostosis, addiction, adreno-leukodystrophy, albinism, ablepharon-macrostomia syndrome, alagille syndrome, alkaptonuria, alpha- 1 antitrypsin 13 / 191#14753190vldeficiency, Alport’s syndrome, Alzheimer’s disease, asthma, autoimmune polyglandular syndrome, androgen insensitivity syndrome, Angelman syndrome, ataxia, ataxia telangiectasia, atherosclerosis, attention deficit hyperactivity disorder (ADHD), autism, baldness, Batten disease, Beckwith-Wiedemann syndrome, Best disease, bipolar disorder, brachy dactyl), breast cancer, Burkitt lymphoma, chronic myeloid leukemia, Charcot-Marie-Tooth disease, Crohn’s disease, cleft lip, Cockayne syndrome, Coffin Lowry syndrome, colon cancer, congenital adrenal hyperplasia, Cornelia de Lange syndrome, Costello syndrome, Cowden syndrome, craniofrontonasal dysplasia, Crigler-Najjar syndrome, Creutzfeldt-Jakob disease, cystic fibrosis, deafness, depression, diabetes, diastrophic dysplasia, DiGeorge syndrome, Down’s syndrome, dyslexia, Duchenne muscular dystrophy, Dubowitz syndrome, ectodermal dysplasia, Ellis- van Creveld syndrome, Ehlers-Danlos, epidermolysis bullosa, epilepsy, essential tremor, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Friedreich’s ataxia, Gaucher disease, glaucoma, glucose galactose malabsorption, glutaricaciduria, gyrate atrophy, Goldberg Shprintzen syndrome (velocardiofacial syndrome), Gorlin syndrome, Hailey-Hailey disease, hemihypertrophy, hemochromatosis, hemophilia, hereditary motor and sensory neuropathy (HMSN), hereditary nonpolyposis colorectal cancer (HNPCC), Huntington’s disease, immunodeficiency with hyper- IgM, juvenile onset diabetes, Klinefelter’s syndrome, Kabuki syndrome, Leigh’s disease, long QT syndrome, lung cancer, malignant melanoma, manic depression, Marfan syndrome, Menkes syndrome, miscarriage, mucopolysaccharide disease, multiple endocrine neoplasia, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, myotonic dystrophy, neurofibromatosis, Niemann-Pick disease, Noonan syndrome, obesity, ovarian cancer, pancreatic cancer, Parkinson’s disease, paroxysmal nocturnal hemoglobinuria, Pendred syndrome, peroneal muscular atrophy, phenylketonuria (PKU), polycystic kidney disease, Prader-Willi syndrome, primary biliary cirrhosis, prostate cancer, REAR syndrome, Refsum disease, retinitis pigmentosa, retinoblastoma, Rett syndrome, Sanfilippo syndrome, schizophrenia, severe combined immunodeficiency, sickle cell anemia, spina bifida, spinal muscular atrophy, spinocerebellar atrophy, sudden adult death syndrome, Tangier disease, Tay-Sachs disease, thrombocytopenia absent radius syndrome, Townes-Brocks syndrome, tuberous sclerosis, Turner syndrome, Usher syndrome, von Hippel-Lindau syndrome, Waardenburg syndrome, Weaver syndrome, Werner syndrome, Williams syndrome, Wilson’s disease, xeroderma pigmentosum, and Zellweger syndrome.

[0040] In some embodiments, the genetic disorder is selected from the group of inflammatory and / or auto-immune genetic conditions consisting of IPEX syndrome (Immune 14 / 191#14753190vlDysregulation, Polyendocrinopathy, Enteropathy, X-linked Syndrome), Autoimmune Lymphoproliferative Syndrome (ALPS), Autoimmune Polyendocrinopathy Syndrome Type 1 (APS-1), Chediak-Higashi Syndrome, Systemic Juvenile Idiopathic Arthritis (SJIA), Chronic Granulomatous Disease (CGD), Shwachman-Diamond syndrome, Fanconi anemia, Familial Hemophagocytic Lymphohistiocytosis (HLH), and Wiskott-Aldrich Syndrome (WAS)Neurological disease

[0041] The term “neurological disease” refers to any disease of the nervous system, including diseases that involve the central nervous system (brain, brainstem and cerebellum), the peripheral nervous system (including cranial nerves), and the autonomic nervous system (parts of which are located in both central and peripheral nervous system). Neurodegenerative diseases refer to a type of neurological disease marked by the loss of nerve cells, including, but not limited to, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, tauopathies (including frontotemporal dementia), and Huntington’s disease. Examples of neurological diseases include, but are not limited to, headache, stupor and coma, dementia, seizure, sleep disorders, trauma, infections, neoplasms, neuro-ophthalmology, movement disorders, demyelinating diseases, spinal cord disorders, and disorders of peripheral nerves, muscle and neuromuscular junctions. Addiction and mental illness, include, but are not limited to, bipolar disorder and schizophrenia, are also included in the definition of neurological diseases. Further examples of neurological diseases include acquired epileptiform aphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy; agenesis of the corpus callosum; agnosia; Aicardi syndrome; Alexander disease; Alpers’ disease; alternating hemiplegia; Alzheimer’s disease; amyotrophic lateral sclerosis; anencephaly; Angelman syndrome; angiomatosis; anoxia; aphasia; apraxia; arachnoid cysts; arachnoiditis; Arnold-Chiari malformation; arteriovenous malformation; Asperger syndrome; ataxia telangiectasia; attention deficit hyperactivity disorder; autism; autonomic dysfunction; back pain; Batten disease; Behcet’s disease; Bell’s palsy; benign essential blepharospasm; benign focal; amyotrophy; benign intracranial hypertension; Binswanger’s disease; blepharospasm; Bloch Sulzberger syndrome; brachial plexus injury; brain abscess; brain injury; brain tumors (including glioblastoma multiforme); spinal tumor; Brown-Sequard syndrome; Canavan disease; carpal tunnel syndrome (CTS); causalgia; central pain syndrome; central pontine myelinolysis; cephalic disorder; cerebral aneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism; cerebral palsy; Charcot-Marie-Tooth disease; chemotherapy-induced neuropathy and neuropathic pain; Chiari malformation; chorea; chronic15 / 191#14753190vlinflammatory demyelinating polyneuropathy (CIDP); chronic pain; chronic regional pain syndrome; Coffin Lowry syndrome; coma, including persistent vegetative state; congenital facial diplegia; corticobasal degeneration; cranial arteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing’s syndrome; cytomegalic inclusion body disease (CIBD); cytomegalovirus infection; dancing eyes-dancing feet syndrome;Dandy -Walker syndrome; Dawson disease; De Morsier’s syndrome; Dejerine-Klumpke palsy; dementia; dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia; dysgraphia; dyslexia; dystonias; early infantile epileptic encephalopathy; empty sella syndrome; encephalitis; encephaloceles; encephalotrigeminal angiomatosis; epilepsy; Erb’s palsy; essential tremor; Fabry’s disease; Fahr’s syndrome; fainting; familial spastic paralysis; febrile seizures; Fisher syndrome; Friedreich’s ataxia; frontotemporal dementia and other “tauopathies”; Gaucher’s disease; Gerstmann’s syndrome; giant cell arteritis; giant cell inclusion disease; globoid cell leukodystrophy; Guillain-Barre syndrome; HTLV-1 associated myelopathy; Hallervorden- Spatz disease; head injury; headache; hemifacial spasm; hereditary spastic paraplegia; heredopathia atactica polyneuritiformis; herpes zoster oticus; herpes zoster; Hirayama syndrome; HIV-associated dementia and neuropathy (see also neurological manifestations of AIDS); holoprosencephaly; Huntington’s disease and other polyglutamine repeat diseases; hydranencephaly; hydrocephalus; hypercortisolism; hypoxia; immune-mediated encephalomyelitis; inclusion body myositis; incontinentia pigmenti; infantile; phytanic acid storage disease; Infantile Refsum disease; infantile spasms; inflammatory myopathy; intracranial cyst; intracranial hypertension; Joubert syndrome; Kearns-Sayre syndrome; Kennedy disease; Kinsboume syndrome; Klippel Feil syndrome; Krabbe disease; Kugelberg-Welander disease; kuru; Lafora disease; Lambert-Eaton myasthenic syndrome; Landau- Kleffner syndrome; lateral medullary (Wallenberg) syndrome; learning disabilities; Leigh’s disease; Lennox-Gastaut syndrome; Lesch-Nyhan syndrome; leukodystrophy; Lewy body dementia; lissencephaly; locked-in syndrome; Lou Gehrig’s disease (aka motor neuron disease or amyotrophic lateral sclerosis); lumbar disc disease; lyme disease-neurological sequelae; Machado-Joseph disease; macrencephaly; megalencephaly; Melkersson-Rosenthal syndrome; Menieres disease; meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly; migraine; Miller Fisher syndrome; mini-strokes; mitochondrial myopathies; Mobius syndrome; monomelic amyotrophy; motor neuron disease; moyamoya disease; mucopolysaccharidoses; multi-infarct dementia; multifocal motor neuropathy; multiple sclerosis and other demyelinating disorders; multiple system atrophy with postural hypotension; muscular dystrophy; myasthenia gravis; myelinoclastic diffuse sclerosis;16 / 191#14753190vlmyoclonic encephalopathy of infants; myoclonus; myopathy; myotonia congenital; narcolepsy; neurofibromatosis; neuroleptic malignant syndrome; neurological manifestations of AIDS; neurological sequelae of lupus; neuromyotonia; neuronal ceroid lipofuscinosis; neuronal migration disorders; Niemann-Pick disease; O’Sullivan-McLeod syndrome; occipital neuralgia; occult spinal dysraphism sequence; Ohtahara syndrome; olivopontocerebellar atrophy; opsoclonus myoclonus; optic neuritis; orthostatic hypotension; overuse syndrome; paresthesia; Parkinson’s disease; paramyotonia congenita; paraneoplastic diseases; paroxysmal attacks; Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodic paralyses; peripheral neuropathy; painful neuropathy and neuropathic pain; persistent vegetative state; pervasive developmental disorders; photic sneeze reflex; phytanic acid storage disease; Pick’s disease; pinched nerve; pituitary tumors; polymyositis; porencephaly; Post-Polio syndrome; postherpetic neuralgia (PHN); postinfectious encephalomyelitis; postural hypotension; Prader-Willi syndrome; primary lateral sclerosis; prion diseases; progressive; hemifacial atrophy; progressive multifocal leukoencephalopathy; progressive sclerosing poliodystrophy; progressive supranuclear palsy; pseudotumor cerebri; Ramsay-Hunt syndrome (Type I and Type II); Rasmussen’s Encephalitis; reflex sympathetic dystrophy syndrome; Refsum disease; repetitive motion disorders; repetitive stress injuries; restless legs syndrome; retrovirus-associated myelopathy; Rett syndrome; Reye’s syndrome; Saint Vitus Dance; Sandhoff disease; Schilder’s disease; schizencephaly; septo-optic dysplasia; shaken baby syndrome; shingles; Shy-Drager syndrome; Sjogren’s syndrome; sleep apnea; Soto’s syndrome; spasticity; spina bifida; spinal cord injury; spinal cord tumors; spinal muscular atrophy; stiff-person syndrome; stroke; Sturge-Weber syndrome; subacute sclerosing panencephalitis; subarachnoid hemorrhage; subcortical arteriosclerotic encephalopathy; Sydenham chorea; syncope; syringomyelia; tardive dyskinesia; Tay-Sachs disease; temporal arteritis; tethered spinal cord syndrome; Thomsen disease; thoracic outlet syndrome; tic douloureux; Todd’s paralysis; Tourette syndrome; transient ischemic attack; transmissible spongiform encephalopathies; transverse myelitis; traumatic brain injury; tremor; trigeminal neuralgia; tropical spastic paraparesis; tuberous sclerosis; vascular dementia (multi-infarct dementia); vasculitis including temporal arteritis; Von Hippel-Lindau Disease (VHL); Wallenberg’s syndrome; Werdnig-Hoffman disease; West syndrome; whiplash; Williams syndrome; Wilson’s disease; and Zellweger syndrome.

[0042] In some embodiments, the neurological disease is selected from the group consisting of Alzheimer’s Disease, Parkinson’s Disease, Amyotrophic Lateral Sclerosis (ALS), multiple17 / 191#14753190vlsclerosis, stroke, traumatic brain injury, epilepsy, myasthenia gravis, Huntington’s disease, Lewy Body Dementia, Frontotemporal Dementia, Vascular Dementia, and prion infections.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1: Representative iTreg and nTreg gating strategy, including a Cells gate and Singlet gate, for the characterization of cellular phenotypes. While not displayed, both single stains and FMOs, alongside unstained controls, were used to determine positive / negative populations.

[0044] Figure 2: Naive T lymphocyte gating strategy. (A) Representative naive T lymphocyte gating strategy, including a Lymphocytes gate and Singlet gate, for the characterization of cellular phenotype. Representative (B) single stains and (C) FMOs for the determination of positive / negative populations also presented.

[0045] Figure 3: Naive CD4+ T lymphocyte isolation and culture, differentiation, and expansion of induced T regulatory cells (iTregs). (A) Percentage (%) of Live Cells positive for CD4. (B) Percentage (%) of CD4+ Cells CD45RO+ CD45RA-, CD45RO+ CD45RA+, CD45RO- CD45RA+, and CD45RO- CD45RA- (of CD4+ cells). (C) Principal component analysis (PCA) of unpaired RNA sequencing comparing Control iTregs to baseline (isolated naive T lymphocytes). (D) Enrichment plot of GSE14415_INDUCED_TREG_VS_TCONV_UP from the C7: ImmuneSigDB gene set enrichment analysis comparing Control iTregs to baseline (isolated naive T lymphocytes). Data derived from n=2 biological duplicates (two human donors) for flow cytometric analysis and n=3 biological triplicates (three human donors) for RNA sequencing analysis. Error bars represent standard error of the mean (SE).

[0046] Figure 4: Flow cytometric analysis of Control, Control_s (30 min, 300 lU / mL IL-2 stimulation), microgravity (“pg”; simulated), and pg_s (30 min, 300 lU / mL IL-2 stimulation) iTreg cellular phenotype. (A) Percentage (%) of Live Cells. (B) Percentage (%) of iTregs (of Live Cells), defined as CD4+ FoxP3+ cells. (C) Percentage (%) of CD4+ FoxP3+ pSTAT5+ cells (of CD4+ FoxP3+ cells). (D) Percentage of CD4+ FoxP3+ CD25+ cells (of CD4+ FoxP3+ cells). (E) Percentage (%) of CD4+ FoxP3+ CD45RO+ cells (of CD4+ FoxP3+ cells). (F) Percentage (%) of CD4+ FoxP3+ CD3+ cells (of CD4+ FoxP3+ cells). Data derived from at least an n=3 biological triplicates (three human donors, two donors repeated at multiple, different times) for a total n=6. All data points used in paired t-test analysis18 / 191#14753190vlpresented on graphs, with outlier(s) removed using the ROUT method. Error bars represent standard error of the mean (SE).

[0047] Figure 5: Flow cytometric analysis of Control, Control_s (30 min, 300 lU / mL IL-2 stimulation), pg (simulated), and pg_s (30 min, 300 lU / mL IL-2 stimulation) iTreg Mean Fluorescence Intensity (MFI). (A) FoxP3 MFI of CD4+ FoxP3+ cells. (B) Delta (A) of FoxP3 MFI between Control and pg groups. (C) pSTAT5 MFI of CD4+ FoxP3+ pSTAT5+ cells. (D) Delta (A) pSTAT5 of pSTAT5 MFI between Control and pg groups. (E) CD25 MFI of CD4+ FoxP3+ CD25+ cells. (F) Delta (A) CD25 of CD25 MFI between Control and pg groups. (G) CD45RO MFI of CD4+ FoxP3+ CD45RO+ cells. (H) Delta (A) CD45RO of CD45RO MFI between Control and pg groups. Data derived from at least an n=3 biological triplicates (three human donors, two donors repeated at multiple, different times) for a total n=6. All data points used in paired t-test analysis presented on graphs, with outlier(s) removed using the ROUT method. Error bars represent standard error of the mean (SE).

[0048] Figure 6: RNA sequencing analysis of Control and simulated pg iTregs. (A) Principal component analysis (PCA) of paired RNA sequencing. (B) Volcano plot highlighting significant differentially expressed genes (DEGs) in the paired RNA sequencing analysis. X-axis represents the log2FoldChange. Y-axis represents the -loglO(p-value). Dashed horizontal and vertical lines represent the cut-off threshold for q- value and NES significance criteria, respectively. Data derived from n=3 biological triplicates (three human donors).

[0049] Figure 7: Short- and long-term stimulation assay reveals differences in IL- 10 and latent TGF-P production from Control and simulated pg iTregs. (A) Short-term (24-hour, 300 lU / mL IL-2 stimulation) IL- 10 production (pg / mL) from Control and pg iTregs. (B) Shortterm (24-hour, 300 lU / mL IL-2 stimulation) latent TGF-P production (ng / mL). (C) Longterm (72-hour, 300 lU / mL IL-2 and CD3 / CD28 bead stimulation) IL- 10 production (pg / mL).(D) Long-term (72-hour, 300 lU / mL IL-2 and CD3 / CD28 bead stimulation) latent TGF-P production (ng / mL). Data derived from n=3 biological triplicates (three human donors, two donors repeated at multiple, different times) for a total n=5 for the short-term stimulation assay and n=3 biological triplicates (three human donors) for the long-term stimulation assay. All data points used in paired t-test analysis presented on graphs. Error bars represent standard error of the mean (SE).

[0050] Figure 8: Co-culture immunosuppressive assay reveals differences in immunosuppressive capacity of Control and pg iTregs. (A) Representative histograms of CellTrace™ Violet (CTV) staining of CD8+ T lymphocytes in unstimulated and stimulated control wells. (B) Percent (%) Suppression at 1:1, 2:1, 4:1, and 8:1 CD8:iTreg cellular ratios 19 / 191#14753190vlfor Control and pg iTregs. Data derived from n=3 biological triplicates (three human donors, one donor repeated at multiple, different times) for a total n=4. All data points used in paired t-test analysis presented on graphs. Error bars represent standard error of the mean (SE).

[0051] Figure 9: Short- (4 day) and long- (10 day) term persistence assay reveals differences in cellular persistence between Control and simulated pg iTregs. (A) Percentage (%) of Live Cells at days 4 and 10 for Control and pg iTregs. (B) Absolute Live Cells per mL at days 4 and 10 for Control and pg iTregs. (C) Percentage (%) of iTreg (CD4+ FoxP3+) cells (of Live Cells) at days 4 and 10 for Control and pg iTregs. (D) Percentage of CD4+ FoxP3+ pSTAT5+ cells (of CD4+ FoxP3+ cells) at days 4 and 10 for Control and pg iTregs. (E) Percentage (%) of CD4+ FoxP3+ CD25+ cells (of CD4+ FoxP3+ cells) at days 4 and 10 for Control and pg iTregs. (F) Percentage (%) of CD4+ FoxP3+ CD45RO+ cells (of CD4+ FoxP3+ cells) at days 4 and 10 for Control and pg iTregs. (G) FoxP3 MFI of CD4+ FoxP3+ cells at days 4 and 10 for Control and pg iTregs. (H) pSTAT5 MFI of CD4+ FoxP3+ pSTAT5+ cells at days 4 and 10 for Control and pg iTregs. (I) CD25 MFI of CD4+ FoxP3+ CD25+ cells at days 4 and 10 for Control and pg iTregs. (J) CD45RO MFI of CD4+ FoxP3+ CD45RO+ cells at days 4 and 10 for Control and pg iTregs. Data derived from n=3 biological triplicates (three human donors) for a total n=3. All data points used in paired t-test analysis presented on graphs. Error bars represent standard error of the mean (SE).

[0052] Figure 10: Flow cytometric analysis of Control, Control_s (30 min, 300 lU / mL IL-2 stimulation), pg (simulated), and pg_s (30 min, 300 lU / mL IL-2 stimulation) nTreg cellular phenotype. (A) Percentage (%) of Live Cells. (B) Percentage (%) of nTregs (of Live Cells), defined as CD4+ FoxP3+ cells. (C) Percentage (%) of CD4+ FoxP3+ pSTAT5+ cells (of CD4+ FoxP3+ cells). (D) Percentage of CD4+ FoxP3+ CD25+ cells (of CD4+ FoxP3+ cells).(E) Percentage (%) of CD4+ FoxP3+ CD45RO+ cells (of CD4+ FoxP3+ cells). (F) Percentage (%) of CD4+ FoxP3+ CD3+ cells (of CD4+ FoxP3+ cells). Data derived from n=l biological replicate (one human donor). Error bars represent standard deviation (SD) of technical replicates.

[0053] Figure 11: Flow cytometric analysis of Control, Control_s (30 min, 300 lU / mL IL-2 stimulation), pg (simulated), and pg_s (30 min, 300 lU / mL IL-2 stimulation) nTreg Mean Fluorescence Intensity (MFI). (A) FoxP3 MFI of CD4+ FoxP3+ cells. (B) Delta (A) of FoxP3 MFI between Control and pg groups. (C) pSTAT5 MFI of CD4+ FoxP3+ pSTAT5+ cells. (D) Delta (A) pSTAT5 of pSTAT5 MFI between Control and pg groups. (E) CD25 MFI of CD4+ FoxP3+ CD25+ cells. (F) Delta (A) CD25 of CD25 MFI between Control and pg groups. (G) CD45RO MFI of CD4+ FoxP3+ CD45RO+ cells. (H) Delta (A) CD45RO of 20 / 191#14753190vlCD45RO MFI between Control and pg groups. Data derived from n=l biological replicate (one human donor). Error bars represent standard deviation (SD) of technical replicates.

[0054] Figure 12: Short- and long-term stimulation assay reveals differences in IL- 10 and latent TGF-P production from Control and simulated pg nTregs. (A) Short-term (24-hour, 300 lU / mL IL-2 stimulation) IL- 10 production (pg / mL). (B) Short-term (24-hour, 300 lU / mL IL-2 stimulation) latent TGF-P production (ng / mL). (C) Long-term (72-hour, 300 lU / mL IL-2 and CD3 / CD28 bead stimulation) IL- 10 production (pg / mL). (D) Long-term (72-hour, 300 lU / mL IL-2 and CD3 / CD28 bead stimulation) latent TGF-P production (ng / mL). Data derived from n=l biological replicate (one human donor). Error bars represent standard deviation (SD) of technical replicates.

[0055] Figure 13: Co-culture immunosuppressive assay reveals differences in suppressive capacity of Control and pg nTregs. (A) Representative histograms of CellTrace™ Violet (CTV) staining of CD8+ T lymphocytes in CTVmaxand CTVmin wells. (B) Percent (%) Suppression at 1:1, 2:1, 4:1, and 8:1 CD8:nTreg cellular ratios for Control and pg nTregs. Data derived from n=l biological replicate (one human donor), conducted with n=2 experimental replicates. Error bars represent standard deviation (SD) of experimental replicates.

[0056] Figure 14: Short- (4 day) and long- (10 day) term persistence assay reveals differences in cellular persistence between Control and simulated pg nTregs. (A) Percentage (%) of Live Cells at days 4 and 10 for Control and pg nTregs. (B) Absolute Live Cells per mL at days 4 and 10 for Control and pg nTregs. (C) Percentage (%) of nTreg (CD4+ FoxP3+) cells (of Live Cells), at days 4 and 10 for Control and pg nTregs. (D) Percentage of CD4+ FoxP3+ pSTAT5+ cells (of CD4+ FoxP3+ cells) at days 4 and 10 for Control and pg nTregs. (E) Percentage (%) of CD4+ FoxP3+ CD25+ cells (of CD4+ FoxP3+ cells) at days 4 and 10 for Control and pg nTregs. (F) Percentage (%) of CD4+ FoxP3+ CD45RO+ cells (of CD4+ FoxP3+ cells) at days 4 and 10 for Control and pg nTregs. (G) FoxP3 MFI of CD4+ FoxP3+ cells at days 4 and 10 for Control and pg nTregs. (H) pSTAT5 MFI of CD4+ FoxP3+ pSTAT5+ cells at days 4 and 10 for Control and pg nTregs. (I) CD25 MFI of CD4+ FoxP3+ CD25+ cells at days 4 and 10 for Control and pg nTregs. (J) CD45RO MFI of CD4+ FoxP3+ CD45RO+ cells at days 4 and 10 for Control and pg nTregs. Data derived from n=l biological replicates (one human donor). Error bars represent standard deviation (SD) of technical replicates.21 / 191#14753190vlDETAILED DESCRIPTION

[0057] The present disclosure generally relates to regulatory T (Treg) cells exposed to microgravity. Aspects of the disclosure relate to producing immunosuppressive Treg cells by exposing Treg cells to microgravity. The Treg cells may be obtained directly from a peripheral blood sample, an organ (e.g., thymus), peripheral tissues (e.g., gut-associated lymphoid tissue, skin, lung, liver, etc.), bone marrow, secondary lymph organs (e.g., spleen and lymph nodes), an non-lymphoid tissues (e.g., adipose tissue, skin, muscle, lung, intestine, tumor tissue). Additionally, or alternatively, CD4+ T cells obtained from a peripheral blood sample may be induced to become Tregs. Other aspects of the disclosure relate to compositions and kits comprising the Treg cells exposed to microgravity. The compositions may comprise other cell types and / or other components, such as cytokines and excipients. In some cases, exposure of the compositions to microgravity increases the immunosuppressive activity of the Treg cells within the compositions. Additional aspects of the disclosure relate to methods for producing immunosuppressive Treg cells by exposing natural Treg cells to microgravity. Additional aspects of the disclosure relate to methods for producing immunosuppressive Tregs cells by exposing induced Treg cells to microgravity. The disclosure further provides methods of treating an autoimmune disease, inflammatory disease, neurological disease, cancer, or a genetic disorder (e.g., disease comprising an inflammatory component) using Treg cells and / or compositions disclosed herein.

[0058] Other aspects relate to pharmaceutical compositions comprising a population of Treg cells exposed to microgravity and / or compositions comprising a plurality of Treg cells exposed to microgravity.

[0059] Other aspects relate to pharmaceutical compositions comprising a population of Treg cells exposed to microgravity and / or compositions comprising a plurality of Treg cells exposed to microgravity for the treatment of a disease.

[0060] In some embodiments, a population of Treg cells in the pharmaceutical composition comprise at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or at least 99.9% Treg cells by total cell number.

[0061] In some embodiments, a population of Treg cells in the pharmaceutical composition comprise at least 0.1%, at least 0.5%, at least 1.0%, at least 2.5%, at least 5%, at least 10%, at least 15%, or at least 20% or at least 99.9% non-Treg cells by total cell number.

[0062] In some embodiments, the total number of cells in a pharmaceutical composition disclosed herein is between 5xl05and 5xl09cells. In some embodimens, the total number of cells in the pharmaceutical composition is greater than or equal to 5xl05, greater than or 22 / 191#14753190vlequal to 5xl06, greater than or equal to 5xl07, greater than or equal to 5xl08, or greater than or equal to 5xl09. In some embodiments, the total number of cells in the pharmaceutical composition is less than or equal to 5xl09, less than or equal to 5xl08, less than or equal to 5xl07, less than or equal to 5xl06, or less than or equal to 5xl05. Combinations are also possible in some embodiments. For example, in some embodiments, the total number of cells in the pharmaceutical composition is greater than or equal to 5xl05and less than or equal to 5xl09.Treg cells and Compositions comprising the same

[0063] Aspects of the disclosure relate to exposing regulatory T cells (Tregs) and / or compositions comprising Treg cells to microgravity. In some embodiments, the term microgravity refers to the gravitational force equivalent to one-millionth (IO-6) of the force of gravity at the Earth’s surface (e.g., 9.8 m / s2). In some embodiments, the term microgravity refers to the gravitational forces equivalent to 90% of the Earth’s gravity (e.g., low Earth orbit, LEO). In some embodiments, microgravity refers to the gravitational forces experienced by an object on the moon, or about 16.5% of Earth’s gravity. In some embodiments, microgravity refers to the gravitational forces experienced by an object on Mars, or about 38% of Earth’s gravity. In some embodiments, the term microgravity refers to an object in free fall (e.g., an object falling solely under the influence of gravity and those gravitational forces are counteracted by orbital motion or other conditions to result in apparent weightlessness).

[0064] Accordingly, in some embodiments, an object experiences accelerations that are greater than or equal to 10’9, greater than or equal to 10’8, greater than or equal to 10’7, greater than or equal to 10’6, greater than or equal to 10’5, greater than or equal to 10’4, greater than or equal to IO’3, greater than or equal to 10’2, greater than or equal to 101of the force of gravity at Earth’s surface. In some embodiments, the object experiences accelerations that are less than or equal to 101, less than or equal to 10’2, less than or equal to IO’3, less than or equal to KF4, less than or equal to KF5, less than or equal to KF6, less than or equal to KF7, less than or equal to KF8, or less than or equal to KF9of the force of gravity at Earth’s surface.Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the object experiences accelerations that are greater than or equal to KF9and less than or equal to 101of the force of gravity at Earth’s surface.

[0065] Those of skill in the art will understand that objects in free fall are exposed to a constant gravitational force but the effects of this force are counteracted by the object’s motion (e.g., simulated microgravity). Any suitable method known to the skilled artisan may 23 / 191#14753190vlbe used to expose the Treg cells and / or compositions disclosed herein to microgravity. For example, in some cases, a rotary cell culture system (e.g., Synthecon RCCS) may be used to simulate the microgravity environment of continuous free fall. In other cases, the Treg cells and / or compositions disclosed herein may be physically brought into space (e.g., LEO or beyond).

[0066] In some embodiments, a population of Treg cells comprise at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% induced Treg (iTreg) cells by total cell number. In some embodiments, a population of Treg cells comprise 99.9% or 100% iTregs by total cell number (methods of producing iTregs from CD4 cells is discussed elsewhere herein).

[0067] In some embodiments, the Treg cells comprise at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% natural Treg (nTreg) cells by total cell number. In some embodiments, a population of Treg cells comprise 99.9% or 100% nTreg s by total cell number.

[0068] In some embodiments, a population of Treg cells comprise at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% Tregs (e.g., iTregs and nTregs) cells by total cell number. In some embodiments, a population of Treg cells comprise 99.9% or 100% Tregs by total cell number (e.g., iTregs and nTregs).

[0069] In some embodiments, the disclosure relates to exposing a population of Treg (e.g., iTreg and / or nTreg) cells to microgravity. In some embodiments, a population of Tregs are exposed to microgravity for between 0.1 hours and 30 days. In some embodiments, a population of Treg cells are exposed to microgravity for at least 2 to 216 hours. In some embodiments, a population of Tregs are exposed to microgravity for greater than or equal to 2 hours, greater than or equal to 10 hours, greater than or equal to 50 hours, greater than or equal to 100 hours, greater than or equal to 125 hours, greater than or equal to 150 hours, greater than or equal to 175 hours, greater than or equal to 200 hours, or greater than or equal to 216 hours. In some embodiments, a population of Tregs are exposed to microgravity for less than or equal to 216 hours, less than or equal to 200 hours, less than or equal to 175 hours, less than or equal to 150 hours, less than or equal to 125 hours, less than or equal to 100 hours, less than or equal to 50 hours, less than or equal to 10 hours, or less than or equal to 2 hours. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, a population of Treg cells are exposed to microgravity for greater than or equal to 2 hours but less than or equal to 216 hours.24 / 191#14753190vl

[0070] In some embodiments, the effects of microgravity on a population of Treg cells (e.g., iTreg and / or nTreg cells) lasts from 1 hour to 24 hours. In some embodiments, the effects of microgravity on a population of Treg cells e.g., iTreg and / or nTreg cells) lasts from 1 day to 30 days. In some embodiments, the effects of microgravity on a population of Treg cells (e.g., iTreg and / or nTreg cells) lasts from 1 month to 1 year. In some embodiments, the effects of microgravity on a population of Treg cells (e.g., iTreg and / or nTreg cells) lasts greater than 1 year.

[0071] In some embodiments, exposure of a population of Treg cells (e.g., iTreg cells and / or nTreg cells) to microgravity (e.g., for between 2 and 216 hours) increases the Treg cells secretion of one or more cytokines. In some embodiments, the one or more cytokines comprise IL- 10 and / or TGF-beta. In some embodiments, the one or more cytokines is IL- 10. In some embodiments, the one or more cytokines is TGF-beta.

[0072] In some embodiments, the one or more cytokines (e.g., IL- 10 and / or TGF-beta) have an immunosuppressive function. For example, it is generally known in the art that efficient immune responses require regulated antigen presentation to CD4+ T cells, and that IL- 10 inhibits the ability of dendritic cells (DCs) and macrophages to stimulate antigen- specific CD4 T cells. Similarly, it is also known in the art that TGF-beta signaling suppresses T-cell proliferation, activation, and effector functions and plays a key role in suppressingimmuno surveillance in tumor microenvironments. Without wishing to be bound by any one particular theory, it is believed that the spatiotemporal delivery of the combination of immunosuppressive cytokines (e.g., as produced and secreted by the Tregs) rather than delivery of a single cytokine at high quantity (e.g., via intravenous cytokine therapy) that produces the desired immunomodulatory effect.

[0073] In some embodiments, exposure of a population of Treg cells (e.g., iTreg cells and / or nTreg cells) to microgravity for between 2 and 216 hours modulates one or more immunosuppressive pathways listed below. Additionally, in some embodiments, exposure of a population of Treg cells (e.g., iTreg cells and / or nTreg cells) to microgravity for between 2 and 216 hours limits proliferation more efficiently than normal Tregs (as measured by the reduction in CD4+ CD25- T responder cell proliferation at various concentration of (responder cells:Tregs) and / or CD8+ T lymphocyte cell proliferation at various concentrations of (CD8+ T lymphocyte: Tregs)).

