Methods and compositions for modulating peripheral immune function

Abstract

To provide cell populations useful for modulating various peripheral immune functions, methods for preparing the cell populations, and methods for their use.SOLUTION: The invention provides cell populations useful for modulating various peripheral immune functions, preparation of the cell populations, and their use.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 61 / 516,637, filed April 6, 2011, and U.S. Provisional Patent Application No. 61 / 541,248, filed September 30, 2011, the disclosures of which are incorporated by reference in their entirety herein for all purposes.

[0002] Statement regarding government support Not applicable.

[0003] The present disclosure is in the field of immunomodulation (eg, immunosuppression). [Background technology]

[0004] Peripheral (i.e., non-CNS) immunity in vertebrates is mediated by two systems: the innate immune system and the adaptive immune system. The innate immune system provides an early, non-specific response to injury and / or infection, whereas the adaptive immune system is utilized later in the course of injury or infection and is specific for invading antigens. The adaptive immune system is active only in vertebrates, while the more evolutionarily older innate immune system is active in plants, invertebrates and vertebrates.

[0005] As mentioned above, the innate immune system becomes active at the site of infection immediately after infection and does not depend on prior exposure to an infectious agent. It therefore provides a set of general defense mechanisms that are not specific to any particular pathogen. Cellular elements of the innate immune system include macrophages, dendritic cells, neutrophils and natural killer (NK) cells. Macromolecular components of the innate immune system include defensin peptides and the complement system. Further elements of innate immunity include physical barriers to infection (e.g., tight junctions between keratinized skin, epithelial cells, gastric acid and mucus secreted by many epithelial tissues) and cell-intrinsic responses, such as phagocytosis (possibly with lysosomal fusion of phagocytosed material) and degradation of double-stranded RNA.

[0006] Activation of the innate immune system is mediated, in part, by the recognition of pathogen-associated molecules, such as N-formylmethionine-containing polypeptides, cell wall peptidoglycans, bacterial flagella, lipopolysaccharides, teichoic acids, and fungal-specific molecules, such as mannans, glucans, and chitin. Also, certain nucleic acid sequences common to microorganisms (e.g., unmethylated CpG dinucleotides) can trigger an innate immune response. Recognition of such pathogen-associated immune stimuli leads to increased inflammatory responses and phagocytosis of pathogens by macrophages, neutrophils, and / or dendritic cells.

[0007] The specific pathogen-associated immune stimulants mentioned above occur in repeating patterns called pathogen-associated molecular patterns (PAMPs) that can be recognized by pattern recognition receptors on the surface of cells of the innate immune system. These receptors include soluble members of the complement system and membrane-bound receptors, such as members of the Toll-like receptor family (TLRs) and the so-called NOD proteins. Membrane-bound receptors can stimulate phagocytosis and activate gene expression programs involved in a variety of innate and adaptive immune responses.

[0008] Finally, the innate immune system is responsible, in part, for activating adaptive immunity by secreting extracellular signaling molecules that stimulate the proliferation and differentiation of cells of the adaptive immune system, and by processing and presenting antigens to cells of the adaptive immune system.

[0009] The adaptive immune system, in contrast to the innate immune system, is not activated immediately after infection and produces a specific, long-term response against pathogens. Activation of the adaptive immune system occurs in lymphoid organs, not at the site of injury, and depends on the presentation of antigens by components of the innate immune system to activate cells of the adaptive immune system. The primary cells of the adaptive immune system are B lymphocytes (B cells) and T lymphocytes (T cells), which synthesize and secrete antibodies.

[0010] There are three major classes of T cells: cytotoxic, helper and regulatory T cells. Cytotoxic T cells are capable of destroying infected host cells. Helper T cells are involved in the activation of macrophages, dendritic cells, B cells and cytotoxic T cells by secreting cytokines and / or by surface expression of one of a number of different co-stimulatory molecules. There are two types of helper T cells: T H T cells are involved in the activation of macrophages, cytotoxic T cells, and B cells to provide immunity against intracellular pathogens, and secrete the macrophage-activating cytokines interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). H 1 cells can also stimulate inflammatory responses. H T helper T cells promote the activation of B cells to produce antibodies, primarily in response to extracellular pathogens, and secrete the cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10). H Growth of T helper cells into IL-1 cells is stimulated by interleukin-12 (IL-12); whereas pathogen-induced expression of Jagged protein by dendritic cells induces naive helper T cells to produce IL-4. H Regulatory T cells (T cells) give rise to T cells that stimulate antibody production by B cells. reg s) inhibit the function of cytotoxic T cells, helper T cells, and dendritic cells, and are unique in their expression of the Foxp3 transcription factor. Thus, the interaction between helper T cells and regulatory T cells helps to keep the immune response in balance, with sufficient activity to eliminate invading pathogens without causing excessive damage to the host.

[0011] A type of lymphocyte in the adaptive immune system known as memory cells retains receptors for pathogens after infection and clearance, allowing for a more rapid adaptive immune response to subsequent exposure to the same pathogen, and providing the basis for natural or vaccination-induced immunity to many infectious diseases. In contrast, the innate immune system does not possess such immunological memory.

[0012] Mesenchymal stem cells (MSCs, also known as "bone marrow stem cells" or "marrow adherent stem cells") transfected with a plasmid expressing the Notch intracellular domain (NICD) are useful for the treatment of numerous diseases and disorders of the central and peripheral nervous system. See, e.g., U.S. Patent No. 7,682,825 (March 23, 2010); U.S. Patent Application Publication No. 2006 / 0216276 (September 28, 2006); U.S. Patent Application Publication No. 2010 / 0034790 (February 11, 2010); U.S. Patent Application Publication No. 2010 / 0310523 (December 9, 2010); WO 08 / 102460 (August 28, 2008); Yasuhara et al., (2009) Stem Cells and Development 18: 1501-1513 and Glavaski-Joksimovic et al., (2009) Cell Transplantation 18: 801-814.

[0013] The ability of these cells (known as SB623 cells) to rescue damaged neural tissue is associated, in part, with their secretion of various trophic factors and their assimilation of various extracellular matrix components (see, e.g., U.S. Patent Application Publication No. 2010 / 0266554 (October 21, 2010) and U.S. Patent Application Publication No. 2010 / 0310529 (December 9, 2010).

[0014] Current cell transplantation therapies have significant disadvantages, including, for example, host peripheral immune responses against transplanted cells. Inflammation is also a hallmark of many neurodegenerative diseases, such as Parkinson's disease and multiple sclerosis. See Villoslada et al. (2008) Clin. Immunol. 128: 294-305. MSCs have been reported to reduce peripheral immune activity through mechanisms including inhibiting the production of antigen-presenting cells and altering the cytokine profile of helper T cells. See Kong et al. (2009) J. Neuroimmunol. 207: 83-91. However, MSCs have limited regenerative potential and exhibit senescence following ex vivo manipulation. See Wagner et al. (2008) PLoS One 3: e2213; Jin et al. (2010) Biochem Biophys Res Commun. 391: 1471-1476. Although senescent cells secrete a number of cytokines that may be beneficial for tissue regeneration, the general senescent cell secretion profile is proinflammatory. See Rodier et al. (2009) Nature Cell Biol. 11: 973-979; Coppe et al. (2008) PLoS Biol. 6: 2853-2868; Freund et al. (2010) Trends Mol. Med. 16(5): 238-246.

[0015] For these and other reasons, there remains a need for methods and compositions for cell transplantation that do not induce a host peripheral immune response and / or reduce inflammation and other immune responses. Summary of the Invention [Means for solving the problem]

[0016] The present inventors have identified a population of senescent cells in cultures of MSCs and their progeny (i.e., SB623 cells) transfected with sequences encoding the Notch intracellular domain. Although SB623 cells have been shown to be capable of treating a number of central nervous system disorders, the present application discloses the surprising ability of SB623 to regulate a number of peripheral immune functions. For example, SB623 cells can inhibit the proliferation of human T cells in both allogeneic and xenogeneic mixed lymphocyte reactions, stimulate IL-10 production by T cells, and block the differentiation of monocytes into dendritic cells. SB623 cells also inhibit dendritic cell maturation compared to parental MSCs, and SB623 cells confer greater inhibition to dendritic cell maturation as evidenced by a greater reduction in surface expression of the costimulatory molecule, CD86. SB623 cells can also alter the cytokine profile of T cell populations from pro-inflammatory to anti-inflammatory. These properties of SB623 cells are even more surprising and unexpected in light of studies reporting that senescent cells secrete proinflammatory cytokines (see Orjalo et al. (2009) Proc. Natl. Acad. Sci. USA 106: 17031-17036).

[0017] Thus, SB623 cells, and / or senescent cell subpopulations thereof, are useful in a number of therapeutic methods, as exemplified in the embodiments below.

[0018] 1. A method for peripheral immune suppression in a subject, the method comprising administering to the subject an effective amount of SB623 cells; wherein the SB623 cells are obtained by: (a) providing a culture of mesenchymal stem cells; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length of a Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection.

[0019] 2. A method for inhibiting a peripheral inflammatory response in a subject, the method comprising administering to the subject an effective amount of SB623 cells; wherein the SB623 cells are obtained by: (a) providing a culture of mesenchymal stem cells; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length of a Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection.

