WO2008150868A1 - Methods for inducing therapeutic t cells for immune diseases - Google Patents

Abstract

A combination treatment of an immunosuppressive adjuvant and a disease-specific antigen provides short and long term benefits for diseases associated with hyperactive immune responses. Such diseases include autoimmune diseases, transplantation rejection, and allergic diseases. The treatment can employ protein or nucleic acid delivery agents for antigen specific immunization. The immunosuppressive adjuvant inhibits the differentiation of dendritic cells and stimulates the expansion of Treg cells, in particular antigen-specific Treg cells.

Description

METHODS FOR INDUCING THERAPEUTIC T CELLS FOR IMMUNE DISEASES

[01] This invention was made using funds from grant RO1CA92243 from the U.S. National Institutes of Health. The U. S. Government therefore retains certain rights in the invention.

[02] This application claims the benefit of U.S. provisional application S. N. 60/940,483 filed May 29, 2007.

TECHNICAL FIELD OF THE INVENTION

[03] This invention is related to the area of pathological immune responses. In particular, it relates to treatment to reduce the pathology of such responses.

BACKGROUND OF THE INVENTION

[04] Pharmaceutical agents with immunosuppressive and anti-inflammatory properties have long provided simple and economic treatment for allergy, autoimmune disease, and transplant rejection ( Allison, A. C. 2000. Immunosuppressive drugs: the first 50 years and a glance forward. Immunopharmacology 47:63-83; Halloran, P. F. 2004. Immunosuppressive drugs for kidney transplantation. N Engl J Med 351 :2715-2729.). In addition to their effects on T cells, various immunosuppressants affect the function of innate immune cells, dendritic cells (DCs) in particular ( Hackstein, H., and A. W. Thomson. 2004. Dendritic cells: emerging pharmacological targets of immunosuppressive drugs. Nat Rev Immunol 4:24-34.). Nonetheless, these agents are unable to distinguish between harmful and beneficial immunity; their prolonged use causes nonspecific immunosuppression, increasing the risk of infection and cancer. Therapeutically, these agents temporarily dampen, but not permanently eliminate, pathogenic immune responses and are, thus, not a cure. Hence, there is a need for approaches that allow long-term control of untoward immunity in an antigen-specific manner. [05] Three major approaches have been explored to achieve this goal. The first one is the use of tolerogenic DCs. DCs are the most potent antigen-presenting cells (APCs). It is generally recognized that the context in which an antigen is presented, but not the antigen per se, determines whether the encounter between the antigen and its cognate T cell leads to immunity or tolerance. This notion has inspired investigators to pulse antigens onto tolerogenic DCs induced and amplified ex vivo, and apply the resultant DCs as a vaccine to induce antigen-specific tolerance. Tolerogenic DCs are marked by a semi-mature phenotype and the ability to produce tolerogenic cytokines such as IL-IO while downregulating pro-inflammatory cytokines such as IL-12 ( Lutz, M. B., and G. Schuler. 2002. Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity? Trends Immunol 23:445-449; Wakkach, A., N. Fournier, V. Brun, J. P. Breittmayer, F. Cottrez, and H. Groux. 2003. Characterization of dendritic cells that induce tolerance and T regulatory 1 cell differentiation in vivo. Immunity 18:605-617.). Importantly, they have been shown to preferentially expand regulatory T cells, including the CD4+CD25+Foxp3+ regulatory T cell subset ( Groux, H., N. Fournier, and F. Cottrez. 2004. Role of dendritic cells in the generation of regulatory T cells. Semin Immunol 16:99-106; Kuwana, M. 2002. Induction of anergic and regulatory T cells by plasmacytoid dendritic cells and other dendritic cell subsets. Hum Immunol 63:11 Soi l 63; Rutella, S., and R. M. Lemoli. 2004. Regulatory T cells and tolerogenic dendritic cells: from basic biology to clinical applications. Immunol Lett 94:11-26; Morelli, A. E., and A. W. Thomson. 2007. Tolerogenic dendritic cells and the quest for transplant tolerance. Nat Rev Immunol 7:610-621), which is immunosuppressive and plays a pivotal role in immune tolerance. Tolerogenic DCs can be induced by the use of anti- inflammatory cytokines or immunosuppressants ( Morelli, A. E., and A. W. Thomson. 2007. Tolerogenic dendritic cells and the quest for transplant tolerance. Nat Rev Immunol 7:610-621; Adorini, L., N. Giarratana, and G. Penna. 2004. Pharmacological induction of tolerogenic dendritic cells and regulatory T cells. Semin Immunol 16:127-134; Bros, M., F. Jahrling, A. Renzing, N. Wiechmann, N. A. Dang, A. Sutter, R. Ross, J. Knop, S. Sudowe, and A. B. Reske-Kunz. 2007. A newly established murine immature dendritic cell line can be differentiated into a mature state, but exerts tolerogenic function upon maturation in the presence of glucocorticoid. Blood 109:3820-3829.). For example, dexamethasone (DEX), a synthetic glucocorticoid (Franchimont, D. 2004. Overview of the actions of glucocorticoids on the immune response: a good model to characterize new pathways of immunosuppression for new treatment strategies. Ann N Y Acad Sd 1024:124-137), alters the normal maturation pathway of DCs in response to maturation signals (Matyszak, M. K., S. Citterio, M. Rescigno, and P. Ricciardi-Castagnoli. 2000. Differential effects of corticosteroids during different stages of dendritic cell maturation. Eur J Immunol 30:1233-1242; Rozkova, D., R. Horvath, J. Bartunkova, and R. Spisek. 2006. Glucocorticoids severely impair differentiation and antigen presenting function of dendritic cells despite upregulation of Toll-like receptors. Clin Immunol 120:260-271); DEX decreases the expression of MHC and costimulatory molecules and the production of IL-12, while stimulating the production of IL-10 ( Piemonti, L., P. Monti, P. Allavena, M. Sironi, L. Soldini, B. E. Leone, C. Socci, and V. Di Carlo. 1999. Glucocorticoids affect human dendritic cell differentiation and maturation. J Immunol 162:6473-6481; Rea, D., C. van Kooten, K. E. van Meijgaarden, T. H. Ottenhoff, C. J. Melief, and R. Offringa. 2000. Glucocorticoids transform CD40-triggering of dendritic cells into an alternative activation pathway resulting in antigen-presenting cells that secrete IL-10. Blood 95:3162-3167; Xia, C. Q., R. Peng, F. Beato, and M. J. Clare-Salzler. 2005. Dexamethasone induces IL-10-producing monocyte-derived dendritic cells with durable immaturity. Scand J Immunol 62:45-54). Despite the feasibility to induce tolerogenic DCs, DC-based therapy is limited by the requirement for isolation and ex vivo manipulation of autologous DCs {i.e., DCs from the very patient to be treated); this necessitates individualized DC preparation, which may be too costly and complex for routine clinical application.