[0074] In some embodiments, exposure of a population of Treg cells (e.g., iTreg cells and / or nTreg cells) to microgravity for between 2 and 216 hours modulates the Treg cells’ expression of one or more immunosuppressive gene expression pathways (as determined by gene set 25 / 191#14753190vlenrichment analyses). In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity include HALLMARK pathways (e.g., HALLMARK_ALLOGRAFT_REJECTION, HALLMARK_APOPTOSIS, HALLMARK_CHOLESTEROL_HOMEOSTASIS, HALLMARK_COAGULATION, HALLMARK_COMPLEMENT, HALLMARK_DNA_REPAIR, HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, HALLMARK_FATTY_ACID_METABOLISM, HALLMARK_GLYCOLYSIS, HALLMARK_HYPOXIA, HALLMARK_IL2_STAT5_SIGNALING, HALLMARK_INFLAMMATORY_RESPONSE, HALLMARK_IL6_JAK_STAT3_SIGNALING, HALLMARK_INTERFERON_ALPHA_RESPONSE, HALLMARK_INTERFERON_GAMMA_RESPONSE, HALLMARK_MITOTIC_SPINDLE, HALLMARK_MTORC 1_SIGNALING, HALLMARK_NOTCH_SIGNALING, HALLMARK_OXIDATIVE_PHOSPHORYLATION, HALLMARK_PI3K_AKT_MTOR_SIGNALING, HALLMARK_PROTEIN_SECRETION, HALLMARK_REACTIVE_OXYGEN_SPECES_PATHWAY, HALLMARK_TGF_BETA_SIGNALING, HALLMARK_TNFA_SIGNALING_VIA_NFKB, HALLMARK_UNFOLDED_PROTEIN_RESPONSE, HALLMARK_UV_RESPONSE_DN, HALLMARK_UV_RESPONSE_UP, HALLMARK_XENOBIOTIC_METABOLISM). In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity include C2 - CGP pathways (e.g.UNTERMAN_IPF_VS_CTRL_TREG_CELL_DN, UNTERMAN_IPF_VS_CTRL_TREG_CELL_UP, BAKER_HEMATOPOESIS_STAT5_TARGETS, FUNG_IL2_TARGETS_WITH_STAT5_BINDING_SITES, FUNG_IL2_TARGETS_WITH_STAT5_BINDING_SITES_T1,WIERENGA_STAT5 A_TARGETS_DN, WIERENGA_STAT5 A_TARGETS_GROUP 1 , WIERENGA_STAT5A_TARGETS_GROUP2, WIERENGA_STAT5A_TARGETS_UP, GAVIN_IL2_RESPONSIVE_FOXP3_TARGETS_DN, GAVIN_IL2_RESPONSIVE_FOXP3_TARGETS_UP, MARZEC_IL2_SIGNALING_DN, MARZEC_IL2_SIGNALING_UP, ZHENG_IL22_SIGNALING_DN, ZHENG_IL22_SIGNALING_UP, DASU_IL6_SIGNALING_DN, DASU_IL6_SIGNALING_SCAR_DN, DASU_IL6_SIGNALING_SCAR_UP, DASU_IL6_SIGNALING_UP, BROCKE_APOPTOSIS_REVERSED_BY_IL6,26 / 191#14753190vlDEBOSSCHER_NFKB_TARGETS_REPRESSED_BY_GLUCOCORTICOIDS, DUTERTRE_ESTRADI0L_RESP0NSE_6HR_UPDUTTA_AP0PT0SIS_VIA_NFKB, GILMORE_CORE_NFKB_PATHWAY, HANSON_HRAS_SIGNALING_VIA_NFKB , HINATA_NFKB_TARGETS_FIBROBLAST_UP, HINATA_NFKB_TARGETS_KERATINOCYTE_DN, HINATA_NFKB_TARGETS_KERATINOCYTE_UP, BREDEMEYER_RAG_SIGNALING_VIA_ATM_NOT_VIA_NFKB_DN, BREDEMEYER_RAG_SIGNALING_VIA_ATM_NOT_VIA_NFKB_UP, RASHI_NFKB 1 -TARGETS, JAIN_NFKB_SIGNALING, MARTIN_NFKB_TARGETS_DN, MARTIN_NFKB_TARGETS_UP). In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity include C2 - CP:BIOCARTA pathways (e.g. BIOCARTA_TGFB_PATHWAY, BIOCARTA_IL10_PATHWAY, BIOCARTA_IL12_PATHWAY, BIOCARTA_IL17_PATHWAY, BIOCARTA_IL1R_PATHWAY, BIOCARTA_IL22BP_PATHWAY, BIOCARTA_IL2_PATHWAY, BIOCARTA_IL2RB_PATHWAY, BIOCARTA_IL3_PATHWAY, BIOCARTA_IL4_PATHWAY, BIOCARTA_IL5_PATHWAY, BIOCARTA_IL6_PATHWAY, BIOCARTA_IL7_PATHWAY, BIOCARTA_INFLAM_PATHWAY, BIOCARTA_STAT3_PATHWAY, BIOCARTA_TCR_PATHWAY). In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity include C2 - CP:KEGG pathways (e.g. KEGG_MEDICUS_REFERENCE_CYTOKINE_JAK_STAT_SIGNALING_PATHWAY, KEGG_MEDICUS_ENV_FACTOR_NNK_NNN_TO_PI3K_SIGNALING_PATHWAY_N013 39, KEGG_MEDICUS_ENV_FACTOR_NNK_NNN_TO_PI3K_SIGNALING_PATHWAY_N013 50, KEGG_MEDICUS_REFERENCE_TGFA_EGFR_PI3K_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_TGFA_EGFR_PLCG_PKC_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_TGFA_EGFR_RAS_ERK_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_EGF_EGFR_ACTIN_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_EGF_EGFR_PI3K_NFKB_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_EGF_EGFR_PI3K_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_EGF_JAK_STAT_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_TLR1_2_4_NFKB_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_TLR2_4_MAPK_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_HORMONE_LIKE_CYTOKINE_TO_JAK_STAT_SIGNA 27 / 191#14753190vlLING_PATHWAY, KEGG_MEDICUS_REFERENCE_IC0SLG_IC0S_PI3K_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_IFN_RIPK1_3_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_IL10_FAMILY_TO_JAK_STAT_SIGNALING_PATHWA Y, KEGG_MEDICUS_REFERENCE_IL 12_23_TO_JAK_STAT_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_IL1_IL1R_JNK_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_IL1_IL1R_P38_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_IL2_FAMILY_T0_JAK_STAT_SIGNALING_PATHWAYKEGG_MEDICUS_REFERENCE_IL6_FAMILY_T0_JAK_STAT_SIGNALING_PATHWAY , KEGG_MEDICUS_REFERENCE_ORGANIZATION_OF_THE_INNER_KINETOCHORE, KEGG_MEDICUS_REFERENCE_ORGANIZATION_OF_THE_OUTER_KINETOCHORE, KEGG_MEDICUS_REFERENCE_CCR2_GNB_G_PI3K_NFKB_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_CCR5_GNB_G_PLCB_G_PKC_SIGNALING_PATHWAYKEGG_MEDICUS_REFERENCE_CCR_CXCR_GNB_G_PI3K_RAC_SIGNALING_PATHW AY, KEGG_MEDICUS_REFERENCE_CX3CR1_GNAI_AC_PKA_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_CXCL12_CXCR4_PKC_ERK_SIGNALING_PATHAWAY , KEGG_MEDICUS_REFERENCE_CXCR4_GNA 12_ 13_RH0_SIGNALING_PATHWA Y, KEGG_MEDICUS_REFERENCE_CXCR4_GNAI_PI3K_BAD_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_CXCR4_GNAQ_PLCB_G_CALCINEURIN_SIGNALING_ PATHWAY, KEGG_MEDICUS_REFERENCE_CXCR4_GNB_G_PLCB_PKC_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_CXCR4_GNB_G_RAC_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_CXCR_GNB_G_ERK_SIGNALING_PATHWAY, KEGG_MEDICUS_REFERENCE_CXCR_GNB_G_PI3K_AKT_SIGNALING_PATHWAY, KEGG_MEDICUS_VARIANT_TGFA_0VEREXPRESSI0N_T0_PI3K_SIGNALING_PATH WAY). In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity include C2 - CP:REACTOME pathways (e.g.REACTOME_SIGNALING_BY_TGF_BETA_RECEPTOR_COMPLEX, REACTOME_RUNX1_AND_FOXP3_CONTROL_THE_DEVELOPMENT_OF_REG, REACTOME_ADORA2B_MEDIATED_ANTI_INFLAMMATORY_CYTOKINES_PRODUC TION, REACTOME_ANTIVIRAL_MECHANISM_BY_IFN_STIMULATED_GENES,28 / 191#14753190vlREACTOME_ADAPTIVE_IMMUNE_SYSTEM, REACTOME_DOWNREGULATION_OF_TGF_BETA, REACTOME_SIGNALING_BY_TGF_BETA_RECEPTOR_COMPLEX, REACTOME_SIGNALING_BY_TGF_BETA_RECEPTOR_COMPLEX_IN_CANCER, REACTOME_SIGNALING_BY_TGFB_FAMILY_MEMBERS, REACTOME_TGF_BETA_RECEPTOR_SIGNALING_ACTIVATES_SMADS, REACTOME_TGF_BETA_RECEPTOR_SIGNALING_IN_EMT_EPITHELIAL_TO_MESEN CHYMAL_TRANSITION, REACT0ME_TGFBR3_EXPRESSI0N, REACT0ME_TGFBR3_PTM_REGULATI0N, REACT0ME_TGFBR3_REGULATES_TGF_BETA_SIGNALING, REACTOME_DOWNREGULATION_OF_TGF_BETA_RECEPTOR_SIGNALING, REACTOME_GENE_AND_PROTEIN_EXPRESSION_BY_JAK_STAT_SIGNALING_AFTE R_INTERLEUKIN_12_STIMULATI0N, REACT0ME_STAT5_ACTIVATI0N, REACTOME_STAT5_ACTIVATION_DOWNSTREAM_OF_FLT3_ITD_MUTANTS, REACTOME_GENE_AND_PROTEIN_EXPRESSION_BY_JAK_STAT_SIGNALING_AFTE R_INTERLEUKIN_12_STIMULATI0N, REACTOME_FCGR3A_MEDIATED_IL10_SYNTHESIS, REACT0ME_AD0RA2B_MEDIATED_ANTI_INFLAMMAT0RY_CYT0KINES_PR0DUC TION). In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity include C2 - CP: WIKIPATHWAYS pathways (e.g.WP_CANCER_IMMUNOTHERAPY_BY_CTLA4_BLOCKADE, WP_CANCER_IMMUNOTHERAPY_BY_PD1_BLOCKADE, WP_CANONICAL_AND_NONCANONICAL_NOTCH_SIGNALING, WP_CANONICAL_AND_NONCANONICAL_TGFB_SIGNALING, WP_CANONICAL_NFKB_PATHWAY, WP_CHEMOKINE_SIGNALING, WP_CONTROL_OF_IMMUNE_TOLERANCE_BY_VASOACTIVE_INTESTINAL_PEPTID E, WP_CYTOKINECYTOKINE_RECEPTOR_INTERACTION, WP_CYTOKINES_AND_INFLAMMATORY_RESPONSE, WP_EPAC1_AND_PKA_REDUCTION_OF_RETINAL_INFLAMMATION, WP_FOXP3_IN_COVID 19, WP_IL 10_ANTIINFLAMMATORY_SIGNALING, WP_IL2_SIGNALING, WP_IMMUNE_RESPONSE_TO_TUBERCULOSIS, WP_INCLUSION_BODY_MYOSITIS, WP_LEUKOCYTEINTRINSIC_HIPPO_PATHWAY_FUNCTIONS, WP_MITOCHONDRIAL_IMMUNE_RESPONSE_TO_SARSCOV2,29 / 191#14753190vlWP.NEUROINFLAMMATION, WP_NEUROINFLAMMATION_AND_GLUTAMATERGIC_SIGNALING, WP_OVERVIEW_OF_PROINFLAMMATORY_AND_PROFIBROTIC_MEDIATORS, WP_T_CELL_MODULATION_IN_PANCREATIC_CANCER, WP_T_CELL_RECEPTOR_AND_COSTIMULATORY_SIGNALING, WP_TGFB_SMAD_SIGNALING, WP_TGFBETA_RECEPTOR_SIGNALING, WP_TGFBETA_RECEPTOR_SIGNALING_IN_SKELETAL_DYSPLASIAS, WP_TGFBETA_SIGNALING_IN_THYROID_CELLS_FOR_EPITHELIALMESENCHYMAL .TRANSITION, WP_TYPE_II_INTERFERON_SIGNALING, WP.TYPE.III.INTERFERON.SIGNALING), In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity include C2 - CP:PID pathways (e.g. PID_CD8_TCR_DOWNSTREAM_PATHWAY, PID_CD8_TCR_PATHWAY, PID.CXCR3.PATHWAY, PID_CXCR4_PATHWAY, PID_FOXO_PATHWAY, PID.IFNG.PATHWAY, PID_IL12_2PATHWAY, PID_IL12_STAT4_PATHWAY, PID.ILl.PATHWAY, PID_IL23_PATHWAY, PID_IL27_PATHWAY, PID.IL2.1PATHWAY, PID_IL2_PI3K_PATHWAY, PID_IL2_STAT5_PATHWAY, PID.IL3.PATHWAY, PID_IL4_2PATHWAY, PID_IL5_PATHWAY, PID.IL6.7.PATHWAY, PID_IL8_CXCR I .PATH WAY, PID_IL8_CXCR2_PATHWAY, PID.MAPK.TRK.PATHWAY, PID.MET.PATHWAY, PID.MTOR.4PATHWAY, PID.NFKAPPAB.ATYPICAL.PATHWA, PID.NFKAPPAB.CANONICAL.PATHWAY, PID.PI3K.PLC.TRK.PATHWAY, PID_PI3KCI_AKT_PATHWAY, PID.PI3KCI.PATHWAY, PID_TCR_CALCIUM_PATHWAY, PID.TCR.JNK.PATHWAY, PID.TCR.PATHWAY, PID.TCR.RAS.PATHWAY, PID.TGFBR.PATHWAY, PID.TNF.PATHWAY, PID.TOLL.ENDOGENOUS.PATHWAY), In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity include C2 - CP:KEGG_LEGACY pathways (e.g. KEGG.ASTHMA, KEGG.AUTOIMMUNE.THYROID.DISEASE, KEGG_CALCIUM_SIGNALING_PATHWAY, KEGG_CELL_ADHESION_MOLECULES_CAMS, KEGG_CHEMOKINE_SIGNALING_PATHWAY, KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION, KEGG_FC_EPSILON_RI_SIGNALING_PATHWAY, KEGG_FC_GAMMA_R_MEDIATED_PHAGOCYTOSIS, KEGG_FOCAL_ADHESION, KEGG_GRAFT_VERSUS_HOST_DISEASE,30 / 191#14753190vlKEGG_INTESTINAL_IMMUNE_NETWORK_FOR_IGA_PRODUCTION, KEGG_JAK_STAT_SIGNALING_PATHWAY, KEGG_LEISHMANIA_INFECTION, KEGG_LEUKOCYTE_TRANSENDOTHELIAL_MIGRATION, KEGG_MAPK_SIGNALING_PATHWAY, KEGG_MATURITY_ONSET_DIABETES_OF_THE_YOUNG, KEGG_MTOR_SIGNALING_PATHWAY, KEGG_NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY, KEGG_OXIDATIVE_PHOSPHORYLATION, KEGG_PARKINSONS_DISEASE, KEGG_PATHOGENIC_ESCHERICHIA_COLI_INFECTION, KEGG_PATHWAYS_IN_CANCER, KEGG_PRIMARY_IMMUNODEFICIENCY, KEGG_REGULATION_OF_ACTIN_CYTOSKELETON, KEGG_SYSTEMIC_LUPUS_ERYTHEMATOSUS, KEGG_T_CELL_RECEPTOR_SIGNALING_PATHWAY, KEGG_TGF_BETA_SIGNALING_PATHWAY, KEGG_TOLL_LIKE_RECEPTOR_SIGNALING_PATHWAY, KEGG_TYPE_I_DIABETES_MELLITUS, KEGG_TYPE_II_DIABETES_MELLITUS). In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity include C3:MIR pathways (e.g. MIR15A_3P, MIR15A_5P, MIR15B_3P, MIR15B_5P, MIR16_1_3P, MIR16_2_3P, MIR16_5P, MIR17_3P, MIR17_5P, MIR155_3P, MIR155_5P, MIR146A_3P, MIR146A_5P, MIR142_3P, MIR142_5P, MIR335_3P, MIR335_5P). In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity include C3:TFT pathways (e.g. GATA1_O1, GATA1_O2, GATA1_O3, GATA1_O4, GATA1_O5, GATA2_01, GATA3_01, GATA4_Q3, GATA6_01, GATA_C, GATA_Q6, IRF1_O1, IRF1_Q6, IRF2_01, IRF5_TARGET_GENES, IRF7_01, IRF_Q6, NFKAPPAB65_01, NFKAPPAB.Ol, NFKB_C, NFKB_Q6, NFKB_Q6_01, NFKBIA_TARGET_GENES, SOX10_TARGET_GENES, SOX11_TARGET_GENES, SOX15_TARGET_GENES, SOX3_TARGET_GENES, SOX5_01, SOX9.B1, STATl.Ol, STAT1_O2, STAT1_O3, STAT3.01, STAT3_02, STAT4.01, STAT5A.01, STAT5A.02, STAT5A_03, STAT5A.04, STAT5B.01, STAT6.01, STAT6_02, STAT.Ol, STAT_Q6), C4:3CA (GAVISH_3CA_METAPROGRAM_CD4_T_CELLS_T_REG). In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity include C5:GO pathways e.g.GOBP_POSmVE_REGULATION_OF_NATURAL_KILLER_CELL_MEDIATED_IMMUNI TY, GOBP_NEGATIVE_REGULATION_OF_LEUKOCYTE_MEDIATED_IMMUNITY,31 / 191#14753190vlGOBP_NEGATIVE_REGULATION_OF_T_CELL_MEDIATED_IMMUNITY, GOBP_REGULATION_OF_LEUKOCYTE_MEDIATED_IMMUNITY, GOBP_REGULATION_OF_NATURAL_KILLER_CELL_MEDIATED_IMMUNITY, GOBP_REGULATION_OF_T_CELL_MEDIATED_IMMUNITY, GOBP T CELL MEDIATED IMMUNITY, GOBP_REGULATION_OF_LEUKOCYTE_MEDIATED_IMMUNITY). In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity include C5:HPO pathways (e.g. HP_ABNORMAL_CD4_CD8_RATIO, HP_ABNORMAL_CIRCULATING_C_REACTIVE_PROTEIN_CONCENTRATION, HP_ABNORMAL_CIRCULATING_IGA_LEVEL, HP_ABNORMAL_CIRCULATING_IGE_LEVEL, HP_ABNORMAL_CIRCULATING_IGG_LEVEL, HP_ABNORMAL_CIRCULATING_IGM_LEVEL, HP_ABNORMAL_CIRCULATING_INTERFERON_CONCENTRATION, HP_ABNORMAL_CIRCULATING_INTERLEUKIN_10_CONCENTRATION, HP_ABNORMAL_CIRCULATING_INTERLEUKIN_CONCENTRATION, HP_ABNORMAL_GASTRIC_MUCOSA_MORPHOLOGY, HP_ABNORMAL_GRANULOCYTE_COUNT, HP_ABNORMAL_GRANULOCYTOPOIETIC_CELL_MORPHOLOGY, HP_ABNORMAL_IMMUNE_SERUM_PROTEIN_PHYSIOLOGY, HP_ABNORMAL_IMMUNE_SYSTEM_MORPHOLOGY, HP_ABNORMAL_LEUKOCYTE_COUNT, HP_ABNORMAL_LEUKOCYTE_ENZYME_CONCENTRATION_OR_ACTIVITY, HP_ABNORMAL_LEUKOCYTE_PHYSIOLOGY, HP_ABNORMAL_LYMPHOCYTE_APOPTOSIS, HP_ABNORMAL_LYMPHOCYTE_MORPHOLOGY, HP_ABNORMAL_LYMPHOCYTE_PROLIFERATION, HP_ABNORMAL_MACROPHAGE_MORPHOLOGY, HP_ABNORMAL_MYELOID_LEUKOCYTE_MORPHOLOGY, HP_ABNORMAL_NEUTROPHIL_COUNT, HP_ABNORMAL_NEUTROPHIL_MORPHOLOGY, HP_ABNORMAL_PHAGOCYTOSIS, HP_ABNORMAL_PROPORTION_OF_CD4_POSmVE_HELPER_T_CELLS, HP_ABNORMAL_PROPORTION_OF_CD4_POSmVE_T_CELLS, HP_ABNORMAL_PROPORTION_OF_CD8_POSmVE_T_CELLS,32 / 191#14753190vlHP_ABNORMAL_PROPORTION_OF_CLASS_SWITCHED_MEMORY_B_CELLS, HP_ABNORMAL_PROPORTION_OF_DOUBLE_NEGATIVE_ALPHA_BETA_REGULATO RY_T_CELL, HP_ABNORMAL_PROPORTION_OF_MEMORY_T_CELLS, HP_ABNORMAL_PROPORTION_OF_NAIVE_T_CELLS, HP_ABNORMAL_T_CELL_ACTIVATION, HP_ABNORMAL_T_CELL_MORPHOLOGY, HP_ABNORMAL_T_CELL_PROLIFERATION, HP_ABNORMAL_T_CELL_SUBSET_DISTRIBUTION, HP_ABNORMAL_THYMUS_MORPHOLOGY, HP_ABNORMALITY_OF_HUMORAL_IMMUNITY, HP_ABNORMALITY_OF_IMMUNE_SYSTEM_PHYSIOLOGY, HP_ABNORMALITY_OF_NEUTROPHIL_PHYSIOLOGY, HP_ABNORMALITY_OF_NEUTROPHILS, HP_ABNORMALITY_OF_T_CELL_PHYSIOLOGY, HP_ABNORMALLY_LOW_T_CELL_RECEPTOR_EXCISION_CIRCLE_LEVEL, HP_ALLERGIC_RHINITIS, HP_ALLERGY, HP_ANTI_ACETYLCHOLINE_RECEPTOR_ANTIBODY_POSITIVITY, HP_ANTI_SMOOTH_MUSCLE_ANTIBODY_POSITIVITY, HP_ANTI_THYROGLOBULIN_ANTIBODY_POSITIVITY, HP_ANTI_THYROID_ANTIBODY_POSITIVITY, HP_ANTIMITOCHONDRIAL_ANTIBODY_POSITIVITY, HP_ANTINEUTROPHIL_ANTIBODY_POSITIVITY, HP_ANTINUCLEAR_ANTIBODY_POSITIVITY, HP_ANTIPHOSPHOLIPID_ANTIBODY_POSITIVITY, HP_ARTERIOSCLEROSIS, HP_ARTHRETIS, HP_ARTHROPATHY, HP_ASTHMA, HP_AUTOIMMUNE_ANTIBODY_POSITIVITY, HP_AUTOIMMUNE_HEMOLYTIC_ANEMIA, HP_AUTOIMMUNE_THROMBOCYTOPENIA, HP_AUTOIMMUNITY, HP_CELLULAR_IMMUNODEFICIENCY, HP_CHRONIC_AXONAL_NEUROPATHY, HP_CHRONIC_BRONCHITIS, HP_CHRONIC_CSF_LYMPHOCYTOSIS, HP_CHRONIC_GASTRITIS, HP_CHRONIC_HEMOLYTIC_ANEMIA, HP_CHRONIC_HEPATITIS, HP_CHRONIC_HEPATITIS_DUE_TO_CRYPTOSPORIDIUM_INFECTION, HP_CHRONIC_INFECTION, HP_CHRONIC_KIDNEY_DISEASE, HP_CHRONIC_LUNG_DISEASE, HP_CHRONIC_LYMPHOCYTIC_MENINGITIS,33 / 191#14753190vlHP_CHRONIC_NEUTROPENIA, HP_CHRONIC_NONINFECTIOUS_LYMPHADENOPATHY, HP_CHRONIC_OTITIS_MEDIA, HP_CHRONIC_PAIN, HP_CHRONIC_PANCREATITIS, HP_CHRONIC_RHINITIS, HP_CHRONIC_SINUSITIS, HP_COLITIS, HP_COMBINED_IMMUNODEFICIENCY, HP_CORONARY_ARTERY_ATHEROSCLEROSIS, HP_CSF_LYMPHOCYTIC_PLEIOCYTOSIS, HP_CSF_PLEOCYTOSIS, HP_CUTANEOUS_AMYLOIDOSIS, HP_DECREASED_CIRCULATING_ACTH_CONCENTRATION, HP_DECREASED_CIRCULATING_FOLLICLE_STIMULATING_HORMONE_CONCENTR ATION, HP_DECREASED_CIRCULATING_FREE_T3, HP_DECREASED_CIRCULATING_FREE_T4_CONCENTRATION, HP_DECREASED_CIRCULATING_IGA_LEVEL, HP_DECREASED_CIRCULATING_IGE, HP_DECREASED_CIRCULATING_IGG2_LEVEL, HP_DECREASED_CIRCULATING_IGG_SUBCLASS_LEVEL, HP_DECREASED_CIRCULATING_TOTAL_IGA, HP_DECREASED_CIRCULATING_TOTAL_IGG, HP_DECREASED_CIRCULATING_TOTAL_IGM, HP_DECREASED_LYMPHOCYTE_APOPTOSIS, HP_DECREASED_LYMPHOCYTE_PROLIFERATION_IN_RESPONSE_TO_ANTI_CD3, HP_DECREASED_LYMPHOCYTE_PROLIFERATION_IN_RESPONSE_TO_MITOGEN, HP_DECREASED_PROPORTION_OF_CD3_POSITIVE_T_CELLS, HP_DECREASED_PROPORTION_OF_CD8_POSITIVE_T_CELLS, HP_DECREASED_PROPORTION_OF_NAIVE_T_CELLS, HP_DECREASED_SPECIFIC_ANTI_POLYSACCHARIDE_ANTIBODY_LEVEL, HP_DECREASED_SPECIFIC_ANTIBODY_RESPONSE_TO_POLYSACCHARIDE_VACCI NE, HP_DEFECTIVE_T_CELL_PROLIFERATION, HP_DEMENTIA, HP_DEMYELINATING_MOTOR_NEUROPATHY, HP_DEMYELINATING_PERIPHERAL_NEUROPATHY, HP_DIABETES_INSIPIDUS, HP_DIABETIC_KETOACIDOSIS, HP_DIFFUSE_DEMYELINATION_OF_THE_CEREBRAL_WHITE_MATTER, HP_ENCEPHALOPATHY, HP_GRANULOMATOSIS, HP_HYPERSEGMENTATION_OF_NEUTROPHIL_NUCLEI, HP_HYPERSPLENISM, HP_IMMUNE_DYSREGULATION, HPJMMUNODEFICIENCY,34 / 191#14753190vlHP_IMMUNOLOGIC_HYPERSENSmVITY, HP_IMPAIRED_T_CELL_FUNCTION, HP_INCREASED_CIRCULATING_IGA_LEVEL, HP_INCREASED_CIRCULATING_IGE_LEVEL, HP_INCREASED_CIRCULATING_IGG_LEVEL, HP_INCREASED_CIRCULATING_IGM_LEVEL, HP_INCREASED_INFLAMMATORY_RESPONSE, HP_INCREASED_T_CELL_COUNT, HP_INFLAMMATION_OF_THE_LARGE_INTESTINE, HP_INFLAMMATORY_ABNORMALITY_OF_THE_EYE, HP_JUVENILE_RHEUMATOID_ARTHRITIS, HP_LEUKOCORIA, HP_LEUKOCYTOSIS, HP_LEUKODYSTROPHY, HP_LEUKOENCEPHALOPATHY, HP_LUPUS_NEPHRITIS, HP_LYMPH ADENITIS, HP_LYMPHOCYTOSIS, HP_MYELOFIBROSIS, HP_MYELOPROLIFERATIVE_DISORDER, HP_OSTEOARTHRITIS, HP_OSTEOARTHRITIS_OF_THE_SMALL_JOINTS_OF_THE_HAND, HP_PANCREATIC_FIBROSIS, HP_PATCHY_ALOPECIA,HP_PREMATURE_OSTEO ARTHRITIS, HP_RHEUMATOID_ARTHRITIS, HP_RHEUMATOID_FACTOR_POSmVE, HP_SEVERE_COMBINED_IMMUNODEFICIENCY, HP_SKIN_PLAQUE, HP_SPONDYLOLISTHESIS, HP_SPONDYLOLYSIS, HP_SYNOSTOSIS_INVOLVING_BONES_OF_THE_FEET, HP_SYNOSTOSIS_INVOLVING_BONES_OF_THE_HAND, HP_SYNOSTOSIS_INVOLVING_BONES_OF_THE_UPPER_LIMBS, HP_SYNOSTOSIS_INVOLVING_THE_ELBOW, HP_SYNOSTOSIS_OF_CARPAL_BONES, HP_SYNOSTOSIS_OF_CARPALS_TARSALS, HP_SYNOSTOSIS_OF_JOINTS, HP_SYNOSTOSIS_OF_METACARPALS_METATARSALS, HP_SYSTEMIC_LUPUS_ERYTHEMATOSUS, HP_TYPE_I_DIABETES_MELLITUS, HP_TYPE_I_TRANSFERRIN_ISOFORM_PROFILE, HP_TYPE_II_DIABETES_MELLITUS), C6 (TGFB_UP.V1_DN, TGFB_UP.V1_UP, IL15_UP.V1_DN, IL15_UP.V1_UP, IL21_UP.V1_DN, IL21_UP.V1_UP, IL2_UP.V1_DN, IL2_UP.V1_UP, JAK2_DN. V1_DN, JAK2_DN.V1_UP). In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity include C7 -IMMUNESIGB pathways (e.g. GSE14308_TH17_VS_INDUCED_TREG_DN, GSE14308_TH17_VS_INDUCED_TREG_UP, GSE14308_TH17_VS_NAIVE_CD4_TCELL_DN, GSE14308_TH17_VS_NAIVE_CD4_TCELL_UP,35 / 191#14753190vlGSE14308_TH17_VS_NATURAL_TREG_DN, GSE14308_TH17_VS_NATURAL_TREG_UP, GSE143O8_TH1_VS_INDUCED_TREG_DN, GSE143O8_TH1_VS_INDUCED_TREG_UP, GSE143O8_TH1_VS_NAIVE_CD4_TCELL_DN, GSE143O8_TH1_VS_NAIVE_CD4_TCELL_UP, GSE143O8_TH1_VS_NATURAL_TREG_DN, GSE143O8_TH1_VS_NATURAL_TREG_UP, GSE143O8_TH1_VS_TH17_DN, GSE143O8_TH1_VS_TH17_UP, GSE14308_TH2_VS_INDUCED_TREG_DN, GSE14308_TH2_VS_INDUCED_TREG_UP, GSE14308_TH2_VS_NAIVE_CD4_TCELL_DN, GSE14308_TH2_VS_NAIVE_CD4_TCELL_UP, GSE14308_TH2_VS_NATURAL_TREG_DN, GSE14308_TH2_VS_NATURAL_TREG_UP, GSE13306_TREG_RA_VS_TCONV_RA_DN, GSE 13306_TREG_RA_VS_TCONV_RA_UP, GSE13306_TREG_VS_TCONV_DN, GSE13306_TREG_VS_TCONV_LAMINA_PROPRIA_DN, GSE13306_TREG_VS_TCONV_LAMINA_PROPRIA_UP, GSE13306_TREG_VS_TCONV_SPLEEN_DN, GSE13306_TREG_VS_TCONV_SPLEEN_UP, GSE13306_TREG_VS_TCONV_UP, GSE14415_ACT_TCONV_VS_ACT_NATURAL_TREG_DN, GSE14415_ACT_TCONV_VS_ACT_NATURAL_TREG_UP, GSE14415_ACT_VS_CTRL_NATURAL_TREG_DN, GSE14415_ACT_VS_CTRL_NATURAL_TREG_UP, GSE14415_FOXP3_KO_NATURAL_TREG_VS_TCONV_DN, GSE14415_FOXP3_KO_NATURAL_TREG_VS_TCONV_UP, GSE14415_INDUCED_TREG_VS_FAILED_INDUCED_TREG_DN, GSE14415_INDUCED_TREG_VS_FAILED_INDUCED_TREG_UP, GSE14415_INDUCED_TREG_VS_FOXP3_KO_INDUCED_TREG_DN, GSE14415_INDUCED_TREG_VS_FOXP3_KO_INDUCED_TREG_IL2_CULTURE DN, GSE14415_INDUCED_TREG_VS_FOXP3_KO_INDUCED_TREG_IL2_CULTURE_UP, GSE14415_INDUCED_TREG_VS_FOXP3_KO_INDUCED_TREG_UP, GSE14415_INDUCED_TREG_VS_TCONV_DN, GSE14415_INDUCED_TREG_VS_TCONV_UP, GSE14415_INDUCED_VS_NATURAL_TREG_DN, GSE14415_INDUCED_VS_NATURAL_TREG_UP, GSE14415_NATURAL_TREG_VS_FOXP3_KO_NATURAL_TREG_DN,36 / 191#14753190vlGSE14415_NATURAL_TREG_VS_FOXP3_KO_NATURAL_TREG_UP, GSE14415_NATURAL_TREG_VS_TCONV_DN, GSE14415_NATURAL_TREG_VS_TCONV_UP, GSE14415_TCONV_VS_FOXP3_KO_INDUCED_TREG_DN, GSE14415_TCONV_VS_FOXP3_KO_INDUCED_TREG_UP, GSE15659_CD45RA_NEG_CD4_TCELL_VS_ACTIVATED_TREG_DN, GSE15659_CD45RA_NEG_CD4_TCELL_VS_ACTIVATED_TREG_UP, GSE15659_CD45RA_NEG_CD4_TCELL_VS_NONSUPPRESSIVE_TCELL_DN, GSE15659_CD45RA_NEG_CD4_TCELL_VS_NONSUPPRESSIVE_TCELL_UP, GSE15659_CD45RA_NEG_CD4_TCELL_VS_RESTING_TREG_DN, GSE15659_CD45RA_NEG_CD4_TCELL_VS_RESTING_TREG_UP, GSE15659_NAIVE_CD4_TCELL_VS_ACTIVATED_TREG_DN, GSE15659_NAIVE_CD4_TCELL_VS_ACTIVATED_TREG_UP, GSE15659_NAIVE_CD4_TCELL_VS_NONSUPPRESSIVE_TCELL_DN, GSE15659_NAIVE_CD4_TCELL_VS_NONSUPPRESSIVE_TCELL_UP, GSE15659_NAIVE_CD4_TCELL_VS_RESTING_TREG_DN, GSE15659_NAIVE_CD4_TCELL_VS_RESTING_TREG_UP, GSE15659_NAIVE_VS_PTPRC_NEG_CD4_TCELL_DN, GSE15659_NAIVE_VS_PTPRC_NEG_CD4_TCELL_UP, GSE15659_NONSUPPRESSIVE_TCELL_VS_ACTIVATED_TREG_DN, GSE15659_NONSUPPRESSIVE_TCELL_VS_ACTIVATED_TREG_UP, GSE15659_RESTING_TREG_VS_NONSUPPRESSIVE_TCELL_DN, GSE15659_RESTING_TREG_VS_NONSUPPRESSIVE_TCELL_UP, GSE15659_RESTING_VS_ACTIVATED_TREG_DN, GSE15659_RESTING_VS_ACTIVATED_TREG_UP, GSE15659_TREG_VS_TCONV_DN, GSE15659_TREG_VS_TCONV_UP, GSE17580_TREG_VS_TEFF_DN, GSE17580_TREG_VS_TEFF_S_MANSONI_INF_DN, GSE17580_TREG_VS_TEFF_S_MANSONI_INF_UP, GSE17580_TREG_VS_TEFF_UP, GSE18893_CTRL_VS_TNF_TREATED_TREG_24H_UP, GSE18893_CTRL_VS_TNF_TREATED_TREG_2H_DN, GSE18893_CTRL_VS_TNF_TREATED_TREG_2H_UP, GSE18893_TCONV_VS_TREG_24H_CULTURE_DN, GSE18893_TCONV_VS_TREG_24H_CULTURE_UP, GSE18893_TCONV_VS_TREG_24H_TNF_STIM_DN,37 / 191#14753190vlGSE18893_TCONV_VS_TREG_24H_TNF_STIM_UP, GSE18893_TCONV_VS_TREG_2H_CULTURE_DN, GSE18893_TCONV_VS_TREG_2H_CULTURE_UP, GSE18893_TCONV_VS_TREG_2H_TNF_STIM_DN, GSE18893_TCONV_VS_TREG_2H_TNF_STIM_UP, GSE18893_CTRL_VS_TNF_TREATED_TREG_24H_UP, GSE18893_CTRL_VS_TNF_TREATED_TREG_2H_DN, GSE18893_CTRL_VS_TNF_TREATED_TREG_2H_UP, GSE18893_TCONV_VS_TREG_24H_CULTURE_DN, GSE18893_TCONV_VS_TREG_24H_CULTURE_UP, GSE18893_TCONV_VS_TREG_24H_TNF_STIM_DN, GSE18893_TCONV_VS_TREG_24H_TNF_STIM_UP, GSE18893_TCONV_VS_TREG_2H_CULTURE_DN, GSE18893_TCONV_VS_TREG_2H_CULTURE_UP, GSE18893_TCONV_VS_TREG_2H_TNF_STIM_DN, GSE18893_TCONV_VS_TREG_2H_TNF_STIM_UP, GSE7852_TREG_VS_TCONV_DN, GSE7852_TREG_VS_TCONV_FAT_DN, GSE7852_TREG_VS_TCONV_FAT_UP, GSE7852_TREG_VS_TCONV_LN_DN, GSE7852_TREG_VS_TCONV_LN_UP, GSE7852_TREG_VS_TCONV_THYMUS_DN, GSE7852_TREG_VS_TCONV_THYMUS_UP, GSE7852_TREG_VS_TCONV_UP, GSE20366_EX_VIVO_VS_HOMEOSTATIC_CONVERSION_TREG_DN, GSE20366_EX_VIVO_VS_HOMEOSTATIC_CONVERSION_TREG_UP, GSE20366_TREG_VS_NAIVE_CD4_TCELL_DE, C205_CONVERSION_DN, GSE20366_TREG_VS_NAIVE_CD4_TCELL_DE, C205_CONVERSION_UP, GSE20366_TREG_VS_NAIVE_CD4_TCELL_DN, GSE20366_TREG_VS_NAIVE_CD4_TCELL_HO, MEOSTATIC_CONVERSION_DN, GSE20366_TREG_VS_NAIVE_CD4_TCELL_HO, MEOSTATIC_CONVERSION_UP, GSE20366_TREG_VS_NAIVE_CD4_TCELL_UP, GSE20366_TREG_VS_TCONV_DN, GSE20366_TREG_VS_TCONV_UP, GSE35543_IN_VITRO_ITREG_VS_CONVERTED_EX_ITREG_DN, GSE35543_IN_VITRO_ITREG_VS_CONVERTED_EX_ITREG_UP, GSE35543_IN_VIVO_NTREG_VS_CONVERTED_EX_ITREG_DN, GSE35543_IN_VIVO_NTREG_VS_CONVERTED_EX_ITREG_UP, GSE34006_A2AR_KO_VS_A2AR_AGONIST_TREATED_TREG_DN,38 / 191#14753190vlGSE34006_A2AR_KO_VS_A2AR_AGONIST_TREATED_TREG_UP, GSE34006_UNTREATED_VS_A2AR_AGONIST_TREATED_TREG_DN, GSE34006_UNTREATED_VS_A2AR_AGONIST_TREATED_TREG_UP, GSE7460_FOXP3_MUT_VS_WT_ACT_WITH_TGFB_TCONV_DN, GSE7460_FOXP3_MUT_VS_WT_ACT_WITH_TGFB_TCONV_UP, GSE7460_TCONV_VS_TREG_LN_DN, GSE7460_TCONV_VS_TREG_LN_UP, GSE7460_TCONV_VS_TREG_THYMUS_DN, GSE7460_TCONV_VS_TREG_THYMUS_UP, GSE7460_TREG_VS_TCONV_ACT_DN, GSE7460_TREG_VS_TCONV_ACT_UP, GSE7460_TREG_VS_TCONV_ACT_WITH_TGFB_DN, GSE7460_TREG_VS_TCONV_ACT_WITH_TGFB_UP, GSE7460_WT_VS_FOXP3_HET_ACT_TCONV_DN, GSE7460_WT_VS_FOXP3_HET_ACT_TCONV_UP, GSE7460_WT_VS_FOXP3_HET_ACT_WITH_TGFB_TCONV_DN, GSE7460_WT_VS_FOXP3_HET_ACT_WITH_TGFB_TCONV_UP,GSE11818_WT_VS_DICER_KO_TREG_DN, GSE11818_WT_VS_DICER_KO_TREG_UP, GSE13306_RA_VS_UNTREATED_TREG_DN, GSE13306_RA_VS_UNTREATED_TREG_UP, GSE14308_INDUCED_VS_NATURAL_TREG_DN, GSE14308_INDUCED_VS_NATURAL_TREG_UP, GSE14308_NAIVE_CD4_TCELL_VS_INDUCED_TREG_DN, GSE14308_NAIVE_CD4_TCELL_VS_INDUCED_TREG_UP, GSE14308_NAIVE_CD4_TCELL_VS_NATURAL_TREG_DN, GSE14308_NAIVE_CD4_TCELL_VS_NATURAL_TREG_UP, GSE14350_IL2RB_KO_VS_WT_TREG_DN, GSE14350_IL2RB_KO_VS_WT_TREG_UP, GSE14350_TREG_VS_TEFF_DN, GSE14350_TREG_VS_TEFF_IN_IL2RB_KO_DN, GSE14350_TREG_VS_TEFF_IN_IL2RB_KO_UP, GSE42021_TCONV_PLN_VS_TREG_PRECURSORS_THYMUS_DN, GSE42021_TCONV_PLN_VS_TREG_PRECURSORS_THYMUS_UP, GSE42021_TREG_PLN_VS_CD24HI_TREG_THYMUS_DN, GSE42021_TREG_PLN_VS_CD24HI_TREG_THYMUS_UP, GSE42021_TREG_PLN_VS_CD24INT_TREG_THYMUS_DN, GSE42021_TREG_PLN_VS_CD24INT_TREG_THYMUS_UP, GSE42021_TREG_PLN_VS_CD24LO_TREG_THYMUS_DN,39 / 191#14753190vlGSE42021_TREG_PLN_VS_CD24LO_TREG_THYMUS_UP, GSE42021_TREG_PLN_VS_TREG_PRECURSORS_THYMUS_DN, GSE42021_TREG_PLN_VS_TREG_PRECURSORS_THYMUS_UP,GSE42021_TREG_VS_TCONV_PLN_DN, GSE42021_TREG_VS_TCONV_PLN_UP GSE28130_ACTIVATED_VS_INDUCEED_TREG_DN,GSE28130_ACTIVATED_VS_INDUCEED_TREG_UP, GSE6681_DELETED_FOXP3_VS_WT_TREG_DN, GSE6681_DELETED_FOXP3_VS_WT_TREG_UP, GSE6875_TCONV_VS_FOXP3_KO_TREG_DN, GSE6875_TCONV_VS_FOXP3_KO_TREG_UP, GSE6875_TCONV_VS_TREG_DN, GSE6875_TCONV_VS_TREG_UP, GSE6875_WT_VS_FOXP3_KO_TREG_DN, GSE6875_WT_VS_FOXP3_KO_TREG_UP,GSE 19512_NAUTRAL_VS_INDUCED_TREG_DN, GSE19512_NAUTRAL_VS_INDUCED_TREG_UP). In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity include C8 pathways (HE_LIM_SUN_FETAL_LUNG_C4_TREG_CELL). In some embodiments, the one or more immunosuppressive gene expression pathways modulated by microgravity is a combination of the above pathways.