[0020] 3. The method of embodiment 2, wherein said peripheral inflammatory response results from allogeneic transplantation, ischemia or necrosis.

[0021] 4. A method for suppressing peripheral T cell activation in a subject; the method comprising administering to the subject an effective amount of SB623 cells; wherein the SB623 cells are obtained by: (a) providing a culture of mesenchymal stem cells; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length of a Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection.

[0022] 5. The method of embodiment 4, wherein said peripheral T cell activation comprises expression of CD69 and / or HLA-DR by said T cells.

[0023] 6. The peripheral T cell activation is characterized by the CD4 + 5. The method of embodiment 4, comprising propagation of the

[0024] 7. A method for suppressing peripheral helper T cell function in a subject; the method comprising administering to the subject an effective amount of SB623 cells; wherein the SB623 cells are obtained by: (a) providing a culture of mesenchymal stem cells; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length of a Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection.

[0025] 8. The method of embodiment 7, wherein said peripheral helper T cell function is cytokine secretion.

[0026] 9. The method of embodiment 7, wherein said peripheral helper T cell function is associated with rheumatoid arthritis pathology.

[0027] 10. Peripheral regulatory T cells (T reg 16. A method for expanding a population of SB623 cells (s) comprising administering to the subject an effective amount of SB623 cells; wherein the SB623 cells are obtained by: (a) providing a culture of mesenchymal stem cells; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full-length Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection.

[0028] 11. A method for regulating peripheral production of a cytokine in a subject; the method comprising administering to the subject an effective amount of SB623 cells; wherein the SB623 cells are obtained by: (a) providing a culture of mesenchymal stem cells; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length of a Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection.

[0029] 12. The method of embodiment 11, wherein the cytokine is a proinflammatory cytokine and the production of the cytokine is reduced.

[0030] 13. The method of embodiment 12, wherein the cytokine is produced by a T cell.

[0031] 14. The method of embodiment 13, wherein the cytokine is interferon-gamma (IFN-γ).

[0032] 15. The method of embodiment 12, wherein the cytokine is produced by monocytes.

[0033] 16. The method according to embodiment 15, wherein the cytokine is tumor necrosis factor-alpha (TNF-α).

[0034] 17. The method of embodiment 11, wherein the cytokine is an anti-inflammatory cytokine and the production of the cytokine is stimulated.

[0035] 18. The method of embodiment 17, wherein the cytokine is interleukin-10 (IL-10).

[0036] 19. The method of embodiment 18, wherein the cytokine is produced by a T cell or a monocyte.

[0037] 20. The method of embodiment 19, wherein the T cells are helper T cells.

[0038] 21. The helper T cells are T H 21. The method of embodiment 20, wherein the cell is a single cell.

[0039] 22. The method of embodiment 19, wherein the cytokine is produced by a regulatory T cell.

[0040] 23. The regulatory T cell is T R 23. The method of embodiment 22, wherein the cell is a single cell.

[0041] 24. A method for inhibiting differentiation of peripheral monocytes into dendritic cells in a subject; the method comprising administering to the subject an effective amount of SB623 cells; wherein the SB623 cells are obtained by: (a) providing a culture of mesenchymal stem cells; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length of a Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection.

[0042] 25. A method for inhibiting maturation of peripheral dendritic cells in a subject; the method comprising administering to the subject an effective amount of SB623 cells; wherein the SB623 cells are obtained by: (a) providing a culture of mesenchymal stem cells; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length of a Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection.

[0043] 26. The method of embodiment 25, wherein said maturation comprises increased expression of CD86 by the dendritic cells.

[0044] 27. A method for treating graft versus host disease (GVHD) in a subject; the method comprises administering to the subject an effective amount of SB623 cells; wherein the SB623 cells are obtained by: (a) providing a culture of mesenchymal stem cells; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length of a Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection.

[0045] 28. A method for inhibiting transplant rejection in a subject; the method comprising administering to the subject an effective amount of SB623 cells; wherein the SB623 cells are obtained by: (a) providing a culture of mesenchymal stem cells; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full-length Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection.

[0046] 29. A method for treating a peripheral autoimmune disease in a subject; the method comprises administering to the subject an effective amount of SB623 cells; wherein the SB623 cells are obtained by: (a) providing a culture of mesenchymal stem cells; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode the full length of a Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection.

[0047] 30. The method of embodiment 29, wherein the peripheral autoimmune disease is selected from the group consisting of multiple sclerosis, ulcerative colitis, chronic obstructive pulmonary disease (COPD), asthma, lupus and type 1 diabetes.

[0048] 31. The method of any one of embodiments 1-30, wherein the subject is an experimental animal.

[0049] 32. The method of any one of embodiments 1-30, wherein the subject is a human. [Brief description of the drawings]

