WO2006052660A9 - Il-7 receptor blockade to suppress immunity - Google Patents

Abstract

The present application describes methods of suppressing an undesired immune response through the administration of an interleukin-7 antagonist to a subject in an amount effective to suppress the undesired immune response in the subject. A further aspect of the present invention describes a method of treating graft-versus-host disease in a mammalian subject through the administration of an interleukin-7 antagonist in an amount effective to treat the graft-versus-host disease in the subject. The present invention further describes the use of an interleukin-7 antagonist active agent for the preparation of a medicament for carrying out a method of treatment.

Description

IL-7 RECEPTOR BLOCKADE TO SUPPRESS IMMUNITY

Statement of Priority

This application claims the benefit of U.S. Provisional Application Serial No. 60/625,318, filed November 4, 2004 and the entire contents of which is incorporated by reference herein.

Government Support

This invention was made with U.S. Government support under grant numbers HL 54729 and AI 50765 from the National Institutes of Health. The U.S. Government has certain rights to this invention.

Field of the Invention

The present invention concerns methods and compounds for the suppression of undesirable immune responses. The methods and compounds are useful for the treatment of autoimmune diseases and medical conditions that result from immune responses that occur in hematopoietic stem cell transplantation and solid organ transplantation.

Background of the Invention

Graft-versus host disease (GVHD) continues to be a potentially serious complication that limits the efficacy of allogeneic bone marrow transplant (BMT). GVHD is initiated when donor T lymphocytes are activated by host alloantigens. Upon activation, inflammatory cytokines produced by donor T cells induce proliferation and differentiation of various effector cells including anti-host helper and cytotoxic T cells (CTLs), macrophages, and natural killer cells, which cause damage to target organs such as liver, gut, lung, and skin (Ferrara, Curr Opin Immunol. 5: 794-799 (1993); Antin et al., Blood. 80: 2964-2968 (1992); Sprent et al., J Immunol. 144: 2946-2954 (1990); Korngold et al., Bone Marrow Transplantation. Ch. 18. pp. 220-230, Blackwell Scientific Publications (1994); Nikolic et al., J. Clin. Invest. 105: 1289-1298 (2000); Sprent et al., J. Exp. Med. 167: 556-569 (1988)). A number of therapeutic measures such as T cell-depletion (TCD) and immunosuppression have been used to prevent GVHD post-BMT (Ferrara et al., N Engl J Med. 324: 667-674 (1991)). The post-BMT period is marked by profound immunodeficiency, making newly transplanted patients susceptible to various bacterial, viral, or fungal infections (Atkinson, Bone Marrow Trans. 5: 209 (1990); Lum. Blood. 69: 369 (1987)). GVHD, immunosuppressive procedures or therapies to prevent or treat GVHD, and the time required for donor stem cells to develop into T lymphocytes contribute to the post-BMT immunodeficiency. T cell depletion, although it can prevent GVHD, increases the degree of post-transplant immune deficiency because of the additional time required for development of new T lymphocytes from donor-derived progenitors. In addition, the non-specific immunosuppressive effects of drugs to prevent or treat GVHD also may contribute to poor immune function. Immune reconstitution after BMT is further hindered by impaired function of the thymic microenvironment caused by age, pre-BMT conditioning, and GVHD itself (Miller et al, Blood. 77: 1845 (1991); Weinberg et al., Biol Blood Marrow Transplant. 1: 18- 23 (1995); Chung et al., Blood. 99: 4592-4600 (2001)). Methods to enhance the development of T lymphocytes and immune reconstitution are critical to solving the problem of post-BMT immune deficiency.

Summary of the Invention

The present invention relates generally to the suppression of immunity through IL-7 blockades and more specifically suppression of immunity through IL-7 monoclonal antibodies.

A first aspect of the present invention is a method of suppressing an undesired immune response in a mammalian subject in need thereof, comprising: administering an interleukin-7 antagonist to said subject in an amount effective to suppress an undesired immune response in said subject.

A further aspect of the present invention is a method of treating graft-versus-host disease in a mammalian subject in need thereof, comprising: administering an interleukin-7 antagonist to said subject in an amount effective to treat said graft-versus-host disease in said subject.

A further aspect of the present invention is the use of an interleukin-7 antagonist active agent as described herein for the preparation of a medicament for carrying out a method of treatment as described herein. Brief Description of the Drawings

Figure 1. Treatment with IL-7 increased GVHD-related mortality in B6.IL-7^7' recipient animals following allogeneic LN BMT. B6 or B6.IL-7^"7" recipients which had received 1300 cGy TBI were transplanted with either IxIO⁶ T -cell depleted BM and 4xlO⁶ LN cells from BALB/c donor mice, or similar cell numbers of LN and BM cells from congenic B6.SJL donors (CD45.1), and then treated with recombinant human IL-7 500 ng BID SQ X 60 days. Survival over the 150 days after BMT is shown. (A) Survival of all wild-type allogeneic B6 recipients is significantly less than that of the congenic recipients (p<0.002). (B) Survival of B6.DL-7^"7" allogeneic recipients is significantly decreased in allogeneic vs recipients (p<0.03), and IL-7 treated vs PBS treated allogeneic recipients (p< 0.002).

Figure 2. Treatment with IL-7 increased GVHD-related morbidity following allogeneic BMT. The severity of GVHD was determined using the GVHD clinical grading system with scoring for five clinical criteria: percentage of weight loss, skin integrity, posture, mobility, and fur texture (Alpdogan et al, J Clin Invest. 112: 1095-1107 (2003)). Clinical signs were graded on a scale of 0 to 2, where 0 was absent, 1 was moderate, and 2 was severe, and the individual signs summed. Shown are GVHD clinical index scores at 4 and 6 weeks for B6 (2A) and B6.IL-7^"7' recipients (2B). Differences between IL-7 and PBS- treated allogeneic recipients are p < 0.05 in both B6 recipients and B6.IL-7^"7" recipients (asterisk).

Figure 3. Histological evidence of an increase in GVHD as a result of IL-7 administration. H & E staining of skin or gut sections from B6 (A) and B6.IL-7^"7" (B) recipients of either allogeneic or congenic LN and TCD BM, sacrificed after 30 days of either IL-7or PBS treatment.

Figure 4. Administration of IL-7 results in increased number of donor CD4 and CD8 T cells in blood, lymph nodes, and spleen following allogeneic BMT. Analyses of donor- derived peripheral lymphocyte numbers in B6 and B6.IL-7^"7" recipients sacrificed 10 or 30 days after allogeneic transplant with BALB/c LN and TCD BM cells. Shown are numbers of donor CD4 and CD8 T cells in peripheral blood (A and B), LN (C and D), and spleen (E and F) of B6 and B6.IL-7^"Λ recipients at day 10 and 30. Asterisk indicates significant differences (p<0.05) between PBS and EL-7 treated groups.

