WO2003008558A2 - Cd4+cd25+ inhibitory hybridoma clones - Google Patents

Abstract

Provided by the invention are immortalized CD4+CD25+ regulatory cells that provide a source of abundant CD4+CD25+ cells. CD4+CD25+ cells are capable of regulating immune tolerance to foreign transplantation antigens, autoimmune reactions, allergic reactions and other undesired immune reactions. Also provided are methods for generating CD4+CD25+ regulatory cells using the immortalized cells of the invention. Furthermore, the instant invention provides methods for alleviating diseases caused bay undesirable/adverse immune reactions, such as autoimmune diseases/conditions, transplantation rejections and allergic diseases, by providing effective amounts of the CD4+CD25+ cells to patients afflicted with such diseases.

Description

CD4⁺CD25⁺ INHIBITORY HYBRIDOMA CLONES

BACKGROUND OF THE INVENTION

The present application claims priority to co-pending U.S. Patent Application Serial Number 60/307,000, filed July 20, 2001, the entire contents of which are incorporated herein by reference. The government owns rights in the present invention pursuant to grant number CA- 45541 from the National Institutes of Health.

1. Field of the Invention

The present invention relates generally to the fields of basic and clinical immunology, immune mediated inflammatory disorders, and immune-tolerance. More particularly, it concerns the development of immortalized CD4⁺CD25⁺ regulatory cells that are capable of regulating immune responses and tolerance to foreign transplantation antigens, autoimmune reactions, allergic reactions and other undesired immune reactions. Methods for generating CD4⁺CD25⁺ regulatory cells using the immortalized cells ofthe invention and methods for using such cells in therapeutic embodiments are also set forth.

2. Description of Related Art

Immunoregulatory abnormalities, where immune tolerance regulation is affected, manifests in a wide variety of immune mediated inflammatory disorders such as autoimmune diseases, chronic inflammatory diseases and allergic diseases, including systemic lupus erythematosis, chronic rheumatoid arthritis, type I and II diabetes mellitus, inflammatory bowel disease, biliary cirrhosis, uveitis, multiple sclerosis and other disorders such as Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, ichthyosis, Grave's ophthalmopathy and asthma. Another area where immune tolerance regulation is actively sought is in tissue and organ transplants where graft rejection reactions cause morbidity. Still another area where immune tolerance regulation is sought is in blood cell or bone marrow transplantation, where graft versus host disease reactions cause high rates of morbidity and mortality.

CD4⁺CD25⁺ cells are known to regulate immune tolerance. In models of spontaneous autoimmunity, the role of regulatory CD4⁺ T-cells has been described (Mason and Powrie, 1998). For example, neonatal BALB/c mice thymectomized within three days of birth spontaneously developed organ-specific autoimmunity (Asano et al, 1996). The adoptive transfer of CD4⁺CD25⁺ spleen cells from normal mice prevented autoimmunity (Asano et al, 1996; Suri-Payer et al, 1998). Sakaguchi and colleagues identified immunoregulatory CD25⁺CD4⁺CD8^~ thymocytes that were capable of controlling self-reactive T-cells (Itoh et al, 1999). While the adoptive transfer of CD25⁺CD4⁺CD8^~ -depleted thymocytes produced various autoimmune diseases in syngeneic athymic nude mice, co-transfer with the CD25⁺CD4⁺CD8^~ thymocyte fraction blocked development of autoimmunity (Itoh et al, 1999). The regulatory thymocytes in this case were CD69^" (Thornton and Shevach, 2000) and expressed high levels of CD62L (Itoh et al, 1999; Thornton and Shevach, 2000). Thus, early thymectomy prevents the development and emergence of important CD4⁺CD25⁺ regulatory cells that function to maintain peripheral tolerance to self-antigens.

CD4⁺CD25⁺ regulatory cells also have been implicated in preventing spontaneous diabetes in NOD mice (Salomon et al, 2000). B7.1/B7.2 double deficient (B7^"/") NOD mice developed an accelerated and more severe form of diabetes compared to their B7^{+ "} or B7^+/+ littermates. Treatment of NOD mice with 5 injections of CTLA4Ig beginning at 6-8 weeks of age, prior to the onset of diabetes, accelerated the progression and severity of diabetes compared to untreated controls. Both B7^{_ "} NOD mice and CTLA4Ig-treated NOD mice had markedly decreased numbers of CD4⁺CD25⁺ T-cells. Moreover, the adoptive transfer of CD4⁺CD25 T- cells from NOD mice blocked the development of spontaneous diabetes when co-injected into NOD.SCID mice along with diabetogenic splenocytes, while the adoptive transfer of CD4⁺CD25^~ cells did not prevent the development of diabetes by the diabetogenic splenocytes (Salomon et al, 2000).

A critical role of CD4⁺CD25⁺ regulatory T-cells in protection from several autoimmune syndromes including gastritis and colitis has been shown (Suri-Payer and Cantor, 2001). CD4⁺CD25⁺ cells are also known to mediate tolerance in tissue and organ transplantation reactions. For example, tolerance to cyclosporin-A induced autologous graft-vs-host disease is actively mediated by CD4⁺CD25⁺ cells (Wu et al, 2001). In addition, CD4⁺CD25⁺ regulatory cells isolated from tolerant mice have been shown to prevent graft rejection in non-tolerant mice (Hara et al, 2001). In fact, Taylor et al, (2001), have shown that CD4⁺CD25⁺ cells are required in order for pharmacological agents that block immune co-stimulation to be effective at preventing graft-vs-host disease.

Despite their importance in regulating peripheral tolerance, especially in the context of controlling autoimmune diseases and transplantation rejections, CD4⁺CD25⁺ cells are present at a very low frequency and comprise <l-5% of the peripheral lymphoid cells. Thus, they are difficult to isolate in sufficient amounts required to provide immunotherapy. Furthermore, due to their low abundance in vivo, sufficient numbers of cells are not available to study the details of their mechanism of action in controlling immune responses important in establishing or maintaining tolerance.

Some special in vitro culture systems have had success in generating CD4⁺CD25⁺ cells. For example, Yamagiwa et al. (2001), have generated CD4⁺CD25⁺ regulatory cells ex vivo by culturing human naive CD4 cells with allogeneic APCs and TGFβ. These ex vivo generated CD4⁺CD25⁺ human regulatory cells are capable of blocking CD8⁺ proliferation and generating CTL when added to MLR cultures. However, although CD4⁺CD25⁺ cells having immune regulatory properties have been generated through ex vivo cultures these culture systems are expensive and time consuming and are not efficient in producing large quantities of CD4⁺CD25⁺ cells.

Thus, the art lacks an efficient and cost-effective method for generating a large population of CD4⁺CD25⁺. The provision of such a method will overcome the major obstacle of obtaining sufficient numbers of immunoregulatory CD4⁺CD25⁺ cells and enable the use of these cells as therapeutic agents or critical reagents to generate therapeutic agents.

SUMMARY OF THE INVENTION

The present invention overcomes the aforementioned defects in the art and provides an immortalized source of the regulatory CD4⁺CD25⁺ cells. The invention also provides methods for generating both immortalized regulatory CD4⁺CD25⁺ cells and non-immortalized regulatory CD4⁺CD25⁺ cells. Methods for the treatment of several immune-related diseases using the regulatory CD4⁺CD25⁺ cells ofthe invention are provided as well.

The invention provides immortalized cells expressing the antigens CD4 and CD25. Several clones of immortalized CD4⁺CD25⁺ cells are provided and include the 3C1 clone and its subclones such as, the RD6 cells, the 3B1 cells, the 3C2 cells, the 2B6 cells, the 1B4 cells, the 2D5 cells, the 2A6 cells, the lDlcells, the 1A6 cells, the 2B1 cells, the 2D3 cells, the 2D4 cells, the 2C5 cells, the 3 A2 cells, and the 1A2 cells.

The immortalized cells of the invention are further defined as hybridoma cells. Hybridoma cells are defined here as hybrid cells formed by a fusion of a first cell population which comprises a regulatory T-cell population, such as a CD4⁺ cell population and/or a CD4⁺CD25⁺ cell population, and a second cell population which is a immortalized fusion partner cell, such as a tumor cell or cell line, to provide a third hybrid/hybridoma cell population. Typically a CD4⁺ cell population comprises about 10% CD4⁺CD25⁺ cells. In some embodiments, the immortalized fusion partner cell is a thymoma cell, a T-lymphoma cell, or a T- cell tumor cell. Thus, the hybridoma cells ofthe invention are also defined as T-cell hybridomas or T-T cell hybridomas.

The invention also provides methods for generating an immortalized cell expressing the antigens CD4 and CD25 and having the ability to regulate immune responses contributing to tolerance comprising: a) obtaining CD4⁺ regulatory cells from a subject; b) providing immortalized fusion partner cells; c) fusing the CD4⁺ cells with the immortalized fusion partner cells; and d) screening for immortalized hybrid cell clones expressing CD4 and CD25 and having the ability to inhibit T-cell proliferation and/or T-cell function. In some embodiments of this method, the CD4⁺ cell is a CD4⁺CD25⁺ cell.

In some embodiments, the CD4⁺CD25⁺ cells are isolated from a subject. The subject can be a mouse or a human or any other mammalian animal. In some specific embodiments, the subject from which the CD4⁺ cell is isolated from is a normal subject. A normal subject is one who is free of any disease or pathological condition. In specific aspects of this embodiment, the CD4⁺ cells from the normal subject can be additionally treated with one or more cytokine(s) and/or antigen(s) in vitro or in vivo prior to the fusion. The antigen may be a foreign transplantation antigen, a self antigen or a peptide fragment of an antigen. In other embodiments, the subject from which the CD4⁺ cells are isolated has tolerance to a foreign transplantation antigen. In yet other embodiments, the subject has tolerance to a particular self- antigen (i.e., resistance to an autoimmune disease), or has resistance to one or more allergens.

The immortalized fusion partner cell can be any cell that has fuseogenic capability and is immortal or transformed. By immortal or transformed it is meant that the cell is capable of proliferating endlessly. Typically, such an immortalized fusion partner cell is a tumor cell line or a tumor cell. For example, a BW5147 cell or a BW5147ofβ^~ fusion partner may be used to generate the T-cell hybridomas of the invention. Of these the BW5147ofβ^~ fusion partner is preferred. BW5147ofβ^~ is a mutated version of BW5147 and the mutation knocks out expression of the endogenous alpha and beta chains of the T-cell receptor. It is contemplated that use ofthe BW5147 ofβ^~ line will facilitate screening of T-cell hybridomas ofthe invention. Additionally, the use of BW5147 lines, MOLT-4, other T-cell tumors such as thymomas and T- cell lymphomas such as lymphoblastic lymphomas, acute lymphoblastic leukemias, T-cell CD30⁺ anaplastic large cell lymphomas, peripheral T-cell lymphomas, T-cell chronic lymphocytic leukemias, angioimmunoblastic T-cell lymphomas, angiocentric T-cell lymphomas, HTLN-related T-cell leukemias, or adult T-cell leukemias are also contemplated.

Cell fusion methodologies are well known in the art and generally entail the use of agents such as polyethyleneglycol (PEG), or Sendai virus, or electrically-induced cell fusion methods. One of skill in the art is generally well versed in these methodologies and variations and modifications thereof.

The invention also provides methods for generating CD4⁺CD25⁺ regulatory cells comprising: a) providing immortalized cells expressing CD4 and CD25; b) contacting the immortalized cells with a population of CD4⁺ cells wherein the contacting causes the differentiation of the CD4⁺ cells into CD4⁺CD25⁺ regulatory cells; and c) separating the immortalized cells from the CD4⁺CD25⁺ regulatory cells, thereby obtaining a population of CD4⁺CD25⁺ regulatory cells. These methods may comprise in vitro methods or may even be in vivo or ex vivo methods. Such methods are described in other parts ofthe specification.

The CD4⁺CD25⁺ regulatory cells generated by this method are useful for human therapy as they are non-immortal. In some embodiments, the population of CD4⁺ cells can be isolated from a human subject with an immune mediated inflammatory disorder, such as an autoimmune disease or condition, or a human subject who is about to receive a tissue or organ transplant, or a human subject who is allergic to an allergen. Procedures such as leukapharesis or plasmapharesis, which are well known in the art, may be used for the isolation.

In further embodiments of this method, the contacting in step b) is carried out in the presence of an antigen to which tolerance is desired. Thus, the CD4⁺CD25⁺ regulatory cells generated by this method can regulate immune responses which provide tolerance to a particular antigen. When CD4⁺CD25⁺ regulatory cells are desired to regulate tolerance to a transplanted tissue/organ and prevent rejection ofthe transplant, the antigen may be in the form of donor cells and/or donor antigen and/or donor peptide. When CD4⁺CD25⁺ regulatory cells are desired to regulate tolerance to a self antigen or auto antigen and to provide therapy in the case of an autoimmune disease or other immune condition the antigen is a self antigen and/or peptide fragment of a self antigen. When CD4⁺CD25⁺ regulatory cells are desired to regulate tolerance to an allergen and to prevent allergic responses and to provide therapy in the case of allergic diseases the antigen will be an allergen and/or peptide fragment of an allergen.

It is contemplated that the contacting in step b) may be performed in the additional presence of other cells or cellular components or cellular extracts from cells such as but not limited to an unfractionated CD4⁺ cell population; and/or CD8⁺ cells; and/or antigen presenting cells including, B-cells, dendritic cells, macrophages, or monocytes; or cells from the peripheral blood or lymphoid tissue or bone marrow or peripheral lymphoid tissue, including spleen cells and bone marrow. In addition, the presence of cytokines, particularly TGFβ, and/or E -10, and/or IL-2, and/or inhibitors of other cytokines such as antibodies against IL-12 may be required.

The present invention contemplates various methods to separate the regulatory CD4⁺CD25⁺ cells. For example, one may use FACS sorting to separate the regulatory CD4⁺CD25⁺ cells from other cells. FACS can be employed to effectively separate out the regulatory CD4⁺CD25⁺ cells from the hybridoma CD4⁺CD25⁺ cells as the inventors have demonstrated that the regulatory CD4⁺CD25⁺ cells are CD69^" whereas the hybridoma CD4⁺CD25⁺ cells are CD69⁺. Other methods for separating the regulatory CD4⁺CD25⁺ cells are also contemplated and some of these include employing monoclonal antibodies. Accordingly, one may contact the cell suspension with one or a combination of monoclonal antibodies which recognize an epitope on the regulatory CD4⁺CD25⁺ cells that is not present on other cells and separating and recovering the regulatory CD4⁺CD25⁺ cells bound by the monoclonal antibodies. Alternatively, the monoclonal antibodies may be specific for epitopes expressed by other cells and not by the regulatory CD4⁺CD25⁺ cell. The monoclonal antibodies may be linked to a solid- phase and utilized to capture the regulatory CD4⁺CD25⁺ cells. The bound cells may then be separated from the solid phase by known methods depending on the nature of the antibody and solid phase. Monoclonal based systems appropriate for preparing the desired cell population include magnetic bead/paramagnetic particle column utilizing antibodies for either positive or negative selection; separation based on biotin or streptavidin affinity; and high speed flow cytometric sorting of immunofluorescent-stained regulatory CD4⁺CD25⁺ cells mixed in a suspension of other cells.

Thus, the invention provides a purified or a substantially pure population of regulatory CD4⁺CD25⁺ cells. The term "purified" indicates that the cell population contains less than 5% impurities, impurities being for example, cells that are not regulatory CD4⁺CD25⁺. By "substantially pure" it is meant that the regulatory CD4⁺CD25⁺ cell population is about 75%, 80%, 85%, 90%, 95% to about 99% free of other impurities.

The invention also provides methods for generating CD4⁺CD25⁺ regulatory cells comprising: a) providing immortalized cells expressing CD4 and CD25; b) contacting the immortalized cells with a population of CD4⁺ cells wherein the contacting causes the differentiation of the CD4⁺ cells into CD4⁺CD25⁺ regulatory cells; and c) separating the immortalized cells from the CD4⁺CD25⁺ regulatory cells, thereby obtaining a population of CD4⁺CD25⁺ regulatory cells. Additionally provided is a method for generating CD4⁺CD25⁺ regulatory cells comprising: a) providing immortalized cells expressing CD4 and CD25; b) contacting the immortalized cells with a population of thymocytes and/or CD4^"CD8^" T-cells wherein the contacting causes the differentiation of the cells into CD4⁺CD25⁺ regulatory cells; and c) separating the immortalized cells from the CD4⁺CD25⁺ regulatory cells, thereby obtaining a population of CD4⁺CD25⁺ regulatory cells.

In some specific embodiments of the methods, the immortalized cells are attached to a solid support prior to or after being contacted with the CD4⁺ cells. Use of a solid support such as, a bead, a magnetic particle, a matrix, or a column, simplifies the process of separating the immortalized CD4⁺CD25⁺ cells from the CD4⁺CD25⁺ regulatory cells. In this context, it is also contemplated that an affinity tag may be further attached to the immortalized cells prior to or after being contacted with the CD4⁺ cells. Some non-limiting examples of affinity tags that may be used include biotin, streptavidin, or an antigen, or an antibody. One of ordinary skill in the art is well versed with methods of separation and purification using solid supports and/or affinity tags.

The methods described above for the generation of the CD4⁺CD25⁺ regulatory cells may be either in vitro methods, ex vivo methods, or in vivo methods. The CD4⁺CD25⁺ cells generated by these methods are suitable for in vivo use by administering to a patient or subject. Methods for large scale cultivation or scaling up the cultivation of the cells of the invention are contemplated to obtain cells in a sufficient quantity for therapeutic methods. These cultivation methods are similar to methods of large scale cultivation of mammalian cells especially other hybridoma cells which are well known in the art.

