WO2010017220A1 - Procédés d’expansion des lymphocytes t régulateurs - Google Patents

Procédés d’expansion des lymphocytes t régulateurs Download PDF

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Publication number
WO2010017220A1
WO2010017220A1 PCT/US2009/052737 US2009052737W WO2010017220A1 WO 2010017220 A1 WO2010017220 A1 WO 2010017220A1 US 2009052737 W US2009052737 W US 2009052737W WO 2010017220 A1 WO2010017220 A1 WO 2010017220A1
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Prior art keywords
regulatory
cells
subject
cell
cell population
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PCT/US2009/052737
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English (en)
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Taku Kambayashi
Gary Koretzky
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The Trustees Of The University Of Pennsylvania
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Publication of WO2010017220A1 publication Critical patent/WO2010017220A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)

Definitions

  • the invention relates to methods for expanding the number of regulatory T-cells and uses thereof. Specifically, the invention relates to methods for selectively expanding a regulatory T-cell population by the use of a granulocyte-macrophage colony stimulating factor (GMCSF).
  • GMCSF granulocyte-macrophage colony stimulating factor
  • CD4 + CD25 + FOXP3 + regulatory T cells are thought to differentiate in the thymus and immigrate from the thymus to the periphery.
  • Treg cells can regulate both acquired and innate immunity through multiple modes of suppression.
  • APCs antigen-presenting cells
  • CD4 + Foxp3 + regulatory T cells have been referred to as "naturally-occurring" regulatory T cells to distinguish them from “suppressor” T cell populations that are generated in vitro.
  • the regulatory T cell field is further complicated by reports of additional suppressive T cell populations, including TrI, CD8 + CD28 " , and Qa-I restricted T cells.
  • TrI TrI
  • CD8 + CD28 " CD8 + CD28
  • Qa-I restricted T cells the contribution of these populations to self-tolerance and immune homeostasis is less well defined.
  • regulatory T cells develop in the thymus.
  • the latest research suggests that regulatory T cells are defined by expression of the forkhead family transcription factor FOXP3 (forkhead box p3). Expression of FOXP3 is required for regulatory T cell development and appears to control a genetic program specifying this cell fate.
  • the large majority of Foxp3 -expressing regulatory T cells are found within the major histocompatibility complex (MHC) class II restricted CD4-expressing (CD4+) helper T cell population and express high levels of the interleukin-2 receptor alpha chain (CD25).
  • MHC major histocompatibility complex
  • CD4+CD25 interleukin-2 receptor alpha chain
  • CD25 interleukin-2 receptor alpha chain
  • CD4 and CD25 Prior to the identification of Foxp3, expression of these two cell surface molecules (CD4 and CD25) was used to define this cell population. As defined by CD4 and CD25 expression, regulatory T cells comprise about 5-10% of the mature CD4 + helper T cell subpopulation in mice and about 1-2% CD4 + helper T cells in humans. Foxp3 is not expressed on activated T cells and the regulatory T cell population as more accurately defined by Foxp3 expression extends beyond the CD4 + CD25 + operational definition.
  • the invention provides a method for selectively expanding the number of regulatory T-cells comprising the step of contacting a leukocyte cell population having antigen-presenting cells with a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), thereby selectively expanding the number of regulatory T-cells while preserving the function of pathogen-reactive T cells
  • a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF)
  • the invention provides a method for selectively expanding the number of regulatory T-cells in a subject, comprising the step of contacting a leukocyte cell population having antigen-presenting cells in the subject with a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF), interleukin-3 (IL-3), interleukin-5 (DL-5), or combinations thereof, thereby selectively expanding the number of regulatory T-cells while preserving the function of pathogen-reactive T cells.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • IL-3 interleukin-3
  • DL-5 interleukin-5
  • the invention provides a method for enriching a T-cell population with regulatory T-cells in a subject, comprising the step of contacting a population of leukocyte cells having antigen-presenting cells with a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), thereby increasing the number of regulatory T-cells and suppressing naive T-cells thus enriching a T-cell population with regulatory T-cells.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • the invention provides a method for enriching a T-cell population with regulatory T-cells in a subject, comprising the step of contacting a leukocyte cell having antigen-presenting cells with a composition comprising: a granulocyte- macrophage colony stimulating factor (GMCSF), interleukin-3 (IL-3), interleukin-5 (EL-5), or combinations thereof, thereby increasing the number of regulatory T-cells and suppressing naive T-cells thus enriching a T-cell population with regulatory T-cells.
  • GMCSF granulocyte- macrophage colony stimulating factor
  • IL-3 interleukin-3
  • EL-5 interleukin-5
  • the invention provides a method for suppressing a T-cell activation mediated response in a subject, comprising the step of administering to the subject a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), thereby increasing the number of regulatory T-cells and suppressing naive T-cells.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • the invention provides a method for suppressing a T-cell activation mediated response in a subject, comprising the step of administering to the subject a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF), interleukin-3 (IL-3), interleukin-5 (IL-5), or combinations thereof, thereby increasing the number of regulatory T-cells and suppressing naive T-cells thus suppressing a T-cell activation mediated response in said subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • IL-3 interleukin-3
  • IL-5 interleukin-5
  • the invention provides a method for suppressing a T-cell activation mediated response in a subject in need thereof, comprising the steps of: obtaining leukocyte cell population having antigen-presenting cells from a subject or pool of subjects; contacting the cell population with a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF), interleukin-3 (IL-3), interleukin-5 (IL-5), or combinations thereof, thereby increasing the number of regulatory T-cells and suppressing na ⁇ ve T-cells; and administering to the subject in need thereof the resulting cell population with increased regulatory T-cell number, thus suppressing a T-cell activation mediated response in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • IL-3 interleukin-3
  • IL-5 interleukin-5
  • the invention provides a method for treating an autoimmune disease in a subject, comprising the step of selectively enriching a T-cell population in the subject with regulatory T-cells by administering to the subject a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), thereby increasing the number of regulatory T-cells and suppressing na ⁇ ve T-cells.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • the invention provides a method for treating an autoimmune disease in a subject, comprising the step of selectively enriching a T-cell population in the subject with regulatory T-cells by administering to the subject a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF), interleukin-3 (IL-3), interleukin-5 (IL-5), or combinations thereof, thereby increasing the number of regulatory T- cells and suppressing na ⁇ ve T-cells and treating an autoimmune pathology in said subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • IL-3 interleukin-3
  • IL-5 interleukin-5
  • the invention provides a method for treating an autoimmune disease in a subject in need thereof, comprising the steps of: obtaining a leukocyte cell population having antigen-presenting cells from a subject or pool of subjects; selectively enriching the leukocyte cell population in the subject with regulatory T-cells by contacting the leukocyte cell population with a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF), interleukin-3 (IL-3), interleukin-5 (TL-5), or combinations thereof, thereby increasing the number of regulatory T-cells and suppressing naive T-cells; and administering to the subject in need thereof the resulting cell population enriched with regulatory T-cells, thereby treating said autoimmune pathology in said subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • IL-3 interleukin-3
  • TL-5 interleukin-5
  • the invention provides a method for inhibiting or suppressing graft-versus-host disease (GVHD) in a bone marrow transplant recipient, comprising the steps of preconditioning bone marrow stem cell mixture with a composition comprising: granulocyte-macrophage colony stimulating factor (GMCSF); and either interleukin-3 (IL-3), interleukin-5 (IL-5) or both prior to transplantation; and transplanting the preconditioned bone marrow into the subject, thereby increasing the number and potency of regulatory T cells and inhibiting or suppressing graft-versus-host disease (GVHD) in a bone marrow transplant recipient.
