Methods of using human mesenchymal stem cells to effect cellular and humoral immunity

Abstract

The present invention provides a method for determining whether treatment of symptoms of non-ischemic dilated cardiomyopathy with isolated allogeneic human bone marrow-derived mesenchymal stem cells (MSCs) is effective by determining the levels of exhausted B cells (CD19+, CD27-, IgD-), the levels of switched memory B cells (CD19+, CD27high, IgD-), the levels of B-cells expressing intracellular TNF-α, the levels of early activated T-cells (CD3+, CD69+), the levels of chronic activated T-cells (CD3+, CD25+), the levels of Temra cells (CD45RA+, CCR7-), the TNF-α concentration in a sample of the subject’s serum using a diagnostic agent.

Description

Methods of Using Human Mesenchymal Stem Cells to Effect Cellular and Humoral ImmunityCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of New Zealand Patent Application No. 753525, the national phase of PCT / US2017 / 061031, and claims priority to US application no 62 / 421,048 filed 11 November 2016. The entire contents of each of these applications is incorporated herein by cross reference.FIELD OF THE INVENTION[0001a] The present invention relates to methods of administering therapeutically effective amounts of isolated populations of allogeneic rnesenchymal stem cells to effect cellular and humoral immunity in subjects suffering from non-ischemic dilated cardiomyopathy. The present invention also relates to methods of administering therapeutically effective amounts of isolated populations of allogeneic mesenchymal stem cells to effect cellular and humoral immunity in subjects suffering from symptoms of aging frailtyBACKGROUND OF THE INVENTIONAging Frailty and Inflammaging

[0002] Aging frailty poses a very concerning problem for the overall health and well-being of individuals and is characterized as a syndrome of multisystem physiological dysregulation. Aging frailty is a geriatric syndrome characterized by weakness, low physical activity, slowed motor performance, exhaustion, and unintentional weight loss. See Yao, X. et al., Clinics in Geriatric Medicine 270):79-87 (2011). Furthermore, there are many studies showing a direct correlation between aging frailty and inflammation. See Hubbard, RE et al., Biogerontology 11(5):635-641 (2010).

[0003] Immunosenescence is characterized by a low grade, chronic systemic inflammatory state known as inflammaging. See Franceshi, C. et al., Annals of the New York Academy of Sciences 908:244-254 (2000). This heightened inflammatory state or chronic inflammation found in aging and aging frailty leads to immw1e dysregulation and a complex remodeling of both innate and adaptive immunity. In immunosenescence, the T cell and B cell repertoire is skewed resulting in an increase in CD8+ T effector memory cells re-expressing CD45ra (TEMRA) and in the CD19+ late / exhausted memory B cells, and a decrease in the CD8; Naive T cells, and in the switched mernory B cells (CD2i). See Blomberg, B.B. et al., Immunologic Research 57(1-3):354-360 (2013); Colonna-Romano, G. et al., Mechanisms of Ageing and Development 130(10):681-690 (2009); and Koch S. et al., Immunity & Ageing: 5:6 (2008). This shift in the T cell and B cell repertoire results in arefractory or less efficient immune status. It is well known that age-associated increase in systemic inflammation (TNF-a, IL-6, IL-8, INFy and CRP) induces impaired B cell function.

[0004] Inflammaging has received considerable attention because it proposes a link betvveen immune changes and a number of diseases a.nd conditions (such as aging frailty) common in old age. Circulating inflarmnatory mediators such as cytokines and acute phase proteins are markers of the low-grade inflammation observed to increase with aging. These pro-inflammatory cytokines (e.g., TNF-a, lL-6) impair the capacity of B cells to make protective antibodies to exogenous antigens and vaccines. This impaired B cell response is measured by reduced class switch recombination (CSR) which is the ability of immunoglobulins to svvitch isotype from IgM to a secondary isotype (lgG, IgA, or IgE). Immunoglobulin isotype switching is crucial for a proper immune response as the effector functions differ in each isotype. A key player in CSR and somatic hypennutation (SHM) is the enzyme, activation-induced cytidine deaminase (AID), encoded by the Aic^a gene. AID's basic function in CSR and SHM is to initiate breaks in the DNA by converting cytosines to macils in the sv,-itch and variable regions of immunoglobulins. E47, encoded by the Tcfe2a (E2A) gene, is a transcription factor belonging to the class I basic helix loop helix (bHLH) proteins, also known as E proteins. Without E47 expression, the B cell specific transcription factors EBFl (early B cell factor) and Pax-5 (paired box protein) are not expressed. Both E47 and Pax-5 are key transcription factors in early development for the B cell lineage and mature B cell function. See Hagman J. et al., Immunity 27(1):8-10 (2007); Horcher M. et al., Immunity 14(6):779-790 (2001): Riley R.L. et al., Seminars in Immunology 17(5):330-336 (2005). The Pax-5 gene encodes the B cell lineage specific activator protein (BSAP) that is expressed at all stages of B cell differentiation, but not in terminally differentiated B cells. Pax-5 controls B cell commitment by repressing B lineage inappropriate genes and activating B cell specific genes making Pax-5 the B cell gatekeeper and is exclusively expressed in the B lymphoid lineage from the committed pro-B cell to the rnature B cell stage. Tiie B cell specific transcription factor, Pax-5, is not only highly important in early B cell development and B cell lineage commitment it is also involved in CSR.[ 0005] Tt has also been shown in humans that the amount of TNF-a made: (l) depends on the amount of system inflammation and (2) impairs the ability of the same B cells to be stimulated with mitogens or antigens. See Frasca, D. et al., Journal of immunology188(1):279-286 (2012). Thus, the immwie response in subjects suffering from aging frailty is impaired for a number of reasons.Non-Ischemic Dilated Cardiomyopathy (NIDCM)

[0006] Non-ischemic dilated cardiomyopathy (NIDCM) is a progressive disorder with no current cure, often culminating in heart transplantation. See Felker, G.M. et al.. The Nevv England Journal of Medicine 3-12: 1077-84 (2000) and Kirklin, J.K. et al., J Heart Lung Transplant. 35:407-412 (2016). NlDCM is a disorder with a major component of immW1e dysregulation as an w1derlying etiology. See Efthimiadis, L et al. . Hippokratia 15:335-342 (2011) and Meng, X. et al, Nature Reviews Cardiology 13: 167-79 (2016). Cell-based therapy for heart disease is a promising new treatment strategy undergoing evaluation, \vith a major challenge and opportunity in developing allogeneic therapy. See PateL A.N. et al. . Lancet 387:2412-21 (2016); Assrnus, B. et al., The New England Journal cfMedicine 355:1222-32 (2006); Hare, J.M. et al.. J Am. Coll. Cardiol. 5-1:2277-86 (2009); Heldman, A.W. et al., JAA1A 311:62-73 (2014): Perin, E.C. et al.. JAlvlA 307:1717-26 (2012); Golpanian, S. et al., Physiol. Rev. 96:1 127-68 (2016); and Hare, J.M. et al., JAi\.1A 308:2369-79 (2012).Mesenchymal Siem Cells

[0007] Mesenchymal stem cells are multipotent cells able to migrate to sites of injury, while also being illlinWloprivileged by not detectably expressing major histocompatibility complex class II (MHC-II) molecules, and expressing MHC-I molecules at low levels. See Le Blanc, K. et al., Lancet 37 j(962-I): J 579-1586 (2008) and Klyushnenkova E. et al., J Biomed. Sci. 12(1):47-57 (2005). As such, allogeneic mesenchymal stem cells hold great promise for therapeutic and regenerative medicine, and have been repeatedly shown to have a high safety and efficacy profile in clinical trials for multiple disease processes. See -fare, J.M. et al.. Journal O the American College of Cardiology 54(24):2277-2286 (2009); Hare, J.M. et al.. Tex. Heart Inst J 36(2):145-147 (2009): and Lalu, M.M. et al., PloS One 7(10):e47559 (2012). They have also been shown to not undergo malignant transformation after transplantation into patients. See Togel F. et al., American Journal of Physiology Renal Physiology 289(i):F31-F42 (2005). Treatment with mesenchymal stem cells has been shown to ameliorate severe graft-versus-host disease, protect against ischemic acute renal failure, contribute to pancreatic islet and renal glomerular repair in diabetes, reverse folimant hepatic failure, regenerate damaged lung tissue, attenuate sepsis, and reverse remodeling and improve cardiac function after myocardial infarction. See Le Blanc K. et al. . Lancet 371(962 1) 15791586 (2008); Hare, J.M. et al., Journal of the American College ol Cardiology 54(24):2277-2286 (2009); Togel F. et al., American Journal of Physiology Rena! Physiology 259<7):F31-F42 (2005); Lee RH. et al. . PNAS 103(-16): 17438-17442 (2006); Parekkadan, B. et al., PloS One 2(9):e941 (2007); Ishizawa K. et al, FEBSLetters 556(1-3):249-252 (2004); Nemeth K. et al, Nature Medicine 15(1).42-49 (2009); Iso Y. et al, Biochem. Biophys. Res. Comm. 351(3):700-706 (2007); Schuleri KJi. et al., Eur. Hearth .J 30(22):2722-2732 (2009); and Heldman AW. et al., JAMA. 311(1):62-73 (2014). Furthermore, mesenchymal stem cells are also a potential source of multiple cell types for use in tissue engineering. See Gong Z. et al., 1\1ethods in 1\.fol. Bio. 698:279-294 (2011); Price, AP. et al., Tissue Engineering Part A J6(8):2581-2591 (201O); and Toge! F. et al., Organogenesis 7(2):96-100 (2011).

[0008] Mesenchymal stem cells have immuno-modulatory capacity. They control int1ammation and the cytokine production of lymphocytes and myeloid-derived immune cells without evidence of immunosuppressive toxicity and are hypo-immunogenic. See Bernardo M.E. et al, Cell Stem Cell J3(4):392-402 (2013).

[0009] Mesenchymal stem cells also have the capacity to differentiate not only into cells of mesodermal origin, but into cells of endodermal and ectodermal origin. See Le Blanc K. et al. Exp. Hematol. 31(10):890-896 (2003) , For example, in vitro, mesenchymal stem cells cultured in airway growth media differentiate to express lung-specific epithelial markers, e.g., surfactant protein-C, Clara cell secretory protein, and thyroid transcription factor-I. See Jiang Y. et al.. Nature 418(6893):41-49 (2002) and Kotton D.N. et al., Development J28(24):5181-5188 (2001) ,

[00010] In vivo studies have shown that human mesenchymal stem cells undergo sitespecific differentiation into various cell types, including myocytes and cardiomyocytes, v,'hen transplanted into fetal sheep. See Airey JA. et al., Circulation 109(JJ):140l-1407 (2004). These mesenchymal stem cells can persist for as long as 13 months in multiple tissues after transplantation in non-immunosuppressed immunocompetent hosts. Other in vivo studies using rodents, dogs, goats, and baboons similarly demonstrate that human mesenchymal stem cells xenografts do not evoke lyrnphocyte proliferation or systemic allo-antibody production in the recipient. See Klyushnenkova E. et al, l Biomed. Sci. 12(1):47-.57 (2005); Aggarwal S et al, Blood 105(1):1815-22 (2005); Augello A et al., Arthritis and Rheumatism 56(4): 1175-86 (2007); Bartholomew A. et al., Exp Hematol. 30(1):42-48. (2002); Dokic let al., European Journal of Immunology 43(7):1862-72 (2013); Gerdoni E. et al., Annals of Neurology 61 (3):219-227 (2007); Lee S.H et al., Respiratory Research 11:16 (2010); Urban V.S. et al., Stem Cells 26(1):244-253 (2008); Yang H. et a!., PfoS One 8(7):e69l29 (2013);Zappia E. et al.. Blood 106(5):1755-1761 (2005); Bonfield T.L. et al., American Journal of Physiology Lung Cellular and Afolecular Physiology 299(6):L760-70 (2010); Glenn J.D. et al., World Journal of Stem Cells. 6(5):526-39 (2014); Guo K. et al, Frontiers in Cell and Developmental Biology 2:8 (2014); Puissant B. et al., British Journal of Haematology 129(1):118-129 (2005); and Sun L. et al., Stem Cells 27(6):1421-32 (2009) . Taken as a whole, these repeated finding of allogeneic safety and efficacy solidify the notion for using mesenchymal stem cells as an allograft for successful tissue regeneration.