The invention also provides CD4⁺CD25⁺ regulatory cells generated by a method comprising: a) providing immortalized cells expressing CD4 and CD25; b) contacting the immortalized cells with a population of CD4⁺ cells or thymocytes, or CD4^"CD8^" T-cells or CD8⁺ cells, wherein the contacting causes the differentiation of the CD4⁺ cells or the thymocytes, or CD4^"CD8^" T-cells or the CD8⁺ cells into CD4⁺CD25⁺ regulatory cells or other T-cells with regulatory properties; and c) separating the immortalized cells from the CD4⁺CD25⁺ regulatory cell or other T-cells with regulatory properties, thereby obtaining a population of CD4⁺CD25⁺ regulatory cells or other T-cells with regulatory properties.

In specific embodiments, the immortalized cells in step a) may be further treated through the process of transfection of a nucleic acid to express proteins or nucleic acids that encode molecules of interest including but not limited to specific T-cell receptors and/or cytokines and/or chemokines prior to contacting in step b). The term "transfection" refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. The term "transformation" as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous nucleic acid. One of ordinary skill in the art is well versed with methods of nucleic acid transfection using mammalian and/or viral expression systems and methods of nucleic acid delivery.

In specific embodiments the contacting in step b) may be carried out in the additional presence of a donor cell, a donor antigen, any allergen, or any immunogenic peptide/antigen to which tolerance is desired. Cytokines and/or antigen presenting cells also may be present.

Additionally, the instant invention provides a method for controlling or preventing an undesirable or adverse immune reaction in a subject comprising administering to the subject a pharmaceutical formulation of CD4⁺CD25⁺ regulatory cells in an amount required to provide therapeutic benefit from the undesirable immune reaction.

The 'amount required' is also referred to as the 'effective amount' in this specification and is defined as an amount ofthe CD4⁺CD25⁺ cell that will decrease, reduce, inhibit, ameliorate or otherwise abrogate the undesirable or adverse immune reaction. In some embodiments, this may be achieved by inhibiting the proliferation of an activated or hyperactivated immune cell such as activated T-cells including, CD4⁺ activated T-cells, CD8⁺ activated T-cells, T-cells activated by any self-antigen, allergen, or foreign transplantation antigen, an activated host T-cell recognizing donor alloantigen or donor antigen or donor peptide, or an activated donor T-cell recognizing host alloantigen or host antigen or host peptide.

Thus, the method is contemplated useful in providing therapy for immune-mediated inflammatory diseases or disorders. These diseases or disorders include: graft rejection, where the host immune system mounts a response against the donor transplanted organ or tissue; graft versus host disease, where the donor immune system mounts a response against the host's organs or tissue; autoimmune disorders, where the immune system mounts a destructive immune response against one's own organs or tissues, and allergic conditions, where the immune system mounts a vigorous but unnecessary response to an innocuous material to which it has become hypersensitive.

The amount of CD4⁺CD25⁺ regulatory cells required to provide therapeutic benefit is from O.lxlO⁷ to 2xl0⁷ cells/kilogram of body weight of the subject receiving the therapy. This includes the doses of O.lxlO⁷, 0.2xl0⁷, 0.3xl0⁷, 0.4xl0⁷, 0.5xl0⁷, O.όxlO⁷, 0.7xl0⁷, 0.8xl0⁷, 0.9xl0⁷, lxlO⁷, l.lxlO⁷, 1.2xl0⁷, 1.3xl0⁷, 1.4xl0⁷, 1.5xl0⁷, 1.6xl0⁷, 1.7xl0⁷, 1.8xl0⁷, 1.9xl0⁷ and 2x10⁷ cells/kilogram of body weight. Intermediate ranges are also contemplated. It is also possible that one may utilize doses less than 0. lxlO⁷ cells/kilogram of body weight. Of course, it will be understood that the exact dose will be decided at the time of therapy depending on the health of the individual patient, taking into account factors such as age, sex, and disease condition, and such adjustments will be made by a skilled physician.

In specific embodiments, the autoimmune disease may be celiac disease, type I diabetes, multiple sclerosis, rheumatoid arthritis, rheumatic fever, ulcerative colitis, autoimmune gastritis and other autoimmune mediated processes.

In some embodiments of the methods, the subject afflicted with the adverse immune reaction is a human being. In other embodiments, the controlling or preventing an undesirable immune reaction comprises inducing immune tolerance. The immune tolerance can be immune tolerance to a transplanted tissue, immune tolerance to a transplanted organ, immune tolerance to an autoimmune disease, immune tolerance to a self antigen, immune tolerance to an allergic reaction, immune tolerance to an allergen.

In yet other embodiments ofthe methods, the immune tolerance is regulated by inhibiting the proliferation of activated T-cells such as CD4⁺-activated T-cells, or CD8⁺-activated T-cells. In still other embodiments, the function of activated T-cells is inhibited, such as the secretion of cytokines, including but not limited to IL-2 or other inflammatory cytokines. In yet other embodiments, the differentiation of T-cells into functional effector cells, such as cells with cytolytic activity or cells that secrete inflammatory cytokines, including but not limited to JFNγ is inhibited. In other embodiments, the immune tolerance is regulated by altering the function of antigen presenting cells, such as macrophages, dendritic cells or monocytes. In still other embodiments, the secretion of inflammatory cytokines by antigen presenting cells, including but not limited to EL- 12 is inhibited.

As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. 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

The following drawings form part of the present specification and are included to further demonstrate certain aspects ofthe present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Expression of markers. Histograms depict constitutive expression of CD4, CD25, CD3, CD40L, CD28, and CD62L on BW5147 cells (thin line) and RD6 CD4⁺CD25⁺ hybridomas (bold line).

FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 21, 2J, 2K, 2L, 2M, 2N, 2O, 2P, 2Q, 2R, 2S, 2T. CD4⁺CD25⁺ hybridoma inhibits anti-CD3-induced T-cell proliferation in vitro. BALB/c spleen cells (10⁶ cells/well) were labeled with CFSE and cultured with increasing concentrations of soluble anti-CD3 mAb 2C11 (0 to 1.0 μg/ml). The CFSE-labeled cells were cultured alone (FIGS. 2A, 2B, 2C, 2D & 2E), with 100 U/ml rLL-2 (FIGS. 2F, 2G, 2H, 21 & 2J), with unlabeled CD4⁺CD25⁺ hybridoma 3C1 (2.5 x 10⁵ cells/well; FIGS. 2K, 2L, 2M, 2N & 2O), or with both rIL-2 and unlabeled 3C1 (FIGS. 2P, 2Q, 2R, 2S & 2T). Flow cytometry was used to examine anti-CD3 -induced proliferation, defined by decreasing concentrations of CFSE label within daughter cells following each cell division. Histograms depict CFSE expression in a gated population containing both CD4⁺ and CD8⁺ T-cells. Gates within each panel indicate the percent of T-cells that have undergone at least one division.

FIGS. 3A, 3B, 3C, 3D. 3C1 hybridoma inhibits anti-TCR stimulated CD8⁺ cell proliferation. Naive CD8⁺ cells were labeled with CFSE and stimulated in vitro with soluble anti-CD3 (FIGS. 3A & 3C) or anti-CD3 with IL-2 (FIGS. 3B & 3D) for 72 hours with syngeneic APCs and either the fusion partner, BW5147cf β^~ (FIGS. 3A & 3B), or CD4⁺CD25⁺ hybridoma, 3C1 (FIGS. 3C & 3D). CD8⁺ cell proliferation was measured using CFSE content and FACS analysis. Histograms represent the relative number of gated CD8VH-2K ^" cells. One of 3 representative experiments.

FIG. 4. The effect of CD4⁺CD25⁺ hybridomas on the proliferation of anti-TCR stimulated CD8⁺ cells. CD8⁺ cells were purified from naive mice, labeled with CFSE, and stimulated in vitro with soluble anti-CD3 (1 μg/ml) with or without IL-2 (100 U/ml) for 72 hours in the presence of syngeneic APCs and either no hybridoma (not shown), CD4⁺CD25^" control hybridoma, or CD4⁺CD25⁺ hybridomas (3C1, DC3, DD9, and RA2). CD8⁺cell proliferation was measured using CFSE content of gated CD8⁺/H-2K^k" cells. Bars represent the % cells with one or more divisions.

FIG. 5. 3C1 alters CD62L expression on anti-TCR stimulated CD8⁺ cells. Naϊve CD8⁺ cells were labeled with CFSE and stimulated in vitro with soluble anti-CD3 or anti-CD3 with IL-2 for 72 hours with syngeneic APCs and either the fusion partner, BW5147, or CD4⁺CD25⁺ hybridoma, 3C1, as in FIG. 3. CFSE-CD8⁺ cells were examined for surface expression of CD62L using multiparameter FACS analysis. Undivided CD8⁺ cells were gated. Bars represent the percent of undivided CD8⁺ cells that are CD62L negative (black) or CD62L positive (hatched). One of three representative experiments.

FIGS. 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 61, 63, 6K & 6L. CD4⁺CD25⁺ hybridoma RD6 inhibits T-cell proliferation in vitro. BALB/c spleen cells were labeled with CFSE and cultured for 3 days (10⁶ cells/well) in media alone (FIGS. 6A, 6B & 6C), with rBL-2 (100 U/ml; FIGS. 6D, 6E & 6F), soluble anti-CD3 mAb 2C11 (1 μg/ml; FIGS. 6G, 6H & 61), or both rIL-2 and 2C11 (FIGS. 6J, 6K & 6L). Additionally, the CFSE-labeled cells were cultured alone (FIGS. 6A, 6D, 6G, 6J), or with either fusion partner (2.5 x 10⁵ cells/well): BW5147α^~β^~ (FIGS. 6B, 6E, 6H, 6K), or CD4⁺CD25⁺ hybridoma (2.5 x 10⁵ cells/well), RD6 (FIGS. 6C, 6F, 61, 6L). Flow cytometry was used to measure T-cell proliferation by decreasing concentrations of CFSE label within the daughter cells following each cell division. Histograms depict CFSE expression in a gated population containing both CD4⁺ and CD8⁺ T-cells. Gates within each panel indicated the percent of T-cells that have undergone at least one cell division.

FIGS. 7A & 7B. CD4+CD25+ hybridoma, RD6, inhibits CD4 and CD8 T-cell proliferation in vitro. BALB/c spleen cells were labeled with CFSE and cultured for 3 days (10⁶ cells/ml) with increasing concentrations of soluble anti-CD3 mAb either alone (circles), with unlabeled BW5147α^"β^" (triangles, 10⁵ cells/well), or with unlabeled CD4⁺CD25⁺ hybridoma, RD6 (squares, 10⁵ cells/well). Flow cytometry was used to examine proliferation of BALB/c CD4⁺ (FIG. 7A) or CD8⁺ (FIG. 7B) T-cells, defined by decreasing concentrations of CFSE label within daughter cells following each cell division. Symbols depict the percentage of proliferating CD8⁺ (left hand panel) or CD4⁺ (right hand panel) BALB/c T cells. Cells proliferation was determined by examining CFSE expression on gated CD8⁺ or CD4⁺ cells. The percentage of proliferating cells was calculated by setting proliferation gates, which were established by examining CFSE-labeled CD8⁺ and CD4⁺ cells that were cultured for 3 days without anti-CD3. Proliferation of unstimulated T cells is <2%.

FIG. 8. CD4⁺CD25⁺ hybridomas inhibit IL-2 production of activated T-cells.

BALB/c spleen cells (10⁶ cells/ml) were cultured unstimulated or stimulated with 1 mg/ml soluble anti-CD3 overnight in the presence of increasing amounts of BW5147α-β- or the CD4+CD25+ hybridoma, RD6. The cultures were transferred to JJL-2 ELISPOT plates and the number of IL-2 producing cells was determined 24 hours later. Spots were counted using the ImmunoSpot™ computerized system and software. Bars represent the number of spots per cultures. The numbers depict the percent suppression versus the control culture. One of three representative experiments.

FIG. 9. RD6 inhibits alloantigen-induced T-cell proliferation. Purified BALB/c CD8 cells were labeled with CFSE and cultured for 6 days in media alone (not shown), with. T- depleted CAF spleen cells, with and without 100 U/ml rIL-2, and either no other cells or with RD6 (RD6:CD8⁺ cell was 1:20). Panels depict histogram of CFSE expression of gated CD8 cells. Less than 2% of CD8 cells show signs of cell division after 6 days in vitro without stimulation. Numbers are the percentages of cells within the gates.

FIGS. 10A & 10B. Inhibition of T cell proliferation by RD6 requires cell-cell contact. BALB/c spleen cells were labeled with CFSE and cultured for 3 days in media containing soluble anti-CD3 (0.1 μg/ml). Cells were cultured either alone (10⁶ cells/well), with BW5147of β^~ (10⁵ cells/well), or with CD4⁺CD25⁺ hybridoma subclone, RD6 (10⁵ cells/well). In FIG. 10 A, spleen cells were cultured separate from BW5147α^~β^~ or RD6 in a Transwell plate. In FIG. 10B, BW5147α^~β^~ or RD6 cells were cultured in the lower well and CFSE-labeled spleen cells were simultaneously added to both upper (separated) and lower (contact) wells. Histograms depict CFSE expression in a gated population containing both CD8⁺ and CD4⁺ BALB/c T-cells. Numbers indicate the percent of T-cells that have undergone at least one cell division. Proliferation of unstimulated T-cells is <2%.

FIG. 11. Ability of CD4⁺CD25⁺ hybridoma RD6 to inhibit proliferation of T-cells that are activated by soluble but not plate-bound anti-CD3. CFSE-labeled BALB/c spleen cells (10⁶ cells/well) were cultured for 3 days in media (unstimulated, not shown), media containing soluble anti-CD3 (0.1 μg/ml), or wells containing plate-bound anti-CD3. The cells were either cultured alone, with unlabeled BW5147of β^~ cells (10⁵ cells/well), or with unlabeled CD4⁺CD25⁺ hybridoma subclone, RD6 (10⁵ cells/well). Histograms depict CFSE expression in a gated population containing both CDS and CD4 cells. Numbers indicate the percent of T-cells that have undergone at least one cell division. Gates were established by examining CFSE- labeled spleen T-cells from unstimulated cultures (< 2% of T-cells had divided).

FIG. 12. CD4⁺CD25⁺ hybridoma, RD6, alters the in vivo immune response to foreign transplantation antigens. BALB/c mice were injected with T-depleted spleen cells from CAFl mice (25x10⁶ cells/mouse) and either no other cells (black bars, N=3) or RD6 hybridoma cells (5x10⁶ cells/mouse, hatched bars, N=3). Seven days later spleen cells were isolated from individual mice and cultured for 72 hours in vitro. Supernatants were collected and the levels of EL-2, DL-4, IFNγ, IL-10, IL-13, LL-12 and TGFβ were measured using cytokine specific ELISA assays. Bars represent the means ± SE of the levels of the depicted cytokines. The symbol * indicates significant differences between the groups, p < 0.05, t-test. NS is no significant difference between groups. Spleen cells from uninjected mice produce background levels of cytokine (not shown). The experiment was performed three times with similar results.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Regulatory CD4⁺CD25⁺ cells play a vital role in the induction and maintenance of self- tolerance and are essential for T-cell homeostasis, for the prevention of autoimmunity, and for preventing transplant rejection reactions. Although the role of immune regulatory CD4⁺CD25⁺ cells in inducing tolerance in undesirable immune reactions has been well documented, the percentage of naturally-occurring CD4⁺CD25⁺ cells is <l-5% of the peripheral lymphoid cell population. Thus, lengthy isolation procedures result in very low yields ofthe CD4⁺CD25⁺ cells which are insufficient for any therapeutic or research purpose.

The present inventors have generated immortalized CD4⁺CD25⁺ regulatory cells to provide a source of CD4⁺CD25⁺ regulatory cells. These immortalized cells are hybridoma cells formed by fusing purified CD4⁺ cells from tolerant-mice with an immortalized fusion-partner cell such as a BW5147 ^"β^" cell.

The fusion products are then screened to identify the immortalized cells/hybridomas by screening for cells that co-express CD4⁺ and CD25⁺ and by screening for the ability of the CD4⁺CD25⁺ cells to inhibit T-cell activation/proliferation and/or T-cell function. The CD4⁺CD25⁺ cells so identified were then subcloned. Characterization of the CD4⁺CD25⁺ immortalized cells of the invention shows that these cells stably express surface CD4⁺CD25⁺ over several months and constitutively secrete cytokines, particularly TGFβ, in a fashion similar to their cellular counterpart CD4⁺CD25⁺ cells.

It is envisioned that these immortalized CD4⁺CD25⁺ regulatory hybridoma cells will regulate the differentiation of CD4⁺ cells and/or precursor CD4⁺ cells and/or thymocytes and/or CD4^"CD8^" cells into non-hybridoma CD4⁺CD25⁺ regulatory cells. These non-hybridoma CD4⁺CD25⁺ regulatory cells can then be used for therapeutic purposes in the prevention and cure of undesirable immune mediated inflammatory conditions such as autoimmune diseases, transplant rejections, allergic reactions etc.

Thus, the present invention also provides methods for generating non-hybridoma CD4⁺CD25⁺ cells. Using the methods provided sufficient numbers of CD4⁺CD25⁺ cells can be generated for therapeutic and/or research purposes. Therapeutic methods for ameliorating, preventing, controlling and/or curing adverse immune reactions by inducing immune tolerance using CD4⁺CD25⁺ cells are also provided.