[0050] [Figure 1] FIG. 1 shows the measurement of CFSE dilution in MSCs and SB623 cells by flow cytometry. [Diagram 2] FIG. 2 shows the change in cell number in cultures of MSCs and SB623 cells as determined by trypan blue exclusion after 3 days in culture. [Diagram 3] Figure 3 shows the results of propidium iodide staining of cultures of MSCs and S623 cells (Figures 3A and 3B). Figure 3A shows representative FACS data. The "M1" labeled peak indicates resting (G0 / G1) cells. Figure 3B shows the percentage of cells in the resting phase of the cell cycle for MSCs and SB623 cells, as determined by measuring the area of ​​the M1 peak in Figure 3A. [Figure 4] FIG. 4 shows the measurement of p16Ink4A levels in MSCs and SB623 cells. [Diagram 5] FIG. 5 shows the levels of certain surface markers in MSCs and SB623 cells. [Figure 6] FIG. 6 shows the measurement of CD54 expression in MSCs and SB623 cells. [Figure 7] FIG. 7 shows the levels of certain cytokines in MSCs and SB623 cells. [Figure 8] FIG. 8 shows the levels of transforming growth factor beta-1 (TGF-β-1) and vascular endothelial growth factor-A (VEGF-A) in MSCs and SB623 cells. [Figure 9] Figure 9 shows the effect of SB623 cells and MSCs on T cell activation in an allogeneic MLR (mixed lymphocyte reaction) (Figures 9A and 9B). Figure 9A shows representative FACS traces gated with CFSE and phycoerythrin-labeled anti-CD69 for unstimulated human T cells (control) (top left panel, indicated by "-"); human T cells stimulated with allogeneic PBMC (top right panel, indicated by "MLR"); a previously described MLR with 104 MSCs (bottom left panel, indicated by "MLR+MSC") and a previously described MLR with 104 SB623 cells (bottom right panel, indicated by "MLR+SB623"). Figure 9B shows quantification of CD69 expression in MLR cultures. Unstimulated T cell cultures (control) are indicated with "serum"; PBMC-stimulated T cells in a mixed lymphocyte reaction are indicated with "MLR"; a mixed lymphocyte reaction as previously described in the presence of mesenchymal stem cells is indicated with "MSC"; and a mixed lymphocyte reaction as previously described in the presence of SB623 cells is indicated with "SB623". "MSC" and "SB623" values ​​are the average of three cultures each containing MSC or SB623 cells from different donors. [Figure 10] FIG. 10 shows a comparison of T cell proliferation rates in allogeneic MLRs, quantified by measuring CFSE dilution (FIGS. 10A and 10B). FIG. 10A shows representative FACS traces of unstimulated human T cells (control) (top left panel, labeled "T cells alone"); human T cells stimulated with allogeneic PBMCs (top right panel, labeled "MLR"); an MLR as previously described with 104 MSCs (bottom left panel, labeled "MLR+MSC") and an MLR as previously described with 104 SB623 cells (bottom right panel, labeled "MLR+SB623"). FIG. 10B shows quantification of CSFE dilution in MLR cultures. Culture composition is as shown in FIG. 10A. [Figure 11]Figure 11 shows a comparison of HLA-DR expression under different culture conditions. Unstimulated T cell cultures (control) are represented by "serum"; PBMC-stimulated T cells in mixed lymphocyte reaction are represented by "MLR"; mixed lymphocyte reaction in the presence of mesenchymal stem cells is represented by "MSC"; and mixed lymphocyte reaction in the presence of SB623 cells is represented by "SB623". "MSC" and "SB623" values ​​are the average of three cultures containing MSC or SB623, respectively, from different donors. [Figure 12] Figure 12 shows the effect of SB623 cells and MSCs on T cell proliferation in a heterologous lymphocyte stimulation response. Proliferation was measured by dilution of PKH26 (a cell-permeable dye). The percentage of CD32+ T cells containing PKH26 was measured for unstimulated T cells ("T cells alone"); T cells co-cultured with mixed glial cells ("heterologous MLR"); T cells co-cultured with mixed glial cells and mesenchymal stem cells ("heterologous MLR+MSC"), and T cells co-cultured with mixed glial cells and SB623 cells (heterologous MLR+SB623"). Preparations of MSCs and SB623 cells were obtained from three different donors as shown in the figure. [Figure 13] FIG. 13 shows an assay for regulatory T cells (Tregs) in in vitro T cell cultures using co-expression of CD4 and CD25 as markers of Tregs (FIGS. 13A and 13B). FIG. 13A shows representative FACS traces measuring CD4 and CD25 for IL-2 stimulated T cells ("T cells"), IL-2 stimulated T cells co-cultured with mesenchymal stem cells for 7 days ("T cells + MSC"), and IL-2 stimulated T cells co-cultured with SB623 cells for 7 days. FIG. 13B shows the average CD4 / CD25 expression levels of five different corresponding lots of MSC and SB623 cells. For "Donor 1 PBL", a significant increase in CD4+CD25+ cells was observed in co-culture with SB623 cells compared to co-culture with MSC (p<0.05). [Figure 14]Figure 14 shows levels of FoxP3 transcription factor in T cells cultured in the presence of IL-2, stained for intracellular FoxP3 with a PE-conjugated anti-FoxP3 antibody and subsequently analyzed by flow cytometry analysis (Figures 14A and 14B). Figure 14A shows representative FACS traces for T cells cultured in the absence of IL-2 (indicated as "RPMI / 10%FBS"), T cells cultured in 10 ng / ml IL-2 (indicated as "+IL-2"), T cells cultured in the IL-2 as above and co-cultured with MSCs (indicated as "+MSC"), and T cells cultured in the IL-2 as above and co-cultured with SB623 cells (indicated as "+SB623"). Figure 14B shows the average percentage of FoxP3-expressing T cells after culture in the presence of IL-2 ("T cells alone"), after co-culture with MSCs in the presence of IL-2 ("T cells + MSC"), or after co-culture with SB623 cells in the presence of IL-2 ("T cells + SB623"). Co-cultures were performed with three different corresponding lots of MSCs and SB623 cells. [Figure 15] FIG. 15 shows the results of measuring intracellular IL-10 levels in CD4+ T cells cultured in the presence of IL-2 (FIGS. 15A and 15B). FIG. 15A shows representative FACS traces for T cells cultured in the presence of IL-2 ("T cells alone"), T cells cultured in IL-2 as above and co-cultured with MSCs (denoted "T cells+MSC"), and T cells cultured in IL-2 as above and co-cultured with SB623 cells (denoted "T cells+SB623"). Alexa 488 fluorescence, which indicates IL-10 levels, is shown on the abscissa. FIG. 15B shows the average percentage of IL-10 positive cells in co-cultures of T cells with three different corresponding lots of MSCs ("T cells+MSC") and SB623 cells ("T cells+SB623") compared to T cells not co-cultured ("T cells alone"). [Figure 16]FIG. 16 shows cytokine levels in T cells cultured in the absence of IL-2 and in the presence of non-maximally inducing levels of PMA and ionomycin (FIGS. 16A and 16B). In FIG. 16A, levels of interferon-gamma (IFN-γ) are shown for T cells freshly isolated before culture ("fresh cells"); T cells cultured for 7 days in the absence of other cells ("culture control"); T cells co-cultured with SB623 cells for 7 days ("SB623"); and T cells co-cultured with MSCs for 7 days ("MSCs"). In FIG. 16B, levels of interleukin-10 (IL-10) are shown for T cells freshly isolated before culture ("fresh cells"); T cells cultured for 7 days in the absence of other cells ("culture control"); T cells co-cultured with SB623 cells for 7 days ("SB623"); and T cells co-cultured with MSCs for 7 days ("MSCs"). Values ​​for "MSC" and "SB623" are the average of three cultures each containing MSC or SB623 cells from different donors. [Figure 17] Figure 17 shows the levels of IL-17 in IL-23 stimulated T cells. The expression percentage was determined by flow cytometry after staining cells for IL-17 with a fluorescent antibody. T cells were cultured with or without IL-23 as indicated, and alone or in co-culture with MSCs or SB623 cells as indicated. [Figure 18]FIG. 18 shows the levels of CD1a and CD14 in monocyte cultures after 7 days of culture or co-culture (FIGS. 18A and 18B). FIG. 18A shows representative FACS traces of cells stained for CD1A and CD14. Monocytes contain a population of CD1A+CD14+ dendritic cell precursors (leftmost panel). When monocytes are cultured for 7 days in the presence of IL-4 and GM-CSF, this dendritic cell precursor population is reduced and replaced by a population of CD1A+CD14- dendritic cells (second panel from the left). When monocytes are co-cultured with MSCs (third panel from the left) or SB623 cells (rightmost panel) in the presence of IL-4 and GM-CSF, the CD1A+CD14- dendritic cell population is reduced and the CD1A+CD14+ precursor cell population is increased. Figure 18B shows the average expression data for monocytes (leftmost pair of bars), monocytes cultured for 7 days in the presence of IL-4 and GM-CSF (second pair of bars from the left), monocytes co-cultured with MSCs for 7 days in the presence of IL-4 and GM-CSF (third pair of bars from the left), or monocytes co-cultured with SB623 cells for 7 days in the presence of IL-4 and GM-CSF (rightmost pair of bars). Results from co-culture experiments were obtained from three different corresponding lots of MSCs and SB623 cells. [Figure 19]FIG. 19 shows the levels of CD86 expressed as mean fluorescence intensity in TNF-α stimulated monocyte cultures. Cultures designated "control" contain PBMCs cultured for 5 days in the presence of IL-4 and GM-CSF followed by an additional 48 hours in TNF-α. Cultures designated "with CsA" contain PBMCs cultured for 5 days in the presence of IL-4 and GM-CSF followed by an additional 48 hours in TNF-α+1 μg / ml cyclosporine A. Cultures designated "MSC" contain PBMCs cultured for 5 days in the presence of IL-4 and GM-CSF followed by an additional 48 hours in TNF-α+104 MSC. Cultures designated "SB623" contain PBMCs cultured for 5 days in the presence of IL-4 and GM-CSF followed by an additional 48 hours in TNF-α+104 SB623 cells. Results for MSC and SB623 cells are the average of three experiments, each using samples from a different donor. The monocyte donor was the same in all cases. All cultures were initiated with 10 5 PBMC. [Figure 20] Figure 20 shows the measurement of cytokine expression in monocytes (Figures 20A and 20B). Figure 20A shows the percentage of monocytes in culture expressing the proinflammatory cytokine TNF-α. Figure 20B shows the percentage of monocytes in culture expressing the anti-inflammatory cytokine IL-10. Monocytes selected based on surface expression of CD14 were cultured with no supplementation ("negative"), macrophage colony-stimulating factor supplementation ("MCSF"), granulocyte / macrophage colony-stimulating factor supplementation ("GMCSF"), MSC supplementation or SB623 cell supplementation. MSC and SB623 cells were obtained from three different donors, shown in the figures as D52, D55 and D65. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] The practice of the present disclosure will employ, unless otherwise indicated, standard methods and conventional techniques in the fields of cell biology, toxicology, molecular biology, biochemistry, cell culture, immunology, oncology, recombinant DNA, and related fields that are within the skill of the art. Such techniques are described in the literature and are thereby available to those of skill in the art. See, for example, Alberts, B. et al., "Molecular Biology of the Cell," 5 th edition, Garland Science, New York, NY, 2008; Voet, D. et al., "Fundamentals of Biochemistry: Life at the Molecular Level," 3 rd edition, John Wiley & Sons, Hoboken, NJ, 2008; Sambrook, J. et al., "Molecular Cloning: A Laboratory Manual," 3 rd edition, Cold Spring Harbor Laboratory Press, 2001; Ausubel, F. et al., "Current Protocols in Molecular Biology," John Wiley & Sons, New York, 1987 and regularly updated; Freshney, RI, "Culture of Animal Cells: A Manual of Basic Technique," Fifth Edition, Wiley, New York, 2005; and "Methods in Enzymology" series, Academic Press, San Diego, CA. Standard techniques in immunology are described, for example, in "Current Protocols in Immunology," (R. Coico, series editor), Wiley, updated August 2010.

[0052] For the purposes of this disclosure, the term "peripheral" is used to mean parts of the body outside the central nervous system. These include, for example, the bone marrow, the peripheral circulation, and the lymphatic organs.

[0053] Preparation of SB623 cells Mesenchymal stem cells (MSCs) can be obtained by selecting adherent cells from bone marrow, and can be induced to form SB623 cells by expressing Notch intracellular domain (NICD) in the adherent cells. In one embodiment, a culture of MSCs is contacted (e.g., by transfection) with a polynucleotide comprising a sequence encoding NICD, followed by drug selection to enrich and further culture the transfected cells. See, e.g., U.S. Patent No. 7,682,825 (March 23, 2010); U.S. Patent Application Publication No. 2010 / 0266554 (October 21, 2010); and WO 2009 / 023251 (February 19, 2009); the disclosures of all of which are incorporated by reference in their entireties for purposes of describing the isolation of mesenchymal stem cells and their conversion into SB623 cells (referred to in those documents as "neural precursor cells" and "neural regenerating cells"). See also Example 1, below.

[0054] In these methods, any polynucleotide (e.g., vector) encoding the Notch intracellular domain can be used, and any method for selection and enrichment of transfected cells can be used. For example, in certain embodiments, the vector containing the sequence encoding the Notch intracellular domain also contains a sequence encoding a drug resistance marker (e.g., resistance to G418). In these embodiments, selection is achieved by adding to the cell culture after transfection of the cell culture with the vector a selection agent (e.g., G418) in an amount sufficient to kill insufficient cells containing the vector but not cells not containing the vector. The absence of selection requires removal of the selection agent or reduction of its enrichment to a level that does not kill cells not containing the vector.

