WO2010129770A1 - Procédés d'expansion de lymphocytes t régulateurs humains et leurs utilisations - Google Patents

Procédés d'expansion de lymphocytes t régulateurs humains et leurs utilisations Download PDF

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WO2010129770A1
WO2010129770A1 PCT/US2010/033869 US2010033869W WO2010129770A1 WO 2010129770 A1 WO2010129770 A1 WO 2010129770A1 US 2010033869 W US2010033869 W US 2010033869W WO 2010129770 A1 WO2010129770 A1 WO 2010129770A1
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cells
cell
treg
treg cells
regulatory
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Xinjian Chen
Xiaohua Guo
Peter E. Jensen
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The University Of Utah Research Foundation
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
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    • C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells

Definitions

  • This disclosure relates generally to compositions and methods for the production of regulatory T cells. More particularly, the disclosure relates to the production of human T regulatory cells using allogeneic B cells and to the production of regulatory T cells using weak stimulation of the T cell receptor (TCR).
  • TCR T cell receptor
  • Treg The naturally arising regulatory FoxP3 + CD4 + T cells (Treg) play an important role in the induction and maintenance of immunological tolerance to self-antigens.
  • Mouse Treg cells prevent allograft rejection and graft versus host disease (GVHD) when adoptively transferred into recipient hosts, unveiling the attractive prospect of using Tregs as therapeutic tools for treatment of transplant rejection and GVHD in humans.
  • Treg cell-based therapy has the potential to provide long lasting, nontoxic and antigen-specific suppression of graft rejection or GVHD without compromising protective immunity in the host.
  • Treg cells Since the number of Treg cells that can be obtained from a donor is limited, freshly isolated Treg cells need to be expanded ex vivo to generate a sufficient number of cells for therapeutic applications. In addition, expanded Treg cells have been reported to be more therapeutically effective than primary Treg cells.
  • the present technology provides methods for expanding Treg cells.
  • the disclosure provides a method for expanding human regulatory T cells comprising co- culturing a human regulatory T cell and a B cell in the presence of a cytokine and a co- stimulatory agent to produce a population of allospecific human regulatory T cells.
  • the cytokine is an interleukin.
  • the interleukin is IL-2.
  • in the co-stimulatory agent activates CD28.
  • the co-stimulatory agent is a CD28 ligand (e.g., CD80 or CD86).
  • the co-stimulatory agent is an anti-CD28 antibody, and in a more specific embodiment, the anti-CD28 antibody is CD28.2.
  • the B cell has been transformed to express the cytokine, the co- stimulatory agent, or both the cytokine and co-stimulatory agent.
  • the co- stimulatory agent is a B cell transformed to express the co-stimulatory agent or membranes derived from these cells.
  • the B cell has been transformed to express CD80 and/or CD86.
  • the technology provides for isolating T cells and B cells from different sources. Accordingly, some embodiments provide for isolating the regulatory T cell from a host, a donor, or a third party who is unrelated to the host and the donor.
  • One specific source of regulatory T cells can be leukopheresis-derived leukocytes.
  • the B cell is isolated from a host, a donor, or a third party who is unrelated to the host and the donor.
  • the B cell expresses a common alloantigen toward which regulatory T cell activity is desired.
  • a similar embodiment provides that the B cell expresses a target HLA haplotype toward which regulatory T cell activity is desired.
  • the regulatory T cell, the B cell, or both the regulatory T cell and the B cell are isolated from a host. In one embodiment, the regulatory T cell, the B cell, or both the regulatory T cell and the B cell are isolated from a donor. In one embodiment, the regulatory T cell, the B cell, or both the regulatory T cell and the B cell are isolated from a third party who is unrelated to the host and the donor. In one embodiment, the regulatory T cell, the B cell, or both the regulatory T cell and the B cell are isolated from a host.
  • the technology provides that the regulatory T cell is a CD25 + CD4 + T cell or that the regulatory T cell is a CD25 CD4 + T cell.
  • the expanded human regulatory T cells express FoxP3 at a high level, remain anergic, and are potent suppressors of allospecific effector T cells.
  • the disclosure provides a method for expanding regulatory T cells, the method comprising: culturing a population of T cells with a T cell receptor agonist (TCR) under conditions wherein the T cell receptor is weakly stimulated, wherein Treg cells in the population of T cells are preferentially expanded compared to non-Treg cells.
  • TCR T cell receptor agonist
  • the preferential expansion includes proliferation of existing Treg cells and conversion of non-Treg cells to Treg cells.
  • the Treg cells have a CD25 + FoxP3 + CD4 + phenotype.
  • the conditions wherein the T cell receptor is weakly stimulated include contacting the population of T cells with a T cell receptor agonist.
  • the T cell receptor agonist is selected from the group consisting of: an anti-CD3 antibody, an antigen, and an allogenic antigen presenting cell.
  • the conditions wherein the T cell receptor is weakly stimulated include contacting the population of T cells with an anti-CD3 antibody.
  • the concentration of the anti-CD3 antibody in the culture is from about 0.0025 ⁇ g/mL to about 0.01 ⁇ g/mL.
  • the conditions wherein the T cell receptor is weakly stimulated include contacting the population of T cells with an antigen.
  • the antigen is an OVA peptide.
  • the concentration of the OVA peptide in the culture is from about 0.01 ⁇ M to about 0.02 ⁇ M.
  • the disclosure provides a method for expanding regulatory T cells, the method comprising: culturing a population of T cells with a phorbol ester, wherein Treg cells in the population of T cells are preferentially expanded compared to non-Treg cells.
  • the phorbol ester is phorbol 12-myristate 13 -acetate (PMA).
  • the concentration of the PMA in the culture is from about 10 nM to about 20 nM.
  • the culture does not include ionomycin.
  • the technology also provides allospecific regulatory T cell compositions produced by the present methods. These compositions may be used in therapeutic applications to treat a patient.
  • allospecific regulatory T cell products are used to treat a patient who suffers from an autoimmune disease, graft versus host disease, transplant rejection, or an immune -related inflammatory disease.
  • the disclosure provides a method for treating an immunological disorder comprising administering to a transplant recipient an effective amount of the allospecific human regulatory T cells prepared in accordance with the disclosed methods.
  • the immunological disorder is an autoimmune disease.
  • allospecif ⁇ c regulatory T cell products are used to treat a patient who suffers from multiple sclerosis, type 1 diabetes, inflammatory bowel disease, cardiovascular disease, Parkinson's disease, Alzheimer's disease, arthritis, IPEX, allergy, gastritis, lung inflammation, or from the destructive effects of an immune response to an infectious agent.
  • Some embodiments provide for using allospecif ⁇ c regulatory T cell products to promote transplant tolerance in a host.
  • the transplant is bone marrow or a solid organ.
  • FIGs. 1A-1H show the results of a flow cytometry experiment to demonstrate that Treg cells were directly expanded with allogeneic B cells.
  • FIGs. 2A-2L show the results of a flow cytometry experiment to demonstrate that B- cell expanded Treg cells are anergic and potent suppressors.
  • FIGs. 3A-3J show the results of a flow cytometry experiment to demonstrate that B- cell expanded Treg cells have specificity for the alloantigens of the B-cells with which the Treg cells were expanded.
  • FIGs. 4A-4D show the results of a flow cytometry experiment to demonstrate that B-cell expanded Treg cells potently inhibit alloproliferation of third-party responder T cells in various Treg and responder cell combinations in an alloantigen-specific manner.
  • FIGs. 5A-5G show the results of a flow cytometry experiment in which Treg cells were expanded using the OVA peptide.
  • FIGs. 6A-6D show the results of a flow cytometry experiment in which Treg cells were expanded using an anti-CD3 antibody.