A. Tolerance

There are three major mechanisms by which T-cells can become "tolerant" to self or acquired antigens. The first two mechanisms are by "deletion" and "anergy," both of which constitute the passive mechanisms of tolerance, as this form of tolerance cannot be transferred from one individual to another. The third mechanism is termed "dominant regulation" and is so defined as this form of tolerance can be adoptively transferred from one individual to another. All three mechanisms play a role in maintaining self-tolerance in vivo. However, in the case of transplantation of non-autologous organs, where one wishes to establish tolerance to a broad set of foreign major and minor MHC antigens, the ability to induce and maintain robust tolerance mainly depends on the ability to establish a network of dominant regulation.

There is substantial evidence for the role of dominant regulation in various models of transplantation tolerance. All models in which tolerance can be adoptively transferred by cells from the primary tolerant recipient to naive secondary recipients involves the generation of a regulatory network (Dorsch and Roser, 1977; Streilein and Gruchalla, 1981; Roser, 1989; Hall et al, 1990; Takeuchi et al, 1992; Scully et al, 1994; Onodera et al, 1998; Gao et al, 1999a). Despite the growing number of tolerance models that evoke regulatory cells and dominant regulation as the mechanism of tolerance, relatively little is know about the actual phenotype of the regulatory cells, other than they appear to be CD4⁺. The mechanisms by which the regulatory cells function to maintain tolerance to foreign transplantation antigens are also not completely understood.

Because tolerance depends on antigen persistence (Onodera et al, 1998; Ramsdell and Fowlkes, 1992; Hamano et al, 1996), the regulatory cells are most likely triggered through their encountering antigen. Additionally, regulatory cells must inhibit immunocompetent T-cells, which will always be present because of the ongoing generation of new thymic emigrants, as they come across donor antigen for the first time in the periphery and develop into alloreactive effector cells which are capable of damaging or destroying the graft. (i) Regulatory Cells in Tolerance

One of the earliest reports that regulatory cells function in transplantation tolerance was by Dorsch and Roser who observed that neonatal tolerance (i.e., the induction of tolerance by exposing neonates to antigen) involved the generation of "suppressor" T-cells (Dorsch and Roser, 1977). Streilein and colleagues later confirmed that regulatory T-cells mediated tolerance in a MHC class II disparate neonatal tolerance mouse model (Streilein and Gruchalla, 1981; Matriano et al, 1994a; Mohler and Streilein, 1989; Powell and Streilein, 1991). Shortly afterward, Waldmann and colleagues reported that CD4⁺ cells from mice, which were made tolerant to minor histocompatibility (MHC) antigens by anti-CD4 and anti-CD8 treatment, could adoptively transfer tolerance to immunocompetent secondary recipient mice (Qin et al, 1993). Furthermore, CD4⁺ cells from the tolerant mice were capable of passing their property of regulation on to other CD4⁺ cells in adoptive transfer recipients, a property that is known as "infectious tolerance" (Bemelman et α/., 1998).

Based on Streilein' s work in neonatal tolerance (Streilein, 1993) and Waldmann' s model of infectious tolerance in adults (Qin et al, 1993; Bemelman et al, 1998), several groups looked for evidence of regulatory cells in more MHC disparate tolerance models and began to examine the phenotype and function ofthe putative regulatory CD4⁺ cells (Onodera et al, 1996; Davies et al, 1996; Bushell et α/., 1995; Saitovitch et /., 1996; Hall et al, 1998).

In one model of neonatal tolerance, a specialized regulatory CD4⁺ T-cell population, which was phenotyped as CD4⁺CD25⁺ cells, prevented development of organ-specific autoimmunity in neonatal BALB/c mice thymectomized within three days of birth (Asano et al, 1996). Additionally, immunoregulatory CD25⁺CD4⁺CD8^~ thymocytes capable of controlling self-reactive T-cells were identified (Itoh et al, 1999). Thus, the role of CD4⁺CD25⁺ regulatory cells in maintaining peripheral tolerance to self-antigens was demonstrated.

More recently, CD4⁺CD25⁺ regulatory cells have been shown to prevent other autoimmune conditions, including, spontaneous diabetes (Salomon et al, 2000), and gastritis and colitis (Suri-Payer and Cantor, 2001) in murine models. The role of CD4⁺CD25⁺ cells has also been confirmed in mediating tolerance in tissue and organ transplantation reactions, for example, tolerance to autologous graft-vs-host disease is actively mediated by CD4⁺CD25⁺ cells (Wu et al, 2001); and CD4⁺CD25⁺ regulatory cells isolated from tolerant mice have been shown to prevent graft rejection in non-tolerant mice (Hara et al, 2001). Additionally, CD4⁺CD25⁺ cells have been shown to be required for pharmacological agents that block immune co-stimulation to be effective at preventing graft-vs-host disease (Taylor et al, 2001). B. Cell Fusion

The present invention provides immortalized CD4⁺CD25⁺ regulatory cells generated by combining two populations of cells, a first cell population comprising regulatory CD4⁺CD25⁺ cells and a second cell population that is an immortalized fusion partner cell population, to obtain a third 'hybrid cell' population that is an immortalized regulatory T-cell that bears the CD4⁺ and CD25⁺ antigens. The first cell population can be a CD4⁺ expressing cell population which typically includes a subpopulation of about 10% of cells that also co-express the CD25⁺ antigen. Alternatively, the first cell population can be an exclusively CD4⁺CD25⁺ cell population or even a cell population enriched in CD4⁺CD25⁺ cells. The cells are combined by cell fusion methods that are well known to the skilled artisan. The resulting hybrid cell expresses both CD4⁺ and CD25⁺ antigens and bears the 'immortal' properties of the fusion- partner cell, resulting in an immortalized CD4⁺CD25⁺ cell.

The term 'fusing' or 'fusion' of two or more cells is defined as a method in which two or more cells are combined to form a single hybrid cell which contains all or part of each individual cell. Fusion may be accomplished by any method of combining cells under fuseogenic conditions well known in the art (See, for example, Current Protocols in Immunology, 3.14.1- 4.14.11 Contributed by Ada M. Kruisbeek 1997 copyright; Harlow and Lane, 1988, incorporated herein by reference). Known methods for fusing cells includes by use with polyethylene glycol (PEG) or Sendai virus or electrically induced fusion methods. Methods for generating hybrids of spleen or lymph node cells and immortal fusion partners cells usually comprise mixing somatic cells with the immortal fusion partners cell in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976, the entire content of both is incorporated herein by reference), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977, incorporated herein by reference). The use of electrically induced fusion methods also is appropriate (Goding, 1986, incorporated herein by reference).

By the term 'hybrid cell' is meant a cell formed by combining two or more cells, e.g., by fusion. The term 'hybridoma' is also used to refer to the hybrid cell ofthe invention.

The present invention provides a method for generating an immortal CD4⁺CD25⁺ cell line that provides a source of CD4⁺CD25⁺ cells. As naturally-occurring CD4⁺CD25⁺ cells comprise a very small population of cells, the present invention allows for the generation of virtually limitless numbers of CD4⁺CD25⁺ cells. Any mammalian cell line that is immortalized and is amenable to fusion may be used as an immortalized fusion partner cell. By immortalized, it is meant that the cell has acquired the ability to divide or proliferation endlessly, or that a cell is transformed into a cell with a cancerous phenotype. Fusion partners for generating T-cell hybridomas are chosen based on rapid growth, ease of cloning, high fusion efficiency, absence of specific T-cell surface antigen, and presence of HGPRT deficiency (sensitivity to aminopterin, required for selection of hybrids) (Current Protocols in Immunology, 3.14.1-4.14.11 Contributed by Ada M. Kruisbeek 1997, incorporated herein by reference). Typically such an immortalized fusion partner cell is a tumor cell line or a tumor cell. T-cell tumor, T-lymphoma and thymoma cells have been used as fusion partners to generate T-T hybridoma cells. It is contemplated that such transformed/immortalized T-cells may be used as fusable host cells in the methods ofthe present invention. For example, a BW5147 cell or a BW5147αTβ^~ or a MOLT-4 fusion partner may be used to generate the T-cell hybridomas or hybrid cells of the invention. Of these the BW5147αfβ^~ cell which is a mutated version of BW5147 with a mutation that knocks out expression ofthe endogenous alpha and beta chains ofthe T-cell receptor is preferred. Use of the BW5147 α^~β^~ line can facilitate screening of T-cell hybridomas ofthe invention.

Fusion procedures usually produce viable hybrid cells at low frequencies of about 1x10^"^ to lxl0^"8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental immortalized fusion partner cells that would normally continue to divide indefinitely by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (hypoxanthine-aminopterin-thymidine (HAT) medium). Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells that comprise functional nucleotide salvage pathways are able to survive in HAT medium. The immortalized fusion partner cells are defective in key enzymes ofthe salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The T-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are successfully fused hybridomas/hybrid cells. Such methods and variations thereof are described in Ada M. Kruisbeek (1997), incorporated herein by reference. This culturing provides a population of hybridomas/hybrid cells from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal populations (after about two to four weeks) for the desired reactivity, such as presence of the CD4⁺ and the CD25⁺ markers and having regulatory properties relating to immune-tolerance. The initial cloning can be performed manually or with the use of the cloning operation of the FACS. The assay should be sensitive, simple and rapid, such as FACS, radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, cytokine ELISA assays, cytokine ELISPOT assays, cell proliferation assays and the like.

The selected hybridomas would then be serially diluted and subcloned into individual cell lines that express CD4⁺CD25⁺ and have immune regulatory properties, which clones can then be propagated indefinitely. The subcloning can be performed manually or with the aid of the cloning operation ofthe FACS.

C. Screening for CD4⁺CD25⁺ Cells

After a period of time sufficient to allow the hybridoma cell of the invention to begin expressing its 'hybrid characteristics' (in most cases, about 2-4 weeks) the hybrid cells are subject to screening to identify those cells expressing both CD4⁺ and CD25⁺ antigens and having the immune-regulatory properties desired. The immune-regulatory properties include inhibition of activated T-cell proliferation and inhibition of T-cell function or T-cell differentiation. This may be accomplished by, for instance, any immunodetection method such as fluorescent activated cell sorting (FACS) to identify the cellular antigens and by immunoassays to detect the ability of the immortalized CD4⁺CD25⁺ cells of the invention to regulate T-cell proliferation and/or function and/or differentiation.

In some embodiments of the present invention, the screening strategy to select for CD4⁺CD25⁺ hybridomas comprises the following: 1) screening of individual wells for cells co- expressing both CD4⁺ and CD25⁺ surface markers, using multiparameter flow cytometric analysis; 2) sub-cloning mixed populations so identified; and 3) screening for hybridomas with inhibitory activity using a functional assay. For example see FIG. 1 through FIG.8. The selected hybridoma candidates can then be further tested to examine the extent to which the candidates resemble naturally occurring CD4⁺CD25⁺ cells or CD4⁺CD25⁺ cells from tolerant subjects (for example, see FIGS. 9, 10 & 11). (i) Immunodetection

The expression of the CD4⁺ and CD25⁺ antigens can be tested using any conventional immunological screening method known in the art, for example, FACS. The cell can be further selected for additional characteristics such as ability to regulate immune tolerance by regulating T-cell proliferation and/or function.

In some embodiments, the present invention contemplates immunodetection methods for binding, purifying, identifying, removing, quantifying or otherwise generally detecting biological components (for example IL-2 as in FIG. 8) or the product of biological components (for example cell proliferation in FIGS. 2, 3, 6, 7, and 9). The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Nakamura et al (1987), incorporated herein by reference. Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs), radioimmunoassays ( IA) and immunobead capture assay. Still other immunoassays involve enzyme linked immunosorbent spot assays (ELISPOTs). Immunohistochemical detection using tissue sections also is particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like also may be used in connection with the present invention.

In general, immunobinding methods include obtaining a sample suspected of containing a protein, peptide or antibody, a putative hybrid cell obtained after the 'fusion' suspected of expressing CD4⁺ and CD25⁺ antigens in the case of the present invention, and contacting the sample with an antibody or protein or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes.

The immunobinding methods of this invention include methods for detecting or quantifying the amount of a reactive component in a sample, which methods require the detection or quantitation of any immune complexes formed during the binding process.

Contacting the chosen biological sample with the protein, peptide or antibody under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any antigens present, such as the CD24⁺ and the CD25⁺ antigens. After this time, the sample-antibody composition, such as a hybridoma cell of the invention, tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. References concerning the use of such labels include U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.

The encoded protein, peptide or corresponding antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount ofthe primary immune complexes in the composition to be determined.

Alternatively, the first added component that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the encoded protein, peptide or corresponding antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by a two step approach. A second binding ligand, such as an antibody, that has binding affinity for the encoded protein, peptide or corresponding antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.

The immunodetection methods of the present invention have evident utility in the in the selection of hybridomas. (a) ELISAs

As noted, it is contemplated that an immunodetection technique such as an ELISA may be useful in conjunction with respect to detecting T-cell function assays and assays for detecting cytokine production by the cells ofthe invention. An Example is shown in Table 3 and FIG. 12.

In one exemplary ELISA, antibodies binding to proteins such as cytokines produced by the hybrid cells of the invention or by the CD4⁺CD25⁺ cells of the invention are immobilized onto a, selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. The cells of the invention suspected of producing the protein/cytokine of interest e.g., TGFβ, EL- 10, etc.,; or some other cleaved or secreted proteins and/or receptor molecules, are added to the wells. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen may be detected.

Detection is generally achieved by the addition of a second antibody that is specific for the target protein and linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection also may be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immunecomplexes. These methods are well known to one of skill in the art.

(ii) ELISPOT

The enzyme-linked immunospot (ELISPOT) assay is a variation of the enzyme-linked immunosorbent assay and is useful for detecting and analyzing individual cells that secrete a particular protein in vitro. Originally developed for analyzing specific antibody-secreting cells, the assay has been adapted for detecting cells that produce and secrete a variety of effector molecules such as cytokines.

Antibodies that recognize a target antigen are coated in a plate. The antigen is typically a cytokine or chemokine. Cells are stimulated in the coated wells and after the incubation period washed away. Alternatively, cells are stimulated for a short period of time in round-bottom tissue culture plates or tubes and then transferred to the ELISPOT well for additional time in culture. A second, enzyme-labeled antibody is added to the wells and the excess (non-binding) antibody is washed away again. The last incubation step is the addition of a chromogen, which will precipitate on the place (spot) of a cytokine (or other antigen) producing cell. Thus, cytokine release can be detected at the single cell level, allowing for direct determination of cytokine-producing cell frequencies. This assay has been found to be more sensitive than ELISA and intracellular staining. The sensitivity of the assay enables measurement of even very low frequencies of cytokine-producing cells (e.g., 1/300,000). Recent developments in assay plate design and in high-throughput ELISPOT plate-reader instrumentation have significantly improved the utility of the ELISPOT method. Objective and rapid analyses of cytokine producing cell numbers (spots), and relative amounts of cytokine produced per cell (spot size) are now possible. ELISPOT assays enable high-sensitivity and high throughput analyses for direct ex vivo monitoring of T-cell function, including Ag-specific T-cells.

(iii) FACS Analyses

Fluorescent activated cell sorting, flow cytometry or flow microfluorometry provides the means of scanning individual cells for the presence of one or more antigens, such as the CD4⁺ and the CD25⁺ antigens in the instant invention. The method employs instrumentation that is capable of activating, and detecting the excitation emissions of labeled cells in a liquid medium.

FACS is unique in its ability to provide a rapid, reliable, quantitative, and multiparameter analysis on either living or fixed cells. Cells would generally be obtained by culturing the hybridoma cells of the invention. FACS analyses is most useful when desiring to analyze a number of antigens at a given time as in the case of the present invention where the hybridoma cells are selected only if they express both the CD4⁺ and the CD25⁺ antigens. It is also contemplated that the hybridomas of the invention will be analyzed for expression of other important T-cell surface receptors and ligands using FACS analysis including TCRγδ, Thy 1.2. Ly49, DX5, CD3ε, H-2K^k, H-2K^d, TCRαβ, CD28, CD40L, CD62L, CTLA-4, and ICOS.

(iv) T-Cell Proliferation Assays

T-cell proliferation assays are generally known to one of skill in the art. The method below describes one ofthe T-cell proliferation assays using CFSE. Use of other assays that use the incorporation of nucleic acids or other labels to quantify cell proliferation are also contemplated. The skilled artisan will recognize that these methods are useful for assaying inhibition of T-cell proliferation and will be used in screening and selecting the hybrid cells or the non-hybrid cells of the invention which induce immune tolerance by inhibiting T-cell proliferation.

For the CFSE assay, spleen is removed from a BALB/c mouse, placed in a petri dish containing 10 ml of complete medium (RPMI 1640 supplemented with fetal calf serum, HEPES, 2-ME, L-Glutamine, and penicillin/streptomycin), and a cell suspension is prepared by gently disrupting the spleen between the frosted ends of glass microscope slides. Red blood cells (RBC) are removed from the cell suspension as described in section 3.1 of Current Protocols in Immunology. The RBC-depleted BALB/c spleen cells are then labeled with CFSE (5,6- carboxyfluorescein diacetate succinimidyl ester; Molecular Probes, Eugene, OR) using an established protocol (Lyons and Parish, 1994). Briefly, cells are resuspended (5 x 10⁷ cells/ml) in CFSE-labeling buffer (10 μM CFSE in phosphate-buffered saline, PBS) for 10 minutes at 37°C, and then washed three times with 10 ml of complete medium. To examine the effect of CD4⁺CD25⁺ hybridomas on T-cell activation, CFSE-labeled BALB/c spleen cells (10⁶) are cultured with a selected CD4⁺CD25⁺ hybridoma (1-2.5 x 10⁵) for 3 days in complete medium containing anti-CD3 T cell-activating mAb, 2C11 (0.1 or 1 μg/ml; ATCC # CRL 1975). After 3 days, cultures are harvested and T cell proliferation is examined by flow cytometry as a measure of T-cell activation (Lyons and Parish, 1994). Cells from each culture are stained for flow cytometry with PE-conjugated anti-CD8 mAb, PE-conjugated anti-CD4 mAb, and biotin- conjugated anti-H-2K^k mAb followed in a second stage by streaptavidin-conjugated Texas red, using commercially available reagents. Cell surface fluorescence staining is performed with a predetermined optimal amount of primary antibody in 100 μl staining buffer (PBS supplemented with fetal calf serum and HEPES) at 4°C for 30 minutes. Staining with secondary reagents is executed in a similar manner after washing cells to remove unbound primary reagent. Flow cytometry analysis is gated exclusively on BALB/c T-cells (CD4⁺/CD8⁺ H-2K^k"). T-cells that have undergone proliferation are identified by the sequential halving of CFSE-label between daughter cells in successive generations of cell division.