  • GVHD graft-versus-host disease
  • the invention provides a method for inhibiting allograft rejection in solid organ transplantation in a subject, comprising the step of selectively enriching a T-cell population in the transplanted subject with regulatory T-cells by administering to the subject a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), thereby increasing the number of regulatory T-cells and suppressing naive T-cells and inhibiting allograft rejection in solid organ transplantation in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • the invention provides a method for inhibiting allograft rejection in solid organ transplantation in a subject, comprising the step of selectively enriching a T-cell population in the transplanted subject with regulatory T-cells by administering to the subject a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF); and either interleukin-3 (IL-3), interleukin-5 (IL-5) or both, thereby increasing the number of regulatory T-cells and suppressing na ⁇ ve T-cells and inhibiting allograft rejection in solid organ transplantation in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • IL-5 interleukin-5
  • the invention provides a method for inhibiting or suppressing allograft rejection in solid organ transplantation in a subject in need thereof, comprising the steps of obtaining a leukocyte cell population having antigen-presenting cells from a subject or pool of subjects; selectively enriching the leukocyte cell population in the subject with regulatory T-cells by contacting the leukocyte cell population with a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF); and either interleukin-3 (IL-3), interleukin-5 (EL-5) or both, thereby increasing the number of regulatory T-cells and suppressing naive T-cells; and administering to the subject in need thereof the resulting cell population enriched with regulatory T-cells, thus inhibiting or suppressing allograft rejection in solid organ transplantation in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • IL-3 interleukin-3
  • EL-5 interleukin-5
  • the invention provides a method for increasing transplant tolerance in a subject, comprising the step of selectively enriching a T-cell population in the transplanted subject with regulatory T-cells by administering to the subject a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF); and either interleukin-3 (IL-3), interleukin-5 (EL-5) or both, thereby increasing the number of regulatory T-cells and suppressing na ⁇ ve T-cells, thus increasing transplant tolerance in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • IL-3 interleukin-3
  • EL-5 interleukin-5
  • the invention provides a method for increasing transplant tolerance in a subject, comprising the steps of selectively enriching a T-cell population in the transplanted subject with regulatory T-cells by extracting leukocyte cells having antigen- presenting cells from the subject; selectively enriching a leukocyte cell population with regulatory T-cells by contacting the leukocyte cell population with a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF) and either interleukin-3 (IL-3), interleukin-5 (IL-5) or both; and administering the resulting cell population enriched with regulatory T-cells into the subject, thereby increasing the number of regulatory T-cells and suppressing naive T-cells and increasing transplant tolerance in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • IL-3 interleukin-3
  • IL-5 interleukin-5
  • the invention provides a method for increasing transplant tolerance in a subject, comprising the steps of: obtaining leukocyte cells having antigen- presenting cells from a subject or pool of subjects; selectively enriching the leukocyte cell population in the subject with regulatory T-cells by contacting the leukocyte cell population with a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF); and either interleukin-3 (IL-3), interleukin-5 (EL-5) or both, thereby increasing the number of regulatory T-cells and suppressing naive T-cells; and administering to the subject in need thereof the resulting cell population enriched with regulatory T-cells, thereby increasing transplant tolerance in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • IL-3 interleukin-3
  • EL-5 interleukin-5
  • the invention provides a method for increasing the suppressive capacity of freshly-isolated regulatory T-cells, comprising the step of contacting the freshly-isolated regulatory T-cells with a composition comprising: a granulocyte- macrophage colony stimulating factor (GMCSF); and either interleukin-3 (IL-3), interleukin-5 (IL-5) or both thereby increasing FoxP3 expression, CD25 expression or both and increasing the suppressive capacity of freshly-isolated regulatory T-cells.
  • GMCSF granulocyte- macrophage colony stimulating factor
  • IL-3 interleukin-3
  • IL-5 interleukin-5
  • the invention provides a method for activating regulatory T- cells in a subject, comprising the steps of: obtaining leukocyte cells having antigen- presenting cells from a subject or pool of subjects; and activating regulatory T-cells by contacting the leukocyte cell population with a composition comprising: a granulocyte- macrophage colony stimulating factor (GMCSF); and either interleukin-3 (IL-3), interleukin-5 (IL-5) or both, thereby activating the regulatory T-cells.
  • GMCSF granulocyte- macrophage colony stimulating factor
  • IL-3 interleukin-3
  • IL-5 interleukin-5
  • Figure 1 shows that leukocyte cell having antigen-presenting cells from Balb/c mice were stained with fluorescently labeled antibodies against CD4 and FoxP3 before (left plot) and after culture in GMCSF (right plot) for 4 days, and analyzed by flow cytometry. The number in the upper right corner represents the proportion of regulatory T cells (CD4+ /FoxP3+, right upper quadrant) out of total CD4+ T cells.
  • Figure 2 shows that leukocyte cell having antigen-presenting cells from Balb/c mice were labeled with CFSE dye and cultured in GMCSF, IL-3, or IL-5 for 4 days. The cells were then stained with fluorescently labeled antibodies against CD4 and FoxP3 and analyzed by flow cytometry. The number in the upper quadrants represents the proportion of proliferating (left quadrant) or non-proliferating (right quadrant) regulatory T cells (CD4+ /FoxP3+) out of total CD4+ T cells.
  • FIG. 3 shows that leukocyte cell having antigen-presenting cells from Balb/c mice were stained with fluorescently labeled antibodies against CD4, FoxP3, and CD25 before (solid line) and after culture in GMCSF (dotted) for 4 days.
  • the regulatory T cells CD4+ /FoxP3+
  • CD25 left plot
  • the mean fluorescence intensity (MFI) of FoxP3 for T cells before and after culture in GMCSF were 428 and 1307, respectively.
  • the MFI of CD25 for T cells before and after culture in GMCSF were 29 and 530, respectively.
  • FIG 4 shows that CD4+CD25- T cells were flow cytometry-sorted from Balb/c spleens and mixed at different ratios with regulatory T cells (Treg) that were flow cytometry-sorted from fresh Balb/c leukocyte cell having antigen-presenting cells (blue line) or Balb/c leukocyte cell having antigen-presenting cells cultured in GMCSF for 4 days (pink line).
  • the T cells were stimulated with anti-CD3 antibody and irradiated Balb/c leukocyte cell having antigen-presenting cells for 4 days. 24 hours before harvest, the cells were pulsed with 3H-thymidine and the incorporation of thymidine was quantified as a measure of proliferation.
  • the invention relates to methods for expanding the number of regulatory T-cells and uses thereof. Specifically, the invention relates to methods for selectively expanding a regulatory T-cell population by the use of a granulocyte-macrophage colony stimulating factor (GMCSF).
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of selectively expanding a regulatory T-cell in a subject comprising the step of contacting a leukocyte cell population having antigen-presenting cells in the subject a composition comprising: a granulocyte- macrophage colony stimulating factor (GMCSF); and either IL-3, IL-5 or both, thereby selectively expanding the number of regulatory T-cells while preserving the function of pathogen-reactive T cells.
  • GMCSF granulocyte- macrophage colony stimulating factor
  • a method of selectively expanding a regulatory T-cell in a subject comprising the step of contacting a leukocyte cell population having antigen-presenting cells in the subject with a composition comprising a granulocyte- macrophage colony stimulating factor (GMCSF), thereby selectively expanding the number of regulatory T-cells while preserving the function of pathogen-reactive T cells.
  • GMCSF granulocyte- macrophage colony stimulating factor
  • treating is directly contacting a regulatory T-cell with a granulocyte-macrophage colony stimulating factor (GMCSF); and interleukin (IL)-3, EL-5, or their combination.
  • treating is indirect contacting a regulatory T- cell with a granulocyte-macrophage colony stimulating factor (GMCSF); and EL-3, IL-5, or their combination.
  • expanding a regulatory T-cell is contacting an antigen presenting cell with a granulocyte-macrophage colony stimulating factor (GMCSF); and EL- 3, EL-5, or their combination.