[0075] In some embodiments, exposure of a population of Treg cells (e.g., iTreg cells and / or nTreg cells) to microgravity for between 2 and 216 hours increases the Treg cells immunosuppressive capacity, as measured by decreased proliferation of CD8+ T cells in a co-culture system. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of CD8+ T cells by 100% at a greater CD8+ T celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of CD8+ T cells by 90% at a greater CD8+ T celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of CD8+ T cells by 80% at a greater CD8+ T celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of CD8+ T cells by 70% at a greater CD8+ T celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of CD8+ T cells by 60% at a greater CD8+ T celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of the Treg cells to microgravity reduces proliferation of CD8+ T cells by 50% at a greater 40 / 191#14753190vlCD8+ T celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of CD8+ T cells by 40% at a greater CD8+ T celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of CD8+ T cells by 30% at a greater CD8+ T celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of CD8+ T cells by 20% at a greater CD8+ T celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of CD8+ T cells by 10% at a greater CD8+ T celkTreg cell ratio than Tregs not exposed to microgravity.

[0076] In some embodiments, exposure of a population of Treg cells (e.g., iTreg cells and / or nTreg cells) to microgravity for between 2 and 216 hours increases the Treg cells immunosuppressive capacity, as measured by decreased proliferation of T responder (Tresp) cells in a co-culture system. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of Tresp cells by 100% at a greater Tresp:Treg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of Tresp cells by 90% at a greater Tresp celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of Tresp cells by 80% at a greater Tresp celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of Tresp cells by 70% at a greater Tresp celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of Tresp cells by 60% at a greater Tresp celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of Tresp cells by 50% at a greater Tresp celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of Tresp cells by 40% at a greater Tresp celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of Tresp cells by 30% at a greater Tresp celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity reduces proliferation of Tresp cells by 20% at a greater Tresp celkTreg cell ratio than Tregs not exposed to microgravity. In some embodiments, exposure of a population of Treg cells to microgravity 41 / 191#14753190vlreduces proliferation of Tresp cells by 10% at a greater Tresp celkTreg cell ratio than Tregs not exposed to microgravity.

[0077] In some embodiments, Treg cells exposed to microgravity are more immunosuppressive relative to Treg cells not exposed to microgravity. Without wishing to be bound by any particular theory, it is generally believed that exposure of Treg cells to microgravity increases intracellular STAT5 expression and phosphorylation of STAT5 (pSTAT5) in Treg cells. As evidenced by the available literature, STAT5 binds to the promoter and enhancer regions of the FOXP3 gene, driving its expression. FOXP3 is the master transcription factor for Tregs, essential for their development, stability, and immunosuppressive function. High levels of FOXP3 enhance the suppressive capacity of Tregs by upregulating key immunosuppressive molecules. Of various mechanisms, these Tregs exert their immunosuppressive function through increased expression of CTLA-4 [blocks costimulatory signals to effector T cells by interacting with CD80 / CD86 on antigen-presenting cells (APCs)], secretion of IL- 10 and / or TGF-p, and increased expression of CD39 / CD73 (catalyze the conversion of ATP to adenosine, which has immunosuppressive effects). STAT5 activation reinforces FOXP3 expression even in inflammatory environments, preventing Tregs from losing their identity or converting into pro-inflammatory effector T cells (e.g., Th 17 cells).

[0078] Without wishing to be bound by any particular theory, it is generally believed that microgravity influences cells through changes in the plasma membrane. Microgravity alters the fluidity of the plasma membrane, disrupting the organization and clustering of membrane proteins. These cytoskeletal disruptions then modulate intracellular signaling, nuclear mechano-sensing (e.g., altered cytoskeletal forces on the nucleus reduce the mechanical regulation of chromatin organization, influencing the accessibility of transcriptional machinery), as well as epigenetic modification (e.g., microgravity induces changes in DNA methylation, histone acetylation, and chromatin structure, leading to long-term changes in transcriptional activity).

[0079] In some embodiments, a population of Treg cells are exposed to a combination of cytokines and / or adjuvants. In some cases, a population of Treg cells are exposed to a combination of cytokines and / or adjuvant prior to, during, or after exposure to microgravity. In some embodiments, the cytokines are IL-2, IL-7, TGF-betal, IL-35, IL-12, IFN-gamma, and / or IL- 15. In some embodiments, the cytokine is IL-7. In some embodiments, the adjuvants comprise metabolic supplements (e.g., amino acids, glucose, human serum, vitamins, lipids, minerals, nucleotides), chemical additives (e.g., antibiotics / antimycotics,42 / 191#14753190vlsmall molecule inhibitors / activators, recombinant albumin, all-trans retinoic acid, taurine, antioxidants) aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate), oil-in-water emulsions (e.g., MF59, AS03), pattern-recognition receptor (PRR) agonists (e.g., TLR agonists such as CpG ODN, MPL / MPLA, or poly(I:C)), saponin-based adjuvants (e.g., QS-21), liposomes, nanoparticles, Freund’s adjuvant, cryoprotectants (e.g., dimethyl sulfoxide), and / or osmoprotectants (e.g., sucrose, mannitol, glucose).

[0080] Other aspects of the present disclosure relate to compositions comprising a microgravity cell culture (MCC) medium, the MCC medium comprising a plurality of Treg cells. In some embodiments, the plurality of Treg cells in the MCC medium have been exposed to microgravity (e.g., for between 2 and 216 hours).

[0081] In some embodiments, the plurality of Treg cells in the MCC medium comprise at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% nTreg cells by total cell number. In some embodiments, the plurality of Treg cells in the MCC medium comprises 99.9% or 100% nTregs by total cell number. In some embodiments, the plurality of Tregs cells comprises one or more undifferentiated T cells (e.g., T helper cells, Thl, Th2, etc.).

[0082] In some embodiments, the plurality of Treg cells in the MCC medium comprise at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% iTreg cells by total cell number. In some embodiments, the plurality of Treg cells in the MCC medium comprises 99.9% or 100% iTregs by total cell number.

[0083] The nTreg cells may be isolated directly from a whole blood sample obtained from a subject or from a peripheral blood mononuclear cell (PBMC) cell suspension, for example, via processing of the whole blood using techniques known in the art (e.g., peripheral blood venipuncture and / or apheresis). nTreg cells may be identified by a combination of surface markers, such as CD4, and intracellular markers, such as FoxP3. Optionally, in some embodiments, the Treg cells may further express CD3, CD4, CD25, and / or FoxP3 (e.g., a CD3+ / CD4+ / CD25+ / FoxP3+ cell). Any suitable technique known to the skilled artisan for isolating nTregs from a blood sample (e.g., whole blood or PBMC suspension) may be used herein (e.g., flow cytometric cell sorting, magnetic bead separation, etc.).

[0084] In contrast, iTreg cells are differentiated from CD4+ T helper cells. In some embodiments, the CD4+ T cells are first isolated from a PBMC suspension. Again, any suitable technique known to the skilled artisan for isolating CD4+ T helper cells may be used, for example, magnetic bead cell separation, flow cytometric cell sorting, or immunoaffinity columns, among others. In some embodiments, adding the CD4+ T helper cells to a 43 / 191#14753190vldifferentiation cell culture (DCC) medium drives differentiation of the CD4+ T helper cells into iTreg cells.

[0085] In some embodiments, the MCC medium further comprises at least one cytokine and / or adjuvant. In some embodiments, at least one cytokine is TGF-betal, IL-2, IL-7, IL-10, IL-35, IL-12, IFN-gamma, and / or IL-15. In some embodiments, at least one cytokine is IL-7.

[0086] In some embodiments, exposing the composition comprising Treg cells to microgravity increases intracellular STAT5 expression and / or phosphorylation of STAT5 (pSTAT5) in the Treg cells (e.g., nTreg cells and / or iTreg cells). Additionally, in some embodiments, exposing the composition to microgravity increases extracellular secretion of one or more cytokines, such as TGF-beta and IL- 10, from the Treg cells in the composition (e.g., nTregs and / or iTregs). As discussed in more detail above, IL-10 and / or TGF-beta, in some embodiments, have an immunosuppressive function. In some embodiments, exposing the composition to microgravity modulates the expression of one or more immunosuppressive gene expression pathways. In some embodiments, exposing the composition to microgravity decreases the proliferation of CD8+ T cells in a co-culture system. In some embodiments, exposing the composition to microgravity decreases the proliferation of T responder cells in a co-culture system. Thus, in some embodiments, compositions comprising Treg cells exposed to microgravity are more immunosuppressive than compositions comprising Treg cells not exposed to microgravity.

[0087] In some embodiments, an increase in the intracellular STAT5 expression and / or phosphorylation of STAT5 in Tregs in the compositions, an increase in extracellular secretion of IL- 10 and / or TGF-beta from Tregs in the compositions, and / or the increase in immunosuppressive properties of Tregs in the compositions are induced by a microgravity environment.

[0088] In some embodiments, the composition comprises a cell type other than Treg cells (e.g., nTreg cells and / or iTreg cells). In some embodiments, the non-Treg cells comprise Thl, Th2, Thl7, and / or follicular T helper cells (Tfh). In some embodiments, the concentration of non-Treg cells in the composition is between 0% and 25% of the total cell number. In some embodiments, the concentration of non-Treg cells is greater than or equal to 0%, greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 15%, greater than or equal to 20%, or greater than or equal to 25% by total cell number. In some embodiments, the concentration of non-Treg cells is less than or equal to 25%, less than or equal to 20%, less than or equal to 15%, less than or equal to 10%, less than or equal to 44 / 191#14753190vl5%, or equal to 0% by total cell number. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the concentration of non-Treg cells in the composition is greater than or equal to 0% and less than or equal to 25% by total cell number.

[0089] In some embodiments, the composition further comprises one or more antibiotics, serums, cytokines, metabolic supplements, chemical additives, stimulators, and / or other compounds. In some embodiments, the one or more cytokines comprises IL-2, IL-7, TGF-betal, IL-10, IL-35, IL-12, IFN-gamma, and / or IL-15. In some embodiments, the one or more metabolic supplements comprise arginine, glutamine, serine, and / or glycine. In some embodiments, the one or more chemical additives comprises taurine, N-acetylcysteine, and / or retinoic acid. In some embodiments, the one or more stimulators comprises anti-CD3 / CD28 stimulatory beads. In some embodiments, the one or more other compounds comprises rapamycin.

[0090] In some embodiments, exposing the Treg cells to microgravity increases cell survival, relative to Treg cells not exposed to microgravity. In some embodiments, the Tregs exposed to microgravity exhibit increased survival in the setting of IL-2 deprivation. In some embodiments, a least a portion of the Treg cells exposed to microgravity are more immunosuppressive, relative to Treg cells not exposed to microgravity. In some embodiments, the Treg cell is an iTreg cell. In some embodiments, the Treg cell is an nTreg cell. In some embodiments, the Treg cells comprises a mixture of iTreg and nTreg cells.

[0091] In some embodiments, Treg cells with increased cell survival (e.g. following exposure to microgravity) express one or more markers of Tregs, including for example, CD4, FoxP3, Stat5, CD45RO, and / or CD25. In some embodiments, Treg cells with increased survival express CD4 / FoxP3. In some embodiments, Treg cells with increased survival express CD4 / FoxP3 / Stat5. In some embodiments, Treg cells with increased survival express CD4 / FoxP3 / CD45RO. In some embodiments, Treg cells with increased survival express CD4 / FoxP3 / CD25.

[0092] In some embodiments, increased Treg cell survival persists for at least 4 days after exposure to microgravity. In some embodiments, increased Treg cell survival persists for at least 4 days after exposure to microgravity. In some embodiments, increased Treg cell survival persists for at least 10 days after exposure to microgravity. In some embodiments, increased Treg cell survival persists for greater than or equal to 1 days, greater than or equal to 2 days, greater than or equal to 3 days, greater than or equal to 4 days, greater than or equal to 5 days, greater than or equal to 6 days, greater than or equal to 7 days, greater than or equal 45 / 191#14753190vlto 8 days, greater than or equal to 9 days, greater than or equal to 10 days, greater than or equal to 11 days, greater than or equal to 12 days, greater than or equal to 13 days, greater than or equal to 14 days, greater than or equal to 15 days, greater than or equal to 16 days, greater than or equal to 17 days, greater than or equal to 18 days, greater than or equal to 19 days, greater than or equal to 20 days, greater than or equal to 21 days, greater than or equal to 22 days, greater than or equal to 23 days, greater than or equal to 24 days, greater than or equal to 25 days, greater than or equal to 26 days, greater than or equal to 27 days, greater than or equal to 28 days, greater than or equal to 29 days or greater than or equal to 30 days. In some embodiments, increased Treg cell survival persists of less than or equal to 30 days, less than or equal to 29 days, less than or equal to 28 days, less than or equal to 27 days, less than or equal to 26 days, less than or equal to 25 days, less than or equal to 24 days, less than or equal to 23 days, less than or equal to 22 days, less than or equal to 21 days, less than or equal to 20 days, less than or equal to 19 days, less than or equal to 18 days, less than or equal to 17 days, less than or equal to 16 days, less than or equal to 15 days, less than or equal to 14 days, less than or equal to 13 days, less than or equal to 12 days, less than or equal to 11 days, less than or equal to 10 days, less than or equal to 9 days, less than or equal to 8 days, less than or equal to 7 days, less than or equal to 6 days, less than or equal to 5 days, less than or equal to 4 days, less than or equal to 3 days, less than or equal to 2 days or less than or equal to 1 days. Combinations are also possible in some embodiments. For example, in some embodiments, increased Treg cell survival persists greater than or equal to 1 days and less than or equal to 30 days. Combinations of other ranges are also possible (e.g., greater than or equal to 1 days and less than or equal to 30 days. Other ranges are also possible.

[0093] In some embodiments, the compositions disclosed herein comprise a plurality of Treg cells exposed to microgravity, for example, for between 2 and 216 hours. In some embodiments, exposing the composition to microgravity increases Treg cell survival, relative to compositions comprising Tregs not exposed to microgravity. In some embodiments, increased Treg cell survival persists for at least 4 days after exposure to microgravity. In some embodiments, increased Treg cell survival persists for at least 10 days after exposure to microgravity. In some embodiments, the Tregs comprise iTregs. In some embodiments, the Tregs comprise nTregs. In some embodiments, the Tregs comprise a mixture of iTregs and nTregs.

[0094] In some embodiments, the composition comprise a plurality of Treg cells exposed to microgravity for treatment of a disease in a subject. In some embodiments, the disease is 46 / 191#14753190vlselected from the group consisting of an autoimmune disease, an inflammatory disease, a neurological disease, proliferative disease (e.g., cancer) or a genetic disorderUse and Methods of Treatment

[0095] Other aspects of the disclosure relate to methods of treating an autoimmune disease, inflammatory disease, neurological disease, or a genetic disorder by administering any one of the Treg cells and / or composition disclosed herein to a subject in need thereof. In some embodiments, the methods further comprise administering one or more adjuvants (e.g., in addition to Treg compositions disclosed herein).

[0096] In some embodiments, the one or more adjuvants comprise an infusion media and excipients (e.g., buffers, salts, polymers, proteins, and preservatives). For example, in some embodiments, the adjuvants comprise human serum albumin (HAS), dimethyl sulfoxide (DMSO) and an infusion media (e.g., 5% human serum albumin, HSA, 7.5% DMSO, 31.25% Plasma-Lyte A, 31.25% of 5% Dextro se / 0.45% sodium chloride, 10% Dextran 40 (LMD) / 5% Dextrose).

[0097] In other aspects, still, the disclosure relates to any one of the Treg cells and / or compositions disclosed herein for use in treating an autoimmune disease, an inflammatory disease, a neurological disease, proliferative disease (e.g., cancer) or a genetic disorder. In some embodiments, the use further comprises administering one or more adjuvants.

[0098] In some embodiments, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, polymyositis, Sjogren syndrome, Hashimoto thyroiditis, Graves’ disease, type 1 insulin dependent diabetes, Addison’s disease, vitiligo, pernicious anemia, glomerulonephritis, myasthenia gravis, pulmonary fibrosis, psoriasis, psoriatic arthritis, Crohn’s disease, celiac disease, ulcerative colitis, autoimmune gastritis, autoimmune hepatitis, ALS, pemphigus vulgaris, bullous pemphigoid, multiple sclerosis, myasthenia gravis, and autoimmune encephalitis.

[0099] In some embodiments, the inflammatory disease is selected from the group consisting of rheumatoid arthritis, psoriasis, asthma, hepatitis, arthritis, cardiovascular disease, Kawasaki disease, chronic obstructive pulmonary disorder, ulcerative colitis, Crohn’s disease, diabetes, vasculitis, gout, sarcoidosis, systemic lupus erythematosus, Hashimoto’s thyroiditis, Grave’s disease, ankylosing spondylitis, antiphospholipid antibody syndrome, chronic recurrent multifocal osteomyelitis, Henoch- Schonlein Purpura, idiopathic thrombocytopenic purpura, juvenile dermatomyositis, juvenile idiopathic arthritis, juvenile lupus, juvenile scleroderma, juvenile vasculitis, mixed connective tissue disease, myositis,47 / 191#14753190vlpoststreptococcal inflammatory syndrome, psoriatic arthritis, reactive arthritis, scleroderma, Sjogren’s syndrome, uveitis, vasculitis , encephalitis, atherosclerosis, myocardial infarction, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, obesity, sepsis, chronic viral infection, aging, wound injury, hemophagocytic lymphohistiocytosis, atopic dermatitis, pemphigus vulgaris, Guillain-Barre syndrome, hemophilia, B cell autoimmunity, acute respiratory distress syndrome, and osteoarthritis.

[0100] In some embodiments, the neurological disease is selected from the group consisting of Alzheimer’s Disease, Parkinson’s Disease, Guillain-Barre syndrome, Amyotrophic Lateral Sclerosis (ALS), multiple sclerosis, stroke, traumatic brain injury, epilepsy, myasthenia gravis, Huntington’s disease, Lewy Body Dementia, Frontotemporal Dementia, Vascular Dementia, and prion disease.

[0101] In some embodiments, the genetic disorder is selected from the group consisting IPEX syndrome (Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked Syndrome), Autoimmune Lymphoproliferative Syndrome (ALPS), Autoimmune Polyendocrinopathy Syndrome Type 1 (APS-1), Chediak-Higashi Syndrome, Systemic Juvenile Idiopathic Arthritis (SJIA), Chronic Granulomatous Disease (CGD), Shwachman-Diamond syndrome, Fanconi anemia, Familial Hemophagocytic Lymphohistiocytosis (HLH), and Wiskott-Aldrich Syndrome (WAS).

[0102] The disclosure further provides for methods of treating a subject during and / or after surgery by administering any one of the Treg cells and / or compositions disclosed herein to a subject in need thereof. In some embodiments, the methods further comprise administering one or more adjuvants.

[0103] The disclosure also provides for any one of the Treg cells and / or compositions disclosed herein for use during and / or after surgery. In some embodiments, the surgery is selected from the group consisting of solid organ transplantation, graft versus host disease, hematopoietic stem cell transplantation, biomedical device implantation, cardiac surgery (aortic aneurysm, bypass), spinal cord surgery, skin grafting, islet cell transplantation, primary tumor resection, recurrent tumor resection, metastatic tumor resection, joint replacement, and fracture repair.

[0104] In some embodiments, any one of the Treg cells and / or compositions disclosed herein may be used as a medicament. In some embodiments, one or more adjuvants is coadministered with the Tregs during and / or after surgery.

[0105] In some embodiments, administration of one or more of the following adjuvants with any one of the immunosuppressive Treg cells or compositions disclosed 48 / 191#14753190vlherein further enhances the therapeutic effect of the Treg cells and / or compositions:Intravenous Immunoglobulin (IVIG), DMARDs (e.g., Chloroquine, Hydroxychloroquine, Leflunomide, Methotrexate, Sulfasalazine, Mesalamine); Immunosuppressants (e.g., Azathioprine, 6-mercaptopurine, Cyclophosphamide, Mycophenolate mofetil); Calcineurin inhibitors (e.g., Cyclosporine, Pimecrolimus, Tacrolimus); mTOR inhibitors (e.g., Everolimus, Sirolimus); Biologies (e.g., Abatacept, Adalimumab, Anakinra, Anifrolumab, Axatilimab, Belimumab, Benralizumab, Brodalumab, Canakinumab, Certolizumab pegol, Dupilumab, Eculizumab, Efgartigimod, Emapalumab, Etanercept, Golimumab, Guselkumab, Infliximab, Ixekizumab, Mepolizumab, Omalizumab, Reslizumab, Rilonacept, Risankizumab, Rituximab, Sarilumab, Secukinumab, Tezepelumab, Tildrakizumab, Tocilizumab, Ustekinumab, Vedolizumab); JAK inhibitors (e.g., Baricitinib, Filgotinib, Tofacitinib, Upadacitinib); Integrin Inhibitors (Natalizumab, Vedolizumab); SIP receptor modulators (e.g., Fingolimod, Ozanimod, Ponesimod, Siponimod); Complement inhibitors (e.g., Eculizumab, Ravulizumab); Phosphodiesterase (PDE) Inhibitors (e.g., Apremilast, Roflumilast); Corticosteroids (e.g., Dexamethasone, Hydrocortisone, Methylprednisolone, Prednisone, Triamcinolone, Budesonide, Fluticasone); Antihistamines (e.g., Cetirizine, Diphenhydramine, Famotidine, Fexofenadine, Loratadine, Ranitidine); NSAIDs (e.g., Aspirin, Celecoxib, Diclofenac, Ibuprofen, Naproxen, Indomethacin); Antifibrotics (e.g., Nintedabib, Pirfenidone); Leukotriene Modifiers (e.g., Montelukast, Zafirlukast);Immunomodulatory Antibiotics (e.g., Clarithromycin, Dapsone, Metronidazole, Minocycline); Probiotic s / Prebiotics (e.g., Bifidobacterium spp., Lactobacillus spp.);Antimetabolites (e.g., 6-Mercaptopurine, Thioguanine); Anti-Protease (e.g., Alpha- 1 antitrypsin); Cholinesterase Inhibitors (e.g., Donepezil, Rivastigmine, Galantamine); NMDA Receptor Antagonist (e.g., Memantine); Alzheimer’s Disease Modifying Therapies (e.g., Lecanemab, Aducanumab, Donanemab, Dopaminergic Therapies, Levodopa / Carbidopa, Pramipexole, Ropinirole, Selegiline, Rasagiline); ALS Disease Modifying Therapies (e.g., Riluzole, Edaravone); MS Disease Modifying Therapies (e.g., Interferon beta- la, Glatiramer acetate, Fingolimod, Dimethyl fumarate, Natalizumab, Ocrelizumab); and kinase inhibitors (e.g., Belumosudil, Ibrutinib, Ruxolitinib).Methods of Producing Immunosuppressive Tregs using Microgravity

[0106] Additional aspects of the disclosure relate to methods for producing immunosuppressive Treg cells. In some embodiments, the methods comprise exposing a plurality of Treg cells (e.g., nTregs and / or iTregs) to microgravity. In some embodiments,49 / 191#14753190vlthe method comprises obtaining a peripheral blood sample from a human subject (e.g., peripheral blood venipuncture or apheresis). In some embodiments, the methods further comprise isolating PBMCs from the peripheral blood sample. In some embodiments, the methods further comprise isolating nTreg cells from the PBMC blood sample to yield a nTreg cell population. In some embodiments, the isolated nTreg cells are CD4+ / CD25+ / CD127dim / - Treg cells. In some embodiments, the isolated nTreg cells are also FoxP3+.

[0107] In some embodiments, the methods relate to stimulating and expanding the nTreg cells in the nTreg cell population e.g., isolated from the PBMC sample). Stimulation of the nTreg cell population may be achieved using any method known to the skilled artisan. For example, in some embodiments, stimulating the nTreg cells is achieved by adding the nTreg cells to a first stimulation cell culture (SCC) media for between 2 and 48 hours. In some embodiments, the first stimulation media comprises cell media comprising one or more antibiotics, serum, CD3 / CD28 activation / expansion beads, cytokines, and / or other compounds. In some embodiments, the stimulation medium comprises a basal media (e.g., a serum free basal media such as TexMACS™ GMP). In some embodiments, the basal media comprises Penicillin-Streptomycin Solution (e.g. 0 to 1%), Human AB serum (or patient derived serum at 0 to 10%), GMP ActiveMax® Human T cell Activation / Expansion CD3 / CD28 Beads (100,000 to 1,000,000 beads for every 105to 106cells), GMP Human IL-2 Protein (e.g. 1 to 300 lU / mL), Retinoic acid (Powder USP) (e.g. 1 to 100 nM), and / or MACS GMP Rapamycin (e.g. 1 to 100 nM).

[0108] In some embodiments, the method further comprises expanding the nTreg cell population by adding the stimulated nTreg cell population to a first expansion cell culture (ECC) media for between 24 and 168 hours. In some embodiments, the first expansion media comprises the first SCC media comprising one or more additional antibiotics, serum, cytokines, and / or other compounds. In some embodiments, the expansion medium comprises a basal media (e.g., a serum free basal media such as TexMACS™ GMP). In some embodiments, the basal media comprises Penicillin-Streptomycin Solution (e.g. 0 to 1%), Human AB serum (or patient derived serum at 0 to 10%), GMP Human IL-2 Protein (e.g. 1 to 300 lU / mL), MACS GMP Rapamycin (e.g. 1 to 50 nM), and / or Retinoic acid (Powder USP) (e.g. 1 to 50 nM).

[0109] In some embodiments, the method further comprises performing a second stimulation and expansion step. For example, in some embodiments, the methods comprise stimulating the expanded nTreg cell population by adding the expanded nTreg cell population 50 / 191#14753190vlto a second SCC media for between 24 and 48 hours. In some embodiments, the second SCC media comprises cell media comprising one or more antibiotics, serum, CD3 / CD28 activation / expansion beads, cytokines, and / or other compounds. In some embodiments, the second SCC medium comprises a basal media (e.g., a serum free basal media such as TexMACS™ GMP). In some embodiments, the basal media comprises Penicillin-Streptomycin Solution (e.g. 0 to 1%), Human AB serum (or patient derived serum at 0 to 10%), GMP ActiveMax® Human T cell Activation / Expansion CD3 / CD28 Beads (100,000 to 1,000,000 beads for every 105to 106cells), GMP Human IL-2 Protein (e.g. 1 to 300 lU / mL), Retinoic acid (Powder USP) (e.g. 1 to 100 nM), and / or MACS GMP Rapamycin (e.g. 1 to 100 nM).

[0110] In some embodiments, the second expansion step comprises adding the stimulated nTreg cell population to a second ECC media for between 24 and 168 hours. In some embodiments, the second ECC media comprises the second SCC media comprising one or more additional antibiotics, serum, cytokines, and / or other compounds. In some embodiments, the second ECC media comprises a basal media (e.g., a serum free basal media such as TexMACS™ GMP). In some embodiments, the basal media comprises Penicillin-Streptomycin Solution (e.g. 0 to 1%), Human AB serum (or patient derived serum at 0 to 10%), GMP Human IL-2 Protein (e.g. 1 to 300 lU / mL), MACS GMP Rapamycin (e.g., 1 to 50 nM), and / or Retinoic acid (Powder USP) (e.g., 1 to 50 nM). In some embodiments, the nTregs expand 200-300 fold within 12-14 days.

[0111] In other aspects, methods related to producing Tregs derived from naive CD4+ T cells are described. As described elsewhere herein, iTregs are derived from naive CD4+ T helper cells isolated, for example, from a PBMC cell suspension to yield a naive CD4+ T helper cell population. In some embodiments, the CD4+ T helper cells are obtained by cell depletion using a plurality of antibodies (or antibody-conjugates such as antibodies bound to a magnetic bead). In some embodiments, the plurality of antibodies comprises CD45RO, CD8, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD56, CD123, TCRy / 6, HLA-DR, and / or CD235a. Accordingly, in some embodiments, naive CD4+ T cells populations are CD45RO-, and / or CD8a-, and / or CD14-, and / or CD15-, and / or CD16-, and / or CD19-, and / or CD25-, and / or CD34-, and / or CD36-, and / or CD56-, and / or CD123-, and / or TCRy / 6-, and / or HLA-DR-, and / or CD235a-. In some embodiments, cells in the isolated naive CD4+ T helper cell population are CD4+ / CD45RA+.

[0112] In some embodiments, the methods further comprise differentiating the CD4+ / CD45RA+ cell population into iTregs by adding them to a differentiation cell culture 51 / 191#14753190vlmedia for between 2 and 48 hours. In some embodiments, the DCC media comprises a basal media (e.g., a serum free basal media such as TexMACS™ GMP). In some embodiments, the basal media comprises Penicillin-Streptomycin Solution (e.g., 0 to 1%), Human AB serum (or patient derived serum at 0 to 10%), GMP ActiveMax® Human T cell Activation / Expansion CD3 / CD28 Beads (e.g., 100,000 to 1,000,000 beads for every 105to 106cells), GMP Human IL-2 Protein (e.g., 1 to 100 lU / mL), GMP Recombinant Human TGF-beta 1 (e.g., 0.001 to 1 ng / mL), and / or Retinoic acid (Powder USP) (e.g., 1 to 100 nM).