[0055] Senescence in SB623 cells As described above, SB623 cells are derived from MSCs due to the expression of NICD in cultured MSCs. Since MSCs undergoing manipulation in culture often undergo senescence; the SB623 cells derived therefrom were tested for senescence.

[0056] SB623 cells do not form colonies in soft agar, indicating that they are not transformed cells. Also, when SB623 cells were prelabeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), a cell-permeable dye that is diluted by cell division, a subpopulation of cells retained high concentrations of CFSE after 5 days in culture (Figure 1). This slowly proliferating (or non-proliferating) subpopulation was not observed in MSC cultures. We observed that certain cells in the SB623 cell population also stained intensely for β-galactosidase (a marker of cellular senescence), and such cells were more abundant in SB623 cultures than in MSC cultures. These results are consistent with the presence of a pool of non-proliferating, senescent cells in the SB623 cell population.

[0057] Cell proliferation was measured by seeding one million MSC or SB623 cells and measuring viable cells by trypan blue exclusion after 3 days of culture. Figure 2 shows that more viable cells were observed in MSC cultures, indicating a lower proliferation index for SB623 cells. Cell cycle status was assessed by propidium iodide staining, which showed a larger population of cells in the resting phase (G0 / G1) in SB623 cultures (Figure 3), providing further support for the reduced proliferation rate in SB623 cells.

[0058] Further evaluation of senescence was performed by staining SB623 cell populations for expression of p16Ink4A protein. p16Ink4A inhibits progression from G1 to S phase of the cell cycle and is expressed in senescent cells. Figure 4 shows that a higher percentage of p16Ink4A-expressing cells was detected in cultures of SB623 cells compared to MSCs. Furthermore, when cells in SB623 cultures that retained high CFSE levels after 5 days of culture were tested for p16Ink4A expression, the subpopulation of SB623 cells expressing p16Ink4A coincided with the fraction containing high CFSE levels. Taken together, these results indicate the presence of a subpopulation of senescent cells in SB623 cultures.

[0059] Surface markers and cytokine expression SB623 cells express many surface markers in common with MSCs. These include CD29, CD44, CD73, CD90, CD105, and vascular endothelial cell adhesion molecule-1 (VCAM-1 or CD106). Compared to MSCs, SB623 cells had higher levels of CD44 and CD73 and lower levels of VCAM-1. SB623 cells also express intercellular adhesion molecule-1 (ICAM-1 or CD54), which is not normally expressed in MSCs. See Figures 5 and 6. MSCs and SB623 cells do not express surface markers CD31, CD34, and CD45.

[0060] SB623 cells also secrete many cytokines and trophic factors. The specific identity of these factors was determined by blocking protein secretion with Brefeldin A and testing for intracellular cytokines by antibody staining and flow cytometry. These studies showed that SB623 cells produce interleukin 1 alpha (IL-1 alpha), interleukin-6 (IL-6), granulocyte / macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor-A (VEGF-A), and transforming growth factor beta-1 (TGF beta-1), among other factors. See Figures 7 and 8. The amounts of IL-6 and GM-CSF produced by SB623 cells are generally greater than those produced by MSCs.

[0061] Since senescent cells have been reported to synthesize and secrete certain growth-stimulating cytokines and trophic factors (Orjalo et al., (2009) Proc. Natl. Acad. Sci. USA 106: 17031-17036), the presence of a population of senescent cells within SB623 cultures suggested the usefulness of SB623 cell transplantation to support various types of tissue regeneration. However, the secretory profile of senescent cells has been reported to be proinflammatory, which, if this is the case for SB623 cells, may reduce the usefulness of SB623 cells for cell transplantation therapy.

[0062] Surprisingly, and despite the presence of a population of senescent cells in SB623 cell cultures, SB623 cells, as disclosed herein, have many immunosuppressive properties: For example, SB623 inhibits T cell proliferation and activation, alters the cytokine profile of T cells, blocks monocyte differentiation into dendritic cells, and is superior to their parental MSCs in reducing dendritic cell maturation.

[0063] Inhibition of T cell activation and proliferation by SB623 cells SB623 cells were cultured at 10 5 CFSE labeled peripheral blood T cells and 10 5SB623 cells were added to a mixed lymphocyte reaction (MLR) containing peripheral blood mononuclear cells. The ability of SB623 cells to modulate T cell activation was examined by measuring the levels of CD69, an early marker of T cell activation. In control MLRs, they potently induced surface expression of CD69. However, 10 4 After 1 day in the presence of SB623 cells, CD4 in the MLR expressing surface CD69 + The fraction of T cells (i.e., helper T cells) was significantly reduced. See Example 4.

[0064] After 5 days in the presence of SB623 cells, prelabeled CD4 + The dilution of CFSE in T cells (an indicator of cell proliferation) was significantly higher than that of CD4 + It was shown that T cell proliferation was decreased in the presence of SB623 cells. See Example 4. Thus, SB623 cells can suppress both T cell proliferation and T cell activation.

[0065] Further effects of SB623 cells on T cell function include reduced surface HLA-DR expression (Example 4 herein), increased production of regulatory T cells in in vitro cultures of naive T cells (Example 6 herein), and altered cytokine secretion (Examples 7 and 8 herein). SB623 cells were also effective in reducing T cell proliferation in a heterologous lymphocyte activation system. See Example 5 herein.

[0066] Inhibition of dendritic cell development by SB623 cells The differentiation of monocytes into dendritic cells (a type of antigen-presenting cell) and further maturation of dendritic cells can be stimulated in vitro by exposure of monocytes to the cytokines interleukin-4 (IL-4) and granulocyte / macrophage colony-stimulating factor (GM-CSF). This differentiation can be inhibited by interleukin-6 (IL-6) or vascular endothelial growth factor (VEGF), both of which are among the cytokines known to be secreted by SB623 cells. See, e.g., Tate et al. (2010) Cell Transplant. 19: 973-984 and WO 2009 / 023251.

[0067] The present inventors have demonstrated that co-culture of monocytes with SB623 cells enhances the expression of CD1a + Dendritic cell differentiation and CD86 + It is shown herein that SB623 cells reduce both the production of new dendritic cells and the maturation of dendritic cells to a state where they are unable to differentiate into host cells (see Examples 9 and 10 below). Due to their ability to reduce the production of new dendritic cells and inhibit the function of existing dendritic cells, SB623 cells can be used to treat and / or ameliorate graft-versus-host disease (GVHD) that results from the activation of T cells by presentation of peptides by antigen-presenting cells, e.g., dendritic cells.

[0068] Due to their various immunosuppressive properties as described herein, SB623 cells can be used in place of other biological and chemical immunosuppressants (e.g., cyclosporine, tacrolimus, sirolimus, interferon, mycophenolic acid, fingolimod, myriocin, azathioprine, mercaptopurine, dactinomycin, mitomycin C, bleomycin, mithramycin, anthracyclines, methotrexate, FK506, cyclophosphamide, nitrosoureas, platinum compounds, and glucocorticoids). Furthermore, the use of immunosuppressants is not necessary with SB623 allografts in cell therapy, for example, for nerve regeneration and treatment of nervous system disorders.

[0069] Progenitor cells The cells that can be transformed into SB623 cells can be any kind of non-terminally differentiated cells.For example, totipotent stem cells, such as those disclosed in U.S. Patent Nos. 5,843,780; 6,200,806 and 7,029,913, can be used as progenitor cells.Totipotent stem cells can be cultured (e.g., U.S. Patent Nos. 6,602,711 and 7,005,252) and differentiated into various kinds of pluripotent cells (e.g., U.S. Patent Nos. 6,280,718; 6,613,568 and 6,887,706), which can also be used as progenitor cells in the implementation of the disclosed method.

[0070] Other exemplary progenitor cell types are bone marrow adherent stromal cells (MASC), also known as bone marrow adherent stem cells, bone marrow stromal cells (BMSC) and mesenchymal stem cells (MSC). Exemplary disclosures of MASC are provided in U.S. Patent Application Publication No. 2003 / 0003090; Prockop (1997) Science 276: 71-74 and Jiang (2002) Nature 418: 41-49. Methods for isolating and purifying MASC can be found, for example, in U.S. Patent No. 5,486,359; Pittenger et al., (1999) Science 284: 143-147 and Dezawa et al., (2001) Eur. J. Neurosci. 14: 1771-1776. Human MASC are commercially available (e.g., Bio Whittaker, Walkersville, MD) or can be obtained from a donor, for example, by bone marrow aspiration followed by selection of adherent bone marrow cells. See, for example, WO 2005 / 100552.

[0071] MASCs can also be isolated from umbilical cord blood (see, e.g., Campagnoli et al. (2001) Blood 98: 2396-2402; Erices et al. (2000) Br. J. Haematol. 109: 235-242 and Hou et al. (2003) Int. J. Hematol. 78: 256-261).

[0072] The conversion of MSCs into SB623 cells is described, for example, in U.S. Pat. No. 7,682,825 (March 23, 2010) and WO 2009 / 023251 (February 19, 2009), the disclosures of both of which are incorporated by reference in their entireties for purposes of describing the isolation of mesenchymal stem cells and the conversion of mesenchymal stem cells into SB623 cells (referred to in these documents as "neural precursor cells" and "neural regenerating cells").