(v) T-Cell Function Assays

Several aspects of T-cell function inhibition by the CD4⁺CD25⁺ cells of the invention, including the immortalized CD4⁺CD25⁺ cells and/or the regulatory CD4⁺CD25⁺ cells, may be assayed such as inhibition of production of cytokines. The differentiation of T-cells into functional effector cells, such at cells with cytolytic activity or cells that secrete inflammatory cytokines including but not limited to EFNγ, may also be inhibited. General procedures for such assays are outlined below. The skilled artisan will recognize that these and modifications of these assays can be used in the screening methods of the invention to select the hybrid cells or the non-hybrid cells with immune regulatory functions of the invention, based on the type of T- cell function inhibited by these cells. (a) T-Cell Cytokine Assay

The following is an example of an assay to analyze the inhibition of cytokine production. This and modifications of such an assay may be used to assay for T-cell function with respect to T-cells ability to secrete cytokines and other proteins. Peripheral blood mononuclear (MNC) cells from healthy donors are separated by density centrifugation with ficoll-hypaque (LSM, Organon Teknika, Durham, N.C.), followed by rosetted with neuraminidase treated sheep red blood cells (SRBC). After another centrifugation with leucocyte separation medium (LSM), the SRBC of the rosetted-T-cells are then lysed with ammonium chloride lysing buffer (GIBCO, Grand Island, N.Y.). Such purified T-cells were resuspended at 3xl0^δ/ml in RPMI 1640 culture medium (GIBCO) supplemented with 10% fetal calf serum (Sigma, St. Louis, Mo.), 100 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, and 1% penn-strep (GIBCO). The cell suspension was immediately distributed into 96 well round-bottom microculture plates (Costar) at 200 μl/well. The various dilutions of test compound are then added in triplicate wells at 25 μl/well, incubated for 30 min at 37°C. Ionomycin (125 ng/ml), and PMA (1 or 5 ng/ml), are added to the appropriate wells. The culture plates were then incubated at 37°C in a humidified atmosphere of 5% CO₂, 95% air for 18-24 hours. The supernatants are removed, and assayed for cytokines such as IL-2, EL-4, EL- 10 etc with an cytokine capture ELISA, using monoclonal anti-cytokine antibodies and biotinylated goat anti- cytokine antibodies. The ELISA is then developed with streptavidin conjugated peroxidase (Zymed, San Francisco, Calif.) and substrate for peroxidase (Sigma). Mean OD and units of cytokine of the replicate wells are calculated from standard curve, created with recombinant cytokine and the results were expressed as concentration of CD4⁺CD25⁺ cells required to inhibit cytokine production of T-cells by 50%. Cytokine producing cells can also be detected using the standard cytokine ELISPOT assay.

D. Nucleic Acid-Based Expression Systems 1. Vectors

In the context ofthe present invention, it is contemplated that the CD4⁺CD25⁺ regulatory cells provided by the invention may be generated by a method comprising: a) providing immortalized cells expressing CD4 and CD25; b) contacting the immortalized cells with a population of CD4⁺ cells or CD25⁺ cells or CD8⁺ cells, wherein the contacting causes the differentiation of the CD4⁺ cells or the CD25⁺ cells or the CD8⁺ cells into CD4⁺CD25⁺ regulatory cells or other T-cells with regulatory properties; and c) separating the immortalized cells from the CD4⁺CD25⁺ regulatory cell or other T-cells with regulatory properties, thereby obtaining a population of CD4⁺CD25⁺ regulatory cells or other T-cells with regulatory properties.

In some embodiments of this method, it is further contemplated that the immortalized cells expressing CD4 and CD25 may be transfected with a nucleic acid to express one or more proteins or nucleic acids of interest. Such a nucleic acid may encode a T-cell receptor and/or a cytokine and/or a chemokine.

Although, one of ordinary skill in the art is well versed with methods of nucleic acid transfection using mammalian and/or viral expression systems, the present section provides a detailed description of such methods.

The nucleic acid encoding the protein or nucleic acid of interest will generally be in a vector. The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell, such as an immortalized cell expressing CD4 and CD25, where it can be replicated. A nucleic acid sequence can be "exogenous," which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Maniatis et al, 1989 and Ausubel et al, 1994, both incorporated herein by reference).

The term "expression vector" refers to any type of genetic construct comprising a nucleic acid coding for an RNA capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.

a. Promoters and Enhancers

A "promoter" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate the specific transcription a nucleic acid sequence. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.

A promoter generally comprises a sequence that functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as, for example, the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SN40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream ofthe start site, although a number of promoters have been shown to contain functional elements downstream ofthe start site as well. To bring a coding sequence "under the control of a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame "downstream" of (i.e., 3' of) the chosen promoter. The "upstream" promoter stimulates transcription ofthe DΝA and promotes expression ofthe encoded RΝA.

The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5' non-coding sequences located upstream ofthe coding segment and/or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. For example, promoters that are most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Patent Nos. 4,683,202 and 5,928,906, each incorporated herein by reference). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression ofthe DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, (see, for example Sambrook etal. 1989, incorporated herein by reference). The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression ofthe introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

Additionally any promoter/enhancer combination (as per, for example, the Eukaryotic Promoter Data Base EPDB, http://www.epd.isb-sib.ch/) could also be used to drive expression. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

Table 1 lists non-limiting examples of elements/promoters that may be employed, in the context of the present invention, to regulate the expression of a RNA. Table 2 provides non- limiting examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus.

The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, is well known to those of skill in the art. Nonlimiting examples of such regions include the human LEMK2 gene (Nomoto et al 1999), the somatostatin receptor 2 gene (Kraus et al, 1998), murine epididymal retinoic acid-binding gene (Lareyre et al, 1999), human CD4 (Zhao-Emonet et al, 1998), mouse alpha2 (XI) collagen (Tsumaki, et al, 1998), D1A dopamine receptor gene (Lee, et al, 1997), insulin-like growth factor II (Wu et al, 1997), and human platelet endothelial cell adhesion molecule- 1 (Almendro et al, 1996).

b. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

In certain embodiments of the invention, the use of internal ribosome entry sites (ERES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). ERES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an ERES from a mammalian message (Macejak and Sarnow, 1991). RES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an ERES, creating polycistronic messages. By virtue of the ERES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patents. 5,925,565 and 5,935,819, each herein incorporated by reference).

c. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector (see, for example, Carbonelli et al, 1999, Levenson et al, 1998, and Cocea, 1997, incorporated herein by reference.) "Restriction enzyme digestion" refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.

d. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression (see, for example, Chandler et al (1997), herein incorporated by reference).

e. Termination Signals

The vectors or constructs of the present invention will generally comprise at least one termination signal. A "termination signal" or "terminator" is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3' end ofthe transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation. f. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, convenient and known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.

g. Origins of Replication

In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

h. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drag resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.

i. Mammalian Expression Systems

Numerous expression systems exist that comprise at least a part or all ofthe compositions discussed above. Typically eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides although prokaryote based systems are also contemplated. Many such systems are commercially and widely available. Examples of mammalian expression systems include STRATAGENE^®' s COMPLETE CONTROL™ Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN^®, which carries the T-REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN^® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high- level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

j. Viral Vectors

The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells). For example, in the present invention it is contemplated that viral vectors may be used to transfect the CD4⁺CD25⁺ hybridoma cells with genes of interest including specific T-cell receptors, chemokines or cytokines. Non-limiting examples of virus vectors that may be used to deliver a nucleic acid in the context ofthe methods of the present invention are described below.

1. Adenoviral Vectors

A particular method for delivery of the nucleic acid involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell-specific construct that has been cloned therein. Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992).

2. AAV Vectors

The nucleic acid may be introduced into the cell using adenovirus assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems (Kelleher and Vos, 1994; Gotten et al, 1992; Curiel, 1994). Adeno-associated virus (AAV) is an attractive vector system for use as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broad host range for infectivity (Tratschin et al, 1984; Laughlin etal, 1986; Lebkowski etal, 1988;

McLaughlin et al, 1988). Details concerning the generation and use of rAAV vectors are described in U.S. Patents. 5,139,941 and 4,797,368, each incorporated herein by reference.

3. Retroviral Vectors

Retroviruses have promise as delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell-lines (Miller, 1992).

In order to construct a retroviral vector, a nucleic acid (e.g., one encoding an T-cell receptor, a chemokine, a cytokine or other protein of interest) is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into a special cell line (e.g., by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al, 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).

Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, Naldini et al, 1996; Zufferey et al, 1997; Blomer et al, 1997; U.S. Patents 6,013,516 and 5,994,136). Some examples of lentivirus include the Human Immunodeficiency Viruses: HEV-1, HEV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and ne/are deleted making the vector biologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Patent 5,994,136, incorporated herein by reference. One may target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell-type. By inserting a sequence (including a regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific.

4. Other Viral Vectors

Other viral vectors may be employed as expression and delivery constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et α/., 1988), sindbis virus, cytomegalovirus and herpes simplex virus may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).

5. Delivery Using Modified Viruses

A nucleic acid to be delivered may be housed within an infective virus that has been engineered to express a specific binding ligand. The virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell. A novel approach designed to allow specific targeting of retroviras vectors was developed based on the chemical modification of a retroviras by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.

Another approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al, 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al, 1989).

2. Vector Delivery and Cell Transformation

Suitable methods for nucleic acid delivery for transformation of an organelle, a cell, a tissue or an organism for use with the current invention are believed to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into an organelle, a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection (Wilson et al, 1989, Nabel et al, 1987), by injection (U.S. Patents 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harlan and Weintraub, 1985; " U.S. Patents 5,789,215, incorporated herein by reference); by electroporation (U.S. Patents 5,384,253, incorporated herein by reference; Tur-Kaspa et al, 1986; Potter et al, 1984); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al, 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et α/., 1979; Nicolau et al, 1987; Wong et al, 1980; Kaneda et α/., 1989; Kato et al, 1991) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Patents 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al, 1990; U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by desiccation/inhibition-mediated DNA uptake (Potrykus et al, 1985), and any combination of such methods. Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed. a. Ex Vivo Transformation

Methods for tranfecting vascular cells and tissues removed from an organism in an ex vivo setting are known to those of skill in the art. For example, canine endothelial cells have been genetically altered by retrovial gene tranfer in vitro and transplanted into a canine (Wilson et al, 1989). In another example, yucatan minipig endothelial cells were tranfected by retroviras in vitro and transplanted into an artery using a double-balloon catheter (Nabel et al, 1987). Thus, it is contemplated that cells or tissues may be removed or isolated and tranfected ex vivo using the nucleic acids of the present invention. In particular aspects, the transplanted cells or tissues may be then placed into an organism. In preferred facets, the nucleic acid that is transfected is expressed in the transplanted cells or tissues.

b. Injection

In certain embodiments, a nucleic acid may be delivered to an organelle, a cell, a tissue or an organism via one or more injections (i.e., a needle injection), such as, for example, subcutaneously, intradermally, intramuscularly, intervenously, intraperitoneally, etc. Methods of injection of nucleic acids are well known to those of ordinary skill in the art (e.g., injection of a composition comprising a saline solution). Further embodiments of the present invention include the introduction of a nucleic acid by direct microinjection. Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985). The amount of nucleic acid used may vary upon the nature of the protein of interest as well as the organelle, cell, tissue or organism used.

c. Electroporation

In certain embodiments of the present invention, a nucleic acid is introduced into an organelle, a cell, a tissue or an organism via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high- voltage electric discharge. In some variants of this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells (U.S. Patent 5,384,253, incorporated herein by reference). Alternatively, recipient cells can be made more susceptible to transformation by mechanical wounding.

Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter et α/., 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al, 1986) in this manner.

d. Calcium Phosphate

In other embodiments of the present invention, a nucleic acid is introduced to the cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique. Also in this manner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NEH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al, 1990).

e. DEAE-Dextran

In another embodiment, a nucleic acid is delivered into a cell using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).

f. Sonication Loading

Additional embodiments of the present invention include the introduction of a nucleic acid by direct sonic loading. LTK" fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et al, 1987).

g. Liposome-Mediated Transfection

In a further embodiment of the invention, a nucleic acid may be entrapped in a lipid complex such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is a nucleic acid complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen).

Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley etα/., 1979; Nicolau etal, 1987). The feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al, 1980). In certain embodiments of the invention, a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaned et al, 1989). In other embodiments, a liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al, 1991). In yet further embodiments, a liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In other embodiments, a delivery vehicle may comprise a ligand and a liposome.

h. Receptor Mediated Transfection

Still further, a nucleic acid may be delivered to a target cell via receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in a target cell. In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present invention.

Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a nucleic acid-binding agent. Others comprise a cell receptor-specific ligand to which the nucleic acid to be delivered has been operatively attached. Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al, 1990; Perales et α/., 1994; Myers, EPO 0273085), which establishes the operability of the technique. Specific delivery in the context of another mammalian cell type has been described (Wu and Wu, 1993; incorporated- herein by reference). In certain aspects of the present invention, a ligand will be chosen to correspond to a receptor specifically expressed on the target cell population.

In other embodiments, a nucleic acid delivery vehicle component of a cell-specific nucleic acid targeting vehicle may comprise a specific binding ligand in combination with a liposome. The nucleic acid(s) to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the liposome membrane. The liposome will thus specifically bind to the receptor(s) of a target cell and deliver the contents to a cell. Such systems have been shown to be functional using systems in which, for example, epidermal growth factor (EGF) is used in the receptor-mediated delivery of a nucleic acid to cells that exhibit upregulation ofthe EGF receptor.

In still further embodiments, the nucleic acid delivery vehicle component of a targeted delivery vehicle may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding. For example, lactosyl-ceramide, a galactose-terminal asialganglioside, have been incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes (Nicolau etal, 1987). It is contemplated that the tissue-specific transforming constructs of the present invention can be specifically delivered into a target cell in a similar manner.

i. Microprojectile Bombardment

Microprojectile bombardment techniques can be used to introduce a nucleic acid into at least one, organelle, cell, tissue or organism (U.S. Patent 5,550,318; U.S. Patent 5,538,880; U.S. Patent 5,610,042; and PCT Application WO 94/09699; each of which is incorporated herein by reference). This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987). There are a wide variety of microprojectile bombardment techniques known in the art, many of which are applicable to the invention.

Microprojectile bombardment may be used to transform various cell(s), tissue(s) or organism(s). Examples of plant species which have been transformed by microprojectile bombardment include monocot species such as maize (PCT Application WO 95/06128), barley (Ritala et al, 1994; Hensgens et al, 1993), wheat (U.S. Patent 5,563,055, incorporated herein by reference), rice (Hensgens et al, 1993), oat (Torbet et al, 1995; Torbet et al, 1998), rye (Hensgens et al, 1993), sugarcane (Bower et al, 1992), and sorghum (Hagio et α/., 1991); as well as a number of dicots including tobacco (Tomes et al, 1990; Buising and Benbow, 1994), soybean (U.S. Patent 5,322,783, incorporated herein by reference), sunflower (Knittel et al. 1994), peanut (Singsit et al, 1997), cotton (McCabe and Martinell, 1993), tomato (VanEck et al. 1995), and legumes in general (U.S. Patent 5,563,055, incorporated herein by reference).

In this microprojectile bombardment, one or more particles may be coated with at least one nucleic acid and delivered into cells by a propelling force. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold particles or beads. Exemplary particles include those comprised of tungsten, platinum, and preferably, gold. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA. DNA-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary. For the bombardment, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into a cell by acceleration is the Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with cells, such as for example, a monocot plant cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing the damage inflicted on the recipient cells by projectiles that are too large.

E. Therapeutic Regimens

The present invention also provides methods for ameliorating, reducing, preventing and controlling immune mediated inflammatory disorders, which result from undesirable immune reactions and lead to such conditions as autoimmune diseases, tissue or organ transplantation rejections, allergies etc. by providing sufficient numbers of CD4⁺CD25⁺ cells to a patient afflicted with such a condition.

The present invention is therefore useful in a mammalian subject for the treatment and prevention of immune mediated inflammatory disorders, including the rejection of transplanted organs or tissue, graft- vs-host diseases brought about by transplantation of a variety of tissues or organs; celiac disease, type I diabetes, multiple sclerosis, rheumatoid arthritis, rheumatic fever, autoimmune ulcerative colitis, autoimmune gastritis and other autoimmune mediated processes; pollen allergies, reversible obstructive airway disease, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma, chronic or inveterate asthma, late asthma and airway hyper-responsiveness; ischemia-reperfusion injury of organs which occurs upon preservation, transplantation or ischemic disease, for example, thrombosis and cardiac infraction. Use in other immune mediated inflammatory disorders not specified above are also contemplated.

