WO2010017220A1

WO2010017220A1 - Methods for expanding regulatory t-cells

Info

Publication number: WO2010017220A1
Application number: PCT/US2009/052737
Authority: WO
Inventors: Taku Kambayashi; Gary Koretzky
Original assignee: University of Pennsylvania Penn
Current assignee: University of Pennsylvania Penn
Priority date: 2008-08-04
Filing date: 2009-08-04
Publication date: 2010-02-11
Anticipated expiration: 2011-02-04

Abstract

The present invention relates a method of expanding and enriching a regulatory T-cell population. Specifically, the invention relates to methods of expanding regulatory T-cells by contacting leukocytes with a composition comprising a regulatory T-cell with a granulocyte-macrophage colony stimulating factor (GMCSF); and interleukin-3 (IL-3), interleukin-5 (IL-5), or their combinations.

Description

METHODS FOR EXPAM)ING REGULATORY T-CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to United States provisional patent application 61/086,068, filed August 4, 2008, which is incorporated herein in its entirety.

FIELD OF THE INVENTION

[002] 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).

BACKGROUND OF THE INVENTION

[003] CD4⁺CD25⁺FOXP3⁺ regulatory T cells (CD4⁺ Treg cells or suppressor 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. The cross-talk between Treg cells and targeted cells, such as antigen-presenting cells (APCs) and T cells, is crucial for ensuring suppression by Treg cells in the appropriate microenvironment.

[004] 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. However the contribution of these populations to self-tolerance and immune homeostasis is less well defined.

[005] Similar to other T cells, 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). In addition to the Foxp3 -expressing CD4+CD25+, there also appears to be a minor population of MHC class I restricted CD8+ Foxp3 -expressing regulatory T cells.

[006] 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.

[007] Accordingly, a need exists to expand the regulatory T cell population in order to treat various auto-immune and other diseases.

SUMMARY OF THE INVENTION

[008] In one embodiment, 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

[009] In another embodiment, 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.

[0010] In another embodiment, 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.

[0011] In another embodiment, 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.

[0012] In another embodiment, 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.

[0013] In another embodiment, 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.

[0014] In another embodiment, 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 naϊve 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.

[0015] hi another embodiment, 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 naϊve T-cells.

[0016] In another embodiment, 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 naϊve T-cells and treating an autoimmune pathology in said subject.

[0017] In another embodiment, 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.

[0018] In another embodiment, 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.

[0019] In another embodiment, 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.

[0020] In another embodiment, 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 naϊve T-cells and inhibiting allograft rejection in solid organ transplantation in a subject.

[0021] In another embodiment, 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.

[0022] In another embodiment, 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 naϊve T-cells, thus increasing transplant tolerance in a subject.

[0023] In another embodiment, 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.

[0024] In another embodiment, 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.

[0025] In another embodiment, 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.

[0026] In another embodiment, 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.

[0027] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The invention will be better understood from a reading of the following detailed description taken in conjunction with the drawings in which like reference designators are used to designate like elements.

[0029] 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. [0030] 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.

[0031] Figure 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+) 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.

[0032] Figure 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 maximal proliferation obtained is found in T cells with no regulatory T cells added (Treg:T cell ratio = 0:1).

DETAILED DESCRIPTION OF THE INVENTION

[0033] 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).

[0034] In one embodiment, provided herein is 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.

[0035] In one embodiment, provided herein is 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.

[0036] In one embodiment, 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. In another embodiment, treating is indirect contacting a regulatory T- cell with a granulocyte-macrophage colony stimulating factor (GMCSF); and EL-3, IL-5, or their combination.

[0037] In another embodiment, 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. In another embodiment, 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.

[0038] In one embodiment, provided herein is 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. In one embodiment, provided herein 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.

[0039] In another embodiment, 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.

[0040] In one embodiment, 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. In another embodiment, provided herein 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.

[0042] In another embodiment, 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. In another embodiment, 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.