[00011] However, despite being a safe therapeutic agent, mesenchymal stem cells are reported in the literature to exert a suppressive effect on antibody production as well as proliferation and maturation of B cells. See Uccelli, A. et al.. Trends in Immunology 28(5):219-226 (2007). Mesenchymal stem cells are also reported to inhibit the generation and function of antigen presenting cells.     See Ifoogduijn M.J et al., Int.hnmunopharmacolog 10(12):1496-1500 (2010). Finally, mesenchymal stem cells are reported to suppress CD4+ and CD8+ T cell proliferation. See Ghannam S. et al., Stem Cell Res. & Ther. 1:2 (2010). Finally, some preclinical data indicates a higher risk of immunological clearance with administration of allogeneic mesenchymal stem cells as compared to the administration of autologous mesenchymal stem cells. See, e.g., Huang, X.P. et al., Circulation 122:2419-29 (2010).SUMMARY

[00012] Surprisingly, despite the reports of mesenchymal stem cells having a suppressive effect on aspects of the immune system, the present inventors discovered a method of treating non-ischemic dilated cardiomyopathy in a subject, comprising administering a therapeutically effective amowit of a population of isolated a!logeneic human mesenchymal stern cells to a subject in need thereof. The present inventors also discovered a method of treating symptoms of aging frailty in a subject comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof The present inventors identified several biomarkers specific for non-ischemic dilated cardiomyopathy and aging frailty including, but not limited to, the levels of exhausted B cells (CD19+, CD2T, lgff), the levels of switched memory B cells (CD19\ CD2?1'igh‘ Igf), the levels of B-cells expressing intracellular TNF-u, the levels of early activated T-cells (CD3\ CD69‘), the levels of chronic activated T-cells (CD3\CD251), the levels of Temra cells (CD45RL-\\ CCRT), the CD4+:CD8+ T cell ratio, and the TNF-a concentration in serum.

[00013] One aspect of the invention relates to a method of treating non-ischemic dilated cardiomyopathy in a subject comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number of exhausted B cells (CD19\ CD2T, lgff) in a sample of the subject' serum decreases by at least 25% as compared to the number of exhausted B cells in a sample of the subject's serwn prior to administration of the population of isolated allogeneic hlm1an rnesenchymal stem cells, thereby treating the non-ischemic dilated cardiomyopathy.

[00014] Another aspect of the invention relates to a method of treating non-ischemic dilated cardiomyopathy in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic hurnan mesenchymal stem cells to a subject in need thereof, wherein the number of switched memory B cells (CD19\ CD2iigh, lgD") in a sample of the subject's serum increases by at least 100%} as compared to the number of sv,-itched memory B cells in a sample of the subject's serum prior to administration of the population of isolated allogeneic human mesenchymal stem cells, thereby treating the nonischemic dilated cardiomyopathy.

[00015] Another aspect of the invention relates to a method of treating non-ischemic dilated cardiomyopathy in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic hurnan mesenchymal stem cells to a subject in need thereof, wherein the number of B-cells expressing intracellular TNF-a in a sample of the subject's serum decreases by at least 30% as compared to the nwnber of B-cel!s expressing intracellular TNF-ci. in a sample of the subject's senm1 prior to administration of the population of isolated allogeneic human mesenchymal stem cells, thereby treating the nonischemic dilated cardiomyopathy.

[00016] Another a<;pect of the invention relates to a method of treating non-ischemic dilated cardiomyopathy in a subject, comprising adrninistering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number of early activated T-cells (CD3+, CD69+) in a sample of the subject's serum decreases by at least 30% as compared to the nwnber of early activated T-cells in a sample of the subject's serum prior to administration of the population of isolated allogeneic human mesenchymal stem cells, thereby treating the non-ischemic dilated cardiomyopathy.

[00017] Another aspect of the invention relates to a method of treating non-ischemic dilated cardiomyopathy in a subject comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof: wherein the nwnber of chronic activated T-ce!ls (CD3+, CD251) in a sample of the subject's serum decreases by at least 70% as compared to the number of chronic activated T-Cells in a sample of the subject’ s serum prior to administration of the population of isolated allogeneic human mesenchymal stem cells, thereby treating the non-ischemic dilated cardiomyopathy.

[00018] Another aspect of the invention relates to a rnethod of treating non-ischemic dilated cardiomyopathy in a subject comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number of Temra cells (CD45RA4, CCRT) in a sample of the subject's serum decreases by at least 40% as compared to the number of Temra cells in a sample of the subjecf s serum prior to administration of the population of isolated allogeneic human mesenchymal stem cells, thereby treating the non-ischemic dilated cardiomyopathy.

[00019] Another aspect of the invention relates to a rnethod of treating non-ischemic dilated cardiomyopathy in a subject comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the TNF-u concentration in a sample of the subject's serum decreases by at least 80% as compared to the TNF-u concentration in a sample of the subject's serum prior to administration of the population of isolated allogeneic human mesenchymal stem cells, thereby treating the non-ischemic dilated cardiomyopathy.

[00020] Another aspect of the invention relates to a method of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number of exhausted B cells (CDl 91, CD2T, Igff) in a sample of the subject' serum decreases by at least 10%i as compared to the number of exhausted B cells in a sample of the subject's serum prior to administration of the population of isolated allogeneic human mesenchymal stem cells, thereby treating the symptoms of aging frailty.

[00021] Another aspect of the invention relates to a method of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number of switched memory B cells (CD19', CD27hig\ Igff) in a sample of thesubject's serum increases by at least 75% as compared to the number of switched memory B cells in a sample of the subject's senmi prior to administration of the population of isolated allogeneic human mesenchymal stem cells, thereby treating the symptoms of aging frailty.

[00022] Another aspect of the invention relates to a method of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number of B-cells expressing intracellular TNF-a in a sample of the subject's serum decreases by at least 60(1 / o a.-: compared to the number of B-cells expressing intracellular TNF-ci. in a sample of the subject's senm1 prior to administration of the population of isolated allogeneic human mesenchymal stem cells, thereby treating the symptoms of aging frailty.

[00023] Another aspect of the invention relates to a method of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number of early activated T-ce!ls (CD31, CD69 1) in a sample of the subject's serum decreases by at least 30% as compared to the number of early activated T-cells in a sample of the subject's serum prior to administration of the population of isolated allogeneic human mesenchymal stem cells, thereby treating the symptoms of aging frailty.

[00024] Another aspect of the invention relates to a method of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number of chronic activated T-cells (CD31, CD25') in a sample of the subject's serum decreases by at lea.st 75% as cornpared to the number of chronic activated T-Cells in a sample of the subject's serum prior to administration of the population of isolated allogeneic human mesenchymal stem cells, thereby treating the symptoms of aging frailty.

[00025] Another aspect of the invention relates to a method of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number of Temra cells (CD45RA', CCR7) in a sample of the subject's serum decreases by at least 20%1 as compared to the number of Ternra cells in a sample of the subject's serum prior to administration of the population of isolated allogeneic human mesenchymal stem cells, thereby treating the symptoms of aging frailty.

[00026] Another aspect of the invention relates to a method of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the TNF-u concentration in a sample of the subject's serum decreases by at least 50%» as compared to the TNF-a concentration in a sarnple of the subject's serum prior to administration of the population of isolated allogeneic human mesenchymal stem cells, thereby treating the symptoms of aging frailty.

[00027] Another aspect of the invention relates to a method of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the CD4+:CD8+ T cell ratio in a sample of the subject's semm increases by at least 100%» as compared to the CD4; :CD8; T cell ratio in a sample of the subject's serum prior to administration of the population of isolated allogeneic human mesenchymal stem cells, thereby treating the symptoms of aging frailty

[00028] In one embodiment of the invention, the subject is a human. In another embodiment ofthe invention, the subject is a human who exhibits inflammaging.

[00029] In one embodiment of the invention, the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells. In one embodiment of the invention, the mesenchymal stem cells do not express STR0-1. In another embodiment of the invention, the mesenchymal stern cells do not express CD45. in another embodiment of the invention, the mesenchymal stem cells do not express fibroblast surface markers or have a fibroblast morphology. In another embodiment of the invention, the mesenchymal stem cells are not genetically manipulated.

[00030] In another embodiment of the invention, the isolated population of allogeneic mesenchymal stem cells is administered in a single dose. In another embodiment of the invention, the isolated population of allogeneic mesenchymal stem cells is administered in multiple doses, e.g., two or more doses. In another embodiment of the invention, the isolated population of allogeneic mesenchymal stem cells is administered at least yearly.

[00031] ln one embodiment, the isolated population of allogeneic mesenchymal stem cells is administered systemically. in one embodiment of the invention, the isolated population of allogeneic mesenchymal stem cells is administered by infusion or direct injection. In one embodiment of the invention, the isolated population of allogeneic mesenchymal stem cells is administered intravenously, intraarterially, intramuscularly,intraperitoneally, subcutaneously, intradermally, orally, transendocardially, or intranasal!y. In a further embodiment, the isolated population of allogeneic mesenchymal stem cells is administered intravenously. In a further embodiment, the isolated population of allogeneic mesenchymal stem cells is administered intramuscularly.

[00032] In one embodiment of the invention, the isolated population of allogeneic mesenchymal stem cells is administered at a dose of about 20xl06 mesenchymal stem cells. ln another embodiment of the invention, the isolated population of allogeneic mesenchymal stem cells is administered at a dose of about 1OOx106 mesenchyma! stem cells. In another embodiment of the invention, the isolated population of allogeneic rnesenchymal stem cells is administered at a dose of about 200x106 mesenchymal stem cells.

[00033] In one embodiment of the invention, the isolated population of allogeneic rnesenchymal stem cells are obtained from a human donor and wherein a step of MHC matching of the human donor to the subject is not employed prior to the administration of the isolated population of allogeneic mesenchymal stem cells to the subject.

[00034] Another aspect of the invention relates to a method of evaluating cellular and hlm1on.tl immunity status in a subject, comprising:(1) obtaining a serum sample from a subject selected for evaluation based on a deternunation that the subject '.Vas previously in need of treatment of non-ischemic dilated cardiomyopathy a.nd the subject had been administered an initial dose of a.n isolated population of allogeneic human mesenchymal stem cells:(2) performing one or more assays configured to detect a non-ischemic dilated cardiomyopathy marker selected from the group of exhausted B cells (CDl91, CD2T Igf f), switched mernory B cells (CDl 9\ CD27hig\ Igff), B-cells expressing intracellular TNF-a, early activated T-cells (CDJ', CD694), chronic activated T-cells (CDS', CD251), Temra cells (CD45RA\ CCRT), and serum TNF-u by introducing the serum sample obtained from the subject into an assay instrument which (i) contacts the serum sample with one or more antibodies which specifically bind for detection the biomarker(s) which are assayed, and (ii) generates one or more assay results indicating of binding of each biomarker which is assayed to a respective antibody to provide one or more assay results;(3) correlating the assay result(s) generated by the assay instrument to the imnnmity status of the subject, \vherein the correlating step comprises assigning a likelihood of one or more future changes in immune status to the subject based on the assay result(s); and(4) treating the subject based on the predetermined subpopulation of individuals to which the subject is assigned, wherein the treatment comprises administration of one or more additional doses of an isolated population of allogeneic human mesenchymal stem cells.

[00035] Another aspect of the invention relates to a method of evaluating cellular and humoral innmmity status in a subject, comprising:(1) obtaining a serum sample from a subject selected for evaluation based on a determination that the subject was previously in need of treatment of symptoms of aging frailty and the subject had been administered an initial dose of an isolated population of allogeneic hwnan mesenchymal stern cells;(2) performing one or more assays configured to detect aging frailty marker selected from the group of exhausted B cells (CD19+, CD2T, Igf'), switched memory B cells (CD! 9\ CD27hig\ Igf), B-cells expressing intracellular TNF-a, early activated T-cells (CD3\ CD69\ chronic activated T-cells (CD3\ CD25+), Temra cells (CD45RA\ CCRT), the CD4+:CD8+ T cell ratio, and serum TNF-a by introducing the serum sample obtained from the subject into an assay instrnment which (i) contacts the serum sample v,'ith one or more antibodies which specifically bind for detection the biomarker(s) which are assayed, and (ii) generates one or more assay results indicating of binding of each biomarker which is assayed to a respective antibody to provide one or more assay results;(3) correlating the assay resu!t(s) generated by the assay instrument to the immw1ity status ofihe subject wherein the correlating step comprises assigning a likelihood of one or more future changes in immune status to the subject based on the assay result(s): and(4) treating the subject based on the predetermined subpopulation of individuals to which the subject is assigned, wherein the treatment comprises administration of one or more additional doses of an isolated population of allogeneic human mesenchymal stem cells.

[00036] In one embodiment of the invention, one or more future changes in immune status comprise one or more of an increase in the number of exhausted B cells (CDl 91, CD2T, Tgff), a decrease in the number of switched memory7 B cells (CDJ 9\ CD27hig\ Igf), an increase in the number of B-cells expressing intracellular TNF-a, an increase in the number of early activated T-cells (CD31, CD69+), an increase in the number of chronic activated T-cells (CD3\ CD25;), an increase in the number of Temra cells (CD45RA\ CCRT), a decrease in the CD4+ :CD8+ T cell ratio, and an increase in serum TNF-a.