[06] Given the importance of regulatory T cells in immune tolerance (O'Garra, A., and P. Vieira. 2004. Regulatory T cells and mechanisms of immune system control. Nat Med 10:801-805; Baecher-Allan, C, and D. A. Hafler. 2006. Human regulatory T cells and their role in autoimmune disease. Immunol Rev 212:203-216; Sakaguchi, S., M. Ono, R. Setoguchi, H. Yagi, S. Hori, Z. Fehervari, J. Shimizu, T. Takahashi, and T. Nomura. 2006. Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol Rev 212:8-27), the second approach is to isolate autologous, antigen-specific regulatory T cells from a patient, expand them ex vivo and, subsequently, adoptively transfer them into the same patient ( June, C. H., and B. R. Blazar. 2006. Clinical application of expanded CD4+25+ cells. Semin Immunol 18:78-88; Masteller, E. L., Q. Tang, and J. A. Bluestone. 2006. Antigen-specific regulatory T cells- ex vzvo expansion and therapeutic potential. Semin Immunol 18:103 -110; Roncarolo, M. G., and M. Battaglia. 2007. Regulatory T-cell immunotherapy for tolerance to self antigens and alloantigens in humans. Nat Rev Immunol 7:585-598; Raimondi, G., M. S. Turner, A. W. Thomson, and P. A. Morel. 2007. Naturally occurring regulatory T cells: recent insights in health and disease. Crit Rev Immunol 27:61-95; Raimondi, G., M. S. Turner, A. W. Thomson, and P. A. Morel. 2007. Naturally occurring regulatory T cells: recent insights in health and disease. Crit Rev Immunol 27:61-95). It is, however, difficult to isolate sufficient quantities of antigen-specific regulatory T cells from a patient because of the lack of a reliable cellular marker for these cells and their low frequency in total circulating T cells. There are also regulatory concerns associated with the use of blood- derived cells. Further, the long-term efficacy of adoptively transferred regulatory T cells, which may depend on the in vivo survival of the infused cells, remains to be assessed. Hence, as with other cell-based modalities, regulatory T cells-based adoptive therapy, while proven effective in animal models, is difficult to be translated into clinical trials due to the technical, regulatory, and economic concerns (Bluestone, J. A., A. W. Thomson, E. M. Shevach, and H. L. Weiner. 2007. What does the future hold for cell- based tolerogenic therapy? Nat Rev Immunol 7:650-654; Verbsky, J. W. 2007. Therapeutic use of T regulatory cells. Curr Opin Rheumatol 19:252-258).

[07] The third approach involves inducing antigen-specific desensitization via cell-free immunization using an antigen or peptide fragments encompassing a relevant epitope ( Larche, M., and D. C. Wraith. 2005. Peptide-based therapeutic vaccines for allergic and autoimmune diseases. Nat Med 11 :S69-76). In theory, peptide immunization works by allowing in vivo presentation of the antigen in the context of nonprofessional APCs or steady-state (quiescent) DCs, both of which have been shown to favor tolerance induction (Steinman, R. M., D. Hawiger, and M. C. Nussenzweig. 2003. Tolerogenic dendritic cells. Annu Rev Immunol 21 :685-711). Although it bypasses limitations associated with cell-based therapies, peptide immunization alone lacks the active control of the context in which the antigen is presented, which critically affects the efficacy in tolerance induction. For instance, while low doses of antigens are found to cause favorable responses, high doses often promote antigen presentation in an inflammatory context, leading to adverse immune responses (Larche, M. 2007. Update on the current status of peptide immunotherapy. J Allergy Clin Immunol 119:906-909). Especially, inevitable exposure of humans to environmental antigens in uncontrolled doses and inflammatory milieu renders antigen-based therapy unreliable for clinical applications.

[08] There is a continuing need in the art to identify safe and effective ways to treat diseases that have an autoimmune component, including classical autoimmune diseases as well as transplant rejection, allergies, and atherosclerosis.

SUMMARY OF THE INVENTION

[09] According to one embodiment of the invention a method is provided for treating a patient with an allo-immune or auto-immune disease. A disease-specific antigen is administered to the patient. A tolerogenic adjuvant is administered to the patient. The tolerogenic adjuvant is selected from the group consisting of glucocorticoids, vitamin D3, and vitamin D3 analogues. Symptoms of the disease are thereby reduced, delayed, or eliminated.

[10] Another aspect of the invention is a kit for treating a patient. The kit comprises a disease- specific antigen or a nucleic acid encoding a disease-specific antigen; and a tolerogenic adjuvant selected from the group consisting of glucocorticoids, vitamin D3, and vitamin D3 analogues. [11] A further aspect of the invention is a method of treating a patient with a chronic inflammatory disorder. A disease-specific antigen is administered to the patient. A tolerogenic adjuvant is administered to the patient. The tolerogenic adjuvant is selected from the group consisting of glucocorticoids, vitamin D3, and vitamin D3 analogues. Symptoms of the disease are thereby reduced, delayed, or eliminated.

[12] An additional aspect of the invention is a population of CDl Ic⁺ dendritic cells (DC) in which the percentage of IL-IO producing DC is at least two-fold greater than in a control population of DC in blood of a human who has not been immunized with an antigen or treated with an immunosuppressant.

[13] Yet another aspect of the invention is a population of CDl Ic⁺ dendritic cells (DC) in which the percentage of IL-IO producing DC is at least two-fold greater than in a control population of DC in blood of a human that has been immunized with an antigen or treated with an immunosuppressant.

[14] A further aspect of the invention is a population of CD4⁺CD25⁺ T cells in which the percentage of CD4⁺CD25⁺Foxp3⁺ Treg is at least twice that in a control population of CD4⁺CD25⁺ T cells in blood of a human who has not been immunized with an antigen or treated with an immunosuppressant.