[0113] Those of skill in the art will understand that not all CD4+ / CD45RA+ cells may become differentiated into iTregs. Thus, in some embodiments, upon exposing the CD4+ / CD45RA+ cells to the DCC media at least a portion of cells are differentiated into iTreg cells (e.g., CD4+ / FoxP3+ cells). In some embodiments, between 80% and 100% of the cells in the DCC media are CD4+ / FoxP3+ iTreg cells after between 24 to 48 hours of culture.

[0114] In some embodiments, the methods comprise further expanding the iTreg cells by adding the differentiated iTreg cell population to a third ECC media for 2 to 120 hours. In some embodiments, the third ECC media comprises the DCC media comprising one or more additional components (e.g., antibiotics, serum, cytokines, and / or other compounds). In some embodiments, the ECC media comprises a basal media (e.g., a serum free basal media such as TexMACS™ GMP). In some embodiments, the basal media comprises Penicillin-Streptomycin Solution (e.g. 0 to 1%), Human AB serum (or patient derived serum at 0 to 10%), GMP Human IL-2 Protein (e.g. 1 to 300 lU / mL), MACS GMP Rapamycin (e.g., 1 to 50 nM), and / or Retinoic acid (Powder USP) (e.g., 1 to 50 nM). In some embodiments, the iTregs expand 10-12 fold within 5 days.

[0115] Following stimulation and expansion of nTregs and / or differentiation and expansion of iTregs, the methods comprise adding the Tregs (e.g., nTregs and / or iTregs) to a microgravity cell culture (MCC) media and exposing them to microgravity for at least 2 hours. In some embodiments, the MCC media comprises one or more cytokines, metabolic supplements, chemical additives, stimulators, and / or other compounds. In some embodiments, the microgravity media comprises a basal media (e.g., a serum free basal media such as TexMACS™ GMP). In some embodiments, the basal media comprises Penicillin-Streptomycin Solution (e.g. 0 to 1%). In some embodiments, the basal media comprises Human AB serum (or patient derived serum at 0 to 10%). In some embodiments, the basal media comprises cytokines [e.g. IL-2 (e.g. 10 to 300 lU / mL), IL-7 (e.g. 5 to 50 ng / mL), TGF-betal (e.g. 1 to 100 ng / mL), IL-10 (e.g. 1 to 100 ng / mL), IL-35 (e.g. 5 to 100 ng / mL), IL-12 (e.g. 0.1 to 100 ng / mL), IFN-gamma (e.g. 0.1 to 100 ng / mL) and / or IL-1552 / 191#14753190vl(e.g. 5 - 50 ng / mL)]. In some embodiments, the basal media comprises nutritional / metabolic supplements [e.g. arginine (e.g. 0.5 - 5 mM), glutamine (e.g. 2 - 6 mM), serine (e.g. 0.5 - 5 mM), glycine (e.g. 0.5 - 10 mM)]. In some embodiments, the basal media comprises chemical additives [e.g. taurine (e.g. 0.1 - 5 mM), N-acetylcysteine (e.g. 0.1 - 10 mM), and / or all-trans retinoic acid (e.g. 1 - 1000 nM). In some embodiments, the basal media comprises stimulators [e.g. anti-CD3 / CD28 stimulatory beads at 100,000 to 1,000,000 beads for every 105to 106cells]. In some embodiments, the basal media comprises other compounds [e.g. rapamycin (10 to 100 nM)].

[0116] Any suitable method for exposing the cells to a microgravity environment may be used by the artisan. Exemplary methods include, but are not limited to, the use of a rotary cell culture system to simulate microgravity through continuous cellular free fall (e.g., via use of Synthecon RCCS systems) or physically bringing said cells into LEO (via space shuttle, capsule, etc.).

[0117] In some embodiments, exposing a CD4+ / FoxP3+ nTreg cell population to microgravity increases IL- 10 and / or TGF-beta secretion from at least a portion of CD4+ / FoxP3+ nTregs within the cell population. Similarly, exposing a CD4+ / FoxP3+ iTreg population to microgravity increases IL- 10 and / or TGF-beta secretion from at least a portion of CD4+ / FoxP3+ iTregs within the cell population.

[0118] In some embodiments, exposing a CD4+ / FoxP3+ nTreg cell population to microgravity modulates gene expression pathways from at least a portion of CD4+ / FoxP3+ nTregs within the cell population. Similarly, exposing a CD4+ / FoxP3+ iTreg cell population to microgravity modulates gene expression pathways from at least a portion of CD4+ / FoxP3+ iTregs within the cell population.

[0119] In some embodiments, exposing a CD4+ / FoxP3+ nTreg cell population to microgravity increases the CD4+ / FoxP3+ nTreg cells immunosuppressive capacity, as measured by decreased proliferation of CD8+ T cells in a co-culture system, from at least a portion of CD4+ / FoxP3+ nTregs within the cell population. Similarly, exposing a CD4+ / FoxP3+ iTreg cell population to microgravity increases the CD4+ / FoxP3+ iTreg cells immunosuppressive capacity, as measured by decreased proliferation of CD8+ T cells in a co-culture system, from at least a portion of CD4+ / FoxP3+ iTregs within the cell population.

[0120] In some embodiments, exposing a CD4+ / FoxP3+ nTreg cell population to microgravity increases the CD4+ / FoxP3+ nTreg cells immunosuppressive capacity, as measured by decreased proliferation of T responder cells in a co-culture system, from at least a portion of CD4+ / FoxP3+ nTregs within the cell population. Similarly, exposing a 53 / 191#14753190vlCD4+ / FoxP3+ iTreg cell population to microgravity increases the CD4+ / FoxP3+ iTreg cells immunosuppressive capacity, as measured by decreased proliferation of T responder cells in a co-culture system, from at least a portion of CD4+ / FoxP3+ iTregs within the cell population

[0121] In some embodiments, a media (e.g., DCC, SCC, ECC, and / or MCC) further comprises one or more cytokines. In some embodiments, the cytokine comprises IL-2, IL-7, IL-10, IL-15, IL-35, IL-12, IFN-gamma, and / or TGF-betal.

[0122] In some embodiments, the one or more cytokines is present in the media (e.g., DCC, SCC, ECC, and / or MCC) at a concentration of between 0.01 and 1000 ng / mL. In some embodiments, the one or more cytokines is present in the media at a concentration of greater than or equal to 0.01 ng / mL, greater than or equal to 1 ng / mL, greater than or equal to 5 ng / mL, greater than or equal to 10 ng / mL, greater than or equal to 50 ng / mL, greater than or equal to 100 ng / mL, greater than or equal to 300 ng / mL, greater than or equal to 500 ng / mL, greater than or equal to 750 ng / mL, or greater than or equal to 1000 ng / mL. In some embodiments, the one or more cytokines is present in the media at a concentration of less than or equal to 1000 ng / mL, less than or equal to 750 ng / mL, 500 ng / mL, 300 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL, or 0.01 ng / mL. Combinations of the recited ranges are also possible in some embodiments. For example, in some embodiments, the one or more cytokines is present in the media at a concentration of greater than or equal to 0.01 ng / mL and less than 1000 ng / mL. In some embodiments, the one or more cytokines is present in the media at a concentration of 5 ng / mL.

[0123] In some embodiments, the one or more cytokines is present in the media (e.g., DCC, SCC, ECC, and / or MCC) at a concentration of between 0.01 and 1000 lU / mL. In some embodiments, the one or more cytokines is present in the media at a concentration of greater than or equal to 0.01 lU / mL, greater than or equal to 1 lU / mL, greater than or equal to 5 lU / mL, greater than or equal to 10 lU / mL, greater than or equal to 50 lU / mL, greater than or equal to 100 lU / mL, greater than or equal to 300 lU / mL, greater than or equal to 500 lU / mL, greater than or equal to 750 lU / mL, or greater than or equal to 1000 lU / mL. In some embodiments, the one or more cytokines is present in the media at a concentration of less than or equal to 1000 lU / mL, less than or equal to 750 lU / mL, 500 lU / mL, 300 lU / mL, 100 lU / mL, 50 lU / mL, 10 lU / mL, 5 lU / mL, 1 lU / mL, or 0.01 lU / mL. Combinations of the recited ranges are also possible in some embodiments. For example, in some embodiments, the one or more cytokines is present in the media at a concentration of greater than or equal to 0.01 lU / mL and less than 1000 lU / mL.54 / 191#14753190vl

[0124] In some embodiments, a media (e.g., DCC, SCC, ECC, and / or MCC) further comprises one or more metabolic supplements. In some embodiments, the one or more metabolic supplements comprises arginine, glutamine, serine, and / or glycine.

[0125] In some embodiments, the one or more metabolic supplements is present in the media e.g., DCC, SCC, ECC, and / or MCC) at a concentration of between 0 and 50 mM. In some embodiments, the one or more metabolic supplements is present in the media at a concentration of greater than or equal to 0.01 mM, greater than or equal to 1 mM, greater than or equal to 5 mM, greater than or equal to 10 mM, greater than or equal to 20 mM, or greater than or equal to 50 lU / mL. In some embodiments, the one or more metabolic supplements is present in the media at a concentration of less than or equal to 50 lU / mL, 20 lU / mL, 10 lU / mL, 5 lU / mL, 1 lU / mL, or 0.01 lU / mL. Combinations of the recited ranges are also possible in some embodiments. For example, in some embodiments, the one or more cytokines is present in the media at a concentration of greater than or equal to 0 mM and less than 50 mM.

[0126] In some embodiments, a media (e.g., DCC, SCC, ECC, and / or MCC) further comprises one or more chemical additives. In some embodiments, the one or more chemical additives comprise taurine, N-acetylcysteine, and / or all-trans retinoic acid.

[0127] In some embodiments, the one or more chemical additives is present in the media (e.g., DCC, SCC, ECC, and / or MCC) at a concentration of between 0 to 1 mM. In some embodiments, the one or more chemical additives is present in the media at a concentration of greater than or equal to 0.000001 mM, greater than or equal to 0.00001 mM, greater than or equal to 0.0001 mM, greater than or equal to 0.001 mM, greater than or equal to 0.01 mM, greater than or equal to 0.1 mM, or greater than or equal to 1 mM. In some embodiments, the one or more chemical additives is present in the media at a concentration of less than or equal to 1 mM, 0.1 mM, 0.01 mM, 0.001 mM, 0.0001 mM, 0.00001 mM, or 0.000001 mM. Combinations of the recited ranges are also possible in some embodiments. For example, in some embodiments, the one or more chemical additives is present in the media at a concentration of greater than or equal to 0 mM and less than 1 mM.

[0128] In some embodiments, a media (e.g., DCC, SCC, ECC, and / or MCC) further comprises one or more stimulators. In some embodiments, the one or more stimulators comprises anti-CD3 / CD28 stimulatory beads.

[0129] In some embodiments, the one or more stimulators is present in the media (e.g., DCC, SCC, ECC, and / or MCC) at a concentration of between 0 to 1,000,000 beads for every 105to 106cells. In some embodiments, the one or more stimulators is present in the 55 / 191#14753190vlmedia at a concentration of greater than or equal to 0 beads, greater than or equal to 1000 beads, greater than or equal to 10,000 beads, greater than or equal to 100,000 beads, greater than or equal to 1,000,000 beads for every 105to 106cells. In some embodiments, the one or more stimulators is present in the media at a concentration of less than or equal to 1,000,000 beads, 100,000 beads, 10,000 beads, or 1,000 beads for every 105to 106cells.

[0130] In some embodiments, a media (e.g., DCC, SCC, ECC, and / or MCC) further comprises one or more other compounds, such as for example rapamycin.

[0131] In some embodiments, the one or more other compounds is present in the media (e.g., DCC, SCC, ECC, and / or MCC) at a concentration of between 0-100 nM. In some embodiments, the one or more other compounds is present in the media at a concentration of greater than or equal to 0 nM, greater than or equal to 1 nM, greater than or equal to 10 nM, or greater than or equal to 100 nM.

[0132] In some embodiments, the methods disclosed herein use microgravity to produce Tregs with increased cell survival (e.g., via live / dead assay and / or flow cytometry), relative to Treg cells not exposed to microgravity. In some embodiments, exposing the Treg cells to microgravity increases cell survival, relative to Treg cells not exposed to microgravity, in the setting of IL-2 deprivation. In some embodiments, the methods result in increased Treg cell survival for at least 4 days after exposure to microgravity. In some embodiments, the methods result in increased Treg cell survival for at least 10 days after exposure to microgravity. In some embodiments, the method comprises iTreg cells. In some embodiments, the method comprises nTreg cells. In some embodiments, the methods comprise a mixture of iTreg cells and nTreg cells.Kits

[0133] The Treg cells and compositions of the present disclosure may be assembled into kits. In some embodiments, the kit comprises immunosuppressive Treg cells and / or compositions comprising the same produced using microgravity. In some embodiments, the kit further comprises one or more adjuvants.

[0134] In some embodiments, the kits comprise a population of Treg cells (e.g., iTregs and / or nTregs) exposed to microgravity or compositions comprising Treg cells (e.g., iTregs or nTregs) exposed to microgravity for the treatment of a disease. The kits, according to some embodiments, are used to treat any one of the diseases contemplated elsewhere herein.

[0135] In some embodiments, the kits are for isolating, expanding, and exposing cells to microgravity. In some embodiments, the kits comprise one or more cell isolation reagents 56 / 191#14753190vlconfigured to isolate a population of cells from a biological sample. In some embodiments, the kits comprises one or more cell culture reagents configured to differentiate and / or expand the isolated population of cells ex vivo. In some embodiments, the kits comprise a microgravity cell culture medium. In some embodiments, the kits are configured such that the population of cells is isolated from a biological sample, expanded ex vivo, and subsequently exposed to microgravity.

[0136] The kit described herein may include one or more containers housing components for performing the methods described herein and optionally instructions for use. Any of the kits described herein may further comprise components needed for performing the methods and / or use. Each component of the kits, where applicable, may be provided in liquid form (e.g., in solution) or in solid form, (e.g., a dry powder). In certain cases, some of the components may be reconstitutable or otherwise processible (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water), which may or may not be provided with the kit.

[0137] In some embodiments, the kits may optionally include instructions and / or promotion for use of the components provided. As used herein, “instructions” can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and / or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use or sale for animal administration. As used herein, “promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with the disclosure. Additionally, the kits may include other components depending on the specific application, as described herein.

[0138] The kits may contain any one or more of the components described herein in one or more containers. The components may be prepared sterilely, packaged in a syringe and shipped refrigerated. Alternatively, it may be housed in a vial or other container for storage.57 / 191#14753190vlA second container may have other components prepared sterilely. Alternatively, the kits may include the active agents premixed and shipped in a vial, tube, or other container.

[0139] The kits may have a variety of forms, such as one or more tubes, containers, a box or a bag. The kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc. Some aspects of this disclosure provide kits comprising any one of the Treg cells and / or compositions comprising the same disclosed herein. In some embodiments, the kits further comprise one or more adjuvants for administration with the Treg cells and / or compositions.

[0140] REFERENCES1. Gu, J., Shao, Q., Zhou, J., Chen, Q., & Lu, L. (2022). Protocol for in vitro isolation, induction, expansion, and determination of human natural regulatory T cells and induced regulatory T cells. STAR protocols, 3(4), 101740.2. Pires, I.S. Hammond, P.T., and Irvine, D.J. (2021). Engineering strategies for immunomodulatory cytokine therapies: Challenges and clinical progress. Advanced Therapeutics, 4, 2100035.3. Passerini, L., Allan, S. E., Battaglia, M., Di Nunzio, S., Alstad, A. N., Levings, M. K., ... & Bacchetta, R. (2008). STAT5-signaling cytokines regulate the expression of FOXP3 in CD4+ CD25+ regulatory T cells and CD4+ CD25- effector T cells. International immunology, 20(3), 421-431.4. Smith, M. R., Satter, L. R. F., & Vargas-Hernandez, A. (2023). STAT5b: A master regulator of key biological pathways. Frontiers in immunology, 13, 1025373.58 / 191#14753190vl5. Jeffery, H. C., Jeffery, L. E., Lutz, P., Corrigan, M., Webb, G. J., Hirschfield, G. M., & Oo, Y. H. (2017). Low-dose interleukin-2 promotes STAT-5 phosphorylation, Treg survival and CTLA-4-dependent function in autoimmune liver diseases. Clinical & Experimental Immunology, 188(3), 394-411.6. Wing, K., Onishi, Y., Prieto-Martin, P., Yamaguchi, T., Miyara, M., Fehervari, Z., & Sakaguchi, S. (2008). CTLA-4 control over Foxp3+ regulatory T cell function. Science, 322(5899), 271-275.7. Zhao, H., Liao, X., & Kang, Y. (2017). Tregs: where we are and what comes next?. Frontiers in immunology, 8, 1578.8. Tsuji-Takayama, K., Suzuki, M., Yamamoto, M., Harashima, A., Okochi, A., Otani, T., ... & Kibata, M. (2008). The production of IL- 10 by human regulatory T cells is enhanced by IL-2 through a STAT5-responsive intronic enhancer in the IL-10 locus. The Journal of Immunology, 181(6), 3897-3905.9. Shen, W., Liang, Y., Lv, D., & Xie, N. (2024). Novel insights into the heterogeneity of FOXP3+ Treg cells in drug-induced allergic reactions through single-cell transcriptomics. Immunologic Research, 1-15.10. Tran, D. Q. (2012). TGF-P: the sword, the wand, and the shield of FOXP3+ regulatory T cells. Journal of molecular cell biology, 4(1), 29-37.11. Chen, X., Feng, L., Li, S., Long, D., Shan, J., & Li, Y. (2020). TGF-pi maintains Foxp3 expression and inhibits glycolysis in natural regulatory T cells via PP2A-mediated suppression of mTOR signaling. Immunology Letters, 226, 31-37.12. Lee, J., Kim, D., & Min, B. (2022). Tissue resident Foxp3+ regulatory T cells:Sentinels and saboteurs in health and disease. Frontiers in immunology, 13, 865593.13. Lu, L., Barbi, J., & Pan, F. (2017). The regulation of immune tolerance by FOXP3. Nature Reviews Immunology, 17(11), 703-717.59 / 191#14753190vl14. Laurence A, et al. Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation. Immunity 2007; 26: 371-381.15. Antov, A., Yang, L., Vig, M., Baltimore, D., & Van Parijs, L. (2003). Essential role for STAT5 signaling in CD25+ CD4+ regulatory T cell homeostasis and the maintenance of self-tolerance. The Journal of Immunology, 171(7), 3435-3441.16. Mamo, T., Hippen, K. L., MacMillan, M. L., Brunstein, C. G., Miller, J. S., Wagner, J. E., & McKenna, D. H. (2022). Regulatory T cells: a review of manufacturing and clinical utility. Transfusion, 62(4), 904.17. Vormittag, P., Gunn, R., Ghorashian, S., & Veraitch, F. S. (2018). A guide to manufacturing CAR T cell therapies. Current opinion in Biotechnology, 53, 164-181.18. Atouf, F. (2016). Cell-based therapies formulations: Unintended components. The AAPS Journal, 18, 844-848.19. van der Walle, C. F., Godbert, S., Saito, G., & Azhari, Z. (2021). Formulation considerations for autologous T cell drug products. Pharmaceutics, 13(8), 1317.EXAMPLES

[0141] Background . A component of the adaptive immune system, T lymphocytes are composed of several different types of cells. Cytotoxic T lymphocytes are responsible for directly attacking cancerous cells or virally infected cells. CD4+ cells are divided into helper T cells (T helper) and regulatory T cells (Tregs), each with unique functions in the immune system. While the CD4+ T helper cells modulate the immune response, Tregs suppress the immune system to prevent overactivation and autoimmunity. Given their role in downregulating immune responses, Tregs have been a focus of cellular therapies for the treatment of autoimmune diseases and organ transplant. Treg therapies can either be isolated as CD4+ CD25+ FoxP3+ natural Tregs (nTregs) or produced from isolated CD4+ peripheral T cells and differentiated ex vivo as induced Tregs (iTregs). These methods have been employed in clinical trials for autoimmune disease, organ transplant, and graft versus host disease.60 / 191#14753190vl

[0142] Due to the power of Tregs in suppressing and modulating the immune response (Pilat, 2023), clinical trials have focused on employing adoptive Tregs as a treatment for autoimmune disease. In the Treg-TID trial (NCT01210664), patients with recent onset type 1 diabetes mellitus (T1DM) received infusion of autologous polyclonal Tregs; however, the Tregs were not detectable in patients after 3 months. A subsequent trial (NCT02772679, TILT) followed the Treg infusion with a 5-day course of low dose interleukin-2 (IL-2). While IL-2 promoted expansion of the infused Tregs, it also promoted growth of CD8+ Cytotoxic T cells (Dong et al., 2021). A Phase II PTreg trial is also being conducted in patients aged 3-18 who have a genetic predisposition for T1DM for PolTREG and Immuthera (NCT06688331). Another Phase 1 trial in systemic lupus erythematous infused autologous Tregs; however, the trial was terminated early due to only enrolling one patient (Dall'Era et al., 2019). A non-randomized, open-label Phase 1 trial of polyTregs in active pemphigus was similarly terminated due to poor enrollment during COVID- 19 (NCT03239470). Several clinical trials infusing Tregs in kidney and liver transplant recipients showed safety and a favorable side effect profile compared to alternatives (Bemaldo-de-Quiros et al., 2022; Chandran et al., 2017; Sawitzki et al., 2020; Tang et al., 2022). Additional clinical trials, including those for Chimeric Antigen Receptor (CAR)-Tregs, are ongoing (NCT03577431; NCT05234190; NCT04817774). Overall, adoptive Treg therapies have shown safety and a potentially favorable side effect profile in early clinical trials, but those existing therapies have failed to achieve tolerance and suppress overactive immune responses (Rodrigues et al., 2025).

[0143] Despite past and ongoing clinical trials, there are no Treg therapies currently FDA approved as standard of care therapy, which is often related to several therapeutic challenges. Treg therapies have proved difficult to manufacture in sufficient numbers for infusion (Rodrigues et al., 2025). However, given their role in treating autoimmune diseases, transplant rejection, and graft versus host disease (GVHD) (Dawson et al., 2017; Dawson et al., 2020; Atif et al., 2020), Tregs have been a point of focus for improved manufacturing methods and techniques. Attempts to enhance Treg therapeutic potential by improving Treg stability, suppressive function, and antigen specificity, using methods such as FOXP3 gene transfer to convert CD4 T cells into functional Tregs, have been assessed (Henschel et al., 2023; Elinav et al., 2009; Imura et al., 2020; Tuomela et al., 2024). Other methods for improving Treg function include genetic engineering to create CAR-Tregs and T cell receptor (TCR)-engineered Tregs, which allows the cells to be precisely targeted to specific antigens and circumvents the problem of nonspecific functionality (Fisher et al., 2025; Vukovic et al.,61 / 191#14753190vl2024; Arjomandnejad et al., 2022; Amini et al., 2021) through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene technology (Pikor et al., 2025;Shimon et al., 2025). Since many studies have shown that metabolic regulation of Tregs through IL-2 signaling, FOXP3 activity, and glycolytic reprogramming and oxidative phosphorylation are critical for controlling their differentiation, stability, and function (Angelin et al., 2017; Niu et al., 2025; Gerriets et al., 2016; Fan & Turka, 2018; Apostolidis et al., 2016; Zhang et al., 2021, Carbone et al., 2024; de Candia et al., 2022), these pathways may also be targets for improvement of Treg therapeutics. In addition to targeting overexpression of FoxP3, modifying the IL-2 receptor, redirecting Treg specificity, and development of an off the shelf Treg therapy that can evade detection through deletion of Human Leukocyte Antigen (HLA) molecules is underway (Sockolosky et al., 2018; Gozalvez et al., 2025; Hassan et al., 2025; McCallion et al., 2025; Du et al., 2025; Robert et al., 2025; Henschel et al., 2023; In dart et al., 2025). Overall, the focus of current research is to generate genetically modified, metabolically optimized, stable, and safe Treg therapies (Amini et al., 2021; Hassan et al., 2025).

[0144] While various methodologies have been employed to improve Treg therapeutic potential, they remain out of reach for most patients suffering from autoimmune disease, transplant rejection, or other inflammatory disorders. Of the methods being investigated to improve Treg therapies, none have explored the utility of microgravity (simulated or zero-gravity - Lower Earth Orbit) as a means for improving adoptive Treg therapies. Presently, both simulated microgravity and Lower Earth Orbit (LEO) are being explored for their ability to manufacture novel therapeutics and understand biological systems. Specifically, use of simulated microgravity for organoid development and 3D culture systems, stem cell generation and optimization, bone regeneration, treatment of heart and vascular disease, and understanding cancer cell biology is being explored (Jasemi et al., 2025; Cui et al., 2023; Ren et al., 2023; Grimm et al., 2022; Moroni et al., 2022; Scott et al., 2022; Ruggiu et al., 2015; Jogdand et al., 2024). Given the promise of microgravity for these therapeutic strategies, LEO is being touted as the next frontier for biomanufacturing on a large scale. Exploiting the zero-gravity environment, LEO biomanufacturing may be useful for protein synthesis, regenerative medicine, organoid development, and antibody and vaccine development (Giulianotti & Low, 2019; Sharma et al., 2022; Marotta et al., 2025; Mozneb et al., 2025). Overall, both simulated microgravity and LEO represent a novel frontier for understanding and developing biologic therapies. Although microgravity has not been exploited for its effects on immune cell function in a therapeutic setting, its62 / 191#14753190vlimmunosuppressive effects have been well documented in the astronaut population during and after spaceflight (Crucian et al., 2013; Hicks et al., 2023; Zhang et al., 2017). Studies investigating the immune system's response to spaceflight and simulated microgravity have established that these conditions generally induce immune system dysregulation and immunosuppression in both humans and animal models (Wu et al., 2024; Miranda et al., 2024; Ismail et al., 2025; Mathyk et al., 2024; Wadhwa et al., 2024; Liu et al., 2014;Ponomarev et al., 2020; Xu et al., 2013; Xu et al., 2016; Buchheim et al., 2019; Chen et al., 2017; Crucian et al., 2013; Hicks et al., 2023; Kelsen et al., 2012; Luo et al., 2013; Sastry et al., 2001). This observation is associated with increased inhibition of T cell activation through suppression of key signaling pathways like Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB) (Calcagno et al., 2023; Chang et al., 2012; Martinez et al., 2015; Paulsen et al., 2010; Tauber et al., 2015; Boonyaratanakomkit et al., 2005; Thiel et al., 2017; Ward et al., 2006), and reduced genetic expression or regulation of IL-2 and its receptor (Cooper & Pellis, 1998; Licato & Grimm, 1999; Walther et al., 1998). Microgravity has been shown to alter the distribution and function of various leukocyte subsets, including T cells, B cells, natural killer (NK) cells, and dendritic cells (Chen et al., 2015; Gallardo-Dodd et al., 2023; Gridley et al., 2009; Spatz et al., 2021; Stervbo et al., 2018; Tackett et al., 2019; Van Walleghem et al., 2017). Research indicates that the microgravity environment may selectively impact Regulatory T cells (FoxP3+ T cells), with simulated microgravity suppressing the expansion of pathogenic T cells in autoimmune models (Bai et al., 2011) and interacting with space radiation to modify FoxP3+ T cell properties (Gridley et al., 2013; Rizvi et al., 2011; Sadhukhan et al., 2021).

[0145] Other related findings include microgravity's influence on the microbiome / immune axis (Li et al., 2015; Zhang et al., 2022), the potential for countermeasures to restore immune function (Hales et al., 2002; Mylabathula et al., 2022), and the relevance of these observations to terrestrial diseases like diabetes and chronic fatigue syndrome (Ahmed et al., 2019; Strassheim et al., 2017). Overall, the unique mechanoimmunomodulatory effects of microgravity on immune cell stability and signaling, especially concerning Treg cells, represent a novel frontier for biomanufacturing next-generation cellular therapies (Dhar et al., 2021; Hauschild et al., 2014; Jacob et al., 2023; Secchi et al., 2015).

[0146] Of the integral works in the field of microgravity immunology, two studies have investigated the effects of microgravity on peripheral blood mononuclear cells (PBMCs), detailing modulations within the Treg cellular population. Kim, et al. (2024)63 / 191#14753190vlutilized multi-omics to explore the effect of zero-gravity on PBMCs isolated both pre- and post-flight from 4 astronauts of the 3-day Inspiration4 mission to LEO. This study demonstrated that within the Treg component of PBMCs, there was an upregulation of both FoxP3 and IL2RA, critical drivers of Treg identity. Similarly, utilizing a rotating wall vessel system, Spatz et al. (2021) demonstrated the effects of simulated microgravity on isolated PBMCs from healthy volunteers. Utilizing single-cell mass cytometry, the authors found that STAT5 signaling was upregulated in the Treg cell component of PBMCs; however, they found no change in the amount of Tregs present. Taken together, these findings suggest that microgravity modulates the functionality and phenotypic profile of Tregs and may have potential to enhance Treg-based cellular therapeutics for treatment of patients. In this work, the functionality of Treg-based cellular therapies was successfully modulated through simulated microgravity exposure of two Treg subpopulations (induced - iTreg and natural -nTreg) following standard isolation and expansion protocols. Through this work, simulated microgravity’s ability to generate a more stable, immunosuppressive Treg therapy with enhanced function and cellular persistence was detailed. Without wishing to be bound by any theory, it is believed that the work described herein may have broad implications in the treatment of autoimmune and inflammatory disorders.

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Crucian B, Stowe R, Mehta S, Uchakin P, Quiriarte H, Pierson D, Sams C. Immune system dysregulation occurs during short duration spaceflight on board the space shuttle. J Clin Immunol. 2013 Feb;33(2):456-65.50. Hicks J, Olson M, Mitchell C, Juran CM, Paul AM. The Impact of Microgravity on Immunological States. Immunohorizons. 2023 Oct l;7(10):670-682.51. Zhang Y, Moreno- Villanueva M, Krieger S, Ramesh GT, Neelam S, Wu H.Transcriptomics, NF-KB Pathway, and Their Potential Spaceflight-Related Health Consequences. Int J Mol Sci. 2017 May 31 ; 18(6): 1166.52. Wu F, Du H, Overbey E, Kim J, Makhijani P, Martin N, Lerner CA, Nguyen K, Baechle J, Valentino TR, Fuentealba M, Bartleson JM, Halaweh H, Winer S, Meydan C, Garrett- Bakelman F, Sayed N, Melov S, Muratani M, Gerencser AA, Kasler HG, Beheshti A, Mason CE, Furman D, Winer DA. Single-cell analysis identifies conserved features of immune dysfunction in simulated microgravity and spaceflight. 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Impairment of antigen- specific cellular immune responses under simulated microgravity conditions. In Vitro Cell Dev Biol Anim. 2001 Apr;37(4):203-8.66. Calcagno G, Jeandel J, Frippiat JP, Kaminski S. Simulated Microgravity Disrupts Nuclear Factor KB Signaling and Impairs Murine Dendritic Cell Phenotype and Function. Int J Mol Sci. 2023 Jan 15;24(2):1720.67. Chang TT, Walther I, Li CF, Boonyaratanakomkit J, Galleri G, Meloni MA, Pippia P, Cogoli A, Hughes-Fulford M. The Rel / NF-KB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity. J Leukoc Biol. 2012 Dec;92(6): 1133-45.68. Martinez EM, Yoshida MC, Candelario TL, Hughes-Fulford M. Spaceflight and simulated microgravity cause a significant reduction of key gene expression in early T- cell activation. Am J Physiol Regul Integr Comp Physiol. 2015 Mar 15;308(6):R480-8.69. Paulsen K, Thiel C, Timm J, Schmidt PM, Huber K, Tauber S, Hemmersbach R, Seibt D, Kroll H, Grote KH, Zipp F. Microgravity-induced alterations in signal transduction in cells of the immune system. Acta Astronautica. 2010 Nov 1 ;67(9- 10): 1116-25.70. Tauber S, Hauschild S, Paulsen K, Gutewort A, Raig C, Hurlimann E, Biskup J, Philpot C, Lier H, Engelmann F, Pantaleo A, Cogoli A, Pippia P, Layer LE, Thiel CS, Ullrich O. Signal transduction in primary human T lymphocytes in altered gravity during parabolic flight and clinostat experiments. Cell Physiol Biochem. 2015;35(3): 1034-51.71. Boonyaratanakomkit JB, Cogoli A, Li CF, Schopper T, Pippia P, Galleri G, Meloni MA, Hughes-Fulford M. Key gravity- sensitive signaling pathways drive T cell activation. FASEB J. 2005 Dec;19(14):2020-2.72. Thiel CS, Hauschild S, Huge A, Tauber S, Lauber BA, Polzer J, Paulsen K, Lier H, Engelmann F, Schmitz B, Schutte A, Layer LE, Ullrich O. Dynamic gene expression response to altered gravity in human T cells. Sci Rep. 2017 Jul 12;7(l):5204.73. Ward NE, Pellis NR, Risin SA, Risin D. Gene expression alterations in activated human T-cells induced by modeled microgravity. J Cell Biochem. 2006 Nov l;99(4):1187-202.71 / 191#14753190vl74. Cooper D, Pellis NR. Suppressed PHA activation of T lymphocytes in simulated microgravity is restored by direct activation of protein kinase C. J Leukoc Biol. 1998 May;63(5):550-62.75. Licato LL, Grimm EA. Multiple interleukin-2 signaling pathways differentially regulated by microgravity. Immunopharmacology. 1999 Nov;44(3):273-9.76. Walther I, Pippia P, Meloni MA, Turrini F, Mannu F, Cogoli A. Simulated microgravity inhibits the genetic expression of interleukin-2 and its receptor in mitogen- activated T lymphocytes. FEBS Eett. 1998 Sep 25;436(1): 115-8.77. Chen H, Euo H, Eiu J, Wang P, Dong D, Shang P, Zhao Y. The distinctive sensitivity to microgravity of immune cell subpopulations. Microgravity Science and Technology. 2015 Nov;27(6):427-36.78. Gallardo-Dodd CJ, Oertlin C, Record J, Galvani RG, Sommerauer C, Kuznetsov NV, Doukoumopoulos E, Ali E, Oliveira MMS, Seitz C, Percipalle M, Nikic T, Sadova AA, Shulgina SM, Shmarov VA, Kutko OV, Vlasova DD, Orlova KD, Rykova MP, Andersson J, Percipalle P, Kutter C, Ponomarev SA, Westerberg LS. Exposure of volunteers to microgravity by dry immersion bed over 21 days results in gene expression changes and adaptation of T cells. Sci Adv. 2023 Aug 25;9(34):eadgl610.79. Gridley DS, Slater JM, Luo-Owen X, Rizvi A, Chapes SK, Stodieck LS, Ferguson VL, Pecaut MJ. Spaceflight effects on T lymphocyte distribution, function and gene expression. J Appl Physiol (1985). 2009 Jan;106(l):194-202.80. Spatz JM, Fulford MH, Tsai A, Gaudilliere D, Hedou J, Ganio E, Angst M, Aghaeepour N, Gaudilliere B. Human immune system adaptations to simulated microgravity revealed by single-cell mass cytometry. Sci Rep. 2021 Jun 7;11(1): 11872.81. Stervbo U, Roch T, Kornprobst T, Sawitzki B, Griitz G, Wilhelm A, Lacombe F, Allou K, Kaymer M, Pacheco A, Vigne J, Westhoff TH, Seibert FS, Babel N. Gravitational stress during parabolic flights reduces the number of circulating innate and adaptive leukocyte subsets in human blood. PLoS One. 2018 Nov 14;13(ll):e0206272.82. Tackett N, Bradley JH, Moore EK, Baker SH, Minter SL, DiGiacinto B, Arnold JP, Gregg RK. Prolonged exposure to simulated microgravity diminishes dendritic cell immunogenicity. Sci Rep. 2019 Sep 25;9(1): 13825.83. Van Walleghem M, Tabury K, Fernandez-Gonzalo R, Janssen A, Buchheim JI, Chouker A, Baatout S, Moreels M. Gravity-Related Immunological Changes in Human Whole Blood Cultured Under Simulated Microgravity Using an In Vitro Cytokine Release Assay. J Interferon Cytokine Res. 2017 Dec;37(12):531-540.72 / 191#14753190vl84. Bai S, Li Y, Wang J, Zhai D, Kong Q, Liu Y, Liu X, Sun B, Xu J, Wang D, Wang G, Mu L, Xu X, Li H. Modeled microgravity suppressed expansion of the MBP- specific T lymphocytes of rats with experimental autoimmune encephalomyelitis. Immunol Invest.201 l;40(5):535-5L85. Gridley DS, Rizvi A, Makinde AY, Luo-Owen X, Mao XW, Tian J, Slater JM, Pecaut MJ. Space-relevant radiation modifies cytokine profiles, signaling proteins and Foxp3+ T cells. Int J Radiat Biol. 2013 Jan;89(l):26-35.86. Rizvi A, Pecaut MJ, Slater JM, Subramaniam S, Gridley DS. Low-dose y-rays modify CD4(+) T cell signalling response to simulated solar particle event protons in a mouse model. Int J Radiat Biol. 2011 Jan;87(l):24-35.87. Sadhukhan R, Majumdar D, Garg S, Landes RD, McHargue V, Pawar SA, Chowdhury P, Griffin RJ, Narayanasamy G, Boerma M, Dobretsov M, Hauer- Jensen M, Pathak R. Simultaneous exposure to chronic irradiation and simulated microgravity differentially alters immune cell phenotype in mouse thymus and spleen. Life Sci Space Res (Amst).2021 Feb;28:66-73.88. Li P, Shi J, Zhang P, Wang K, Li J, Liu H, Zhou Y, Xu X, Hao J, Sun X, Pang X, Li Y, Wu H, Chen X, Ge Q. Simulated microgravity disrupts intestinal homeostasis and increases colitis susceptibility. FASEB J. 2015 Aug;29(8):3263-73.89. Zhang P, Shao L, Zhang J, Wang L, Pang X, Tao W, Fan G, Peng L, Kan G, Li W, Liang X. Microbiota-muscle / immune interactions in rhesus macaque under simulated microgravity revealed by integrated multi-omics analysis. JCSM Rapid Communications.2022 Jul;5(2):212-25.90. Hales NW, Yamauchi K, Alicea A, Sundaresan A, Pellis NR, Kulkami AD. A countermeasure to ameliorate immune dysfunction in in vitro simulated microgravity environment: role of cellularnucleotide nutrition. In Vitro Cell Dev Biol Anim. 2002 Apr;38(4):213-7.91. Mylabathula PL, Diak DM, Baker FL, Niemiro GM, Markofski MM, Crucian BE, Katsanis E, Simpson RJ. IL-2 and Zoledronic Acid Therapy Restores the In Vivo Anti- Leukemic Activity of Human Lymphocytes Pre-Exposed to Simulated Microgravity. Front Biosci (Landmark Ed). 2022 Jul 8;27(7):215.92. Ahmed AS, Ramachandran V, O'Conor DK, Antonsen EL. Immune dysfunction in spaceflight and diabetes mellitus-translating space observations to terrestrial disease. McGill Journal of Medicine. 2019 Jul 6; 17(1).73 / 191#14753190vl93. Strassheim V, Ballantine R, Hackett KL, Frith J, Newton JL. Understanding severely affected chronic fatigue syndrome (CFS): the gravity of the situation. Physical Therapy Reviews. 2017 Jul 4;22(3-4): 197-201.94. Dhar S, Kaeley DK, Kanan MJ, Yildirim-Ayan E. Mechano-Immunomodulation in Space: Mechanisms Involving Microgravity-Induced Changes in T Cells. Life (Basel). 2021 Oct 3;ll(10):1043.95. Hauschild S, Tauber S, Lauber B, Thiel CS, Layer LE, Ullrich O. T cell regulation in microgravity-The current knowledge from in vitro experiments conducted in space, parabolic flights and ground-based facilities. Acta Astronautica. 2014 Nov 1 ; 104(l):365- 77.96. Jacob P, Oertlin C, Baselet B, Westerberg LS, Frippiat JP, Baatout S. Next generation of astronauts or ESA astronaut 2.0 concept and spotlight on immunity. NPJ Microgravity.2023 Jun 28;9(1):51.97. Secchi C, Crescio C, Pantaleo A, Pippia P. Recent advances in human T lymphocyte biology in space. Journal of Biological Research. 2015;88(l): 1.98. Kim J, Tierney BT, Overbey EG, Dantas E, Fuentealba M, Park J, Narayanan SA, Wu F, Najjar D, Chin CR, Meydan C, Loy C, Mathyk B, Klotz R, Ortiz V, Nguyen K, Ryon KA, Damle N, Houerbi N, Patras LI, Schanzer N, Hutchinson GA, Foox J, Bhattacharya C, Mackay M, Afshin EE, Hirschberg JW, Kleinman AS, Schmidt JC, Schmidt CM, Schmidt MA, Beheshti A, Matei I, Lyden D, Mullane S, Asadi A, Lenz JS, Mzava O, Yu M, Ganesan S, De Vlaminck I, Melnick AM, Barisic D, Winer DA, Zwart SR, Crucian BE, Smith SM, Mateus J, Furman D, Mason CE. Single-cell multi-ome and immune profiles of the Inspiration4 crew reveal conserved, cell-type, and sex-specific responses to spaceflight. NatCommun. 2024 Jun 11;15(1):4954.Example 1. Isolation of PBMCs from whole blood