Figure 5. Progressive disappearance of donor-derived allogeneic T lymphocytes in absence of IL-7. Following allogeneic transplant with BALB/c LN and TCD BM cells, donor T cells from the peripheral blood of B6.BL-7^"-^" recipient mice treated with JL-I or PBS were gated and stained for CD4 and CD8 at days 10 and 30.

Figure 6. IL-7 is required for survival and expansion of activated donor T cells, but not their activation. IxIO⁶ TCD BM and 4 X 10⁶ CFSE-labeled LN cells from either congenic or allogeneic donors were transplanted into either lethally irradiated B6 or B6.IL-7^7" recipients. At day 10, most of the proliferating CFSE-labeled congenic (CD45.1+) donor CD4 (6A) and CD8 (6B) T cells in lymph nodes of the B6 recipients did not express the CD69 activation marker, while the allogeneic (H2k^d) donor cells were predominantly CD69+. The frequency of donor-derived CD4 or CD8 T cells in the lymph nodes, which expressed CD69 after allogeneic transplantation, was not significantly changed by the EL-7 treatment of either B6 (6C) or B6.EL-7^7" (6D) recipients. JL-I treatment of the B6.IL-7^7" allogeneic recipients increased the absolute number of activated (CD69+) donor-derived CD4 and CD8 T lymphocytes (6E) (p<0.02 for PBS vs EL-7).

Detailed Description of the Preferred Embodiments

"Antibody" or "antibodies" as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The term "immunoglobulin" includes the subtypes of these immunoglobulins, such as IgG₁, IgG₂, IgG₃, IgG₄, etc. Of these immunoglobulins, IgM and IgG are preferred, and IgG is particularly preferred. The antibodies may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al, Molec. Immunol. 26, 403-11 (1989). Such monoclonal antibodies are produced in accordance with known techniques. The term "antibody" or "antibodies" as used herein includes antibody fragments which retain the capability of binding to a target antigen, for example, Fab, F(ab')₂, and Fv fragments, and the corresponding fragments obtained from antibodies other than IgG. Such fragments are also produced by known techniques.

"Interleukin-7 antagonist" as used herein refers to any compound or active agent which inhibits the activity of interleukin-7 in a subject, including but not limited to compounds that specifically bind to interleukin-7 receptors (i.e., "interleukin-7 receptor antagonists") and compounds that specifically bind to endogenous interleukin-7 itself and hence make it unavailable for binding to interleukin-7 receptors.

"Immunosuppressive agent" as used herein for adjunct therapy refers to substances that act to suppress or mask the immune system of the host into which the graft is being transplanted. This would include substances that suppress cytokine production, downregulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines {See, U.S. Pat. No. 4,665,077); azathioprine (or cyclophosphamide, if there is an adverse reaction to azathioprine); bromocryptine; glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; steroids such as glucocorticosteroids, e.g., prednisone, methylprednisolone, and dexamethasone; cytokine or cytokine receptor antagonists including anti-interferon-gamma, -beta, or -alpha antibodies; anti-tumor necrosis factors antibodies; anti-tumor necrosis factor-s antibodies; anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, preferably anti-CD3 or anti- CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding domain (WO 90/08187, published JuI. 26, 1990); streptokinase; TGF-beta; streptodomase; RNA or DNA from the host; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptor (U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offner et al., Science 251:430-432 (1991); WO 90/11294; and WO 91/01133); and T cell receptor antibodies (EP 340,109) such as T10B9. These agents are administered at the same time or at separate times from the active agent, and are used at the same or lesser dosages than as set forth in the art. The preferred adjunct immunosuppressive agent will depend on many factors, including the type of disorder being treated including the type of transplantation being performed, as well as the patient's history, but a general overall preference is that the agent be selected from cyclosporin A, a glucocorticosteroid (most preferably prednisone or methylprednisolone), OKT-3 monoclonal antibody, azathioprine, bromocryptine, heterologous anti-lymphocyte globulin, or a mixture thereof.

Additional examples of immunosuppressive agents include polysubstituted pteridine- 2,4-diones (lumazines), as well as mon- and poly-substituted 2-thiolumazines, 4- thiolumazines and 2,4-dithiolumazines, as described in U.S. Patent No. 6,946,465; methotrexate; mycophenolate (mofetil); thalidomide; mizoribine; riboflavin; tiazofurin; zafurin; tacrolimus; cyclophophamide; sulfasalazine; and Janus tyrosine kinase 3(Jak3) antagonists, as described in U.S. Pat. Appl. No. 20050203177;

Graft- versus-host disease is known and described in, for example, Jardieu, Method of Treatment using Humanized anti-CDl IA Antibodies, US Patent No. 6,703,018 (Genentech).

The terms "graft" and "transplant" (when used as a noun), as used herein refer to biological material derived from a donor for transplantation into a recipient. Grafts include such diverse material as, for example, isolated cells such as islet cells and neural-derived cells (e.g. Schwann cells), tissue such as the amniotic membrane of a newborn, bone marrow, hematopoietic precursor cells, and organs such as skin, heart, liver, spleen, pancreas, thyroid lobe, lung, kidney, tubular organs (e.g., intestine, blood vessels, or esophagus), etc. The tubular organs can be used to replace damaged portions of esophagus, blood vessels, or bile duct. The skin grafts can be used not only for burns, but also as a dressing to damaged intestine or to close certain defects such as diaphragmatic hernia. The graft is derived from any mammalian source, including human, whether from cadavers or living donors. In some embodiments, the graft is preferably bone marrow or an organ such as heart and the donor of the graft and the host are matched for HLA class II antigens. (See, e.g., US Patent No. 6,703,018).

The term "donor" as used herein refers to the mammalian species, dead or alive, from which the graft is derived. Preferably, the donor is human. Human donors are preferably volunteer blood-related donors that are normal on physical examination and of the same major ABO blood group, because crossing major blood group barriers possibly prejudices survival of the allograft. It is, however, possible to transplant, for example, a kidney of a type O donor into an A, B or AB recipient.

The term "transplant" and variations thereof, refers to the insertion of a graft into a host, whether the transplantation is syngeneic (where the donor and recipient are genetically identical), allogeneic (where the donor and recipient are of different genetic origins but of the same species), or xenogeneic (where the donor and recipient are from different species). Thus, in a typical scenario, the host is human and the graft is an isograft, derived from a human of the same or different genetic origins. In another scenario, the graft is derived from a species different from that into which it is transplanted, such as a baboon heart transplanted into a human recipient host, and including animals from phylogenically widely separated species, for example, a pig heart valve, or animal beta islet cells or neuronal cells transplanted into a human host.