[15] Another embodiment or the invention is a population of CD4⁺CD25⁺Foxp3⁺ Treg in which antigen-specific Treg comprise at least a two-fold greater proportion than in a population of CD4⁺CD25⁺Foxp3⁺ Treg in blood of a human that not been immunized with the antigen or treated with an immunosuppressant.

[16] A further aspect of the invention is a population of memory CD4⁺CD25⁺Foxp3⁺ Treg that remain viable for at least 120 days after discontinuation of antigenic stimulation and that are capable of proliferation in response to recurring antigenic stimulation. [17] These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with methods of treating diseases and populations of cells induced in patients by the treatment methods.

BRIEF DESCRIPTION OF THE DRAWINGS

[18] Fig. IA-ID. Suppressed immunization induces immune tolerance against established DTH. Fig. IA, BALB/c mice with pre-established DTH were divided into four groups and each was treated with an indicated combination of DEX and OVA_323-33C,. All groups were then retested for DTH at a footpad. Fig. IB, The test and negative control groups were tested again 4-5 months later. Fig. 1 C, Treg (CD4⁺CD25⁺Foxp3⁺) in blood samples, taken immediately before (open bar) and 48 h after (filled bar) the second DTH test, were quantified relatively to total CD4⁺ cells. Fig. ID, The same blood taken at 48 h from non- treated mice (indicated as "Control") or mice treated with DEX and peptide ("Test") was further analyzed for the presence of OVA_323-339-specific Treg. White cells were labeled with CFSE, stimulated in culture with OVA_323-33C), and analyzed for CFSE dilution (cell division) by flow cytometry, gating on CD4⁺CD25⁺Foxp3⁺ cells (Treg). The observed cell division was antigen-dependent because no CFSE dilution was seen in the absence of OVA₃₂₃-₃₃₉ (data not shown). Bar, mean and SD of 2-3 independent experiments, each employing 6 mice per group (n = 6); *, P < 0.05 between the indicated pair.

[19] Fig. 2A-2E. Suppressed immunization blocks DC maturation and preferentially expands antigen- specific Treg. Fig. 2A, DOl 1.10 mice were divided into four groups, each injected at a hind footpad with the indicated combination of DEX and OVA_323-33C,. Three days later, draining LN (popliteal) were recovered and analyzed by flow cytometry, gating on CDl Ic⁺ cells (total DC). Immature (CD83^"CD86^low) and mature (CDSS⁺CDSo¹¹¹⁸¹¹) DC were quantified relatively to total DC. Fig. 2B, IL-10-producing (IL-IO⁺) DCs were quantified relatively to total DC. Fig. 2 C, Activated effector T cells (Teff, CD4⁺CD25⁺Foxp3^") and Treg (CD4⁺CD25⁺Foxp3⁺) in same LN were quantified relatively to total CD4⁺ T cells. Fig. ID, CFSE-labeled DOl 1.10 CD4⁺ T cells were co- injected with OVA323-339 into BALB/c mice that had been either pretreated (indicated as "DEX + peptide") or non-pretreated ("peptide") with DEX. As a negative control, the T cells were also injected without OVA₃₂₃_₃₃₉ into non-pretreated mice ("PBS"). Four days later, total cells from draining LN were stained for KJ1-26, CD25, and Foxp3, and analyzed by flow cytometry, gating on KJl-26⁺/CD25⁺ cells. Proliferating Treg are shown in the upper (Foxp3⁺) left quadrant; proliferating Teff, the lower (Foxp3^~) left quadrant. Fig. 2E, Treg from DOl 1.10 mice treated with DΕX and OVA_323-339 were co-cultured with Teff (CD4⁺CD25^~) from naϊve DOl 1.10 mice, along with syngeneic accessory cells and OVA_323-339. Proliferation was assessed by ³H-thymidine incorporation. Treg¹ and Treg² denote Treg from immunized and nonimmunized DOl 1.10 mice, respectively. Bar, mean and SD from 2-4 independent experiments, each using at least 2 mice per group (n > 2); *, P < 0.05 between the indicated pair.

[20] Fig. 3A-3C. Suppressed immunization protects animals from predisposed autoimmune diseases. Fig. 3A, Pre-diabetic NOD mice (n = 6 per group) were treated with PBS (open circle), DΕX alone (triangle), B:9-23 alone (square), or both DΕX and B:9-23 (filled circle). Mice were checked weekly for glycosuria. Shown is one of two independent experiments with similar results. The difference between the "DΕX and B:9-23" and "DΕX alone" or "B:9-23 alone" groups is statistically significant (P < 0.002). Fig. 3B, Mice that were treated with DΕX and B: 9-23 and subsequently remained diabetes-free at 61 weeks of age were rechallenged with B: 9-23. Splenic CD4+ T cells were isolated 5 day later, labeled with CFSΕ, and stimulated in culture with B:9-23 (indicated as "Test") or OVA_323-339 ("Control Ag"). Control splenic T cells were obtained from nonimmunized prediabetic NOD mice that had been similarly rechallenged with B: 9-23. The control cells were stimulated in culture with B:9-23 ("Control"). CFSΕ dilution was analyzed 3 days later by flow cytometry, gating on CD4+CD25+ cells (Treg and activated Teff). Shown are quadrant plots separating Treg (Foxp3+), Teff (Foxp3-), antigen-specific (CFSΕ-low), or nonantigen-specific (CFSΕ-high) cells. The percentage of cells in each quadrant is indicated. Shown is 1 of 2 independent experiments with similar results. Fig. 3 C, Prediabetic NOD mice were treated with PBS (indicated as "Control") or DΕX and B:9-23 ("Test"), as described in A. At the completion of the treatment, blood samples were taken from both groups. White cells were labeled with CFSE and stimulated with B:9-23 in culture. As control for antigen specificity, an aliquot of white cells from the test mice were stimulated with OVA_323-339 ("Control antigen"). Cells were analyzed by flow cytometry as described in B. Antigen-specific cells were identified by CFSE dilution and quantified relatively to total CD4+CD25+ cells gated. Bar, mean and SD of 2 independent experiments employing 2-3 mice per group; *, P < 0.0016 between the indicated pair.