  • FIGs. 7A-C is a series of bar graphs showing that at high concentration, the anti- CD3 antibody 2c 11 induced vigorous T cell proliferation dominated by FoxP3 " non-Treg cells. At low concentrations, the antibody only induced modest T cell proliferation which is, however, dominated by FoxP3 + Treg cells.
  • FIG. 8A is a graph showing the number of cells cultured with the anti-CD3 antibody 2c 11 in the presence or absence of a TGF ⁇ neutralizing antibody.
  • FIG. 8B is a series of graphs of flow cytometry data showing that when TGF ⁇ neutralizing antibody was supplemented in the culture, the Treg cell conversion was diminished.
  • FIG. 9A-9C show a series of Western blots of CD25 " non-Treg (CD25n) and CD25 + Treg (CD25p) cells in whole cell lysate (A), cytoplasm (B) and nuclear extract (C).
  • FIG. 9D shows analysis of these cells by immuno fluorescent microscopy.
  • FIG. lOA-lOF show the results of a flow cytometry experiment in which CFSE- labeled splenocytes were cultured with PMA alone or PMA plus ionomycin for five days.
  • FIGs. 1 IA-I ID shows the results of differential DNA targeting of NFAT in Treg and activated non-Treg cells.
  • FIGs. 12A-D show the results of a flow cytometry experiment to demonstrate that third-party Treg cells prevent rejection of allogeneic bone marrow grafts in MHC-unrelated hosts.
  • the present technology generally provides methods for the ex vivo expansion of Treg cells.
  • the methods involve ex vivo expansion of allospecif ⁇ c human Treg cells.
  • the methods involve preferential expansion of Treg cells in a mixed population of T cells using weak stimulation of the TCR.
  • the methods produce human regulatory T cells in sufficient amounts for therapeutic use in treating diseases and in reducing complications associated with tissue grafts and transplants. Accordingly, various aspects of the technology provide culture components and conditions that allow one to culture large amounts of Treg cells.
  • Cultured human Treg cells are useful, alone or in combination with other therapies, for treating patients suffering from an autoimmune disease, graft versus host disease, transplant rejection, an immune-related inflammatory disease, or for promoting transplant tolerance in a host who is to receive a bone marrow, solid organ, or other transplant.
  • large amounts of allospecific human regulatory T cells may be produced by culturing a human regulatory T cell with a B cell in the presence of a cytokine and a stimulatory molecule.
  • Treg cells were produced that expressed FoxP3 at a high level that was three-fold higher than that of the nondividing input CD25 + CD4 + T cells.
  • activated human non-Treg cells typically express a low level of FoxP3
  • Mouse primary B cells have the capacity to preferentially expand Treg cells in the absence of co-stimulation (Chen and Jensen, Cutting Edge, J Immunol, 2007). Moreover, addition of anti-CD28 stimulatory antibody to mouse B cells and mixtures of FoxP3 + and FoxP3 " cells resulted in T cell proliferation dominated by expansion of FoxP3 " T cells (not Tregs). Attempts to reproduce preferential expansion of human Tregs using human primary B cells did not produce the same result. The present inventors found that exogenous stimulation of CD-28 is required for expansion of human Tregs. In some embodiments, this stimulation can be provided using anti-CD28 exposure or exposure to CD-28 ligands.
  • B-cell expanded Treg cells produced according to the methods described herein display other properties that are characteristic of Treg cells.
  • the B-cell expanded Treg cells remain anergic, while activated non-Treg cells do not.
  • activated Treg cells produce levels of IL-2 and IFN- ⁇ associated with an immune response, while the B-cell expanded Treg cells do not.
  • the B-cell expanded Treg cells suppress responder T cells.
  • B-cell expanded Treg cells suppress the response of CD25 responder T cells to allogeneic monocyte-derived dendritic cells.
  • the proliferation of responder T cells is abrogated at Treg to responder T cell ratios of from about 1 :5 to about 1 :20, and partially inhibited at a Treg to responder T cell ratio of from about 1 :50 to about 1 : 100.
  • the stable anergic phenotype and high suppressive activity of B-cell expanded human Treg cells is related to their high expression of FoxP3.
  • the B-cell expanded, allospecific Treg cells are much more potent than polyclonal Treg cells expanded with anti-CD3 and anti-CD28 antibodies plus IL-2.
  • the high inhibitory potency of B-cell expanded Treg cells is the result of a high frequency of alloantigen-specific Treg cells in the expanded product and the high inhibitory capacity of the individual Treg cells.
  • weak TCR stimulation preferentially activates Tregs over Fox P3 ⁇ CD4 + T cells in the culture.
  • the weak TCR stimulation can be induced by low amounts of a TCR agonist, including antigen- specific or anti-CD3 -mediated TCR stimuli.
  • the TCR agonist is selected from the group consisting of: ovalbumin, anti-CD3 antibody, and allogenic APCs.
  • a weak versus strong stimulation is provided by low or low high concentration of antigen or anti-CD3 antibody, respectively.
  • alloantigen refers to an antigen that differs from an antigen expressed by the recipient.
  • an alloantigen is a MHC polymorphism between a host individual and a donor individual of the same species, or between two populations of cells.
  • alloantigens are the nonself MHC expressed by the cells of allografted tissue that can induce an intense immune response in the recipient host and which is aimed at eliminating the transplanted cells.
  • the immune reaction is the result of the host immune cells recognizing the alloantigenic cells or tissue as originating from a nonself source. If an alloantigen is presented to a member of the same species that does not have the alloantigen, it will be recognized as foreign and induce an immune response.
  • allogeneic refers to two or more individuals, cells, tissues, or other biological materials that differ at the MHC. Host rejection of grafted tissues from unrelated donors usually results from T-cell responses to allogeneic MHC molecules expressed by the grafted tissues. As used herein, a B cell and a T cell are allogeneic when they differ at the MHC as a result of originating from different individuals. In some contexts, these individuals are a transplant host and donor.
  • the term "allograft” refers to a graft of cells or tissue from a donor transplanted to a genetically dissimilar recipient, or host, of the same species.
  • allospecif ⁇ c refers to being reactive to, identifying, or binding cells or other biological components from genetically disparate individuals within the same species. Allospecif ⁇ c T cells can have effector or regulatory functions, and the relative proportions of the two populations activated following alloantigen presentation is one of the factors that determine the clinical outcome of a tissue graft or transplant, namely, graft rejection or persistence.
  • anergic refers to a state of being nonresponsive to an antigen. T cells and B cells are said to be anergic when they cannot respond to their specific antigen under optimal conditions of stimulation. Anergic Treg cells do not mount an immune response, but suppress the response of other effector T cells.
  • antibody refers to an immunoglobulin protein that binds specifically to a particular substance, which is called an antigen.
  • an antigen binds specifically to a particular substance, which is called an antigen.
  • Each antibody molecule has a unique structure that enables it to bind specifically to its corresponding antigen, but all antibodies have the same general gross structure.
  • antigen refers to any molecule that can bind specifically to an antibody. Antigens typically provoke an immune response in an individual, and this immune response may involve either antibody production or the activation of specific immunologically competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.
  • the term "antigen presenting cell” refers to a cell that can process antigens and display antigen peptide fragments on the cell surface together with molecules required for T-cell activation.
  • the main antigen-presenting cells for T cells are dendritic cells, macrophages, and B cells.
  • autoimmune disease refers to a condition that results from an adaptive immune response directed at an individual's own cells and tissues expressing self antigens. Autoimmunity can also be described as a loss of self-tolerance. The resulting immune response against self tissues and cells can lead to various acute and chronic disease states as a result of injury to vital organs and tissues.
  • autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, type I diabetes, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves's disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, among others.
  • autologous refers to any material derived from the same individual to which it is later to be re -introduced into the same individual.