[00037] Another aspect of the invention relates to an in vitro method of detemrning efficacy of treatment of non-ischemic dilated cardiomyopathy in a subject comprising:determining the levels of one or more biomarkers selected from the group consisting of exhausted B cells (CDJ. 9\ CD2T, Igf f), switched memory B cells (CDJ. 9\ CD27high, Igff), B-cells expressing intracellular TNF-a, early activated T-cells (CD3\ CD69+), chronic activated T-cells (CD3\ CD25+), Terma cells (CD45RA+, CCRT), and the TNF-a concentration in semm obtained from the subject before and after administration of a population of isolated allogeneic human mesenchymal stem cells to the subject, and comparing the levels of the one or more biomarkers in the serum obtained before and after administration of the population of isolated human mesenchymal stem cells, wherein treatrnent is efficacious if(1) the number of exhausted B cells (CD19\ CD2T, Igf") decreases by at least 25% as compared to the number of exhausted B cells prior to administration of the population of isolated allogeneic human mesenchymal stem cells,(2) the number of switched memory B cells (CD194', CD2iigh, IgU) increases by at least l00%> as compared to the number of switched memory B cells prior to administration of the population of isolated a!logeneic human mesenchymal stem cells,(3) the number of B-cells expressing intracellular TNF-a. decreases by at least 30%i as compared to the number of B-cells expressing intracellular TNF-a prior to administration of the population of isolated allogeneic human mesenchymal stem cells,(4) the nwnber of early activated T-ce!ls (CDJ\ CD69+) decreases by at least J0% as cornpared to the number of early activatedT-cells prior to administration of the population of isolated allogeneic human mesenchymal stem cells,(5) the number of chronic activated T-cel!s (CD3\ CD25') decreases by at least 70% as compared to the mm1ber of chronic activated T-Cells prior to administration of the population of isolated allogeneic human mesenchymal stem cells,(6) the number ofTemra cells (CD45RA\ CCRT) decreases by at least 40% as compared to the number of Temra cells prior to administration of the population of isolated allogeneic human mesenchymal stem cells, and / or(7) the TNF-a concentration in a sample of the subject's serum decreases by at least 80% as compared to the TNF-a concentration in a sample of the subject's serum prior to administration of the population of isolated allogeneic hum,m mesenchymal stem cells.

[00038] Another aspect of the invention relates to an in vitro method of detemrning efficacy of treatment of symptoms of aging frailty in a subject comprising: determining thelevels of one or more hiomarkers selected from the group consisting of exhausted B cells (CD19\ CD2T', IgD), switched memory B cells (CD19 +, CD27high, Igff), B-cells expressing intracellular TNF-a, early activatedT-cells (CD3+, CD69+), chronic activated T-cells (CD3+, CD25+), Terma cells (CD45RA\ CCRT), the CD4+:CD8+ T cell ratio, and the TNF-a concentration in senm1 obtained from the subject before and after administration of a population of isolated allogeneic human mesenchymal stem cells to the subject, and comparing the levels of the one or more hiomarkers in the serum obtained before and after administration of the population of isolated human mesenchymal stem cells, wherein treatrnent is efficacious if(1) the number of exhausted B cells (CD19\ CD2T, lgf") decreases by at least 10% as compared to the number of exhausted B cells prior to administration of the population of isolated allogeneic hum,m mesenchymal stem cells,(2) the number of switched memory B cells (CD19+, CD2iigh' IgU) increases by at least 75° / o as compared to the number of switched memory B cells prior to adrninistration of the population of isolated allogeneic human mesenchymal stem cells,(3) the number of B-cells expressing intracellular TNF-a. decreases by at least 60%i as compared to the number of B-cells expressing intracellular TNF-a prior to administration of the population of isolated allogeneic human mesenchymal stem cells,(4) the nwnber of early activatedT-cells (CDJ\ CD69+) decreases by at least 30(1 / o as cornpared to the number of early activatedT-cells prior to adrninistration of the population of isolated allogeneic human mesenchymal stem cells,(5) the number of chronic activatedT-cel!s (CD3\ CD25') decreases by at least 75% as compared to the mm1ber of chronic activatedT-Cells prior to adrninistration of the population of isolated allogeneic human mesenchymal stem cells,(6) the number ofTemra cells (CD45RA+, CCRT) decreases by at least 20% as compared to the number ofTemra cells prior to administration of the population of isolated allogeneic human mesenchymal stem cells,(7) the TNF-a concentration in a sample of the subject's serum decreases by at least 50% as compared to the TNF-a concentration in a sample of the subject's serum prior to administration of the population of isolated allogeneic hum,m mesenchymal stem cells, and / or(8) the CD4+:CD8+ T cell ratio in a sample of the subjecf s serum increases by at least 100(% as compared to the CD4+:CD8+ T cell ratio in a sample of the subject's serumprior to administration of the population of isolated allogeneic human mesenchymal stem cells.

[00039]     Another aspect of the invention relates to the use in vitro of the levels ofexhausted B cells (CD19\ CD2T, Igff), the levels of switched memory B cells (CD19 1', CD27hig\ Igff), the levels of B-cells expressing intracellular TNF-u, the levels of early activated T-cells (CDJ1, CD69\ the levels of chronic activated T-cells (CD3\ CD251), the levels of Temra cells (CD45RA\ CCRT), and the TNF-a concentration in serum for determining whether treatment for non-ischemic dilated cardiomyopathy is efficacious.

[00040] Another aspect of the invention relates to the use in vitro of the levels of exhausted B cells (CD19+, CD2T, lgff), the levels of switched memory- B cells (CD19\ CD27high' lgff), the levels of B-cells expressing intracellular TNF-a, the levels of early activated T-cells (Cff, CD69\ the levels of chronic activated T-cells (CD3\ CD25r\ the levels of Temra cells (CD45RA\ CCRT), the CD44 :CD8+ T cell ratio, and the TNF-a concentration in serum for determining whether treatment for aging frailty is efficacious.BRIEF DESCRIPTION OF THE FIGURES

[00041] HG. l is a Consort Diagram for a randomized, double-blinded, placebo-controlled study investigating the use of allogeneic mesenchymal stem cells for the treatment of older individuals with frailty.

[00042] FIG. 2 provides baseline patient characteristics for subjects enrolled in the randomized, double-blinded, placebo-controlled study investigating the use of allogeneic mesenchymal stem cells for the treatment of older individuals with frailty.

[00043] FIG. 3 shows a decrease in TNF-ct in older subjects administered allogeneic hlm1an rnesenchymal stem cells.

[00044] FIG. 4 shows a decrease in early activated T cells expressing CD69 in older subjects administered allogeneic human mesenchymal stem cells.

[00045] FIG. 5 shows a decrease in the numbers of chronic / late activated T cells expressing CD25 in older subjects administered allogeneic human rnesenchymal stem cells.

[00046] FIG 6 shovvs measurements of CDS+ T cells in older subjects administered allogeneic human mesenchymal stem cells.

[00047] HG. 7 shows an increase in the ratio of CD41 / CD8; T cells in older subjects administered allogeneic human mesenchymal stem cells.

[00048] FIG. 8 shows an increased immunosenescence score m patients with aging frailty who received two infusions of human mesenchymal stem cells.

[00049] FIG. 9 shows a correlation between baseline serum TNF-u and baseline B cells expressing IC TNF-a.

[00050] FIG. 10 provides the % of B cells expressing IC TNF-a in Young Control BL, Low TNF:IC TNF BL, and High TNF:IC TNF BL.[0005 l] FIG. 11 provides the% of Switch Memory B cells in Young Control BL, Low TNF:SwMem B cells BL and High TNF:SwMem B cells BL.

[00052] FlG. 12 provides the % of Exhausted B cells in Young Control BL, Low TNF:Exh B cells BL, and High TNF:Exh B cells BL.

[00053] HG. 13 provides the% of Temra cells in Young Control BL, Low TNF:Temra cells BL, and High TNF:Temra cells BL.

[00054] FIG. 14 provides the CD47CD8' ’ T cell ratio in Young Control BL, Low TNF:CD4:CD8 BL, and High TNF:CD4:CD8 BL.

[00055] FIG 15 provides the absolute change of serum TNF-a in pg / ml in Placebo Control, Low TNF-a, and High TNF-u .

[00056] FIG. 16 shows a correlation between baseline serum TNF-a and the change in serum TNF-cc

[00057] FIG. 17 shows the absolute change of B cells expressing IC TNF-a in Placebo Control, Low TNF-a, and High TNF-a.

[00058] FIG. 18 show's a correlation between serum TNF-a and change in B cells expressing TC TNF-u.

[00059] FIG 19 shows the absolute change of% of Switch Memory B cells in Placebo Control, Low TNF-ct, and High TNF-u.

[00060] HG. 20 shows the absolute change of % of Exhausted B cells rn Placebo Control. Low TNF-a, and High TNF-a.

[00061] FIG. 21 shows the absolute change of% of Temra T cells in Placebo Control, Low TNF-a, and High TNF-a.

[00062] FIG. 22 shows a correlation between the change in serurn TNF-a. and the change in Temra T cells.

[00063] FlG. 23 shows the absolute change in the CD4+ / CD8+ T cell ratio in Placebo Control, Low TNF-a, and High TNF-a.

[00064] FIG. 24 shows a correlation between the change in CD4+ / CD8+ T cell ratio and the change in Temra T cells.DETAILED DESCRIPTION

[00065] Tn certain embodiments, the present invention is directed to methods of treating non-ischemic dilated cardiomyopathy in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic hwnan mesenchymal stem cells to a subject in need thereof. The examples demonstrate that in vivo administration of isolated populations of allogeneic human mesenchymal stem cells result in an increase in the percentage of switched memory B cells and a decrease in exhausted B cells in subjects. The examples also demonstrate that in vivo administration of isolated allogeneic human mesenchyrnal stern cells results in an improvement in the CD4+:CD8+ T cell ratio in subjects. Also, as shmvn in the examples, the levels of B-cells expressing intracellular TNF-cc the levels of early activated T-cells (CD3+, CD69+), the levels of chronic activated T-cells (CD3', CD25 +), the levels of Temra cells (CD45RA', CCRT), and the TNF-a concentration in serum is reduced in subjects having received infusions of allogeneic human mesenchymal stem cells. From these unexpected results, the present inventors determined that isolated allogeneic hwnan mesenchymal stem cells favorably altered several immunologic markers typically elevated in chronic inflammation. Restoration of immune competence has clinical relevant in subject vvho are of higher ri.sk for co-morbid infectious disease.

[00066] In other embodiments, the present invention is directed to methods of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic hurnan mesenchymal stem cells to a subject in need thereof The examples demonstrate that in vivo administration of isolated populations of allogeneic human mesenchymal stem cells result in an increase in the percentage of switched memory B cells and a decrease in exhausted B cells in subjects. The exarnples also demonstrate that in vivo administration of isolated allogeneic human mesenchymal stem cells results in an improvement in the CD4+:CD8+ T cell ratio in subjects. Also, as shown in the examples, the levels ofB-cells expressing intracellular TNF-a, the levels of early activated T-cells (CD3', CD69\ the levels of chronic activated T-cells (CD3\ CD25 +), the levels of Temra cells (CD45RA+, CCRT), and the TNF-a concentration in serum is reduced in subjects having received infusions of allogeneic human mesenchymal stem cells. From these unexpected results, the present inventors determined that isolated allogeneic human mesenchymal stem cells are effective at reducing intlammaging, a prevalent feature in aging frailty.Definitions[ 00067] Embodiments may be practiced without the theoretical aspects presented. Moreover, the theoretical aspects are presented with the lmderstanding that the embodiments are not bound by any theory presented.

[00068] Unless othernise defined, all terms (including technical and scientific terms) used herein have the sarne meaning as commonly understood by one of ordinary skill in the art. It will be further understood that tenns, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and ,vill not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[00069] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the temis “including”, "includes", "having", •'has", "with", or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[00070] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, tmless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., "such as"') provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[00071] The term "about' or •'approximately"’ means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e, the limitations of the measurement system For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of ±10% of the referenced value.Dosage, Duration and Subjects

[00072] "A therapeutically effective amounf' means an amount that stimulates a B- or T-cell dependent immune response. Such a response is characterized by the ability to elicitsignificant levels of IgG and opsonic activity. The dosage a.nd number of doses (e.g., single or multiple dose) administered to the subject will vary depending upon a variety of factors, including the route of administration, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired, and the like.