DETAILED DESCRIPTION OF THE INVENTION

[21] The inventors have developed methods for regulating undesired antigen-specific T cell responses. The methods employ a combination of agents which can be packaged as a kit. Upon practice of the method, populations of cells are expanded within the body that have different compositions from those in untreated bodies. The populations of cells can be extracted for analysis.

[22] Unlike most known T cells exerting immune effector functions (i.e., so-called "conventional" or effector T cells), regulatory T cells are those which exert regulatory, immunosuppressive activities instead (Shevach, E. M. 2006. From vanilla to 28 flavors: multiple varieties of T regulatory cells. Immunity 25:195-201). Regulatory T cells play a pivotal role in governing peripheral tolerance (i.e., tolerance to self or auto antigens) by inhibiting overactive T cells, and by dampening B cell, DC, and natural killer cell activities (Raimondi, G., M. S. Turner, A. W. Thomson, and P. A. Morel. 2007. Naturally occurring regulatory T cells: recent insights in health and disease. Crit Rev Immunol 27:61-95). While such immunosuppressive activities serve as the functional definition of a regulatory T cell, there are many different types of such cells based on cell lineage, phenotypic markers, and mode of action (Shevach, E. M. 2006. From vanilla to 28 flavors: multiple varieties of T regulatory cells. Immunity 25:195-201). The most well studied type (also the subject of this invention) is the CD4⁺CD25⁺Foxp3⁺ cells (called Treg), which are developed in the thymus, constitute 5-10% of total CD4+ T cells, and function through direct cell-cell contact. Studies in animal models show that adoptive transfer of Treg prevents or cures transplant rejection or autoimmune disease (Roncarolo, M. G., and M. Battaglia. 2007. Regulatory T-cell immunotherapy for tolerance to self antigens and alloantigens in humans. Nat Rev Immunol 7:585-598), suggesting that this type of cells may be manipulated for therapy. Other types of regulatory T cells may have a similar potential but are currently less well established.

[23] As mentioned earlier, a unique therapeutic advantage of regulatory T cells in general over conventional immunosuppressive drugs is that the former are antigen-specific. That is because like any T cell, a regulatory T cell is able to recognize a specific antigen via T cell receptors (TCRs). Triggering of the TCRs leads to activation and clonal expansion of the regulatory T cell, the outcome of which is increased numbers of antigen-specific regulatory T cells capable of migrating to, and suppressing immune reactions at, the site inflammation without general immunosuppression. It is the last two properties, being antigen-specific and antigen-responsive, that makes these cells particularly well-suited for treating autoimmunity, transplantation rejection, and allergy, for example, where untoward immunity causes disease.

[24] Suppressed immunization is a method for in vivo manipulation of regulatory T cells using a chemically-defined antigen and tolerogenic adjuvant combination. This method makes isolation and ex vivo expansion of regulatory T cells unnecessary. Instead, it enables direct sensitization and expansion of antigen-specific Treg in vivo via simple immunization. Thus, it is highly useful for human therapy. The two agents, antigen and adjuvant can be administered together at the same time, or separated in time over a course of 1-10 minutes, 1-72 hours, or 1-14 days. If administered at the same time, they may be mixed together or administered in separate vehicles.

[25] Suppressed immunization uses a pharmaceutical immunosuppressant as adjuvant that exerts three effects. First, the immunosuppressant preferentially dampens the proliferation of conventional T cells (Fig. 2) and induces their apoptosis (programmed cell death) (Chen, X., T. Murakami, J. J. Oppenheim, and O. M. Howard. 2004. Differential response of murine CD4+CD25+ and CD4+CD25- T cells to dexamethasone-induced cell death. Eur J Immunol 34:859-869), while preserving the activity of Treg (Fig. T). Hence, the immunosuppressant works to block ongoing pathogenic immune reactions by conventional T cells and prevent them from responding to the immunogen applied. Second, the immunosuppressant is anti-inflammatory and blocks inflammation- stimulated maturation of DC. Third, the immunosuppressant induces differentiation of IL- 10-producing, tolerogenic DC, which, in conjunction with an immunogen (antigen), can preferentially stimulate the proliferation and expansion of antigen-specific Treg (Fig. T). Hence, the immunosuppressant serves as an adjuvant for active control of the antigen- presentation context, thereby ensuring that antigenic immunization forcefully drives the induction of Treg.

[26] To induce Treg that respond to and are capable of suppressing a particular immune disease, suppressed immunization also uses a disease-specific immunogen derived from a disease-specific antigen. The disease-specific antigen can be either a disease-causing antigen, such as an autoimmune antigen (Fig. 3), or a disease-induced antigen, such as a tissue-specific antigen released to circulation due to autoimmune destruction of the tissue. In both cases, the use of disease-specific immunogen in conjunction with the adjuvant described above allows induction of disease-specific Treg.

[27] Once induced, disease-specific Treg block pathogenic immunity and prevent it from causing overt disease (Fig. 3). The induced Treg also develop into "memory-like" cells that persist over a long period of time and are capable of recall expansion in response to antigenic rechallenge (Figs. 1 and 3). These memory-like cells are novel and may be unique to suppressed immunization. They are also particularly useful because most immune diseases are chronic or recurrent in nature, and, thus, there is compelling need for long-term protection against disease recurrence. As such recurrence is likely preceded or followed by the disease- specific antigen, the presence of memory-like Treg may provide a surveillance and suppression mechanism that blocks overt recurrence.

[28] In summary, suppressed immunization works by selectively expanding disease-specific Treg that suppress ongoing pathogenic immunity as well as providing long-term surveillance against disease recurrence. Further, although our studies have thus far examined only Treg, suppressed immunization, in principle, may also be useful for in vivo expansion of other regulatory T cell types that respond similarly to the adjuvant and immunogen combination.

[29] Autoimmune diseases include but are not limited to Acute disseminated encephalomyelitis (ADEM); Addison's disease; Ankylosing spondylitis; Antiphospholipid antibody syndrome (APS); Diabetes mellitus type 1 ; Guillain-Barre syndrome (GBS); Hashimoto's disease; Idiopathic thrombocytopenic purpura; Goodpasture's syndrome; Graves' disease; Lupus erythematosus; Multiple sclerosis; Myasthenia gravis; Rheumatoid arthritis; Pemphigus; Sjogren's syndrome; Temporal arteritis; Aplastic anemia; Autoimmune hepatitis; Autoimmune Oophoritis; Celiac disease; Crohn's disease; Gestational pemphigoid; Kawasaki's Disease; Mixed Connective Tissue Disease; Opsoclonus myoclonus syndrome (OMS); Ord's thyroiditis; Pernicious anaemia; Polyarthritis in dogs; Primary biliary cirrhosis; Reiter's syndrome; Takayasu's arteritis; Vitiligo (also known as leukoderma); Warm autoimmune hemolytic anemia; and Wegener's granulomatosis.