[0043] In another embodiment, a regulatory T-cell is a suppressor T cell. In another embodiment, a regulatory T-cell is a cell of the immune system that suppresses immune responses of other cells. In another embodiment, a regulatory T-cell is a CD8⁺ T cell. In another embodiment, a regulatory T-cell is a CD4⁺CD25⁺ T cell. In another embodiment, a regulatory T-cell is a CD4⁺CD25⁺Foxp3⁺ T-cell. In another embodiment, a regulatory T-cell is a CD4⁺Foxp3⁺ T-cell. In another embodiment, a regulatory T-cell has a larger T-cell receptor (TCR) diversity than effector T cells, biased towards self-peptides. In another embodiment, a regulatory T-cell demonstrates a profound state of in vitro and in vivo anergy, refractory to conventional T cell stimulation.

[0044] In another embodiment, enriching a regulatory T-cell population is selectively enriching a regulatory T-cell. In another embodiment, enriching a regulatory T-cell population is enriching a regulatory T-cell and not a T-helper cell, a cytotoxic T-cell, or both. In another embodiment, enriching a regulatory T-cell population comprises breaking

CD4⁺CD25⁺ regulatory T-cell anergy. In another embodiment, 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.

[0045] In another embodiment, 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.

[0046] In another embodiment, expanding a regulatory T-cell is selectively expanding a regulatory T-cell. In another embodiment, expanding a regulatory T-cell is expanding a regulatory T-cell and not a T-helper cell, a cytotoxic T-cell, or both. In another embodiment, expanding a regulatory T-cell comprises breaking CD4⁺CD25⁺ regulatory T- cell anergy. In another embodiment, expanding a regulatory T-cell comprises triggering proliferation of a regulatory T-cell. In another embodiment, activated regulatory T-cell is induced to produce IL-2. In another embodiment, expanding regulatory T-cell results in induction of CD25 expression in a regulatory T-cell. In another embodiment, expanding a regulatory T-cell is inducing the capacity of a regulatory T-cell. In another embodiment, 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).

[0047] In another embodiment, 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.

[0048] In another embodiment, a regulatory T-cell population is not an isolated regulatory T-cell population. In another embodiment, a regulatory T-cell population is comprised within a mixed T-cell population. In another embodiment, a regulatory T-cell population is comprised within a mixed cell population. In another embodiment, a regulatory T-cell population is comprised within a leukocyte cell having antigen-presenting cells cell population. In another embodiment, a regulatory T-cell population is comprised within a mixed immune cell population.

[0049] In another embodiment, enriching a regulatory T-cell population is selectively enriching a regulatory T-cell population. In another embodiment, enriching a regulatory T- cell population is selectively inducing proliferation of a regulatory T-cell population. In another embodiment, 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. In another embodiment, enriching a regulatory T-cell population is selectively inducing proliferation of a regulatory T-cell population. In another embodiment, 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.

[0050] 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.

[0051] In another embodiment, 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.

[0052] The 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.

[0053] In one embodiment, GMCSF is a recombinant GMCSF. In another embodiment, GMCSF is in the form of Leukine. In another embodiment, Leukine is the trade name of sargramostim manufactured by Berlex Laboratories, a subsidiary of Schering AG.

[0054] In another embodiment, an activated regulatory T-cell steers monocyte differentiation toward alternatively activated macrophages (AAM). In another embodiment, an activated regulatory T-cell inhibits the production of pro-inflammatory mediators such as EL- lβ, EL-6, EL-8, MEP- lα, TNF-α by monocytes and/or macrophages. In another embodiment, an activated regulatory T-cell produces EL-10, EL-4, and EL-13.

[0055] In another embodiment, an enriched regulatory T-cell population steers monocyte differentiation toward alternatively activated macrophages (AAM). In another embodiment, an enriched regulatory T-cell population inhibits the production of pro-inflammatory mediators such as EL-lβ, EL-6, EL-8, MEP-lα, TNF-α by monocytes and/or macrophages. Ln another embodiment, an enriched regulatory T-cell population produces EL-IO, EL-4, and EL-13.