[00073]     In one embodiment of the invention, the isolated population of allogeneicmesenchymal stem cells is administered as a single dose. In another embodiment, the isolated population of allogeneic mesenchymal stem cells is administered in multiple doses, e.g.. two or more doses. In other embodiments, the isolated population of allogeneic mesenchymal stem cells is administered at least yearly.

[00074] In another embodiment of the invention. the administration of the isolated population of allogeneic mesenchymal stem cells is repeated, such as at least l, 2, 3, 4, 5, 6, 7, 8, 9, 10, IL 12, 13, 14, 15, 16, 17, or 18 months after the first administration of the isolated population of allogeneic mesenchymal stem cells, or repeated between 2-4, 2-6, 2-8, 2-10, 3-4, 3-6, 3-8, 3-10, 4-6, 4-8, 4-10, 6-8, 6-10, 6-12, or 12-18 months after the first administration of the isolated population of allogeneic mesenchymal stem cells.

[00075] In one embodiment of the invention, the isolated population of allogeneic mesenchymal stem cells is administered at a dose of about lxl06, 2xl06, 5xl06, 10xl06, 20xl0 6, 30xl06, 40xl()6, 50xl06, 60xl06, 70xl06, 80xl06, 90xl06, 100xl0 6, 110xl06, 120xl 06, 130xl 06, 140xl 06, 150x106, 160x106, J70x106, J80x106, 190x106, 200x106, 300xl06, 400xHi, .500x106, or 10x107 mesenchymal stem cells. In a further embodiment, the isolated population of allogeneic mesenchymal stem cells is adrninistered at a dose of about 20xl06 mesenchymal stem cells. In a further embodiment, the isolated population of allogeneic mesenchymal stem cells is administered at a dose of about 100xl06 mesenchymal stem cells. In yet a further embodiment, the isolated population of allogeneic mesenchymal stem cells is administered at a dose of about 200x106 mesenchymal stem cells. In further embodiments, the isolated population of allogeneic mesenchymal stem cells is administered at a dose of from about 1-400xl06, 10-400x106, 100-4 00x106, 20-200x106, 20-400xHl, 0. 1-5xHi, 0. l-10xHi,O. 1-100X106, 1-50xHi, l-100xl06• 0.0l-10x106 or 0.0l-100x106 mesenchyrnal stem cells.

[00076] In some embodiments, the therapeutically effective amount of the isolated population of allogeneic mesenchymal stem cells is sufficient to increase the ratio of CD4+ :CD8+ T cells in a subject, such as to increase the ratio of CD4+ :CD8+ T cells by at leasttwo-, three, four-, five-, or six-fold as compared to the ratio prior to administration of the isolated population of allogeneic mesenchymal stem cells, i.e.. an increase in the ratio of CD4+:CD8+ T cells by at least l 00%, 200%), 300%), 400%, or 500%.

[00077]     In some embodiments, the therapeutically effective amount of the isolatedpopulation of allogeneic mesenchymal stern cells is sufficient to increase the number of switched memory B cells (CD19\ CD27hig\ Igf) in a subject, such as to increase the number of switched memory B cells by at least two-, three-, four-, or five-fold as compared to the number prior to administration of the isolated population of allogeneic mesenchymal stem cells, i.e., an increase in the number of switched rnemory B cells (CDl 9+, CD27hig\ lgf") by at least l 00%, 200%, 300%, or 400%. In other embodiments, the number of switched memory B cells as compared to the number of such B cells prior to administration of the isolated population of allogeneic mesenchyrnal stem cells increases by at least l 0(l;o, 15%, 20%, 25%, 30%, 35%, 40%, 45%), 50%, 5.5%, 60%, 6.5%), 70%), 75%, 80%, 85%, 90%, or 95%.

[00078] In some embodiments, the therapeutically effective amount of the isolated population of allogeneic mesenchymal stem cells is sufficient to decrease the number of B cells expressing intracellular TNF-a in a subject such as to decrease the number by at least t\VO-, three-, four-, five-, or six-fold as compared to the number of such B cells prior to administration of the isolated population of al!ogeneic mesenchymal stem cells. In other embodiments, the number of B cells expressing intracellular TNF-a as compared to the number of such B cells prior to administration of the isolated population of allogeneic mesenchymal stem cells decreases by at least l 0%, 20%i, 30%, 40%), 50%, or 60%.

[00079] In some embodiments, the therapeutically effective amount of the isolated population of allogeneic mesenchymal stem cells is sufficient to decrease the number of exhausted B cells (CD19\ CD2T, lgff) in a subject, such as to decrease the number of exhausted B cells by at least two- or three-fold as compared to the nun1ber prior to administration of the isolated population of allogeneic mesenchymal stem cells. in other embodiments, the number of exhausted B cells as compared to the number of such B cells prior to administration of the isolated population of allogeneic mesenchymal stem cells decreases by at least 10%, 15%, 20%, 25%i, 30%, 35%, 40%, 45%, or 50%1.

[00080] In some embodiments, the therapeutically effective amount of the isolated population of allogeneic mesenchymal stem cells is sufficient to decrease the number of early activated T-cel!s (CD3‘, CD69+) in a subject, such as to decrease the number of earlyactivated T-cells by at least two- or three-fold as compared to the number prior to administration of the isolated population of allogeneic mesenchymal stem cells. In other embodiments, the number of early activated T-cells as compared to the number of such T cells prior to administration of the isolated population of allogeneic mesenchymal stem cells decreases by at least lO%, 20%, 30%, 40%, or 50%.

[00081] In some embodiments, the therapeutically effective amount of the isolated population of allogeneic mesenchymal stem cells is sufficient to decrease the number of chronic activated T-cells (CD3', CD25') in a subject, such as to decrease the nwnber of chronic activated T-cells by at least two- or three-fold as compared to the number prior to administration of the isolated population of allogeneic mesenchymal stem cells. In other embodiments, the number of chronic activated T-cells as compared to the number of such T cells prior to administration of the isolated population of allogeneic mesenchymal stern cells decreases by at least 10%, 15%, 20%, 25%, 30%, 35%), 40%, 45%, 50%), 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[00082] In some embodiments, the therapeutically effective amow1t of the isolated population of allogeneic mesenchymal stem cells is sufficient to decrease the number of Temra cells (CD45RA+, CCRT) in a subject, such as to decrease the number of Temra cells by at least two- or three-fold as compared to the number prior to adrninistration of the isolated population of allogeneic mesenchymal stem cells. In other embodiments, the number of Temra cells as compared to the number of such Temra cells prior to administration of the isolated population of allogeneic mesenchymal stem cells decreases by at least 10%, 20%, 30%, 40%, or 50%.

[00083]     In some embodiments, the therapeutically effective amount of the isolatedpopulation of allogeneic mesenchymal stem cells is sufficient to decrease the TNF-a concentration in a sample of the subject's serum, such as to decrease the TNF-a concentration by at least two- or three-fold as compared to the TNF-a. concentration prior to administration of the isolated population of allogeneic mesenchymal stem cells. In other embodiments, the concentration of TNF-a decreases by at least l 0%, 20%, 30%), 40%, 50%, 60%, 70%, 80%, or 90%, as compared to the concentration of TNF-a prior to administration of the isolated population of allogeneic mesenchymal stem cells.

[00084] Other aspects of the invention include methods for evaluating the cellular and humoral immunity status in subjects to determine if the subjects \Vould benefit from the administration of an isolated population of allogeneic mesenchymal stem cells a.nd treating apredetermined subpopulation of subjects based on hiomarker data. For example, one or more assays could be performed to detect a non-ischemic dilated cardiomyopathy marker selected from the group of exhausted B cells (CD19+, CD27‘, Igf), switched memory B cells (CD19\ CD27'1igh, Igff), B-cells expressing intracellular TNF-a, early activated T-ce!ls (CD34, CD69+), chronic activated T-cells (CD31', CD254), Ternra cells (CD45RA4, CCRT), and serum TNF-a. Similarly, one or more assays could be performed to detect an aging frailty marker selected from the group of exhausted B cells (CD19+, CD2T, Igff), switched memory B cells (CDl 9\ CD27hig\ lgD), B-ce!ls expressing intracellular TNF-a, early activated T-cells (CD3+, CD69+), chronic activated T-cells (CD3+, CD25+), Ternra cells (CD45RA+, CCRT), the CD4+:CD8+ T cell ratio, and serum TNF-a.

[00085] In some embodiments, treatment is demonstrated to be efficacious \Vhen the isolated population of allogeneic mesenchymal stem cells is sufficient to increase the ratio of CD4+ :CD8+ T cells in a subject, such as to increase the ratio of CD4+ :CD8+ T cells by at least two-, three, four-, five-, or six-fold as compared to the ratio prior to administration of the isolated population of al!ogeneic mesenchyma! stem cells, i.e .. an increase in the ratio of CD4+:CD8+ T cells by at least l 00%, 2(00%, 300%, 400%, or 500%.^0008<6]     In some embodiments, treatment is demonstrated to be efficacious when theisolated population of allogeneic mesenchymal stem cells is sufficient to increase the number of switched memory B cells (CD19\ CD(7hig\ Igff) in a subject, such as to increase the number of switched memory- B cells by at least two-, three-, four-, or five-fold as compared to the number prior to administration of the isolated population of allogeneic mesenchymal stem cells, i.e., an increase in the number of switched memory B cells (CDl 9\ CD(iigh' Igff) by at least l 00%, 2OO%i>, 30(0%, or 4(00%. In other embodiments, the number of switched memory B cells as compared to the number of such B cells prior to administration of the isolated population of allogeneic mesenchymal stem cells increases by at least l 0%, 15%1, (0%1, (5%, 30(%, 35%i, (O%i, (5%, 50%, 55%, 60%, 65%;, 70%;, 75%1, 80%1, 85%, 90%, or 95%.

[00087]     In some embodiments, treatment is demonstrated to be efficacious when, theisolated population of allogeneic mesenchymal stem cells is sufficient to decrease the number of B cells expressing intracellular TNF-ci. in a subject, such as to decrease the number by at least two-, three-, four-, five-, or six-fold as compared to the number of such B cells poor to administration of the isolated population of allogeneic mesenchymal stem cells. ln other embodiments, the nwnher of B cells expressing intracellular TNF-a as compared to thenumber of such B cells prior to administration of the isolated population of allogeneic mesenchymal stem cells decreases by at least lO%, 20%, 30%, 40%, 50%, or 6)0%.

[00088] In some embodiments, treatment is demonstrated to be efficacious when the isolated population of a!logeneic mesenchymal stem cells is sufficient to decrease the nwnber of exhausted B cells (CDJ 9\ CD2T, Tgff) in a subject such as to decremse the number of exhausted B cells by at least two- or three-fold as compared to the number prior to adrninistration of the isolated population of allogeneic mesenchymal stem cells. In other embodiments, the number of exhausted B cells as compared to the number of such B cells prior to adrninistration of the isolated population of allogeneic mesenchymal stem cells decreases by at least 10%, 15%), 20%, 25%, 30%, 35%, 40%), 45%, or 50%.

[00089] In some embodiments, treatment is demonstrated to be efficacious vvhen the isolated population of allogeneic mesenchymal stem cells is sufficient to decrease the number of early activated T-cells (CDJ+, CD69+) in a subject, such as to decrease the number of early activated T-cells by at least two- or three-fold as compared to the number prior to administration of the isolated population of allogeneic mesenchy mal stem cells. In other embodiments, the number of early activated T-cells as compared to the number of such T cells prior to administration of the isolated population of allogeneic mesenchymal stem cells decreases by at least 10%, 20%, 30%, 40%, or 50%.

[00090] In some embodiments, treatment is demonstrated to be efficacious when the isolated population of allogeneic mesenchymal stem cells is sufficient to decrease the number of chronic activated T-cells (CD3+, CD25+) in a subject, such as to decrease the number of chronic activated T-cells by at least tvvo- or three-fold as compared to the number prior to administration of the isolated population of allogeneic mesenchymal stem cells. In other embodiments, the number of chronic activated T-cells as compared to the number of such T cells prior to administration of the isolated population of allogeneic mesenchymal stem cells decreases by at least 10(%, 15(1 / o, 20%, 25%, 30%, 35%i, 40%, 4Y%, 5O%i, 55%, 6O%i, 65%, 7O%i, 75%, 80%, 85%, 90%1, or 95%i.

[00091] In some embodiments, treatment is demonstrated to be efficacious when, the isolated population of allogeneic mesenchymal stem cells is sufficient to decrease the number of Temra cells (CD45RA1, CCRT) in a subject, such as to decrease the number of Temra cells by at least two- or three-fold as compared to the number prior to administration of the isolated population of allogeneic mesenchymal stem cells. In other embodiments, the number of Temra cells as compared to the number of such Temra cells prior to administration of theisolated population of allogeneic mesenchyma! stem cells decrea<;es by at least 10%1, 20%, 30%, 40%, or 50%.