  • B cell refers to one of the two major types of lymphocytes. Each B cell expresses a particular antigen receptor on its cell surface. On activation by an antigen, B cells differentiate into cells producing antibody molecules of the same antigen specificity as this receptor.
  • co-stimulatory agent refers to an agent (e.g., an antibody or ligand) that can bind to the cell surface to promote proliferation and activation of the cells in culture.
  • cytokine refers to a protein made by cells that affects the behavior of other cells. Cytokines made by lymphocytes are often called lymphokines or interleukins (abbreviated IL). Cytokines act via specific cytokine receptors on the cells that they affect.
  • lymphokines or interleukins (abbreviated IL). Cytokines act via specific cytokine receptors on the cells that they affect.
  • DC dendritic cell
  • lymphoid or non-lymphoid tissues These cells are characterized by their distinctive morphology and high levels of MHC expression.
  • DCs can be isolated from a number of tissue sources. DCs have a high capacity for sensitizing MHC -restricted T cells and are very effective at presenting antigens to T cells.
  • the antigens may be self-antigens that are expressed during T cell development of tolerance or foreign antigens.
  • the term "effective amount" of a composition refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in, the symptoms associated with a disease that is being treated.
  • the amount administered to a subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight, and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the Treg cells can also be administered in combination with one or more additional therapeutic compounds.
  • effector cell refers to a cell which mediates an immune response against an antigen.
  • An example of an effector cell includes, but is not limited to, a T cell or a B cell.
  • expansion refers to growing cells in culture to achieve a larger homogenous population of the cells.
  • Cells can be expanded in the presence of antigen presenting cells to produce a population of cells that is allospecif ⁇ c for the antigen presented by the antigen presenting cells.
  • Regulatory T cells represent a small fraction of all T cells, so expansion in culture is required to produce enough regulatory T cells for an effective therapy.
  • expression refers to, but is not limited to, one or more of the following: transcription of a gene into precursor mRNA; splicing and other processing of a precursor mRNA to produce mature mRNA; mRNA stability; translating a mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of a translation product, if required for proper expression and function.
  • graft versus host disease refers to a condition that occurs when T cells present in donor tissue attack the host, or recipient, of the grafted cells or tissue.
  • HLA is an acronym for "human leukocyte antigen” and refers to the human MHC.
  • HLA haplotype refers to a linked set of genes associated with one haploid genome, which determines the HLA of cells from an individual.
  • the linked genes of the HLA are usually inherited as one haplotype from each parent. This set of genes resides on chromosome 6, and encodes cell-surface antigen-presenting proteins and many other genes.
  • host refers to an individual to whom transplanted cells, tissues, organs, or other biological material is transplanted.
  • Recipient and “host” are used interchangeably with an equivalent meaning.
  • immune response refers to the concerted action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by these cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from an individual's body of cells that originate from a source other than that individual's body.
  • the immune response is directed to the normal cells or tissues of the same individual rather than to nonself cells.
  • leukocyte refers generally to a white blood cell. Leukocytes include lymphocytes, polymorphonuclear leukocytes, and monocytes.
  • lymphocyte refers a class of white blood cells that bear variable cell-surface receptors for antigens. The two main classes of lymphocytes are B lymphocytes (B cells) and T lymphocytes (T cells), which mediate humoral and cell-mediated immunity, respectively.
  • a preferential expansion of Treg cells refers to conditions where the number of Treg cells in a culture increase (on a percentage basis) to a greater extent than non-Treg cells in the culture.
  • a preferential expansion of Treg cells may be an increase in the cell number that is at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, or at least 100% greater than the increase in the number of non-Treg cells.
  • only Treg cells proliferate (and non-Treg cells do not proliferate) in response to the culture conditions.
  • Treg cell refers to a naturally occurring subtype of T cell that can inhibit T-cell immune responses to an antigen.
  • Treg cells represent a distinct T-cell lineage that has a key role in an individual's tolerance of self-antigens and the prevention of autoimmune disease and inappropriate immune responses. When activated, they are anergic and suppress the proliferation and cytokine production of conventional T cells. Like all T cells, Treg cells require T cell receptor activation and costimulation to become fully active.
  • self-antigen refers to an antigen that is expressed by a host cell or tissue.
  • T cell refers to an immune cell defined by its development in the thymus and having heterodimeric receptors associated with the proteins of the CD3 complex. Overview
  • the present technology provides methods for expanding T cells ex vivo.
  • the methods involve the preferential expansion ex vivo of Treg cells in a mixed population of Treg cells and non-Treg cells.
  • the methods also involve expanding Treg ex vivo with allogenic B cells.
  • the regulatory T cells express very high levels of FoxP3, maintain an anergic phenotype, and potently suppress responder T cell alloproliferation in an alloantigen- specific manner.
  • CD25 + FoxP3 + CD4 + T cells represent a distinct lineage of CD4 T cells that have suppressive function. Although constituting approximately only 5 to 10% of total CD4 T cells, the Tregs are required for the maintenance of immunological tolerance to self-antigens.
  • Transfer of CD25 + cell-depleted T cell or thymocyte suspensions from normal mice into syngeneic T cell-deficient nude mice results in various autoimmune diseases in the recipient mice, and transfer of CD25 CD4 + T cells or thymocytes together with the CD25 + cell-depleted population prevents those diseases.
  • Treg cells are present in a relatively low frequency, their marked efficacy in suppressing the autoimmunity suggests that Treg cells are highly potent suppressors. Despite their remarkable potency in suppressing autoimmunity, Tregs appear not to compromise protective immunity against common microbial infections, as indicated by the fact that a Treg-intact host is fully immunocompetent. Only with certain chronic viral, fungal, special bacterial or parasitic infections, do Tregs appear to play a role in attenuating protective immunity. Techniques for isolating T cells and B cells
  • T cells and B cells for use in the methods described may be isolated from a biological sample taken from a mammalian subject, such as a human subject, originating from a number of sources, including for example, peripheral blood mononuclear cells, bone marrow, thymus, tissue biopsy, tumor, lymph node tissue, gut associated lymphoid tissue, mucosal-associated lymph node tissue, spleen tissue, or any other lymphoid tissue.
  • T cells and B cells can be isolated as peripheral blood mononuclear cells (PBMC) from a blood sample obtained from the peripheral blood of a subject.
  • PBMCs are prepared from peripheral blood by centrifugation on a gradient of Ficoll and Hypaque (metrizamide).
  • T cells and B cells may be isolated from leukocytes derived from blood using an apheresis or leukopheresis procedure.
  • Leukocytes derived from leukopheresis filters can be enriched for lymphocytes using density gradient centrifugation, e.g. , through a Ficoll- metrizamide gradient.
  • CD25 + CD4 T cells and CD25 CD4 T cells may be isolated from a lymphocyte- enriched sample comprising human T cells through the use of gradient centrifugation and positive/negative selection techniques well known to those of skill in the art.
  • Total CD4 T cells can be isolated by panning, affinity separation, cell sorting using antibodies specific for CD4, and other techniques that provide enrichment of CD4 T cells.
  • CD4 T cells may be prepared by magnetic depletion of other lineage -positive cells. For example, using a magnetic depletion kit, CD4 T cells may be prepared by depleting cells expressing CD8, CDl Ib, CD 16, CD 19, CD36, and CD56.
  • CD4 + No-Touch T cell isolation kit is one example of such a kit manufactured by Miltenyi Biotec (Auburn, CA).
  • CD25 + CD4 T cells and CD25 CD4 T cells can be prepared by positive and negative selection, respectively, of CD4 T cells using anti-CD25 microbeads.
  • B cells and monocytes may be prepared from Ficoll-enriched lymphocytes by positive selection with anti-CD 19 and anti-CD 14 microbeads, respectively.
  • Anti-CD25, anti-CD 19, and anti-CD 14 microbeads are available from Miltenyi Biotec. If desired, the purity of cell preparations can be determined by flow cytometric analyses.