  • activating a regulatory T-cell is contacting an antigen presenting cell with a granulocyte-macrophage colony stimulating factor (GMCSF); and IL-3, IL-5, or their combination.
  • a method of activating a regulatory T-cell comprising the step of contacting a regulatory T-cell with a granulocyte-macrophage colony stimulating factor (GMCSF); and IL-3, EL-5, or their combination, thereby activating a regulatory T-cell.
  • a method of activating a regulatory T-cell comprising the step of treating a regulatory T-cell with a granulocyte-macrophage colony stimulating factor (GMCSF); and IL-3, EL-5, or their combination, thereby activating a regulatory T-cell.
  • activating a regulatory T-cell is contacting an antigen presenting cell with a granulocyte-macrophage colony stimulating factor (GMCSF); and EL- 3, EL-5, or their combination.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • provided herein is a method of expanding a regulatory T-cell population comprising a set of specific phenotypic markers as described herein. In one embodiment, provided herein a method of selective expansion of a regulatory T-cell population. In one embodiment, provided herein a method of selective expansion of a regulatory T-cell population by using GMCSF. [0041] In another embodiment, provided herein is a method of enriching a regulatory T-cell population, comprising the step of treating a regulatory T-cell population with a granulocyte-macrophage colony stimulating factor (GMCSF); and IL-3, IL-5, or their combination, thereby enriching a regulatory T-cell population.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of enriching a regulatory T-cell population comprising the step of contacting a regulatory T-cell population with a granulocyte-macrophage colony stimulating factor (GMCSF); and EL-3, IL-5, or their combination, thereby enriching a regulatory T-cell population.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • enriching a regulatory T-cell population is contacting an antigen presenting cell with a granulocyte-macrophage colony stimulating factor (GMCSF); and IL-3, IL-5, or their combination.
  • the step of treating a regulatory T-cell population comprises contacting an antigen presenting cell with a granulocyte-macrophage colony stimulating factor (GMCSF); and IL-3, IL-5, or their combination.
  • a regulatory T-cell is a suppressor T cell.
  • a regulatory T-cell is a cell of the immune system that suppresses immune responses of other cells.
  • a regulatory T-cell is a CD8 + T cell.
  • a regulatory T-cell is a CD4 + CD25 + T cell.
  • a regulatory T-cell is a CD4 + CD25 + Foxp3 + T-cell.
  • a regulatory T-cell is a CD4 + Foxp3 + T-cell.
  • a regulatory T-cell has a larger T-cell receptor (TCR) diversity than effector T cells, biased towards self-peptides.
  • TCR T-cell receptor
  • enriching a regulatory T-cell population is selectively enriching a regulatory T-cell.
  • enriching a regulatory T-cell population is enriching a regulatory T-cell and not a T-helper cell, a cytotoxic T-cell, or both.
  • enriching a regulatory T-cell population comprises breaking
  • enriching a regulatory T- cell population comprises triggering proliferation of a regulatory T-cell population. In another embodiment, enriching a regulatory T-cell population comprises triggering proliferation of a mature regulatory T-cell population.
  • enriching a regulatory T-cell comprises triggering proliferation of a regulatory T-cell. In another embodiment, enriching a regulatory T-cell comprises triggering proliferation of a mature regulatory T-cell. In another embodiment, expanding a regulatory T-cell comprises triggering proliferation of a regulatory T-cell. In another embodiment, expanding a regulatory T-cell comprises triggering proliferation of a mature regulatory T-cell.
  • expanding a regulatory T-cell is selectively expanding a regulatory T-cell.
  • expanding a regulatory T-cell is expanding a regulatory T-cell and not a T-helper cell, a cytotoxic T-cell, or both.
  • expanding a regulatory T-cell comprises breaking CD4 + CD25 + regulatory T- cell anergy.
  • expanding a regulatory T-cell comprises triggering proliferation of a regulatory T-cell.
  • activated regulatory T-cell is induced to produce IL-2.
  • expanding regulatory T-cell results in induction of CD25 expression in a regulatory T-cell.
  • expanding a regulatory T-cell is inducing the capacity of a regulatory T-cell.
  • inducing the capacity of a regulatory T-cell comprises induction of CD25 expression, breaking CD4 + CD25 + regulatory T-cell anergy, induction of IL2 production (expression).
  • enriching a regulatory T-cell population comprises inducing CD25 expression in a regulatory T-cell population. In another embodiment, enriching a regulatory T-cell population comprises inducing Foxp3 expression in a regulatory T-cell population.
  • a regulatory T-cell population is not an isolated regulatory T-cell population.
  • a regulatory T-cell population is comprised within a mixed T-cell population.
  • a regulatory T-cell population is comprised within a mixed cell population.
  • a regulatory T-cell population is comprised within a leukocyte cell having antigen-presenting cells cell population.
  • a regulatory T-cell population is comprised within a mixed immune cell population.
  • enriching a regulatory T-cell population is selectively enriching a regulatory T-cell population.
  • enriching a regulatory T- cell population is selectively inducing proliferation of a regulatory T-cell population.
  • enriching a regulatory T-cell population is enriching a regulatory T- cell population but not T helper cell population, cytotoxic T-cell population, or both.
  • enriching a regulatory T-cell population is selectively inducing proliferation of a regulatory T-cell population.
  • enriching a regulatory T-cell population is ex-vivo culturing a mixed primary cell culture and contacting a mixed primary cell culture with a granulocyte-macrophage colony stimulating factor (GMCSF); and IL-3, IL-5, or their combination.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • Selectively enriching the T-cell population refers in one embodiment to a relative increase in regulatory T-cell accompanied with a decrease in naive T-cell while preserving the function of pathogen-reactive T-cells.
  • enriching a regulatory T-cell population is ex-vivo culturing a mixed primary cell culture and treating a mixed primary cell culture with a granulocyte- macrophage colony stimulating factor (GMCSF); and EL-3, EL-5, or their combination.
  • GMCSF granulocyte- macrophage colony stimulating factor
  • nucleic acid and amino acid sequences and their variants for GMCSF, IL-3, and EL-5 are well known in the art. These sequences are publicly available in the databases of National Center for Biotechnology Information (NCBI). In one embodiment, any variant of GMCSF, EL-3, and/or EL-5 that retains their function can be used in the invention.
  • GMCSF is a recombinant GMCSF.
  • GMCSF is in the form of Leukine.
  • Leukine is the trade name of sargramostim manufactured by Berlex Laboratories, a subsidiary of Schering AG.
  • an activated regulatory T-cell steers monocyte differentiation toward alternatively activated macrophages (AAM).
  • AAM alternatively activated macrophages
  • an activated regulatory T-cell inhibits the production of pro-inflammatory mediators such as EL- l ⁇ , EL-6, EL-8, MEP- l ⁇ , TNF- ⁇ by monocytes and/or macrophages.
  • an activated regulatory T-cell produces EL-10, EL-4, and EL-13.
  • an enriched regulatory T-cell population steers monocyte differentiation toward alternatively activated macrophages (AAM).
  • AAM alternatively activated macrophages
  • an enriched regulatory T-cell population inhibits the production of pro-inflammatory mediators such as EL-l ⁇ , EL-6, EL-8, MEP-l ⁇ , TNF- ⁇ by monocytes and/or macrophages.
  • an enriched regulatory T-cell population produces EL-IO, EL-4, and EL-13.
  • a regulatory T-cell is grown in a cell culture-in vitro.
  • a regulatory T-cell is grown in an isolated regulatory T-cell culture-in vitro.
  • a regulatory T-cell is grown in an enriched regulatory T-cell culture-in vitro.
  • a regulatory T-cell is grown in a mixed cell culture-in vitro.
  • a regulatory T-cell is grown ex-vivo in a primary cell culture.
  • a regulatory T-cell is present within the immune system of a subject.
  • a subject is a human being.