[0148] To isolate Peripheral Blood Mononuclear Cells (PBMCs), blood was first collected from healthy, male donors aged (20-35), through venipuncture using common venipuncture technique. The subject was seated, their arm gently extended. The skin (e.g. antecubital fossa of arm) was prepared using Medline Sterile Alcohol Prep Pads before a Medline Disposable Tourniquet was applied 6 inches above venipuncture site. The tourniquet was applied to visualize the veins (e.g., the median cubital vein of the forearm). Vacutainer Eclipse Blood Collection Needles with Pre-attached Holder and Luer Adapter, 21G x 1.25” were used to collect 20-75 mL of blood into 6 mL BD Vacutainer EDTA Blood Collection 74 / 191#14753190vltubes. A sterile needle was inserted with precision into the vein, and whole blood flows into vacuum-sealed tubes, pre-filled with anticoagulants (e.g., EDTA, heparin, or citrate). Blood tubes were inverted 5 times, with blood processed immediately for PBMC isolation.Following collection of peripheral blood, PBMCs were isolated using density gradient centrifugation. For PBMC isolation, Fymphoprep™ was used according to manufacturer protocol. Briefly, Fymphoprep™ was warmed to room temperature (15 - 25 °C) and gently inverted to mix before use. Collected whole blood was diluted 1:1 with TexMACS™ culture medium. This diluted mixture (e.g. 30 mL) was gently layered on top of 15 mF of Fymphoprep™ in a 50 mF Falcon tube, being careful to minimize mixing of blood with Fymphoprep™. Here, a sterile conical tube e.g., 15 or 50 mF) was prepared by adding a high-density solution (e.g., Ficoll-Paque, Fymphoprep™) into the bottom of the tube. Whole blood (diluted 1:1 with sterile PBS w / HSA or suitable cell culture media) is carefully layered on top of the high-density solution. Samples were centrifuged at 800 x g for 20 minutes at room temperature (20°C) using a benchtop centrifuge (e.g. Thermo Scientific ST Plus centrifuge). The tubes were placed into a centrifuge and spun at 800 x g for 20 minutes at room temperature. Following centrifugation, three distinct layers (top layer: plasma, middle layer: PBMCs or buffy coat, bottom layer: granulocytes and red blood cells) were visualized. The PBMC layer at the plasma:Fymphoprep™ interface was collected without disturbing the erythrocyte / granulocyte pellet using a Fisherbrand™ Pipet Controller and 10 mF pipettes, placing the collected PBMC layer into a new 50 mF Falcon Tube. Once each layer was collected, the PBMCs were washed by adding equal volume of TexMACS™ media to the PBMC layer (e.g. 25 mF TexMACS™ to 25 mF of PBMCs), again spinning at 800 x g for 20 minutes at room temperature (20°C). Supernatant was removed by gently pouring it off, with pelleted PBMCs then resuspended in TheraPEAK PBS, placed on ice, and prepared for counting on a hemocytometer. The buffy coat containing PBMCs was carefully aspirated and washed 2-3 times in PBS w / HSA or culture medium through gentle centrifugation to remove residual high-density solution (e.g. Ficoll-Paque, Fymphoprep™) and platelets. After washing, the PBMC pellet was resuspended, and cell counting was performed using a hemocytometer or automated counter, with Trypan Blue staining for visualization of cell viability. Here, a dilution of resuspended PBMCs (e.g. 1:100) was prepared before mixing in 1:1 ratio with 0.4% Trypan Blue. This mixture was pipetted into a clean hemocytometer chamber w / coverslip. Five (unstained) cells were then counted, with recording using a hand tally counter. Total number of live PBMCs was then determined by multiplying the averaged cell count (from hemocytometer quadrants) by dilution factor(s) and 10,000. Alternatively,75 / 191#14753190vlleukapheresis, using a specialized apheresis machine and two intravenous catheters (one for blood extraction and another for returning components to the body) can be used to isolate PBMCs. Blood was continuously drawn and separated into its components (plasma, red blood cells, and leukocytes). The machine selectively retains the buffy coat, a leukocyte-rich layer containing PBMCs, while returning both plasma and red blood cells to the subject.Example 2. Isolation ofnTregs from whole blood

[0149] The isolation of natural T regulatory cells (nTregs, CD4+ CD25+ CD127dim / -) was carried out using a two-step procedure. First, non-CD4+ and CD127high cells of an isolated PBMC suspension were indirectly magnetically labeled using a cocktail of biotin-conjugated primary antibodies (e.g., CD8, CD19, CD123, and CD127) and anti-biotin monoclonal antibodies conjugated to microbeads [e.g., monoclonal anti-biotin antibody] as the secondary labeling reagent. The labeled cells were depleted from the PBMC suspension by passing them through a column with a magnetic field e.g., MACS® Column placed within the field of a MACS Separator), thus removing the unwanted cell populations. In the second step, the pre-enriched CD4+ T cell fraction was directly labeled with CD25 microbeads [e.g., microbeads conjugated to monoclonal anti-CD25 antibody] to specifically isolate CD4+ CD25+ CD127dim / - regulatory T cells through positive selection. These cells were retained within a separate column placed within a magnetic field (e.g., MACS Column under the magnetic field of the MACS Separator). Once the column was removed from the magnetic field, the retained CD4+ CD25+ CD127dim / - regulatory T cells were eluted as the positively selected cell fraction into a separate falcon tube. To further enhance purity, the positively selected cell fraction was passed through an additional column within a magnetic field (e.g., MACS Column under the magnetic field of the MACS Separator) for an additional separation step. In some cases, an automated magnetic separation machine (e.g., autoMACS Pro Separator) was be used for magnetic cellular separation steps. In some cases, the steps were performed as follows: Human CD4+ CD25+ CD127dim / - natural regulatory T cells were isolated using the CD4+CD25+CD127dim / - Regulatory T Cell Isolation Kit II, human (Miltenyi Biotec, 130-094-775) according to manufacturer protocol. Briefly, for every 107total PBMCs isolated, the PBMC cellular pellet was resuspended in 40 pL of TheraPEAK PBS in a 50 mL Falcon Tube. Then, 10 pL of CD4+CD25+CD127dim / - T Cell Biotin-Antibody Cocktail II was added per 107total PBMCs, mixed well, and incubated for 5 minutes in the refrigerator (4°C). Next, 30 pL of TheraPEAK PBS per 107 total PBMCs was added to the same 50 mL Falcon Tube before 20 pL of Anti-Biotin MicroBeads per 107total76 / 191#14753190vlPBMCs to the same 50 mL Falcon Tube. The suspension was resuspended to ensure full mixing and incubated for another 10 minutes at 4°C. During the 10 minutes incubation, an LD column was prepared by placing in the magnetic field of a Miltenyi Biotec MidiMACS™ Separator and washing with 2 mL of TheraPEAK PBS. After the 10-minute incubation, the cell suspension was applied to the column and unlabeled cells that passed through the column (representing the unlabeled CD4+ T cells) were captured in a 50 mL Falcon Tube. The column was washed twice with 1 mL of TheraPEAK PBS, again collecting unlabeled cells that passed through (representing the unlabeled CD4+ T cells), and combined with previous effluent. The number of CD4+ T cells isolated were counted using a hemocytometer. Next, the CD4+ T cell suspension was centrifuged at 300 x g for 10 minutes, with supernatant discarded. The cellular pellet was resuspended in 90 pL of TheraPEAK PBS per 107total CD4+ cells in a 50 mL Falcon Tube before 10 pL of CD25 MicroBeads II per 107total cells were added. The cellular suspension was mixed well and incubated for 15 minutes in the refrigerator (4 °C). Cells were washed by adding 2 mL of TheraPEAK PBS and then centrifuged at 300 x g for 10 minutes. Supernatant was discarded, and cells were resuspended in 1 mL of TheraPEAK PBS. An LS column was placed in the magnetic field of the Miltenyi Biotec MidiMACS™ Separator and washed with 3 mL of TheraPEAK PBS. The cell suspension was applied to the column, with flow through collected in a waste tube. The column was washed 3 times with 1 mL of TheraPEAK PBS, again collecting unlabeled cells with the previous effluent. The column was removed from the magnet, placed into a 50 mL Falcon Tube, and 1 mL of TheraPEAK PBS was then placed onto the column. The magnetically labeled cells were flushed out immediately by pushing the plunger into the column. The number of isolated CD4+ CD25+ CD127dim / - nTregs was determined by counting with a hemocytometer as previously described. Cells were centrifuged at 500 x g for 5 min, supernatant was discarded, and cellular pellet was resuspended in TexMACS™ Medium for further culture. In some cases, unused nTregs were resuspended in CryoStor® at a concentration of 5 million cells / mL, frozen slowly in Mr. Frosty Freezing Container™ at -80°C, and stored at -200°C until use. Cells were thawed slowly in a 37°C water bath and moved to a 50mL Falcon tube. TexMACS™ medium (1% Penicillin-Streptomycin Solution, 10% Akron Bio Human AB serum) was added slowly before cells were spun at 500 x g for 5 minutes at RT. Cells were allowed to rest for 24 hours in 15 mL TexMACS™ medium with 300IU / mL IL- 12 in a T75 flask before experimentation as below.77 / 191#14753190vlExample 3. Isolation of naive CD4+ T cells from PBMCs

[0150] The isolation of naive CD4+ T cells from PBMCs was carried out using a one-step negative selection procedure. First, non-CD4+ cells of an isolated PBMC mixture were indirectly magnetically labeled using a cocktail of biotin-conjugated monoclonal antibodies [e.g., CD8, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD45RO, CD56, CD123, TCRy / 6, HLA-DR, and CD235a (Glycophorin A)] and monoclonal antibodies e.g., antibiotin, anti-CD61) conjugated to microbeads as the secondary labeling reagent. The labeled cells were depleted from the PBMC mixture by passing them through a column with a magnetic field (e.g., MACS® Column placed within the field of a MACS Separator), thus removing the unwanted cell populations. The column eluent (containing unlabeled cells, representing the enriched naive CD4+ CD45RA+ T cells) was collected into a falcon tube. The column was washed with 3 mL of buffer (e.g. PBS w / HSA or suitable cell culture media) with unlabeled cells that pass through (representing another enriched naive CD4+ CD45RA+ T cells population) collected and combined with the previous column eluent. In some cases, an automated magnetic separation machine (e.g., autoMACS Pro Separator) was be used for magnetic cellular separation steps. In some cases, the steps were performed as follows: Human CD4+ CD45RA+ (naive) T lymphocytes were isolated using the Naive CD4+ T Cell Isolation Kit II, human (Miltenyi Biotec, 130-094-131) according to manufacturer protocol. Briefly, for every 107total PBMCs isolated, the PBMC cellular pellet was resuspended in 40 pL of TheraPEAK PBS in a 50 mL Falcon Tube. Then, 10 pL of Naive CD4+ T Cell Biotin-Antibody Cocktail II was added per 107total PBMCs, mixed well, and incubated for 5 minutes in the refrigerator (4°C). Next, 30 pL of TheraPEAK PBS per 107total PBMCs was added to the same 50 mL Falcon Tube before 20 pL of Naive CD4+ T Cell MicroBeads Cocktail II per 107total PBMCs to the same 50 mL Falcon Tube. The suspension was mixed well and incubated for another 10 minutes at 4°C. During the 10-minute incubation, an LS column was prepared by placing in the magnetic field of a Miltenyi Biotec MidiMACS™ Separator and washing with 3 mL of TheraPEAK PBS. After the 10-minute incubation, the cell suspension was applied to the column and unlabeled cells that passed through the column (representing the enriched CD4+ T cells) were captured in a 50 mL Falcon Tube. The column was washed with 3 mL of TheraPEAK PBS, again collecting unlabeled cells that passed through (representing the enriched CD4+ T cells), and combined with previous effluent. Collected naive CD4+ T lymphocytes were counted by hemocytometer as previously described. Cells were then centrifuged at 500 x g for 5 min,78 / 191#14753190vlsupernatant was discarded, and cellular pellet was then resuspended in TexMACS™ Medium for culture, differentiation, and expansion to induced T regulatory (iTreg) cells.Example 4. Stimulation and Expansion of nTregs

[0151] The natural T regulatory cells (nTregs, CD4+ CD25+ CD127dim / -), isolated from PBMCs of human peripheral blood, were stimulated and expanded using a combination of various medias as previously described by Gu et al. (STAR Protoc. 2022). First, for 48 hours, 1 x 106nTregs were cultured in 1 mL of stimulation media [serum free media (e.g., complete TexMACS™ GMP media) containing 1% Penicillin- Streptomycin Solution, 10% Akron Bio Human AB serum (or patient derived serum), 20 pL GMP ActiveMax® Human T cell Activation / Expansion CD3 / CD28 beads (for every 106cells), 300 lU / mL GMP Human IL-2 Protein (Aero Bio, GMP-L02H14-1X106IU), and 100 nM Retinoic acid (Powder USP) (Avantor, 7000009-310)] for 48 hours in an appropriate cell culture plate (e.g. 6-, 12-, or 24-well) or in a suitable cell culture flask (e.g. 25 cm2or 75 cm2rectangular canted neck cell culture flask with plug seal cap), in standard cell culture conditions (37°C and 5% CO2). Prior to addition, GMP Active Max Human T Cell Activation / Expansion CD3 / CD28 beads were washed with TheraPEAK PBS on a magnetic stand (e.g. Aero Bio, MB-02-1EA) for 3 minutes before being resuspended in original bead volume of complete TexMACS™ medium. During the first 48 hours, the quiescence of the culture was maintained to ensure cell to bead interactions. Following 48 hours of culture, an equal volume of complete TexMACS™ medium [serum free media (e.g., TexMACS™ GMP) containing 1% Penicillin-Streptomycin Solution, 10% Human AB serum (or patient derived serum)] with 600 lU / mL GMP Human IL-2 Protein and 100 nM Retinoic acid (final concentration of 300 lU / mL GMP Human IL-2 Protein and 50 nM Retinoic acid (Powder USP)) was added and cells were cultured for 168 hours (7 days). During this time, if the media turned yellow, an additional 1 mL of expansion medium (one volume of complete TexMACS™ medium with 600 lU / mL GMP Human IL-2 Protein and 100 nM Retinoic acid) was added. Alternatively, if no color change occurred, 300 lU / mL IL-2 (corrected to total volume) was added every 48 hours. Flasks were changed once the culture volume equals or exceeds % the maximum flask volume. Following the first expansion, an additional 1 mL of stimulation media [serum free media (e.g., TexMACS™ GMP) containing 1% Penicillin-Streptomycin Solution, 10% Human AB serum (or patient derived serum), 20 pL GMP ActiveMax® Human T cell Activation / Expansion CD3 / CD28 Beads (for every 106cells), 300 lU / mL GMP Human IL-2 Protein, 100 nM MACS GMP Rapamycin] was added and cells were cultured for another 4879 / 191#14753190vlhours. After 48 hours, a second 1 mL of expansion media [serum free media (e.g., TexMACS™ GMP) containing 1% Penicillin-Streptomycin Solution, 10% Human AB serum (or patient derived serum), 300 lU / mL GMP Human IL-2 Protein, 50 nM MACS GMP Rapamycin] was added to the stimulation media and cells were cultured for another 168 hours. Again, during this time, if the media turned yellow, an additional 1 mL of expansion medium was added. Flasks were changed once the culture volume equals or exceeds % the maximum flask volume. The CD4+ / FoxP3+ nTregs expand 200-300 fold within 12-14 days. Cells were either stored in a suitable cell cryopreservation solution e.g., CellStar®, 10% DMSO in 90% Human AB serum) or prepared for downstream applications. In some cases, at day 10, cell suspensions were transferred to a 50 mL Falcon Tube and counted by hemocytometer before centrifugation at 500 x g, room temperature (RT) for 5 minutes. Next, nTregs were cultured at 1 x 106 / mL in complete TexMACS™ medium (1% Penicillin-Streptomycin Solution, 10% Akron Bio Human AB serum) with 20 pL GMP Active Max Human T Cell Activation / Expansion CD3 / CD28 beads (Aero Bio, GMP-MBSOOl-lOmg) for every 106cells, 300 lU / mL GMP Human IL-2 Protein (Aero Bio, GMP-L02H14-1X106IU), and 100 nM MACS GMP Rapamycin (Miltenyi Biotec, 170-076-308) for 48 hours in an appropriate cell culture flask (e.g., T25, T75) in normal cell culture conditions (37°C and 5% CO2). Prior to addition, GMP Active Max Human T Cell Activation / Expansion CD3 / CD28 beads were washed with TheraPEAK PBS on a magnetic stand (e.g., Aero Bio, MB-02-1EA) for 3 minutes before being resuspended in original bead volume of complete TexMACS™ medium. Following 48 hours of culture, an equal volume of complete TexMACS™ medium with 600 lU / mL GMP Human IL-2 Protein and 100 nM Rapamycin (final concentration of 300 lU / mL GMP Human IL-2 Protein and 50 nM Rapamycin) was added. Over the next 7 days, one volume of complete TexMACS™ medium with 600 lU / mL GMP Human IL-2 Protein and 100 nM Rapamycin was added whenever the media turned yellow. Alternatively, if no color change occurred, 300 lU / mL IL-2 (corrected to total volume) was added every 48 hours. After 18 days of expansion, nTregs were transferred to a 50 mL Falcon tube, counted by hemocytometer, and prepared for downstream applications by centrifuging at 500 x g for 5-minutes, RT, discarding supernatant, and resuspending in 1 mL of TheraPEAK PBS.CD3 / CD28 were removed beads by placing the cell suspension on the magnetic stand for 3-minutes, and centrifuging once again at 500 x g for 5-minutes, RT.80 / 191#14753190vlExample 5. Differentiation and Expansion ofCD4+ T cells into iTres cells

[0152] The naive CD4+ CD45RA+ T cells, isolated from PBMCs of human peripheral blood, were differentiated and expanded using a combination of various medias. First, for 48 hours, 5 x 105naive CD4+ CD45RA+ T cells were cultured in 1 mL of differentiation media [serum free media (e.g., TexMACS™ GMP) containing 1% Penicillin-Streptomycin Solution, 10% Human AB serum (or patient derived serum), 20 pL GMP ActiveMax® Human T cell Activation / Expansion CD3 / CD28 Beads (for every 106cells), 100 lU / mL GMP Human IL-2 Protein, 1 ng / mL GMP Recombinant Human TGF-beta 1, 100 nM Retinoic acid (Powder USP)] in a suitable cell culture flask (e.g., 25 cm2 or 75 cm2 rectangular canted neck cell culture flask with plug seal cap) for 48 hours. Next, to the differentiation media, 1 mL of expansion media [serum free media e.g., TexMACS™ GMP) containing 1% Penicillin- Streptomycin Solution, 10% Human AB serum (or patient derived serum), 300 lU / mL GMP Human IL-2 Protein, 50 nM MACS GMP Rapamycin] was added and cells were cultured for 72 hours. During this time, if the media turned yellow, an additional 1 mL of expansion medium was added. Flasks were changed once the culture volume equals or exceeds % the maximum flask volume. The CD4+ / FoxP3+ iTregs expand 10-12 fold within 5 days. Cells can then either be stored in a suitable cell cryopreservation solution e.g., CellStar®, 10% DMSO in 90% Human AB serum) or prepared for downstream applications. In some cases, the following steps were used: Following naive CD4+ CD45RA+ T lymphocyte isolation, cells were cultured as previously described by Gu et al. (STAR Protoc. 2022) with modification at a density of 5 x 105 / mL in 5 mL of complete TexMACS™ medium (1% Penicillin-Streptomycin Solution, 10% Akron Bio Human AB serum) with 20 pL GMP Active Max Human T Cell Activation / Expansion CD3 / CD28 beads (Aero Bio, GMP-MBSOOl-lOmg) for every 106cells, 100 lU / mL GMP Human IL-2 Protein (Aero Bio, GMP-L02H14-1X106IU), 5 ng / mL Recombinant Human TGF-beta 1 GMP Protein, CF (R&D Systems, 240-GMP-010), and 100 nM Retinoic acid (Powder USP) (Avantor, 7000009-310) for 48 hours in a Corning® 25 cm2U-shape cell culture flask, canted neck (Millipore Sigma, CLS430168-100EA) in standard cell culture conditions (37°C and 5% CO2). Prior to addition, GMP Active Max Human T Cell Activation / Expansion CD3 / CD28 beads were washed with TheraPEAK PBS on a magnetic stand (e.g. Aero Bio, MB-02-1EA) for 3 minutes before being resuspended in original bead volume of complete TexMACS™ medium. During the first 48 hours, the quiescence of the culture was maintained to ensure cell to bead interactions. After 48 hours of incubation, 5 mL of complete TexMACS™ medium (1% Penicillin-Streptomycin Solution, 10% Akron Bio 81 / 191#14753190vlHuman AB serum) was added to the 25 cm2U-shape cell culture flask, bringing the volume to 10 mL. To the volume of 10 mL, 300 lU / mL GMP Human IL-2 and 50 nM MACS® GMP Rapamycin were added, and the culture was incubated for an additional 48 hours at 37 °C and 5% CO2. After 4 days of culture, differentiation, and expansion, iTregs were transferred to a 50 mL Falcon tube, counted by hemocytometer, and prepared for downstream applications by centrifuging at 500 x g for 5-minutes, RT, discarding supernatant, and resuspending in 1 mL of TheraPEAK PBS.CD3 / CD28 beads were removed by placing the cell suspension on the magnetic stand for 3-minutes, and centrifuging once again at 500 x g for 5 minutes, RT.Example 6. Exposure ofnTreg cells or iTreg cells to Microgravity

[0153] To expose the nTregs or iTregs to simulated microgravity, a 10 mL single cell suspension of nTregs or iTregs at a concentration of 1 x 106cells / mL in microgravity cell culture media [TexMACS™ GMP culture media comprising Penicillin- Streptomycin Solution (e.g. 0 to 1%), Human AB serum (or patient derived serum at 0 to 10%), cytokines [e.g. IL-2 (10 to 300 lU / mL), IL-7 (5 to 50 ng / mL), TGF-betal (1 to 100 ng / mL), IL-10 (1 to 100 ng / mL), IL-35 (5 to 100 ng / mL), IL- 12 (0.1 to 100 ng / mL), IFN-gamma (0.1 to 100 ng / mL) and / or IL-15 (5 to 50 ng / mL)], nutritional / metabolic supplements [e.g. arginine (0.5 to 5 mM), glutamine (2 to 6 mM), serine (0.5 to 5 mM), glycine (0.5 to 10 mM)], chemical additives [e.g. taurine (0.1 to 5 mM), N-acetylcysteine (0.1 to 10 mM), all-trans retinoic acid (1 to 1000 nM)], stimulators [e.g. anti-CD3 / CD28 stimulatory beads at 100,000 to 1,000,000 beads for every 105to 106cells], and / or other compounds [e.g. rapamycin (10 to 100 nM)] was prepared. Next, 3 x 10 mL (1 x 106cells / mL) high-aspect ratio vessels (HARVs) were prepared according to Synthecon manufacturer guidelines for simulated microgravity exposure by rinsing with 10 mL sterile TexMACS™ GMP culture media and allowing the vessel to incubate for at least 5 minutes. Next, 10 mL of iTreg or nTreg cellular suspension in microgravity culture medium was pipetted into the vessel through the fill port, filling until the vessel was completely full, and replacing the port cap. After replacing the fill port cap, bubbles from the vessel were removed by placing a syringe on each syringe port, one syringe contains 3 mL of TexMACS™ GMP media, one syringe should be empty. The ports were opened and bubbles began to maneuver underneath the port with the empty syringe attached. The bubbles were pulled into the empty syringe while approximately the same volume of TexMACS™ GMP media was injected through the other syringe port. Once all bubbles were removed, the valves were closed and any residual media was removed with a Pasteur pipet. The HARV was attached onto the rotator base (while it was in the incubator) by slowly turning in a clockwise direction with the 82 / 191#14753190vlincubator set at standard cell culture conditions (37C, 5% CO2). Finally, the power supply was turned on, ensuring that the multicolored ribbon cable was attached to both the power supply and rotator base, and the rotator base started with a rotation rate of 15 RPMs. nTregs or iTregs were then cultured in simulated microgravity for 2, 4, 6, 12, 18, 24, 36, or 48 hours. Cells were removed from the HARVs and prepared for injection into subjects. Alternatively, the CD4+ / FoxP3+ nTreg or iTregs could be exposed to microgravity in low Earth orbit. The nTregs or iTregs, again suspended within a microgravity cell culture medium, were loaded into specially designed biocontainers which maintain select conditions (temperature, pH, and nutrient supply) during spaceflight. These containers were secured in a space capsule designed for travel in low Earth orbit, ensuring protection from vibration, radiation, and temperature fluctuations during launch, orbital trajectory, travel within low Earth orbit, and / or docking to a space station. In low Earth orbit, the cells were exposed to microgravity conditions for a predetermined period. After microgravity exposure, the biocontainers were prepared for reentry, with rigorous measures taken to preserve the cells' viability during the descent back to Earth. Cells were removed from the biocontainers and prepared for injection into subjects. In some cases, the following steps were used: Simulated pg exposure of iTregs and nTregs was conducted using a Synthecon® RCCS-8D Disposable Vessel Systems and 10 mL High Aspect Ratio Vessels (HARVs) according to manufacturer protocol. First, the 10 mL HARV was prepared according to manufacturer guidelines by filling the vessel with 10 mL of complete TexMACS™ medium (1% Penicillin-Streptomycin Solution, 10% Akron Bio Human AB serum) and incubating for 30 minutes. After the incubation, media was then removed using a sterile Pasteur pipette. T regulatory cells (iTregs or nTregs) were counted using a hemocytometer as previously described before a single-cell suspension at the desired concentration (500,000 cells / mL) was prepared in complete TexMACS™ medium. Next, ~10 mL of the cell suspension was pipetted into the HARV, filling until the vessel was completely full (to the brim of fill port). After replacing the fill port, two syringes were placed on each syringe port (one empty 3 mL syringe, one 3 mL syringe filled with 3 mL of the cell suspension). Bubbles were removed from the HARV as per manufacturer guidelines. Here, the syringe ports were opened, and bubbles were maneuvered underneath the port with the empty syringe attached. The bubbles were pulled into the empty syringe while approximately the same volume of cell suspension was injected through the other syringe port. Once all bubbles were removed, the valves were closed (with syringes remaining attached); valve stems and covers were wiped with alcohol swabs and the covers were replaced. The HARV was attached onto83 / 191#14753190vlthe rotator base and placed in the incubator at standard culture conditions (37 °C and 5% CO2) by slowly turning the HARV in a clockwise direction. The rotator base was turned on, with rotations per minute (RPM) slowly increased to the desired 15 RPM (simulated pg). After at least 1 hour of rotation, initial bubbles were removed by decreasing the RPM back to the lowest limit, turning off the rotator base, removing the HARV by turning in a clockwise direction, and removing bubbles as previously described. HARVs were reattached to the rotator base and rotated at 15 RPMs as previously described. Bubble formation was monitored every 6 to 12 hours and removed as needed. Simulated pg exposure was then conducted for 24 hours. Alongside the simulated pg group(s), Control group(s) were incubated in standard culture conditions in T25 flasks at the same concentration (500,000 cells / mL) for the same period (24 hours). At the end of the 24-hour exposure (pg and Control), cells were harvested, counted by hemocytometer, and prepared for downstream applications. This process was conducted for at least an n=3 biological triplicates (three human donors, two donors repeated multiple, different times) for iTregs and n=l biological replicate (one human donor) for nTregs. Experimental replicates (i.e., multiple HARVs or control cultures containing cells from the same experimental donor) were combined before downstream analysis. Statistical analysis comparing the post-simulated pg and Control proliferation index, calculated as below, was conducted using GraphPad Prism 10 and a two-tailed, paired t-test.Recovered Live Cell Count in millions) Proliferation Index = — -77- - - - - — Starting Live Cell Count (in millions)Example 7. Short-term T regulatory cell stimulation assay

[0154] To evaluate differences in flow cytometric parameters (e.g. P-STAT-5, CD25, CD45RO) and protein production (e.g., IL- 10, latent TBG-P), a short-term stimulation assay was conducted as previously described (Li & Park, 2020) with modification. Here, T regulatory cells (iTregs or nTregs) were cultured at a concentration of 1 million cells / mL in complete TexMACS™ medium (1% Penicillin- Streptomycin Solution, 10% Akron Bio Human AB serum) with 300 lU / mL of GMP Human IL-2 Protein (Aero Bio, GMP-L02H14-lxlOA6IU) and incubated in standard culture conditions (37°C and 5% CO2). Following a 30-minute incubation, cell suspensions were transferred to a 50 mL Falcon Tube and centrifuged at 500 x g for 5 minutes. Next, two 1 mL supernatant samples were collected without disturbing the cellular pellet. Each supernatant sample was flash frozen in liquid nitrogen before being transferred to a -80°C freezer for long-term storage. Additionally, the cellular84 / 191#14753190vlpellet was resuspended in TheraPEAK PBS and prepared for flow cytometric analysis as described below (groups, Control_s and pg_s). This process was conducted for at least an n=3 biological triplicates (three human donors, two donors repeated at different times) for iTregs and n=l biological replicate (one human donor) for nTregs.Example 8. Lons-term T regulatory cell stimulation assay