[0056] In another embodiment, a regulatory T-cell is grown in a cell culture-in vitro. In another embodiment, a regulatory T-cell is grown in an isolated regulatory T-cell culture-in vitro. Ln another embodiment, a regulatory T-cell is grown in an enriched regulatory T-cell culture-in vitro. In another embodiment, a regulatory T-cell is grown in a mixed cell culture-in vitro. In another embodiment, a regulatory T-cell is grown ex-vivo in a primary cell culture. In another embodiment, a regulatory T-cell is present within the immune system of a subject. In another embodiment, a subject is a human being. In another embodiment, a subject is a primate. In another embodiment, a subject is a pet. In another embodiment, 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.

[0057] In one embodiment, the composition comprising GMCSF, EL-3, EL-5 or their combination, is used in the methods provided herein. Accordingly, in one embodiment, provided herein is 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 naϊve T-cells. In another embodiment, provided herein is 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. In one embodiment, provided herein is 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 naϊve T-cells. In yet another embodiment, provided herein is 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 naϊve T-cells in the T-cell population. Likewise, in one embodiment provided herein is 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 naϊve T-cells. In another embodiment, provided herein is 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.

[0058] In one embodiment, 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. "Autoantigen" or "self-antigen" as used herein refers in certain embodiments to an antigen or epitope which is native to the mammal and which is immunogenic in said mammal disease.

[0059] In another embodiment, a regulatory T-cell population is grown in a cell culture-in vitro. In another embodiment, a regulatory T-cell population is an isolated regulatory T-cell population in culture-in vitro. In another embodiment, a regulatory T-cell population is grown in culture with other cell types. In another embodiment, a regulatory T-cell population is grown ex-vivo. In another embodiment, a regulatory T-cell population is enriched ex-vivo. In another embodiment, a regulatory T-cell population is present within the immune system of a subject.

[0060] 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 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..

[0061] Methods of segregating CD8 T cells into + and - categories are known to persons of ordinary skill in the art. In some embodiments, 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. In some embodiments, the CD8⁺ cells stain two to three fold more intensely than the CD8^" cells. Particularly preferred methods are also exemplified in the Examples.

[0062] In one embodiment, regulatory T cells (Treg) are critical regulators of immune tolerance. In another embodiment, 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.

[0063] In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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 (Tregs) are either generic in one embodiment, or antigen specific in another embodiment. In another embodiment, regulatory T cells produce TGF-β and/or IL-10 through which Tregs can suppress immune responses.

[0064] In another embodiment, 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. In another embodiment, 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.

[0065] In another embodiment, GMCSF is a cytokine that functions as a white blood cell growth factor. In another embodiment, GMCSF is glycosylated. In another embodiment, the glycosylation sites are at amino acid residues 23 (leucine), 27 (asparagine), 39 (glutamic acid), or any combination thereof.

[0066] 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 50-5000 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 50-200 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 100-500 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 300-800 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 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. 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 2000-4000 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 3000-5000 mcg/mL.

[0067] 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 1 x 10⁴ to 1 x 10^s IU/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 1 x 10⁴ to 1 x 10⁵ IU/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 1 x 10⁵ to 1 x 10⁶ IU/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 x 10⁵ to 5 x 10⁷ IU/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 1 x 10⁷ to 1 x 10⁸ IU/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 1 x 10⁴ to 1 x 10⁶ IU/mL.

[0068] 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 0.5-250 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. 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 25-75 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.

[0069] In another embodiment, 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. 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 20 to 400 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 20 to 100 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 50 tol50 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 100 to 200 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 150 to 250 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 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.

[0070] In another embodiment, 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.

[0071] In one embodiment 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. In one embodiment 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.

[0072] Accordingly and in one embodiment, provided herein is 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.

[0073] In another embodiment, provided herein is 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.

[0074] In another embodiment, provided herein is 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.

[0075] 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 naϊve T-cells thus enriching a T-cell population with regulatory T-cells.

[0076] In another embodiment, provided herein is 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 naϊve T-cells.

[0077] In another embodiment, provided herein is 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 naϊve T-cells thus suppressing a T-cell activation mediated response in a subject.

[0078] In another embodiment, provided herein is 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 naϊve 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.

[0079] In another embodiment, provided herein is 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 naϊve T-cells.