  • a subject is a primate.
  • a subject is a pet.
  • a subject is a farm animal. In another embodiment, a subject is a lab animal. In another embodiment, a subject is a rat. In another embodiment, a subject is a mouse. In another embodiment, a subject is a guinea pig.
  • the composition comprising GMCSF, EL-3, EL-5 or their combination, is used in the methods provided herein.
  • a method of inhibiting allograft rejection in solid organ transplantation in a subject comprising the step of selectively enriching a T-cell population in the transplanted subject with regulatory T-cells by contacting the T-cell population with a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), thereby increasing the number of regulatory T-cells and suppressing na ⁇ ve T-cells.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of inhibiting or suppressing graft-versus- host disease (GVHD) in a bone marrow transplant recipient comprising the step of preconditioning bone marrow stem cell mixture with a composition comprising granulocyte-macrophage colony stimulating factor (GMCSF), IL-3 and IL-5 prior to transplantation thereby increasing the number and potency of regulatory T cells.
  • GVHD graft-versus- host disease
  • a method of treating an autoimmune pathology in a subject comprising the step of selectively enriching a T-cell population in the subject with regulatory T-cells by contacting the T-cell population with a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), thereby increasing the number of regulatory T-cells and suppressing na ⁇ ve T-cells.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of suppressing a T-cell activation mediated response comprising the step of contacting the T-cell population with a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), thereby increasing the number of regulatory T-cells and suppressing na ⁇ ve T-cells in the T-cell population.
  • a method of enriching a regulatory T-cell population in a subject comprising the step of contacting the population with a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), thereby increasing the number of regulatory T-cells and suppressing na ⁇ ve T-cells.
  • a method of increasing the suppressive capacity of freshly- isolated regulatory T-cells comprising the step of contacting the freshly-isolated regulatory T-cells with a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), IL-3 and EL-5 thereby increasing FoxP3 expression, CD25 expression or both.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • autoimmunity refers to a persistent and progressive immune reactions to non infectious self antigens, as distinct from infectious non self antigens from bacterial, viral, fungal, or parasitic organisms which invade and persist within mammals and humans.
  • Autoimmune conditions include scleroderma, Grave's disease, Crohn's disease, Sjorgen's disease, multiple sclerosis, Hashimoto's disease, psoriasis, myasathenia gravis, Autoimmune Polyendocrinopathy syndromes, Type I diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveoretinitis, polymyositis, colitis, and thyroiditis, as well as in the generalized autoimmune diseases typified by human Lupus.
  • TIDM Type I diabetes mellitus
  • autoimmune gastritis autoimmune uveoretinitis
  • polymyositis polymyositis
  • colitis colitis
  • thyroiditis as well as in the general
  • a regulatory T-cell population is grown in a cell culture-in vitro.
  • a regulatory T-cell population is an isolated regulatory T-cell population in culture-in vitro.
  • a regulatory T-cell population is grown in culture with other cell types.
  • a regulatory T-cell population is grown ex-vivo.
  • a regulatory T-cell population is enriched ex-vivo.
  • a regulatory T-cell population is present within the immune system of a subject.
  • the disclosed methods and compositions are useful for: 1. Induction of tolerance to autoimmune target antigens to prevent/treat various autoimmune conditions; 2. Induction of tolerance to allo- or xeno- antigens to prevent rejection of transplanted tissue like, pancreatic islets, kidney, heart, liver, intestine, skin, and the like; 3. Induction of tolerance to allergens; 4. Induction of tolerance to the product of transgenes and their carrier vectors in gene therapy applications; 5. Treatment of graft versus host disease; 6. Induction of tolerance to tolerance to inflammatory conditions such as inflammatory bowel disease; 7. Induction of tolerance to bacterial or viral pathogens that act as inducers of autoimmune disease or inflammatory immune mediated diseases through molecular mimicry such as ulcerative colitis in one embodiment..
  • CD8 T cells Methods of segregating CD8 T cells into + and - categories are known to persons of ordinary skill in the art.
  • the frequency distribution of the CD8 staining is obtained for all the cells and the population curve fit to a higher staining and lower staining population, and cells assigned to the population to which they most statistically are likely to belong in view of a statistical analysis of the respective population distributions.
  • the CD8 + cells stain two to three fold more intensely than the CD8 " cells. Particularly preferred methods are also exemplified in the Examples.
  • regulatory T cells are critical regulators of immune tolerance.
  • Treg are defined based on expression of CD4, CD25 and the transcription factor, FoxP3. Recent information has identified the importance of the gene Foxpro3 which is induced in one embodiment by thymus epithelium to cause T cells to develop CD25 + CD4 + regulatory T cells, in animal models of autoimmune diseases.
  • Deficiency of this gene lead in certain embodiments to wide spread autoimmune phenomena and diseases.
  • treating a regulatory T-cell or a regulatory T-cell population with a compound of the invention such as but not limited to GMCSF activates a regulatory T-cell.
  • treating a regulatory T-cell or a regulatory T-cell population comprises activating an APC with a compound of the invention such as but not limited to GMCSF.
  • contacting a regulatory T-cell or a regulatory T-cell population with a compound of the invention such as but not limited to GMCSF activates a regulatory T-cell.
  • indirect contacting a regulatory T-cell or a regulatory T-cell population with a compound of the invention such as but not limited to GMCSF comprises administering the compound mixed in a cell culture media to a cell culture comprising a regulatory T-cell.
  • indirect contacting a regulatory T-cell or a regulatory T-cell population with a compound of the invention such as but not limited to GMCSF is known to one of skill in the art.
  • Regulatory T cells are either generic in one embodiment, or antigen specific in another embodiment.
  • regulatory T cells produce TGF- ⁇ and/or IL-10 through which Tregs can suppress immune responses.
  • treating a regulatory T-cell or a regulatory T-cell population by activating an APC with a compound of the invention such as but not limited to GMCSF is known to one of average skill in the art.
  • activating a regulatory T-cell or a regulatory T-cell population is activating an APC, with a compound of the invention such as but not limited to GMCSF, within the immune system of a subject.
  • GMCSF is a cytokine that functions as a white blood cell growth factor.
  • GMCSF is glycosylated.
  • the glycosylation sites are at amino acid residues 23 (leucine), 27 (asparagine), 39 (glutamic acid), or any combination thereof.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 50-5000 mcg/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 50-200 mcg/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 100-500 mcg/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 300-800 mcg/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 400-1000 mcg/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 800-1500 mcg/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 1000-2000 mcg/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 1500-3000 mcg/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 2000-4000 mcg/mL.
  • a regulatory T- cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 3000-5000 mcg/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 1 x 10 4 to 1 x 10 s IU/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 1 x 10 4 to 1 x 10 5 IU/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 1 x 10 5 to 1 x 10 6 IU/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 5 x 10 5 to 5 x 10 7 IU/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 1 x 10 7 to 1 x 10 8 IU/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 1 x 10 4 to 1 x 10 6 IU/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 0.5-250 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 0.5-10 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 5-25 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 25-50 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 25-75 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 50-100 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 75-150 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 100-200 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with GMCSF at a concentration of 175-250 ng/mL.
  • a regulatory T-cell present within the immune system of a subject is contacted with GMCSF by administering to a subject a dose of GMCSF ranging from 20 to 400 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with GMCSF by a subcutaneous or intravenous injection of GMCSF in a dose ranging from 20 to 400 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with GMCSF by a subcutaneous or intravenous injection of GMCSF in a dose ranging from 20 to 100 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with GMCSF by a subcutaneous or intravenous injection of GMCSF in a dose ranging from 50 tol50 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with GMCSF by a subcutaneous or intravenous injection of GMCSF in a dose ranging from 100 to 200 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with GMCSF by a subcutaneous or intravenous injection of GMCSF in a dose ranging from 150 to 250 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with GMCSF by a subcutaneous or intravenous injection of GMCSF in a dose ranging from 200 to 300 mcg/kg/day. In another embodiment, a regulatory T-cell present within the immune system of a subject is contacted with GMCSF by a subcutaneous or intravenous injection of GMCSF in a dose ranging from 250 to 400 mcg/kg/day.