In the context of the present invention, it is contemplated that the CD4⁺CD25⁺ cells therapy may comprise providing the cells alone or may comprise a combination therapy where the CD4⁺CD25⁺ cells are provided in conjunction with other immunosupressive therapeutic agents. In the case of organ/tissue transplantation and graft vs host disease other immunosupressive agents such as cyclosporin, rapamycin, FK506, or mycophenolic acid, immunosupressive steriods such as prednisone may be used. In addition, chemical immunosuppression in mammals can be produced by any of a variety of reagents including myelosuppressive alkylating agents such as cyclophosphamide, anti-metabolites such as 5- ffuoro-uracil, or methotrexate, plant alkaloids such as vinblastin, antibiotics such as doxorubicin, triamcinolone acetonide, cyclosporins, cytochalasin and a wide variety of steroids such as hydrocortisone acetate, betamethazone, cortisone acetate. Alternatively, the use of other T-cell suppressive agents such as anti-T-lymphocyte globulin (ATG), and/or nucleoside analogs (e.g. fludarabine), and/or immunophilins (e.g. Cyclosporin A or rapamycin) are also contemplated. Alternatively, the use of other biologies, such as antibodies to cytokines, cytokine receptor agonists or cytokine receptor antagonists are also contemplated. These agents are typically administered i.p, i.m., i.v. or s.c, depending on the pharmacological properties of the agent. Administration is carried out on a regular basis, the frequency of which is sufficient to maintain the human/other mammal in a constant state of immunosuppression over the time frame required.

The administration ofthe other immunotherapeutic may precede, coincide with or follow the therapy using CD4⁺CD25⁺ cells ofthe invention by intervals ranging from minutes to days to weeks. In embodiments where the other immunotherapeutic and the CD4⁺CD25⁺ cells are administered together, one would generally ensure that a significant period of time did not expire between the time of each delivery. In such instances, it is contemplated that one would administer to a patient both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

It also is conceivable that more than one administration of either the other therapeutic and the CD4⁺CD25⁺ cells will be required to achieve amelioration of the undesirable immune- reaction or the immune mediated inflammatory disorder or condition. Various combinations may be employed, where the other therapeutic agent is "A" and the CD4⁺CD25⁺ cells of the invention is "B", as exemplified below:

A/B/A B/A/B B/B/A A/A B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations also are contemplated. The exact dosages and regimens of each agent can be suitable altered by those of ordinary skill in the art.

F. Pharmaceutical Formulations and Delivery

In some embodiments of the present invention, methods for treatment for undesirable or adverse immune reactions or immune mediated inflammatory disorders, such as an autoimmune disease, a tissue or organ transplantation rejection episode, or an allergic condition are provided and comprise administering to a patient in need thereof an effective amount of a pharmaceutical formulation of CD4⁺CD25⁺ regulatory cells wherein the amount of the CD4⁺CD25⁺ cells is effective in ameliorating the undesirable/adverse immune reaction or immune mediated inflammatory disorder.

An effective amount of the pharmaceutical composition, generally, is defined as that amount sufficient to detectably and repeatedly ameliorate, reduce, inhibit, minimize or limit the extent of the disease or its symptoms, or the extent of the condition, such as an undesirable/adverse immune response or immune mediated inflammatory disorder. More rigorous definitions may apply, including elimination, eradication or cure of disease, or elimination or eradication ofthe unwanted/ undesirable/adverse immune response.

Some useful doses contemplated for the amelioration of an immune mediated inflammatory disorder range from O.lxlO⁷ to 2xl0⁷ cells/kilogram of body weight of the subject receiving the therapy. This includes the doses of O.lxlO⁷, 0.2xl0⁷, 0.3xl0⁷, 0.4xl0⁷, 0.5x10⁷, 0.6xl0⁷, 0.7xl0⁷, O.SxlO⁷, 0.9xl0⁷, lxlO⁷, l.lxlO⁷, 1.2xl0⁷, 1.3xl0⁷, 1.4xl0⁷, 1.5xl0⁷, 1.6xl0⁷, 1.7xl0⁷, 1.8xl0⁷, 1.9xl0⁷ and 2x10⁷ cells/kilogram of body weight. Intermediate ranges are also contemplated. Infusion of lower doses may also be possible.

The routes of administration will vary, naturally, with the location and nature of the disease or the condition, and include, e.g., parenteral, intravenous, intralesional, intra-portal, intra-arterial, intramuscular, intranasal, intradermal, subcutaneous, percutaneous, intratracheal, intraperitoneal, direct injection, or in a graft prior to transplant. Intratracheal, intrabronchial or endobronchial administration is especially contemplated for treatment of asthmatic conditions. Local, regional or systemic administration also may be appropriate. In the case of transplantation related immune reactions, the CD4⁺CD25⁺ cells may be administered before surgery, at the time of surgery, and/or thereafter, to prevent, control and treat any transplant rejection reaction. For example, a transplanted tissue/organ and vasculature may be injected or perfused with a formulation comprising the CD4⁺CD25⁺ cells as provided by this invention prior to surgery. The perfusion may be continued post-transplant, for example, by leaving a catheter implanted post-surgery. Periodic post-surgical treatment also is envisioned.

Continuous administration also may be applied where appropriate, for example, in ongoing rejection or pre-existing autoimmune diseases. Delivery may be via syringe or catherization. Such continuous perfusion may take place for a period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 wk or longer following the initiation of treatment. Generally, the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs.

Treatment regimens may vary as well, and often depend on disease type, location, disease progression, and health and age of the patient. Obviously, certain types of diseases and conditions will require more aggressive treatment, while at the same time, certain patients cannot tolerate more taxing protocols. The clinician will be best suited to make such decisions based on the known efficacy and toxicity (if any) ofthe therapeutic formulations.

Solutions comprising the regulatory CD4⁺CD25⁺ cells may be constituted in pharmacologically acceptable solvents. Pharmacologically acceptable solvents for constituting cells are well known in the art. For example, one can use solvents used in blood stem cell transplantation, bone marrow transplantation methods and CD34 selected cell transplantation (O'Donnell et al, 1998; Yanovich et al, 2000; Michallet et al, 2000). These cells may further be suspended in an isotonic solution such as phosphate buffered saline and may be optionally supplemented with human serum albumin prior to infusion to patients. Such methods are well known in the art. For example, U.S. Patent 5,443,983 describes methods for suspending and intravenously introducing LAK cells into patients. U.S. Patent 5,057,423, which describes methods of adoptive immunotherapy utilizing LAK also describe administration and pharmaceutical formulations that may be used for cell based therapy. The regulatory CD4⁺CD25⁺ cells obtained according to the method ofthe present invention may be administered according to any of the known prior art methods including those set forth in Mule et al, 1985; Rosenberg et al, 1987; Rosenberg et al, 1985.

It is also contemplated that prior to usage the cells may be stored cryopreserved either in dimethylsulfoxide (DMSO) or other solvents by methods analogous to those used for preserving and reconstituting CD34 selected cells and/or cells used for bone marrow transplantation procedures and/or blood stem cells. Such methods are well known in the art. For example, the regulatory CD4 CD25 cells may be frozen and stored using the same techniques previously developed for bone marrow hematopoietic cells. General parameters include cryopreservation in DMSO and a source of plasma protein with or without hydroxyethylstarch (HES) followed by cooling at 1 to 3°C/minute and storage at -80°C or colder. This may be achieved by use of computer controlled devices or by direct immersion of bags of about 50 ml into a -80°C freezer. Cooling by immersion into a -80°C freezer is not limited to HES-containing solutions, but may be used for cells frozen with DMSO alone. Although, the maximal tolerated dose of DMSO is not known for humans, the LD50 of DMSO for dogs has been reported to be 2.5 gm per kilogram body weight or about 22 ml/kg of a 10% DMSO solution. These limits will be kept in consideration while using cryopreservation methods. lit is expected that the cells will be washed after thawing, to remove most ofthe DMSO.

Additionally the pharmaceutical compositions may be mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under certain conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile isotonic aqueous solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable- under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, saline, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intraportal, intramuscular, subcutaneous, intratracheal and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light ofthe present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the function ofthe CD4⁺CD25⁺ cells, its use in the therapeutic compositions is contemplated.

The phrase "pharmaceutically-acceptable" or "pharmacologically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.

G. Clinical Trials

This section is concerned with the development of human treatment protocols for providing therapy to undesirable/adverse immune reactions in a human patient using the CD4⁺CD25⁺ cells as described herein.

The various elements of conducting a clinical trial, including patient treatment and monitoring, will be known to those of skill in the art in light of the present disclosure. The following information is being presented as a general guideline for use in establishing the CD4⁺CD25⁺ cells, either alone or in combination with other standard therapies used in the art for the treatment of the immune mediated inflammatory disorders and other related conditions in clinical trials.

Candidates for the phase 1 clinical trial will be patients who have a history of immune mediated inflammatory disorders, such as graft rejection, or an organ specific autoimmune condition like insulin dependent diabetes for which all conventional therapies have failed. Approximately 100 patients will be treated initially. Patients will be treated, and samples obtained, without bias to sex, race, or ethnic group. Research samples will be obtained from peripheral blood or marrow under existing approved projects and protocols. Some ofthe research material will be obtained from specimens taken as part of patient care. This material will be used to monitor the level of CD4⁺CD25⁺ cells in the periphery and to monitor alterations in immune function mediated by the CD4⁺CD25⁺ cells.

The treatments described above will be administered to the patients regionally or systemically on a tentative weekly basis. A typical treatment course may comprise about six doses delivered over a 7 to 21 day period. Upon election by the clinician the regimen may be continued with six doses every three weeks or on a less frequent (monthly, bimonthly, quarterly, etc.) basis. Of course, these are only exemplary times for treatment, and the skilled practitioner will readily recognize that many other time-courses are possible.

The modes of administration may be intravenous, intraportal, intratracheal, and/or any of the other routes described elsewhere in this specification depending on the nature of the disease or condition that the patient is afflicted with.

In one embodiment, CD4 CD25⁺ cells will be administered at dosages in the range of 0.1 x 10⁷ to 2 x 10⁷ cells/kilogram body weight, by intravenous, intraportal, intratracheal, intranasal, or intralesional routes or to graft tissue before transplantation. In some embodiments the CD4⁺CD25⁺ cells may be administered as liposomal formulations. Of course, the skilled artisan will understand that while these dosage ranges, provide useful guidelines appropriate adjustments in the dosage depending on the needs of an individual patient factoring in disease, gender, age and other general health conditions will be made at the time of administration to a patient by a trained physician. The same is true for means of administration, routes of administration as well.

To monitor disease course and evaluate the amelioration ofthe disease it is contemplated that the patients should be examined for appropriate tests every month or more often, depending on the schedule of the treatments. To assess the effectiveness of the CD4⁺CD25⁺ cells, the physician will determine parameters to be monitored depending on the type of disease and will involve methods to monitor reduction in the unwanted immune response, such as the measurement of auto-antibodies (in the case of autoimmune diseases), T-cell responses to the self antigen or the target foreign antigen to which tolerance is desired, decrease of graft vs host reactions such as tissue necrosis (in transplant patients) and the like. Tests that will be used to monitor the progress of the patients and the effectiveness of the treatments include: physical exam, X-ray, blood work, and other clinical laboratory methodologies, including immune responses to the target antigen by ELISA, ELISPOT, in vitro proliferation assays, engraftment studies, studies of endothelial integrity, measurement of various lymphocyte subsets by flow cytometry, intracytoplasmic cytokine analysis or ELISPOT analysis. The doses given in the phase 1 study will be escalated as is done in standard phase 1 clinical phase trials, i.e. doses will be escalated until maximal tolerable ranges are reached.

Clinical responses may be defined by acceptable measure. For example, a complete response may be defined by complete disappearance of the immune mediated inflammatory disorder or condition, whereas a partial response may be defined by a 50% reduction of the disease or condition.

H. Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Generation of CD4⁺CD25⁺ Hybridomas

Generation of MHC Tolerant Mice

Pregnant BALB/c (H-2^d) mice, normal BALB/c, A/J, CAFi (BALB/c x A/3), and C57B1/6J (B6) mice were purchased from Charles River Laboratories (Wilting, MA) and The Jackson Laboratory (Bar Harbor, ME). Newborn BALB/c mice (< 24 hours old) were injected via the intraperitoneal route with 10⁸ live semi-all ogeneic spleen cells that were isolated from adult CAFi mice (H-2^dxa). Four to six weeks after the injection, the BALB/c mice were grafted with A J (H-2^a) tail skin. Bandages were removed seven days after skin grafting, and grafts were inspected daily. BALB/c mice were deemed tolerant to H-2^a antigen if they accepted A/J skin grafts for >40 days. These tolerant mice were than used as a source of tolerant-cells.

Fusion of Hybridomas

Three days before the fusion, CD4⁺ cells were isolated from tolerant mice by negative bead selection and magnetic sorting according to manufacturer's specifications. The CD4⁺ cells were activated by incubating the cells overnight in tissue culture plates that were pre-coated with anti-T3 (2C11, anti-TCR-ε, 10 μg/ml). CD4⁺ cells from tolerant mice were fused to BW5147of β^~ cells (provided by P. Marrack) (White et al, 1989) using standard procedures for generating T-cell hybridomas (Current Protocols in Immunology, 3.14.1-4.14.11 Contributed by Ada M. Kruisbeek 1997 copyright). The BW5147of β^~ cells were grown in complete DMEM- 10% FCS at 37°C at concentrations below 1 xlO⁶ cells/ml and split the day before the fusion to provide >1 x 10⁸ cells.

The T-cells and the BW5147α^~β^~ cells were harvested and washed in DMEM. A suspension of thymocytes from BALB/c mice was prepared at a concentration of 5 x 10⁶ cells/ml in 50 ml complete DMEM-20% FCS. The BW5147α^~β^~ cells and the CD4+ cells were mixed at a 1:1 ratio. 20 ml of 37°C DMEM was added and then the cells were centrifuged for 5 minutes at 2000 rpm, room temperature. The supernatant was removed. 1ml of prewarmed 50% PEG solution (50% w/v polyethylene glycol in unsupplemented DMEM) was added dropwise over a period of one minute stirring gently after each drop. The mixture was stirred for another minute. 2 ml of prewarmed DMEM was added dropwise in a similar fashion over a period of 2 minutes. 7 ml of prewarmed DMEM was added over a period of 2-3 minutes. The cells were centrifuged for 5 minutes at 2000 rpm, room temperature. 10 ml of the thymocyte suspension was added forcefully onto the cell pellet. The remaining thymocytes were added while stirring until the desired volume was reached (10 ml per plate). A 10 ml pipette was used to add 2 drops of the cell suspension to each well of a 96 well flat-bottom plate. Five plates were made and placed in the incubator (37°C, 7.5% C0₂). After 1 day of incubation 2 drops of 2X HAT plating medium diluted in DMEM 20% FCS) was added to each well (100X HAT is lOmM sodium hypoxanthine, 40 μM aminopterin, 1.6 mM thymidine). The cells were viewed under a microscope after 3 days to ensure cell death of nonhybrid cells. After 7 days, half the volume of the wells was removed with a pipet and the hybridomas were fed with 2 drops of fresh 2X HAT plating medium. After 6-7 days the medium turned slightly yellow. The cells were then transferred into a 24 well plate and 1 ml of IX HT plating medium (100X is 10 nM sodium hypoxanthine, 1.6 mM thymidine) diluted in complete DMEM-10% FCS medium) was added to wells. The cells were fed 1 or 2 days later with IX HT plating medium. Duplicate cultures were made of wells that were growing well by resuspending the content of one well and pipetting 1 ml into wells on a fresh plate and adding 1 ml of compete DMEM-10% FCS to the two new wells. Screening of Hybridoma Cell Lines

The hybridomas were screened for CD4⁺CD25⁺ expressing hybridomas by FACS analysis of one of the duplicate wells using the following monoclonal antibodies: H57-597 (TCRαβ) FITC, anti-CD4 Texas Red, and anti-CD25 PE (EL-2R, alpha chain). A total of 114 hybridomas were screened and 15 CD4⁺CD25⁺ hybridomas were isolated. From these a single CD4⁺CD25⁺ hybridoma line was chosen for future study. This was then subcloned by FACS to yield CD4⁺CD25⁺ hybridoma clones.

Cloning

The double positive population was cloned by plating 1 cell/well into flat-bottomed 96 well plates with DMEM-10%. The plates were incubated and allowed to grow. When needed the clones were expanded onto a 24 well plate, and duplicates made. The clones were screened to select clones with high expression of CD4⁺CD25⁺.

Phenotypic Analysis of Hybridomas

CD4⁺CD25⁺ hybridomas (lxl0⁵/well) were cultured alone or with irradiated BALB/c or A/J spleen cells (5x10⁵) for three days. Supernatants were harvested and analyzed for cytokine production for the following cytokines: interleukin-4 (EL-4), interferon-γ (IFN-γ), interleukin-2 (EL-2), and transforming growth factor βi (TGF-βi), using commercially available ELISA assays. The cytokine studies revealed that all the hybridomas constitutively secreted cytokines, particularly TGFβ. The CD4⁺CD25⁺ hybridomas could be grouped according to their cytokine profile (Table 3). Of these clone 3C1 was selected for further functional studies and additional subcloning because of its high constitutive production of TGFβ i.