[00092] In some embodiments, treatment is demonstrated to be efficacious vvhen the isolated population of allogeneic mesenchymal stem cells is sufficient to decrease the TNF-a concentration in a sample of the subject's serurn, such as to decrease the TNF-a concentration by at least two- or three-fold as compared to the TNF-a concentration prior to administration of the isolated population of allogeneic mesenchymal stem cells. In other embodiments, the concentration of TNF-a decreases by at least 10%, 20%, 30%i, 40%, 50%, 60%, 70%, 80%, or 90%, as compared to the concentration of TNF-u prior to administration of the isolated population of allogeneic mesenchymal stem cells.

[00093] "Administering" a composition may be accomplished by oral administration, injection, infusion, parenteral, intravenous, mucosal, sublingual, intramuscular, intradennal, intranasal, intraperitoneal. intraarterial, subcutaneous absorption or by any method in combination with other knmvn techniques. ln one embodiment of the invention, the isolated population of allogeneic mesenchy mal stem cells is administered systemically. In another embodiment of the invention, the isolated population of allogeneic rnesenchymal stem cells is administered by infusion or direct injection. ln one embodiment of the invention, the isolated population of allogeneic mesenchymal stem cells is administered intramuscularly. intravenously, intraarteerial!y, intraperitoneally, subcutaneously, intradermally, orally, transendocardially, or intra.nasally. in a further embodiment, the isolated population of allogeneic mesenchymal stem cells is administered intramuscularly. In a further embodiment, the isolated population of a!logeneic mesenchymal stem cells is administered intravenously.

[00094] The term "subject" as used herein includes, but is not limited to, humans and non-human vertebrates such as wild, domestic, and farm animals. ln some embodiments, the term refers to humans, such as elderly humans 2:65 years of age, or elderly humans 60-95 years of age. In some embodiments, the human subject exhibits symptoms of aging frailty. In some embodiments, the human subject exhibits intlammaging.[ 00095] The term "allogeneic" refers to a cell that is of the same animal species but genetically different in one or more genetic loci as the animal that becomes the "recipient host"' This usually applies to cells transplanted from one animal to another non-identical animal of the same species.

[00096] As used herein, the phrase "in need thereof" means that the subject has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the subject can be in need thereof. In some embodiments, the subject is in an environment or will be traveling to an environment in which a particular disease, disorder, or condition is prevalent

[00097] Cells are referred to herein as being positive or negative for certain markers. For example, a cell can be negative for CD45, which can also be referred to as CD45‘. The superscript notation "" refers to a cell that is negative for the marker linked to the superscript In contrast a marker with the “+” refers to a cell that is positive for that marker. For example, a cell that is referenced as “CD8+’- is positive for CDS. A •'+" can also be used to reference the marker as positive. A "-" can also be used to reference the i:narker as negative.

[00098] As used herein, the term "stem cell" refers to a cell from the embryo, fetus, or adult that has, under certain conditions, the ability to reproduce itself for long periods or, in the case of adult stem cells, throughout the life of the organism. It also can give rise to specialized cells that make up the tissues and organs of the body.