  • the Treg cells administered to a transplant patient should selectively inhibit pathological immunity against allografts, or the host tissue in the case of allogeneic bone marrow transplantation, without compromising protective immunity.
  • the B-cell expanded Treg cells have a high alloantigen specificity that is not determined by the HLA haplotypes of the Treg cells, but that is induced and determined by the haplotype of the B cells used to expand them. Consequently, using the methods described herein, Treg cells from a given individual can be made specific for a particular alloantigen by expanding them with allogeneic B cells presenting that particular alloantigen.
  • the B cells can be isolated from a transplant or tissue graft donor to produce Treg cells that are allospecific for the transplant or tissue graft donor, the B cells can be isolated from a transplant or tissue graft recipient to produce Treg cells that are allospecific for the transplant or tissue graft recipient, or B cells from an individual having a target HLA haplotype can be used to produce Treg cells that are allospecific for that HLA haplotype.
  • a B cell bearing a synthetic genetic construct could be used to present an antigen with a defined amino acid sequence toward which Treg cell activity is desired.
  • Methods for expressing antigen constructs by B cells are known in the art. See, e.g., MeIo, et al, Gene Transfer of Ig-Fusion Proteins into B cells Prevents and Treats Autoimmune Diseases, The Journal of Immunology , 168:4788-95 (2002); Lei & Scott, Induction of Tolerance to Factor VIII Inhibitors by Gene Therapy with Immunodominant A2 and C2 Domains Presented by B cells as Ig-Fusion Proteins, Blood, 105:4865-70 (2005).
  • Antigenic peptides useful in the methods may be identified by eluting peptides from MHC molecules known to be associated with autoimmunity, for example the HLA-DQ and DR molecules that confer susceptibility to several common autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis.
  • Antigenic peptides useful in the present methods also include synthesized peptides predicted to bind to MHC molecules associated with autoimmune diseases. Suitable antigenic peptides for use in the methods herein and for producing the pharmaceutical compositions are known in the art.
  • Treg cells may be isolated from a transplant or tissue graft recipient, or from a transplant or tissue graft donor.
  • Treg cells can be isolated from a "third-party" individual other than the donor or the recipient.
  • B-cell expanded Treg cells can potently inhibit the antigen-specific alloproliferation of HLA- unrelated responder T cells, even when the HLA haplotypes of the Treg and responder cells differ at both the class I and class II HLA alleles. Effective inhibition is obtained with either antigen-specific or nonspecific Treg cells at a high Treg cell to responder T cell ratio. At a low Treg to responder cell ratio, potent inhibition is associated with antigen-specific Treg cells.
  • B-cell expanded Treg cells inhibit alloproliferation of responder T cells at a Treg to responder cell ratio of at least or about 1 :5, regardless of the HLA haplotypes of the Treg and responder cells.
  • the B-cell expanded Treg cells when the B-cell expanded Treg cells are stimulated with the same alloantigens against which the Treg cells were expanded, the B-cell expanded Treg cells can inhibit alloproliferation of responder T cells at a Treg to responder cell ratio of at least or about 1 :50, regardless of the HLA haplotypes of the Treg and responder cells.
  • Treg cells when B-cell expanded Treg cells are stimulated with dendritic cells expressing different alloantigens from which the Treg cells were expanded, the Treg cells inhibit proliferation of responder cells at a Treg to responder cell ratio of at least or about 1 :5. Under the same conditions, but at a Treg to responder cell ratio of at least or about 1 :50, only partial or marginal inhibition occurs.
  • Treg cells from any individual unrelated to the donor or recipient can be directed or enriched toward a desired specificity and unwanted reactivity can be attenuated through B-cell expansion of alloantigen specific T cell clones.
  • Isolating and expanding "third-party" Treg cells that are allospecific to a particular alloantigen may provide "off the shelf Treg cell pharmaceutical compositions that can be rapidly employed in therapy.
  • Such a Treg cell product overcomes several limitations of producing allospecific Treg cells from a transplant or tissue graft host or donor. For example, because at least two weeks are needed to expand Treg cells on a large scale, the expansion of allospecific Treg cells from unexpected hosts, such as cadaver donors, is not possible. Also, isolating and expanding allospecific Treg cells from a donor or host for immediate use in an emergency transplantation or tissue graft is not possible because of this time requirement.
  • Treg cells from a single donor or cord blood source might also constitute a barrier to providing enough Treg cells for therapy.
  • an "off the shelf Treg cell product is useful to treat, for example, acute graft- versus-host disease or acute allograft rejection under conditions where the availability of donor or recipient T cells is limiting, or when an insufficient time period is available to expand the Treg cells.
  • the pharmaceutical compositions comprise an enriched antigen specific Treg cell population in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans; mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA; adjuvants and preservatives.
  • Compositions are suitably formulated for intravenous administration.
  • the composition contains a therapeutically effective amount of the CD4 CD25 + Treg cells in combination with an effective amount of another bioactive material.
  • isolated CD25 + CD4 T cells are cultured in a suitable culture medium with isolated allogeneic B cells in the presence of a cytokine and a co-stimulatory agent.
  • a suitable culture medium is MEM- ⁇ plus GlutaMAX MEM supplemented with 50 ⁇ M 2-ME, HEPES, pyruvate, nonessential amino acids, penicillin/streptavidin, and 10% fetal calf serum.
  • the co-stimulatory agent activates CD28.
  • any agent capable of stimulating or cross-linking CD28 can be used to stimulate T cells, such as an anti-CD28 antibody or a ligand for CD28 (e.g., CD80 and/or CD86).
  • exemplary anti-CD28 antibodies or fragments thereof include monoclonal antibody 93 (IgG2; Bristol Myers Squibb, Princeton, NJ), monoclonal antibody KOLT-2 (IgGl), CD28.2 (Pharmigen, San Diego, CA).
  • Exemplary ligands include the B7 family of proteins such as B7-1 (CD80) and B7-2 (CD86) (Freedman et al, J. Immunol.
  • the method uses a CD28.2 antibody concentration of about 1 ⁇ g/ml.
  • the cytokine is an interleukin.
  • the interleukin is IL-2.
  • the interleukin is IL-15.
  • isolated T cells are cultured in a suitable culture medium under conditions for the weak stimulation of the TCR.
  • a suitable culture medium is MEM- ⁇ plus GlutaMAX MEM supplemented with 50 ⁇ M 2-ME, HEPES, pyruvate, nonessential amino acids, penicillin/streptavidin, and 10% fetal calf serum.
  • weak stimulation of the TCR is achieved using a low concentration of a TCR agonist.
  • weak stimulation of the TCR is achieved using an anti-CD3 antibody.
  • weak stimulation of the TCR is achieved using an antigen.
  • isolated T cells are cultured in a suitable culture medium containing a phorbol ester, including, but not limited to, phorbol 12-myristate 13-acetate (PMA).
  • a suitable culture medium is MEM- ⁇ plus GlutaMAX MEM supplemented with 50 ⁇ M 2-ME, HEPES, pyruvate, nonessential amino acids, penicillin/streptavidin, and 10% fetal calf serum.
  • CD25 + CD4 T cells are cultured in standard laboratory culture plates, dishes, bottles, or other containers at an appropriate cell density.
  • the CD25 + CD4 T cells can be cultured in 96-well round- bottom plates at a cell density from 2x 10 3 to 1 x 10 4 cells per well.
  • CD25 + CD4 T cells can be expanded at a ratio of B cells to T cells ranging from about 1 to about 4, from about 1 to about 1 , or from about 4 to about 1.
  • the cytokine (e.g., IL-2) concentration is from 4 to 100 units/ml.
  • a 20- to 40-fold expansion of the Treg cells may be accomplished within 14 days without further addition of B cells to the cultures.