[0155] To evaluate differences in protein production (e.g., IL-10, latent TBG-P), a long-term stimulation assay was conducted as previously described (Procaccini et al., 2016) with modification. Here, T regulatory cells (iTregs or nTregs) were cultured at a concentration of 1 million cells / mL in complete TexMACS™ medium (1% Penicillin-Streptomycin Solution, 10% Akron Bio Human AB serum) with 300 lU / mL of GMP Human IL-2 Protein (Aero Bio, GMP-L02H14-1X106IU) and incubated in standard culture conditions (37°C and 5% CO2) for 24 hours. Following the 24-hour incubation, 20 pL of GMP Active Max Human T Cell Activation / Expansion CD3 / CD28 beads (Aero Bio, GMP-MBSOOl-lOmg) for every 106cells was added and the culture was incubated for an additional 48 hours. Following 72 hours of incubation, cell suspensions were transferred to a 50 mL Falcon Tube and centrifuged at 500 x g for 5 minutes. Next, two 1 mL supernatant samples were collected without disturbing the cellular pellet. Each supernatant sample was flash frozen in liquid nitrogen before being transferred to a -80°C freezer for long-term storage. This process was conducted for n=3 biological triplicates (three human donors) and n=l biological replicate (one human donor) for nTregs.Example 9. Flow cytometric analysis ofiTreg and nTreg phenotypes

[0156] For evaluation of both surface and intracellular markers of T regulatory cell phenotype, flow cytometric analysis was conducted using the 5-laser Cytek Aurora as previously described (Lacinski et al., 2024). Briefly, 2 x 105cells per well (experimental, no stain control, single stains, and fluorescence minus one (FMO) controls were plated in a 300 pL Falcon™ 96-Well, non-treated, V-shaped-bottom microplate. Cells were centrifuged at 500 x g for 5 min, RT, with supernatant discarded by flicking. Cells were washed by adding 200 pL of Dulbecco’s Phosphate Buffered Saline (DPBS) and again centrifuged at 500 x g for 5 min, RT, with supernatant removed by flicking. Next, experimental cells, the Live / Dead single stain control, and FMOs were stained for live / dead using the Zombie NIR™ live / dead stain at a dilution of 1:1000 by resuspending in a volume of 100 pL; all other controls (no stain, single stains, FMO controls) were resuspended in equal volume of PBS. The plate was 85 / 191#14753190vlincubated at 4°C, in the dark, for 30 min. Following incubation, 100 |aL of PBS was added to all wells before centrifugation at 500 x g for 5 min, RT, with supernatant removed by flicking. All wells were blocked by resuspending cells in 5 pL of Human TruStain FcX™ (per million cells) in 100 pl staining volume of DPBS, with incubation at RT for 10 min. Cells were then washed by adding 100 pL of DPBS to each well, the cells were centrifuged at 500 x g, 5 min, RT, followed by supernatant removal by flicking. Next, surface staining was conducted for experimental, single stain, and FMO wells. First, a master mix containing 50 pL of BD Horizon™ Brilliant Stain Buffer (per sample) and 2 pL of each surface stain (AF 488 CD45RA, BV 510 CD45RO, BV 650 CD3, PE-Fire 810 CD4, BV 785 CD8a, PE / Dazzle 594 CD25, and AF 647 CD127) was made for each experimental well (plus one).Experimental wells were resuspended in the appropriate volume of master mix (e.g., 64 pL total volume). Remaining wells (single stains, FMOs, no stain controls) were resuspended in 50 pL BD Horizon™ Brilliant Stain Buffer before adding the appropriate surface stain to single stain and FMO wells. While all surface markers were analyzed with a corresponding single stain, only those with less definitive positive / negative populations (CD4, CD45RO, CD45RA, CD25) were analyzed with corresponding FMOs. The plate was incubated at 4°C, in the dark, for 30 min. Following surface stain incubation, 100 pL of DPBS was added to each well before centrifugation at 500 x g, 5 min, RT, followed by supernatant removal by flicking; this process was repeated for a total of two washing steps. All wells were then fixed by resuspending cells in 200 pL of Invitrogen™ eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set Fixation / Permeabilization Concentrate (1 part concentrate with 3 parts fixation / permeabilization diluent) followed by incubation for 30 min at 25°C (or overnight 4°C) in the dark. Following fixation, the plate was centrifuged at 500 x g for 5 minutes at RT followed by supernatant removal by flicking. Next, cells were permeabilized by adding 200 pL of IX Permeabilization Buffer (1-part Permeabilization buffer, 9-parts deionized water) before resuspension. Cells were centrifuged at 500 x g for 5 minutes at RT followed by supernatant removal by flicking. Next, intracellular staining was performed by making an intracellular antibody master mix containing in 50 pL IX Permeabilization Buffer and 5 pL of each intracellular antibody (BV 421 FoxP3, PE P-STAT-5) for each experimental well (plus one). Experimental wells were resuspended in the appropriate volume of master mix (e.g., 60 pL volume). Remaining wells (single stains, FMOs, no stain controls) were resuspended in 50 pL IX Permeabilization Buffer before adding the appropriate intracellular stain to single stain and FMO wells. All intracellular markers were analyzed with a corresponding single stain and FMO stained at the same concentration as the experimental 86 / 191#14753190vlwells. The plate was incubated for a minimum of 30 min, in the dark, at 25°C. All wells were washed by adding 100 pL of IX Permeabilization Buffer followed by centrifugation at 500 x g for 5 minutes at RT and supernatant removal by flicking; the wash step was repeated with 200 pL of IX Permeabilization Buffer. Wells were then resuspended in 200 pL of DPBS for analysis. Flow cytometric analysis was conducted using FlowJo 10 or 11 according to manufacturer guidelines. A representative gating strategy, including a cells gate and singlet gate with use of both single stains and FMOs to determine positive / negative populations, is presented in Figure 1. Single stains and FMOs, comprised of a mix of Control, Control_s, pg, and pg_s groups, were conducted with each experimental staining run to allow for best comparisons across groups and flow sessions. Both % of parent population (Live cells or CD4+ FoxP3+ cells) and mean fluorescence intensity (MFI) for each surface and intracellular marker were exported from FloJo 10 / 11 before biologically relevant statistical comparisons between Control and pg cohorts were analyzed in GraphPad Prism 10 using a two-tailed, paired t-test where applicable. This process was conducted for at least an n=3 biological triplicates (three human donors, two donors repeated multiple, different times) for iTregs and n=l biological replicate (one human donor) for nTregs, with each replicate conducted in at least n=3 technical replicates for Control, Control_s (short-term stimulation), pg, and pg_s (short-term stimulation), unless otherwise stated in Figure Legend.Example 10. RNA Isolation, Bulk RNA sequencing, and analysis

[0157] To evaluate differences in gene expression profiles between Control and pg groups, RNA was isolated from T regulatory cells (iTregs, nTregs) using the RNeasy Plus Mini Kit (Qiagen, 74134) according to manufacturer guidelines. Briefly, fewer than 1 x 107cells were pelleted in 1.5 mL Eppendorf tubes before adding 350 pL of Buffer RLT and vortexing cells for 30 seconds for cellular lysis. Next, 350 pL of 70% ethanol was added to the lysate and mixed well by pipetting before transferring the complete volume to an RNeasy Mini spin column placed in a 2 mL collection tube; the column was centrifuged for 15 seconds at 8000 x g with the flow through discarded. Then, 700 pL of Buffer RW1 was added to the column, which was again centrifuged for 15 seconds at 8000 x g with flow through discarded. To the column, 500 pL of Buffer RPE was added, the mixture was centrifuged for 15 seconds at 8000 x g with flow through discarded; this step was repeated, with centrifugation at 8000 x g for 2 minutes in a new 2 mL collection tube. The RNeasy column was placed in a new 1.5 collection tube, 30 pL of RNase-free water was added, and the tube was then centrifuged for 1 minute at 8000 x g for RNA elution. RNA was nano- 87 / 191#14753190vldropped using the Thermo / Spectronic BioMate3 to determine both concentration (ng / pL) and purity (A260 / 280) and then stored at -80°C. Bulk RNA sequencing was conducted by Admera Health, with pre-library DNase treatment, library preparation using the NEBNext Ultra II Directional with Poly- A selection kit, and sequencing with the Illumina 2 x 150 sequencing for 40M PE reads (20M reads each side) per sample. This process was conducted for at least an n=3 biological triplicates (three human donors) for iTregs and n=l biological replicate (one human donor) for nTregs. Raw FASTQ files were assessed for quality using FastQC v0.12.0. Reads were aligned to the human reference genome (hg38) using STAR v2.7.9a with default parameters and the corresponding GENCODE / Ensembl gene annotation. Only uniquely mapped primary alignments were retained. Gene-level quantification was performed using featureCounts with the same annotation. Differential expression was assessed using DESeq2 (Love et al., 2014) with the standard normalization and both unpaired (CD4 naive v Control) and paired (pg v Control) statistical testing workflow. Pathway enrichment was performed using GSEA 7.5.1 gene sets and ranked gene list from DESeq2Genes. Normalized enrichment scores (NES) > 1.5 and false discovery rate (FDR) q-values < 0.05 were used to identify significantly enriched pathways. In the nTreg dataset (n = 1 per condition), raw FASTQ files were processed using the same standard RNA-seq pipeline as described above through gene-level quantification with featureCounts, after which DESeq2 was applied using its standard normalization framework; however, due to the lack of biological replication, dispersion estimates and adjusted p-values were not interpreted, and differential expression was evaluated based on effect size (log2 fold change - log2FC) only.Example 11. IL-10 and TGF-fi quantification

[0158] To quantify differences in IL- 10 and latent TGF-P production from T regulatory cells (iTregs, nTregs), both the LEGEND MAX™ Human IL- 10 ELISA Kit (BioLegend, 430607) and Human Latent TGF-P ELISA Kit (BioLegend, 432907) were used according to manufacturer guidelines. Briefly, all reagents were brought to RT and diluted immediately prior to use. Additionally, supernatant samples (short- and long-term protein stimulation assay) and standards were run in technical duplicates. First, the standard was prepared by performing six two-fold serial dilutions in the appropriate Assay Buffer. The LEGEND MAX™ plate was then washed 4 times with 300 pL of IX Wash Buffer per well, with buffer removed by flicking and residual buffer removed by firmly tapping the plate on absorbent paper. Next, 50 pL of the appropriate Assay Buffer was added to each well along with 50 pL of standard dilutions or samples; each sample was run undiluted and 1:1 (iTregs)88 / 191#14753190vland undiluted, 1:1, 1:5, 1:10, 1:25, 1:100, and 1:250 (nTregs) with the appropriate Assay Buffer. The plate was sealed and incubated at RT for 2 hours on a plate shaker set at 200 RPM. The contents of the plate were discarded, and the plate was washed 4 times with IX Wash Buffer as previously described. Then, 100 pL of the appropriate Detection Antibody solution was added to each well, with the plate incubated for 1 hour at RT while shaking at 200 RPM. The plate contents were discarded, and the plate was washed 4 times with IX Wash Buffer as previously described before 100 L of Avidin-HRP solution was added to each well and incubated for 30 minutes at RT while shaking at 200 RPM. Again, the contents of the plate were discarded, and the plate was washed 5 times with IX Wash Buffer as previously described, with the final wash step including 1 minute well soaking to minimize background. Following the wash, 100 pL of Substrate Solution was added to each well, with the plate incubated for 20 minutes (IL-10) or 10 minutes (Latent TGF-P). The reaction was stopped by adding 100 pL of Stop Solution, and absorbance was immediately read at 450 nm using the Mini ELISA Plate Reader™ (BioLegend, 423555) or Tecan Infinite Microplate Spectrophotometer and corresponding software according to manufacturer protocol. Data were then exported, with comparisons between Control and pg conducted in GraphPad Prism 10 using a two-tailed, paired t-test where applicable. This process was conducted for at least an n=3 biological triplicates (three human donors, two donors repeated multiple, different times) for iTregs and n=l biological replicate (one human donor) for nTregs for short-term protein analysis, and n=3 biological triplicates (three human donors) for iTregs and n=l biological replicate (one human donor) for nTregs for long-term protein analysis, with each specimen and standard run as a technical duplicate on each plate.Example 12. Co-culture immunosuppression assay

[0159] To compare the immunosuppressive capacity of Control and pg T regulatory cells (iTregs, nTregs), an in vitro CD8+ T lymphocyte co-culture suppression assay was conducted as previously described by Miltenyi Biotec (Human CD4+ CD25+ Regulatory T Cell Isolation, In Vitro Suppression Assay and Analysis; Application Protocol) with modification. First, donor-matched CD8+ T lymphocytes were isolated using the CD8+ T Cell Isolation Kit, human (Miltenyi Biotec, 130-096-495) per manufacturer protocol. These cells were then counted by hemocytometer and cultured in T25 flasks at a density of 500,000 cells / mL in complete TexMACS™ medium (1% Penicillin-Streptomycin Solution, 10% Akron Bio Human AB serum) with 20 pL GMP Active Max Human T Cell Activation / Expansion CD3 / CD28 beads (Aero Bio, GMP-MBSOOl-lOmg) for every 106cells 89 / 191#14753190vland 300 lU / mL of GMP Human IL-2 Protein (Aero Bio, GMP-L02H14-1X106IU) for 5 days prior to use in co-culture suppression assay. The CD3 / CD28 beads were washed as previously described before their addition. After 48 hours, an equal volume of complete TexMACS™ medium with 300 lU / mL of GMP Human IL-2 was added to the culture.During this activation and expansion, if media turned yellow, an equal volume of complete TexMACS™ medium with 300 lU / mL of GMP Human IL-2 was also added. After 5 days of CD8+ T lymphocyte expansion and activation, cells were counted by hemocytometer and then prepared for downstream applications by centrifuging at 500 x g for 5 minutes, RT, discarding supernatant, and resuspending in 1 mL of TheraPEAK PBS. CD3 / CD28 beads were removed by placing the cell suspension on the magnetic stand for 3minutes, and centrifuging once again at 500 x g for 5 minutes, RT. Next, to monitor CD8+ T lymphocyte proliferation in the co-culture assay, CD8+ T lymphocytes were stained with the CellTrace™ Violet Cell Proliferation Kit, for flow cytometry (Thermo Fisher, C34557) according to manufacturer guidelines with a 0.5 pM CellTrace™ Violet (CTV) staining solution. Both T regulatory cells (iTregs or nTregs) and CTV+ CD8 T lymphocytes were then prepared at a concentration of 500,000 cells / mL in complete TexMACS™ medium. GMP Active Max Human T Cell Activation / Expansion CD3 / CD28 beads were washed and placed in the original volume of complete TexMACS™ medium as previously described. A triplicate of the following ratios of CTV+ CD8 T lymphocytes to T regulatory cells (iTregs or nTregs) and stimulated / unstimulated controls were then plated in a 96-well tissue culture plate for both Control and pg T regulatory groups:

[0160] Following plating, 5 lU / mL GMP Human IL-2 was added to all wells to ensure cell survival and reflect cytokine concentrations encountered by Tregs in inflamed tissues, as described by Westacott et al. (1990). The co-culture plate was incubated in standard cell culture90 / 191#14753190vlconditions (37°C and 5% CO2) for 4 days. After 4 days of culture, cells were harvested from each well, CD3 / CD28 beads were removed as previously described, and cells were transferred to a 300 pL Falcon™ 96-Well, non-treated, V-shaped-bottom microplate for flow cytometric analysis using the 5-laser Cytek Aurora as previously described with modification. Here, cells were blocked and surface stained with PE-Fire 810 CD4 and BV 785 CD8a. Stimulated / unstimulated CTV+ CD8 T lymphocyte controls were used as CellTrace™ Violet single stain controls for downstream analysis.

[0161] FlowJo 11 was used to identify CTV+ CD8s versus CD4+ CTV- Tregs in a bivariate plot. To measure the immunosuppressive capacity of Control and pg T regulatory cells (iTregs, nTregs), percent (%) Suppression was calculated as previously described (Eggenhuizen et al., 2024; Collison and Vignali, 2012) with modification for each ratio of CD8s:Tregs as follows:% Suppression= 100CTV MF 1 experimental co-culture CTV MF 1 sttm.ula.ted CD8+T lymphocyte controlX CTV MFIunstimuiatedCD8+T lymphocyte control CTV MF 1 stimulate CD8+T lymphocyte control

[0162] Alternatively, for the nTreg co-culture experimental replicates, % Suppression was calculated using the CTVmaxand CTVmin from all wells in replacement of the CTV MFIunstimulated CD8+ T lymphocyte control and CTV MFIstimulated CD8+ T lymphocyte control, respectively, tO better represent maximum and minimum CD8+ T lymphocyte proliferation.

[0163] For each technical replicate, % Suppression was capped between 0-100% before cohort averages were determined. The % Suppression for both Control and pg T regulatory cells (iTregs, nTregs) were graphed and compared using GraphPad Prism 10 and a two-tailed, paired t-test for each cellular ratio (iTregs). This process was conducted for at least an n=3 biological triplicates (three human donors, one donor repeated at different times) for iTregs and n=l biological replicate (one human donor), two experimental replicates for nTregs.Example 13. Short- and long-term persistence assay

[0164] To compare the cellular persistence of Control and pg T regulatory cells (iTregs, nTregs), an in vitro short- and long-term persistence assay was conducted as previously91 / 191#14753190vldescribed (Fleischer et al., 2007) with modification. Briefly, both Control and pg T regulatory cells were prepared at a cellular density of 3 x 106cells / mL in complete TexMACS™ medium (1% Penicillin- Streptomycin Solution, 10% Akron Bio Human AB serum) with 300 lU / mL of GMP Human IL-2 Protein (Aero Bio, GMP-L02H14-1X106IU). These cells were incubated at standard cell culture conditions (37°C and 5% CO2) for 48 hours. After the 48-hour incubation, one volume of complete TexMACS™ medium was added (to promote IL-2 deprivation) before cells were incubated for an addition 48 hours (short-term persistence) or 8 days (long-term persistence). After either 4 or 10 days of culture, cells were harvested by transferring to a 50 mL Falcon tube, centrifuged at 500 x g, 5 min, RT, and then prepared for downstream analysis by flow cytometry as previously described. Persistence of both Control and pg T regulatory cells was compared by analyzing the percent (%) of live cells and absolute live cells per mL, calculated as follows:Absolute Live CellsLive Cell Percentage (% — as determined by flow cytometry) x Total Cell Count Starting Culture Volume (mL)Total Cell Count = Live Cells + Trypan Positive Dead Cells (as determined by hemocytometer)

[0165] Additionally, flow cytometry was used to assess cellular phenotype by surface and intracellular markers using FlowJo 11 as previously described. This process was conducted for at least an n=3 biological triplicates (three human donors) for iTregs and n=l biological replicate (one human donor) for nTregs, with each replicate conducted with at least n=5 (iTreg) or n=2 (nTreg) technical replicates during flow cytometric analysis for Control and pg groups. All data, where applicable, were analyzed using GraphPad Prism 10 and a two-tailed, paired t-test.Example 14. Analysis of short-term human spaceflight data using the Weill Cornell Spatial Omics and Medical Atlas (SOMA) browser

[0166] Data for gene expression were sourced from the Space Omics and Medical Atlas (SOMA), a unified database containing clinical, cellular, and multi-omic information from the three-day, LEO Inspiration4 (14) expedition (Overbey et al., 2024). The SOMA browser (soma.weill.cornell.edu / apps / SOMA_Browser / ), was employed to examine singlecell RNA sequencing (scRNA-seq) information derived from peripheral blood mononuclear cells (PBMCs) sampled from the four 14 participants across various time points: prior to launch (for instance, L-92, L-44, L-3), during the mission, and following return (such as R+l,92 / 191#14753190vlR+45, R+82). Differential expression for targeted genes was evaluated through the browser's (14 PBMC Multiome Data, Pseudobulk RNA expression from single-cells) integrated features, involving computations of log2 fold changes by contrasting expression at designated intervals (e.g., R+l against pre-flight, R+45 versus R+l, or other pairs noted in the figures) across categorized immune cell groups. Significance was determined via adjusted p-values (q- values). Importantly, differential expression of various T regulatory cell related genes (e.g., phenotype - FOXP3, IL2RA, CTLA4, TNFRSF18, IKZF2, IKZF4, NRP1, SATB1, BACH2, IRF4; function - IL10, TGFB1, EBI3, IL12A, ENTPD1, NT5E, LAG3, TIGIT, IL10RA, PDCD1) and DEGs highlighted in the simulated pg experimentation e.g., HSPA6, HSPA1B, HPD, MALAT1, GBP5, SOCS2, CISH, EGR1, EGR2, EGR3) on the CD4_T cell and other_T cell populations were analyzed, with particular focus on the following time point comparisons: R+l versus all pre-flight (1 day after return to Earth), R+45 versus all pre-flight (45 days following spaceflight), and R+82 versus all pre-flight (82 days following spaceflight), and all post-flight (R+l, R+45, R+82) versus all pre-flight.Example 15. Statistical analysis

[0167] Data were organized using Microsoft Excel before statistical analysis was conducted using GraphPad Prism 10 and a two-tailed, paired t-test to compare various Control and pg cohorts (iTregs). Outlier testing was conducted using GraphPad Prism 10 and the ROUT method with Q = 1%. iTreg analyses were conducted with a minimum of n=3 human donors, while exemplary nTreg analyses were conducted with n=l human donor. All data points included in analyses are present on Figures and / or noted in Figure Legends.Human donors collected at multiple, different times were treated as independent experimental replicates due to intra-donor variability and inherent differences in the human immune system between assessments, which often spanned weeks to months between experimentation (Lakshmikanth et al., 2020).References (Examples 1-15}1. Gu J, Shao Q, Zhou J, Chen Q, Lu L. Protocol for in vitro isolation, induction, expansion, and determination of human natural regulatory T cells and induced regulatory T cells. STAR Protoc. 2022 Dec 16;3(4): 101740.2. Li C, Park JH. Assessing IL-2-Induced STAT5 Phosphorylation in Fixed, Permeabilized Foxp3+Treg Cells by Multiparameter Flow Cytometry. STAR Protoc. 2020 Dec l;l(3):100195.93 / 191#14753190vl3. Procaccini C, Carbone F, Di Silvestre D, Brambilla F, De Rosa V, Galgani M, Faicchia D, Marone G, Tramontane D, Corona M, Alviggi C, Porcellini A, La Cava A, Mauri P, Matarese G. The Proteomic Landscape of Human Ex Vivo Regulatory and Conventional T Cells Reveals Specific Metabolic Requirements. Immunity. 2016 Feb 16;44(2):406-21. doi: 10.1016 / j.immuni.2016.01.028. Erratum in: Immunity. 2016 Mar 15;44(3):712. Erratum in: Immunity. 2016 Mar 15;44(3):712.4. Lacinski RA, Dziadowicz SA, Stewart A, Chaharbakhshi E, Akhter H, Pisquiy JJ, Victory JH, Hardham JB, Chew C, Prorock A, Bao Y, Sol-Church K, Hobbs GR, Klein E, Nalesnik MA, Hu G, de Oliveira A, Santiago SP, Lindsey BA. Nanosphere pharmacodynamics improves safety of immuno stimulatory cytokine therapy. iScience.2024 Jan 9;27 (2): 108836.5. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550.6. Miltenyi Biotec. (n.d.). Human CD4+CD25+ regulatory T cell isolation, in vitro suppression assay, and analysis. Retrieved December 04, 2024, from https: / / www.miltenyibiotec.com / US-en / applications / all-protocols / human-cd4-cd25- regulatory-t-cell-isolation-in-vitro-suppression-assay-and-analysis.html.7. Westacott CI, Whicher JT, Barnes IC, Thompson D, Swan AJ, Dieppe PA. Synovial fluid concentration of five different cytokines in rheumatic diseases. Ann Rheum Dis. 1990 Sep;49(9):676-81.8. Eggenhuizen PJ, Cheong RMY, Lo C, Chang J, Ng BH, Ting YT, Monk JA, Loh KL, Broury A, Tay ESV, Shen C, Zhong Y, Lim S, Chung JX, Kandane-Rathnayake R, Koelmeyer R, Hoi A, Chaudhry A, Manzanillo P, Snelgrove SL, Morand EF, Ooi JD. Smith-specific regulatory T cells halt the progression of lupus nephritis. Nat Commun.2024 Feb 6;15(1):899.9. Collison LW, Vignali DA. In vitro Treg suppression assays. Methods Mol Biol.2011;707:21-37.10. Fleischer A, Duhamel M, Lopez-Fernandez LA, Munoz M, Rebollo MP, Alvarez-Franco F, Rebollo A. Cascade of transcriptional induction and repression during IL-2 deprivation-induced apoptosis. Immunol Lett. 2007 Sep 15;112(l):9-29. doi:10.1016 / j.imlet.2007.06.004. Epub 2007 Jul 24. Erratum in: Immunol Lett. 2009 Jun 4; 124(2): 112-3.11. Overbey EG, Ryon K, Kim J, Tierney BT, Klotz R, Ortiz V, Mullane S, Schmidt JC, MacKay M, Damle N, Najjar D, Matei I, Patras L, Garcia Medina JS, Kleinman AS,94 / 191#14753190vlWain Hirschberg J, Proszynski J, Narayanan SA, Schmidt CM, Afshin EE, Innes L, Saldarriaga MM, Schmidt MA, Granstein RD, Shirah B, Yu M, Lyden D, Mateus J, Mason CE. Collection of biospecimens from the inspiration4 mission establishes the standards for the space omics and medical atlas (SOMA). Nat Commun. 2024 Jun 11;15(1):4964.12. Lakshmikanth T, Muhammad SA, Olin A, Chen Y, Mikes J, Fagerberg L, Gummesson A, Bergstrom G, Uhlen M, Brodin P. Human Immune System Variation during 1 Year. Cell Rep. 2020 Jul 21;32(3):107923.Example 16. Human peripheral blood collection, PBMC isolation from healthy donors

[0168] For downstream analysis of T regulatory cells (Tregs), human peripheral blood was successfully collected from healthy, male donors by venipuncture before PBMC isolation using Lymphoprep™ as described above. Average milliliters of blood collected, PBMCs isolated (in millions), and number of PBMCs isolated (in millions of cells per mL of blood) are presented for each donor analyzed (Table 1). Individual replicates for those donors whose blood was collected for multiple, independent experiments are also presented (ex., Donor 1, separate replicates of 1.1, 1.2, 1.3, etc).Table 1: Human peripheral blood collection, PBMC isolation from healthy, male donors. Milliliters of blood collected, PBMCs isolated (in millions), and number of PBMCs isolated (in millions) per mL of blood for all donors analyzed, with average (AVG) ± standard error of the mean (SE) presented.95 / 191#14753190vlExample 17. Naive CD4+ T lymphocyte isolation and culture, differentiation, and expansion of induced T regulatory cells (iTregs

[0169] Following PBMC isolation, naive CD4+ T lymphocytes were isolated by negative selection using a magnetic bead separation kit before culture, differentiation, and expansion to induced T regulatory cells (iTregs). On average, 9.130 ± 1.167 x 106naive CD4+ T Eymphocytes, or 0.270 ± 0.031 x 106cells / mL blood, were isolated from donors (Table 2). Using the representative gating strategy presented in Figure 2, the identity of the isolate (naive CD4+ T lymphocytes) was confirmed (Figure 3). Here, of all live cells, 97.556 ± 0.184% were determined to be CD4+ (Figure 3A). Of this CD4+ cellular subset, the majority were determined to be “naive” or CD45RA+ [CD45RO+ CD45RA-: 0.460 ± 0.287%; CD45RO+ CD45RA+: 11.903 ± 7.680%; CD45RO- CD45RA+: 87.085 ± 8.125%; CD45RO- CD45RA-: 0.534 ± 0.156%] (Figure 3B). These naive CD4+ T lymphocytes were then differentiated and expanded to an average of 26.012 ± 4.974 x 106iTregs, representing an average expansion fold of 2.638 ± 0.236 over the total 4-day culture (Table 2).Table 2: Naive CD4+ T lymphocyte isolation and culture, differentiation, and expansion of induced T regulatory cells (iTregs). CD4+ Naive T cells count (in millions), CD4+ Naive T cells count (in millions) per mL of blood, differentiated and expanded iTreg count (in millions), and Expansion Fold for all donors analyzed, with average (AVG) ± standard error of the mean (SE) presented.

[0170] Additionally, an unpaired RNA-seq analysis between naive CD4+ T lymphocytes and the Control iTreg group supported successful differentiation and expansion of iTregs. Here, principal component analysis (PCA) demonstrated adequate separation of sequenced naive (baseline - CD4+ T lymphocyte) and Control (iTreg) samples (Figure 3C).96 / 191#14753190vlGene set enrichment analysis (GSEA) using C7: ImmuneSigDB confirmed a statistically significant enrichment (NES = 2.834, p-value < 0.001) of the gene set GSE14415_INDUCED_TREG_VS_TCONV_UP or genes upregulated in iTregs compared to conventional T cells (Figure 3D), with top 10 leading genes and their core enrichment presented (Table 3). Overall, these data support the successful differentiation and expansion of iTregs.Table 3: Rank in Gene List, Gene Symbol, Rank Metric Score, Running ES, and Core Enrichment for the top 10 leading genes of C7: ImmuneSigDB gene set GSE14415_INDUCED_TREG_VS_TCONV_UP in iTregs compared to naive T lymphocytes.Example 18. Simulated microgravity (us) exposure of iTregs

[0171] Following 4 days of culture, differentiation, and expansion, iTregs were then either placed in standard culture (group - Control) or exposed to simulated pg (group - pg) using the Synthecon® RCCS-8D Disposable Vessel for 24 hours as previously described. Cell counts by hemocytometer following exposure to either Control or pg conditions supported no statistically significant difference in proliferation index (p = 0.9558) between groups (Table 4), suggesting both cellular viability and proliferative capacity during simulated pg exposure were no different than Controls.Table 4: Simulated microgravity (pg) exposure of iTregs versus Control. Control iTregs cell count (in millions), Control Proliferation Index, pg iTregs cell counts (in millions), and pg Proliferation Index for all donors analyzed, with average (AVG) ± standard error of the mean (SE) presented. For comparison between Control and pg iTregs, cell counts were normalized to equal starting volume.97 / 191#14753190vlExample 19. Flow cytometric analysis of Control and simulated ug iTregs

[0172] Flow cytometry was used to compare iTreg cellular phenotypes between Control, pg, Control_s, and pg_s groups, with both stimulation (_s) groups exposed to 300 lU / mL of IL-2 for 30 minutes before analysis. To begin, there was a significant decrease in the percentage (%) of Live cells in pg_s when compared to Control_s (p = 0.008), as well as a trending decrease in the pg versus the Control cohort (p = 0.099) (Figure 4A, Table 5), suggesting that simulated pg exposure causes cellular apoptosis in weaker iTregs following differentiation and expansion. However, there was no difference in % of iTregs, defined as CD4+ FoxP3+ cells, between groups (Figure 4B, Table 5). Importantly, the minimum percentage of iTregs for all experimental cohorts was 88.76% (Figure 4B), further supporting adequate differentiation and expansion of iTregs from naive CD4+ T lymphocytes. While there was an evident trend toward a significant decrease in % pSTAT5+ iTregs between both the Control and pg groups (p = 0.1985), there was a stronger trend toward a significant increase in % pSTAT5+ iTregs between pg and pg_s cohorts (p = 0.1364), suggesting an enhanced response to IL-2 stimulus in pg iTregs (Figure 4C, Table 5). Additionally, for % CD25+ iTregs, a trend toward a significant increase (p = 0.2156) was evident between only Control and Control_s groups (Figure 4D, Table 5). While there was no significant difference in % CD45RO+ iTregs between cohorts, a trending increase was evident for both the Control vs Control_s (p = 0.2018) and pg vs pg_s (p = 0.1799) comparisons (Figure 4E, Table 5). There was no significant difference in % CD3+ iTregs for all comparisons analyzed; however, there were trending increases evident for the Control vs pg (p = 0.2503), Control vs Control_s (p = 0.0845), and Control_s vs pg_s (p = 0.2359) comparisons (Figure 4F, Table 5).98 / 191#14753190vlTable 5: Paired, two-tailed t-test results from flow cytometric analysis of Control and simulated jag iTregs, with cellular gate (Gate), statistical comparison (Control vs pg, Control vs Contro s, Control_s vs pg_s, and pg vs pg_s), and p-value for percentage (%) data of cellular populations analyzed.

[0173] Mean fluorescence intensity (MFI) was also measured as a surrogate for expression level of the various markers analyzed in iTreg populations (Figure 5). The AMFI was calculated by subtracting the baseline MFI value (Control or pg) from the stimulated MFI value for each respective group (Control_s or pg_s). Importantly, there was a significant increase in FoxP3 MFI in the Control_s (p = 0.0033) and pg_s (p = 0.0202) groups relative to their respective baselines (Figure 5A, Table 6). However, there was no difference in AFoxP3 between Control and pg groups (Figure 5B, Table 6). Additionally, while there was a significant decrease in pSTAT5 MFI in both pg v Control (p = 0.0320) and pg_s v Control_s (p = 0.0132), there was a significant increase in pSTAT5 MFI in pg_s iTregs in comparison to its respective baseline (p = 0.0487). This finding was further supported by analysis of ApSTAT5, which revealed a greater increase in the pg group when compared to Control, albeit non- significant (Figure 5D, Table 6). With respect to CD25 MFI, there was a trend toward decreased MFI for Control_s relative to Control (p = 0.0834) and pg_s relative to ControLs (p = 0.0511). Importantly, there was a significant (p = 0.0017) decrease in CD25 MFI for pg_s relative to pg (Figure 5E, Table 6). While ACD25 was negative for both the99 / 191#14753190vlControl and pg cohorts (Figure 5F), the MFI change was greater for pg iTregs (Table 6, p = 0.2695). Finally, while there was no significant difference in CD45RO MFI or ACD45RO between comparative cohorts, there were trending increases in Control_s v Control (p = 0.1017) and Control_s v pg_s (p = 0.2477) (Figure 5G / H, Table 6).Table 6: Paired, two-tailed t-test results from flow cytometric analysis of Control and simulated jag iTregs, with cellular gate (Gate), statistical comparison (Control vs pg, Control vs Contro s, Control_s vs pg_s, and pg vs pg_s), and p-value for MFI or delta (A) MFI data of cellular markers analyzed.