[00099] Mesenchymal stem cells are the formative pluripotent blast cells found inter alia in bone marrow, blood, dermis, and periosteum that are capable of difforentiating into any kind of the specific types of mesenchymal or connective tissues (i.e, the tissues of the body that support the specialized elements; particularly adipose, osseous, cartilaginous, elastic, and fibrous connective tissues) depending upon various influences from bioactive factors, such as cytokines.[000100] Certain methods of isolating and / or purifying mesenchymal stem cells have been described herein and are known in the mi. ln some embodiments, mesenchymal stem cells are isolated from bone marrow of adult hwnans. In some embodiments, the cells are passed through a density gradient to eliminate undesired cell types. The cells can be plated and cultured in appropriate media. In some embodiments, the cells are cultured for at least one day or about three to about seven days, and removing non-adherent cells. The adherent cells can then be plated and expanded.[000101] Other methods for isolating and culturing stem cells are also known. Placenta is an excellent readily available source for mesenchymal stem cells. Moreover, mesenchymal stem cells can be derivable from adipose tissue and bone marrmv stromal cells are speculatedto be present in other tissues. While there are dramatic qualitative and quantitative differences in the organs from which adult stem cells can be derived, the initial differences between the cells may be relatively superficial and balanced by the similar range of plasticity they exhibit.[000 l 02] Homogeneous human mesenchymal stem cell compositions are provided which serve as the progenitors for all mesenchymal cell lineages. Mesenchymal stem cells are identified by specific cell surface markers which are identified with unique monoclonal antibodies. The homogeneous mesenchymal stem cell compositions are obtained by positive selection of adherent marrow or periosteal cells which are free of markers associated with either hematopoietic or differentiated mesenchymal cells. These isolated mesenchymal cell populations display epitopic characteristics associated '.vith only mesenchymal stem cells, have the ability to regenerate in culture without differentiating, and have the ability to differentiate into specific mesenchymal lineages when either induced in vitro or placed in vivo at a site of inflammation.[ 000103] In order to obtain the human mesenchymal stem cells for the compositions, methods, and kits disclosed herein, pluripotent mesenchymal stem cells are separated from other cells in the bone marrow or other mesenchymal stem cell source. Bone marrow cells may be obtained from iliac crest, femora, tibiae, spine, rib, or other medulla!),; spaces. Other spaces of human mesenchymal stem cells include embryonic yolk sac, placenta, wnbilical cord, fetal and adolescent skin, and blood.[000104] In some embodiments, the human mesenchymal stem cells are identified by the absence of markers. For example, human mesenchymal stem cells useful in the invention include those that are negative for STRO-l and / or negative for CD45. Similarly, human mesenchymal stem cells useful in the invention include those that do not express fibroblast surface markers or have a fibroblast morphology.Methods of Enhancing Immw1e Responses[000105] As discussed above, the present invention is directed to a method of enhancing a subject's cellular or humoral immune response, comprising administering to the subject therapeutically effective amounts of an isolated population of human mesenchymal stem cells. In some embodiments of the invention, the mesenchyrnal stem cells are not genetically manipulated. In some embodiments of the invention, the mesenchymal stem cells are obtained from a human donor and wherein a step of MHC matching of the human donor tothe subject is not employed prior to the administration of the isolated population of human mesenchyrnal stem cells.[000106] Compositions for use in the invention may be fonnulated using any suitable method. Fommlation of cells with standard pharmaceutically acceptable carriers and / or excipients may be carried out using routine methods in the pharmaceutical art Tiie exact nature of a formulation will depend upon several factors including the cells to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.[000107] Compositions may be prepared together with a physiologically acceptable carrier or diluent. Typically, such compositions are prepared as liquid suspensions of cells. The cells may be mixed with an excipieni which is phannaceuiically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, of the like and combinations thereof[000108]     In addition, if desired, the pharmaceutical compositions of the invention maycontain minor an10unts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and / or adjuvants which enhance effectiveness. In one embodiment of the invention, the adjuvant comprises human serum albumin (HSA).[000109] One suitable carrier or diluent is PlasmaLyte ATM_ This is a sterile, nonpyrogenic isotonic solution for intravenous administration. Each J 00 mL contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg of Sodium Gluconate (C6H11Na0--); 368 mg of Sodium Acetate Trihydrate, USP (C2H,Na021H20); 37 mg of Potassium Chloride, USP (KC!): and 30 mg of Magnesium Chloride, USP (MgCh6H20). H contains no antimicrobial agents. The pH is adjusted with sodium hydroxide. 'The pH is 7.4 (6.5 to 8.0) .[000110] In one embodiment of the invention the mesenchymal stem cells are not genetically manipulated. In another embodiment of the invention, the mesenchymal stem cells are cryopreserved. For example, the mesenchymal stem cells can be suspended in cryoprotectant consisting of Hespan@ (6% hetastarch in 0.9% sodium chloride) supplemented with 2% HSA and 5% DMSO and then aliquoted into cryopreservation containers for placement in vapor phase nitrogen freezers. In another embodiment, the mesenchymal stem cells may be provided in PlasrnaLyte ATM supplemented with l % HSA.EXAMPLESExample 1Treatment of Chronic Non-Ischemic Dilated Cardiomyopathy[000111] Briefly, thirty-seven patients were randomized to receive either allogeneic or autologous human mesenchymal stem cells in a 1: 1 ratio. Patients were recruited between December 2011 and July 201.5 at the University of Miami Hospital. Patients (age: 55.8 ,l: 11.2: 32%) female) received human mesenchymal stem cells (100 million) by transendocardial stem cell injection (TESl) in ten left ventricular sites by NOGA Catheter. Treated patients were evaluated at baseline, 30 days, 3-, 6- and 12-months for immw1e biornarkers.Patient Population[000112] Patient eligibility \Vas determined after confirmation of diagnosis of NIDCM with an EF of less than 40% and either a left ventricular end diastolic diameter greater than 5.9 cm in male subjects and greater than 5.6 cm in female subjects or a left ventricular end diastolic volume index > 125 mL / nl. Thirty-seven patients were randomized to either auto!ogous human mesenchyme stem cells (hMSCs) or allogeneic hMSCs in a ratio of 1.1. Thirty-frmr patients received study injection; 16 and 18 patients in the auto- and allo-hMSC, respectively. Three patients did not receive the study injection. One patient withdrew consent before treatment. .Another patient was recruited but did not receive treatment due to automatic implantable cardioverter-defibrillator placement (1Fl), and one patient died before treatment (n l), The mean age of injected participants was 55.8 ± 11.2, 29% were female, and 35% were Hispanic. The mean years ofNIDCM diagnosis before the TESI was 6.1 ± 6.2 years for al!o and 6.9 ± 7.3 years for auto patients (p 0.5 between groups). Fifty percent of patients had NYHA class n symptoms, mean baseline global EF was 26.5 ± 9.64%, mean 6M\VT was 422 ± 86.8 M, and median baseline MLHFQ scores were 36 (IQR 18.0, 64,0). Study Procedures and Timeline[000113] Baseline assessments included chemistry and hematology laboratories, echocardiography, and chest, abdominal and pelvic computed tomography scans.Human Mesenchymal Stem Cells (hMSCs) for Cell Therapy[000114] All allogeneic and autologous human mesenchymal stem cells '.Vere manufactured at the University of Miami ISCI. See, e.g. Golpanian, S. et al.. Physiol. Rev. 96:1127-68 (2016) and Mushtaq, M. et al., J Cardio. Trans. Res. 7:769-80 (2014). Allogeneic human mesenchymal stem cells '.Vere derived from Caucasian male donors mean age 25.4 ± 3.3 years and ,vere between 80 to 90%; viable at the time of TESL The auto-hMSCs were from 11 males with a mean age of 58.0 ± 9.9 and six females with a mean age of 55.0 i 12.4 years.Transendocardial Stem Cell Injection[000115] Injection sites were selected to prioritize safety of the TESI procedure and to distribute sites throughout the accessible myocardial territories. Considerations for site selection included avoidance of the ventricular apex, and optimization of catheter stability prior to needle extension.Immune Monitoring[000116] Calculated panel reactive antibodies (cPRA) were measured at baseline and at six-months using Luminex 200. Serum TNF-a, was measured using human TNF-a ELISA high sensitivity kit (eBiosciences). Lymphocytes were stained for T-cell markers of activation. Late / exhausted T-cells, B-cell subsets (Switched memory and Late / Exhausted B-cells) and TNF-a by B-cells. All samples were acquired using the LSR-Fortessa-HTS analyzer (BD Phamiigen) and analyzed with the FlowJo VlO software, i.e., all lymphocyte populations v,-ere assessed by flow cytometry and expressed as a percent from gated (B-cell parameters gated on CD191 and T-cell parameters gated on CD3\[000117] cPRA results showed that 67% of allo and 92% of auto recipients had no reaction to low cPRA (0-20% cPRA). Twenty-seven % of allo and 8%i of auto had a moderate cPRA (21-79(% cPRA), and one subject (7%;) receiving allo MSCs had a high cPRA response (+80% cPRA).[000118] Elevated baseline levels of TNF-(1 decreased from baseline to 6-months in both groups (allo: -10.6 ± 1.6 pg / ml, p<0.0001, auto: -6.8 ± 1.4 pg / ml, p<0.0001, Table 1; between group, p 0.05). Ternra T-cells (exhausted T-cell phenotype) were reduced in both groups \vith a greater decrease in allo (allo: -15.9±5.4%, p<0.0001, auto: -9.3±3.3%, p<0.0001, Table l; between group, p 0.Cll11; Table 1). Suppressed %Switch Memory B-cells (predictive biomarker for antibody response) at baseline were significantly increased at six-months in both groups with a greater improvement in allo (allo: +!0.2±4.9%, p<0.0001 vs. auto: +4.3±3.9%, p 0.0014, between group, p<0.0001; Table 1). Finally, intracellular TNF-a expression in B23 cells \Vas also decreased at 6-months relative to baseline in both groups (allo: -l l.2±3.3%, p<0.0001, auto: -8.5+3.0%, p<0.0001; between group, p 0.174; Table 1). In contrast Late / exhausted B-cells decreased significantly in both groups (allo: -5.4±1.03%, p<0.0001, auto: -5.9±2S%, p 0.003; between group difference, p 0.57; Table 1). Early T-cell activation (al!o: -5.57±1.03%, p<0.0001, auto: -2.92±1S%, p 0.02; betweengroup, p 0.08; Table 1) decreased to similar degrees in both group. Whereas !ate / chronic T-cell activation did not significantly decrease in either group (allo: -2.3,.±:J .3%, P 0.4, and auto: -3.4±2.1%, p 0.7).Table 1. Effects of Human Mesenchymal Stem Cells on Cellular and Humoral Immunity at 6-months.Immune Biomarkers AHogenek Autologous Baseline 6-Months Baseline 6-Months Serum TNF-a (pg / ml) 13.5 ± 15 2.3 ± 0.2 "".t 11.8±0.9 4.8 ± 0.8'” %Early T-cell Activation (CD3\ CD69+) 13.4±4.4 7.9± :Lf" 13.3 ± 4.8 10.6 ± 5_.f %Late / Chronic T-cen activation (CD3\ 0125^ 8.9, IQR (5.9, 10.4) 3.4, IQR (3.0, 5.0)* 8.1, lQR (5.2, 14.1) 4.4, lQR (3.6, 11.1) % Temra (CDJ\ Cll45RA7, CCRT) 33.7 J 7.6 J 7.94c 5.2 ) 30.1 ,+, 10.8 2 l.3 ,+, 7.5 '” %Late / Exhausted B-cells (CD19\ CD2T, Igff) 19.3, IQR (17.9, 28.6) 14.7, IQR (14.2, 17.3)44' 1 20.0, IQR (17 . 7 ‘    • 0) 15.5, IQR (14.0, 19.0)* %Switched Memory B-ceHs (ClH9\ CJ)27hig\ lgff) 10.0 ± 3.6 20.2 ± 3.f"n 9.6± 3.1 14. l ± 4.5' % B-ceUs expressing intracellular TNF'-a 32.l ± 7.3 20.4 ± 6.:f' 28.2±6.8 19.7±4.8 ” ’ indicates p .:::: 0.05 wnhm group; "" mdicates p .:::: 0.001 within group; t indicates between group p< 0.5; H indicates between group p::::; 0.001 .Example 2Treatment of Symptoms of Aging Frailty Study l[000119] Thirty subjects aged 60 years of age or older who met the study inclusion / exclusion criteria for frailty by the Canadian Study of Health and Aging (CSHA) were randomized. Ten subjects received 100 million MSCs, ten subjects received 200 million MSCs, and ten subjects received placebo. Subjects were followed for one year for safety and efficacy. Assessments included at least the following: a study of inflammatory' biomarkers and a review of immunologic effects. FIG. 1 is a Consort Diagram. FIG. 2 provides baseline patient characteristics. FIGs. 3-7 illustrate the impact of allogeneic human MSCs on immune biomarkers.[000120] Figure 3 shows a decrease TNF-a in subjects administered human MSCs. Both early and late / chronic T cell activation decreases after allogeneic MSC treatment. FIG. 4 shows a decrease in early activated T cells expressing CD69 in subjects administered human MSCs. FIG. 5 shows a decrease in the numbers of chronic / late activated T cells expressing CD25 in subjects administered human MSCs. FIG. 7 shows an increase in the ratio of CD4+ / CD8+ T cells in subjects administered human MSCs.Example 3Treatment of Symptoms of Aging Frailty Study 2[000121] Fifteen aged subjects were included in an open-label study and administered human mesenchymal stem cells in two separate infusions. Subjects \Vere evaluated for changes in various immune biomarkers to determine a response to the injected hwnan rnesenchymal stem cells. These immune biomarkers included the levels of exhausted B cells (CD19\ CD2T, Igf), the levels of switched memory-' B cells (CD19\ CD27high, Igf), the levels of B-cells expressing intracellular TNF-a, the levels of early activated T-cells (CD3\ CD69'), the levels of chronic activated T-cells (CD31, CD25\ the levels of Temra cells (CD45RA\ CCRT), the CD4+:CD8+ T cell ratio, and the TNF-a concentration in serum. Subjects were evaluated 6 months after the first injection and 1, 3, and 6 months after the second injection. Provided in Tables 2-9 are the measured values for each of the immune biomarkers discussed above. Also provided in Tables 2-9 are Scores assigned to each of the immune biomarkers assessed in each patient at different time-points that indicate whether an increase or decrease was observed in the imrmme biomarker measured. Finally, in Table 10 are the total Scores for each patient that includes different time-points as well as averages among the patients. As can be observed in the last row' of Table 10 and in FIG. 8, the immunosenescence Score improves in subjects with aging frailty after the first injection ofhuman mesenchymal stem cells and continues to stay in an improved state after a second injection of human mesenchymal stem cells.Table 2. Effects of Human Mesenchymal Stem Cells on Switched Memory B CellsPatien t# BL {i Mo 2"ri inject .BL 2nd Inject .     1- Mo. 2nd Inject .    3- Mo. 2"d Inject .     6- Mo. BL Scor e 6- Mo. Scor e 2nd Inject . BL Score 2nd Inject: .      1- Mo. Score 2"d inject .    3- Mo. Score 2nd inject .     6- Mo. Score 1 6.6 1 2l. 2 14.2 16.5 22.8 21.3 l 2 2 2 2 2 ,. ;. 21. 3 41 249 24 25.4 30.8 2 4 3 3 3 3 3 5,3 4 9.3 1 7.14 l6.6 7.58 9.8[ l l l 2 1 l 4 10. 5 l7 30. l 36.6 187 40.3 l 2 3 4 2 2 5 10. 24 25. 33.3 28.8 31.5 1 3 3 3 3 3 6 7.5 1 14. 6 1 ,'7 / 14.7 l9.7 ]9.8 1 2 2 ,. ,.., ., L 2 7 7.3 2 20. — ]2,3 13.3' 2l2 27.l l 2 2 2 2 2 8 7.6 9 9.8 9 8.31 11.7 26.4 21.5 l i 1 2 2 2 9 10. 1 17. 8 158 15.3 25.8 35.9 1 2 " L 2 3 2 rn 2l. "" ! 35. 3 23. l 23.6 23.8 25.7 ,. ,.., 4 ., L 3 3 3 15 9.2 5 13. 5 22.3 28.l 28 l 29.7 l 2 2 3 3 3 Improvement Scale: No Change O; Improved »<l fold l; Improved >1<2 fold 2; Improved >2::;3 fold 3; Improved >3 fold 4; Worsened >0:::.:1 fold -1: Worsened >l::;2 fold -2; Worsened >2::;3 fold -3; and Worsened >3 fold -4.Table 3. Effects of Human Mesenchymal Stem Cells on ° / 4,B-CeHs ExpressingIntracellular TNI<'-aPatien t# BL 6- Mo 2nd Inject .BL 2"cl Inject .        1-- Mo. 2"rl Inject .     3- Mo. 2nd Inject .    6 Mo. BL Scor e 6- Mo. Scor e 2"'! Inject . BL Score 2"rl Inject . l-Mo. Score 2nd Inject .     3 Mo. Score 2nd Inject .       6- Mo. Score 1 L,,..,J. 4 9.6 5 15.6 9.59 5.35 8.21 1 3 2 2 4 3 2 34. 8 9.3 3 10 9.83 6.88 6.71 l 3 2 3 3 3 3 l4. '.-i 7.6 2 40.4 21.2 l5.7 8.02 2 3 1 2 3 -, 1 4 !4. 3 7.6 3 13.7 12 5 25 3.63 2 2 2 2 4 4 5 19. 5 10. 9 9.52 8.89 8.33 7.59 2 2 ,; L 3 3 3 6 ..,..., ' ! . ..., ! L2 2 5.57 4 4.04 4.33 i 4 4 4 4 4 7 13. 2 2.7 1 41 3.67 4.42 2.52 2 4 4 4 4 4 8 30. s 4.6 6 6.82 7.01 7.05 7.42 l 4 3 3 3 3 9 11. 1 6.9 4 11.2 5.76 7.79 5. l 2 3 2 4 3 4 10 l3. 4.2 3.55 3.87 3.72 3.42 2 4 4 4 4 4 15 7.0 4 .',7 5 5.75 3.84 6.46 2.04 3 4 4 4 4 4 Table 4. Effects of Human Mesenchymal Stem Cells on Exhausted B CellsPatien t# BL 6- Mo 2"rl Inject .BL 2"rl Inject .     1- Mo. ,,ml ;. Inject .    3- Mo. 2"'! Inject .     6- Mo. BL Scor e 6- Me. Scor e znd Inject . BL Score ,,ml ;. Inject .      1- Mo. Score 2"'! Inject .     3- l\fo. Score 2"rl Inject .     6- Mo. Score 1 23. ,; L 40. 4 15.7 9.88 10.7 15.6 l l ., L 3 3 2 2 26. s 2'A 6 6.59 3.33 6.67 7.5 l l l 4 4 4 4 Patien t# BL 6- Mo 2nd Inject .BL 2"cl Inject .     1- Mo. 2"rl Inject .      3-- Mo. 2nd Inject .     6- Mo. BL Scor e 6- Mo. Scor e 2"'! Inject . BL Score 2"rl Inject . l- Mo. Score 2nd Inject .     3- Mo. Score 2nd Inject .      6- Mo. Score 3 76 3 18 2 15.7 15.7 9.01 14.2 4 2 2 2 3 2 1J 29. 1 14. 8 11.9 7.12 9.86 lO.l l 2 3 4 3 ,.1, 5 '.-i 15. 2 ]9.2 8.39 l3.6 l2.9 l 2 2 4 2 -, 1 6 34. 6 14. l 9.47 9.09 8.01 5.26 l 2 3 3 4 4 7 44. 1 51. 9 66 5 57.2 43.1 40.3 1 1 1 l 1 1 8 9.2 3 7.l 12.6 i 1.6 8.05 12 3 4 3 3 4 3 9 15. 4 n . 8 15.6 7.85 9.51 17.8 2 2 2 4 3 2 10 12. 24 6 717 11.9 13.4 742 2 2 4 3 '1 L 4 15 32. 4 26. 6 24.4 16.2 ll.7 l2.4 l i 2 2 3 ,., 1 Table 5. Effects of Human Mesenchymal Stem Cells on Temra CellsPatien t# BL 6- Mo 2nd inject .BL 2nd Inject .     1- Mo. znd Inject' .     3- Mo. znd Inject .     6- Mo. BL Scor e 6-- Mo. Scor e 2nd Inject . BL Score znd Inject .      1- Mo. Score 2nd inject .     3- Mo. Score 2nd Inject .     6- Mo. Score 1 30. 1 42. 6 ]4.6 17.4 l9.4 15.4 l l 2 2 1 2 2 25. 6 L"7, . s 25 2 -,7 ,., / 26.4 37.1 1 1 1 l 1 1 3 7.5 2.8 14.4 13.3 10.l l l.8 3 4 ., L 2 2 2 4 15. -, ' 3.0 6 [5 l2.7 9.5 12.5 2 4 2 2 '.-i 2 Patien t# BL 6- Mo 2nd Inject .BL 2"cl Inject .        1-- Mo. 2"rl Inject .     3- Mo. 2nd Inject .     6- Mo. BL Scor e 6- Mo. Scor e 2"'! Inject . BL Score 2"rl Inject .      l- Mo. Score 2nd Inject .     3- Mo. Score 2nd Inject .     6- Mo. Score 5 10 2.8 9.86 8.43 11 10.4 2 4 2 3 ,; L,; 2 6 39. 18. L., 21.6 16.4 20 18.1 1 2 1 ,..,') l 2 7 l3. '.-i 14. 2 l l.4 10.3 [5 l7.7 2 2 2 2 2 2 8 31. 12. 2 118 7.09 l l.6 33,3 l 2 2 3 2 l 9 16. 1 11. 1 24 25.1 34.3 38.8 2 2 1 l 1 1 rn 7.1 4.8 9 12.2 19 9.52 13.2 3 4 ., L 2 3 2 15 15. 2 13. '.-i 30.2 40.6 46 l 34.9 2 2 l l 1 i Table 6. Effeds of I-Imnan MesenchymaJ Stem Cells on Eady Activation of T Cells(Cll69)Patien t# BL 6- Mo znd inject .BL znd Inject .     1- Mo. znd Inject .    3- Mo. znd Inject .    6- Mo. BL Scor e 6- Mo. Scor e znd Inject . BL Score znd Inject' .      1- Mo. Score znd inject .    3- Mo. Score ZUd Inject .     6- Mo. Score 1 l6 16. 6 ]2.98 8.03 8.74 6.07 2 2 2 3 3 4 2 5.8 7.5 10.3l 13.82 l0.77 12.21 4 4 3 2 3 2 3 29 13 14.76 3.36 4.83 9.l4 1 2 2 4 4 ,., 1 4 65 30 7,87 5 4.33 17.07 l l 3 4 4 2 5 4.4 11. 7 13.15 3.42 20.88 6.75 4 2 '1 L 4 2 4 6 42 22. 4 13.23 13.56 8.6 13.29 l 2 ., L 2 3 2 7 25. 8 22. 1 l3.73 10.26 10.87 37.35 l 2 2 3 '.-i 1 8 28. 3 17 . 1 422 5.1 4.93 12.32 l 2 4 4 4 2 9 4.4 6.2 20.12 7.63 [2.06 11.9 4 4 2 3 ., L 2 rn 19. 4 1' / ', 9 14.18 [3.53 6.22 13.85 2 2 2 2 4 2 15 10. 9 6.l 8 14. [8 12.79 142 13.1 3 4 2 2 2 2 Table 7. Effects of Human MesenchymaJ Stem Cells on Late / Chronic Activation of TCells (Cll25)Patien t# BL 6- Mo znd ’ inject .BL zml Inject .     1- Mo. znrl Inject' .    3- Mo. znd Inject .    6- Mo. BL Scor e 6- Mo. Scor e znd Inject . BL Score znrl Inject' .     1- Mo. Score znd inject .    3- Mo. Score zml Inject .     6- Mo. Score 1 1 ,I, 2.9 5.74 8.31 l0.98 7.63 4 4 '.-i 2 2 2 2 2.2 3 425 4.69 3.84 4 .',7 4 4 3 3 4 3 3 20 5 10.83 4.93 14.32 5.87 l ,., 1 L., 3 1 3 4 53 3.2 4.54 5.88 7.44 6.97 l 4 '.-i 3 2 2 5 10 1.9 5.69 7.9 7.91 8.9 2 4 3 2 2 2 Patien t# BL 6- Mo 2nd Inject .BL 2"cl Inject .     1- Mo. 2"rl Inject .     3- Mo, 2nd Inject .    6- Mo. BL Scor e 6- Mo. Scor e 2"'! Inject . BL Score 2"rl Inject .      l- Mo. Score 2nd Inject .     3- Mo. Score 2nd Inject .       6-- Mo. Score 6 16 1.4 569 6.95 6.69 2 53 l 4 3 2 2 4 7 0.9 2.3 7.35 8.38 0r .ry / rI 8.19 4 4 ., L, 2 2 2 8 20. 5 3.7 6.81 9.41 l0.74 4.26 l 4 2 2 2 3 9 2.1 13 3 4.47 3.88 3.82 4 4 4 3 4 4 10 6.6 2.9 l 1.61 5.29 3.88 5.08 2 4 2 3 4 -, •' 15 1.4 l.l 1 11.61 2.2] 3.14 2.11 4 4 2 4 4 4 Table 8. Effects of Human Mesenchymal Stem Cells on Ratio of CD4+:CD8r T CellsPatien t# BL Rati 0 6- Mo. Rati 0 2"cl Inject . BL Ratio ,,ml ;. Inject .     1- Mo. Ratio 2nd Inject .    3- Mo. Ratio znd Inject .    6- Mo. Ratio BL Scor e 6- Mo. Srnr e ,,ml ;. Inject . BL Score 2nd Inject .     1- Mo. Score znd Inject .    3- Mo. Score 2"rl Inject .    6- Mo. Score 1 n_,. . / ,.,I, 0.69 0.48 1.03 0.68 0.66 i l l l l i 2 2.l7 2.48 0.98 1.16 1.48 ().96 2 2 1 1 2 l 3 1 66 1.94 2.24 1.95 2.98 2.32 2 2 2 2 3 2 4 0.96 3.43 6.49 2.l8 3.67 4.59 i 3 4 2 3 4 5 2.34 5.32 5.05 4,27 445 3.79 2 4 4 4 4 3 6 0 94 3.47 4.38 3.54 3.50 4.42 l 3 4 2 3 4 7 0.51 1.60 0.95 0.92 0.92 0.97 l 2 l l l l 8 0.83 2.26 2.49 2.49 2 58 021 l 2 2 ') L 2 l 9 l.57 2.68 i.56 2.48 1.25 0.67 2 2 ') ,_ 2 1 l 10 3.02 4.76 3.97 3.26 4.66 2.65 3 4 3 3 4 2 15 0.68 1.94 0.84 0.62 047 110 1 " L l 1 1 1 Table 9. Effects of Human Mesenchymal Stem Cells on Serum TNF-a ConcentrationPatient# BL 6--Mo. 6-Mo. Score* 1 3.5 2.4 2 2 3.6 2 ,., ., , 3 2.8 L. 3 2 Patient# BL 6-Mo. 6-1\fo, Score 4 2.8 0.9 2 5 8 6.8 0 6 .., ., _1, ,L, u 2 7 3.5 () J 4 2 8 ,..,I .J.., 0 2 9 6.8 4.1 2 10 5.7 2J{ 2 11 6.3 2.7 2 12 4.l 2.;.,., ., ,., 13 3.5 () 6 2 H 9.2 3.8 2 15 6.3 l.2 2 Table 10. Total Immunosenescence Scores of PatientsPatient# BL 6-Mo. zml Jn,ject. BL 2nd Inject. 1-Mo. Score 2nd Inject. 3-Mo. Score 2nd Inject. 60-Mo. Score 1 11 14 14 15 16 16 2 15 19 17 17 20 17 3 14 17 12 17 17 16 4 9 18 20 2] 21 21 5 ]4 21 18 23 18 20 6 7 19 19 17 19 ...,..., Ld., 7 12 17 14 15 15 14 8 9 19 17 19 19 16 9 17 19 15 19 17 18 10 ]6 24 19 20 24 20 15 15 19 14 17 18 18 Average 12.64 18.73 16.27 18.18 18.55 18