  • GMCSF induces the expansion of a regulatory T-cell but not of a T helper cell and/or a cytotoxic T-call. In another embodiment, GMCSF induces the proliferation of a regulatory T-cell but not of a T helper cell and/or a cytotoxic T-call. In another embodiment, GMCSF activates a regulatory T-cell but not a T helper cell and/or a cytotoxic T-call. In another embodiment, GMCSF activates a mature regulatory T-cell but not a mature T helper cell and/or a mature cytotoxic T-call.
  • a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF) and either EL-3 or BL-5 or both is used in the methods provided herein.
  • the composition comprises a granulocyte-macrophage colony stimulating factor (GMCSF) and IL-3, or in another embodiment a granulocyte-macrophage colony stimulating factor (GMCSF) and IL-5 or in another embodiment, a granulocyte- macrophage colony stimulating factor (GMCSF) and IL-3 and EL-5.
  • a method of selectively expanding a regulatory T-cell in a subject comprising the step of contacting a leukocyte cell population having antigen-presenting cells in the subject with a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), thereby selectively expanding the number of regulatory T-cells while preserving the function of pathogen-reactive T cells.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of selectively expanding a regulatory T-cell in a subject comprising the step of contacting a leukocyte cell population having antigen-presenting cells in the subject a composition comprising: a granulocyte- macrophage colony stimulating factor (GMCSF); and either IL-3, IL-5 or both, thereby selectively expanding the number of regulatory T-cells while preserving the function of pathogen-reactive T cells.
  • GMCSF granulocyte- macrophage colony stimulating factor
  • a method of enriching a T-cell population with regulatory T-cells in a subject comprising the step of contacting a population of leukocyte cells having antigen-presenting cells with a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), thereby increasing the number of regulatory T-cells and suppressing naive T-cells thus enriching a T-cell population with regulatory T-cells.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • A. method of enriching a T-cell population with regulatory T-cells in a subject comprising the step of contacting a leukocyte cell having antigen-presenting cells with a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF); and either BL-3, IL-5 or both, thereby increasing the number of regulatory T-cells and suppressing na ⁇ ve T-cells thus enriching a T-cell population with regulatory T-cells.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of suppressing T-cell activation mediated response in a subject comprising the step of administering to the subject a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), thereby increasing the number of regulatory T-cells and suppressing na ⁇ ve T-cells.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of suppressing a T-cell activation mediated response in a subject comprising the step of administering to the subject a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF); and either DL-3, IL-5 or both, thereby increasing the number of regulatory T- cells and suppressing na ⁇ ve T-cells thus suppressing a T-cell activation mediated response in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of suppressing a T-cell activation mediated response in a subject in need thereof comprising the steps of: obtaining leukocyte cell population having antigen-presenting cells from a subject or pool of subjects; contacting the cell population with a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF) and either IL-3, BL-5 or both, thereby increasing the number of regulatory T-cells and suppressing na ⁇ ve T-cells; and administering to the subject in need thereof the resulting cell population with increased regulatory T-cell number, thus suppressing a T-cell activation mediated response in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of treating an autoimmune pathology in a subject comprising the step of selectively enriching a T-cell population in the subject with regulatory T-cells by administering to the subject a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), thereby increasing the number of regulatory T-cells and suppressing na ⁇ ve T-cells.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of treating an autoimmune pathology in a subject comprising the step of selectively enriching a T-cell population in the subject with regulatory T-cells by administering to the subject a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF); and either EL- 3, EL-5 or both, thereby increasing the number of regulatory T-cells and suppressing na ⁇ ve T-cells and treating an autoimmune pathology in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of treating an autoimmune pathology in a subject in need thereof comprising the steps of: obtaining a leukocyte cell population having antigen-presenting cells from a subject or pool of subjects; selectively enriching the leukocyte cell population in the subject with regulatory T-cells by contacting the leukocyte cell population with a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF); and either IL-3, IL-5 or both, thereby increasing the number of regulatory T-cells and suppressing naive T-cells; and administering to the subject in need thereof the resulting cell population enriched with regulatory T-cells, thereby treating an autoimmune pathology in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of inhibiting or suppressing graft-versus-host disease (GVHD) in a bone marrow transplant recipient comprising the steps of preconditioning bone marrow stem cell mixture with a composition comprising: granulocyte-macrophage colony stimulating factor (GMCSF); and either IL-3, BL-5 or both prior to transplantation; and transplanting the preconditioned bone marrow into the subject, thereby increasing the number and potency of regulatory T cells and inhibiting or suppressing graft-versus-host disease (GVHD) in a bone marrow transplant recipient.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of inhibiting allograft rejection in solid organ transplantation in a subject comprising the step of selectively enriching a T- cell population in the transplanted subject with regulatory T-cells by administering to the subject a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF), thereby increasing the number of regulatory T-cells and suppressing naive T- cells and inhibiting allograft rejection in solid organ transplantation in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of inhibiting allograft rejection in solid organ transplantation in a subject comprising the step of selectively enriching a T- cell population in the transplanted subject with regulatory T-cells by administering to the subject a composition comprising: a granulocyte-macrophage colony stimulating factor
  • GMCSF GMCSF
  • EL-3, BL-5 or both thereby increasing the number of regulatory T- cells and suppressing naive T-cells and inhibiting allograft rejection in solid organ transplantation in a subject.
  • a method of inhibiting or suppressing allograft rejection in solid organ transplantation in a subject in need thereof comprising the steps of obtaining a leukocyte cell population having antigen-presenting cells from a subject or pool of subjects; selectively enriching the leukocyte cell population in the subject with regulatory T-cells by contacting the leukocyte cell population with a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF); and either IL- 3, IL-5 or both, thereby increasing the number of regulatory T-cells and suppressing naive T-cells; and administering to the subject in need thereof the resulting cell population enriched with regulatory T-cells, thus inhibiting or suppressing allograft rejection in solid organ transplantation in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of increasing transplant tolerance in a subject comprising the step of selectively enriching a T-cell population in the transplanted subject with regulatory T-cells by administering to the subject a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF); and either IL- 3, IL-5 or both, thereby increasing the number of regulatory T-cells and suppressing naive T-cells, thus increasing transplant tolerance in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of increasing transplant tolerance in a subject comprising the steps of selectively enriching a T-cell population in the transplanted subject with regulatory T-cells by extracting leukocyte cells having antigen-presenting cells from the subject; selectively enriching a leukocyte cell population with regulatory T-cells by contacting the leukocyte cell population with a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF) and either IL-3, IL-5 or both; and administering the resulting cell population enriched with regulatory T- cells into the subject, thereby increasing the number of regulatory T-cells and suppressing na ⁇ ve T-cells and increasing transplant tolerance in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of increasing transplant tolerance in a subject comprising the steps of: obtaining leukocyte cells having antigen- presenting cells from a subject or pool of subjects; selectively enriching the leukocyte cell population in the subject with regulatory T-cells by contacting the leukocyte cell population with a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF); and either EL-3, IL-5 or both, thereby increasing the number of regulatory T- cells and suppressing na ⁇ ve T-cells; and administering to the subject in need thereof the resulting cell population enriched with regulatory T-cells, thereby increasing transplant tolerance in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of increasing the suppressive capacity of freshly-isolated regulatory T-cells comprising the step of contacting the freshly- isolated regulatory T-cells with a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF); and either EL-3, BL-5 or both thereby increasing FoxP3 expression, CD25 expression or both and increasing the suppressive capacity of freshly- isolated regulatory T-cells.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of activating regulatory T-cells in a subject comprising the steps of: obtaining leukocyte cells having antigen-presenting cells from a subject or pool of subjects; and activating regulatory T-cells by contacting the leukocyte cell population with a composition comprising: a granulocyte-macrophage colony stimulating factor (GMCSF); and either IL-3, IL-5 or both, thereby activating the regulatory T-cells.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • GMCSF enriches a regulatory T-cell population but not a T helper cell population and/or a cytotoxic T-cell population. In another embodiment, GMCSF enriches a mature regulatory T-cell population but not a mature T helper cell population and/or a mature cytotoxic T-cell population.