"Increasing tolerance of a transplanted graft" by a host refers to prolonging the survival of a graft in a host, in which it is transplanted, i.e., suppressing the immune system of the host so that it will better tolerate a foreign transplant.

The present invention is primarily concerned with the treatment of human subjects, but the invention may also be carried out on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, livestock and horses for veterinary purposes, and for drug screening and drug development purposes. Human subjects may be any race and either gender. Subjects may be any age including neonate, infant, juvenile, adolescent, adult, and geriatric or aged subjects.

The disclosures of all United States patents cited herein are to be incorporated by reference herein in their entirety.

A. Active agents.

Active agents for carrying out the present invention are interleukin-7 antagonists, as noted above. Numerous interleukin-7 antagonists are known.

In one embodiment of the invention, the interleukin-7 antagonist is an anti- interleukin-7 receptor antibody (e.g., an anti-interleukin-7 receptor monoclonal antibody). Such antibodies are known and described in, for example, US Patent No. 5,194,375, titled DNA encoding interleukin-7 receptors and methods of use.

In another embodiment of the invention, the interleukin-7 antagonist is an exogenous interleukin-7 receptor, for example a soluble interleukin-7 receptor, administered to bind endogeneous interleukin 7 and make it unavailable or less available for binding to the endogenous interleukin-7 receptor. Interleukin-7⁵. receptors are known and available and described in, U.S. Patent No. 5,264,416 , titled Interleukin-7 receptors.

In another embodiment, the interleukin-7 antagonist is an antibody (e.g., a monoclonal antibody) that specifically binds to interleukin 7. Such antibodies are known and described in, for example, U.S. Patent No. 5,714,585, titled Antibodies that are immunoreactive with interleukin-7.

Monoclonal antibody active agents may be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 to Reading, or U.S. Pat. No. 4,816,567 to Cabilly et al. The antibodies may also be chemically constructed by specific antibodies made according to the method disclosed in U.S. Pat. No. 4,676,980 to Segel et al. (Applicants specifically intend that the disclosure of all U.S. patent references cited herein be incorporated herein by reference).

Monoclonal antibody active agents may be chimeric antibodies produced in accordance with known techniques. (See, e.g., U.S Patent No. 6,808,901). For example, chimeric monoclonal antibodies may be complementarity determining region-grafted antibodies (or "CDR-grafted antibodies") produced in accordance with known techniques. Monoclonal antibodies can be humanized by methods known in the art, e.g., MAbs with a desired binding specificity can be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, Calif.).

Monoclonal antibody Fab fragment active agents may be produced in Escherichia coli by recombinant techniques known to those skilled in the art. {See, e.g., W. Huse, Science 246, 1275-81 (1989)).

B. Pharmaceutical Formulations.

Therapeutic formulations of the active compound are prepared for storage by mixing the active compound having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol;>3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an immunosuppressive agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in a microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsion;, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, non- degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the Lupron Depot™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 ⁰C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S--S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

C. Therapeutic Administration.

Suitable subjects for the methods described herein include, but are not limited to, subjects afflicted with an autoimmune disease, subjects afflicted with type I diabetes, subjects afflicted with multiple sclerosis, subjects afflicted with systemic lupus erythematosus, and subjects afflicted with thyroiditis. Suitable subjects include, but are not limited to, subjects afflicted with or at risk of developing graft-versus-host disease, organ transplant recipients, particularly allogenic transplant recipients (e.g., lung, heart, kidney, liver transplant recipients), allogenic bone marrow transplant recipients, hematopoietic stem cell transplant recipients. The active compound is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration (including perfusing or otherwise contacting the graft with the antibody before transplantation). Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the active compound is suitably administered by pulse infusion, e.g., with declining doses of the active compound. Preferably the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

For the prevention or treatment of disease, the appropriate dosage of active compound will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the active compound, and the discretion of the attending physician. The active compound is suitably administered to the patient at one time or over a series of treatments.

Depending on the type and severity of the disease or condition, about 1 μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of active compound is an initial candidate dosage for ^administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

The active compound composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The "therapeutically effective amount" of the antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat the mediated disorder, including preventing an immune response that would result in rejection of a graft by a host or vice- versa, or prolonging survival of a transplanted graft. Such amount is preferably below the amount that is toxic to the host or renders the host significantly more susceptible to infections.

The active compound need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. For example, the active compound may be given in conjunction with a glucocorticosteroid. For transplants, the antibody may be administered concurrently with or separate from an immunosuppressive agent as defined above, e.g., cyclosporin A, to modulate the immunosuppressant effect. The effective amount of such other agents depends on the amount of active compound present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.

The present invention is explained in greater detail in the following non-limiting Examples.

EXAMPLE 1

IL-I, along with c-kit ligand (KL, stem cell factor [SCF]), is the major thymopoietic cytokine (Rodewald et al., Immunity. 3: 313-319 (1995); Murray et al., Int Immunol. 169: s 707-716-1(1989)). TL-I induces proliferation, differentiation, and survival of immature T • ' lymphocytes. During normal T cell development in the thymus, IL-7 produced by thymic epithelial cells (TECs) binds to the cognate IL-7 receptors expressed on the surface of immature T lymphoid progenitor cells. IL-7 stimulates the differentiation of immature CD3^" CD4^" CD8^" (triple negative, TN) thymocytes to later stages, ultimately resulting in the development of mature CD4⁺CD8^" or CD4^"CD8⁺ T cells. The importance of IL-7 for thymopoiesis is demonstrated by mice with targeted mutations of the IL-7, IL-7 receptor α, or common γ chain (γc) genes, dogs with X-SCDD (γc mutations), or humans with X-SCID or IL-7RD(-) SCID, all of which have defective thymopoiesis and impaired ability to produce T lymphocytes (Pechon et al., J Exp Med. 180: 1955-1960 (1994); Von Freeden-Jeffry et al., J Exp Med. 181: 1519-1526 (1995); Somberg et al., J Immunol. 153: 4006 (1994); Noguchi et al., Cell. 73: 147 (1993)). It has been shown that administration of recombinant human IL-7 corrects the thymopoietic defects observed following histocompatible BMT, suggesting that post-BMT IL-7 administration may be a potential therapy for post-transplant immunodeficiency. However, other effects of IL-7 have raised concerns about its safety in the context of allogeneic BMT. Besides common lymphoid progenitors and thymocytes, the IL-7 receptor is expressed by mature T lymphocytes (Sudo et al., Proc. Natl. Acad. Sci. USA. 90: 9125- 9129 (1993)). The IL-7R expressed by mature T lymphocytes mediates several important biological effects that are likely to be clinically relevant. Homeostatic proliferation of naϊve CD4⁺ and CD8⁺ T lymphocytes depends on combined TCR recognition of self-ligands and BL-7R signaling. ΣL-7Rα^"Λ T lymphocytes do not proliferate normally in a normal host while conversely, normal T lymphocytes do not undergo homeostatic proliferation in JL-T^1' hosts (Maraskovsky et al., J Immunol. 157: 5315-5323 (1996); Tan et al., Proc Natl Acad Sci USA. 98: 8732-8737 (2001); Schluns et al., Nature Immunology. 1: 426-432 (2000)). Stimulation through IL-7R also increases expression of the bcl-2 anti-apoptotic protein, thereby increasing survival of mature T lymphocytes (Schluns et al., Nature Immunology. 1: 426-432 (2000); Kondrack et al., J. Exp. Med. 198: 1797-1806 (2003); Vella et al., Proc Natl Acad Sci USA. 95: 3810-3815 (1998)). Furthermore, IL-7 may function as a co-factor for T lymphocyte activation by stimulating production of ThI cytokines such as EL-2, IFN-γ, and BL-12 (Gringhuis et al., Blood. 90: 2690-2700 (1997); van Roon et al., Ann Rheum Dis. 62: 113-119 (2003); Fukui et al., Immunol Lett. 59: 21-28 (1997)).