Claims

WHAT IS CLAIMED IS:

1. A method oftreating non-ischemic dilated cardiornyopathy in a subject, comprising administering a therapeutically effective amount ofa population ofisolated allogeneic human mesenchymal stem cells to a suqject in need thereof: wherein the nwnber ofexhausted B cells (CDJ. 9\ CD27'. Igf) in a sample ofthe subject's serum decreases by at least 25% as compared to the number of exhausted B cells in a sample of the subject's serum prior to administration of said population of isolated allogeneic human mesenchymal stem cells, thereby treating said non-ischemic dilated cardiomyopathy.

2. A method oftreating non-ischemic dilated cardiornyopathy in a subject, comprising administering a therapeutically effective amount ofa population ofisolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number ofS'.vitched rnemory B cells (CD19\ CD27high' Igff) in a sample ofihe subject's serum increases by at least 100% as compared to the number of switched memory B cells in a sample of the subject's serum prior to administration of said population of isolated allogeneic human mesenchymal stem cells, thereby treating said non-ischemic dilated cardiomyopathy.

3. A method of treating non-ischemic dilated cardiornyopathy in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number ofB-cells expressing intracellular TNF-a in a sample of the subject's serum decreases by at least 30(1 / o as cornpared to the number of B-cells expressing intracellular TNF-a. in a sample ofthe subject's serum prior to administration of said population of isolated allogeneic human mesenchymal stem cells, thereby treating said non-ischemic dilated cardiomyopathy.

4. A method of treating non-ischemic dilated cardiornyopathy in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number ofearly activated T-cells (CD3+, CD69+) in a sample of the subject's serwn decreases by at least 30% as compared to the number of early activated T-cells in a sarnple ofthe subject's serum prior to administration of said population of isolated allogeneic human mesenchymal stem cells, thereby treating said non-ischemic dilated cardiomyopathy.

5. A method oftreating non-ischemic dilated cardiornyopathy in a subject, comprising administering a therapeutically effective amount ofa population ofisolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number ofchronic activated T-cells (CD3+, CD25+) in a sample of the subject's serwn decreases by at least 70% ascompared to the number of chronic activated T-Cells in a sample of the subject's serum prior to administration of said population of isolated allogeneic human mesenchymal stem cells, thereby treating said non-ischemic dilated cardiomyopathy.

6. A method of treating non-ischemic dilated cardiomyopathy in a subject, comprising administering a therapeutically effective arnmmt of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number of Temra cells (CD45llA+, CCRT) in a sample of the subject's serum decreases by at least 40% as compared to the number of Temra cells in a sample of the subject's serwn prior to administration of said population of isolated allogeneic human mesenchymal stem cells, thereby treating said non-ischemic dilated cardiomyopathy.

7. A method of treating non-ischemic dilated cardiomyopathy in a subject, comprising administering a therapeutically effective arnmmt of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the TNF-a, concentration in a sample of the subj ecf s serum decreases by at least 80% as compared to the TNF-a concentration in a sample of the subject's serum prior to administration of said population of isolated allogeneic human mesenchymal stem cells, thereby treating said non-ischemic dilated cardiomyopathy .

8. A method of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effoctive a.mount of a population of isolated allogeneic human mesenchyma! stem cells to a subject in need thereof, wherein the number of exhausted B cells (CD l 9\ CD2T Igf f) in a sample of the subject' serum decreases by at least 10% as compared to the number of exhausted B cells in a sample of the subject's serum prior to administration of said population of isolated allogeneic human mesenchymal stem cells, thereby treating said symptoms of aging frailty.

9. A method of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effoctive amount of a population of isolated allogeneic human mesenchyma! stem cells to a subject in need thereof wherein the number of switched memmy B cells (CD19\ CD2711ig\ lgff) in a sample of the subject's serum increases by at least 75% as compared to the number of switched memory B cells in a sample of the subject's serwn prior to administration of said population of isolated allogeneic human mesenchymal stem cells, thereby treating said symptoms of aging frailty.

10. A method of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effoctive amount of a population of isolated allogeneic human mesenchyma!stem cells to a subject in need thereof, wherein the number ofB-cells expressing intracellular TNF-a in a sample of the subject's semm decreases by at least 60% as compared to the number of B-cells expressing intracellular TNF-a in a sample of the subject’s serum prior to administration ofsaid population ofisolated allogeneic hwrn:m mesenchy mal stem cells, thereby treating said symptoms of aging frailty ,11. A method of treating symptoms of aging frailty in a subject comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number of early activated T-cells (CD31, CD694) in a sample of the subject's serum decreases by at least 30% as compared to the number of early activated T-cells in a sample of the subject's serum prior to administration of said population ofisolated allogeneic human mesenchymal stem cells, thereby treating said symptoms of aging frailty.

12. A method of treating symptoms of aging frailty in a subject comprising administering a therapeutically effective amount ofa population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number ofchronic activated T-cells (CD3\ CD25+) in a sample of the subject's serum decreases by at least 75% as compared to the number of chronic activated T-Cells in a sample of the subject's serum prior to administration ofsaid population ofisolated allogeneic human mesenchymal stem cells, thereby treating said symptoms of aging frailty.

13. A method of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the number ofTernra cells (CD45RA+, CCRT ) in a sample of the subject's senmi decreases by at least 20% as compared to the number of Temra cells in a sample of the subject’s serum priorto administration of said population of isolated allogeneic human mesenchymal stem cells, thereby treating said symptoms of aging frailty.

14. A method of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchymal stem cells to a subject in need thereof, wherein the TNF-a concentration in a sample of the subject's serum decreases by at least 50% as compared to the TNF-a concentration in a sample of the subject's serum prior to administration of said population ofisolated allogeneic human mesenchymal stem cells, thereby treating said symptoms ofaging frailty.

15. A method of treating symptoms of aging frailty in a subject, comprising administering a therapeutically effective amount of a population of isolated allogeneic human mesenchyrnal stem cells to a subject in need thereof, wherein the CD4+:CD8+ T cell ratio in a sample of the subject's serum increases by at least 100% as compared to the CD4':CD8' T cell ratio in a sample of the subject's serum prior to administration of said population of isolated allogeneic human mesenchymal stem cells, thereby treating said symptoms of aging frailty.

16.    The method of any one of claims 1-7, wherein the subject is a human.

17.    The method of any one of claims 8-15, wherein the subject exhibits inflammaging.

18.    The method of any one of claims 1-17, vvherein the mesenchymal stem cells are bonemarrow-derived mesenchymal stem cells.