  • the alloantigen-specif ⁇ c Treg cells suitably possess the following characteristics.
  • the alloantigen-specif ⁇ c Treg cells may express the cell surface markers CD4 + and CD25 + . These markers may be measured, for example, using anti-CD4 and anti-CD25 antibody reagents.
  • the alloantigen-specif ⁇ c Treg cells express FoxP3. Fox P3 may be measured, for example, as a function of protein expression as measured by a Western blot; as a function of FoxP3 mRNA transcription measured using a Northern blot, RT-PCR, or another method of mRNA quantification; or by flow cytometry.
  • the alloantigen- specif ⁇ c Treg cells may maintain an anergic state, which may be measured, for example, by quantifying IL-2 and IFN - ⁇ production as in Example 2.
  • the alloantigen-specif ⁇ c Treg cells may suppress proliferation of autologous responder T cells stimulated in culture by exposure to the antigen used for expansion. The suppression may be measured, for example, using a proliferation assay as in Example 2.
  • FoxP3 is a forkhead family transcription factor that is necessary, but not sufficient, for human Treg cell suppressive activity. Accordingly, FoxP3 expression is a useful marker for verifying the presence of and quantifying the number of regulatory T cells present in the expanded population, for example, by quantifying FoxP3 protein, FoxP3 mRNA, or FoxP3 -expressing cells. FoxP3 protein expression can be quantified by western blot analysis.
  • An example of an exemplary protocol consists of the following steps.
  • T cell populations are washed in PBS and lysed and sonicated in lysis buffer comprising 25 mM Tris pH 8.5, 2% lithium dodeccyl sulfate, 1 mM EDTA, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 x Roche Complete protease inhibitors and protein levels are quantified (or example, by using a BCA assay; Pierce). Lysates are separated on 4- 12% gradient bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes.
  • Membranes are blocked for 3 hours in TBS/0.1% Tween-20 with 5% nonfat dry milk, probed with polyclonal rabbit-anti-FoxP3 antiserum (1 :2000) overnight at 4°C in the same buffer and developed using standard protocols. Western blots are stripped and re-probed with TFIIB (Santa Cruz) for a loading control. For a positive control, 293T cells are transfected with a human FoxP3 cDNA clone.
  • FoxP3 levels can be assayed by a quantitative PCR assay.
  • An example of an exemplary protocol consists of the following steps. RNA is extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions, and cDNA is prepared with 2.5 ⁇ M random hexamers (Applied Biosystems Inc., Foster City, CA). Message levels are quantified by real-time PCR using the ABI 7000 Sequence Detection System (Applied Biosystems Inc.). Amplification is carried out in a total volume of 25 ⁇ l for 40 to 50 cycles of 15 seconds at 95°C, 1 minute at 60 0 C, and product can be detected using SYBR Green I dye (Molecular Probes Inc., Eugene, OR).
  • the relative expression can be determined by normalizing expression of each target to GAPDH, and then comparing this normalized value to the normalized expression in a reference sample to calculate a fold-change value.
  • Primers can be designed so that amplicons spanned intron/exon boundaries to minimize amplification of genomic DNA.
  • FoxP3 -expressing cells can also be quantified using, for example, flow cytometry and an anti-FoxP3 antibody.
  • an anti-FoxP3 antibody One specific protocol is described in Roncador et ah, Eur. J. Immunol. 35:1681-1691, 2005. Generally, cells are washed and suspended in phosphate- buffered saline containing 2% FCS and 0.02% sodium azide, then incubated with an anti- FoxP3 antibody using standard protocols and manufacturers' instructions.
  • the anti-FoxP3 antibody can be fluorescently labeled, or a second fluorescently-labeled antibody that recognizes the anti-FoxP3 antibody can be used.
  • the anti-FoxP3 antibody 236A/E7 from eBioscience is one such antibody that can be used to label cells expressing FoxP3. Stained cells can be analyzed using a flow cytometry analysis machine or a fluorescence-activated cell sorter such as a FAC S aria (Becton Dickinson, Franklin Lakes, NJ) or FACScalibur and the data can be analyzed with appropriate software such as FIoJo.
  • the present technology provides a pharmaceutical composition comprising a T cell population containing antigen-specific CD4 CD25 + Treg cells in a formulation which is suitable for administration to a patient in need thereof.
  • the antigen-specific CD4 CD25 + Treg cells are specific for a self-antigen associated with an autoimmune or inflammatory disease.
  • the Treg cells are useful for promoting transplant tolerance.
  • the methods of generating antigen- specific CD4 CD25 + Treg cells described herein are useful for generating the T cell population for use in the composition according to this embodiment.
  • the pharmaceutical composition comprising CD4 CD25 + antigen- specific regulatory T cells is administered to a subject in need thereof in a manner appropriate to the disease to be treated and/or prevented.
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient and the type and/or severity of the patient's disease. Appropriate dosages may also be determined by clinical trials.
  • An effective amount of the composition can be determined by a physician with consideration of individual differences in age, weight, disease severity, condition of the patient, route of administration and any other factors relevant to treatment of the patient.
  • a pharmaceutical composition comprising Treg cells may be administered at a dosage of about 10 5 to 10 8 cells/kg body weight, suitably 10 5 to 10 6 cells/kg body weight, including all integer values within these ranges.
  • the compositions may also be administered multiple times at these dosages.
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • the cells can be administered by using infusion techniques that are commonly used in immunotherapy, and may be administered to a patient subcutaneously, intradermally, intramuscularly, or by intravenous injection.
  • the methods described herein may be useful in a therapeutic application to treat a patient.
  • the methods are useful for treating immunological disorders, such as various autoimmune diseases and immune-related inflammatory disease, and in promoting transplant tolerance in a transplant recipient.
  • the expanded Treg cells are useful in promoting tolerance of bone marrow or a solid organ transplanted from a donor to a recipient.
  • the expanded Treg cells are also useful in preventing or treating graft- versus-host disease associated with a transplant or tissue graft.
  • Treg cells include multiple sclerosis, type 1 diabetes, inflammatory bowel disease, cardiovascular disease, Parkinson's disease, Alzheimer's disease, arthritis, IPEX, allergy, gastritis, lung inflammation, or the destructive effects of an immune response to an infectious agent.
  • HLA haplotype- shared third-party B cells for expansion of alloantigen-specif ⁇ c Treg cells are also useful for bone marrow transplantation situations where the recipients cannot be a source of the B cells because of ongoing hematological malignancies such as leukemia or multiple myeloma.
  • the present technology provides methods for treating and/or preventing an autoimmune disease or inflammatory condition.
  • the pathogenesis of a number of autoimmune diseases is believed to be caused by autoimmune T cell responses to self- antigens present in the organism.
  • autoreactive T cells have been implicated in the pathogenesis of multiple sclerosis (MS), rheumatoid arthritis (RA), type 1 diabetes (TlD), and Pemphigus.
  • MS multiple sclerosis
  • RA rheumatoid arthritis
  • TlD type 1 diabetes
  • Pemphigus Pemphigus
  • Treg In humans, the ability of Treg to regulate T cells in an antigen-specific manner has been demonstrated in the context of various diseases, including regulation of T cells specific to tumor antigens (Viguier et al., J. Immunol., 173:1444-53 (2004)); alloantigens in the setting of bone marrow transplantation (Ng et al., Blood, 98:2736-44 (2001)); and the nonself alloantigens as described herein and in Walker et al, PNAS, 102:4103-08 (2005)). Therefore, immunotherapy with Treg cells obtained from T cells of a human subject is useful in the context of a cellular therapy for regulating the immune response in the subject.
  • the Treg cells may be used for preventing and/or treating a disease or condition such as an autoimmune disease, inflammatory disease, or in the treatment and/or prevention of transplant rejection and also to prevent graft-versus-host reactions.