[30] Allergic diseases include allergic rhinitis, allergic asthma, atopic dermatitis, allergic gastroentheropathy, anaphylaxis, urticaria and angioedema.

[31] Allo-immune diseases include transplantation rejection and graft versus host disease, as well as fetus, neonates, and pregnant mothers. Immune rejection of tissue transplants, including lung, heart, liver, kidney, pancreas, bone marrow, and other organs and tissues, is mediated by immune responses in the transplant recipient directed against the transplanted organ. Allogeneic transplanted organs contain proteins with variations in their amino acid sequences when compared to the amino acid sequences of the transplant recipient. Because the amino acid sequences of the transplanted organ differ from those of the transplant recipient they frequently elicit an immune response in the recipient against the transplanted organ. Rejection of transplanted organs is a major complication and limitation of tissue transplant, and can cause failure of the transplanted organ in the recipient. The chronic inflammation that results from rejection frequently leads to dysfunction in the transplanted organ.

The antigen for an future or past transplant recipient may include one or more major histocompatibility antigens, e.g. HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, HLA- DP, etc., and may comprise a cocktail of such antigens, where the antigens will include those not matched between the recipient and the donor.

[32] Antigens may be protein or nonprotein, including other biomolecules such as pyrophosphates and gangliosides. Protein antigens for treating allergic diseases may include proteins of Alternaria altemata (Alt a I), Artemisia vulgaris (Art v II), Aspergillus fumigatus (Asp f II), Dermatophagoides far. (Der fl), Dermatophagoides pteron. (Der p I, Der p III, Der p IV, Der p VI and Der p VIII). Other antigens may include proteins from domestic and farm animals, fungal antigens including from Basidiomycetes such as Ustilago, Ganoderma, Alternaria, Cladosporium, Aspergillus, Sporobofomyces, Penicillium, Epicoccum, Fusarium, Phoma, Borrytis, Helminthosporium, Stemphylium and Cephalosporium; Phycomycetes such as Mucor and Rhizopus; and Ascomycetes such as Eurotium and Chaetomium. Plant antigens associated with allergies may be used as antigens including club mosses, ferns, conifers, flowering plants, grasses, sedges, palms, cattails, nettles, beeches, chenopods, sorrels, willows, poplars, maples, ashes, ragweeds (antigen E, antigen K and Ra3) and sages, or proteinaceous plant products such as those found in latex products. Insect antigens with allergies include proteins from the honeybee, yellow jacket, hornet, wasp and fire ant. Food allergens can be used as antigens, including those from crustaceans, mollusks, fish, legumes, seeds, nuts, berries, egg white, buckwheat, and milk.

[33] Any antigen associated with such diseases as described can be used, including acetylcholine receptor, oxidized LDL, heat shock protein, proteolipid protein (PLP), myelin basic protein (MBP), myelin oligodendrocyte protein (MOG), cyclic nucleotide phosphodiesterase (CNPase), myelin-associated glycoprotein (MAG), and myelin- associated oligodendrocytic basic protein (MBOP), alpha-B-crystalin (a heat shock protein), OSP (oligodendrocyte specific-protein), type II collagen, hnRNP, A2/RA33, Sa, filaggrin, keratin, citruUine, cartilage proteins including gp39, collagens type I, III, IV, V, IX, XI; HSP-65/60, IgM (rheumatoid factor), RNA polymerase, hnRNP-Bl, hnRNP-D, cardiolipin, aldolase A, tyrosine phosphatase IA-2, IA-2β, glutamic acid decarboxylase (GAD), carboxypeptidase H, insulin, proinsulin, heat shock proteins (HSP), glima 38, islet cell antigen 69 KDa (ICA69), p52, two ganglioside antigens (GT3 and GM2-1), an islet cell glucose transporter (GLUT 2), desmoglein-3, SSA (Ro); SSB (La); and fodrin. Antigens can be administered by any route useful for immunizations, including but not limited to subcutaneous, intramuscular, intradermal, oral, etc. The antigens can be administered in a carrier, such as mineral oil, or Freund's adjuvant.

[34] Tolerogenic adjuvants which can be used in the methods described include but are not limited to glucocorticoids, vitamin D3, vitamin D3 analogues which are tolerogenic adjuvants, such as lα,25(OH₂)D3, short chain fatty acids, such as butyrate, rapamycin, sanglifehrin, 15-desoxyspergualin, and mycophenolate mofetil. Glucocorticoids which may be used include Hydrocortisone (Cortisol), Cortisone acetate, Prednisone, Prednisolone, Methylprednisolone, Dexamethasone, Betamethasone, Triamcinolone, Beclometasone, Fludrocortisone acetate, Deoxycorticosterone acetate (DOCA), Aldosterone, and dexamethasone. Many analogs of vitamin D3 are known. These can readily be tested to determine that they retain the tolerogenic properties of vitamin D3 itself.

[35] Kits for practicing the methods comprise both a disease-specific antigen as well as a tolerogenic adjuvant. These may be packaged together in a single or divided container. They may be in the same or different format, such as liquid, frozen, dried, tablet, etc. They may optionally require reconstitution prior to administration. The kit may comprise instructions regarding dosages, side effects, scheduling, etc.

[36] Populations of cells as are induced using the methods described above include those that are induced in the body and those that are later removed, for example for analysis. The populations can be stored, saved, and used for re-infusion at a later time. The method described above induces changes in populations of cells and their relative proportions of subsets of cells. Subsets can increase relative to larger sets or relative to control populations by at least 1.5 fold, at least 2 fold, at least 3 fold, at least 5 fold, or at least 10 fold.