In allogeneic transplantation, the effects of IL-7 on mature T lymphocytes might cause or exacerbate GVHD by either promoting the proliferation or survival of alloreactive donor mature T cells, or increasing their activation state. Using genetic models of IL-7 deficiency, either endogenously produced or exogenously administered IL-7 are demonstrated to be necessary for the development of GVHD, most likely by promotion of the survival and proliferation of alloreactive T lymphocytes. The results suggest that the therapeutic use of IL-7 to improve immune reconstitution in an allogeneic setting may exacerbate GVHD. Material and Methods: Mice

Female C57BL/6J (H-2k^b, CD45.2), B6.SJL (H-2k^b, CD45.1), and male BALB/c (H- 2k^d) mice (aged 8 to 10 weeks) were purchased from the Jackson Laboratory (Bar Harbor, ME). A breeding colony of IL-7^"Λ mice on a C57BL/6J background (H-2k^b, CD45.2, B6.IL- T^1') was established from founder mice kindly provided by Dr. Richard Murray (DNAX Research Institute, Palo Alto, CA). Protocols for animal care and BMT were approved by the Childrens Hospital Los Angeles Research Institute Animal Care Committee. Bone marrow transplantation procedure

Female C57BL/6J (H-2K^b) recipient mice were given two separate doses of radiation from a ¹³⁷Cs source at 128 cGy/minute (700 cGy on day -1 and 600 cGy on day 0) prior to transplantation. In each experiment, control mice were irradiated without subsequent BMT to verify that the doses of radiation were marrow-ablative. Bone marrow (BM) cells were harvested from the femur and tibia of either male congenic (histocompatible) B6.SJL (H-2K^b, CD45.1) or allogeneic BALB/c (H-2K^d) donor mice by perfusion, after the mice were sacrificed by CO₂ narcosis. The donor marrow was depleted of mature T lymphocytes by immunomagnetic depletion, using rat anti -mouse Thy 1.2, CD4, and CD8 monoclonal antibodies (PharMingen, San Diego, CA) and sheep anti-rat antibodies conjugated to beads (Dynal, Great Neck, NY). The purity of the T-lymphocyte depleted cells was determined by flow cytometric analysis (FACS) to ensure that the frequency of mature T lymphocytes in the marrow was less than 0.1%. GVHD was induced by administration of T lymphocytes derived from lymph nodes (LN) of donor mice at the same time as BMT. Mesenteric, axillary, and inguinal LN were collected, minced, and filtered through nylon mesh in order to eliminate adherent cells. After completion of pre-transplant irradiation on day 0, 1 x 10⁶ T cell-depleted (TCD) BM cells and 4 x 10⁶ LN cells were resuspended in phosphate buffered saline (PBS)(BiO Whittaker_/ Walkerville, Maryland) and transplanted into each recipient via tail vein injection (0.3 ml total volume). Following transplantation, mice were housed in sterilized microisolator cages and given normal chow and autoclaved water containing tetracycline HCl 20ug/ml (Goldline, Miami, FL) for the first two weeks post-transplant and filtered water thereafter. Administration of rhIL-7

Recombinant human TL-I (R&D Systems, Minneapolis, MN), resuspended in PBS, was aseptically administered to the transplanted recipients by subcutaneous injection at a dose of 500 ng, twice daily, for 30 or 60 days, or until sacrifice. Human IL-7 was chosen because of its availability as a purified, endotoxin-free product, biological cross activity in vitro with murine thymocytes, and previous data demonstrating enhancement of thymopoiesis after BMT (Cooke et al., Blood. 88: 3230-3239 (1996)). The control mouse groups were injected with normal saline according to the same schedule. Assessment of GVHD

The degree of GVHD severity was assessed using the clinical scoring system described by Cook et al (Cooke et al., Blood. 88: 3230-3239 (1996)). Each transplanted animal was scored weekly for five parameters (weight loss, skin integrity, fur texture, mobility, and posture), using a scale of 0 to 2, with 0 for absent or normal, 1 for mildly abnormal, and 2 for severely abnormal. The GVBTO clinical index was the sum of the scores for individual criteria. Histological analysis

Wax coated tissue sections from small intestine and skin were cut into 5-μm thick sections, mounted onto slides, fixed in 10% formalin, and stained with hematoxylin and eosin (H&E). The tissue sections were independently examined in a blinded manner for evidence of GVHD (Nikolic et al., J. Clin. Invest. 105: 1289-1298 (2000)). Flow cytometry

Peripheral blood lymphocytes (PBL) or single cell suspensions of splenocytes or LN cells were prepared by lysing the red blood cells with lysis buffer containing ammonium chloride (Gibco Life Technologies, Carlsbad, CA). IxIO⁵ cells were stained with optimal concentrations of fluorescein (FITC), phycoerithrin (PE), allophycocyanin (APC), or PerCP conjugated anti-Thyl, CD3, CD4, CD8, CD69, H2K^b, H2K^d, or isotype control monoclonal antibodies (Pharmingen, San Diego, CA). Following staining, cells were washed twice in PBS and analyzed on the FACS Calibur (Becton-Dickinson, San Diego, CA). In some experiments, donor LN cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) prior to transplantation to measure the proliferation in vivo of the cells after transplant. Donor LN cell proliferation was assessed by measuring separate peaks of decreased intensity of CFSE fluorescence upon successive cell division by FACS analysis of the labeled donor CD4 or CD8 T cell population. Statistical analyses

Analyses of survival rates were performed by Wilcoxon log-rank test. Comparisons of donor cell recovery, weight loss, and GVHD scoring were made with a two-way analysis of variance. Differences between groups of different immunophenotypic populations of cells after transplant were analyzed by 2-tailed t-test with unequal distributions. P-values that were less than or equal to 0.05 were considered statistically significant.