  • a disease or condition such as an autoimmune disease, inflammatory disease, or in the treatment and/or prevention of transplant rejection and also to prevent graft-versus-host reactions.
  • Methods of Treating and/or Preventing Graft Versus Host Disease One problem in hematopoietic stem cell transplantation is graft-versus-host disease (GVHD), which is caused by alloreactive T cells present in the infused hematopoietic stem cell preparation.
  • GVHD graft-versus-host disease
  • Studies in mice have demonstrated that adoptive transfer of Treg can block graft-versus-host disease without affecting the graft- versus-leukemia response (Edinger et ah, Nat.
  • the technology provides a method for reducing the risk of, or the severity of, an adverse GVHD effect in a patient who is undergoing a hematopoietic stem cell transplant, comprising administering to the patient an amount of regulatory T cells specific for mismatched antigens between the recipient and donor according to the methods described herein effective to reduce the risk or severity of an adverse GVHD effect in the patient.
  • the technology provides methods for reducing the risk of, or the severity of, an adverse immune response in a patient that has undergone, is undergoing, or will undergo, an organ transplant, comprising administering to the patient an amount of a population of transplant- specific Treg cells according to the methods described herein effective to reduce the risk or severity of an adverse immune response in the patient.
  • the transplant-specific Treg cells may be generated using the methods described herein.
  • the method comprises obtaining a sample containing T cells from the patient.
  • a population of CD4 CD25 + T cells is isolated from the sample and transplant-specific regulatory T cells are produced by contacting the isolated T cells in a culture vessel with B cells isolated from the transplant donor and using the culture conditions disclosed herein.
  • B cells can be used that comprise at least one antigenic peptide specific to the transplant organ or tissue. The cells are administered in an amount effective to reduce the risk and/or the severity of an adverse immune response in the patient.
  • Cyclosporin is the mainstay drug used in treating GVHD and solid organ rejection. From time to time, the drug is ineffective for reasons not yet understood, and its prolonged use often causes multi-organ damage or toxicity.
  • the present inventors discovered that CsA when used in conjunction with IL-2 selectively inhibited activation of effector cells while promoting expansion of Treg cells (FIG. 4). This finding suggests CsA used alone could worsen GVHD by damaging Treg cells through inhibiting IL-2 secretion from effector T cells. Adding IL-2 to the regimen will promote Treg cell propagation in the patients while the inhibition of effector T cells is maintained.
  • low doses of IL-2 with CsA are adiminstered to prevent GVHD in allogeneic BMT patients. If CsA is used with IL-2 for treatment of solid organ rejection, it is predicted that patients may no longer need to take CsA for a lifetime but do well without taking the drug because of generation of a stable population of Treg cells in the immune system.
  • the methods described in this aspect are useful for reducing the risk of, or the severity of, any adverse immune response in a transplant recipient, such as graft-versus-host disease.
  • the methods may be applied to solid organ (e.g., kidney(s), heart, lung(s), liver, and pancreas) transplant recipients, to allogeneic bone marrow, or to autoimmune patients with autologous or allogeneic bone marrow.
  • a reduction of severity of an adverse immune response may be measured by any suitable method. Nonlimiting examples include the reduction or elimination of acute graft rejection, the reduction or elimination of chronic rejection, the reduction or elimination of graft-versus-host disease, and/or the reduction or elimination of the need for high doses of immunosuppressive drugs.
  • compositions can be further approximated through analogy to cells and compositions known to exert the desired effect.
  • Example 1 Provided by way of illustration and are not intended to be limiting of the present methods.
  • B cells were isolated from Ficoll-enriched lymphocytes by positive selection with anti-CD 19 beads (Miltenyi Biotec). Monocytes were positively selected with CD 14 beads (Miltenyi Biotec), and cultured with 50 ng/ml each of GM-CFS and IL-4 for 6 days with addition 500 ng of LPS on day 5 to generate mature DCs.
  • CD25 + cells were cultured with anti-CD3 antibody (OKT3 ) (1 ⁇ g/ml) plus CD28.2 (0.5 ⁇ g/ml).
  • the cells were cultured for 4 hours with phorbol 12-myristate 13- acetate (PMA) (10 ng/ml) plus ionomycin (300 ng/ml).
  • PMA phorbol 12-myristate 13- acetate
  • Inhibition assays were performed by culturing CD25 CD4 responder cells with allogeneic DCs at a 1 to 5 DC to T cell ratio in the presence of various numbers of Treg cells expanded with B cells of the same or different donor origin as the DCs.
  • the Treg cells in the cultures were enumerated by combining cell counts and flow cytometry.
  • B cells constituted only a small fraction ( ⁇ 10%) of the cells in the expanded product at the end of culture, and were not removed from expanded Treg cells used in suppressor assays.
  • OKT3 plus CD28.2 was also used to stimulate the cultures as indicated.
  • Antibodies for flow cytometry were purchased from BD Bioscience, except for anti-FoxP3 (236A/E7), which was from eBioscience. Intracellular cellular staining was performed using Perm/Fix kits (eBioscience). The data were collected on FACS Calibur and analyzed with FlowJo software.
  • HLA-I HLA-A, -B
  • HLA-II HLA-DR
  • CD25 CD4 non-Treg cells were cultured for 3 days with allogeneic DCs (FIG. ID).
  • FIG. ID To examine expression of costimulatory molecules (FIG. IE), primary B cells (solid thick lines) were stained with anti-CD80, CD86, or isotype-control antibodies (dotted line); EBV-transformed B cells were used as positive controls.
  • CD25 + CD4 T cells were cultured for 7 days with allogeneic B cells at a B to T cell ratio of 1 to 1 in the presence of 0.5 ⁇ g/ml CD28.2 plus various concentrations of IL-2 (FIG.
  • CD25 + CD4 T cells were cultured with allogeneic B cells at B to T cell ratio of 1 to 1 in the presence of 200 u/ml of IL-2 and 1 ⁇ g/ml of CD28.1, and enumerated at various days of culture (FIG. IH). The results are representative of more than three experiments.
  • Treg cells were cultured with allogeneic B cells alone (FIG. IA), and supplementation of IL-2 resulted in only minimal Treg cell proliferation (FIG. IB) in multiple attempts.
  • the lack of significant Treg cell expansion under these culture conditions raised the possibility that human Treg cells might require costimulation, which was not required by mouse Treg cells, for activation and proliferation.
  • Freshly isolated human B cells were analyzed for expression of CD80 and CD86, and neither of these costimulatory molecules was detected (FIG. IE).
  • stimulatory anti-CD28 antibody CD28.2 was supplemented in the cultures along with exogenous IL-2, and this resulted in robust proliferation of FoxP3 + cells (FIG.
  • the concentration of IL-2 and the B cell to Treg cell ratio influenced the degree of Treg cell expansion, but the impact was modest (FIGs. IF & IG), since a 25-fold change in IL-2 concentration and 16-fold change in B to T cell ratio resulted in less than 2-fold change in expansion of Treg cells.
  • the kinetics of allogeneic B-cell induced Treg cell proliferation was slow (FIG. IH). Very little cell division was detected at day 3, with minimal division observed on day five. Robust cell proliferation was observed at later time points. A 20- to 40- fold expansion of the cells could be obtained within 14 days without further addition of B cells to the cultures.
  • the expanded Treg cells expressed high levels of FoxP3 (with a mean fluorescence intensity, MFI, 1630) (FIG. 1C), which was three-fold higher than that of the non-dividing input CD25 + CD4 cells (MFI, 550).