[0174] Overall, these data provide integral support for the successful differentiation of naive CD4+ T lymphocytes to induced T regulatory cells (iTregs), defined as CD4+ FoxP3+ cells. Additionally, % Live Cell data support that simulated pg applies a stressor to iTregs, driving apoptosis in likely weaker iTregs following differentiation and expansion. Furthermore, these data support that pg exposure generates a more responsive and efficient T regulatory phenotype. Of note, increased % pSTAT5+, increased pSTAT5 MFI, and greater ApSTAT5 in pg iTregs suggests heightened IL-2 signal transduction and activation (Mahmud et al., 2013). The more drastic decrease in CD25 MFI following IL-2 stimulation suggests that pg iTregs have increased receptor (CD25) - ligand (IL-2) engagement (Permanyer et al., 2021), leading to more rapid internalization of the IL-2 / IL-2R complex. Enhanced internalization could facilitate sustained signal transduction (e.g., via endosomal JAK-STAT activation) for survival and proliferation. Further, increased IL-2 sensitivity and binding100 / 191#14753190vlcould limit cytokine availability, contributing to Treg function as IL-2-sequesters for effector immune cell suppression (Seddu et al., 2025; Chinen et al., 2016). Together, these data suggest that pg exposed iTregs display heightened IL-2 signal transduction and CD25 regulation for induction of an enhanced Treg phenotype, possibly improving their immunosuppressive capabilities, stability, and / or resistance to inflammatory environments when compared to Control iTregs.Example 20. RNA-seguencing analysis of Control and simulated us iTregs

[0175] Following 24 hours of standard cellular culture or simulated pg exposure, RNA was isolated from Control and pg iTregs for paired RNA sequencing analysis of differential gene expression. To begin, PC A supported pg-induced changes in gene expression, as depicted by a shift downward on the PC2 axis compared to the matched donor Control (Figure 6A). Further analysis identified 59 (11 upregulated, 48 downregulated) differentially expressed genes (DEGs) when comparing pg to Control iTreg cohorts (Figure 6B, Table 7). Of note, the most significantly upregulated gene (HSPA6 - human heat shock protein HSP70B') in pg iTregs reported a log2FC = 10.363 ± 1.346 (an approx. 1200-fold increase) and adjusted p-value = 7.99 x 1017. Additionally, the genes EGR1 / 2 / 3 (Early growth response proteins) were the most significantly downregulated genes in pg iTregs with reported log2FCs of -5.501 ± 0.456, -4.222 ± 0.498, and -3.933 ± 0.420 and adjusted p-values of 1.65 x IO’30, 4.48 x 1016, and 2.91 x 1019, respectively (Table 7).Table 7: Differentially expressed genes including the Gene symbol, baseMean, log2FC, log2FC standard error (IfcSE), p-value, and adjusted p-value (p-adj) from the pg versus Control iTreg paired RNA sequencing analysis.101 / 191#14753190vl

[0176] Further analysis and categorization of these DEGs reveals major shifts toward a more potent and stable suppressive profile of pg iTregs compared to Controls. First, simulated pg exposure, as previously reported (Manna et al., 2024), upregulated stress response genes (HSPA6, HSPA7, HSPA1B, MT1A). Literature suggests that Heat shock proteins (HSPs) play a critical role in Treg homeostasis (Kolinski et al., 2016) including induction, suppressive function (Brenu et al., 2013), and anti-inflammatory cytokine production (van Eden et al., 2005). This upregulation likely protects pg iTregs from102 / 191#14753190vlapoptosis, ensuring cellular integrity, likely enhancing pg iTregs resilience in harsh, low IL-2 inflammatory microenvironments encountered in vivo, while also supporting enhanced immunosuppressive function. Additionally, downregulation of transient, immediate-early genes (EGR1, EGR2, EGR3) and transcription factors (NR4A1, NR4A3, E2F7, ZBED2) likely reduce activation and proliferation signals in pg iTregs compared to Controls, promoting a more quiescent Treg phenotype. While literature has shown a critical role for the EGR family in early Treg differentiation and function (Yang et al., 2025; Sumitomo et al., 2013), this downregulation may suggest a shift in pg iTregs toward a more differentiated and suppressive state, reflecting phenotype stabilization as opposed to functional impairment.

[0177] Downregulation of cell cycle and proliferation genes (RRM2, CCNF, TRIP13, FBXO5, POLE2, TICRR, CCNE2, FAM111B), common for lymphocytes exposed to simulated pg (Lv et al., 2023), may promote iTreg phenotypic stability through modulation of cellular replication. Upon T-cell receptor (TCR) stimulation, both stored-operated calcium entry and NF AT translocation to the nucleus, mechanisms not seen in nTregs, can promote proliferation and induction of inflammatory genes in iTregs, causing phenotypic instability and Thl conversion (Lyu et al., 2023). While TCR stimulation in the presence of IL-2 helps promote FoxP3 expression in iTregs in vitro, TCR stimulation in vivo (with only endogenous IL-2 levels) drives Treg-specific demethylated region (TSDR) methylation at the FoxP3 locus and loss of FoxP3 expression (Chen et al., 2011). Together, suppression of iTreg proliferation machinery may then enhance in vivo phenotypic stability, reduce plasticity, and prevent Thl effector shift in non-optimal IL-2 conditions. Correspondingly, downregulation of DNA replication and repair genes (EXO1, FEN1, DMC1, BRIP1, DSCC1), as seen previously following simulated pg exposure (Hauschild et al. 2014), reveals slowed replication / repair in pg iTregs in comparison to the Control cohort, favoring quiescence and survival over proliferation. Altogether, this downregulation likely reflects further maturation of pg iTregs, shifting cells from predominately a proliferative state to a stable, suppressive state.

[0178] Additionally, a set of immune-related genes were also identified in this analysis. First, downregulation of negative regulators of the IL-2 / STAT5 signaling pathway (CISH, SOCS2) likely enhances IL-2 signaling capacity and responsiveness for sustained pSTAT5 expression without overstimulation in pg iTregs. This modulation may lead to a more stable T regulatory phenotype (pSTAT5 / FoxP3) and potent suppressive capacity, as overexpression of SOCS2 was previously shown to inhibit Treg activity (Lan et al., 2025).103 / 191#14753190vlWhen coupled with the downregulation of IL2RA, as previously seen in the flow cytometry data and other microgravity studies (Gallardo-Dodd et al., 2023), pg iTregs may display more efficient IL-2 / IL2R signaling and resultantly greater resilience in dynamic IL-2 environments in vivo. Furthermore, downregulation of interferon-inducible inflammatory response genes (GBP5, SLFN12L, SLFN13) may translate to a more immunosuppressive phenotype with reduced pro-inflammatory bias. Of note, inhibited activation of Gbp5 by loganic acid was previously shown to ameliorate Gbp5-induced pyroptosis (inflammatory cell death) of Treg cells in hemorrhagic shock injury (Huang et al., 2024). Further, while CCR2 has been shown to play a critical role in migration of Tregs toward inflamed sites (Loyher et al., 2016), more recent studies highlight that the use of the dual CCR2 / CCR5 antagonist Cenicriviroc promotes type 1 or IL-10-producing Treg development (Madan et al., 2024).

[0179] Analysis of differentially expressed metabolic genes including upregulation of HPD and downregulation of HK2, CYP1B1, HILPDA, PAQR4, TLCD3A reveals a shift from glycolysis to oxidative metabolism alongside lipid and amino acid reprogramming in pg iTregs. Importantly, reduced reliance on rapid, glucose-fueled energy (suited for proliferating cells) and a pivot toward TCA / OXPHOS / FAO may allow for a more sustainable iTreg metabolic profile for enhanced function and survival under nutrient / IL-2 limitations. Recent literature suggests that while glycolysis may enhance Treg proliferation / migration, balance with OXPHOS and FAO for the generation of TCA cycle intermediates is necessary for FoxP3 lineage stability and suppressive function (Shi and Chi, 2019). This finding aligns with literature which suggests that glycolysis tends to support pro-inflammatory cell function while OXPHOS and FAO are the predominant metabolic drivers in anti-inflammatory cells such as Tregs (Kempkes et al., 2019). This shift may indicate the pg iTregs possess a metabolic profile more like stable, suppressive nTregs than Controls, leading to enhanced anti-inflammatory effects.

[0180] Various epigenetic modifications were also apparent in pg iTregs and likely support heightened immunosuppressive capacity. Of note, low expression of the long noncoding RNA (IncRNA) MIR210HG, an epigenetic modulator and the fourth most negative log2FC = -3.887 ± 0.811 of the DEG analysis (Table 7), in esophageal squamous cell carcinoma tumors was previously associated with increased Tregs in the tumor microenvironment (Wang et al. 2025). Additionally, the IncRNA MALAT1, upregulated in pg iTregs versus Controls (log2FC = 1.028 ± 0.291, Table 7), has been positively associated 104 / 191#14753190vlwith differentiation of CD4+ T cells towards a Treg phenotype while promoting antiinflammatory responses in autoimmune encephalomyelitis (Masoumi et al., 2019). These IncRNA changes in pg iTregs suggest improved anti-inflammatory function versus Controls. Additionally, the RING-finger type E3 ubiquitin ligase, UHRF1, is a potent epigenetic regulator decreased in pg iTregs (Table 7). Previous reports suggest that ablation of Uhrfl in naive T cells before adoptive transfer results in significant colitis suppression due to increased iTreg differentiation. Through TGF-P-mediated proteasomal degradation, Uhrfl driven DNA methylation of Treg- specific genes is halted, revealing a critical epigenetic mechanism for iTreg differentiation (Sun et al. 2019). Furthermore, downregulation of E3 ubiquitin ligase RNF157 (Ring finger protein 157) has been shown to promote Thl development in CD4+ T cells through HDAC1 ubiquitination / degradation (Wang et al., 2023), suggesting that pg iTregs have a less prone pro-inflammatory skew in comparison to Controls. Overall, these epigenetic modulations support a cellular phenotype of enhanced immunosuppressive capacity in pg iTregs. All together, these DEGs reveal that pg exposure likely reprograms iTregs for enhanced stress resilience (HSPs), IL-2 sensitivity, metabolic reprogramming, and epigenetic modifications for more potent immunosuppressive capacity.

[0181] Gene set enrichment analysis (GSEA) was conducted to identify enriched biological pathways in pg iTregs compared to Controls, with the top 10 positively and negatively enriched gene sets highlighted in Tables 8-21. First, analysis revealed that there was no significant positive or negative enrichment of Hallmark pathways (Table 8A, 8B). Of note, for HALLMARK_CHOLESTEROL_HOMEOSTASIS, which had the highest positive NES, literature suggests that cholesterol transport and its cellular metabolism is essential for Treg proliferation and function (Grimaldos et al., 2022). The gene set HALLMARK_P53_PATHWAY was also positively modulated and previously associated with a role in Treg differentiation by promoting shifts toward Treg fate in inflammatory conditions through pSTAT5 activity (Park et al., 2013). Importantly, HALLMARK_UNFOLDED_PROTEIN_RESPONSE (UPR) was also positively enriched in pg iTregs, aligning with the differential expression of HSPs, as previously noted.Additionally, trending negative enrichment was evident for HALLMARK_E2F_TARGETS, HALLMARK_MYC_TARGETS_V2, and HALLMARK_G2M_CHECKPOINT, likely highlighting microgravity-induced reduction in proliferative capacity (Lv et al., 2023); however, this trend could reflect a shift for recently expanded iTregs to a more stable, suppression dominant phenotype. Trending negative enrichment of the gene set105 / 191#14753190vlHALLMARK_WNT_BETA_CATENIN_SIGNALING could be beneficial in pg iTregs, as Wnt-P-catenin activation has been shown to drive a pro-inflammatory phenotype in Tregs (Quandt et al., 2021). Finally, trending negative enrichment of HALLMARK_ALLOGRAFT_REJECTION, or enrichment of genes downregulated during in immune activation and rejection response, suggests that pg iTregs have improved suppressive capacity when compared to Controls (Pasquet et al., 2013) (Table 8B).Table 8A: Gene set enrichment analysis (GSEA) using Hallmark pathways for pg versus Control iTregs. Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presentedTable 8B: Gene set enrichment analysis (GSEA) using Hallmark pathways for pg versus Control iTregs. Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.

[0182] Next, C2-CGP (chemical and genetic perturbations) pathway analysis revealed no significant positive enrichment in pg iTregs (Table 9A). Of note, while not trending, positive enrichment of the MCBRYAN_PUBERTAL_TGFB1_TARGETS_DN and PLASARI_TGFBl_SIGNALING_VIA_NFIC_10HR_UP suggests heightened TGFB1 signaling in pg iTregs, known to be critical for Treg development, induction, and immunosuppressive functions (Moreau et al., 2023). For those negatively enriched pathways,106 / 191#14753190vla statistically significant negative enrichment of the THALER_BONE60 gene set (NES = -2.08, q = 0.034) was present, however, its relation to Treg biology is not apparent (Table 9B). Of the numerous trending negatively enriched pathways, negative enrichment of WANG_LSD1_TARGETS_DN suggests that those genes known to be downregulated by LSD1 are negatively enriched in pg iTregs. Literature suggests that LSD1 modulates suppressive programs in T cell development, with LSD1 knockout driving upregulation of pro-inflammatory gene signatures (Stamos et al., 2021). Again, this analysis revealed further support for p53-induced Treg fate, as evidenced by negative enrichment of the SCIAN_CELL_CYCLE_TARGETS_OF_TP53_AND_TP73_DN pathway (Table 9B).Table 9A: Gene set enrichment analysis (GSEA) using C2-CGP pathways for pg versus Control iTregs. Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.Table 9B: Gene set enrichment analysis (GSEA) using C2-CGP pathways for pg versus Control iTregs. Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.

[0183] GSEA using C2-CP: BIOCARTA revealed no significant positively or negatively enriched pathways (Table 10A, 10B). Of note, analysis revealed positive107 / 191#14753190vlenrichment of BIOCART A_PPARA_PATHW AY, indicating heightened PPARa pathway activity in pg iTregs. PPARa signaling has been associated with enhanced Treg responses in tumors (Zeng et al., 2021). Additionally, positive enrichment of the BIOCARTA_NUCLEARRS_PATHWAY indicates elevated nuclear signaling activity, possibly indicating greater persistence and suppressive potency as nuclear receptors are responsible for regulating Treg development, Foxp3 expression, and tolerance by integrating environmental cues into transcriptional programs (Table 10A). For those negatively enriched pathways, analysis revealed many gene sets associated with cell cycle regulation (Table 10B). Additionally, negative enrichment of BIOCARTA_TH1TH2_PATHWAY may reinforce that pg iTregs display enhanced suppression of Thl / Th2 polarization and potent immunoregulatory properties (Venuprasad et al., 2010; Yang et al., 2013).Table 10A: Gene set enrichment analysis (GSEA) using C2-CP: BIOCARTA pathways for pg versus Control iTregs. Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.Table 10B: Gene set enrichment analysis (GSEA) using C2-CP: BIOCARTA pathways for pg versus Control iTregs. Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.108 / 191#14753190vl

[0184] Furthermore, GSEA revealed no significant positive or negative enrichment of gene sets within C2-CP: KEGG_MEDICUS (Table 11A, 11B). Of those positively enriched pathways, 8 are associated with proteasome-median protein degradation (Table 11 A), likely related to stress response in pg iTregs in comparison to Controls. Of those negatively enriched pathways, a trending negative enrichment of KEGG_MEDICUS_REFERENCE_IL2_FAMILY_TO_JAK_STAT_SIGNALING_PATHW AY gene set was evident (Table 11B). This negative enrichment further supports the resolution of transient early response genes within the IL-2 / JAK / STAT pathway and transition to a stable Treg phenotype, findings relayed in prior analyses.Table 11A: Gene set enrichment analysis (GSEA) using C2-CP: KEGG_MEDICUS pathways for pg versus Control iTregs. Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.Table 11B: Gene set enrichment analysis (GSEA) using C2-CP: KEGG_MEDICUS pathways for pg versus Control iTregs. Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.109 / 191#14753190vl

[0185] C2-CP: Reactome pathway analysis revealed no significant positively or negatively enriched pathways in pg exposed iTregs compared to Controls (Table 12A, 12B). Of note, positive enrichment of REACTOME_ATTENUATION_PHASE, REACTOME_HSF1_ACTIVATION, and REACTOME_HSF1_DEPENDENT_TRANSACTIVATION support coordinated activation of stress-response programs of Heat shock factor 1 (HSF1) (Table 12A). Elevated HSF1 has been shown to drive Treg-specific gene expression and function, with HSF1 deficiency hindering Treg development and immunosuppressive responses (Collins et al., 2024).Positive enrichment of these pathways may indicate the formation of a resilient, activated Treg with an enhanced survival phenotype.Table 12A: Gene set enrichment analysis (GSEA) using C2-CP: Reactome pathways for pg versus Control iTregs. Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.Table 12B: Gene set enrichment analysis (GSEA) using C2-CP: Reactome pathways for pg versus Control iTregs. Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.110 / 191#14753190vl

[0186] Furthermore, C2-CP: WikiPathways analysis revealed significant positive enrichment of WP_ZINC_HOMEOSTASIS (Table 13A). Importantly, zinc homeostasis is crucial for Treg differentiation and function, as it modulates Foxp3 expression, suppresses Thl7 polarization, and enhances immunosuppressive capacity, with deficiency impairing Treg stability and IL-10 production (Kulik et al., 2019). Again, positive enrichment of PPAR signaling, unfolded protein response, and lipid metabolism were evident (Table 13A). While no negative gene sets displayed significance (Table 13B), the non-significant negative enrichment of WP_IL2_SIGNALING (NES = -1.91) and WP_JAKSTAT_SIGNALING_IN_THE_REGULATION_OF_BETA_CELLS (NES = -1.91) further supports negative feedback relief of JAK-STAT signaling in pg iTregs.Table 13A: Gene set enrichment analysis (GSEA) using C2-CP: WikiPathways pathways for pg versus Control iTregs. Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.111 / 191#14753190vlTable 13B: Gene set enrichment analysis (GSEA) using C2-CP: WikiPathways pathways for pg versus Control iTregs. Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.

[0187] Additionally, analysis of the C2-CP: PID (Pathway Interaction Database) revealed only a trending positive enrichment of the PID_DELTA_NP63_PATHWAY (NES = 2.51 , q = 0.172, Table 14A) and a trending negative enrichment of PID_IL3_PATHWAY (NES = -1.97, q = 0.284, Table 14B). Importantly, autocrine signaling of IL-3 has been shown to negatively regulate Treg activity, with anti-IL-3 treatment increasing Treg counts and suppressive function in models of sepsis (Zhao et al., 2021). Reduced pathway expression could therefore limit autocrine signaling and drive improved immunosuppressive capacity in pg iTregs (Table 14B). Furthermore, negative enrichment of PID_MAPK_TRK_PATHWAY (NES = -1.92) could have functional implications. Downregulation of Trk-mediated MAPK / ERK signaling is a recognized molecular signature of enhanced Treg stability and resistance to inflammatory conversion. This finding suggests that simulated pg exposure induces a protective, anti-plasticity reprogramming, as ERK / MAPK signaling has been associated with active destabilization of Foxp3 expression and Treg conversion toward effector or Thl7-like phenotypes. Mechanistically, ERK phosphorylates Foxp3 at Ser418, leading to its ubiquitination and degradation via the proteasome and is exacerbated by pro-inflammatory cytokines (e.g., IL-6 / IL-ip via STAT3 / RORyt) (Liu et al., 2013). This significant downregulation of ERK protects Foxp3 from degradation, prevents Treg to Thl7 / effector conversion, and is likely reflective of a stable, inflammation-resistant T regulatory cell.112 / 191#14753190vlTable 14A: Gene set enrichment analysis (GSEA) using C2-CP: PID pathways for pg versus Control iTregs. Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.Table 14B: Gene set enrichment analysis (GSEA) using C2-CP: PID pathways for pg versus Control iTregs. Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.

[0188] Analysis of the C2-CP: KEGG_LEGACY gene set highlighted a trending positive enrichment of KEGG_ALANINE_ASPARTATE_AND_GLUTAMATE_METABOLISM, KEGG_PPAR_SIGNALING_PATHWAY, KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION, KEGG_PYRUVATE_METABOLISM, and KEGG_ABC_TRANSPORTERS (Table 15 A). Of note, PPARs, particularly PPARy, promote Treg differentiation and anti-inflammatory functions by regulating lipid metabolism and suppressing effector T cells (Choi and Bothwell, 2012). Additionally, positive enrichment of genes associated with antigen processing and presentation in pg iTregs (in comparison to Controls) suggests enhanced cellular machinery for Treg-APC interactions for enhanced tolerogenic responses (Wardell et al., 2020). Further, positive enrichment of the KEGG_SYSTEMIC_LUPUS_ERYTHEMATOSUS may seem counterintuitive, as upregulated genes associated with an autoimmune disease may denote113 / 191#14753190vldecreased immunosuppressive and tolerogenic activity in pg iTregs. Importantly, functionally suppressive Treg cells are found in patients with systemic lupus erythematous (SLE), and failure of their immunosuppressive capacity in active disease is due to cellular crosstalk and a persistent inflammatory cytokine milieu (Ohl & Tenbrock, 2014). Regarding those negatively enriched pathways (Table 15B), a strong, trending negative enrichment was evident for KEGG_PRION_DISEASES (NES = -1.99, q = 0.09); however, its biological relevance in Tregs is unknown. Trending negative enrichment of KEGG_TYPE_II_DIABETES_MELLITUS (NES -1.88, q = 0.264) suggests that pg iTregs lack the dysregulated phenotype of Tregs commonly seen in this disease, which features decreased FOXP3 mRNA expression and pSTAT5 as well as reduced immunosuppressive function (Sheikh et al., 2018).Table 15A: Gene set enrichment analysis (GSEA) using C2-CP: KEGG_LEGACY pathways for pg versus Control iTregs. Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.Table 15B: Gene set enrichment analysis (GSEA) using C2-CP: KEGG_LEGACY pathways for pg versus Control iTregs. Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.

[0189] Furthermore, GSEA using the C3-MIR (microRNA - miRNA targets) database (Table 16A, 16B) revealed no significant or trending positive enrichment of miRNA114 / 191#14753190vlpathways. Conversely, significant negative enrichment of TTCCGTT_MIR191 (NES = -2.03, q = 0.028) and a strong, trending negative enrichment of GC AAGAC_MIR431 (NES = -2.03, q = 0.057) was present in pg iTregs. Interestingly, increased miR-191 repressive activity in pg iTregs likely reflects enhanced homeostatic maintenance in the exposed cohort, with conditional deletion of miR-191 resulting in loss of Treg cells through loss of STAT5 activation and deficient cytokine signaling (Lykken and Li, 2016). These observations from literature suggest that miR-191 activity supports pg iTreg phenotypic stability and function following pg-induced stress. Additionally, while no known biological function of miRNA-431 in Tregs is documented to date, this miRNA has been associated with various disease states (Pan et al., 2022).Table 16A: Gene set enrichment analysis (GSEA) using C3-MIR pathways for pg versus Control iTregs. Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.Table 16B: Gene set enrichment analysis (GSEA) using C3-MIR pathways for pg versus Control iTregs. Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.

[0190] C3-TFT (transcription factor targets) gene set analysis revealed no significant positive or negative enrichment (Table 17A, 17B) in pg iTregs. Trending positive enrichment115 / 191#14753190vlof RUVBL2_TARGET_GENES (NES = 2.52, q = 0.248) was noted and may possibly play a role in regulating proliferative capacity of Tregs through repression of Cdkn2c, as previously documented in Th2 cells (Hosokawa et al., 2013). Trending negative enrichment of various pathways was observed in pg iTreg (Table 17B). Of note, downregulation of STAT3 signaling in pg iTreg is likely beneficial, as STAT3 has been previously associated with phenotypic instability of Tregs; STAT3 prevents polarization of naive CD4+ T lymphocytes to iTreg phenotypes (Laurence et al., 2013) and epigenetically silences FoxP3 (Hou et al., 2018).Table 17A: Gene set enrichment analysis (GSEA) using C3-TFT pathways for pg versus Control iTregs. Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.Table 17B: Gene set enrichment analysis (GSEA) using C3-TFT pathways for pg versus Control iTregs. Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.

[0191] Next, C5-GO analysis revealed significant positive enrichment of GOBP_PROTEIN_REFOLDING (NES = 2.70, q = 0.037), further reflecting enrichment and activity of chaperones like HSPs (Brenu et al., 2013) following simulated pg exposure for likely more potent suppressive function and cytokine production (Table 18 A). Additionally,116 / 191#14753190vlnumerous trending negatively enriched pathways were also noted, but they were not biologically relevant to Treg physiology. However, negative enrichment of the GOBP_NEGATIVE_REGULATION_OF_RECEPTOR_SIGNALING_PATHWAY_VIA_S TAT further suggests modulated negative JAK-STAT feedback, as indicated previously by the significantly decreased differential expression of both CISH and SOCS2 (Table 7, 18B).Table 18A: Gene set enrichment analysis (GSEA) using C5-GO pathways for pg versus Control iTregs. Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.Table 18B: Gene set enrichment analysis (GSEA) using C5-GO pathways for pg versus Control iTregs. Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.

[0192] C5-HPO pathway analysis highlighted no significant positively or negatively enriched pathways (Table 19A, 19B). Of note, positive enrichment of HP_DECREASED_SPECIFIC_ANTIBODY_RESPONSE_TO_PROTEIN_VACCINE suggests that pg iTregs possess an expression profile associated with reduced antibody response to vaccines, with vaccine- specific Tregs previously identified with repeated117 / 191#14753190vlinfluenza vaccination (Lin et al., 2023). For those negatively enriched pathways, analysis highlighted downregulation of genes found in various autoimmune diseases including HP_PATCHY_ALOPECIA and HP_RHEUMATOID_ARTHRITIS pathways, both previously associated with dysregulated Treg function (Wan et al., 2023; Yan et al., 2022). These results further suggest enhanced immunomodulatory capacity of pg iTregs when compared to Controls.Table 19A: Gene set enrichment analysis (GSEA) using C5-HP0 pathways for pg versus Control iTregs. Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.Table 19B: Gene set enrichment analysis (GSEA) using C5-HPO pathways for pg versus Control iTregs. Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.

[0193] C7-IMMUNSIGB analysis identified various trending positive enrichments relevant to Treg biology including GSE37532_TREG_VS_TCONV_CD4_TCELL_FROM_VISCERAL_ADIPOSE_TISSUE UP (NES = 2.40, q = 0.173),118 / 191#14753190vlGSE37532_VISCERAL_ADIPOSE_TISSUE_VS_LN_DERIVED_TCONV_CD4_TCELL_ DN (NES = 2.33, q = 0.210), GSE13887_RESTING_VS_ACT_CD4_TCELL_UP (NES = 2.28, q = 0.230), and GSE40493_BCL6_KO_VS_WT_TREG_DN (NES = 2.23, q = 0.252) (Table 20A). These pathways reflect that pg iTregs appear phenotypically similar to classic visceral adipose tissue Tregs (VAT-Tregs), often regarded as among the most stable, inflammation-resistant, and highly suppressive T regulatory cells (Zeng et al., 2018).Additionally, the positive enrichment of GSE40493_BCL6_KO_VS_WT_TREG_DN provides supporting evidence that pg iTregs upregulate those genes downregulated in BCL6-KO Tregs. Previous studies highlight that BCL6 deletion in Tregs is associated with impaired suppressive function alongside increased phenotypic drift (Wen et al., 2024; Li et al., 2020; Sawant et al., 2012). Therefore, simulated pg exposure may enhance BCL6-dependent transcriptional programs associated with highly suppressive, tissue-adapted regulatory T cells.

[0194] Further C7-IMMUNSIGB analysis also revealed various trending negative enrichments relevant to Treg biology (Table 20B). Of note, negative enrichment ofGSE11818_WT_VS_DICER_KO_TREG_UP indicates reduced Dicer- deficient Treg-like activity in pg iTregs. Importantly, the Dicer-controlled miRNA pathway is critical for Treg stabilization and immunosuppressive function (Liston et al., 2008), again suggesting that pg iTregs possess a more stable and suppressive phenotype when compared to Controls.Furthermore, negative enrichment of the GSE40274_FOXP3_VS_FOXP3_AND_SATB1_TRANSDUCED_ACTIVATED_CD4_TC ELL_UP highlights that pg iTregs have increased SATB1 -modified FoxP3 expression, with SATB 1 known to be a crucial regulator of Treg cell lineage and possibly associated with autoimmune disease manifestation when disrupted (Kitagawa et al., 2017).Table 20A: Gene set enrichment analysis (GSEA) using C7-IMMUNESIGB pathways for pg versus Control iTregs. Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.119 / 191#14753190vlTable 20B: Gene set enrichment analysis (GSEA) using C7-IMMUNESIGB pathways for pg versus Control iTregs. Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.

[0195] Finally, while the C8 analysis revealed significant and trending positive and negative enrichment of various pathways, most pathways’ relevance to Treg physiology is unknown (Table 21 A, 2 IB). Of note, the negative enrichment of HE_LIM_SUN_FETAL_LUNG_C4_TH17_CELL (NES -1.9, q = 0.341) may suggest less Thl7-skew in pg iTregs when compared to Controls and prevent conversion to this pro-inflammatory phenotype in vivo (Yousefi et al., 2015).Table 21A: Gene set enrichment analysis (GSEA) using C8 pathways for pg versus Control iTregs. Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.120 / 191#14753190vlTable 21B: Gene set enrichment analysis (GSEA) using C8 pathways for pg versus Control iTregs. Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.Example 21. Short- and long-term stimulation assay reveals differences in IL-10 and latent l'GE-1) production from Control and simulated ug iTregs

[0196] To evaluate differences in IL- 10 and latent TGF-P production from Control and pg iTregs, both a short- (30 minutes of 300 lU / mL IL-2 stimulation) and long- (72 hours of 300 lU / mL IL-2 and CD3 / CD28 bead stimulation) term stimulation study was conducted (Figure 7). First, for short-term stimulation, while mean IL-10 production was increased in pg iTregs (0.7184 ± 0.5978 pg / mL) versus Controls (0.1374 ± 0.1374 pg / mL), with a trending significance of p = 0.2778 (Figure 7A), production of IL-10 overall was limited with only 30 minutes of IL-2 stimulation (< 1 pg / mL). Conversely, there was no significant difference in short-term latent TGF-P production between groups (p = 0.7373) (Figure 7B). In response to long-term stimulation, mean IL- 10 production was again increased in pg iTregs (50.53 ± 27.58 pg / mL) versus Controls (48.61 ± 26.43 pg / mL), with a trending significance of p = 0.2381 (Figure 7C). Conversely, there was no significant difference in long-term latent TGF-P production between groups (p = 0.9030) (Figure 7D). Overall, these data suggest that pg iTregs, in response to stimulus, have enhanced ability for IL- 10 release and likely improved immunosuppressive capacity in comparison to Controls, reflecting the development of a more effector Treg (eTreg) profile (Teh et al., 2015) following simulated microgravity exposure.121 / 191#14753190vlExample 22. Co-culture immunosuppressive assay reveals differences in suppressive capacity of Control and simulated OR iTregs

[0197] To directly evaluate differences in immunosuppressive capacity of pg versus Control iTregs, an effector CD8+ T lymphocyte co-culture suppression assay was conducted using the CellTrace™ Violet Cell Proliferation Kit (Figure 8). Representative histograms of CellTrace™ Violet (CTV) staining revealed adequate proliferation of CD8+ T lymphocytes in stimulated control wells, with an average CTV MFI dilution from unstimulated controls of 26.448 ± 7.842% across donor assays (Figure 8A). Importantly, analysis revealed enhanced immunosuppressive capacity of pg iTregs in comparison to Controls at 1:1 (44.90 ± 14.66% vs 38.46 ± 18.70%, p = 0.6304), 2:1 (55.16 ± 9.379% vs 37.75 ± 12.93%), with a strong trending significance of p = 0.0634, and 4:1 (49.85 ± 6.480% vs 48.98 ± 16.39%, p = 0.9492) ratios of CD8s to iTregs (Figure 8B). These data suggest that pg iTregs have enhanced suppressive capacity of effector CD8+ T lymphocyte proliferation in vitro versus Controls, likely through both increased secretion of immunosuppressive cytokines (IL- 10) in combination with direct cell-cell interactions through immunomodulatory cellular surface receptors, as previously reported (von Boehmer et al., 2005; Nguyen et al., 2014).Example 23. Short- and long-term persistence assay reveals differences in cellular persistence between Control and simulated pg iTregs

[0198] To compare the cellular persistence of Control and pg iTregs, an in vitro short-and long-term persistence assay with IL-2 deprivation was conducted (Figure 9). First, while there was a significant decrease in % Live Cells for pg iTregs in comparison to Control at day 4 (p = 0.0169), there was no significant difference (p = 0.9910) observed at day 10 (Figure 9A). While Absolute Live Cells per mL were decreased in pg iTregs, at days 4 and 10, there was no statistically significant differences when compared to the Control group (Figure 9B). These data suggest that pg exposure causes a transient decrease in iTregs that stabilizes in comparison to the Control cohort after 10 days of IL-2 deprivation.

[0199] Next, the phenotype of iTregs from pg and Control cohorts was compared at both days 4 and 10 using flow cytometry. Importantly, there was a trending (p = 0.1630) increase in % CD4+ FoxP3+ for the pg iTreg group in comparison to Controls at day 4, with no difference measured at day 10 (Figure 9C). No significant difference in % CD4+ FoxP3+ pSTAT5+ was measured between groups at either timepoint (Figure 9D). For % CD4+ FoxP3+ CD25+, there were trending increases at both day 4 (p = 0.2342) and day 10 (p = 0.3597) for122 / 191#14753190vlpg iTregs in comparison to Control (Figure 9E). Furthermore, while there was a trending (p = 0.2951) decrease in % CD4+ FoxP3+ CD45RO+ at day 4, there was a stronger trending (p = 0.1047) increase in this cellular population at day 10 in the pg iTreg cohort (Figure 9F).

[0200] The expression level (inferred through MFI) of various markers was also investigated for pg and Control iTregs at both timepoints. While there was no significant difference at day 10, there was a trending (p = 0.1152) increase in FoxP3 MFI for the pg iTregs in comparison to Controls at day 4 (Figure 9G). Regarding pSTAT5 MFI, no difference was observed at day 4, while a trending (p = 0.2397) decrease was apparent at day 10 for pg iTregs (Figure 9H). Furthermore, there were trending increases in CD25 MFI in the pg group at both days 4 (p = 0.2620) and 10 (p = 0.3397) in comparison to Control (Figure 91). Finally, a significant (p = 0.0431) increase in CD45RO MFI was evident for pg iTregs at day 4, but no difference was detected at day 10 (Figure 9 J).

[0201] Overall, these data indicate that while there is minimal difference in cell counts at days 4 and 10 of IL-2 deprivation, there are phenotypic and expression differences between groups. Importantly, these differences suggest that pg iTregs maintain a more mature (increased % CD4+ FoxP3+ CD45RO+, CD45RO MFI), IL-2 responsive (increased % CD4+ FoxP3+ CD25+, CD25 MFI), and stable (increased % CD4+ FoxP3+, FoxP3 MFI) phenotype in comparison to Controls, suggesting improved phenotypic persistence and immunosuppressive function in the setting of long-term IL-2 deprivation. These data support that the more efficient IL-2 signaling of pg iTregs in comparison to Control, as suggested by prior flow cytometric and RNA sequencing analysis, appears essential to phenotype maintenance in this setting.Example 24. Natural T regulatory cell isolation, culture, and expansion

[0202] Following PBMC isolation, CD4+ CD25+ CD127dim / - nTregs were then isolated by a combined negative and positive selection using a magnetic bead separation kit before culture and expansion. Here, 1.110 x 106CD4+ CD25+ CD127- / dim nTregs, or 0.0222 x 106cells / mL blood, were isolated from an exemplary, n=l human donor. Those nTregs were expanded 36.266-fold to 40.255 x 106cells using the previously detailed 18-day expansion protocol (Table 22).123 / 191#14753190vlTable 22: Isolation, culture, and expansion of natural T regulatory cells (nTregs). CD4+ CD25+ CD127- / dim nTreg cell count (in millions), CD4+ CD25+ CD127- / dim nTreg cells count (in millions) per mL of blood, expanded nTreg count (in millions), and Expansion Fold presented for donor analyzed.Example 25. Simulated micro gravity (pg) exposure of nTregs

[0203] Following 18 days of culture and expansion, the exemplary nTregs were placed in either standard 2D culture (group - Control) or exposed to simulated pg (group -pg) using the Synthecon® RCCS-8D Disposable Vessel for 24 hours as previously described. Cell counts by hemocytometer following exposure to either the Control (1.239 ± 0.5766) or simulated pg (0.8766 ± 0.0216) condition revealed a decreased proliferation index for the pg group in comparison to Control, which itself was variable between experimental replicates (Table 23).Table 23: Simulated microgravity (pg) exposure of nTregs versus Control. Control nTregs cell count (in millions), Control Proliferation Index, pg nTregs cell counts (in millions), and pg Proliferation Index, with average (AVG) ± standard deviation (SD) from experimental replicates presented for donor analyzed. For comparison between Control and pg nTregs, cell counts were normalized to equal starting volume.Example 26. Flow cytometric analysis of Control and simulated ,ug nTregs

[0204] Flow cytometry was used to investigate nTreg cellular phenotype between Control, pg, Control_s, and pg_s groups, with findings from the exemplary nTreg analysis compared to previous iTreg experimentation. Regarding cellular phenotype (Figure 10, Table 24), the % Five Cells was similar between all groups and greater than 90% following either standard culture (Control, Control_s) or simulated microgravity (pg, pg_s) exposure (Figure 10A, Table 24). The % of nTregs, defined as CD4+ FoxP3+ cells, was overall decreased in comparison to previous iTregs; however, the population still displayed an approximate 80% cellular purity across all groups (Figure 10B, Table 24). The % CD4+ FoxP3+ pSTAT5+ decreased in the Control_s and pg_s groups compared to their respective unstimulated124 / 191#14753190vlcohorts (Figure 10C, Table 24), differing from experimentation with iTregs. Interestingly, unlike iTregs, there was no substantial difference in % CD4+ FoxP3+ CD25+ between groups (Figure 10D, Table 24), suggesting that these naturally derived Tregs likely maintain their CD25 expression and dependence on IL-2 following robust expansion. The % CD4+ FoxP3+ CD45RO+ population was also increased in the pg_s compared to the pg group, a finding not evident in the Control group (Figure 10E, Table 24). Further, % CD4+ FoxP3+ CD3+ was increased in the Control_s and pg_s groups relative to their unstimulated baselines, with the greatest increase in the pg_s group (Figure 10F, Table 24).Table 24: Flow cytometric analysis of Control and simulated jag nTregs, with cellular gate (Gate), group (Control, Control_s, pg, pg_s), and average (AVG) ± standard deviation (SD) of technical replicates for percentage (%) data of cellular populations analyzed.