[0073] Notch intracellular domain Notch proteins are transmembrane receptors found in all metazoans that affect cell differentiation through intracellular signaling. Contact of the Notch intracellular domain with Notch ligands (e.g., Delta, Serrate, Jagged) results in two proteolytic cleavages of the Notch protein, the second of which is catalyzed by γ-secretase, and releases the Notch intracellular domain (NICD) in the cytoplasm. In mouse Notch protein, this cleavage occurs between amino acids gly 1743 and val 1744. NICD translocates to the nucleus where it acts as a transcription factor and recruits additional transcriptional regulatory proteins (e.g., MAM, histone acetylases) to relieve transcriptional repression of various target genes (e.g., Hes1).

[0074] Further details and information regarding Notch signaling can be found, for example, in Artavanis-Tsakonas et al. (1995) Science 268: 225-232; Mumm and Kopan (2000) Develop. Biol. 228: 151-165 and Ehebauer et al. (2006) Sci. STKE 2006 (364), cm7. [DOI: 10.1126 / stke.3642006cm7].

[0075] Cell culture and transfection Standard methods for cell culture are known in the art, see, for example, RI Freshney "Culture of Animal Cells: A Manual of Basic Technique," Fifth Edition, Wiley, New York, 2005.

[0076] Methods for introducing foreign DNA into cells (i.e., transfection) are also well known in the art. See, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual," Third Edition, Cold Spring Harbor Laboratory Press, 2001; Ausubel et al., "Current Protocols in Molecular Biology," John Wiley & Sons, New York, 1987 and regular updates.

[0077] Autoimmune diseases and allergic reactions Autoimmune diseases result from an immune response that attacks normal healthy tissue. Exemplary autoimmune diseases include, but are not limited to, amyotrophic lateral sclerosis, ankylosing spondylitis, thrombocytopenic purpura, Hashimoto's thyroiditis, Guillain-Barre syndrome, pernicious anemia, dermatomyositis, Addison's disease, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus ("lupus"), dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, polymyositis, biliary cirrhosis, psoriasis, reactive arthritis, Graves' disease, ulcerative colitis, inflammatory bowel disease, vasculitis, Crohn's disease, and celiac sprue (gluten-sensitive enteropathy).

[0078] Allergies result from immune hypersensitivity to external substances that do not normally stimulate an immune response. Common allergies include pollen, mold, pet dander and dust. Certain foods and medications can also trigger allergic reactions.

[0079] As disclosed herein, the immunosuppressive properties of SB623 cells are useful for the treatment of immune diseases and allergies.

[0080] Formulations, kits and routes of administration Also provided are therapeutic compositions comprising SB623 cells as disclosed herein. Such compositions typically comprise cells and a pharma- ceutically acceptable carrier.

[0081] The therapeutic compositions disclosed herein are useful, inter alia, for immune modulation (e.g., reducing immune activation) and reversing the progression of various immune diseases. Thus, a "therapeutically effective amount" of a composition comprising SB623 cells may be an amount that prevents or reverses immune activation. Depending on, for example, body weight, route of administration, severity of disease, etc., dosages may vary from about 100; 500; 1,000; 2,500; 5,000; 10,000; 20,000; 50,000; 100,000; 500,000; 1,000,000; 5,000,000; to 10,000,000 cells or more; for example, administration frequencies may be once a day, twice a week, once a week, twice a month, or once a month.

[0082] Supplementary active compounds can also be incorporated into the composition. For example, SB623 cells are useful in combination with other immunomodulators, such as cyclosporine, for treating autoimmune diseases, or preventing transplant rejection and / or GVHD. Thus, the therapeutic compositions disclosed herein can include both SB623 cells and cyclosporine (or any other immunosuppressant). When the composition of SB623 cells is used in combination with other therapeutic agents, it can refer to a therapeutically effective amount of the combination, which is the combined amount of SB623 cells and other agents that results in immunomodulation, administered in combination, either sequentially or simultaneously. Two or more combinations of concentrations may be therapeutically effective.

[0083] Various pharmaceutical compositions and techniques for their preparation and use are known to those skilled in the art in light of the present disclosure. For detailed listings of suitable pharmacological compositions and techniques for their administration, reference may be made to texts such as Remington's Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., "Goodman and Gilman's The Pharmacological Basis of Therapeutics," McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds.), "Remington: The Science and Practice of Pharmacy," Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), "Remington: The Principles of Pharmacy Practice," Lippincott Williams & Wilkins, 2008.

[0084] The cells described herein may be suspended in a physiologically compatible carrier for transplantation. As used herein, the term "physiologically compatible carrier" refers to a carrier that is compatible with other components of the formulation and is not harmful to the recipient. Those skilled in the art are familiar with physiologically compatible carriers. Examples of suitable carriers include cell culture media (e.g., Eagle's minimum essential medium), phosphate buffered saline, Hank's balanced salt solution + / - glucose (HBSS), and multiple electrolyte solutions, such as Plasma-Lyte™ A (Baxter).

[0085] The dose of SB623 cell suspension administered to a patient varies depending on the site of implantation, the therapeutic goal, and the number of cells in the solution. Typically, the amount of cells administered to a patient is a therapeutically effective amount. As used herein, "therapeutically effective amount" or "effective amount" refers to the number of implanted cells required to achieve treatment of a particular disease, i.e., to produce a reduction in the amount and severity of symptoms associated with the disease. A therapeutically effective amount further refers to an amount of a composition sufficient to cause a complete or partial improvement of symptoms of the relevant medical condition, or an increase in the rate of treatment, cure, prevention, or improvement of such a condition. For example, in the case of treatment for graft-versus-host disease, implantation of a therapeutically effective amount of SB623 cells typically results in immunosuppression of the implanted cells. If the disease is transplant rejection, for example, a therapeutically effective amount is the number of SB623 that, when implanted, results in sufficient immunosuppression in the host so that the graft is tolerated. A therapeutically effective amount varies with the type of disease or disorder, the extent of the disease or disorder, and the size of the organ affected by the disease or disorder.

[0086] The disclosed therapeutic compositions further comprise a pharma- ceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, i.e., a carrier. These carriers can, for example, stabilize SB623 cells and / or facilitate survival of SB623 cells in the body. Each carrier should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject. Some examples of materials which can serve as pharma- ceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxycellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifying agents and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0087] Another aspect of the present disclosure relates to a kit for administering SB623 cells, optionally in combination with other therapeutic agents, to a subject. In one embodiment, the kit includes a composition of SB623 cells formulated in a pharmaceutical carrier, optionally including, for example, one or more appropriate cyclosporine or other immunosuppressant agents formulated as separate pharmaceutical agents.

[0088] Exemplary formulations include, but are not limited to, those suitable for parenteral administration, e.g., intrapulmonary, intravenous, intraarterial, intraocular, intracranial, sub-meningial or subcutaneous administration, formulations encapsulated in micelles, liposomes or drug-release capsules (active agent incorporated in a biocompatible coating designed for sustained release); ingestible formulations; formulations for topical use, e.g., eye drops, creams, ointments and gels; and other formulations, e.g., inhalation, aerosols and sprays. The dosage of the disclosed compositions will vary according to the extent and severity of the need for treatment, the activity of the composition administered, the overall health of the subject, and other considerations well known to those skilled in the art.

[0089] In further embodiments, the compositions described herein are delivered locally. Local delivery allows the composition to be delivered non-systemically, thereby reducing the burden of the composition on the body compared to systemic delivery. Such local delivery can be achieved, for example, through the use of various medical implantation devices, including but not limited to stents and catheters, or by inhalation, phlebotomy, injection or surgery. Coating, implantation, embedding methods, and other methods of attaching desired agents to medical devices, such as stents and catheters, are established in the art and are contemplated herein. EXAMPLES

[0090] Example 1: Preparation of MSCs and SB623 cells Bone marrow aspirates from adult donors were obtained from Lonza Walkersville, Inc. (Walkersville, MD) and seeded in α-MEM (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine (Invitrogen, Carlsbad, CA), and penicillin / streptomycin (Invitrogen). Cells were cultured at 37°C and 5% CO2 for 3 days to obtain a monolayer of adherent cells. After removal of non-adherent cells, culture was continued under the same conditions for 2 weeks. During this time, cells were passaged twice using 0.25% trypsin / EDTA. A portion of cells from the second passage was frozen as MSCs.

[0091] The remaining cells from the second passage were seeded and transfected with the cytomegalovirus promoter (pCMV-hNICD1-SV40-Neo R The cells were transfected using Fugene6 (Roche Diagnostics, Indianapolis, IN) with a plasmid containing a sequence encoding the Notch intracellular domain operably linked to SV40 promoter. This plasmid also contains sequences encoding resistance to neomycin and G418 under the transcriptional control of the SV40 promoter. The transfected cells were cultured at 37°C, 5% CO2 in the growth medium described in the previous paragraph supplemented with 100 μg / ml G418 (Invitrogen, Carlsbad, CA). After 7 days, G418-resistant clones were expanded and the culture was passaged twice. After two passages, the cells were harvested and frozen as SB623 cells.

[0092] MSCs and SB623 prepared as described herein were thawed as necessary and used in further studies.

[0093] Example 2: Proliferation capacity of MSCs and SB623 cells To quantify cell proliferation, one million MSC or SB623 cells were seeded and cultured for 3 days. On day 3, viable cells were counted by trypan blue exclusion. Figure 2 shows that fewer viable cells were present in the SB623 cultures compared to the MSC cultures.