[0080] In another embodiment, provided herein is 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 naϊve T-cells and treating an autoimmune pathology in a subject.

[0081] In another embodiment, provided herein is 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.

[0082] In another embodiment, provided herein is 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.

[0083] In another embodiment, provided herein is 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.

[0084] In another embodiment, provided herein is 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); and either 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.

[0085] In another embodiment, provided herein is 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.

[0086] In another embodiment, provided herein is 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.

[0087] In another embodiment, provided herein is 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 naϊve T-cells and increasing transplant tolerance in a subject.

[0088] In another embodiment, provided herein is 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 naϊve 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.

[0089] In another embodiment, provided herein is 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.

[0090] In another embodiment, provided herein is 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.

[0091] In another embodiment, 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.

[0092] In another embodiment, interleukin (IL)-3 is a recombinant IL-3. In another embodiment, EL-3 is a human Interleukin 3 protein. In another embodiment, IL-3 is a mouse Interleukin 3 protein. In another embodiment, 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. In another embodiment, hIL-3 multiple mutation polypeptides retain one or more activities of native hTL-3. In another embodiment, hIL-3 multiple mutation polypeptides show improved regulatory T-cell stimulating activity and/or an improved activity profile, and/or proliferation.

[0093] 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 50-5000 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 50-200 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 75-150 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 100-200 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 150-300 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 200-400 ng/mL. In another embodiment, 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. 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 400-800 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 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. In another embodiment, 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. 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 4000-5000 ng/mL.

[0094] Ln another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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.

[0095] In another embodiment, EL-3 activates a regulatory T-cell but not a T helper cell and/or a cytotoxic T-cell. In another embodiment, IL-3 activates a mature regulatory T-cell but not a mature T helper cell and/or a mature cytotoxic T-cell.

[0096] In another embodiment, 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.

[0097] In another embodiment, interleukin (DL)-5 is a recombinant EL-5. In another embodiment, EL-5 is a human Interleukin 3 protein. In another embodiment, EL-5 is a mouse Interleukin 3 protein. In another embodiment, 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. In another embodiment, hEL-5 multiple mutation polypeptides retain one or more activities of native hEL-5. In another embodiment, hEL-5 multiple mutation polypeptides show improved regulatory T-cell stimulating activity and/or an improved activity profile, and/or proliferation.

[0098] 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 50-5000 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 50-200 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 75-150 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 100-200 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 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. 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 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. 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 3000-4000 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 4000-5000 ng/mL.

[0099] hi another embodiment, 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. 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 0.5 to 50 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 0.5 to 2.5 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 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. 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 10 to 20 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 15 to 30 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 20 to 40 mcg/kg/day. In another embodiment, 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.

[00100] In another embodiment, BL-5 activates a regulatory T-cell but not a T helper cell and/or a cytotoxic T-call. In another embodiment, IL-5 activates a mature regulatory T-cell but not a mature T helper cell and/or a mature cytotoxic T-call.

[00101] In another embodiment, BL-5 enriches a regulatory T-cell population but not a T helper cell population and/or a cytotoxic T-cell population. In another embodiment, 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.

[00102] In another embodiment, 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. In another embodiment, 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.

[00103] In another embodiment, 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. In another embodiment, contacting BL-5,

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. In another embodiment, 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.

[00104] In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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.

[00105] In another embodiment, provided herein 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. In another embodiment, a T cell is a T helper cell (CD4+) or cytotoxic T cell (CD8+). In another embodiment, provided herein 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. In another embodiment, a T cell is a T helper cell (CD4+) or cytotoxic T cell (CD8+). In another embodiment, provided herein 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. In another embodiment, provided herein 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. In another embodiment, a T cell is a T helper cell (CD4+) or cytotoxic T cell (CD8+).