  • CD25 CD4 T cells were cultured with allogeneic DCs for 3 days, which resulted in a clear increase in FoxP3 expression (MFI 80) in proliferating CD4 T cells (FIG. Id) as compared to the non-dividing CD4 T cells (MFI 9). This level, however, was much lower (>20 fold) than that detected in the B-cell expanded Treg cells or in the non-dividing input Treg cells. Therefore, the expression of FoxP3 in B-cell expanded Treg cells was quantitatively different from that of activated non-Treg cells.
  • B-cell expanded Treg and activated non-Treg cells were rested in IL-2 containing media for 2 days and then re-stimulated with PMA plus ionomycin.
  • Expanded CD25 + CD4 Treg cells (FIGs. 2A & 2B) or CD25 CD4 non-Treg cells (FIGs. 2C & 2D) were rested and then re-stimulated with PMA plus ionomycin, and analyzed for IL-2 and IFN- ⁇ production by intracellular flow cytometry.
  • CFSE-labeled CD25 CD4 responder T cells were cultured with allogeneic DCs alone (FIG. 2E) or in the presence of B-cell expanded Treg cells at a Treg to responder cell ratio of 1 to 5 (FIG. 2F), 1 to 20 (FIG. 2g) or 1 to 80 (FIG. 2H), or in the presence of OKT3/CD28.2-expanded Treg cells at Treg to responder ratio of 1 to 5 (FIG. 21), 1 to 20 (FIG. 2J) or 1 to 80 (FIG. 2K).
  • FIG. 21 To provide polyclonal stimulation (FIG.
  • OKT3 plus CD28.2 instead of allogeneic DCs was used to stimulate the cultures consisting of CD25 CD4 responder T cells with various numbers of either B cell-expanded (open square) or OKT3/CD28.1 -expanded (open circle) Treg cells for 6 days.
  • the total number of proliferating responder cells was enumerated by flow cytometry, and the number presented in each histogram represents the average of duplicates or triplicates. The results are representative of at least two experiments.
  • the B-cell expanded Treg cells were much more potent than OKT3 -expanded polyclonal Treg cells in suppressing alloreactivity of responder T cells. Since the low inhibitory capacity of OKT3 -expanded Treg cells could be due to a low frequency of alloantigen-specific Treg cells in the expanded cells, OKT3 plus CD28.2 instead of allogeneic DCs were used as a global stimulus to provide polyclonal activation of both Treg and responder T cells in the cultures.
  • the Treg cells administered to a transplant patient should selectively inhibit pathological immunity against allografts or the host tissue in the case of allogeneic bone marrow transplantation (BMT) without compromising protective immunity. This might be accomplished by generating Treg cells that are specific for donor or host alloantigens.
  • CD25 + CD4 T cells from a given donor #32 were divided and expanded separately with B cells from two unrelated donors designated Y and Z, whose HLA-DR haplotypes were DRBl *0701/*0301 and DRBl *1201/*0101, respectively (Table 1).
  • CFSE-labeled CD25 CD4 responder T cells were cultured for 5 days with allogeneic DCs from donor Y either alone (FIG. 3A) or in the presence of Treg cells expanded by B cells from either donor Y at Treg to responder cell ratio of 1 to 5 (FIG.
  • Each expanded Treg population was used to inhibit alloproliferation of the CD25 CD4 responder T cells from an unrelated donor #40 (DRB * 04 AM AD/* 0407) elicited by mature DCs derived from either donor Y or Z.
  • the DCs of both donors Y and Z elicited vigorous proliferation of the responder T cells in mixed leukocyte cultures (MLCs) (FIG. 3 A & F).
  • MLCs mixed leukocyte cultures
  • Treg cells expanded by B cells of donor Y imposed only minimal inhibition to the MLCs elicited by DCs of donor Z at a Treg to responder ratio of 1 to 50 (FIG. 3J), in contrast to strong inhibition in MLCs elicited by DCs from donor Y (FIG. 3C).
  • FIG. 4 Similar results were obtained in multiple cultures involving different allogeneic BC/DC and Treg cell combinations (FIG. 4).
  • the results demonstrate that allogeneic B cell-expanded Treg cells are highly suppressive and, at a relatively high Treg to responder cell ratio (1 to 5), they are capable of imposing potent inhibition on alloreactive, third-party responder T cells in a haplotype nonspecific manner.
  • Treg cells lose their capacity to potently inhibit alloreactivity elicited by non-specific alloantigens while remaining highly potent in inhibiting alloproliferation of responder cells elicited by specific alloantigens.
  • the alloantigen specificity demonstrated by B-cell expanded Treg cells is not determined by the HLA-haplotypes of the Treg cells but induced and determined by the haplotype of the B cells used to expand them.
  • Treg cells While the information regarding the availability of Treg cells from a single donor is scant in the literature, in our experience, with magnetic beads sorting, only a very small number (1 to 3 ⁇ 10 5 ) of CD25 + cells of purity greater than 90% FoxP3 + cells could be obtained from a leuko-f ⁇ lter of 1 unit (500 ml) of blood. Although the number of Treg cells could be expanded ex vivo substantially, excessive expansion has been shown to increase the frequency of FoxP3 T cells in the expanded product.
  • Treg cells Since transfer of activated effector T cells is potentially harmful to the recipients, the requirement for highest quality of expanded Treg cells might pose a limit to the extent which Treg cells could be expanded ex vivo, and thus make it necessary to have a sufficient number of freshly isolated Treg cells as starting input cells for expansion.
  • Another potential barrier to the use of Treg cells in transplantation could be related to the time needed to expand Treg cells. According to all current protocols, at least two weeks are needed to expand Treg cells on a large scale, which makes expansion of Treg cells specific for alloantigens of unexpected donors, such as cadaver donors, impossible.
  • the use of third-party Treg cells expanded for specificity for various major target HLA haplotypes opens the potential for "off the shelf cell products that could be rapidly employed in therapy.
  • CD25 CD4 T cells from three HLA-unrelated blood donors, #38, #39 and #40 were used in cultures with CD25 + CD4 T cells from each of two HLA- unrelated donors, #31 and #32, which had been expanded separately with B cells from donors Y and Z (Table 1).
  • the B cell-expanded Treg cells potently inhibited alloproliferation of the responder T cells in all Treg and responder cell combinations at a Treg to responder T cell ratio of 1 to 5, or at 1 to 50 when the Treg cells were stimulated with the same alloantigens against which the Treg cells were expanded (FIG. 4A & 4B).
  • the B cell-expanded Treg cells were stimulated with DCs expressing different alloantigens from which they were expanded (FIG.
  • the Treg cells still substantially inhibited alloproliferation of the responder cells at Treg to responder cell ratio of 1 to 5. At Treg to responder cell ratio of 1 to 50, however, only partial or marginal inhibition was observed.
  • the B cell-expanded Treg cells can potently inhibit alloproliferation of HLA-unrelated responder T cells. Effective inhibition was obtained with either antigen-specific or nonspecific Treg cells at relatively high Treg to responder cell ratio. At a low Treg to responder cell ratio, however, potent inhibition was only associated with antigen-specific Treg cells.
  • DRB1 '0701 is a shared allele between donor Y and #32
  • # DRB1 '0404 is a shared allele between donor #31 and #39
  • CD45.1 + Treg cells underwent only mild proliferation, with a small number of CD45.1 " converted Treg cells contributing to the FoxP3 CD4 + T cell pool.
  • CD45.1 + Treg cells under more vigorous expansion, and at the same time, more FoxP3 + CD4 + T cells are converted from CD45.1 " non-Treg cells.
  • TGF ⁇ concentration for each culture condition. More TGF ⁇ was detected in low 2Cl 1 stimulated cultures (FIG. 8A). When TGF ⁇ neutralizing antibody was supplemented in the culture, the Treg cell conversion was diminished (FIG. 8B).