TGFβ is immunosuppressive and has been shown to play a role in other models of tolerance which involve regulatory pathways (Chen et al, 1994; Powrie et al, 1996; Groux et al, 1997; Chen et al, 1996; Weiner, 1997; Bridoux et al, 1997). TGFβ can alter the accessory signals of APCs (Takeuchi et al, 1998) and skew the development of antigen reactive Th2 (Takeuchi et al, 1998; King et al, 1998). It is contemplated that TGFβ producing CD4⁺CD25⁺ cells may regulate the development of anti-donor "allo-reactive" CD8⁺ T-cells in mice with acquired tolerance to donor MHC in a similar fashion. TABLE 3

Cytokine Profile of Hybridoma Cells

Hybridoma TGFβ IFNγ ΓL-4

Group 1 + + +

3B1 435 114 72

3C2 294 157 69

2B6 203 143 28

Group II + - -

1B4 294 <38 <3.2

Group IH + + -

2D5 203 116 <3.2

2A6 618 73 <3.2

Group IV ++ - -

1D1 3799 <38 <3.2

1A6 3092 <38 <3.2

2B1 1136 <38 <3.2

Group V ++ + -

2D3 3165 115 <3.2

2D4 1157 345 <3.2

2C5 2793 73 <3.2

3A2 1719 229 <3.2

1A2 9741 59 <3.2

Group VI +++ ++ -

3C1 15,588 1145 <3.2

Hybridoma cells (1x10 ) were cultured in media for three days. Supernatants were harvested and the amount of cytokine was determined using cytokine specific ELISA assays. Detection limits: 31 pg/ml, TGFβl; 38 pg/ml, EFNγ; 3 pg/ml, EL-4. EL-2 was not detected and EL- 10 was not tested.

CD4⁺CD25⁺ hybridomas were also analyzed for expression of important T-cell surface receptors and ligands using FACS analysis to evaluate: TCRγδ, Thy 1.2. Ly49, DX5, CD4, CD25, CD3ε, H-2K^k, H-2K_d, TCRαβ, CD28, CD40L, and CD62L. EXAMPLE 2 Generation of CD4+CD25+ Hybridomas

Tolerant mice generated as described above in Example 1 with healthy skin grafts for > 40 days were used as a source of the tolerant CD4⁺ cell population. CD4⁺ cells were purified from tolerant mice and fused to the BW5147 (TCRofβ-) fusion partner (kindly provided by P Marrack (White et al, 1989) ) using conventional methodology. 115 hybridomas were screened for co-expression of CD4⁺CD25⁺ and the CD4⁺CD25⁺ candidates were subcloned to yield a series of CD4⁺CD25⁺ hybridomas. FIG. 1 shows the FACS profile of one of the CD4⁺CD25⁺ hybridoma subclones, RD6. Other hybridoma clones show similar phenotype, including 3C1. The CD4⁺CD25⁺ hybridomas are CD62L positive, comparable to other conventional CD4⁺CD25⁺ regulatory cells (Herbelin et al, 1998; Itoh et al, 1999, Lepault et al, 2000; Thornton and Shevak, 2000). In addition, the CD4⁺CD25⁺ hybridomas are CD28 positive, but CD40L negative and CD3 negative.

EXAMPLE 3 CD4+CD25+ Hybridomas Inhibit T-cell Proliferation

To determine whether CD4⁺CD25⁺ hybridoma cells have regulatory function their ability to inhibit T-cell proliferation was examined. For this, unfractionated spleen cells from naive BALB/c mice were labeled with CFSE (5,6-carboxyfluorescein diacetate succinimidyl ester) and 10⁶ cells per tube were stimulated with increasing concentrations of anti-TCR (0 to 1.0 μg/ml) with or without recombinant EL-2 (100 U/ml) in the presence or absence of CD4⁺CD25⁺ hybridoma clone, 3C1 (2.5xl0⁵), for 1-3 days. Stimulation by anti-TCR in the absence of 3C1 induced T-cell proliferation by day 3, as detected by decreasing content of CFSE on gated T- cells (FIGS. 2A-2E) The addition of EL-2 to the anti-TCR stimulated cultures further enhanced the proliferation (FIGS. 2F-2J). In contrast, co-culture with 3C1 inhibited the number of T-cells that proliferated and decreased the number of rounds of proliferation, at all concentrations of anti-TCR (FIGS. 2K-20). Moreover, the addition of EL-2 did not restore T-cell proliferation in the presence ofthe CD4⁺CD25⁺ hybridoma, 3C1 (FIGS. 2P-2T).

CD4⁺CD25⁺ hybridomas could also directly inhibit proliferation of anti-CD3 activated purified CD8⁺ cells (FIGS. 3A-D). For this, CD8⁺ cells were purified from whole spleen cell preparations from naive BALB/c mice, using magnetic bead separation. Naϊve CD8⁺ cells were labeled with CFSE and stimulated in vitro with soluble anti-CD3 (1 μg/ml) with and without EL- 2 (100 U/ml) for 72 hours with syngeneic APCs and either the fusion partner, BW5147ofβ^~ or CD4⁺CD25⁺ hybridoma, 3C1. Purified CD8⁺ cells proliferate in response to soluble anti-CD3 in the presence ofthe control cell, BW5147ofβ^~, and the proliferation is enhanced with the addition of EL-2. In contrast, CD8⁺ cells show less proliferation when stimulated with soluble anti-CD3 in the presence of the CD4⁺CD25⁺ hybridoma, 3C1, and proliferation is only partially restored with the presence of EL-2.

FIG. 4. shows the extent to which the CD4⁺CD25⁺ hybridoma 3C1 and several of the 3C1 subclones, DC3, DD9, and RA2, inhibit the proliferation of purified CD8+ cells following activation with soluble anti-CD3. FIG. 4. also shows that in the presence of a control hybridoma that expresses CD4⁺ but is CD25 negative, CD8⁺ cells proliferate normally following stimulation with soluble anti-CD3.

The inhibition of T-cell proliferation was not secondary to media exhaustion from the hybridomas. There was no decrease in the number of T-cells recovered from the unstimulated cultures when CD4⁺CD25⁺ hybridomas were added (see FIGS. 2K-T). In addition, co-culture with either the BW5147 fusion partner (FIGS. 3A-B) or with a CD4⁺CD25⁺ negative control hybridoma, which was isolated from the original fusion (FIG. 4) did not inhibit anti-CD3 mediated T-cell proliferation. These results demonstrate that CD4⁺CD25⁺ hybridomas inhibit the proliferation of T-cells following activation via their T cell receptors.

EXAMPLE 4 CD4⁺CD25⁺ Hybridomas Block Development of Alloreactive CD8⁺ Cells

The ability of the CD4⁺CD25⁺ hybridomas, 3C1 and DC3, to block the development of alloreactivity in vitro was also examined. For this, CD8⁺ cells were purified from naϊve mice and cultured in vitro with T-cell-depleted-A/J cells for 4 days with or without CD4⁺CD25⁺ hybridomas. Cells from these primary cultures were centrifuged over Lympholyte-M, separated from the hybridomas and stimulatory cells by column purification, and then were re-stimulated with fresh A/J antigen presenting cells and the number of EFNγ-producing CD8⁺ cells was determined using intracytoplasmic cytokine detection by flow cytometry. Brefeldin A (10 mg/ml, Epicentre echnologies) was added for the last 2 hours of culture. Cells were stained with anti-CD8+ and then fixed and permeabalized using a commercial Fix & Perm kit (Caltag) according to manufactures directions. Intracellular cytokine staining was performed with both RPE conjugated rat anti-mouse EFNγ mAb (XMG-1.2, Pharmingen) and RPE conjugated rat IgGl isotype control mAb. Analysis was performed with a Coulter EPICS FACS. Table 4 depicts an experiment in which CD4⁺CD25⁺ hybridomas inhibited the differentiation of alloreactive EFNγ-producing CD8⁺ cells.

TABLE 4 CD4⁺CD25⁺ Hybridomas Inhibit the Development of IFNγ⁺ CD8⁺ Cells

harvested and restimulated for 6 hours with T-depleted A/J spleen cells. Brefeldin A was added for 12 hours. ^cCells were stained with anti-CD4, anti-CD8 and anti-H-2^k mAbs. Intracellular staining was performed using either RPE-IgG isotype control or RPE-anti-EFNγ mAb. Numbers depict the percent of IFNγ⁺ cells of gated CD8⁺/H-2K^k~.

EXAMPLE 5 CD4⁺CD25⁺ Hybridomas Alter Expression of CD62L on Activated CD8⁺ Cells

CD62L (L-selectin) is an important homing receptor for leukocytes. Trafficking of lymphocytes across high endothelial venules of peripheral lymph nodes depends on the expression of CD62L (Butcher et al, 1996). CD62L knock-out mice have impaired homing to peripheral lymph node and decreased primary T-cell responses to antigen (Xu et al, 1996; Steeber et al, 1996). It is known that activation through the TCR induces an early shedding of surface CD62L on T-cells (Chao et al, 1997). This rapid shedding is caused by proteolytic cleavage of CD62L at the cell surface by a metalloproteinase (Preece et al, 1996). Following the initial down-regulation of CD62L, surface CD62L expression markedly increases over the following 48 hours, due to increased CD62L mRNA, and the lymphocytes proceed to proliferate (Chao et al, 1997). Cultures that were stimulated with anti-CD3 in the presence of CD4⁺CD25⁺ hybridomas contained more CD62L negative CD8⁺ cells (FIG. 5) as compared to controls. This indicates that the CD4⁺CD25⁺ hybridomas may allow the first phase of rapid metalloproteinase dependent shedding of CD62L but inhibit the second phase of CD62L re-expression. The down- regulated CD62L on CD8⁺ cells may alter CD8⁺ cell homing and interfere with the ability of CD8⁺ cells to participate in immune responses in vivo. Down-regulation of CD62L has been described in an in vitro model of CD4⁺ T-cell "tolerance" (Marschner et al, 1999). Crosslinking of the CD4 co-receptor on CD4⁺ cells in the absence of antigen inhibits TCR-dependent signaling (Haughn et al, 1992) and triggers activation-induced cell death after subsequent crosslinking of the TCR (Newell et al, 1990). CD4⁺ cells that are treated in this manner also demonstrate rapid proteolytic cleavage of surface CD62L and impaired homing to peripheral lymph nodes (Marschner et al, 1999).

EXAMPLE 6 Isolation and Functional Characterization of CD4⁺CD25⁺ Hybridoma Cells

Although the cytokine profile produced constitutively by Group IV hybridomas (see Table 3) resembled most closely the cytokine profile of A/J activated CD4⁺CD25⁺ cells that were isolated from neonatal tolerant mice, hybridoma 3C1 was selected for further functional analysis as this hybridoma secreted the highest level of TGFβ.

Hybridoma 3C1 was further subcloned, using FACS sorting, to select for cells with the highest expression of CD4⁺CD25⁺. Ten subclones were selected. These clones, along with the parent T-cell hybridoma line (3C1) were examined over 3-4 months for stability of CD4⁺CD25⁺ expression and cytokine profile. The CD4⁺CD25⁺ expression was stable for several months, but the cytokine profile varied considerably between the 3C1 subclones and over time. One of these subclones, RD6, was tested in various functional assays. The phenotype of this subclone is shown in FIG. 1.

Inhibition of T-Cell Proliferation by KD6 CDf^rCD25 Hybridoma Subclone

Work by Shevach and colleagues showed that CD4⁺CD25⁺ cells that were isolated from DOl l. lO mice (transgenic for the T-cell receptor against OVA peptide) could regulate other CD4⁺ cells in vitro (Thornton and Shevach, 2000). The DOl l.lO CD4⁺CD25⁺ cells required a specific antigen (OVA) to be activated, but once activated, they exhibited non-specific inhibition of CD4⁺ cell proliferation. In view of this, 3C1 subclones were examined in relation to their ability to inhibit anti-TCRε stimulated proliferation of naϊve CD4+ and CD8⁺ cells.

To determine whether CD4⁺CD25⁺ hybridoma subclone, RD6, has regulatory function their ability to inhibit T-cell proliferation was examined. For this, unfractionated spleen cells from naϊve BALB/c mice were labeled with CFSE and cultured for 1-3 days with no stimulation or with anti-CD3 monoclonal antibody, 2C11 (1 μg/ml) with or without EL-2 in (100 U/ml, recombinant mouse EL-2), either alone or in the presence of the CD4⁺CD25⁺ hybridoma subclone, RD6, or the control BW5147α^~β^~ fusion partner (see FIGS. 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 61, 6J, 6K & 6L). T-cells that are not stimulated with anti-CD3 show little or no proliferation after 3 days (FIGS. 6A-6C). Stimulation with 1 μg/ml of soluble anti-CD3 induces maximal stimulation of the T-cells after day 3 (FIGS. 6G, 63). Stimulation in the presence of control cells BW5147cf β^~ did not alter the extent of T-cell proliferation (FIGS. 6H, 6K). In contrast, co-culture with RD6 inhibited the number of T-cells that proliferated and decreased the number of rounds of proliferation (FIGS. 6G-6I), and the addition of EL-2 could partially overcome the inhibition of T-cell proliferation by the CD4⁺CD25⁺ hybridomas in some experiments (FIGS. 6A-L).

EXAMPLE 7 CD4⁺CD25⁺ Hybridoma Subclone, RD6, Inhibits Proliferation of Anti-CD3 Stimulated CD4⁺ and CD8⁺ Cells

The CD4⁺CD25⁺ hybridoma, RD6, inhibits the proliferation of CD4⁺ and CD8 cells that have been activated with soluble anti-CD3. Spleen cells from naϊve BALB/c mice were labeled with CFSE and were either left unstimulated or were stimulated with soluble anti-CD3 (1 μg/ml) for three days. The CFSE-labeled spleen cells (10⁶/culture tube) were cultured alone, or in the presence of either BW5147α^~β^~ cells (10⁶/culture tube) or CD4⁺CD25⁺ hybridoma, RD6, (10⁶/culture tube). After three days the cultures were harvested and the number of proliferating CD4⁺ and CD8⁺ cells was determined using flow cytometry (FIGS. 7A-7B). CD4⁺ or CD8⁺ spleen cells proliferate after three days of stimulation with soluble anti-CD3, and proliferation is not affected when BW5147of β^~ cells are present in the cultures. In contrast, co-culture with the CD4⁺CD25⁺ hybridoma subclone, RD6, inhibits proliferation of both the CD4⁺ and the CD8⁺ splenic T-cells (FIGS. 7A-7B).

EXAMPLE 8 CD4⁺CD25⁺ Hybridoma Subclone, RD6, Inhibits IL-2 Production by Anti-CD3

Stimulated Splenic T-Cells

T-cells, particularly CD4⁺ cells secrete the cytokine, EL-2 following stimulation through their T-cell receptor or stimulation with cognate antigen. The CD4⁺CD25⁺ hybridoma subclone, RD6, was tested for its ability to inhibit EL-2 secretion by activated T-cells. Spleen cells from naive BALB/c mice were left unstimulated or were stimulated with soluble anti-CD3 (1 μg/ml) overnight in the presence of no other cells, increasing numbers of BW5147α^"β^~ cells or increasing numbers ofthe CD4⁺CD25⁺ hybridoma subclone, RD6. The ratio of spleen cells o- cultured cells was 1:0.2; 1:0.1; 1:0.05. Spleen cells were then transferred to EL-2 ELISPOT wells (10⁶ cells/well) for another 24 hours, after which time the EL-2 ELISPOTS were developed. The EL-2 ELISPOT used JES6-1A12 for the capture and JES6-5H4-biotin and goat anti-biotin-horse radish peroxidase (Sigma, St. Louis, MO) for the detection according to methods well known in the field (Field & Rouse, 1995). Spots were detected using the ImmunoSpot analyzer (Cellular Technologies). Spleen cells that are not stimulated do not secrete EL-2, and no EL-2 secreting cells can be detected by ELISPOT assay. When spleen cells are stimulated with soluble anti-CD3 in the presence of control BW5147α^~β^~ cells, 200-400 EL-2 producing cells per well are detected. Stimulation in the presence of CD4⁺CD25⁺ hybridoma subclone, RD6, decreases the number of EL-2 producing cells by 60-80%. Therefore, CD4⁺CD25⁺ hybridoma subclone, RD6, inhibits the function of activated T-cells, EL-2 secretion (FIG.8). FIG. 8 further shows that the RD6 hybridoma itself does not constitutively secrete EL-2, because no EL-2 ELISPOTS were detected in the co-cultures of unstimulated spleen cells and RD6 hybridoma cells.

EXAMPLE 9 CD4⁺CD25 Hybridoma Subclone, RD6, Inhibits Proliferation of Alloantigen

Stimulated T-Cells

To be effective regulators of immune response to transplanted tissue, the CD4⁺CD25⁺ hybridomas must inhibit T-cell responses to foreign major histocompatibility antigens, the major transplantation antigens. The extent to which CD4⁺CD25⁺ hybridoma subclone, RD6, inhibits the response to foreign transplantation antigens, the following system was examined. CD4⁺CD25⁺ hybridomas were derived from CD4⁺ cells from mice with acquired tolerance to donor cells from CAFI mice; therefore, their ability to inhibit responses to CAFI cells was examined. CD8⁺ T-cells were purified by column purification of naϊve BALB/c spleen cells. Purified CD8⁺ cells were labeled with CFSE and were cultured for 6 days either by themselves, with EL-2 (100 U/ml), with T-depleted spleen cells from CAFI mice, or with the combination of EL-2 and T-depleted spleen cells from CAFI mice. In some of the cultures, CD4⁺CD25⁺ hybridoma subclone, RD6, was added at a ratio of RD6:CD8⁺ cells of 1:20. After six days the ceils were harvested and the level of proliferation of the CD8⁺ cells was determined using flow cytometry, gating on the CD8⁺ cells. About 10% of CD8⁺ cells proliferate after stimulation with allogeneic CAFI cells, and the addition of EL-2 increases the level of proliferation of CD8⁺ cells to 72%. Co-culture with the CD4⁺CD25⁺ hybridoma subclone, RD6, inhibits the amount of proliferation ofthe CAFI stimulated CD8⁺ cells from 10% to 1% and the amount of proliferation of CAFI and EL-2 stimulated CD8⁺ cells from 72% to 20%. This indicates that the CD4⁺CD25⁺ hybridomas are capable of regulating T -cell responses to foreign transplantation antigens.