  • interleukin (IL)-3 is a recombinant IL-3.
  • EL-3 is a human Interleukin 3 protein.
  • IL-3 is a mouse Interleukin 3 protein.
  • IL-3 is a mutant or a variant of human interleukin-3 which contain multiple amino acid substitutions and which may have portions of the native hIL-3 molecule deleted.
  • hIL-3 multiple mutation polypeptides retain one or more activities of native hTL-3.
  • hIL-3 multiple mutation polypeptides show improved regulatory T-cell stimulating activity and/or an improved activity profile, and/or proliferation.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-3 at a concentration of 50-5000 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-3 at a concentration of 50-200 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-3 at a concentration of 75-150 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-3 at a concentration of 100-200 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-3 at a concentration of 150-300 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with IL-3 at a concentration of 200-400 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with BL-3 at a concentration of 300-500 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with IL-3 at a concentration of 400-800 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with IL-3 at a concentration of 600-1000 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with IL-3 at a concentration of 800-1500 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-3 at a concentration of 1000-2000 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-3 at a concentration of 1500-3000 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with BL-3 at a concentration of 3000-4000 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-3 at a concentration of 4000-5000 ng/mL.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-3 by administering to a subject a dose of EL-3 ranging from 0.5 to 50 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-3 by a subcutaneous or intravenous injection of EL- 3 in a dose ranging from 0.5 to 50 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-3 by a subcutaneous or intravenous injection of EL-3 in a dose ranging from 0.5 to 2.5 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-3 by a subcutaneous or intravenous injection of EL-3 in a dose ranging from 1 to 5 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-3 by a subcutaneous or intravenous injection of EL-3 in a dose ranging from 3 to 10 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-3 by a subcutaneous or intravenous injection of IL-3 in a dose ranging from 8 to 12 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-3 by a subcutaneous or intravenous injection of EL-3 in a dose ranging from 10 to 20 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-3 by a subcutaneous or intravenous injection of EL- 3 in a dose ranging from 15 to 30 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-3 by a subcutaneous or intravenous injection of EL-3 in a dose ranging from 20 to 40 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-3 by a subcutaneous or intravenous injection of IL-3 in a dose ranging from 40 to 50 mcg/kg/day.
  • EL-3 activates a regulatory T-cell but not a T helper cell and/or a cytotoxic T-cell.
  • IL-3 activates a mature regulatory T-cell but not a mature T helper cell and/or a mature cytotoxic T-cell.
  • IL-3 enriches a regulatory T-cell population but not a T helper cell population and/or a cytotoxic T-cell population. In another embodiment, IL-3 enriches a mature regulatory T-cell population but not a mature T helper cell population and/or a mature cytotoxic T-cell population.
  • interleukin (DL)-5 is a recombinant EL-5.
  • EL-5 is a human Interleukin 3 protein.
  • EL-5 is a mouse Interleukin 3 protein.
  • EL-5 is a mutant or a variant of human interleukin-3 which contain multiple amino acid substitutions and which may have portions of the native hEL-5 molecule deleted.
  • hEL-5 multiple mutation polypeptides retain one or more activities of native hEL-5.
  • hEL-5 multiple mutation polypeptides show improved regulatory T-cell stimulating activity and/or an improved activity profile, and/or proliferation.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-5 at a concentration of 50-5000 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-5 at a concentration of 50-200 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-5 at a concentration of 75-150 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-5 at a concentration of 100-200 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-5 at a concentration of 150-300 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-5 at a concentration of 200-400 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-5 at a concentration of 300-500 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with IL-5 at a concentration of 400-800 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with IL-5 at a concentration of 600-1000 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with IL-5 at a concentration of 800-1500 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with IL-5 at a concentration of 1000-2000 ng/mL. In another embodiment, a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with IL-5 at a concentration of 1500-3000 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with IL-5 at a concentration of 3000-4000 ng/mL.
  • a regulatory T-cell or a regulatory T-cell population in a cell culture is contacted with EL-5 at a concentration of 4000-5000 ng/mL.
  • a regulatory T-cell present within the immune system of a subject is contacted with IL-5 by administering to a subject a dose of IL-5 ranging from 0.5 to 50 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-5 by a subcutaneous or intravenous injection of EL- 5 in a dose ranging from 0.5 to 50 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-5 by a subcutaneous or intravenous injection of EL-5 in a dose ranging from 0.5 to 2.5 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-5 by a subcutaneous or intravenous injection of EL-5 in a dose ranging from 1 to 5 mcg/kg/day. In another embodiment, a regulatory T-cell present within the immune system of a subject is contacted with EL-5 by a subcutaneous or intravenous injection of EL-5 in a dose ranging from 3 to 10 mcg/kg/day. In another embodiment, a regulatory T-cell present within the immune system of a subject is contacted with EL-5 by a subcutaneous or intravenous injection of IL-5 in a dose ranging from 8 to 12 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-5 by a subcutaneous or intravenous injection of EL-5 in a dose ranging from 10 to 20 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-5 by a subcutaneous or intravenous injection of EL- 5 in a dose ranging from 15 to 30 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with EL-5 by a subcutaneous or intravenous injection of EL-5 in a dose ranging from 20 to 40 mcg/kg/day.
  • a regulatory T-cell present within the immune system of a subject is contacted with BL-5 by a subcutaneous or intravenous injection of IL-5 in a dose ranging from 40 to 50 mcg/kg/day.
  • BL-5 activates a regulatory T-cell but not a T helper cell and/or a cytotoxic T-call.
  • IL-5 activates a mature regulatory T-cell but not a mature T helper cell and/or a mature cytotoxic T-call.
  • BL-5 enriches a regulatory T-cell population but not a T helper cell population and/or a cytotoxic T-cell population.
  • EL-5 enriches a mature regulatory T-cell population but not a mature T helper cell population and/or a mature cytotoxic T-cell population.
  • EL-5, IL-3, GMCSF, or any combination thereof activate a regulatory T-cell but not a T helper cell and/or a cytotoxic T-call.
  • IL-5, IL-3, GMCSF, or any combination thereof activate a mature regulatory T-cell but not a mature T helper cell and/or a mature cytotoxic T-cell.
  • contacting BL-5, IL-3, GMCSF, or any combination thereof with a mixed cell population enrich a regulatory T-cell population but not a T helper cell population and/or a cytotoxic T-cell population.
  • contacting BL-5, IL-3, GMCSF, or any combination thereof with a mixed cell population enrich a regulatory T-cell population but not a T helper cell population and/or a cytotoxic T-cell population.
  • IL-3, GMCSF, or any combination thereof with a mixed cell population induces the expansion of a regulatory T-cell population but not a T helper cell population and/or a cytotoxic T-cell population.
  • administration of BL-5, IL-3, GMCSF, or any combination thereof to a leukocyte cell having antigen-presenting cells cell culture enrich a mature regulatory T-cell population but not a mature T helper cell population and/or a mature cytotoxic T-cell population.
  • contacting a mixed cell population comprising a plurality of cells and a regulatory T-cell population with IL-5, IL-3, GMCSF, or any combination thereof results in enrichment of the regulatory T-cell population.