  • NFATcI The total amount of NFATcI, NFATc2 and c-Fos (AP-I) was found to be similar in the total cell lysates of freshly isolated Treg and non-Treg CD4 + cells as detected by Western blots (FIG. 9), but two to three folds more c-Rel (NF- ⁇ B) was identified in Treg than in non-Treg cells in all (five) experiments. In non-Treg cells, all these factors were detected only in the cytoplasm but not in the nucleus, consistent with previous findings.
  • Treg and non-Treg cells were isolated and rested. Some non-Treg cells were stimulated with PMA plus ionomycin for 30 minutes. Chromatin Immunoprecipitation (ChIP) was performed as described previously. Antibodies against NFATcI and NFATc2 (Santa Cruz) were mixed and used to precipitate NFAT-bound DNA. PCR were performed with primer sets for 112, Pde3b-20, Irf4, miR-155, FasL, 114 promoter and enhancer regions.
  • NFAT bound to IL-2, Pde-20 and IrF4 in both Treg and activated non-Treg cells, but IL-4 promoter and enhancer and Pde3b only in activated non-Treg cells (FIG. 11).
  • the binding of miR-155 was detected only in Treg but not in activated non-Treg cells.
  • NFAT did not bind to FasL in either Treg or activated non-Treg cells (FIG. 11).
  • Example 7 Third-Party Treg Cells Prevent Rejection of Allogenic Bone Marrow Grafts in a MHC-related Host.
  • B6 mice were sub-lethally irradiated (with 500 rads of X-ray), then transplanted with bone marrow cells from BALB/c mice (2 x 10*7 cells/mouse) with or without the expanded Treg cells described above).
  • the BM-transplanted mice were followed of their peripheral blood cells by flow cytometry for evidence of BM engrafment.
  • FIG. 12 show that the that mice received B6 bone marrow alone rejected the bone marrow cells (FIG .12C) while the mice that received the bone marrow plus the Treg cells did not reject (FIG 12D) (as indicated by the co-presence of H2-Kd+ and H2-Kb+ cells in the blood.
  • a range includes each individual member.
  • a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

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Abstract

L'invention porte sur des procédés d'expansion de lymphocytes T régulateurs. Les lymphocytes T régulateurs humains sont expansés par culture d'un lymphocyte T régulateur humain avec un lymphocyte B afin de produire une population de lymphocytes T régulateurs humains allospécifiques. Les procédés comprennent la mise en culture d'un lymphocyte T et d'un lymphocyte B en présence d'une cytokine et d'un anticorps stimulateur. Les lymphocytes T régulateurs peuvent également être expansés à l'aide d'une stimulation faible du récepteur de lymphocyte T ou d'un traitement par l'ester de phorbol (phorbol-12-myristate 13-acétate). L'invention porte en outre sur des méthodes de traitement de troubles liés à l'immunité par administration des lymphocytes T régulateurs humains expansés à un sujet en ayant besoin.
PCT/US2010/033869 2009-05-07 2010-05-06 Procédés d'expansion de lymphocytes t régulateurs humains et leurs utilisations WO2010129770A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013131045A1 (fr) 2012-03-02 2013-09-06 The Regents Of The University Of California Expansion de lymphocytes t régulateurs réagissant avec des alloantigènes
WO2017062035A1 (fr) * 2015-10-09 2017-04-13 Abt Holding Company Procédés d'activation de la prolifération de cellules régulatrices t
US10092597B2 (en) 2014-01-14 2018-10-09 The University Of Hong Kong Human CD8+ regulatory T cells inhibit GVHD and preserve general immunity in humanized mice
US11384336B2 (en) 2016-12-07 2022-07-12 East Carolina University Compositions and methods for in vitro cultivation and/or expansion of regulatory T cells

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006052844A2 (fr) * 2004-11-04 2006-05-18 Medical College Of Georgia Research Institute Vaccin tolerogenique et sa methode
WO2007033291A2 (fr) * 2005-09-14 2007-03-22 The Trustees Of Columbia University In The City Of New York Lymphocytes t cd8+ regulateurs induits par des anticorps anti-cd3
WO2010000127A1 (fr) * 2008-06-30 2010-01-07 Versitech Limited Procédé d’induction et d’expansion de lymphocytes t régulateurs humains spécifiques d’un alloantigène thérapeutiques à grande échelle

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006052844A2 (fr) * 2004-11-04 2006-05-18 Medical College Of Georgia Research Institute Vaccin tolerogenique et sa methode
WO2007033291A2 (fr) * 2005-09-14 2007-03-22 The Trustees Of Columbia University In The City Of New York Lymphocytes t cd8+ regulateurs induits par des anticorps anti-cd3
WO2010000127A1 (fr) * 2008-06-30 2010-01-07 Versitech Limited Procédé d’induction et d’expansion de lymphocytes t régulateurs humains spécifiques d’un alloantigène thérapeutiques à grande échelle

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALBERT, M. H. ET AL.: "Ethylenecarbodiimide-coupled allogeneic antigen presenting cells induce human CD4+ regulatory T cells", CLINICAL IMMUNOLOGY, vol. 129, 2008, pages 381 - 393 *
CHEN, L. C. ET AL.: "Direct expansion of Human allospecific FoxP3+ CD4+ regulatory T cells with allogeneic B cells for therapeutic application", THE JOURNAL OF IMMUNOLOGY, vol. 183, no. 6, 2009, pages 4094 - 4102 *
CHEN, T.-G. ET AL.: "Induction of dominant transplantation tolerance by an altered peptide ligand of the male antigen Dby", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 113, no. 12, 2004, pages 1754 - 1762 *
TU, W. ET AL.: "Efficient generation of human alloantigen-specific CD4+ regulatory T cells from naive precursors by CD40-activated B cells", BLOOD, vol. 112, no. 6, 2008, pages 2554 - 2562 *
ZHENG, J. ET AL.: "Efficient induction and expansion of human alloantigen-specific CD8 regulatory T cells from naive precursors by CD40-activated B cells", THE JOURNAL OF IMMUNOLOGY, vol. 183, no. 6, 2009, pages 3742 - 3750 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013131045A1 (fr) 2012-03-02 2013-09-06 The Regents Of The University Of California Expansion de lymphocytes t régulateurs réagissant avec des alloantigènes
CN104245923A (zh) * 2012-03-02 2014-12-24 加州大学评议会 同种异型抗原-反应性调节t细胞的增殖
JP2015513403A (ja) * 2012-03-02 2015-05-14 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 同種抗原反応性の制御性ｔ細胞を増大させる方法
EP2820125A4 (fr) * 2012-03-02 2015-07-29 Univ California Expansion de lymphocytes t régulateurs réagissant avec des alloantigènes
US9801911B2 (en) 2012-03-02 2017-10-31 The Regents Of The University Of California Expansion of alloantigen-reactive regulatory T cells
CN104245923B (zh) * 2012-03-02 2018-04-20 加州大学评议会 同种异型抗原‑反应性调节t细胞的增殖
AU2013225721B2 (en) * 2012-03-02 2018-06-14 The Regents Of The University Of California Expansion of alloantigen-reactive regulatory T cells
EP3366768A1 (fr) * 2012-03-02 2018-08-29 The Regents of The University of California Expansion de lymphocytes t régulateurs réagissant avec des alloantigènes
US10092597B2 (en) 2014-01-14 2018-10-09 The University Of Hong Kong Human CD8+ regulatory T cells inhibit GVHD and preserve general immunity in humanized mice
US10653722B2 (en) 2014-01-14 2020-05-19 The University Of Hong Kong Human CD8+ regulatory T cells inhibit GVHD and preserve general immunity
WO2017062035A1 (fr) * 2015-10-09 2017-04-13 Abt Holding Company Procédés d'activation de la prolifération de cellules régulatrices t
US11384336B2 (en) 2016-12-07 2022-07-12 East Carolina University Compositions and methods for in vitro cultivation and/or expansion of regulatory T cells

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