EXAMPLE 10 Inhibition of T-Cell Proliferation by RD6, Requires Cell: Cell Contact

CD4⁺CD25⁺ regulatory cells that are isolated from normal mice require cell: cell contact in order to inhibit proliferation of polyclonally activated T-cells (Takahashi et al. 1998; Thornton and Shevach 2000; Maeda et al. 2000). The CD4⁺CD25⁺ hybridoma, RD6, was examined to determine whether they demonstrate similar regulatory function. CFSE-labeled spleen cells from naϊve BALB/c mice were cultured for three days in transwell plates, which contain an upper and a lower chamber that are separated from each other by a membrane filter. CFSE- labeled spleen cells (10⁶ cells) were cultured in the upper chamber and either control BW5147α^"β^~ cells (10⁶ cells) or the CD4⁺CD25⁺ hybridoma subclone RD6 (10⁶ cells) were added to the lower chamber. In the same experiment, CFSE-labeled spleen cells were cultured in both the upper and lower chambers (10⁶ cells/chamber) and either control BW5147α^~β^~ cells (10⁵ cells) or the CD4⁺CD25⁺ hybridoma subclone RD6 (10⁵ cells) were added to the lower chamber. Soluble anti-CD3 (0.1 μg/ml) was added to the transwell plates. After three days the cells in the upper chambers were harvested and the amount of T-cell proliferation was determined using flow cytometry and CFSE expression of gated T-cells. In the control cultures, about 60% of the activated splenic T-cells underwent proliferation. When CD4⁺CD25⁺ hybridoma cells were cultured separate from the CFSE-labeled spleen cells, then the hybridomas did not inhibit proliferation (FIG. 10A, and FIG. 10B, lower left panel). When CD4⁺CD25⁺ hybridoma cells were cultured in contact with the CFSE-labeled spleen cells, then the hybridomas inhibited the proliferation of the activated splenic T-cells (FIG. 10B, lower right panel). CD4⁺CD25⁺ hybridomas require contact with the target T-cell in order to mediate their regulatory effect. Thus, the CD4⁺CD25⁺ hybridomas resemble naturally occurring CD4⁺CD25⁺ cells in this regard as well. CD4⁺CD25⁺ regulatory cells most likely exert their inhibitory activity via the interactions of receptor/ligand(s) between the two cells. The inventors also contemplate studies to discover the nature of these receptor/ligand pair using the CD4⁺CD25⁺ hybridomas as reagents to facilitate the exploration of important functional receptor/ligand interactions.

EXAMPLE 11 RD6 Inhibits Proliferation of T-Cells That are Activated by Soluble but not Plate- Bound Anti-CD3

CD4⁺CD25⁺ regulatory cells that are isolated from normal mice inhibit proliferation of T- cells that have been activated by soluble but not plate-bound anti-CD3 (Thornton and Shevach 2000). The CD4⁺CD25⁺ hybridoma, RD6, was examined to determine whether they demonstrate similar regulatory function. CFSE-labeled spleen cells from naϊve BALB/c mice were cultured for three days with either no anti-CD3 or soluble 0.1 μg/ml anti-CD3, or in wells with plate- bound anti-CD3. Anti-CD3 was bound to the wells by pre-incubating the wells overnights with solution containing anti-CD3 (0.1 μg/ml) using standard operating procedure for coating plastic tissue culture wells with protein. The CFSE-labeled cells (10⁶ cells/well) were cultured with no other cells, or with BW5147α^~β^~ control cells (10⁵ cells/well), or with CD4⁺CD25⁺ hybridoma subclone, RD6 (10⁵ cells/well). After three days the extent of T-cell proliferation was determined by measuring the level of CFSE expression on gated T-cells using flow cytometry. In unstimulated cultures, only about 2% ofthe T-cells undergo proliferation. When cultures are stimulated with soluble anti-CD3, roughly 60% ofthe T-cells have undergone proliferation three days later (FIG. 11, upper left panel); the addition of BW5147α^~β^~ cells at the beginning of the culture period has no effect on the level of T-cell proliferation (FIG. 11, middle left panel). However, the addition of CD4⁺CD25⁺ hybridoma subclone, RD6 at the beginning of the three- day culture decreases the amount of T-cell proliferation by about 50% (FIG.11, lower left panel). When cultures are stimulated with plate-bound anti-CD3, more than 80% of the T-cell have undergone proliferation three days later (FIG.11, upper right panel). The proliferation of the T- cells is not decreased by either the addition of BW5147α^~β^~ cells (FIG. 11, middle right panel) or the addition of CD4⁺CD25⁺ hybridoma subclone, RD6 (FIG.11, lower right panel), at the beginning of the three day culture. Thus, the CD4⁺CD25⁺ hybridomas resemble naturally occurring CD4⁺CD25⁺ cells in regard to their ability to inhibit proliferation of soluble but not plate-bound anti-CD3 stimulated T-cells. EXAMPLE 12 Injection of Normal Mice with CD4+CD25+ RD6 Alters the Immune Response to

Transplanted Foreign Cells

Naϊve BALB/c mice were primed by injecting i.p. with T-depleted allogeneic spleen cells from either fully allogeneic (A/J strain) or semi-allogeneic (CAFI strain) mice (25x10⁶ cells/mouse). Control mice received no other cells, whereas the test mice were co-injected with 5x10⁶ CD4⁺CD25⁺ hybridoma cells. Seven to ten day later, the spleens were removed from individual mice. Spleen cells from individual mice were cultured for 72 hours. Supernatants were collected and assayed for cytokine levels (EL-2, EL-4, EFNγ, EL-10, EL-13, EL-12) using cytokine specific ELISA assays. FIG. 12 shows that the pattern of cytokine produced by the control primed mice was significantly different from the pattern of cytokines produced by the primed mice that were treated with CD4⁺CD25⁺ hybridoma cells. Control primed mice made higher levels of EL-12 cytokine, whereas primed mice that were treated with CD4⁺CD25⁺ hybridoma made higher levels of anti-inflammatory cytokines, EL-4, EL-10, and EL- 13. There was no difference in the level of EFNγ that was constitutively produced. The results demonstrate that treatment with CD4⁺CD25⁺ hybridoma at the time of injection of donor cells alters the in vitro recall immune response to the donor cells. This alteration is not due to the presence of CD4⁺CD25⁺ regulatory cells, because none are detected by flow cytometry. The alteration in anti-donor immune response afforded by treatment with CD4⁺CD25⁺ hybridomas may improve graft survival. One week after injection of donor cells, the percentage of donor B cells was higher in animals that were treated with CD4⁺CD25⁺ hybridomas at the time of injection compared to animals that did not received CD4⁺CD25⁺ hybridomas at the time of injection (29% ± 4 Vs. 16% ± 3, mice primed with CD4⁺CD25⁺ hybridomas vs mice primed without CD4⁺CD25⁺ hybridomas).

EXAMPLE 13 Generation of Non-Immortal CD4⁺CD25⁺ Cells

As demonstrated in the previous Examples, the present invention shows that CD4⁺CD25⁺ cells inhibit several kinds of immune responses, examples being the inhibition of in vitro immune responses to alloantigen antigen (see Example 9) or the inhibition of in vivo immune response of mice injected with donor cells plus the present invention (see Example 12). The invention also provides methods for generating a source of CD4⁺CD25⁺ regulatory cells which are not immortalized. This provides a source of non-transformed or non-cancerous CD4⁺CD25⁺ regulatory cells which are useful for the purposes of human therapy.

The method comprises providing immortalized cells expressing CD4 and CD25 and contacting the immortalized cells with a population of T-cells, such as CD4⁺ cells and/or thymocytes and/or CD4^"CD8^" cells.

In some embodiments, these T-cells may be isolated from a human patient who needs such a therapy. Isolation of regulatory T-cells from individuals will generally comprise performing leukopharesis or plasmapharesis to yield sufficient numbers of cells for the ex vivo cell culture method described above. One of ordinary skill in the art is well versed with leukopharesis or plasmapharesis and these procedures typically yields about 50x10⁸-l 00x10⁸ cells to start the culture process. The immortalized CD4⁺CD25⁺ cells can be obtained from one ofthe hybridoma cells ofthe invention or by the methods ofthe invention.

Contacting the immortalized CD4⁺CD25⁺ regulatory cells with the T-cell population causes their differentiation into the CD4⁺CD25⁺ regulatory cells. As a final step the immortalized CD4⁺CD25⁺ cells are separated from the CD4⁺CD25⁺ regulatory cells generated by the method. Thus, a population of non-hybrid and non-immortal CD4⁺CD25⁺ regulatory cells are obtained.

In some embodiments, the step of contacting the immortalized CD4⁺CD25⁺ regulatory cells with the T-cell population may be carried out in the additional presence of a donor cell, a donor antigen, any immunogenic peptide/antigen, or any allergen, to which tolerance is desired. The presence of such an antigenic molecule induces tolerance to that molecule in the resulting CD4⁺CD25⁺ regulatory cell population generated. Therefore, in specific embodiments the donor cell, a donor antigen, or the immunogenic peptide/antigen may be from a potential tissue/organ donor and the resulting CD4⁺CD25⁺ regulatory cell population generated will provide or confer immune tolerance to that donor antigen/immunogen/cell when administered therapeutically to a transplant recipient. In other similar specific embodiments the immunogenic peptide/antigen can be any self antigen to which autoimmunity is generated in an individual. The resulting CD4⁺CD25⁺ regulatory cell population generated, when administered therapeutically, will provide or confer immune tolerance or resistance to the individual suffering from the autoimmune disease. For example, one may isolate regulatory T-cells from an individual suffering from type I diabetes and contact these T-cells with an immortalized CD4⁺CD25⁺ cell population in the presence of the antigenic peptide or protein or cell, that causes the immune- mediated diabetes and thereby generate CD4⁺CD25⁺ cells that confer tolerance to the antigenic peptide, protein or cell, that causes diabetes. It is also contemplated that in some embodiments the step of contacting the immortalized CD4⁺CD25⁺ regulatory cells with the regulatory T-cell population will be performed in the additional presence of other cells or cellular components or cellular extracts from cells such as but not limited to an unfractionated CD4⁺ cell population, and/or CD8⁺ cells and/or CD25⁺ cells, and/or antigen presenting cells including, B-cells, dendritic cells, macrophages, or monocytes, or cells from the peripheral blood or lymphoid tissue or bone marrow or peripherial lymphoid tissue, including spleen cells. In addition, the presence of cytokines, particularly TGFβ, and/or EL-10, and/or EL-2, and/or inhibitors of other cytokines such as antibodies against EL-12 may also be required.

A. Generation of Non-Immortal CD4^rCO25^v Cells by In Vivo Methods

Generation of de novo non-immortal CD4⁺CD25⁺ regulatory cells can be performed in vivo by injecting CD4⁺CD25⁺ hybridomas together with responder CD4⁺ cells and/or thymocytes and/or CD4^"CD8^" cells into mice along with the antigen to which tolerance is desired. CD4⁺CD25⁺ cells can be purified from the recipient mice examined for their ability to inhibit T- cell responses against the target and antigen to which tolerance is desired. The purification can be performed using sterile cell sorting and flow cytometry methodology, magnetic-bead purifcation, or column purification. The inventors contemplate using the expression of a combination of unique phenotypic markers on the CD4^TCD25⁺ regulatory cell in the purification methods. Examples of such markers include but are not limited to co-expression of CD4⁺, CD25⁺, CD122⁺, and the lack of expression of CD69, i.e, CD69^".

For example, naϊve BALB/c mice can be primed by injecting i.p. with T-depleted allogeneic spleen cells from fully allogeneic or semi-allogeneic mice (50xl0⁶ cells/mouse) along with 5x10⁶ CD4⁺CD25⁺ hybridoma cells. Control groups comprise: 1) mice primed with spleen cells alone or 2) mice primed with spleen cells along with 5x10⁶ hybridoma fusion partner, BW5147ofβ^"; and 3) unprimed mice injected with 5xl0⁶ CD4⁺CD25⁺ hybridoma cells. Mice that are sensitized by transplanting allogeneic or semi-allogeneic skin grafts can also be examined by injecting with 5xl0⁶ CD4⁺CD25⁺ hybridoma cells right after transplantation. For these experiments the controls will typically include: 1) mice transplanted with allogeneic or semi-allogeneic skin alone; 2) mice transplanted with allogeneic or semi-allogneic skin along with 5xl0⁶ hybridoma fusion partner, BW5147ofβ^~; and 3) mice with transplanted with syngeneic skin and then injected with 5xl0⁶ CD4⁺CD25⁺ hybridoma cells. Both fully allogeneic and semi-allogeneic stimulator cells are used to enable examination of the effect of direct or indirect antigen presentation on the generation of CD4⁺CD25⁺ regulatory cells. Some examples of strains ofthe donor mice include: A/J, CAFI, C57B1/6 and CB6F1. Ten to fourteen days after priming, the spleen and draining lymph nodes of primed mice will be harvested and examined for the presence of regulatory cells. Fourteen to twenty-one days after skin grafting, the spleen, draining lymph nodes, and the graft infiltrating cells from the skin grafts on the sensitized mice will be isolated and examined for the presence of regulatory cells (see functional assays in the Example below). For each test and control condition 5-6 mice/group are typically used. Alternatively, mice will be immunized with antigen to which tolerance is desired along with 5xl0⁶ CD4⁺CD25⁺ hybridoma cells. Control groups comprise: 1) mice immunized with antigen alone or 2) mice immunized with antigen along with 5x10⁶ hybridoma fusion partner, BW5147of β^~; and 3) unimmunized mice injected with 5xl0⁶ CD4⁺CD25⁺ hybridoma cells.

Alternative Approaches to In Vivo Generation of CD4 CD25⁺ Regulatory Cells (i) Graft- Vs-Host Disease Model

An alternative approach to generating CD4⁺CD25⁺ regulatory cells in vivo is to utilize a graft-vs-host disease model. For this, sublethally irradiated CAFI mice (500-700 rads, whole body irradiation) can be injected with BALB/c spleen cells (50x106 per mouse) along with 5x10⁶ CD4⁺CD25⁺ hybridomas (spleemhybridoma 10: 1). Control groups include: 1) mice given BALB/c cells alone; 2) mice given BALB/c cells and 5xl0⁶ hybridoma fusion partner, BW5147α^~β^~; and 3) mice given only 5xl0⁶ CD4⁺CD25⁺ hybridoma cells (no BALB/c cells). Each group will consist of 5-6 mice. The CAFI hosts should not reject the BALB/c donor cells, but the donor cells will become activated against the host. An advantage of this system is that the alloantigen is ubiquitously expressed on the host endothelium. As the endothelium is capable of initiating alloresponses, the CD4⁺CD25⁺ regulatory cells generated will be those that specifically function to block proliferation to allogeneic endothelium. After 1 or 2 weeks, mice can be sacrificed and the spleen and lymph node cells can be isolated for analysis. Spleen and lymph node cells can be analyzed separately, in case CD4⁺CD25⁺ regulatory cells preferentially home to the spleen. Cell phenotype can then be determined using multiparameter FACS and staining for CD4, CD8, CD25, CD69, CD19, CD16 expressing cells of both donor (BALB/c) and host (CAFI) strain. The absolute numbers of CD4⁺CD25⁺CD69^" cells can be compared from the spleen and lymph node populations in the test and control groups of mice. CD4⁺CD25⁺CD69^" cell can then be purified by high speed sorting and examined in functional assays. (ii) Sponge Graft Model

In another alternative approach, the sponge graft model may be used to generate and concentrate regulatory cells. The sponge graft model is well described (Chiang, et al, 2001; Gu, et al, 2001). Naϊve BALB/c mice can be transplanted with 1 by 1 cm pieces of sponge under the skin on the flanks. The sponges can be injected with the following combinations: Group 1, T- depleted allo- or semi-allogeneic spleen only; Group II, T-depleted allo or semi-allogeneic spleen and CD4⁺CD25⁺ hybridoma; Group EH, T-depleted allo or semi-allogeneic spleen and BW5147α-β~; or Group IV, syngeneic spleen and CD4⁺CD25⁺ hybridoma. After 7 days, sponges can be removed and cells can be harvested from the sponges. The composition of the harvested cells can be characterized using multiparamater flow cytometry. Differences in the number and phenotype of the sponge infiltrating cells between the different groups of mice can be compared. Cell phenotype can be determined using multiparameter FACS and staining for CD4, CDS, CD25, CD69, CD 19, CD 16 expressing cells of both host (BALB/c) and donor strain origin. As it may take longer than 7 days to generate new CD4⁺CD25⁺ regulatory cells, sponges will also be examined at 14, 21 and 28 days after injection. CD4⁺CD25⁺CD69^" cells that are purified from the sponge infiltrating cells can then be tested for their ability to inhibit response to the immunizing antigen using one or more of the in vitro and in vivo assay systems described below. In other experiments where one desires to generate CD4⁴CD25⁺ regulatory cells to a specific self-antigen, peptide, peptide fragment, or allergen, the sponge grafts can be injected with the following test and control combinations: Group 1, antigen only; Group π, antigen plus CD4⁺CD25⁺ hybridoma; Group HI, antigen plus BW5147α^"β^"; or Group IV, irrelevant antigen (to which tolerance is NOT desired) and CD4⁺CD25⁺ hybridoma.