  • contacting a mixed cell population comprising a plurality of cells and a regulatory T-cell population with IL-5, IL-3, GMCSF, or any combination thereof results in expansion of the regulatory T-cell population.
  • contacting a mixed cell population comprising a plurality of cells and a regulatory T-cell population with BL-5, EL-3, GMCSF, or any combination thereof results in the proliferation of the regulatory T-cell population.
  • contacting a mixed cell population comprising a plurality of cells and a regulatory T-cell population with IL-5, EL-3, GMCSF, or any combination thereof, results in activation of the regulatory T-cell population.
  • contacting a mixed cell population consisting: a regulatory T-cell population, a T helper cell population, and/or a cytotoxic T-cell population with EL-5, JL-3, GMCSF, or any combination thereof, results in enrichment of the regulatory T-cell population.
  • contacting a mixed cell population consisting: a regulatory T-cell population, a T helper cell population, and/or a cytotoxic T-cell population with IL-5, EL-3, GMCSF, or any combination thereof, results in selective enrichment of the regulatory T-cell population.
  • a method of suppressing a T cell activation response comprising the step of treating a regulatory T-cell or a regulatory T-cell population with a granulocyte-macrophage colony stimulating factor (GMCSF); and JL-3, EL-5, or their combination, thereby suppressing a T cell activation response.
  • a T cell is a T helper cell (CD4+) or cytotoxic T cell (CD8+).
  • a method of suppressing a T cell activation response comprising the step of contacting a regulatory T-cell or a regulatory T-cell population with a granulocyte-macrophage colony stimulating factor (GMCSF); and IL-3, IL-5, or their combination, thereby suppressing a T cell activation response.
  • a T cell is a T helper cell (CD4+) or cytotoxic T cell (CD8+).
  • a method of suppressing a T cell activation response comprising the step of indirect contacting a regulatory T-cell or a regulatory T-cell population with a granulocyte- macrophage colony stimulating factor (GMCSF); and IL-3, EL-5, or their combination, thereby suppressing a T cell activation response.
  • a method of suppressing a T cell activation response comprising the step of contacting an APC with a granulocyte-macrophage colony stimulating factor (GMCSF); and EL-3, EL-5, or their combination, thereby suppressing a T cell activation response.
  • a T cell is a T helper cell (CD4+) or cytotoxic T cell (CD8+).
  • suppressing a T cell activation response comprises suppressing the interactions of the T cell receptor with pMHC molecules. In another embodiment, suppressing a T cell activation response comprises suppressing a T cell activation response to pathogen-derived pMHC molecules. In another embodiment, suppressing a T cell activation response comprises suppressing a T cell activation response to endogenous pMHC molecules. In another embodiment, suppressing a T cell activation response comprises suppressing the engagement of both the T cell receptor and CD28 on the T cell by the Major histocompatibility complex peptide and B7 family members on the APC, respectively.
  • suppressing a T cell activation response comprises suppressing the differentiation of CD4 positive T helper (Th) cells into either ThI or Th2 cells.
  • suppressing a T cell activation response comprises suppressing a humoral immune response.
  • suppressing a T cell activation response comprises suppressing a cytotoxic immune response.
  • suppressing a T cell activation response comprises suppressing a cell- mediated immune response.
  • suppressing a T cell activation response comprises suppressing a delayed type of hypersensitivity (DTH) reaction.
  • suppressing a T cell activation response comprises suppressing macrophage activation.
  • suppressing a T cell activation response comprises antagonizing pro-inflammatory cytokines such as IL-6, TNF and BL-I.
  • suppressing a T cell activation response comprises suppressing T-cell division. In another embodiment, suppressing a T cell activation response comprises suppressing T-cell secretion of cytokines that upregulate the immune response. In another embodiment, suppressing a T cell activation response comprises suppressing co-stimulation by CD80 and CD86 proteins. In another embodiment, suppressing a T cell activation response comprises suppressing T-cell expression of OX40 and/or ICOS. In another embodiment, suppressing a T cell activation response comprises suppressing ITAM motifs phosphorylation. In another embodiment, suppressing a T cell activation response comprises suppressing PKC ⁇ . In another embodiment, suppressing a T cell activation response comprises suppressing the activation of the transcription factor NFAT.
  • a method of inducing transplant tolerance, counteracting autoimmunity, or inhibiting a graft versus host disease in a subject comprising the step of contacting an APC with a granulocyte-macrophage colony stimulating factor (GMCSF); and BL-3, IL-5, or their combination, thereby inducing transplant tolerance, counteracting autoimmunity, or inhibiting a graft versus host disease in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of inducing transplant tolerance, counteracting autoimmunity, or inhibiting a graft versus host disease in a subject comprising the step of treating a regulatory T-cell or a regulatory T-cell population with a granulocyte-macrophage colony stimulating factor (GMCSF); and EL-3, EL-5, or their combination, thereby inducing transplant tolerance, counteracting autoimmunity, or inhibiting a graft versus host disease in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of inducing transplant tolerance, counteracting autoimmunity, or inhibiting a graft versus host disease in a subject comprising the step of contacting a regulatory T-cell or a regulatory T-cell population with a granulocyte-macrophage colony stimulating factor (GMCSF); and BL-3, EL-5, or their combination, thereby inducing transplant tolerance, counteracting autoimmunity, or inhibiting a graft versus host disease in a subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • a method of suppressing overactivation of T cells comprising the step of contacting an APC with a granulocyte-macrophage colony stimulating factor (GMCSF); and DL-3, IL-5, or their combination, thereby suppressing overactivation of T cells.
  • a method of suppressing overactivation of T cells comprising the step of contacting a regulatory T-cell or a regulatory T-cell population with a granulocyte-macrophage colony stimulating factor (GMCSF); and IL-3, IL-5, or their combination, thereby suppressing overactivation of T cells.
  • transplant tolerance is the lack of a destructive immune response toward the graft in the absence of ongoing immunosuppressive therapy.
  • transplant tolerance is manifested clinically by normal graft function in the absence of acute and chronic rejection.
  • transplant tolerance coexists with general immune competence.
  • transplant tolerance coexists with normal immune responses to pathogens.
  • transplant tolerance comprises clonal deletion, hi another embodiment, transplant tolerance comprises clonal anergy. In another embodiment, transplant tolerance comprises clonal regulation/suppression. In another embodiment, transplant tolerance comprises profound reduction in clonal T cell expansion, hi another embodiment, transplant tolerance comprises profound reduction in clonal T cell expansion accompanied by active immune regulation.
  • transplant tolerance is the complete and successful withdrawal of treatment with immunosuppressive medications.
  • transplant tolerance suppresses acute rejection episodes, hi another embodiment, transplant tolerance suppresses development of chronic allograft rejection, hi another embodiment, transplant tolerance promotes the graft's function.
  • transplant tolerance suppresses recognition of alloantigen occurs.
  • transplant tolerance suppresses CD4 + T-cell differentiaion and/or maturation.
  • autoimmunity is the failure of an organism to recognize its own constituent parts (down to the sub-molecular levels) as self, which results in an immune response against its own cells and tissues.
  • autoimmunity comprises autoimmune disease such as but not limited to Coeliac disease, diabetes mellitus type 1 (IDDM), systemic lupus erythematosus (SLE), Sjogren's syndrome, Churg-Strauss Syndrome, multiple sclerosis (MS), Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, and rheumatoid arthritis (RA).
  • IDDM diabetes mellitus type 1
  • SLE systemic lupus erythematosus
  • MS multiple sclerosis
  • Graves' disease idiopathic thrombocytopenic purpura
  • RA rheumatoid arthritis
  • a graft-versus-host disease is a common complication of allogeneic bone marrow transplantation in which functional immune cells in the transplanted marrow recognize the recipient as "foreign" and mount an immunologic attack.
  • a graft-versus-host disease is the acute or fulminant form of the disease (aGVHD).