[37] The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLE 1 -Materials and Methods

Mice and reagents

[38] DO 11.10 TCR- transgenic B ALB/c mice were bred in the University of Illinois College of Medicine at Rockford animal facility. Other mice were from the Jackson Laboratory. All animals were maintained in pathogen-free rooms and used in accordance with the institutional guidelines for animal care. The OVA_323-339 (ISQAVHAAHAEINEAGR; SEQ ID NO:1) and B:9-23 (SHLVEALYLVCGERG; SEQ ID NO: 2) peptides were from AnaSpec. DEX, hen ovalbumin (OVA; grade VII), and hen lysozyme were from Sigma- Aldrich. Incomplete Freund's adjuvant was from Pierce Biotechnology. All antibodies were from eBioscience. The mouse CD4⁺ T cell negative selection kit was from Miltenyi Biotec. Carboxyfluorescein succinimidyl ester (CFSE) was from Invitrogen.

Antigen sensitization

[39] OVA in incomplete Freund's adjuvant was injected subcutaneously into BALB/c mice (100 μg/mouse) twice in a two-week interval. The mice were also similarly sensitized for hen lysozyme. Delayed-type hypersensitity (DTH) to either sensitizing antigen was determined by rechallenge at a footpad (10 μg/injection) and measuring the net increase in footpad thickness (swelling) at 24 h. Suppressed immunization

[40] Mice were injected four times (on days 1, 4, 7, and 10) with DEX into the two hind footpads (8 μg/footpad). For the day-7 injection, OVA_323-339 (1 μg/footpad) was co- injected with DEX. This regimen was given twice in a two-week interval.

Analysis of Tr eg in the blood

[41] White cells from tail blood were blocked with anti-CD16/32 mAb, immunostained for CD4 and CD25, fixed (1% paraformaldehyde) and permeablized (0.5% Tween-20), and intracellularly immunostained for Foxp3. CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg), were counted relatively to total CD4⁺ cells by flow cytometry. To determine the antigen specificity of blood Treg, white cells were labeled with CFSE (Invitrogen) and stimulated in a 96-well plate with the immunizing peptide (10 μg/ml), or an irrelevant peptide as control, in the presence of syngeneic accessory cells (2.5 x 10⁴/well) and IL-2 (800 IU/ml). Three days later, cells were stained for CD4, CD25, and Foxp3 as described above, and analyzed for CFSE dilution by flow cytometry, gating on Treg.

Analysis of DC and T cells in draining lymph nodes

[42] Total cellular content of popliteal lymph nodes (LN) was blocked with anti-CD16/32 mAb, immunostained with anti-CDl lc, anti-CD83, and anti-CD86 mAbs (for DC analysis); or with anti-CD4, anti-CD25, and anti-Foxp3 mAbs (for T cell analysis); and analyzed by flow cytometry. To detect IL-IO⁺ DC, LN cells were stained with anti- CDl lc, fixed (1% paraformaldehyde) and permeablized (0.5% Tween-20), and intracellularly immunostained for IL-10. In vivo proliferation assay

[43] CFSE-labeled DOl 1.10 TCR-transgenic CD4⁺ T cells (2.5 ~ 5 x 10⁵/footpad) were co- injected with OVA₃₂₃_₃₃₉ (1 μg) into a footpad of BALB/c mice that had been pretreated with DEX on days -6, -3, and 0. Draining LN were recovered on day 4; total cellular content was immunostained with KJ 1-26, anti-CD25, and anti-Foxp3 mAbs and analyzed by flow cytometry.

In vitro suppression assay

[44] The assay was performed as previously described (3).

Treatment of NOD mice

[45] Female NOD mice (6 weeks of age) were injected 4 times (on days 1, 4, 1, and 10) with DEX into the two hind footpads (8 μg/footpad). For the day-7 injection, B:9-23 (1 μg/footpad) was co-injected with DEX. This regimen was given twice in a two-week interval. Glycosuria was checked weekly using Diastix strips (Bayer Corp.). Mice tested positive (> 250 mg/dl) twice consecutively were deemed diabetic.

Analysis of Tr eg in the spleen

[46] Mice were rechallenged (day 0) at a footpad with the immunizing peptide emulsified in incomplete Freund's adjuvant (50 μg/footpad). On day 5, splenic CD4⁺ T cells were isolated, labeled with CFSE, and stimulated in a 96-well plate with the immunizing peptide (10 μg/ml) or an irrelevant peptide (as control), in the presence of naϊve syngeneic accessory cells (2.5 x 10⁴/well) and IL-2 (800 IU/ml). On day 8, cells were stained for CD4, CD25, and Foxp3 and analyzed for CFSE dilution by flow cytometry, gating on CD4⁺CD25⁺ cells. Statistic analysis

[47] Two-sided Student's t test was used, except for analysis of diabetic incidence, where the Kaplan-Meier method and Log-Rank test were applied. Differences are deemed significant if/^J> < 0.05.

EXAMPLE 2— LONG-TERM AND SPECIFIC DESENSITIZATION TO DTH ANTIGEN

ASSOCIATED WITH INCREASED T_REG AND ANTIGEN SPECIFIC T_REG

[48] To test the idea of suppressed immunization, we first sensitized BABL/c mice for hen ovalbumin (OVA). Using the resulting delayed-type hypersensitivity (DTH) to OVA as a model for recall response, we tested suppressed immunization in these mice using an OVA-derived, MHC Il-restricted peptide (OVA₃₂₃_₃₃₉) (4) as immunogen and DEX as adjuvant (test group). Some of the DTH-positive mice were control-immunized, with either or both of the immunosuppressant and the peptide replaced by PBS (control groups).

[49] Two weeks after the completion of the immunization, all groups were retested for DTH. While the control groups remained fully sensitive to the recall antigen, the test group was markedly desensitized to OVA (Fig. IA). The desensitization was OVA-specific because the test group showed normal DTH to an irrelevant protein, hen lysozyme (data not shown). Importantly, the test group remained hyporesponsive to OVA when rechallenged 4-5 months later (Fig. IB), suggesting the establishment of long-term tolerance to the antigen. Consistent with such tolerance, blood samples taken from the test group at 48 h following the antigen rechallenge showed an elevated count of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) compared with that of the non-treated control group (Fig. 1C). At least part of the increased Treg count consisted of OVA₃₂₃_₃₃₉-specific Treg, because they proliferated in culture in response to OVA_323-33Q (Figure ID), but not to an irrelevant peptide (data not shown). In comparison, no such Treg were detected in the blood of the non-treated control group (Fig. ID).