The results of the study are described below: IL-7 is necessary for GVHD-related mortality following allogeneic BMT

In order to assess whether EL-7 is necessary for the development of GVHD, we measured the survival rate of recipient B6 or B6.TL-T'^' mice, following transplantation of congenic or allogeneic BM and LN cells. Irradiated (1300 cGy) B6 and B6.IL-7^'/' mice (H- 2K^b) were co-transplanted with Ix 10⁶ TCD bone marrow cells and 4 x 10⁶ LN T lymphocytes from either congenic B6.SJL (H-2K^b) or allogeneic BALB/c mice (H-2K^d). Following BMT, either PBS or rhIL-7 (500 ng BID SQ) was administered to the transplanted recipients for.60 days. A schedule of continuous administration of EL-7 was chosen to assure that EL-7 was present at all times post-transplant, since it was unknown when IL-7 might exert effects on the co-transplanted mature T lymphocytes.

Figures IA and IB show survival of the congenic and allogeneic B6 and B6.EL-7^"7" recipients. The survival of the congenic recipients was 100%, regardless of whether the recipient mice were normal B6 or B6.TL-7^''^' mice. Administration of IL-7 to the congenic B6 or B6.EL-7^"'^" recipients did not decrease survival. Thus, neither the presence nor absence of endogenous IL-7, nor the administration of exogenous IL-7 had any effect on survival in the congenic setting.

In contrast to the results observed in congenic recipients, the presence or absence of IL-7 affected the outcome of allogeneic transplant. In the first 25 days after allogeneic BMT, there was no difference between the B6 and B6.IL-7^"/" recipients. As expected with the fully H2-incompatible BALB/c to B6 model, the mortality of either EL-7 or PBS treated normal B6 recipients was approximately 70%, with deaths continuing to occur throughout the 150-day observation period. Although IL-7 injections* induced a higher mortality in normal allogeneic B6 recipients (75% vs 65%), the difference between the IL-7 and PBS treated mice was not statistically significant in this recipient group. However, in the absence of IL-7, GVHD- related mortality was diminished to 41% at day 150 in PBS treated B6.IL-7^"/" recipients. Similar to the normal B6 allogeneic recipients, there was mortality of the PBS-treated B6.EL- T^1' allogeneic recipient mice in the first 25 days after transplant. However, after this initial post-BMT period, no further deaths were observed. In contrast, the mortality rate of EL-7 treated allogeneic B6.IL-7^"/" and B6 recipients continually increased with time.

To further quantify the effects of EL-7 treatment on the clinical status of transplanted mice, they were evaluated for GVHD with a clinical scoring system for 6 weeks following transplantation (Cooke et al, Blood. 88: 3230-3239 (1996)). As expected, both congenic B6 and B6.IL-7^"/" recipients treated with either EL-7 or PBS showed no signs of GVHD-related morbidity (Figures 2A, 2B). In contrast, allogeneic normal B6 recipients displayed significant weight loss and other evidence of GVHD, compared to the congenically transplanted controls. The overall clinical grading of GVHD in the IL-7-treated B6 mice was more severe (p<0.05) (Fig 2A). Similar to the results observed in the congenic recipients, allogeneic B6.IL-7^"'^' recipient mice treated with PBS showed no clinical evidence of GVHD (Fig 2B). However, administration of IL-7 to the allogeneic B6.IL-7^"7" recipients resulted in

GVHD-related morbidity that resembled that observed in the allogeneically transplanted B6 recipients (p<0.012). The difference in mortality and GVHD score in the JL-I vs PBS treated allogeneic B6.JL-T'^' recipients, combined with the lack of clinical toxicity of JLl after congenic transplantation, indicates that JL-I was necessary for the development of GVHD symptoms.

Tissue inflammation caused by GVHD requires IL-7

GVHD-target organs were examined to determine whether IL-7 caused histologic damage. The irradiated animals received either IL-7 or PBS from day 1 to day 30 following allogeneic BMT. On day 30, sections from the small intestine and skin were analyzed for evidence of GVHD. As expected, the tissue samples from congenic recipients showed no evidence of tissue inflammation. The results differ somewhat from Shinha et al., who noted increased tissue inflammation in syngeneic BMT recipients treated with IL-7 compared to non-transplanted mice treated with IL-7 (Blood. 100: 2642-2649 (2002)). Compared to the congenic recipients, however, the IL-7 or PB S -treated allogeneic normal B 6 recipients showed histological evidence of GVHD, and the severity of the tissue inflammation in both IL-7-treated and PBS-treated hosts did not appear significantly different (Figure 3A). The intestines had villus blunting, lamina propria inflammation, crypt destruction, and mucosal atrophy in both IL-7 and PBS treated groups of allogeneic B6 recipients. The animals also had cutaneous GVHD, with infiltration of lymphocytes, hyperkeratosis, and hair loss. In the absence of IL-7 in the B6.IL-7^7" hosts, the histological samples from the allogeneic recipients did not appear to be different from those of the congenic B6 recipients. The B6.IL-7^7" allogeneic recipients treated with IL-7 displayed the same GVHD-related histological features seen in allogeneic B6 recipients. (Figure 3B). Therefore, the data illustrate that IL-7 is necessary for GVHD-related tissue damage and inflammation, but was not intrinsically pathogenic in the histocompatible setting. IL-7 is necessary for maintenance of donor T cells in the periphery of recipient mice

Since the development of GVHD depends on donor mature T cells, we determined whether IL-7 treatment altered the number of donor-derived mature CD4 and CD8 T cells in the spleen, LN, and peripheral blood (PBL) after allogeneic transplantation. IL-7 or PBS- treated allogeneic recipients were analyzed at day 10 and 30 following transplantation. Figure 4 shows the number of donor-derived CD4 and CD8 T cell populations at both day 10 and day 30 in the peripheral blood (4A and 4B), LN (4C and 4D), and spleen (4E and 4F) of the IL-7 or PBS-treated mice. Administration of IL-7 to normal B6 recipients increased the numbers of donor-derived mature T cells 1.2 - 3-fold in all sites (Figures 4A, 4C, 4E). The effects of IL-7 on mature T cell numbers was observed as early as Day 10, before thymic- derived T cells would have been expected to be generated. Therefore, the increase in T cell numbers likely reflected IL-7 induced proliferation of the transplanted mature T cells.