[00106] In another embodiment, 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. [00107] In another embodiment, suppressing a T cell activation response comprises suppressing the differentiation of CD4 positive T helper (Th) cells into either ThI or Th2 cells. In another embodiment, suppressing a T cell activation response comprises suppressing a humoral immune response. In another embodiment, suppressing a T cell activation response comprises suppressing a cytotoxic immune response. In another embodiment, suppressing a T cell activation response comprises suppressing a cell- mediated immune response. In another embodiment, suppressing a T cell activation response comprises suppressing a delayed type of hypersensitivity (DTH) reaction. In another embodiment, suppressing a T cell activation response comprises suppressing macrophage activation. In another embodiment, suppressing a T cell activation response comprises antagonizing pro-inflammatory cytokines such as IL-6, TNF and BL-I.

[00108] In another embodiment, 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 PKCΘ. In another embodiment, suppressing a T cell activation response comprises suppressing the activation of the transcription factor NFAT.

[00109] In another embodiment, provided herein 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.

[00110] In another embodiment, provided herein 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. In another embodiment, provided herein 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.

[00111] hi another embodiment, provided herein 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. In another embodiment, provided herein 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.

[00112] In another embodiment, transplant tolerance is the lack of a destructive immune response toward the graft in the absence of ongoing immunosuppressive therapy. In another embodiment, transplant tolerance is manifested clinically by normal graft function in the absence of acute and chronic rejection. In another embodiment, transplant tolerance coexists with general immune competence. In another embodiment, transplant tolerance coexists with normal immune responses to pathogens.

[00113] In another embodiment, 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.

[00114] In another embodiment, transplant tolerance is the complete and successful withdrawal of treatment with immunosuppressive medications. In another embodiment, 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.

[00115] In another embodiment, transplant tolerance suppresses recognition of alloantigen occurs. In another embodiment, transplant tolerance suppresses CD4⁺ T-cell differentiaion and/or maturation.

[00116] In another embodiment, 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. In another embodiment, 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).

[00117] In another embodiment, a graft-versus-host disease (GVHD) 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.

[00118] In another embodiment, a graft-versus-host disease is the acute or fulminant form of the disease (aGVHD). In another embodiment, a graft-versus-host disease is the chronic form of graft-versus-host-disease (cGVHD).

[00119] In another embodiment, 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.

[00120] In another embodiment, provided herein a method for inducing the proliferation of regulatory T cells with enhanced function. In another embodiment, provided herein a method for activating a regulatory T-cell comprising contacting an APC with GMCSF, IL- 5, IL-3, or any combination thereof. In another embodiment, provided herein a method for expanding and/or enriching regulatory T cells (FoxP3+CD4+ T cells) ex vivo. In another embodiment, provided herein a method for expanding and/or enriching regulatory T cells (FoxP3+CD4+ T cells) ex vivo by contacting an APC with GMCSF, IL-5, BL-3, or any combination thereof.

[00121] In another embodiment, provided herein a method for inducing the proliferation of regulatory T cells with enhanced function. In another embodiment, provided herein a method comprising contacting a regulatory T-cell with GMCSF, BL-5, EL-3, or any combination thereof. In another embodiment, provided herein a method for expanding and/or enriching regulatory T cells (FoxP3+CD4+ T cells) ex vivo. In another embodiment, provided herein 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). In another embodiment, provided herein 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.

[00122] In one embodiment, 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. In another embodiment, 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. In another embodiment, 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.

[00123] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

EXAMPLE 1: EXPANDING REGULATORY T CELLS

[00124] Isolated leukocyte cell having antigen-presenting cells cultures were treated with granulocyte-macrophage colony stimulating factor (GMCSF) and enriched, by selective proliferation, the regulatory T cells (FoxP3+CD4+ T cells) (Fig. 1). GMCSF proteins used in the experiment were obtained from Peprotech, Inc and R&D Systems, Inc. These are recombinant GMCSF containing the amino acid sequence of SEQ ID NO: 2 (Ala 18 - Lys 141, with an N-terminal Met - Accession No.: Q14AD9). [00125] As shown in Figure 1, 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.

[00126] Since 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). As shown in Figure 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.

[00127] Further the studies conducted revealed that 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.

[00128] As shown in Figure 3, 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. Figure 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 maximal proliferation obtained is found in T cells with no regulatory T cells added (Treg:T cell ratio = 0:1).