B. Generation of Non-Immortal CD4*CD25 Regulatory Cells by In Vitro Methods

CD4⁺CD25⁺ hybridomas can also be used to generate CD4⁺CD25⁺ regulatory cells de novo by m vitro methods by adding CD4^f CD25⁺ hybridomas to standard cultures of respόnder CD4⁺ cells and/or CD4^"CD8^" cells and/or thymocytes, APCs and antigen. CD4⁺CD25⁺CD69^" cells can then be isolated from the in vitro cultures and examined for their ability to inhibit T-cell responses against the target and the antigen. Various antigens, such as A/J, CAFI, B6, and CB6F1 stimulator cells, or host antigen presenting cells that express the antigen to which tolerance is desired.

To generate CD4⁺CD25⁺ regulatory cells against transplantation antigens, bulk cultures can be set up with BALB/c CD4⁺ spleen cells as responder cells (lxl0⁶/ml), T-depleted allogeneic spleen cells (from A/J, CAFI, B6, or CB6F1 donors) as stimulator cells (5xlO°7ml) along with CD4⁺CD25⁺ hybridoma cells (5x10⁵ cell/ml). Control cultures include: 1) responder and stimulator cells alone; 2) responder cells, stimulator cells and BW5147 (5xl0⁵/ml); 3) syngeneic responder and stimulator cells (each from BALB/c) and CD4⁺CD25⁺ hybridoma cells (5x10⁵ cells/ml). Responder cells can be harvested and analyzed for the presence of regulatory cells using multiparameter FACS and the in vitro and in vivo functional assays described in the Example below. Cultures can be set up for 7, 14, 28 or more days as required.

To generate CD4⁺CD25⁺ regulatory cells against a self-antigen, peptide, peptide fragment or allergen, bulk, cultures can be set up with CD4⁺ host spleen, lymph node or thymus cells as responder cells (1x107ml), T-depleted host antigen presenting cells (5xl0⁶/ml), such as B cells macrophages or dendritic cells, antigen to which tolerance is desired, and CD4⁺CD25⁺ hybridoma cells (5xl0⁵ cell/ml). Control cultures include: 1) host CD4⁺ cells (lxl0°7ml) and host antigen presenting cells (5xl0⁶/ml) only; 2) host CD4⁺ cells (1x107ml), host antigen presenting cells (5x107ml) and BW5147α^~β^~ (5xl0⁵/ml); 3) host CD4⁺ cells (1x107ml), host antigen presenting cells (5x107ml), irrelevant antigen, CD4⁺CD25⁺ hybridoma cells (5x10⁵ cells/ml). .

EXAMPLE 14 Isolation and Functional Assays to Detect Regulatory CD4⁺CD25⁺ Cells

Previous studies by the inventors on mice with acquired MHC tolerance show that regulatory cells may be identified by their CD4 CD25⁺CD69^" phenotype as tolerant mice showed expansion of CD4⁺CD25⁺CD69^" cells in MLRs against the tolerizing antigen, whereas naϊve or non-tolerant mice showed expansion of the CD4⁺CD25⁺CD69⁺ cells (Gao, et al, 1999). Isolation ofthe CD4⁺CD25⁺CD69^" subset from the tissues ofthe primed and/or sensitized groups of mice or from the in vitro cultures may be achieved by high speed FACS sorting. This purification step concentrates the non-immortal CD4⁺CD25⁺ regulatory subset and also insures that the CD4⁺CD25⁺ hybridomas used as a starting material are eliminated. The CD4⁺CD25⁺ hybridomas can be easily distinguished from the non-hybridoma CD4⁺CD25⁺CD69^" subset using forward and side scatter and expression of H-2kk of the hybridoma cells. The purified CD4⁺CD25⁺CD69^" cells (i.e., the non-immortal regulatory CD4⁺CD25⁺ cells generated by the methods of the invention) can be further assayed to detect their antigen specificity and other T- cell inhibitory actions by a variety of functional in vitro and in vivo assays. A. In Vitro Inhibition of Proliferation of Standard MLR

The extent to which CD4⁺CD25⁺CD69^" cells inhibit T-cell proliferation to a foreign cell or an antigen to which tolerance is desired may be determined by this assay. CFSE-labeled T- cells from naϊve BALB/c mice are cultured with T-depleted allogeneic or semi-allogeneic spleen cells (A/J, CAFI, B6, or CB6F1) along with purified CD4⁺CD25⁺CD69^" cells from the test or control groups of mice for seven days. Alternatively, CFSE-labeled T-cells from naϊve BALB/c mice are cultured with the antigen to which tolerance is desired along with purified CD4⁺CD25⁺CD69^" cells from the test or control groups of mice for seven days. At the end ofthe culture period the extent of T-cell proliferation (total T-cells, CD4+ cells or CD8+ cells) in response to foreign cells or an antigen can be determined using FACS analysis. Various ratios of effector T cells: CD4⁺CD25⁺CD69^" cells such as 100:0.1; 100:1; 50:1; 20:1; 10:1 and 5:1 etc., can be examined to observe the inhibition of T cell responses. In some of the above assays the cultures can be supplemented with exogenous EL-2, to determine whether EL-2 can overcome any inhibition by CD4⁺CD25⁺CD69^" cells. The CFSE proliferation assay has been used by the inventors to demonstrate that CD4⁺CD25⁺ hybridomas inhibit proliferation of alloreactive CD8⁺ cells (see Example 9). In such experiments, it has been shown that the CD4⁺CD25⁺ hybridomas inhibit proliferation of CD8⁺ cells in an MLR response despite addition of exogenous EL-2.

B. In Vitro Inhibition of IL-2 Production in α Standard MLR

The ability of CD4⁺CD25⁺CD69^" cells to inhibit EL-2 production in a standard MLR can be performed using an EL-2 MLR ELISPOT assays. T-cells from naϊve BALB/c mice can be cultured overnight in round bottom plates with T-depleted allogeneic or semi-allogeneic spleen cells (A/J, CAFI, B6, or CB6F1) along with purified CD4⁺CD25⁺CD69^" cells from the test or control groups of mice. Effector T-cells and CD4⁺CD25⁺CD69^" cells can be added at various ratios (100:1; 50:1; 20:1; 10:1; 5:1). Alternatively, the CD4⁺CD25⁺CD69^" putative regulatory cell population can be added with T-depleted host antigen presenting cells and an antigen to which tolerance is desired. The cultures can then be transferred to EL-2 ELISPOTs for an additional 24 hours of culture and spots can be determined using the ImmunoSpot analyzer. Cytokine analysis may also be alternatively be performed by intracytoplasmic staining.

C. In Vitro Inhibition of Endothelial Function

The CD4⁺CD25⁺CD69^" cells may also be tested for their ability to inhibit endothelium- induced proliferation of T-cells and their ability to sustain endothelial barrier function. Freshly isolated allogeneic endothelial cells can be grown to confluence and cultured with CFSE-labeled purified T-cells from naϊve BALB/c mice for seven days as previously described (Krupnick, et al, 2001). CD4⁺CD25⁺CD69^" cells from the test and control groups of mice can be added to the cultures at various ratios (T-cell: CD4⁺CD25⁺CD69^" at 100:0.1; 100:1; 50:1; 20: 1; 10:1; and 5:1). At the end of seven days, cultures can be harvested and CD4⁺ and CD8⁺ T-cell proliferation in response to alloantigen can be determined using FACS analysis, by gating on CFSE positive CD4⁺ and CD8⁺ cells. In some assays cultures may be supplemented with exogenous EL-2, to determine whether IL-2 can overcome inhibition by CD4⁺CD25⁺CD69^" test cells.

In vitro inhibition of endothelial barrier function can be carried out as follows. Endothelial cells can be grown on a microelectrode to which a small alternating current is applied which allows continuos monitoring of endothelial barrier function by recording transendothelial resistance of the confluent monolayer (Moy et al, 1996). Quantitative and dynamic changes in barrier function are monitored over time by recording the change in transendothelial resistance. These experiments will allow the determination of whether CD4⁺CD25⁺CD69^" cells prevent the loss of endothelial integrity. The interaction between T- cells and allogeneic endothelium is expected to result in loss of endothelial barrier function over 3 to 7 days of co-culture.

In this system, referred to as electric cell-substrate impedance sensing (ECIS), cells are cultured on a small gold electrode (5 x 10^"4 cm²), using culture medium as the electrolyte, and barrier function is measured dynamically by determining the electrical impedance of a cell covered electrode. The total impedance of the monolayer is composed of the impedance between the ventral surface of the cell and the electrode, the impedance between the cells, and . the impedance of the cell membranes which is dominated by the membrane capacitance. Membrane impedance is very large, and, thus, most of the current flows under and between the cells. Furthermore, membrane impedance is not expected to change upon the initial addition of lymphocytes. Thus, measured changes in impedance represent alterations primarily in cell-cell adhesion and/or cell-matrix adhesion. A 1 volt, 4000 Hz AC signal is supplied through a resistor to approximate a constant-current source. Voltage and phase data are measured with a SRS830 lock-in amplifier (Stanford Research Systems) stored and processed with a personal computer. The same computer also controlled the output of the amplifier and relay switches to different electrodes. Critical features ofthe setup are the current frequency of 4000 Hz and the small area of the active electrode (a surface area of 10^"4 cm²). For experiments, electrodes are coated with adsorbed fibronectin by exposure to a 100 μg/ml solution for 30 minutes. Cultured endothelial cells are inoculated on electrodes at a confluent density of 10⁵ cells/cm². The in-phase voltage (proportional to the resistance) and the out-of-phase voltage (proportional to the capacitive reactance) are measured. Barrier integrity is expressed as a function of resistance, normalized to the initial value and expressed as a fractional change as there were greater changes in resistance than impedance or reactance. Thus, a 10 percent decline in resistance, for example, would represent a fractional resistance of 0.9. Electrical resistance increases after cells attach and cover the electrodes, and the resistance achieves a steady state level by 24 hrs.

D. Functional In Vivo Assays to Detect Regulatory CD4 CD25⁺ Cells

The purified CD4⁺CD25⁺CD69^" cells from the test and control mice may also be examined for their ability to inhibit allospecific rejection in vivo. This may be accomplished by modifying the SCED adoptive transfer model, a model previously used to demonstrate that CD4⁺ regulatory cells from mice with acquired tolerance suppress rejection of skin grafts by immunocompetent CD8⁺ cells (Field et al, 2001). Briefly, SCED mice can be transplanted with A/J and B6 skin on opposite flanks at -21 day. On day 0, groups of mice (N=5-8 per group) can be co-injected IV with 5xl0⁶ unfractionated spleen cells from syngeneic BALB/c mice and either no other cells or 5x10⁵ or purified 2.5xl0⁵ CD4⁺CD25⁺CD69^" cells that were generated under the various test and control conditions. Grafts are then inspected daily for signs of rejection of the A/J and the B6 grafts. The day of complete rejection is noted when 100% of the graft bed is involved. Data may be analyzed using Kaplan-Meier survival statistics. Some ofthe SCED mice may also be sacrificed and examined for anti-donor T-cells responses, using ELISPOT assays to detect Thl, Th2 and TCI cell subsets.

Λ A A A Λ Λ A A A Λ Λ A A A A A A A A A A A A A A A A A A A A

All of the COMPOSITIONS and/or METHODS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the COMPOSITIONS and/or METHODS and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept ofthe invention as defined by the appended claims. REFERENCES

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Claims

1. An immortalized cell expressing the antigens CD4 and CD25.

2. The immortalized cell of claim 1, wherein the cell is a 3B1 cell, a 3C2 cell, a 2B6 cell, a 1B4 cell, a 2D5 cell, a 2A6 cell, a IDlcell, a 1A6 cell, a 2B1 cell, a 2D3 cell, a 2D4 cell, a 2C5 cell, a 3A2 cell, a 1 A2 cell, or a 3C1 cell.

3. The immortalized cell of claim 1, wherein the immortalized cell is a hybridoma cell.

4. A method for generating an immortalized cell expressing the antigens CD4 and CD25 and having the ability to regulate immune tolerance comprising: a) obtaining CD4⁺CD25⁺ regulatory cells from a subject; b) providing immortalized fusion partner cells; c) fusing the CD4^~rCD25⁺ cells with the immortalized fusion partner cells; and d) screening for immortalized hybrid cell clones expressing CD4 and CD25 and having the ability to inhibit T-cell proliferation and/or function.

5. The method of claim 4, wherein the immortalized fusion partner cell is a tumor cell line or a tumor cell.

6. The method of claim 5, wherein the tumor cell is a BW5147α^"β^" cell, a BW5147α^"β^" cell line, a BW5147 cell, or a MOLT4 cell.

7. The method of claim 4, wherein said subject is a mouse.

8. The method of claim 4, wherein said subject is a human.

9. The method of claim 4, wherein the CD4⁺CD25⁺ regulatory cells are treated with at least one cytokine prior to the fusion.

10. The method of claim 4, wherein the CD4⁺CD25⁺ regulatory cells are treated with at least one antigen prior to the fusion.

11. The method of claim 10, wherein said antigen is a foreign transplantation antigen or a self antigen.

12. The method of claim 4, wherein the CD4⁺CD25⁺ regulatory cells are isolated from a subject with tolerance to a foreign transplantation antigen.

13. The method of claim 4, wherein the CD4⁺CD25⁺ regulatory cells are isolated from a subject with tolerance a self-antigen.

14. The method of claim 4, wherein the CD4⁺CD25⁺ regulatory cells are isolated from a subject with resistance to an autoimmune disease.

15. The method of claim 4, wherein the CD4⁺CD25⁺ regulatory cells are isolated from a subject with resistance to an allergen.

16. A method for generating CD4⁺CD25⁺ regulatory cells comprising: a) providing immortalized cells expressing CD4 and CD25; b) contacting said immortalized cells with a population of CD^ cells and/or thymocytes and/or CD4^"CD8^" cells wherein the contacting causes the differentiation of the CD4⁺ cells and/or thymocytes and/or CD4^"CD8^"cells into CD4⁺CD25⁴ regulatory cells; and c) separating the immortalized cells from the CD4⁺CD25⁺ regulatory cells, thereby obtaining a population of CD4⁺CD25⁺ regulatory cells.

17. The method of claim 16, wherein the immortalized cells expressing CD4 and CD25 are made by the method of claim 4.

18. The method of claim 16, wherein the immortalized cells are attached to a solid support prior to being contacted with the CD4⁺ cells and/or thymocytes and/or CD4^"CD8^"cells.

19. The method of claim 16, wherein the immortalized cells are attached to a solid support after being contacted with the CD4⁺ cells and/or thymocytes and/or CD4^"CD8^" cells.

20. The method' of claim 18 or claim 19, wherein the solid support is a bead, a matrix, or a column.

21. The method of claim 16, wherein an affinity tag is further attached to the immortalized cells prior to being contacted with the CD4⁺ cells and/or thymocytes and/or CD4^'CD8^" cells.

22. The method of claim 16, wherein an affinity tag is further attached to the immortalized cells after being contacted with the CD4⁺ cells and/or thymocytes and/or CD4^'CD8^" cells.

23. The method of claim 21 or claim 22, wherein the affinity tag is biotin, streptavidin, an antigen, or an antibody.

24. A CD4⁺CD25⁺ regulatory cell generated by a method comprising: a) providing immortalized cells expressing CD4 and CD25; b) contacting said immortalized cells with a population of CD4⁺ cells and/or thymocytes and/or CD4^"CD8^" cells wherein the contacting causes the differentiation of the CD4⁺ cells and/or thymocytes and/or CD4^"CD8^" cells into CD4⁺CD25⁺ regulatory cells; and c) separating the immortalized cells from the CD4⁺CD25^"r regulatory cells, thereby obtaining a population of CD4⁺CD25⁺ regulatory cells.

25. The CD4⁺CD25⁺ regulatory cell of claim 24, wherein the immortalized cells of step a) are transfected with one or more nucleic acid expression vector to express one or more protein and/or nucleic acid prior to step b).

26. The CD4⁺CD25⁺ regulatory cell of claim 25 wherein the nucleic acid expression vector encodes a T-cell receptor, a cytokine, or a chemokine.

27. The CD4⁺CD25⁺ regulatory cell of claim 25 wherein the nucleic acid expression vector is a mammalian expression vector or a viral expression vector.

28. A method for controlling or preventing an undesirable immune reaction in a subject comprising administering to said subject a pharmaceutical formulation of CD4⁺CD25⁺ regulatory cells in an amount required to provide therapeutic benefit from the undesirable immune reaction.

29. The method of claim 28, wherein the amount of CD4⁺CD25⁺ regulatory cells required to

7 1 provide therapeutic benefit is from about 0.1x10 to about 2x10 cells per kilogram weight ofthe subject.

30. The method of claim 28, wherein the undesirable immune reaction is a tissue transplant rejection, an organ transplant rejection, an autoimmune disease, an allergy, an asthamatic reaction, or an immune-mediated inflammatory disorder.

31. The method of claim 30, wherein the autoimmune disease is celiac disease, autoimmune ulcerative colitis, type I diabetes, autoimmune gastritis, multiple sclerosis, or rheumatoid arthritis.

32. The method of claim 28, wherein the subject is a human being.

33. The method of claim 28, wherein the controlling or preventing an undesirable immune reaction comprises inducing immune tolerance.

34. The method of claim 33, wherein the immune tolerance is immune tolerance to a transplanted tissue, immune tolerance to a transplanted organ, immune tolerance to an autoimmune disease, immune tolerance to an allergic reaction, or immune tolerance to an immune-mediated inflammatory disorder.

35. The method of claim 33, wherein immune tolerance is regulated by inhibiting the proliferation of activated T-cells.

36. The method of claim 35, wherein the inhibited T-cells are CD4⁺-activated T-cells, or CD8⁺-activated T-cells.

37. The method of claim 33, wherein function of activated T-cells is inhibited.

38. The method of claim 33, wherein differentiation of activated T-cells is inhibited.