EXAMPLE 3— DEX BLOCKS DC MATURATION AND CONTRIBUTES TO T_REG

EXPANSION

[50] Treg expansion is known to be linked to the function of immature dendritic cells (DC) (5, 6), and DEX was previously reported to prevent DC maturation in vitro (7, 8) suggesting that DEX might help expand Treg by increasing immature DC. We therefore analyzed the change in the ratio of mature to immature DC in the draining lymph node (LN) (popliteal) following footpad injection of DEX and OV A_323-33^ CD83 is a maturation marker for DC (9, 10). Using CD83 in conjunction with CD86, a costimulator upregulated in DC upon maturation, we were able to identify immature and mature DC as CO\ \c⁺CDS3^'CO86^hw and CDI I C⁺CDSS⁺CDSO¹¹'⁸¹¹ cells, respectively. Initial experiments in BALB/c mice showed that DEX blocked DC maturation in the draining LN (data not shown). However, it was difficult to detect corresponding changes in antigen-specific Treg as they represented only a tiny fraction of total Treg in the LN.

[51] Consequently, to allow simultaneous observation of DC and antigen-specific Treg, we switched to DOl 1.10 T cell receptor (TCR)-transgenic mice, where OVA_323-339-specific CD4⁺ T cells are abundant (4). Injecting the mice with DEX alone increased immature DC count while decreasing mature DC count, indicating that DEX blocks DC maturation in vivo (Fig. 2A). Injecting the mice with OVA₃₂₃_₃₃₉ alone, on the other hand, stimulated DC maturation, as was indicated by an increase in mature DC count and decrease in immature DC count. Co-injection of both DEX and OVA_323-339 led to an effect similar to that from DEX alone, indicating the dominance by DEX. Additional experiments showed that DEX augmented the differentiation of IL-IO⁺ DC in the presence of OVA_323-339 (Fig. 2B), suggesting that DEX not only blocks DC maturation, but also alters DC function. In parallel to the immature DC count, total Treg count was also increased in a DEX- dependent manner (Fig. 2C). Importantly, DEX and OVA_323-339 combined gave rise to more Treg than did DEX alone, suggesting antigen-specific expansion of Treg even in the presence of DEX.

EXAMPLE 4- DEX AND OVA₃₂₃-S_3O CAUSE ANTIGEN-SPECIFIC EXPANSION OF T_REG

[52] To determine whether the combination of DEX AND OV A_323-330 indeed cause antigen- specific expansion of Treg, we isolated CD4⁺ T cells from DOl 1.10 mice, labeled them with carboxyfluorescein succinimidyl ester (CFSE) to allow tracking of cell division (11) and adoptively transferred the cells into syngeneic BALB/c mice, where they were stimulated with DEX and OVA_323-339. Upon recovery and subsequent flow cytometric analysis of the donor cells, we found that both the Foxp3⁺ (Treg) and Foxp3^" (Teff) subsets (12) proliferated in the absence or the presence of DEX (Fig. 2D). However, the fraction of proliferating Treg in the presence of DEX rose to twice of that in the absence of DEX. At the same time, the presence of DEX slightly dampened the proliferation of Teff. Thus, the overall effect of combining DEX and antigen is preferential proliferation of Treg over Teff Further experiments showed that the Treg expanded in the presence of DEX are functional, as they effectively inhibited the proliferation of Teff in co-culture (Fig. 2E). In aggregate, these data established the capability of DEX to expand antigen- specific Treg, which may at least partly account for the efficacy of suppressed immunization.

[53] We further tested suppressed immunization in the NOD autoimmune diabetes model (13) using DEX and the insulin-derived, MHC II-restricted peptide antigen B:9-23 (14, 15). While non-treated mice developed the disease with a median incidence of 19 weeks, mice treated with either DEX or B:9-23 showed a delayed median incidence of 24.5 and 26 weeks, respectively (Fig. 3A). In contrast, most mice treated with both DEX and B:9-23 remained disease- free during the entire period of the experiment (35 weeks), indicating that suppressed immunization is effective for delaying or preventing the progression of autoimmune diabetes. Moreover, about 40% of the animals (5 out of 12 mice) in this group lived for 61 weeks without diabetes; and upon rechallenge of these animals with B: 9-23, antigen-specific Treg could be detected in the spleen, the primary site of memory T cells (Fig. 3B). These Treg showed greater sensitivity to restimulation with B:9-23, but not with control antigen, in comparison with control Treg (naϊve Treg isolated from non- immunized, prediabetic mice). They also dominated the proliferative response to the recall antigen over splenic Teff (Foxp3^~), suggesting that these Treg might be able to elicit adaptive response to recall stimulation.

EXAMPLE 5- SUPPRESSED IMMUNIZATION PREFERENTIALLY PRIMES ANTIGEN-

SPECIFIC TREG

[54] To ascertain that suppressed immunization can preferentially prime antigen-specific Treg during the immunization phase and is therefore responsible for the generation of the "memory-like" Treg, we monitored the appearance of antigen-specific Treg and Teff in two different groups of prediabetic NOD mice that had just been treated with either DEX and B:9-23 (test) or PBS (control). At the completion of the treatment, peripheral blood was taken from the animals, and white cells were restimulated in culture with B: 9-23. Compared with control group, the test group showed a significant increase in antigen- specific Treg, but not in Teff, that proliferated specifically to B:9-23 in culture (Fig. 3C), confirming that antigen-specific Treg are preferentially expanded during the immunization phase. Collectively, these results suggest that suppressed immunization may stop the progression of autoimmune diseases by preferential priming of antigen- specific Treg and provide long-term protection by generating long-term persistent Treg.