[00129] These results provide a method for expansion and activatation of regulatory T cells ex vivo (to be reinfused into patients) or in vivo by direct therapeutic administration. An increase in regulatory T cells can help patients achieve transplant tolerance, or to counteract autoimmunity or GVHD.

[00130] Having described preferred embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to the precise embodiments, and that various changes and modifications may be effected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. 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.

2. The method of claim 1, whereby expanding said regulatory T-cells comprises increasing the expression of FoxP3, CD25, or both.

3. The method of claim 1, whereby expanding said regulatory T-cells comprises inducing proliferation, enhancing functional capacity of said regulatory T-cells, or a combination thereof.

4. The method of claim 1, whereby said regulatory T-cells comprise a FoxP3⁺CD4⁺ T cell.

5. The method of claim 1, whereby said regulatory T-cells comprise a T helper cell (CD4⁺) or cytotoxic T cell (CDS⁺).

6. 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 (IL-5), or combinations thereof, thereby selectively expanding the number of regulatory T-cells while preserving the function of pathogen-reactive T cells.

7. 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 naϊve T-cells thus enriching a T-cell population with regulatory T-cells.

8. 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 (IL-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.

9. 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.

10. 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 (EL-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.

11. 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 (JL-5), or combinations thereof, thereby increasing the number of regulatory T-cells and suppressing naϊve 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.

12. 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 naive T-cells.

13. 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 naϊve T-cells and treating an autoimmune pathology in said subject.

14. 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 (IL-5), or combinations thereof, thereby increasing the number of regulatory T-cells and suppressing naϊve 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.

15. 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.

16. 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 naϊve T-cells and inhibiting allograft rejection in solid organ transplantation in a subject.

17. 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 (DL-3), interleukin-5 (IL-5) or both, thereby increasing the number of regulatory T-cells and suppressing naϊve T-cells and inhibiting allograft rejection in solid organ transplantation in a subject.

18. 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 (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.

19. 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 (IL-5) or both, thereby increasing the number of regulatory T-cells and suppressing naive T-cells, thus increasing transplant tolerance in a subject.

20. 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 (EL-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.

21. 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 (EL-3), interleukin-5 (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 increasing transplant tolerance in a subject.

22. 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 (DL-5) or both thereby increasing FoxP3 expression, CD25 expression or both and increasing the suppressive capacity of freshly-isolated regulatory T-cells.

23. 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.

24. The method of any one of claims 6 - 19, whereby said regulatory T-cell is a FoxP3⁺CD4⁺ T cell.

25. The method of any one of claims 9-21, whereby the step-of administering is via oral, topical, intraluminal or by inhalation or parenteral administration.

26. The method of claim 25, whereby said parenteral administration is intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, submucosal or intradermal.

27. The method of claim 7 or 8, whereby enriching said regulatory T-cell comprises increasing FoxP3 expression, CD25 expression or both.

28. The method of claim 7 or 8, whereby said enriching is selectively inducing proliferation of said regulatory T-cell population.

29. The method of any one of claims 9-11, whereby said T cell is a T helper cell (CD4⁺) or cytotoxic T cell (CD8⁴).

30. The method of any one of claims 9-11, whereby suppressing T-cell activation, further comprises increasing FoxP3 expression, CD25 expression or both.

31. The method of any one of claims 9-11, whereby suppressing T-cell activation, further comprises inducing regulatory T-cell proliferation, and capacity.

32. The method of any one of claims 12-14, 16-18 and 20-21, whereby selectively enriching a T-cell population with regulatory T-cell comprises increasing FoxP3 expression, CD25 expression or both.

33. The method of any one of claims 12-14, 16-18 and 20-21, whereby selectively enriching a T-cell population with regulatory T-cell results in the induction of proliferation, induction of the capacity of said regulatory T-cell, or a combination thereof.

34. The method of any one of claims 12-14, whereby the autoimmune disease is a multiple sclerosis, a rheumatologic disease, an inflammatory bowel disease, a dermatitis, a Lupus or a combination thereof.

35. The method of any one of claims 12-14, whereby the subject having an autoreactive autoimmune disease, is an allograft recipient having a graft-versus-host disease (GVHD).