PBS-treated B6.JL-T^1' recipients exhibited significantly lower donor T cell numbers in all three lymphoid compartments compared to the normal recipients (Figures 4B, 4D, 4F). Low numbers of donor CD4 and CD8 cells were detected in the peripheral blood of Bό.IL^^" mice treated with PBS at day 10, but were 2-8-fold less frequent than in normal B6 recipients (Figures 4A, 4B). The number of T cells detected in the lymph nodes of the B6.TL-1^''^' recipients was 100- to 1000-fold lower than in the normal B6 recipients (Figures 4C, 4D). Similarly, the number of splenic T cells was 10-30-fold lower in the B6.EL-7^"7" mice. By day 30 after transplantation, the number of donor-derived T cells in the PBS-treated B6.IL-7^7" mice was almost nil. Thus, in the absence of IL-7, adoptively transferred mature T lymphocytes did not survive (Figures 4B, 4D, 4F, 5).

It was then determined whether the defect in lymphocyte survival could be rescued by administration of exogenous IL-7. IL-7 treatment significantly increased the numbers of donor-derived mature T cells in the B6.IL-7^"/" recipients (Figures 4B, 4D, 4F, 5). The number of donor-derived T lymphocytes after IL-7 treatment ^! was, lower in the B6.IL-7^"7" recipients than in the normal B6 recipients treated with either PBS, or IL-7. This suggests that the bolus schedule of IL-7 administration is not as efficient at mediating T cell survival as endogenously produced IL-7. Overall, the data support the hypothesis that IL-7 is necessary to maintain transplanted mature T cells, including allogeneic cells, in the periphery (Tan et al., Proc Natl Acad Sci USA. 98: 8732-8737 (2001); Schluns et al., Nature Immunology. 1: 426-432 (2000); Kondrack et al., J. Exp. Med. 198: 1797-1806 (2003); Alpdogan et al., J Clin Invest. 112: 1095-1107 (2003); Mackall et al., Blood. 97: 1491-1497 (2001)). Importance of IL-7 in proliferation of allogeneic T lymphocytes

Because naϊve mature T lymphocytes are known to undergo homeostatic proliferation after interactions with self-peptides, we determined whether there were phenotypic differences between T lymphocytes that proliferated after allogeneic or congenic transplantation (Tan et al., Proc Natl Acad Sci USA. 98: 8732-8737 (2001); Schluns et al., Nature Immunology. 1: 426-432 (2000); Kondrack et al., J. Exp. Med. 198 (12): 1797-1806 (2003)). The expression of CD69 as an activation marker was analyzed, since up-regulation of CD69 has been reported during activation-induced T cell proliferation, including that of alloreactive cells (Mackall et al., Blood. 97: 1491-1497 (2001); Brochu et al., Blood. 94: 390- 400 (1999)). Mice were co-transplanted with TCD BM and CFSE-labelled donor LN T cells. The expression of CD69 was analyzed using H2k^d to gate on the donor cells and CFSE to mark the proliferating T cells in the allogeneic recipients. In the congenic recipients, expression of CD69 was low to absent in the proliferating donor-derived (CD45.1+) T cells (Figures 6A, 6B). The expression of CD69 in the dividing CD4 and CD8 LN-derived T cells was also analyzed in the allogeneic recipients (Figure 6A and B). Most proliferating donor CD4⁺ or CD8⁺ T cells in the allogeneic recipients had increased levels of CD69 expression. The differences in CD69 expression are consistent with CD69 expression marking proliferating alloreactive T cells, while CD69 expression was not induced during homeostatic proliferation of non-alloreactive cells, a concept proposed by Alpdogan et al. (Alpdogan et al. J Clin Invest. 112: 1095-1107 (2003)).

IL-7 has been shown to act both as a co-factor for T cell activation and to increase the survival of activated T lymphocytes (Vella et al., Proc Natl Acad Sci USA. 95: 3810-3815 (1998)). The effects of IL-7 on the survival of activated alloreactive T cells after transplant was studied to determine whether IL-7 enhanced the survival of activated allogeneic T cells in vivo. Allogeneic marrow and CFSE-labeled LN cells from BALB/c donors were transplanted into B6 and B6.TL-T^1' recipients, and treated with either IL-7 or PBS. At day 10, donor-derived T cells in the lymph nodes of the recipient mice were analyzed by gating on H2k^d -positive cells. The frequency of donor-derived T lymphocytes expressing CD69 was used to measure the effects of IL-7 on activation of T lymphocytes in vivo. There were no differences observed in the frequency of activated CD4 or CD8 T lymphocytes in the B6 or B6. TL-T^1" recipients (Figure 6C & D). In normal B6 recipients, DL-7 treatment did not increase the frequency of activated T cells. The absolute number of activated T lymphocytes was higher in the IL-7 treated B6 recipients, but this was not statistically significant. In the B6.IL-7^"7" recipients treated with PBS, the total number of T lymphocytes was lower than in either B6 or IL-7 treated B6.IL-7^"7" recipients (Figure 4). However, the frequency of activated T lymphocytes in the PBS-treated B6.IL-7^'7" recipients was similar to that of both the B6 mice and the IL-7 treated B6.IL-7^"7" recipients (Figure 6D). The absolute number of activated T lymphocytes in the PBS-treated B6.TL-T'^' recipients was significantly lower than that of the TL-I treated B6.IL-7^"7" mice (Figure 6E). Therefore, IL-7 was necessary for the survival and proliferation of the alloreactive T lymphocytes, but did not appear necessary for their activation. Discussion In the present study, it was determined whether JL-I is necessary for the development of GVHD. Previous data showed that naϊve T lymphocytes require JL-I for proliferation and survival cells, thus, BL-7 may be required for the maintenance of donor T lymphocytes that cause GVHD. Normal B6 and B6.EL-7^7" mice were treated with IL-7 or PBS after myeloablative radiation and transplantation of either congenic or allogeneic BM and LN cells. Similar to the results observed in the congenic recipients, no evidence of GVHD was detected in the B6.EL-7^7" allogeneic recipients, which did not receive exogenous IL-7. On the other hand, administration of JL-I to B6.IL-7^"7" recipients restored their sensitivity to the allogeneic cells, thereby decreasing their survival from 59% to 15%. IL-7 treatment also modestly decreased the survival of the normal B6 mice after allogeneic transplant. Although the survival rate was similar in all groups of mice for the first 25 days after transplantation, only the congenic recipients and the PBS treated B6.IL-7^"7" allogeneic recipients survived without GVHD afterwards. Histological examination at day 30 showed that skin and gut tissues from IL-7 and PBS treated congenic recipients and PBS treated allogeneic B6.IL-7^7" recipients displayed no evidence of acute GVHD. The overall GVHD clinical index score of PBS treated B6.ΣL-7^7" was significantly lower than that of the IL-7 treated B6.IL-7^"7" recipients. The mechanism by which IL-7 deprivation prevented GVHD was decreased proliferation and survival of all transplanted CD4+ and CD8+ T lymphocytes, presumably including alloreactive T lymphocytes that cause GVHD. The death of the transplanted cells is slow with complete disappearance of the donor-derived cells from the periphery not occurring until approximately days 25-30 post-BMT.