[0205] MFI, as a proxy for expression level, was also evaluated in the exemplary nTreg cohorts. Similar to iTreg experimentation, FoxP3 MFI was increased in both the Control_s and pg_s groups compared to the Control and pg groups, respectively (Figure 11 A, Table 25). A greater increase in AFoxP3 MFI was seen in the Control group compared to the pg group (Figure 11B, Table 25), much like the trend evident in iTregs. While the pSTAT5 MFI increased in the Control_s and pg_s groups during iTreg experimentation, this trend was less125 / 191#14753190vlapparent in nTregs, with a small decrease in pSTAT5 MFI evident in pg nTregs after stimulation (Figures 11C / D, Table 25). This likely reflects the more stable and less dynamic nature of pSTAT5 in nTregs versus iTregs. Additionally, there was also a decrease in CD25 MFI in both Control_s and pg_s compared to the unstimulated baselines (Figure HE, Table 25), much like findings apparent in iTregs from the same groups. The ACD25 MFI was, however, slightly more negative in the Control than in pg nTregs (Figure 11F, Table 25). Finally, for CD45RO MFI (Figure 11G), there was an increase in ACD45RO for the pg_s group alongside a slight decrease in Control_s (Figure 11H, Table 25), much like observations in previous iTreg experimentation. Overall, these findings suggest that nTregs were successfully isolated, expanded, and then exposed to either Control or pg conditions. While differences exist, many of the trends previously evident in Control and pg iTreg cohorts were also present in this exemplary nTreg analysis. Ultimately, these findings suggest that pg exposure may also promote nTreg resilience and reduced reliance on IL-2 stimulus for enhanced immunosuppressive function.Table 25: Flow cytometric analysis of Control and simulated pg nTregs, with cellular gate (Gate), group (Control, Control_s, pg, pg_s), and average (AVG) ± standard deviation (SD) of technical replicates for MFI or average (AVG) delta (A) MFI data of cellular markers analyzed.126 / 191#14753190vlExample 27. RNA sequencing analysis of Control and simulated us nTregs

[0206] Following 24-hours of standard cellular culture or simulated pg exposure, RNA was isolated from Control and pg nTregs for paired RNA sequencing analysis of differential gene expression (n=l). The statistically significant DEGs identified during the previous RNA sequencing analysis of pg versus Control iTregs were further investigated for expression changes in these exemplary nTregs (Table 26), as statistical significance cannot be achieved with n=l replicate per group. To begin, simulated pg exposure was again associated with upregulation of stress response genes including HSPA6 and HSPA1B. This increased gene expression in pg nTregs in comparison to Controls likely promotes enhanced suppressive function and anti-inflammatory cytokine production while protecting cells from apoptosis in harsh, low IL-2 environments (Table 26). This analysis also revealed a similar downregulation of transient, immediate-early genes (EGR1, EGR2, EGR3), transcription factors (NR4A1, NR4A3, E2F7, ZBED2), cell cycle and proliferation genes (RRM2, CCNF, TRIP13, POLE2), as well as DNA replication and repair genes (FEN1, DMC1, DSCC1), again, likely promoting nTreg phenotypic stability through modulation of cellular replication, with pg nTregs favoring quiescence and survival over proliferation.Table 26: Analysis of previous iTreg differentially expressed genes including the Gene symbol, baseMean, and log2FC from the pg versus Control nTreg paired RNA sequencing analysis. Bolded genes represent those with similar gene expression changes (between pg and Control) to the prior iTreg analysis.127 / 191#14753190vl

[0207] Additionally, investigation of the previously identified set of immune-related DEGs (from iTreg analysis) revealed similar trends in this exemplary nTreg analysis. Here, strong downregulation of negative regulators of the IL-2 / STAT5 signaling pathway (CISH, SOCS2) was again identified in pg nTregs and likely supports enhanced IL-2 signaling capacity and responsiveness for sustained STAT5 expression. Coupling this downregulation with similar modulation of IL2RA, as exhibited in previous flow cytometric analysis, pg nTregs may also exhibit more efficient IL-2 / IL2R signaling and resultantly greater resilience in dynamic IL-2 environments in vivo. Furthermore, similar downregulation of interferon-inducible inflammatory response genes (SLFN12L, SLFN13) and CCR2 may translate to a more immunosuppressive phenotype with reduced pro-inflammatory bias. Regarding128 / 191#14753190vlmetabolic genes, this exemplary analysis again supported a shift from glycolysis to oxidative metabolism alongside lipid and amino acid reprogramming in pg nTregs, as evidenced by similar modulation of the metabolic genes HK2, CYP1B1, HILPDA, PAQR4, TLCD3A. Altogether, this exploratory analysis of prior iTreg DEGs reveals that pg exposure likely reprograms nTregs for enhanced stress resilience (HSPs), IL-2 sensitivity, and metabolic reprogramming for more potent immunosuppressive function.

[0208] Gene set enrichment analysis (GSEA) was again conducted to identify enriched biological pathways in pg nTregs compared to Controls, with the top 10 positively and negatively enriched gene sets highlighted in Tables 27-40. For this analysis, an emphasis was placed on those conserved gene sets which were enriched in both both pg nTregs and iTregs. First, analysis revealed that there was no significant positive or negative enrichment of Hallmark pathways overall (Table 27A, 27B). Of note, HALLMARK_UV_RESPONSE_UP and HALLMARK_P53_PATHWAY were positively enriched in both pg nTregs and iTregs. Additionally, the Hallmark pathways HALLMARK_WNT_BETA_CATENIN_SIGNALING, HALLMARK_HEDGEHOG_SIGNALING, HALLMARK_ALLOGRAFT_REJECTION, and HALLMARK_MYC_TARGETS_V2 were of the top 10 negatively enriched pathways present in pg nTregs and iTregs, highlighting modulation of similar gene sets across both cell types. Of note, negative enrichment of the HALLMARK_IL2_STAT5_SIGNALING pathway in pg nTregs further supports modulation of the negative regulators of IL-2 / STAT5 pathway and likely enhanced IL-2 signaling capacity versus Controls.Table 27A Gene set enrichment analysis (GSEA) using Hallmark pathways for pg versus Control nTregs (n=l). Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.129 / 191#14753190vlTable 27B: Gene set enrichment analysis (GSEA) using Hallmark pathways for pg versus Control nTregs (n=l). Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q- value presented.

[0209] Next, C2-CGP (chemical and genetic perturbations) pathway analysis revealed no significant positive or negative pathway enrichment in pg nTregs (Table 28A, 28B). There were also no conserved positively or negatively enriched pathways present between both pg nTreg and iTreg analyses. Of note, positive enrichment of the WELCH_GATA1_TARGETS pathway (NES = 1.51) likely reveals enhanced FoxP3 expression and phenotypic stability in pg nTregs versus Controls, as previously described (Fu et al., 2012).Table 28A: Gene set enrichment analysis (GSEA) using C2-CGP pathways for pg versus Control nTregs (n=l). Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presentedTable 28B: Gene set enrichment analysis (GSEA) using C2-CGP pathways for pg versus Control nTregs (n=l). Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.130 / 191#14753190vl

[0210] GSEA using C2-CP: BIOCARTA revealed no significant positively or negatively enriched pathways in pg nTregs (Table 29A, 29B). However, the BIOCARTA_MTA3_PATHWAY was found to be positively enriched in both pg nTregs and iTregs. Chromatin remodeling resulting from MTA3 activity, a component of the Nucleosome Remodeling and Deacetylase (NuRD) complex, has been shown to regulate genetic programming of follicular Tregs in lymph nodes and germinal centers, preventing excessive immune activation of B cells and T follicular helper cells at those sites (Shen et al., 2018). This positive enrichment may support that pg nTregs appear much like potent follicular Tregs which are critical for preventing autoimmune disease formation. Additionally, the positive enrichment (NES = 1.42) of the BIOCART A_MEF2D_PATHW AY likely reveals the development and stabilization of an effector Treg phenotype found to be critical in long-term allograft survival (Di Giorgio et al., 2020).Table 29A: Gene set enrichment analysis (GSEA) using C2-CP: BIOCARTA pathways for pg versus Control nTregs (n=l). Top 10 positively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.Table 29B: Gene set enrichment analysis (GSEA) using C2-CP: BIOCARTA pathways for pg versus Control nTregs (n=l). Top 10 negatively enriched pathways with pathway identifier (Pathway), Size, normalized enrichment score (NES), and FDR q-value presented.131 / 191#14753190vl

[0211] Furthermore, GSEA revealed no significant positive or negative enrichment of gene sets within C2-CP: KEGG_MEDICUS, in addition to no overlapping pathways between pg nTreg and iTreg analyses (Table 30A, 30B).Table 30A: Gene set enrichment ana...

Claims

CLAIMSWhat is claimed is:

1. A population of Regulatory T (Treg) cells exposed to microgravity for between 2 to 216 hours.

2. The population Treg cells of claim 1, wherein the Treg cells comprise at least 80% natural Treg (nTreg) cells by total cell number.

3. The population Treg cells of claim 1, wherein the Treg cells comprise at least 80% induced Treg (iTreg) cells by total cell number.

4. The population Treg cells of any one of claims 1-3, wherein exposure of the population of Treg cells to microgravity for between 2 and 216 hours increases the population of Treg cells secretion of IL- 10 and / or TGF-beta.

5. The population Treg cells of any one of claims 1-4, wherein exposure of the Treg cells to microgravity for between 2 and 216 hours modulates the population of Treg cells immunosuppressive gene expression pathways.

6. The population Treg cells of any one of claims 1-5, wherein exposure of the population of Treg cells to microgravity for between 2 and 216 hours decrease proliferation of CD8+ T cells in a co-culture system.

7. The population Treg cells of any one of claims 1-6, wherein exposure of the population of Treg cells to microgravity for between 2 and 216 hours decreases proliferation of T responder cells in a co-culture system.

8. The population Treg cells of any one of claims 1-7, wherein the Treg cell is more immunosuppressive relative to a Treg cell not exposed to microgravity.

9. The population Treg cells of any one of claims 1-8, wherein the Treg cells are further exposed to a combination of cytokines and / or adjuvants.176 / 191#14753190vl10. The population Treg cells of claim 9, wherein the cytokine is IL-7.

11. The population Treg cells of any one of claims 1-10, wherein exposing the Treg cells to microgravity increases intracellular STAT5 expression and / or phosphorylation of STAT5 in the Treg cells.

12. The population Treg cells of any one of claims 1-11, wherein exposing the population of Treg cells to microgravity increases cell survival, in a setting of IL-2 deprivation, relative to Treg cells not exposed to microgravity.

13. The population Treg cells of claim 12, wherein at least a portion of the Treg cells exposed to microgravity are more immunosuppressive, relative to Treg cells not exposed to microgravity.

14. The population Treg cells of claim 12 or 13, wherein the Treg cells exposed to microgravity express higher levels of CD4 / FoxP3, CD4 / FoxP3 / Stat5, CD4 / FoxP3 / CD45RO, and / or CD4 / FoxP3 / CD25, relative to Treg cells not exposed to microgravity.

15. The population Treg cells of any one of claims 12-14, wherein the increased Treg cell survival persists for at least 4 days after exposure to microgravity.

16. The population Treg cells of any one of claims 12-14, wherein the increased Treg cell survival persists for at least 10 days after exposure to microgravity.

17. The population Treg cells of any one of claims 12-16, wherein the Treg cells are iTreg cells.

18. The population Treg cells of any one of claims 12-16, wherein the Treg cells are nTreg cells.

19. A composition comprising a microgravity cell culture (MCC) medium comprising a plurality of Treg cells, wherein the composition has been exposed to microgravity for between 2 to 216 hours.177 / 191#14753190vl20. The composition of claim 19, wherein the plurality of Treg cells comprise at least 80% nTreg cells by total cell number.

21. The composition of claim 20, wherein the nTregs were isolated from a peripheral blood mononuclear cell (PBMC) suspension.

22. The composition of claim 19, wherein the plurality of Treg cells comprise at least 80% iTreg cells by total cell number.

23. The composition of claim 22, wherein the iTreg cells are produced from a suspension of CD4+ helper T cells in a differentiation medium.

24. The composition of claim 23, wherein the CD4+ helper T cells were isolated from a peripheral blood mononuclear cell (PBMC) suspension.

25. The composition of claim 21 or 24, wherein the PBMC suspension was obtained via peripheral blood venipuncture and / or apheresis instrument from a human subject.

26. The composition of any one of claims 19-25, wherein the MCC medium further comprises at least one cytokine and / or adjuvant.

27. The composition of claim 26, wherein at least one cytokine is selected from the group consisting of IL-2, IL-7, TGF-betal, IL-10, IL-12, IFN-gamma, and / or IL-35.

28. The composition of claim 26 or 27, wherein at least one cytokine is IL-7.

29. The composition of any one of claims 26-28, wherein at least one cytokine is present in the MCC medium at a concentration of between 0.01 and 1000 ng / mL.

30. The composition of any one of claims 19-29, wherein exposing the Treg cells to microgravity increases intracellular STAT5 expression and phosphorylation of STAT5 in the Treg cells.178 / 191#14753190vl31. The composition of any one of claims 19-30, wherein exposing the Treg cells to microgravity increases extracellular secretion of IL- 10 and / or TGF-beta from the Treg cells.

32. The composition of any one of claims 19-31, wherein exposure of the Treg cells to microgravity modulates a Treg cells immunosuppressive gene expression pathways.

33. The composition of any one of claims 19-32, wherein exposure of the Treg cells to microgravity decreases proliferation of CD8+ T cells in a co-culture system.

34. The composition of any one of claims 19-33, wherein exposure of the Treg cells to microgravity decreases proliferation of T responder cells in a co-culture system.

35. The composition of any one of claims 19-34, wherein the Treg cells are more immunosuppressive relative to Treg cells not exposed to microgravity.

36. The composition of claim 30, wherein the increase in intracellular STAT5 expression, increase in extracellular secretion of IL- 10 or TGF-beta, modulation of immunosuppressive gene expression pathways, decrease in CD8+ T cell proliferation in a coculture system, decrease in T responder cell proliferation in a co-culture system and / or the increase in immunosuppression are induced by a microgravity environment.

37. The composition of any one of claims 19-36, wherein exposure of the Treg cells to microgravity increases cell survival, in a setting of IL-2 deprivation, relative to Treg cells not exposed to microgravity.

38. The composition of claim 37, wherein the increased Treg cell survival persists for at least 4 days after exposure to microgravity.

39. The composition of claim 37 or 38, wherein the increased Treg cell survival persists for at least 10 days after exposure to microgravity.

40. The composition of any one of claims 37-39, wherein the Treg cells are iTreg cells.

41. The composition of any one of claims 37-39, wherein the Treg cells are nTreg cells.179 / 191#14753190vl42. The composition of any one of claims 19-41, further comprising one or more CD4+ helper T cells.

43. The composition of any one of claims 19-42, wherein a rotary cell culture system is used to simulate a microgravity environment via continuous free fall.

44. The composition of any one of claims 19-43, wherein a MCC media further comprises one or more antibiotics, serum, cytokines, metabolic supplements, chemical additives, stimulators, and / or other compounds.

45. The composition of claim 44, wherein the one or more cytokines comprises IL-2, IL-7, TGF-betal, IL-10, IL-35, IL-12, IFN-gamma and / or IL-15.

46. The composition of claim 44 or 45, wherein the one or more metabolic supplements comprises arginine, glutamine, serine, and / or glycine.

47. The composition of any one of claims 44-46 wherein the one or more chemical additives comprises taurine, N-acetylcysteine, and / or retinoic acid.

48. The composition of any one of claims 44-47, wherein the stimulators comprise anti-CD3 / CD28 stimulatory beads.

49. The composition of any one of claims 44-48, wherein the one or more other compounds comprises rapamycin.

50. The population of Treg cells of any one of claims 1-18 or the composition of any one 19-49 for use as a medicament.

51. The population of Treg cells of any one of claims 1-18 or the composition of any one 19-49 for use in treating an autoimmune disease, inflammatory disease, a proliferative disease, a neurological disease, or a genetic disorder.180 / 191#14753190vl52. The use of claim 51, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, polymyositis, Sjogren syndrome, Hashimoto thyroiditis, Graves’ disease, type 1 insulin dependent diabetes, Addison disease, vitiligo, pernicious anemia, glomerulonephritis, myasthenia gravis, pulmonary fibrosis, psoriasis, psoriatic arthritis, Crohn’s disease, celiac disease, autoimmune hepatitis, ALS, pemphigus vulgaris, bullous pemphigoid, ulcerative colitis, autoimmune gastritis, multiple sclerosis, myasthenia gravis, and autoimmune encephalitis.

53. The use of claim 51, wherein the inflammatory disease is selected from the group consisting of rheumatoid arthritis, psoriasis, asthma, hepatitis, arthritis, cardiovascular disease, Kawasaki disease, chronic obstructive pulmonary disorder, ulcerative colitis, Crohn’s disease, diabetes, vasculitis, gout, sarcoidosis, systemic lupus erythematosus, Hashimoto’s thyroiditis, Grave’s disease, ankylosing spondylitis, antiphospholipid antibody syndrome, chronic recurrent multifocal osteomyelitis, Henoch- Schonlein Purpura, idiopathic thrombocytopenic purpura, juvenile dermatomyositis, juvenile idiopathic arthritis, juvenile lupus, juvenile scleroderma, juvenile vasculitis, mixed connective tissue disease, myositis, poststreptococcal inflammatory syndrome, psoriatic arthritis, reactive arthritis, scleroderma, Sjogren’s syndrome, uveitis, vasculitis, encephalitis, atherosclerosis, myocardial infarction, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, obesity, sepsis, chronic viral infection, aging, wound injury, hemophagocytic lymphohistiocytosis, atopic dermatitis, pemphigus vulgaris, Guillain-Barre syndrome, hemophilia, B cell autoimmunity, acute respiratory distress syndrome, and osteoarthritis.

54. The use of claim 51, wherein the neurological disease is selected from the group consisting of Alzheimer’s Disease, Parkinson’s Disease, Guillain-Barre syndrome, Amyotrophic Lateral Sclerosis (ALS), multiple sclerosis, stroke, traumatic brain injury, epilepsy, myasthenia gravis, Huntington’s disease, Lewy Body Dementia, Frontotemporal Dementia, Vascular Dementia, and prion infections.

55. The use of claim 51, wherein the genetic disorder is selected from the group consisting IPEX syndrome (Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked Syndrome), Autoimmune Lymphoproliferative Syndrome (ALPS), Autoimmune Polyendocrinopathy Syndrome Type 1 (APS-1), Chediak-Higashi Syndrome, Systemic 181 / 191#14753190vlJuvenile Idiopathic Arthritis (SJIA), Chronic Granulomatous Disease (CGD), Shwachman-Diamond syndrome, Fanconi anemia, Familial Hemophagocytic Lymphohistiocytosis (HLH), and Wiskott-Aldrich Syndrome (WAS).

56. The use of any one of claims 51-55, further comprising administering one or more adjuvants alongside Treg therapy.

57. The use of claim 56, wherein the one or more adjuvants are selected from the group consisting of disease-modifying antirheumatic drugs (DMARDs), immunosuppressants, calcineurin inhibitors, mTOR inhibitors, biologies, JAK inhibitors, integrin inhibitors, SIP receptor modulators, complement inhibitors, phosphodiesterase inhibitors, corticosteroids, antihistamines, non-steroidal anti-inflammatory drugs (NSAIDS), antifibrotics, leukotriene modifiers, immunomodulatory antibiotics, probiotics / prebiotics, antimetabolites, antiprotease, cholinesterase inhibitors, N-methyl-D-aspartate (NMD A) receptor antagonist, Alzheimer’s Disease Modifying Therapies, Dopaminergic Therapies, amyotrophic lateral sclerosis (ALS) disease modifying therapies, multiple sclerosis (MS) disease modifying therapies and kinase inhibitors.

58. The Treg cells of any one of claims 1-18 or the composition of any one 19-49 for use during and / or after surgery.

59. The composition of claim 58, wherein the surgery is selected from the group consisting of solid organ transplantation, graft versus host disease, hematopoietic stem cell transplantation, biomedical device implantation, cardiac surgery, spinal cord surgery, skin grafting, islet cell transplantation, primary tumor resection, recurrent tumor resection, metastatic tumor resection, joint replacement, and fracture repair.

60. A method for producing immunosuppressive Treg cells by exposing a plurality of Treg cells to microgravity.

61. The method of claim 60, further comprising obtaining a peripheral blood sample or a bone marrow sample from a human subject.182 / 191#14753190vl62. The method of claim 60 or 61, further comprising isolating a peripheral blood mononuclear cell (PBMCs) suspension from a peripheral blood sample.

63. The method of claim 62, further comprising isolating nTreg cells from the PMBC blood sample to yield a nTreg cell population.

64. The method of claim 63, wherein the nTreg cell population is CD4+CD25+CD127dim / -.

65. The method of claim 63 or 64, further comprising stimulating the nTreg cells by adding the nTreg cells to a first stimulation cell culture (SCC) media for between 24 and 48 hours.

66. The method of claim 65, wherein the first SCC media comprises cell media comprising one or more antibiotics, serum, CD3 / CD28 activation / expansion beads, cytokines, and / or other compounds.

67. The method of claim 65 or 66, further expanding the nTreg cell population by adding the stimulated nTreg cell population to a first expansion cell culture (ECC) media for between 24 and 168 hours.

68. The method of claim 67, wherein the first ECC media comprises the SCC media comprising one or more additional antibiotics, serum, cytokines, and / or other compounds.

69. The method of claim 67 or 68, further stimulating the expanded nTreg cell population by adding the expanded nTreg cell population to a second SCC media for between 24 and 48 hours.

70. The method of claim 69, wherein the second SCC media comprises cell media comprising one or more antibiotics, serum, CD3 / CD28 activation / expansion beads, cytokines, and / or other compounds.

71. The method of claim 69 or 70, further expanding the nTreg cell population by adding the stimulated nTreg cell population to a second ECC media for between 24 and 168 hours.183 / 191#14753190vl72. The method of claim 71, wherein the second ECC media comprises the second SCC media comprising one or more additional antibiotics, serum, cytokines, and / or other compounds.

73. The method of any one of claims 60-62, further comprising isolating naive CD4+ T helper cells from the PMBC suspension to yield a naive CD4+ T helper cell population.

74. The method of claim 73, wherein the cells in the isolated naive CD4+ T helper cell population are CD45RO-, CD8-, CD14-, CD15-, CD16-, CD19-, CD25-, CD34-, CD36-, CD56-, CD123-, TCRy / 6-, HLA-DR-, and / or CD235a-.

75. The method of claim 73 or 74, wherein the cells in the isolated naive CD4+ T helper cell population are CD4+ / CD45RA+.

76. The method of claim 75, further comprising adding the CD4+ / CD45RA+ T cell population to a differentiation cell culture (DCC) media for between 2 and 48 hours.

77. The method of claim 76, wherein the DCC media comprises IL-2, TGF-beta 1, retinoic acid, anti-CD3 / CD28 stimulation beads, and / or rapamycin.

78. The method of claim 76 or 77, wherein upon exposing the CD4+ / CD45RA+ cells to the DCC media at least a portion of cells are differentiated into iTreg cells.

79. The method of claim 78, wherein the iTreg cells are CD4+ / FoxP3+ cells.

80. The method of any one of claims 76-79, wherein between 80% and 100% of the cells in the DCC media are CD4+ / FoxP3+ iTreg cells after between 24 to 48 hours of culture.

81. The method of any one of claims 76-79, further comprising expanding the iTreg cells by adding the differentiated iTreg cell population to a third ECC media.

82. The method of claim 81, wherein the third ECC media comprises the DCC media comprising one or more additional antibiotics, serum, cytokines, and / or other compounds.184 / 191#14753190vl83. The method of any one of claims 63-72, further comprising adding the expanded nTregs to a microgravity media and exposing the nTreg cells to microgravity for at least 2 hours.

84. The method of any one of claims 73-81, further comprising adding the expanded iTregs to a microgravity media and exposing the iTreg cells to microgravity for at least 2 hours.

85. The method of claim 83 or 84, wherein the microgravity media comprises one or more cytokines, metabolic supplements, chemical additives, stimulators, and / or other compounds.

86. The method of claim 85, wherein the one or more cytokines comprise IL-2, IL-7, IL-10, IL-35, IL15, IL-12, IFN-gamma, and / or TGF-beta.

87. The method of claim 85 or 86, wherein the one or more metabolic supplements comprise arginine, glutamine, serine, and / or glycine.

88. The method of any one of claims 85-87, wherein the one or more chemical additives comprise taurine, N-acetylcysteine, and / or retinoic acid.

89. The method of any one of claims 85-88, wherein the one or more stimulators comprise anti-CD3 / CD28 stimulatory beads.

90. The method of any one of claims 85-89, wherein the one or more compounds comprises rapamycin.

91. The method of any one of claims 60-90, wherein a rotary cell culture system is used to simulate microgravity through continuous cellular free fall.

92. The method of any one of claims 63-72, 83 or 85-91, wherein exposing a CD4+ / FoxP3+ nTreg population to microgravity increases IL- 10 and / or TGF-beta secretion from at least a portion of CD4+ / FoxP3+ nTregs within the cell population.185 / 191#14753190vl93. The method of any one of claims 63-72, 83 or 85-91, wherein exposing a CD4+ / FoxP3+ nTreg cell population to microgravity modulates gene expression pathways in at least a portion of CD4+ / FoxP3+ nTregs within the cell population.

94. The method of any one of claims 63-72, 83 or 85-91, wherein exposing a CD4+ / FoxP3+ nTreg cell population to microgravity decreases CD8+ T cell proliferation in at least a portion of CD4+ / FoxP3+ nTregs within the cell population.

95. The method of any one of claims 63-72, 83 or 85-91, wherein exposing a CD4+ / FoxP3+ nTreg cell population to microgravity decreases T responder cell proliferation in at least a portion of CD4+ / FoxP3+ nTregs within the cell population.

96. The method of any one of claims 73-81, 84 or 85-91, wherein exposing a CD4+ / FoxP3+ iTreg cell population to microgravity increases IL- 10 and / or TGF-beta secretion from at least a portion of CD4+ / FoxP3+ iTregs within the cell population.

97. The method of any one of claims 73-81, 84 or 85-91, wherein exposing a CD4+ / FoxP3+ iTreg cell population to microgravity modulates immunosuppression gene expression pathways in at least a portion of CD4+ / FoxP3+ iTregs within the cell population.

98. The method of any one of claims 73-81, 84 or 85-91, wherein exposing a CD4+ / FoxP3+ iTreg cell population to microgravity decreases CD8+ T cell proliferation in at least a portion of CD4+ / FoxP3+ iTregs within the cell population.

99. The method of any one of claims 73-81, 84 or 85-91, wherein exposing a CD4+ / FoxP3+ iTreg cell population to microgravity decreases T responder cell proliferation in at least a portion of CD4+ / FoxP3+ iTregs within the cell population.

100. The method of any one of claims 60-99, wherein exposing the Treg cells to microgravity increases in vitro cell survival, relative to Treg cells not exposed to microgravity.186 / 191#14753190vl101. The method of claim 100, wherein the Treg cell survival is increased in the setting of IL-2 deprivation.

102. The method of claim 100 or 101, wherein the increased Treg cell survival persists for at least 4 days after exposure to microgravity.

103. The method of claim 100 or 101, wherein the increased Treg cell survival persists for at least 10 days after exposure to microgravity.

104. The method of any one of claims 100-103, wherein the Treg cells are iTreg cells.

105. The method of any one of claims 100-103, wherein the Treg cells are nTreg cells.

106. A method of treating an autoimmune disease, inflammatory disease, a proliferative disease, neurological disease, or a genetic disorder, the method comprising administering the Treg cells of any one of claims 1-18 or the composition of any one of claims 19-49 to a subject.

107. The method of claim 106, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, polymyositis, Sjogren syndrome, Hashimoto thyroiditis, Graves’ disease, type 1 insulin dependent diabetes, Addison disease, vitiligo, pernicious anemia, glomerulonephritis, myasthenia gravis, pulmonary fibrosis, psoriasis, psoriatic arthritis, Crohn’s disease, celiac disease, autoimmune hepatitis, ALS, pemphigus vulgaris, bullous pemphigoid, ulcerative colitis, autoimmune gastritis, multiple sclerosis, and myasthenia gravis, and autoimmune encephalitis.

108. The method of claim 106, wherein the inflammatory disease is selected from the group consisting of rheumatoid arthritis, psoriasis, asthma, hepatitis, arthritis, cardiovascular disease, Kawasaki disease, chronic obstructive pulmonary disorder, ulcerative colitis, Crohn’s disease, diabetes, vasculitis, gout, sarcoidosis, systemic lupus erythematosus, Hashimoto’s thyroiditis, Grave’s disease, ankylosing spondylitis, antiphospholipid antibody syndrome, chronic recurrent multifocal osteomyelitis, Henoch- Schonlein Purpura, idiopathic thrombocytopenic purpura, juvenile dermatomyositis, juvenile idiopathic arthritis, juvenile 187 / 191#14753190vllupus, juvenile scleroderma, juvenile vasculitis, mixed connective tissue disease, myositis, poststreptococcal inflammatory syndrome, psoriatic arthritis, reactive arthritis, scleroderma, Sjogren’s syndrome, uveitis, and vasculitis, encephalitis, atherosclerosis, myocardial infarction, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, obesity, sepsis, chronic viral infection, aging, wound injury, hemophagocytic lymphohistiocytosis, atopic dermatitis, pemphigus vulgaris, Guillain-Barre syndrome, hemophilia, B cell autoimmunity, acute respiratory distress syndrome, osteoarthritis.

109. The method of claim 106, wherein the neurological disease is selected from the group consisting of Alzheimer’s Disease, Parkinson’s Disease, Guillain-Barre syndrome, Amyotrophic Lateral Sclerosis (ALS), multiple sclerosis, stroke, traumatic brain injury, epilepsy, myasthenia gravis, Huntington’s disease, Lewy Body Dementia, Frontotemporal Dementia, Vascular Dementia, and prion infections.

110. The method of claim 106, wherein the genetic disorder is selected from the group consisting IPEX syndrome (Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked Syndrome), Autoimmune Lymphoproliferative Syndrome (ALPS), Autoimmune Polyendocrinopathy Syndrome Type 1 (APS-1), Chediak-Higashi Syndrome, Systemic Juvenile Idiopathic Arthritis (SJIA), Chronic Granulomatous Disease (CGD), Shwachman-Diamond syndrome, Fanconi anemia, Familial Hemophagocytic Lymphohistiocytosis (HLH), and Wiskott-Aldrich Syndrome (WAS).

111. The method of any one of claims 106-110, further comprising administering one or more adjuvants alongside Treg therapy.

112. The method of claim 111, wherein the one or more adjuvants are selected from the group consisting of disease-modifying antirheumatic drugs (DMARDs), immunosuppressants, calcineurin inhibitors, mTOR inhibitors, biologies, JAK inhibitors, integrin inhibitors, SIP receptor modulators, complement inhibitors, phosphodiesterase inhibitors, corticosteroids, antihistamines, non-steroidal anti-inflammatory drugs (NSAIDS), antifibrotics, leukotriene modifiers, immunomodulatory antibiotics, probiotics / prebiotics, antimetabolites, anti-protease, cholinesterase inhibitors, N-methyl-D-aspartate (NMD A) receptor antagonist, Alzheimer’ s Disease Modifying Therapies, Dopaminergic Therapies,188 / 191#14753190vlamyotrophic lateral sclerosis (ALS) disease modifying therapies, multiple sclerosis (MS) disease modifying therapies and kinase inhibitors.

113. A method of treating a subject during and / or after surgery, the method comprising administering the Treg cells of any one of claims 1-18 or the composition of any one of claims 19-49 to a subject.

114. The method of claim 113, wherein the surgery is selected from the group consisting of solid organ transplantation, graft versus host disease, hematopoietic stem cell transplantation, biomedical device implantation, cardiac surgery (aortic aneurysm, bypass), spinal cord surgery, skin grafting, islet cell transplantation, primary tumor resection, recurrent tumor resection, metastatic tumor resection, joint replacement, fracture repair.

115. A kit comprising a population of Treg cells of anyone of claims 1-18 or the composition of any one of claims 19-49 for the treatment of a disease.

116. The kit of claim 115, further comprising one or more adjuvants.

117. The kit of claim 116, wherein the one or more adjuvants are selected from the group consisting of disease-modifying antirheumatic drugs (DMARDs), immunosuppressants, calcineurin inhibitors, mTOR inhibitors, biologies, JAK inhibitors, integrin inhibitors, SIP receptor modulators, complement inhibitors, phosphodiesterase inhibitors, corticosteroids, antihistamines, non-steroidal anti-inflammatory drugs (NSAIDS), antifibrotics, leukotriene modifiers, immunomodulatory antibiotics, probiotics / prebiotics, antimetabolites, antiprotease, cholinesterase inhibitors, N-methyl-D-aspartate (NMD A) receptor antagonist, Alzheimer’s Disease Modifying Therapies, Dopaminergic Therapies, amyotrophic lateral sclerosis (ALS) disease modifying therapies, multiple sclerosis (MS) disease modifying therapies and kinase inhibitors.

118. A kit for isolating, expanding, and exposing cells to microgravity, comprising: one or more cell isolation reagents configured to isolate a population of cells from a biological sample, one or more cell culture reagents configured to differentiate and / or expand the isolated population of cells ex vivo, and a microgravity cell culture medium, wherein the kit189 / 191#14753190vlis configured such that the population of cells is isolated from the biological sample, expanded ex vivo, and subsequently exposed to microgravity.

119. A kit comprising a population of Treg cells of anyone of claims 1-18 or the composition of any one of claims 19-49.

120. A pharmaceutical composition comprising a population of Treg cells of anyone of claims 1-18 or the composition of any one of claims 19-49 for the treatment of a disease.

121. A pharmaceutical composition comprising a population of Treg cells of anyone of claims 1-18 or the composition of any one of claims 19-49 and an excipient.

122. The pharmaceutical composition of claim 121, wherein the Treg cells comprise at least 80% Treg cells by total cell number.

123. The pharmaceutical composition of claim 122, wherein the total number of cells in the composition is between 5 x 105and 5 x 109cells.

124. The pharmaceutical composition of claim 121 or 123, wherein the number of non-Treg cells is less than 20% by total cell number.190 / 191#14753190vl

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