[55] We have shown in this study that DEX can function as a tolerogenic adjuvant when applied in conjunction with a peptide immunogen. The particular effect of DEX on Treg expansion is consistent with prior findings by Chen et al. that DEX alone could alter the ratio of CD4⁺CD25⁺ Treg to CD4⁺CD25^" conventional T cells in vivo in favor of the former (16) and that DEX could also amplify IL-2-dependent expansion of functional CD4⁺CD25⁺Foxp3⁺ Treg in vivo (17). The novel finding from the present study is that when combined with antigenic immunization, the use of DEX can lead to preferential induction of Treg that are antigen-specific and long-term persistent. Furthermore, based on the mechanism revealed here, one may speculate that DEX, or glucocorticoids in general, may not be unique in its ability to serve as a tolerogenic adjuvant. Other small- molecule immunosuppressive drugs, such as vitamin D3 and analogues, cyclosporine A, FK506, and rapamycin, have been shown to have similar pharmacological effects on DC maturation and function (18) and might therefore also be useful for tolerogenic immunization. Lastly, Van Overtvelt et al. recently reported their search for IL-IO- inducing adjuvants and the observation that a combination of 1,25-dihydroxyvitamin D3 and dexamethasone augmented the efficacy of sublingual immunotherapy in a murine asthma model, in association with the induction of CD4⁺CD25⁺Foxp3⁺ Treg (19). Both their work and ours support the notion of tolerogenic adjuvants.

References The disclosure of each reference cited is expressly incorporated herein.

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Claims

WE CLAIM:

1. A method of treating a patient with an allo-immune or auto-immune disease, comprising: administering a disease-specific antigen to the patient; administering to the patient a tolerogenic adjuvant selected from the group consisting of glucocorticoids, vitamin D3, and vitamin D3 analogues; whereby symptoms of the disease are reduced, delayed, or eliminated.

2. The method of claim 1 wherein the disease is an allo-immune disease.

3. The method of claim 1 wherein the disease is an autoimmune disease.

4. The method of claim 1 wherein the disease is an allergic disease.

5. The method of claim 1 wherein the disease is transplant rejection.

6. The method of claim 1 wherein the disease is type I diabetes.

7. The method of claim 1 wherein the disease is multiple sclerosis.

8. The method of claim 1 wherein the disease is irritable bowel disease.

9. The method of claim 6 wherein the transplant is of bone marrow.

10. The method of claim 6 wherein the transplant is of a solid organ.

11. The method of claim 1 wherein the tolerogenic adjuvant is a glucocorticoid.

12. The method of claim 1 wherein the tolerogenic adjuvant is dexamethasone.

13. The method of claim 1 wherein the tolerogenic adjuvant is vitamin D3 or an analogue of vitamin D3.

14. The method of claim 1 wherein the tolerogenic adjuvant is vitamin D3.

15. The method of claim 1 wherein the antigen is administered subcutaneously.

16. The method of claim 1 wherein the antigen is administered by delivering a protein or polypeptide.

17. The method of claim 1 wherein the antigen is administered by delivering a nucleic acid encoding a protein or polypeptide.

18. A kit for treating a patient, comprising: a disease-specific antigen or a nucleic acid encoding a disease-specific antigen; a tolerogenic adjuvant selected from the group consisting of glucocorticoids, vitamin D3, and vitamin D3 analogues.

19. The kit of claim 18 wherein the disease-specific antigen is an allo-immune disease- specific antigen.

20. The kit of claim 18 wherein the disease-specific antigen is an autoimmune disease- specific antigen.

21. The kit of claim 18 wherein the disease-specific antigen is an allergy-specific antigen.

22. The kit of claim 18 wherein the disease-specific antigen is transplant rejection- specific antigen.

23. The kit of claim 18 wherein the disease-specific antigen is type I diabetes-specific antigen.

24. The kit of claim 18 wherein the disease-specific antigen is multiple sclerosis-specific antigen.

25. The kit of claim 18 wherein the disease-specific antigen is irritable bowel disease-specific antigen.

26. The kit of claim 18 wherein the disease-specific antigen is an antigen specific for a disease selected from the group consisting of: atherosclerosis, systemic lupus erythematosus, rheumatoid arthritis, human immunodeficiency virus, periodontitis, systemic inflammation, and Crohn's disease.

27. The kit of claim 22 wherein the transplant is of bone marrow.

28. The kit of claim 22 wherein the transplant is of a solid organ

29. The kit of claim 18 wherein the tolerogenic adjuvant is a glucocorticoid.

30. The kit of claim 18 wherein the tolerogenic adjuvant is dexamethasone

31. The kit of claim 18 wherein the tolerogenic adjuvant is vitamin D3 or an analogue of vitamin D3.

32. The kit of claim 18 wherein the tolerogenic adjuvant is vitamin D3.

33. The kit of claim 18 wherein the kit comprises a disease-specific antigen

34. The kit of claim 18 wherein the kit comprises a nucleic acid encoding a disease-specific antigen.

35. A method of treating a patient with a chronic inflammatory disorder, comprising: administering a disease-specific antigen to the patient; administering to the patient a tolerogenic adjuvant selected from the group consisting of glucocorticoids, vitamin D3, and vitamin D3 analogues; whereby symptoms of the disease are reduced, delayed, or eliminated.

36. The method of claim 35 wherein the disorder is atherosclerosis.

37. The method of claim 35 wherein the disorder is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, human immunodeficiency virus, periodontitis, systemic inflammation, and Crohn's disease.

38. A population of CDl Ic⁺ dendritic cells (DC) in which the percentage of IL-IO producing DC is at least two-fold greater than in a control population of DC in blood of a human who has not been immunized with an antigen or treated with an immunosuppressant.

39. A population of CDl Ic⁺ dendritic cells (DC) in which the percentage of IL-IO producing DC is at least two-fold greater than in a control population of DC in blood of a human that has been immunized with an antigen or treated with an immunosuppressant.

40. A population of CD4⁺CD25⁺ T cells in which the percentage of CD4⁺CD25⁺Foxp3⁺ Treg is at least twice that in a control population of CD4⁺CD25⁺ T cells in blood of a human who has not been immunized with an antigen or treated with an immunosuppressant.

41. The population of claim 40 wherein the population of CD4+CD25+ T cells is antigen- specific.

42. A population of CD4⁺CD25⁺Foxp3⁺ Treg in which antigen-specific Treg comprise at least a two-fold greater proportion than in a population of CD4⁺CD25⁺Foxp3⁺ Treg in blood of a human that not been immunized with the antigen or treated with an immunosuppressant.

43. A population of memory CD4⁺CD25⁺Foxp3⁺ Treg that remain viable for at least 120 days after discontinuation of antigenic stimulation and that are capable of proliferation in response to recurring antigenic stimulation.

4. The method of claim 1 or 35 further comprising the step of monitoring a symptom of the disease to determine that it has been reduced, delayed, or eliminated.