[0094] The cell cycle profile of MSC and SB623 cultures was assessed by propidium iodide staining. Propidium iodide is a DNA intercalating dye that stains cells in the resting phase of the cell cycle more intensely than cells in the proliferating phase. After 3 days of culture, 1 million MSC or SB623 cells were fixed overnight in 70% ethanol at 4°C. After washing twice with PBS / 2% FBS, cells were incubated for 30 minutes in the dark with 1 ml of staining buffer (50 μg / ml propidium iodide, 50 μg / ml RNAse) (Sigma, St. Louis, MO) in PBS / 2% FBS. Acquisition and analysis were performed on a FACSCalibur™ flow cytometer (BD Biosciences) using the CellQuestPro™ program (BD Biosciences, San Jose, CA) in the FL-2 linear channel. FIG. 3 shows greater propidium iodide staining of SB623 cells compared to MSCs, indicating a higher fraction of cells in the G0 / G1 resting phase of the cell cycle in SB623 cell cultures.

[0095] Dilution of the cell-autonomous stain, 5-(-6-) carboxyfluorescein diacetate (CFSE), was used as an additional measure of proliferation kinetics. In this analysis, equal numbers of MSCs and SB623 cells were labeled with 5 μM 5-(-6-) carboxyfluorescein diacetate (Invitrogen, Carlsbad, CA) for 2 min at room temperature and then cultured for 5 days. Flow cytometry acquisition and analysis (for CFSE) were performed on a FACSCalibur™ flow cytometer (BD Biosciences) using the FL-1 log channel. The results (Figure 1) show that SB623 cell cultures contain a cell population with a high CFSE content compared to MSCs, indicating the presence of a population of non-dividing or slowly dividing cells in SB623 cell cultures.

[0096] The levels of intracellular p16Ink4A protein in MSCs and SB623 cells were assessed as follows. Cells were cultured for 3 days, then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. After washing twice with 2% fetal bovine serum in PBS (PBS / 2%FBS), the cell pellets were resuspended in 0.2 ml of PBS / 2%FBS and divided into two samples. One cell sample was stained with phycoerythrin (PE)-conjugated anti-p16Ink4A antibody (BD Biosciences, San Jose, CA), and the other sample was incubated with PE-conjugated mouse IgG as an isotype control. Samples were analyzed by flow cytometry on a FACSCalibur™ flow cytometer (BD Biosciences) and data converted to the percentage of cells in the culture expressing p16Ink4A by gating on p16Ink4A positive and IgG negative staining cells. Figure 4 shows that SB623 cell cultures contain a significantly higher fraction of cells expressing p16Ink4A.

[0097] Example 3: Surface marker and cytokine expression by MSCs and SB623 cells To measure surface markers, MSCs or SB623 cells were harvested from the medium using 0.25% trypsin / EDTA (Invitrogen, Carlsbad, CA), washed with PBS / 2% FBS, and resuspended in 1 ml of PBS / 2% FBS. Cells were incubated with fluorochrome-conjugated antibodies to CD29, CD31, CD34, CD44, CD45, CD73, CD90 (all BD Biosciences, San Jose, CA), or fluorochrome-conjugated antibodies to CD105 (Invitrogen, Carlsbad, CA) for 15 min on ice. Cells were then washed once with PBS / 2% FBS and run on a FACSCalibur™ flow cytometer (BD Biosciences, San Jose, CA). CellQuestPro™ software (BD Biosciences) was used for data analysis. Results were expressed as dMFI ("delta mean fluorescence intensity") using IgG as a control; i.e., the MFI for IgG was subtracted from the MFI obtained for a given surface marker to obtain the dMFI.

[0098] The results are shown in Figures 5 and 6. Figure 5 shows that both MSCs and SB623 cells express CD44, CD73 and CD105, but SB623 cells consistently express higher levels of these surface markers. Figure 6 shows that SB623 cells also consistently express higher levels of CD54 than MSCs.

[0099] For detection of intracellular cytokines, cells were cultured for 3 days and treated with a 1:1,000 dilution of Brefeldin A (eBioscience, San Diego, CA, final concentration 3 μg / ml) for 6 hours before harvesting. Cells were fixed, permeabilized as above for measurement of intracellular pInk4A, and incubated with fluorochrome-conjugated antibodies (human GM-CSF (BD), IL-1α (eBioscience, San Diego, CA), IL-6 (BD), or TGFβ-1 (R&D Systems, Minneapolis, MN)) for 1 hour, followed by two washes with PBS / 2% FBS. Data collection and analysis were performed on a BD FACSCalibur™ instrument using CellQuestPro™ software.

[0100] The results of these analyses, shown in Figure 7, show roughly similar levels of expression of IL-1α, IL-6, and GM-CSF by MSCs and SB623 cells; while Figure 8 shows that similar levels of TGF-β-1 and VEGF-A are produced by MSCs and SB623.

[0101] Example 4: Allogeneic mixed lymphocyte reaction (allo-MLR) Cells for allogeneic mixed lymphocyte reaction were obtained from 10 ml samples of peripheral blood from healthy, unrelated donors. To obtain responder T cells, the RosetteSep T-cell enrichment kit (Stemcell Technologies, Vancouver, BC, Canada) was used according to the manufacturer's instructions. The enriched T cells (responder cells) were labeled with 5 μM 5-(-6-) carboxyfluorescein diacetate (CFSE) for 2 min at room temperature (Invitrogen, Carlsbad, CA). After serum quenching and washing three times with PBS, the labeled responder cells were plated at 10 5 Cells were seeded in 0.1 ml of complete lymphocyte medium (RPMI (Mediatech, Manassas, VA)) + 10% FBS (Lonza, Allendale, NJ).

[0102] To prepare stimulator cells, peripheral blood buffy coat mononuclear cells were harvested after Ficoll™ density gradient centrifugation. Red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO) was added for 10 min at 37°C; cells were then washed twice with PBS / 2% heat-inactivated FBS. Mononuclear stimulator cells were added to wells containing responder cells (10 5 cells) or 10 5 10 stimulator cells 4 SB623 cells or 10 4 MSCs, centrifuged and pelleted cells were then resuspended in 0.1 ml of complete lymphocyte medium (as above) which was added to the wells of CFSE-labeled responder cells prepared as above.

[0103] Two days after the start of the reaction, CD4 + Presentation of CD69 (an early T cell activation marker) on the surface of T cells was used as an assay for T cell activation. For analysis of CD69 expression, cells were harvested by pipette after 2 days, stained with peridinin chlorophyll protein (PerCP)-conjugated anti-CD69 antibody (eBioscience, San Diego, CA), and analyzed for CD4+ / CD69 expression using a FACSCalibur™ flow cytometer (Becton, Dickinson & Co., San Jose, CA). + Analysis was performed by gating on lymphocytes.

[0104] For measurement of T cell proliferation, after 7 days of culture, cells were harvested and incubated with phycoerythrin (PE)-conjugated anti-CD40 antibodies. 4 Staining was performed with antibodies (BD). A BD FACSCalibur flow cytometer was used for data collection.

[0105] For allo-MLR controls, CD4 + The T cell fraction within the population, which had been induced to express surface CD69, was significantly increased after 2 days (Figures 9A and 9B).

[0106] The effect of co-culture with MSCs and SB623 cells in the MLR on T cell activation was also evaluated. In these experiments, 10,000 MSCs or 10,000 SB623 cells were added to the cultures at the initiation of the MLR. Under these conditions, after 2 days, the increase in surface CD69 expressing cells observed in control cultures was significantly reduced in co-cultures with MSCs or SB623 cells (p<0.05; Figures 9A and 9B).

[0107] Another measure of T cell activation is CD4 + The proliferation rate of T cells was assayed 7 days after the initiation of the MLR. For these experiments, cells were taken from the MLR with a pipette and stained with PE-labeled anti-CD4 antibody. Flow cytometry was performed using a Becton-Dickinson FACSCalibur™ instrument to measure the proliferation and proliferation of CD4 + Gating was performed on CD4+ cells; and dilutions of CSFE were + The proliferation rate of responder T cells was evaluated. + More than 80% of responder T cells proliferated after 7 days. In the presence of SB623 cells or MSCs, T cell proliferation was significantly reduced (i.e., higher levels of CFSE staining were observed (Figure 10)).

[0108] Induction of surface HLA-DR expression is also a measure of T cell activation. Both SB623 cells and MSCs reduced the percentage of HLA-DR expressing T cells in allo-MLR (Figure 11).

[0109] Thus, by a number of different and independent criteria, SB623 cells suppress T cell activation. The ability to inhibit T cell activation indicates the utility of SB623 cells for immunosuppression.

[0110] Example 5: Heterologous Lymphocyte Activation Reaction The immunosuppressive properties of SB623 cells were also demonstrated in a xenograft model system. Xenogeneic lymphocyte responses were established using Sprague-Dawley rat glial mixed cells (including astrocytes and microglia) as stimulator cells and human peripheral blood T cells labeled with PKH26 according to the manufacturer's instructions as responder cells. To obtain glial mixed cells, 9-day-old rat brains were harvested and ground for 30 min before being treated with 0.25% trypsin. The cell suspension was filtered through a 70 μM cell strainer and overlaid with Ficoll™ before centrifugation. Glial mixed cells were cultured in DMEM / F12 / 10% FBS / pen-strep for 14 days before use in the assay. Xenogeneic reactions were performed over 5 days using the same cell ratios as used in the allogeneic MLR (100,000 mixed glial cells:100,000 CFSE-labeled human T cells; and, optionally, 10,000 MSC or SB623 cells). Human CD3-gated T cells (CD4 + and CD8 + PKH26 dilution in cultured and non-cultured T cells (including both cultured and non-cultured T cells) was assessed by flow cytometry.