The development of GVHD and the increased presence of activated T cells in IL-7 treated B6.JL-7^~'^~ recipients demonstrate the importance of JL-I in survival and proliferation of mature T lymphocytes. Previous experiments have shown that most allogeneic donor T cells undergo activation-induced cell death (AICD) following massive proliferation early in GVHD pathogenesis (Fujioka et al., Trans Immunol. 11: 187-195 (2003); Rathmell et al., J Immunol. 167: 6869-6876 (2001)). However, IL-7 signaling has been reported to up-regulate anti-apoptotic proteins such as Bcl-2 and lung Kruppel-like factor (LKLF), down-regulate the pro-apoptotic protein p27, and promote survival of activated donor T cells following allogeneic BMT (Fukui et al., Immunol Lett. 59: 21-28 (1997); Schober et al., J Immunol. 163: 3662-3667 (1999); Barata et al., Blood. 98: 1524-1531 (2001); Tan et al., J Exp Med. 195: 1523-32 (2002)). In the present invention, PBS treated B6.IL-7^"7' recipients had few detectable donor T lymphocytes in the periphery by day 30. Thus, the presence of either endogenously produced or exogenously administered JL-I was required for the survival of the transplanted T lymphocytes.

Almost all donor T lymphocytes disappeared from the PBS treated B6.IL-7^"7" recipients, indicating that both alloreactive and non-host reactive T lymphocytes were susceptible to cell death in the absence of JL-I. Alpdogan et al. recently demonstrated that alloreactive T lymphocytes either express no or little IL-7Rα from hour 16 to day 8 post- transplant (J Clin Invest., 112: 1095-1107 (2003)). However, results from the present invention indicate that the alloreactive T lymphocytes in the fully MHC-mismatched BALB/c to B6 model are dependent on JL-I. Although the discrepancy might reflect technical differences in the GVHD models used in the respective experiments, the results can be reconciled with those of Alpdogan et al. if either IL-7Rα^Iow/" alloreactive T lymphocytes are still dependent on JL-I or if a later differentiation stage of the alloreactive T lymphocytes are IL-7-dependent. It is possible that the monoclonal antibody staining used to detect IL-7Rα positive cells is unable to detect low levels of biologically important IL-7Rα. Survival signals through the IL-7R may require a smaller number of receptors than the limits of detection of the anti-EL-7Rα antibody used by Alpdogan et al. It is also possible that IL-7Rα was re-expressed on alloreactive T cells at a later time point than the day 8 analyses reported by Alpdogamet ak

Besides the pro-survival effects of IL-7 on naive T lymphocytes, IL-7 has also been reported to be necessary for proliferation of naϊve T cells in conditions of homeostatic expansion. Previous reports have demonstrated that JL-I -mediated signals are necessary for the survival and proliferation of T lymphocytes (Tan et al., J Exp Med. 195: 1523-32 (2002); Schluns et al., Nat Rev Immunol. 3: 269-79 (2003)). For example, adoptive transfer of congenic T cells to JL-T^1' mice resulted in decreased proliferation and eventual loss of the cells (Tan et al., Proc Natl Acad Sci USA. 98: 8732-8737 (2001)). Further, IL-7 may also play a crucial part in the peripheral expansion of donor allogeneic mature T cells. The frequency of activated CD69+ T lymphocytes was found to be similar in the allogeneically transplanted B6 and B6.IL-7^'7" recipients, indicating that IL-7 was not required for activation of the alloreactive T lymphocytes. However, the absolute number of donor-derived T lymphocytes, including the activated CD69+ cells, declined over the first 25 days after transplant in the absence of IL-7, at which point the T cell number was too low for further analysis. The recovery of proliferating activated donor T cells from the lymph nodes of PBS treated B6 JL-T^1' mice at day 10 was significantly lower than in the IL-7 treated B6/ΣL-7^"7" recipients. The results are consistent with experiments that have demonstrated that IL-7 was not needed for generation of primary effector cells but was needed for formation of memory cells (Kondrack et al., J Exp Med. 198:1797-1806 (2003)). Thus, the role of IL-7 in the development of the GVHD response is likely to be its proliferative and pro-survival effects, not a co-activator function.

The experiments extend and contrast with those of Alpdogan et al. and Shinha et al., who evaluated the effects of post-transplant IL-7 administration on GVHD in various models of murine BMT recipients (Alpdogan et al., Blood. 98: 2256-2265 (2001); Shinha et al., Blood. 100: 2642-2649 (2002)). While the former observed no effects of IL-7 treatment on GVHD, the latter observed striking differences in the frequency of GVHD and increased sensitivity to differing doses of alloreactive T lymphocytes. There are potentially confounding technical differences in the studies of Alpdogan et al., Shinha et al., and the present invention. While Alpdogan et al. administered IL-7 in their GVHD experiments from day -1 to +13 via miniosmotic pump, Shinha et al. administered IL-7 intraperitoneally. In the present invention, subcutaneous injection of IL-7 was used rather than miniosmotic pump, because previous studies with minipump delivery of IL-7 failed to show thymopoietic effects in a histocompatible BMT model, although such effects are readily observed after subcutaneous injection (Bolotin et al., Blood. 88: 1887-1894 (1996); Mathur et al., Bone Marrow Transplant. 16:119-124 (1995)). The discrepant results seen in analyses of thymopoiesis lead to questions about whether the IL-7 is stably delivered via minipump over 14 days. Furthermore, in the present invention, IL-7 was administered every day post- transplant, because of the uncertainty as to when JL-I dependence of alloreactive T lymphocytes might occur.