  • a graft-versus-host disease is the chronic form of graft-versus-host-disease (cGVHD).
  • GVHD is grade I GVHD. In another embodiment, GVHD is grade II GVHD. In another embodiment, GVHD is grade m GVHD. In another embodiment, GVHD is grade IV GVHD. In another embodiment, GVHD is a transfusion- associated GVHD.
  • a method for inducing the proliferation of regulatory T cells with enhanced function comprising contacting an APC with GMCSF, IL- 5, IL-3, or any combination thereof.
  • a method for expanding and/or enriching regulatory T cells comprising contacting an APC with GMCSF, IL- 5, IL-3, or any combination thereof.
  • a method for expanding and/or enriching regulatory T cells (FoxP3+CD4+ T cells) ex vivo.
  • a method for inducing the proliferation of regulatory T cells with enhanced function comprising contacting a regulatory T-cell with GMCSF, BL-5, EL-3, or any combination thereof.
  • a method for expanding and/or enriching regulatory T cells (FoxP3+CD4+ T cells) ex vivo.
  • a method comprising the use of GMCSF/IL-5/EL-3 as an administrative therapy to expand, proliferate or enrich human regulatory T cells FoxP3+CD4+ T cells).
  • a method for administration of human regulatory T cells treated with GMCSF, DL-5, BL-3, or any combination thereof to treat autoimmune disorders and to prevent tissue and organ transplant rejection in another embodiment, provided herein a method no prior separation of regulatory T cells is necessary.
  • contacting the T-cell population in which regulatory T-cells are sought to be selectively expanded comprising contacting the T-cell population with the composition described herein.
  • contacting the T-cell population with a composition comprising a granulocyte-macrophage colony stimulating factor (GMCSF) is carried out ex-vivo and is preceded by a step of obtaining T-cells from the leukocyte cell having antigen-presenting cellss of the subject and further comprises a step of reintroducing a regulatory T-cell enriched T-cell population into the transplanted subject.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • contacting the T-cell population with the compositions described herein comprising a granulocyte-macrophage colony stimulating factor (GMCSF) is carried out in- vivo via oral, topical, intraluminal or by inhalation or parenteral administration, wherein parenteral administration is intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, submucosal or intradermal administration.
  • GMCSF granulocyte-macrophage colony stimulating factor
  • GMCSF granulocyte-macrophage colony stimulating factor
  • leukocyte cell having antigen-presenting cells from Balb/c mice were stained with fluorescently labeled antibodies against CD4 and FoxP3 before (left plot) and after culture in GMCSF (right plot) for 4 days, and analyzed by flow cytometry.
  • the number in the upper right corner represents the proportion of regulatory T cells (CD4+ /FoxP3+, right upper quadrant) out of total CD4+ T cells.
  • GMCSF shares signal transduction mechanism, by way of a common receptor subunit, with interleukin (IL)-3 and IL-5
  • both IL-3 and IL-5 also induce selective proliferation of regulatory T cell similar to GMCSF, but at a lower level (Fig. 2).
  • leukocyte cell having antigen-presenting cells from Balb/c mice were labeled with CFSE dye and cultured in GMCSF, IL-3, or IL-5 for 4 days. The cells were then stained with fluorescently labeled antibodies against CD4 and FoxP3 and analyzed by flow cytometry.
  • the number in the upper quadrants represents the proportion of proliferating (left quadrant) or non-proliferating (right quadrant) regulatory T cells (CD4+ /FoxP3+) out of total CD4+ T cells.
  • GMCSF-expanded regulatory T cells display a stronger suppression of naive T cell proliferation compared to freshly isolated regulatory T cells (Fig. 3), indicating that not only did GMCSF increase numbers but also enhanced the functional capacity of regulatory T cells.
  • leukocyte cell having antigen-presenting cells from Balb/c mice were stained with fluorescently labeled antibodies against CD4, FoxP3, and CD25 before (solid line) and after culture in GMCSF (dotted) for 4 days.
  • the regulatory T cells (CD4+ /FoxP3+) were analyzed for expression of FoxP3 (right plot) or CD25 (left plot) by flow cytometry.
  • the mean fluorescence intensity (MFI) of FoxP3 for T cells before and after culture in GMCSF were 428 and 1307, respectively.
  • the MFI of CD25 for T cells before and after culture in GMCSF were 29 and 530, respectively.
  • FIG 4 shows that CD4+CD25- T cells were flow cytometry-sorted from Balb/c spleens and mixed at different ratios with regulatory T cells (Treg) that were flow cytometry-sorted from fresh Balb/c leukocyte cell having antigen-presenting cells (blue line) or Balb/c leukocyte cell having antigen-presenting cells cultured in GMCSF for 4 days (pink line).
  • the T cells were stimulated with anti-CD3 antibody and irradiated Balb/c leukocyte cell having antigen- presenting cells for 4 days. 24 hours before harvest, the cells were pulsed with 3H- thymidine and the incorporation of thymidine was quantified as a measure of proliferation.

Abstract

La présente invention concerne un procédé d’expansion et d’enrichissement d’une population de lymphocytes T régulateurs. Spécifiquement, l'invention concerne des procédés d’expansion de lymphocytes T régulateurs par mise en contact de leucocytes avec une composition comprenant un lymphocyte T régulateur avec un facteur stimulant les colonies de granulocytes-macrophages (GMCSF); et l’interleukine-3 (IL-3), l’interleukine-5 (IL-5) ou leurs combinaisons.
PCT/US2009/052737 2008-08-04 2009-08-04 Procédés d’expansion des lymphocytes t régulateurs WO2010017220A1 (fr)

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US9732134B2 (en) 2009-01-21 2017-08-15 Amgen Inc. Method of treating graft versus host disease using IL-2 muteins
US11560415B2 (en) 2009-01-21 2023-01-24 Amgen Inc. Method of promoting regulatory T-cell proliferation
US11090363B2 (en) 2009-07-10 2021-08-17 University of Pittsburgh—of the Commonwealth System of Higher Education Vasoactive intestinal peptide release from microparticles
US8846098B2 (en) 2009-07-10 2014-09-30 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Artificial cell constructs for cellular manipulation
US9757339B2 (en) 2009-07-10 2017-09-12 University of Pittsburgh—of the Commonwealth System of Higher Education Artificial cell constructs for cellular manipulation
US10449151B2 (en) 2009-07-10 2019-10-22 University of Pittsburgh—of the Commonwealth System of Higher Education Artificial cell constructs for cellular manipulation
US11413245B2 (en) 2009-07-10 2022-08-16 University of Pittsburgh—of the Commonwealth System of Higher Education Artificial cell constructs for cellular manipulation
US9624469B2 (en) 2010-07-22 2017-04-18 Cellect Biotherapeutics Ltd. Regulatory immune cells with enhanced targeted cell death effect
WO2012011113A2 (fr) 2010-07-22 2012-01-26 Shai Yarkoni Cellules immunitaires régulatrices possédant un effet de destruction cellulaire ciblé accru
WO2012064657A1 (fr) * 2010-11-08 2012-05-18 The Board Of Trustees Of The Leland Stanford Junior University L'amyloïde a sérique (saa) annule l'anergie des lymphocytes t régulateurs (trég)
US10092597B2 (en) 2014-01-14 2018-10-09 The University Of Hong Kong Human CD8+ regulatory T cells inhibit GVHD and preserve general immunity in humanized mice
US10653722B2 (en) 2014-01-14 2020-05-19 The University Of Hong Kong Human CD8+ regulatory T cells inhibit GVHD and preserve general immunity
US10851144B2 (en) 2015-04-10 2020-12-01 Amgen Inc. Interleukin-2 muteins for the expansion of T-regulatory cells
US11976103B2 (en) 2015-04-10 2024-05-07 Amgen Inc. Interleukin-2 muteins for the expansion of T-regulatory cells

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