[0111] As in the allogeneic MLR, the addition of SB623 cells or MSCs to the xenogeneic system, otherwise observed after stimulation with mixed glial cells, reduced the extent of responder T cell proliferation (Figure 12). Thus, the immunosuppressive properties of MSCs and SB623 cells are not limited to autologous or allogeneic environments.

[0112] Example 6: Effect of SB623 cells on the growth of regulatory T cells Regulatory T cells (T reg s) can weaken or suppress the immune response. reg The ability of SB623 cells to support the development of s was examined. To this end, peripheral blood-derived enriched T cells, purified as described in Example 2, were cultured in the presence of interleukin-2 (IL-2), which inhibited the T development of naive T cells. regWe have shown that T cells stimulate differentiation into s and assessed the effect of co-culture with MSCs or SB623 cells on this process. Co-cultures were performed at a ratio of 10:1 T cells to SB623 cells or a ratio of 10:1 T cells to MSCs (10 5 T cells: 10 4 MSCs or SB623 cells. Co-expression of the surface markers CD4 and CD25, secretion of the cytokine interleukin-10 (IL-10), and endogenous production of the transcription factor FoxP3 were detected in T reg was used as a marker for s.

[0113] For these experiments, human T cells were enriched from peripheral blood using a T cell isolation kit (StemCell Technologies, Vancouver, Canada) according to the manufacturer's instructions. Enriched T cells were cultured overnight in RPMI-1640 / 10% heat-inactivated FBS / pen / strep before use. One day prior, 10,000 MSC or SB623 cells were seeded per well in a 96-well U-bottom plate. On the day of the coculture assay, 100,000 enriched T cells were transferred to each well of the pre-prepared MSC or SB623 cell monolayer, which also contained 10 ng / ml IL-2. As an internal control, T cell cultures were also maintained in the absence of MSC or SB623 cells. On day 7, cells were stained for surface CD4 (helper T cell marker) and CD25 (IL-2 receptor alpha chain), and intracellular FoxP3.

[0114] The results of the assay for the surface markers CD4 and CD25 are shown in Figure 13. Co-culture of SB623 cells with IL-2 stimulated T cells increased CD4 + CD25 + T reg The number of cells was significantly increased (compare the left and right panels of FIG. 13A), and this T reg Growth stimulation was greater when T cells were co-cultured with SB623 cells than when T cells were co-cultured with MSCs (compare middle and right panels in FIG. 13A).

[0115] Assays for the forkhead box P3 (FoxP3) protein support these results. FoxP3 is involved in the regulation of T reg FoxP3 is a transcription factor that regulates the growth and function of S. Intracellular FoxP3 was detected by culturing or co-cultured T cells for 7 days (as described above), then fixing and permeabilizing the cells with CytoFix / Perm (eBioscience, San Diego, CA). PE-conjugated anti-FoxP3 antibody (clone PCH101, eBioscience, 1:5 dilution) was used to stain the cells for 30 min, and the stained cells were analyzed by flow cytometry gating on lymphocytes based on cell size. The results shown in Figure 14 show that co-culture with MSCs and SB623 cells in the presence of IL-2 increased FoxP3 expression by T cells compared to its expression in T cells that were not co-cultured.

[0116] T reg One mechanism of immunosuppression by s is through the secretion of anti-inflammatory cytokines, such as interleukin-10 (IL-10). Thus, the percentage of IL-10-producing T cells in T cell cultures with IL-2 or co-cultures with MSCs or SB623 cells was measured after 7 days of culture or co-culture by staining intracellular IL-10 with a fluorescent dye-conjugated anti-IL-10 antibody.

[0117] Thus, after 7 days of culture or coculture, cells were treated with a 1:1,000 dilution of Brefeldin A (eBioscience, San Diego California) for 6 hours (to prevent secretion of intracellular proteins), fixed with 2% paraformaldehyde for 15 minutes, and then permeabilized with 0.05% (v / v) Triton-X-100 in PBS / 2% FBS for 15 minutes on ice. Alexa488-conjugated anti-human IL-10 antibody (eBioscience, San Diego CA) was then added, and cultures were incubated on ice for 30 minutes. Wells were washed twice with 2% fetal bovine serum / 0.01% (v / v) Tween 20; cells were obtained with a pipette and analyzed using a FACSCalibur™ flow cytometer (Becton, Dickinson & Co., San Jose, CA). Data analysis was performed using CellQuestPro™ software (Becton, Dickinson & Co., San Jose, Calif.).

[0118] The results of this analysis showed that T cells cultured in the presence of IL-2 did not express intracellular IL-10; whereas, when they were cocultured with either SB623 cells or MSCs in the presence of IL-2, they expressed low levels of IL-10 in the CD4 + We show that slightly more IL-10 was produced by T cells co-cultured with SB623 cells (Figure 15).

[0119] Example 7: Conversion of an inflammatory to an anti-inflammatory cytokine profile by SB623 cells The effect of co-culture of MSC and SB623 cells on the relative amounts of inflammatory and anti-inflammatory cytokines produced by T cells was assessed by measuring IL-10 (an anti-inflammatory cytokine) and interferon-gamma (INF-γ, a pro-inflammatory cytokine) in T cells suboptimally activated by treatment with phorbol myristate acetate (PMA) and ionomycin. For these experiments, T cells were enriched from peripheral blood and cultured or co-cultured with MSC or SB623 as described above (Example 6), except that the cultures were performed in the absence of IL-2. On day 7, a non-activating dose of 25 ng / ml phorbol myristate acetate (PMA) / 0.5 μM ionomycin (Io) (both from Sigma-Aldrich, St Louis, MO) was added in the presence of 3 μg / ml brefeldin A (eBioscience, San Diego, CA), and 6 hours later, cells were harvested and analyzed for intracellular expression of IL-10 and IFN-γ. The non-activating doses of PMA and ionomycin used in these experiments did not induce T cell proliferation but were sufficient to induce cytokine synthesis by T cells. IL-10 levels were measured using an Alexa488-conjugated anti-human IL-10 antibody (eBioscience, San Diego, CA) as described in Example 6. IFN-γ levels were measured by FACS using a PE-labeled anti-human IFN-γ antibody (eBioscience, San Diego, CA).

[0120] The results of this analysis are shown in Figure 16. After suboptimal stimulation with PAM / ionomycin, more than 20% of freshly isolated T cells expressed IFN-γ, while less than 1% expressed IL-10 (i.e., of the cells that expressed either IFN-γ or IL-10, more than 95% expressed IFN-γ and less than 5% expressed IL-10). However, after 7 days of culture with either SB623 cells or MSCs, more than 95% of the cells that expressed either IFN-γ or IL-10 expressed IL-10, while less than 5% expressed IFN-γ. Thus, coculture with either MSCs or SB623 cells shifted the T cell secretome from an inflammatory to an anti-inflammatory one.

[0121] Secretion of the inflammatory cytokine IFN-γ is mediated by T helper T cells. H 1 subset; whereas IL-10 secretion was characteristic of T H 2. Cells and T reg Thus, the shift from IFN-γ secretion to IL-10 secretion observed upon coculture of naive T cells with SB623 cells or MSCs was due to the T H 1-rich population, large amounts of T H 2 cells, T reg The results are consistent with the conversion of SB623 cells or co-culture with MSCs to a population containing proinflammatory T cells, or both, in part through alterations in cytokine production by T cells. H 1 cells) to a more anti-inflammatory population (T H 2. Cells and / or T reg We show that the antibody directs T cell differentiation toward T cells that are characterized by IL-1 and IL-2.

[0122] Example 8: Effect of co-culture of MSCs and SB623 cells on the production of IL-17 by T cells Two known cytokines secreted by MSCs and SB623 cells, TGFβ-1 and IL-6 (see Example 3 above), also play a role in the development of Th17 helper T cells (i.e., helper T cells that secrete IL-17). Therefore, T cells were cultured in the presence of IL-23, which is known to stimulate the development of Th17 helper T cells, and the effect of co-culture with MSCs or SB623 cells on the number of Th17 cells was measured.

[0123] For these experiments, human T cells were isolated and cultured with 10 ng / ml IL-23 (Peprotech, Rocky Hill, NJ) as described in Example 7. After treatment with brefeldin A for 6 hours, cells were harvested, fixed and permeabilized as described in Example 7, stained with PE-conjugated anti-IL-17 antibody (eBioscience), and analyzed by flow cytometry. Results showed that culture of T cells in the presence of IL-23 increased the number of IL-17-expressing cells. Coculture of T cells with MSCs or SB623 cells also resulted in a small increase in the number of IL-17-expressing cells both in the absence and presence of IL-23.