It is likely that the limited survival and expansion of alloreactive T cells in the absence of IL-7 can be overcome by transplantation of sufficient numbers of alloreactive cells. Shinha et al. demonstrated that the increase in severity of GVHD following JL-I treatments was directly proportional to the number of allogeneic T cells transplanted. Although clinical and histological GVHD was not detected in PBS-treated IL-7^"7" allogeneic recipients, their mortality rate (41%) was still significantly greater than that of the comparable congenic recipients. Similar to the results of the present experiments, Tan et al. have demonstrated that the complete disappearance of transplanted T cells in an IL-7^7" recipient takes at least two weeks (Proc Natl Acad Sci USA. 98: 8732-8737 (2001)). Therefore, it is possible that the early mortality observed in the PBS-treated IL-7^";" allogeneic recipients was due to these animals having received higher numbers of alloreactive T cells that overcame the decreased T cell survival caused by lack of IL-7. Consistent with this explanation, all PBS-treated JL-J^''^' allogeneic recipients that survived the first 25 days became long-term survivors and had no evidence of GVHD.

In the present invention, the survival rate of IL-7 treated normal B6 allogeneic mice was lower than that of the PBS treated control mice, but was not statistically significant. Nevertheless, IL-7 treatment increased the number of T lymphocytes and activated T lymphocytes in the normal B6 recipients. The relatively modest effect on mortality suggests that the endogenous production of IL-7 from the PBS treated allogeneic B6 recipients was sufficient to maintain the survival of alloreactive T cells. Peripheral IL-7 levels due to endogenous production have been reported to be elevated after allogeneic BMT (Gendelman et al., J Immunol. 172: 3328-3336 (2004)).

The experimental demonstration of the importance of IL-7 for both survival and proliferation of allogeneic T lymphocytes may allow synthesis of previous observations regarding IL-7 levels, lymphopenia, and GVHD. Several studies have demonstrated that peripheral IL-7 levels are significantly increased in lymphopenic hosts (Bolotin et al., Bone Marrow Transplantation. 23: 783-788 (1999); Llano et al., J Virol. 75: 10319-25 (2001); Napolitano et al., Nat Med. 7: 73-79 (2001)). For example, Bolotin et al. noted that SCID patients undergoing BMT had high circulating levels of IL-7, which normalized after engraftment and development of normal donor T lymphocytes (Bolotin et al., Bone Marrow Transplantation. 23: 783-788 (1999)). Subsequent studies by Napoliano et al. and Llano et al. demonstrated that circulating IL-7 levels were also increased in lymphopenic patients with HIV and (Llano et al., I Virol. 75: 10319-25 (2001); Napolitano et al., Nat Med. 7: 73-79 (2001)). The mechanisms by which lymphopenia results in increased circulating levels of EL- 7 are unclear. While not wishing to be limited by any particular theory, two likely mechanisms may be either that IL-7 levels vary because of consumption by DL-7R-bearing lymphocytes or that peripheral cells that regulate IL-7 production are actively inhibited by a factor produced by T lymphocytes. The variation in levels of IL-7 is probably an important mechanism for regulation of homeostatic proliferation, which has been noted to occur under lymphopenic conditions, i.e., when levels of EL-7 are high.

GVHD or GVHD-like autoimmune illnesses have been noted to occur more readily in lymphopenic hosts than in non-lymphopenic hosts. For example, lymphocytic infiltration of organs has been observed in neonatally thymectomized mice and nude mice that have been engrafted with neonatal thymuses from a normal donor (Sakajuchi et al., J Exp Med. 156: 1577-1586 (1982); Sakaguchi et al., J Exp Med. 172:537-545 (1990)). Assuming that the inverse relationship between T lymphocyte numbers and IL-7 levels can be generalized, then it is likely that there are higher levels of IL-7 in lymphopenic than in non-lymphopenic hosts. The higher TL-I levels in lymphopenic hosts may increase the proliferation of T lymphocytes and pre-dispose to expansion of alloreactive or autoreactive T cells.

The dependence of GVHD on the presence of IL-7 and the apparent increased risk of GVHD in recipients of extrinsic IL-7 suggest potential risks in the use of IL-7 to enhance immune reconstitution after hematopoietic stem cell transplant (HSCT). Although the positive effects of IL-7 on thymopoiesis and expansion of naive T lymphocyte populations by homeostatic proliferation would be expected to increase immune function, the results here indicate that use of rhIL-7 following allogeneic BM and mature T cell transplantation leads to maintenance of donor mature T cells and likely to be responsible for the development of GVHD. The clinical testing of IL-7 therapies to enhance immune reconstitution in the allogeneic setting must be carefully designed to minimize the risk of GVHD, e.g., by testing in the autologous transplant or T cell-depleted (TCD) allogeneic settings. Thus, blockade of the IL-7 signaling pathways provides a useful strategy to eliminate donor alloreactive T cells after transplant.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. A method of suppressing an undesired immune response in a mammalian subject in need thereof, comprising: administering an interleukin-7 antagonist to said subject in an amount effective to suppress an undesired immune response in said subject.

2. The method of claim 1, wherein said subject is afflicted with an autoimmune disease.

3. The method of claim 1, wherein said subject is afflicted with type I diabetes.

4. The method of claim 1, wherein said subject is afflicted with multiple sclerosis.

5. The method of claim 1, wherein said subject is afflicted with systemic lupus erythematosus.

6. The method of claim 1, wherein said subject is afflicted with thyroiditis.

7. The method of claim 1, wherein said subject is an organ transplant recipient.

8. The method of claim 1, wherein said interleukin-7 antagonist is an anti-interleukin- 7 receptor antibody.

9. The method of claim 1, wherein said interleukin-7 antagonist is an anti-interleukin- 7 antibody.

10. The method of claim 1, wherein said interleukin-7 antagonist is an interleukin-7 receptor.

11. The method of claim 1, wherein said administering step is carried out by parenteral injection.

12. The method of claim 1, further comprising administering at least one immunosupressive agent to said subject in a treatment-effective amount.

13. The method of claim 1, wherein said subject is a human subject.

14. A method of treating graft-versus-host disease in a mammalian subject in need thereof, comprising: administering an interleukin-7 antagonist to said subject in an amount effective to treat said graft-versus-host disease in said subject.

15. The method of claim 14, wherein said subject is an allogenic transplant recipient.

16. The method of claim 14, wherein said subject is an allogenic bone marrow transplant recipient.

17. The method of claim 14, wherein said subject is a hematopoietic stem cell transplant recipient.

18. The method of claim 14, wherein said interleukin-7 antagonist is an anti- interleukin-7 receptor antibody.

19. The method of claim 14, wherein said interleukin-7 antagonist is an anti- interleukin-7 antibody.

20. The method of claim 14, wherein said interleukin-7 antagonist is an interleukin-7 receptor.

21. The method of claim 14, wherein said administering step is carried out by parenteral injection.

22. The method of claim 14, further comprising administering at least one immunosupressive agent to said subject in a treatment-effective amount.

23. The method of claim 14, wherein said subject is a human subject.