19. The method of any one of claims 1-18, \Vherein the mesenchymal stem cells do not express STRO-J.

20. The method of any one of claims 1-8, wherein the mesenchymal stem cells do not express CD45.

21. The method of any one of claims 1-20, wherein the mesenchymal stem cells do not express fibroblast surface markers or have a fibroblast morphology.

22. The method of any one of claims 1-21, wherein the mesenchymal stem cells are not genetically manipulated.

23. The method of any one of claims 1-22, wherein the isolated population of allogeneic rnesenchymal stem cells is administered in a single dose.

24. The method of any one of claims 1-22, wherein the isolated population of allogeneic mesenchymal stem cells is administered in two or more doses.

25. The method of any one of clairns 1-22, wherein the isolated population of allogeneic mesenchymal stem cells is administered at least yearly.

26. The method of any one of claims 1-25, wherein the isolated population of allogeneic mesenchymal stem cells is administered systemically.

27. The method of any one of claims l-26, wherein the isolated population of allogeneic-mesenchymal stem cells is administered by infusion or direct injection.

28. The method of any one of claims 1-27, wherein the isolated population of allogeneic mesenchyrnal stem cells is administered intravenously, intraarterially, or intraperitoneally.

29. The method of claim 28, wherein the isolated population of allogeneic mesenchymal stem cells is administered intravenously.

30. The method of any one of claims 1-25, wherein the isolated population of allogeneic mesenchymal stem cells is administered intramuscularly, intravenously, intraarterially, intraperitoneally, subcutaneously, intradermally, orally, tranendocardially, or intranasally.

31. The method of claim 30, wherein the isolated population of allogeneic mesenchymal stem cells is administered intramuscularly.

32. The method of any one of claims 1-31, wherein the isolated population of allogeneic mesenchymal stem cells is administered at a dose of about 20xl06 mesenchymal stem cells.

33. The method of any one of claims 1-31, wherein the isolated population of allogeneic mesenchyrnal stem cells is adrninistered at a dose of about 1OOx106 mesenchymal stem cells.

34. The method of any one of claims 1-31, wherein the isolated population of allogeneic mesenchymal stem cells is administered at a dose of about 200xl cf mesenchymal stem cells.

35. The method of any one of claims l -34, wherein the isolated population of allogeneic mesenchymal stem cells are obtained from a human donor and wherein a step of MHC matching of the human donor to the subject is not employed prior to the administration of the isolated population of al!ogeneic mesenchymal stem cells to the subject.

36. A method of evaluating cellular and humoral immunity status in a subject compnsmg:(1) obtaining a serum sample from a subject selected for evaluation based on a determination that the subject ,vas previously in need of treatment of non-ischemic dilated cardiomyopathy and said subject had been administered an initial dose of an isolated population of allogeneic human mesenchymal stem cells:(2) performing one or more assays configured to detect a non-ischemic dilated cardiomyopathy marker selected from the group of exhausted B cells (CD191, CD2T, Igf f), switched memory B cells (CD19\ CD27hig\ IgD"), B-cells expressing intracellular TNF-a, early activated T-cells (CD3\ CD69+), chronic activated T-cells (CD3\ CD25+), Temra cells (CD45RA\ CCRT), and serwn TNF-(l by introducing the serum sample obtained from the subject into an assay instnmient which (i) contacts the serum sample with one or more antibodies which specifically bind for detection the biomarker(s) which are assayed, and (ii) generates one or more assay results indicating of binding of each biomarker which is assayed to a respective antibody to provide one or more assay results;(3) correlating the assay result(s) generated by the assay instrument to the immunity status of the subject, wherein said correlating step comprises assigning a likelihood of one or more future changes in immune status to the subject based on the assay result(s); and(4) treating the subject based on the predetermined subpopulation of individuals to which the subject is assigned, wherein the treatment comprises administration of one or more additional doses of an isolated population of allogeneic human mesenchymal stem cells.

37. A method of evaluating cellular and humoral immunity status in a subject, compnsmg:(1) obtaining a serum sample from a subject selected for evaluation based on a determination that the subject was previously in need of treatment of symptoms of aging frailty and said subject had been administered an initial dose of an isolated population of allogeneic hwnan mesenchymal stern cells;(2) performing one or more assays configured to detect aging frailty marker selected from the group of exhausted B cells (CD19+, CD2T, Igf), switched memory B cells (CD1 9\ CD2itig\ Igf), B-cells expressing intracellular TNF-a, early activated T-cells (CD3\ CD69\ chronic activated T-cells (CD3\ CD25+), Temra cells (CD45RA\ CCRT), the CD4+:CD8+ T cell ratio, and serum TNF-a by introducing the serum sample obtained from the subject into an assay instrument which (i) contacts the serum sample v,'ith one or more antibodies which specifically bind for detection the biomarker(s) which are assayed, and (ii) generates one or more assay results indicating of binding of each biomarker which is assayed to a respective antibody to provide one or more assay results;(3) correlating the assay resu!t(s) generated by the assay instrument to the immw1ity status ofihe subject, wherein said correlating step comprises assigning a likelihood of one or more future changes in immune status to the subject based on the assay result(s): and(4) treating the subject based on the predetermined subpopulation of individuals to which the subject is assigned, wherein the treatment comprises administration of one or more additional doses of an isolated population of allogeneic human mesenchymal stem cells.

38. The method of claims 36 or 37, wherein said one or more future changes in immune status comprise one or more of an increase in the number of exhausted B cells (CD19\ CD2T, Igff), a decrease in the nwnber of switched memory B cells (CDJ 9\ CD27hig\ Igff), an increase in the number of B-cells expressing intracellular TNF-a, an increase in the number of early activated T-cells (CD3\ CD69'), an increase in the number of chronic activated T-cells (CD3‘, CD25\ an increase in the number of Temra cells (CD45RA\ CCRT), a decrease in the CD4+ :CD8+ T cell ratio, and an increase in serum TNF-a.

39. An in vitro method of determining efficacy of treatment of non-ischemic dilated cardiomyopathy in a subject comprising: determining the levels of one or more biornarkers selected from the group consisting of exhausted B cells (CD19\ CD2T, Igf f), Switched memory B cells (CD19+, CD27high' Igff), B-cells expressing intracellular TNF-u, early activated T-cells (CD3\ CD69+), chronic activated T-cells (CD31', CD25\ Ternra cells (CD45RA+, CCRT), and the TNF-a concentration in serum obtained from the subject before and after administration of a population of isolated allogeneic human mesenchymal stem cells to the subject, and comparing the levels of the one or more biomarkers in the serum obtained before and after administration of the population of isolated human mesenchymal stem cells, wherein treatment is efficacious if(l) the number of exhausted B cells (CD19+, CD2T, Igf) decreases by at least 25% as cornpared to the number of exhausted B cells prior to administration of said population of isolated allogeneic human mesenchymal stem cells,(2) the number of s,vitched memory B cells (CD19+, CD2iig\ Igff) increases by at least 10()(% as compared to the number of switched memory B cells prior to administration of said population of isolated allogeneic human mesenchymal stem cells,(3) the number of B-cells expressing intracellular TNF-a decreases by at least 30% as compared to the number of B-cells expressing intracellular TNF-u prior to administration of said population of isolated allogeneic human mesenchymal stem cells,(4) the nwnber of early activatedT-cells (CD3\ CD69+) decreases by at least JO();o as compared to the number of early activated T-cells prior to administration of said population of isolated allogeneic human mesenchymal stem cells,(5) the number of chronic activated T-cells (CD3‘, CD25') decreases by at least 70%-) as compared to the number of chronic activated T-Cells prior to administration of said population of isolated allogeneic human mesenchymal stem cells,(6) the nunber ofTemra cells (CD45RA\ CCRT) decreases by at least 40%; as compared to the number ofTemra cells prior to administration of said population of isolated allogeneic human mesenchymal stem cells, and / or(7) the TNF-a concentration in a sample of the subject's serum decreases by at least 80% as compared to the TNF-a concentration in a sample of the subject's serun prior to administration of said population of isolated allogeneic human mesenchymal stem cells.

40. .An in vitro method of determining efficacy of treatment of symptoms of aging frailty in a subject comprising: determining the levels of one or more hiomarkers selected from thegroup consisting of exhausted B cells (CD19\ CD2T, Igff), switched memory B cells (CDl 9\ CD27hig\ igff), B-cells expressing intracellular TNF-a, early activated T-cells (CD3+, CD69+), chronic activated T-cells (CD3\ CD25+), Temra cells (CD45RA\ CCRT), the CD4+:CD8+ T cell ratio, and the TNF-u concentration in serum obtained from the subject before and after administration of a population of isolated aliogeneic human mesenchymal stem cells to the subject, and comparing the levels of the one or more biomarkers in the serum obtained before and after administration of the population of isolated human mesenchymal stem cells, wherein treatment is efficacious if(l) the number of exhausted B cells (CD1 9\ CD2T\ Igff) decreases by at least 10)% as compared to the number of exhausted B cells prior to administration of said population of isolated allogeneic human mesenchymal stem cells,(2) the number of switched memory B cells (CDl91, CD27hig\ igff) increases by at least 75% as compared to the number of switched memory B cells prior to administration of said population of isolated allogeneic human mesenchymal stem cells,(3) the number of B-cells expressing intracellular TNF-(1e decreases by at least 60%1 as compared to the number of B-cells expressing intracellular TNF-a prior to administration of said population of isolated allogeneic human mesenchymal stem cells,(4) the number of early activated T-cells (CD3+, CD69+) decreases by at least 30%i as compared to the number of early activated T-cells prior to administration of said population of isolated allogeneic hlmHm mesenchymal stem cells,(5) the number of chronic activated T-cells (CD3+, CD25+) decreases by at least 75% as compared to the number of chronic activated T-Cells prior to administration of said population of isolated allogeneic human mesenchymal stem cells,(0) the number of Temra cells (CD45RA\ CCRT) decreases by at least 20% as compared to the number of Temra cells prior to administration of said population of isolated a!logeneic human mesenchymal stem cells,(7) the TNF-a concentration in a sample of the subject's serum decreases by at least 50% as compared to the TNF-a concentration in a sample of the subject's serum prior to adrninistration of said population of isolated allogeneic human mesenchymal stem cells, and / or(8) the CD4+:CD8+ T cell ratio in a sample of the subject's sernm increases by at least 100 °ii as compared to the CD4+:CD8+ T cell ratio in a sample of the subject's serumprior to administration of said population of isolated allogeneic human mesenchymal stem cells.

41. Use in vitro of the levels of exhausted B cells (CD19\ CD2T, lgD} the levels of switched memory B cells (CD19\ CD2?1'ig\ Igf), the levels of B-cells expressing intracellular TNF-u, the levels of early activated T-cells (CD3\ CD69'), the levels of chronic activated T-cells (CD3\ CD25+), the levels of Temra cells (CD45RA', CCRT), and the TNF-a concentration in serum for detem1ining \Vhether treatment for non-ischemic dilated cardiomyopathy is efficacious.

42. Use in vitro of the levels of exhausted B cells (CD l 94', CD2T, IgD} the levels of switched memory B cells (CD19+, CD27hlg\ Igf), the levels of B-cells expressing intracellular TNF-u, the levels of early activated T-cells (CD3+, CD69+), the levels of chronic activated T-cells (CD3\ CD25+), the levels of Temra cells (CD45RA’, CCRT), the CD4+ :CD8+ T cell ratio, and the TNF-u concentration in serum for determining whether treatment for aging frailty is efficacious.Assessed for eligibility (n=38)Excluded (n=8):• 4 patients withdrew consent® 1 was excluded for malignancy® 3 did not meet inclusion criteriaI Randomized and Infused (n~38)Allogeneic hMSCs - 1X1Q8 cells | ® n=10 administered study product \ c No infusions stopped o No persistent cardiorespiratory signs / symptoms following infusionAllogeneic hMSCs - 2X108 cells« n=10 administered study product o No infusions stopped o No persistent cardiorespiratory signs / symptoms following infusionPlacebo® n=10 administered study producto No infusions stoppedo No persistent cardiorespiratory signs / symptoms following infusion1 / 2410 completed one year follow-up* 0 withdrawn» 0 lost to follow-up* 0 died» 0 pulmonary embolisms® 0 strokes9 completed one year follow-up* 0 withdrawn® 0 lost to follow-up• 1 died of congestive heart failure 327 days post-infusion» 0 pulmonary embolisms« 0 strokesv10 completed one year follow-up® 0 withdrawn® 0 lost to follow-up® 0 died® 0 pulmonary embolisms® 1 experienced a stroke307 davs cost-infusion

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