[0124] Example 9: Inhibition of monocyte differentiation into dendritic cells by co-culture with SB623 cells The normal progression of monocyte (expressing CD14) development into dendritic cells (expressing CD1a) can be reproduced in vitro by culturing monocytes in the presence of interleukin-4 (IL-4) and granulocyte / macrophage colony-stimulating factor (GM-CSF). When co-cultured with monocytes in vitro, MSCs can block monocyte differentiation into dendritic cells, an effect mediated in part by secretion of interleukin-6 (IL-6) by MSCs. Chomarat et al. (2000) Nature Immunology 1:510-514; Djouad et al. (2007) Stem Cells 25: 2025-2032. See US Patent Application Publication No. 2010 / 0266554 (October 21, 2010). VEGF, also secreted by MSCs and SB623, is also involved in dendritic cell differentiation. Therefore, the effect on monocytic differentiation of SB623 cells was examined.

[0125] Monocytes were cultured in RPMI-1640 (Meidatech, Manassas, VA) containing 10% fetal bovine serum (Lonza, Allendale, NJ), 2 mM L-glutamine, 2 mM sodium L-pyruvate, 100 units / ml penicillin, 100 μg / ml streptomycin, 40 ng / ml GM-CSF (Peprotech, Rocky Hill, NJ), and 20 ng / ml IL-4 (Peprotech, Rocky Hill, NJ). Cocultures with SB623 cells (or MSCs, as a control) were performed at a monocyte to SB623 cell (or MSC) ratio of 10:1; i.e., 100,000 monocytes to 10,000 SB623 cells or MSCs. After 7 days of culture (co-culture), a portion of the cells was harvested using trypsin / EDTA (as above) and incubated with PE-conjugated anti-CD-14 antibody and FITC-labeled anti-CD1a antibody (both from eBioscience, San Diego, CA). Collection and analysis were performed using a FACSCalibur™ cell sorter (both from Becton, Dickinson & Co., San Jose, CA) with CellQuestPro™ software. The other portion of the culture was observed by phase contrast microscopy.

[0126] The results of the cell sorting analysis (Figure 18) showed that CD14 + In addition, the effect was greater when monocytes were co-cultured with SB623 compared to co-culture with MSCs. Also, less CD1a + Dendritic cells were observed in the co-culture. These results indicate that SB623 cells (and to a lesser extent MSCs) can inhibit the differentiation of monocytes into dendritic cells.

[0127] Microscopic analysis confirmed these observations: in monocyte cultures, populations of dendritic cells were easily observed by microscopy; however, few such populations were observed in co-cultures with MSCs or SB623 cells.

[0128] Example 10: Inhibition of dendritic cell maturation by co-culture with SB623 cells After differentiation from monocytes, dendritic cells mature into cells expressing the CD86 surface marker. This maturation can be reproduced in vitro by culturing dendritic cells in the presence of tumor necrosis factor-alpha (TNF-α). IL-6 and VEGF have been shown to block dendritic cell maturation. Park et al. (2004) J. Immunol. 173: 3844-3854; Takahashi et al. (2004) Cancer Immunol. Immunother 53: 543-550. Since SB623 cells secrete both of these cytokines, the effect of SB623 co-culture on dendritic cell differentiation was examined.

[0129] To assess the effect of co-culture of SB623 cells on dendritic cell maturation, monocytes were obtained from peripheral blood and differentiated in vitro into dendritic cells as described in Example 9. After 5 days of culture, human TNF-α (Peprotech, Rocky Hill, NJ) was added to the cultures at a final concentration of 10 ng / ml. In some cultures, SB623 cells or MSCs were also added at this time. All samples were incubated for 10 min at 4°C. 5 Monocytes and co-cultures 4 MSC or SB623 cells were included. As a control, CD86 + Cyclosporine A, which inhibits maturation to the TNF-α state, was added to TNF-α stimulated cultures at a final concentration of 1 μg / ml. After 2 days, cells were stained with PE-conjugated anti-CD86 antibody (Becton Dickinson & Co., San Jose, Calif.) and analyzed on a FACSCalibur cell sorter and using CellQuestPro™ software (both Becton Dickinson & Co., San Jose, Calif.).

[0130] The results shown in Figure 19 indicate that a significant fraction of TNF-α matured dendritic cells express CD86, and this fraction is reduced by treatment with cyclosporine, as expected. Co-culture of SB623 cells and MSCs also reduced the expression of CD86+ Notably, SB623 cells have a stronger inhibitory effect on dendritic cell maturation than MSCs, as measured by CD86 expression.

[0131] Example 11: Changes in secretory profile of monocytes / macrophages by co-culture with SB623 cells Human peripheral blood monocytes (i.e., macrophage precursors) expressing the CD14 cell surface marker were obtained from buffy coat cells by magnetic selection using anti-CD14 coated magnetic beads (Miltenyi Biotec, Auburn, CA). + Isolated cultures of monocytes were exposed to granulocyte / macrophage colony-stimulating factor (GM-CSF) to convert them into M1 (pro-inflammatory) macrophages; or to macrophage colony-stimulating factor to convert them into M2 (anti-inflammatory) macrophages; or co-cultured with either SB623 cells or MSCs.

[0132] The percentage of cells expressing tumor necrosis factor-alpha (TNF-α, a proinflammatory cytokine that characterizes M1 macrophages) and interleukin 10 (IL-10, an anti-inflammatory cytokine that characterizes M2 macrophages) was determined in these cultures as follows: Cultures were exposed to 100 ng / ml bacterial lipopolysaccharide (LPS, Sigma, St. Louis, MO) for 24 hours. During the final 6 hours of exposure to LPS, brefeldin A and monensin (both eBioscience San Diego, CA; and both used at a 1:1,000 dilution) were added to the cultures. Cells were then stained with either PE-conjugated anti-TNFα or FITC-conjugated anti-IL-10 and analyzed by flow cytometry.

[0133] The results of these studies, shown in Figure 20, showed that co-culture with MSCs or SB623 cells increased the fraction of monocytes that produced anti-inflammatory cytokines in the cultures. Co-culture with MSCs and SB623 cells did not increase the percentage of cells that produced TNF-α, as did exposure to GM-CSF (Figure 20A). Notably, the percentage of cells expressing the anti-inflammatory cytokine IL-10 was increased when monocytes were co-cultured with MSCs, and even more so when co-cultured with SB623 cells (Figure 20B).

Claims

1. 1. A method for preparing a medicament for modulating a peripheral immune response in a subject, comprising: (a) contacting a culture of mesenchymal stem cells with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD), wherein the polynucleotide does not encode a full-length Notch protein; (b) selecting cells containing the polynucleotide of step (a); and (c) further culturing the selected cells of step (b) without selection. and wherein the peripheral immune response comprises: (i) Suppression of T cell activation, (ii) suppression of peripheral helper T cell function; (iii) peripheral regulatory T cells (T ｒｅｇ S) population expansion, (iv) reduced peripheral production of inflammatory cytokines; (v) stimulation of peripheral production of anti-inflammatory cytokines; (vi) inhibiting the differentiation of peripheral monocytes into dendritic cells; and (vii) Inhibition of dendritic cell maturation The method is selected from one or more of the following:

2. The method of claim 1 , wherein the modulation comprises inhibition of T cell activation.

3. The T cell activation is characterized by expression of CD69, CD4 + The method of claim 2, comprising T cell proliferation or HLA-DR expression.

4. The method of claim 1 , wherein the modulation comprises inhibition of helper T cell function.

5. 5. The method of claim 4, wherein the helper T cell function comprises cytokine secretion or is associated with rheumatoid arthritis pathology.

6. The regulation is performed by regulatory T cells (T reg s) population expansion.

7. The method of claim 1 , wherein the modulation comprises a decrease in the production of inflammatory cytokines.

8. The method of claim 7, wherein the proinflammatory cytokine is interferon-gamma (IFN-γ).

9. The method of claim 8, wherein the IFN-γ is produced by a T cell.

10. The method of claim 7, wherein the proinflammatory cytokine is tumor necrosis factor-alpha (TNF-α).

11. The method of claim 10, wherein the TNF-α is produced by monocytes.

12. The method of claim 1 , wherein the modulation comprises increased production of anti-inflammatory cytokines.

13. 13. The method of claim 12, wherein the anti-inflammatory cytokine is interleukin-10 (IL-10).

14. The method of claim 13, wherein the IL-10 is produced by a helper T cell, a regulatory T cell, or a monocyte.

15. 13. The method of claim 12, wherein the modulation comprises increased production of interleukin-17 (IL-17).

16. The method of claim 1 , wherein the modulation comprises inhibition of differentiation of monocytes into dendritic cells.

17. The method of claim 1 , wherein the modulation comprises inhibition of dendritic cell maturation.

18. The method of claim 17, wherein the maturation comprises increased expression of CD86 by the dendritic cells.

19. The method of claim 1 , wherein the modulation results in conversion of a T cell population from pro-inflammatory to anti-inflammatory.

20. The method of claim 1 , wherein the medicament is used to treat graft-versus-host disease.

21. The method of claim 1 , wherein the medicament is used to inhibit transplant rejection.

22. The method of claim 1 , wherein the medicament is used for the treatment of an autoimmune disease.

23. 23. The method of claim 22, wherein the autoimmune disease is selected from the group consisting of multiple sclerosis, ulcerative colitis, chronic obstructive pulmonary disease (COPD), asthma, lupus and type 1 diabetes.

24. The method of claim 1 , wherein the subject is a human.

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