A METHOD OF PROPHYLAXIS AND TREATMENT

Abstract: The present invention relates generally to a method of prophylaxis and treatment of autoimmune disease conditions and agents useful for same. In a related embodiment, the present invention contemplates the use of mucosal antigens and/or agents capable of blocking or otherwise delaying cytotoxic T-lymphocyte (CTL) induction and/or maturation to prevent or at least reduce the likelihood or risk of CTL-mediated autoimmune disease. More particularly, the present invention contemplates mucosa-mediated tolerance to protect against or ameliorate the symptoms associated with autoimmune pathology. Even more particularly, the present invention provides a method for preventing clinical insulin-dependent diabetes mellitus (IDDM) or preventing or reducing or ameliorating the effects of clinical IDDM by the aerosol administration of IDDM-associated autoantigens to mucosal surfaces or agents which block CTL induction and/or maturation.

Description: A METHOD OF PROPHYLAXIS AND TREATMENT FIELD OF THE INVENTION The present invention relates generally to a method of prophylaxis and treatment of autoimmune disease conditions and agents useful for same. In a related embodiment, the present invention contemplates the use of mucosal antigens and/or agents capable of blocking or otherwise delaying cytotoxic T-lymphocyte (CTL) induction and/or maturation to prevent or at least reduce the likelihood or risk of CTL-mediated autoimmune disease. More particularly, the present invention contemplates mucosa-mediated tolerance to protect against or ameliorate the symptoms associated with autoimmune pathology. Even more particularly, the present invention provides a method for preventing clinical insulin- dependent diabetes mellitus (IDDM) or preventing or reducing or ameliorating the effects of clinical IDDM by the aerosol administration of IDDM-associated autoantigens to mucosal surfaces or agents which block CTL induction and/or maturation. BACKGROUND OF THE INVENTION Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country. Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. The increasing knowledge of the immune system in general and cellular immune mechanisms in particular is greatly facilitating the design of therapeutic agents and alternative routes of their administration. One important area of research is the mechanisms underlying cellular immune hypo-responsiveness induced by particular antigens in autoimmune disease conditions. An autoantigen can be assumed to be pathogenic if its administration modifies the natural history of autoimmune disease. Autoantigen-specific strategies of immune tolerance induction have been shown to favourably modify the natural history of experimental autoimmune disease in rodents (1-6). The presentation of soluble protein antigen to mucosal surfaces, classically via the oral route, results in selective suppression of antigen- specific T cell-mediated, delayed-type hypersensitivity (DTH) and IgE responses (1,7,8). "Oral tolerance" has been associated with deviation of immunity away from T-cell (Thl) to antibody (Th2) responses, with the induction of regulatory T cells and, at higher antigen doses, with both T-cell anergy and T-cell deletion (1,9). A particularly debilitating autoimmune condition is insulin-dependent diabetes mellitus (IDDM) which results from the selective destruction of insulin-producing β cells in the islets of the pancreas, within an autoimmune inflammatory "insulitis" lesion (10,11). The primary role of autoreactive T cells in mediating β-cell destruction has been shown directly in two spontaneous animal models of IDDM, the Bio-Breeding (BB) (12) rat and the non- obese diabetic (NOD) mouse (2). Target autoantigens that trigger or drive immune reactivity to β cells not only have diagnostic applications but are potential agents for specific immunotherapy (3-6). Several potentially pathogenic islet/β-cell autoantigens have been identified by their reactivity with circulating antibodies or T cells in rodents and humans with sub-clinical or clinical IDDM, in particular insulin, glutamic acid decarboxylase (GAD) and tyrosine phosphatases of the IA-2 family (13). However, insulin and its precursor, pre-proinsulin, are the only IDDM autoantigens that are β-cell specific. In humans, insulin autoantibodies (IAN) are a risk marker for the development of clinical IDDM (14) and have been detected before autoantibodies to other islet antigens in the offspring of diabetic mothers (15). Increased proliferation of peripheral blood T cells to human insulin can be demonstrated in up to half of sub-clinical and recently-diagnosed IDDM subjects (16), but responses are relatively low. This is possibly because the dominant human T-cell epitope is in proinsulin. A peptide that spans the natural cleavage site between the B chain of insulin and the connecting (C) peptide in proinsulin was reported to elicit T-cell proliferation in a majority of at-risk IDDM relatives (17). In the NOD mouse, IAN are reported to be a risk marker for the development of diabetes (18) and the majority of T-cell clones generated from the insulitis lesion reacted to insulin B- chain, amino acids 9-23 (19). Several studies have evaluated mucosa-mediated tolerance to insulin in the NOD mouse model. For example, Zhang et al. (20) found that oral porcine insulin (1 mg twice weekly) delayed the onset and reduced the incidence of diabetes, and was associated with splenic T cells that partially blocked the transfer of diabetes to young, non-diabetic mice by spleen cells from diabetic mice. Subsequently, Bergerot et al. (21) reported that the regulatory cells induced by oral insulin were CD4+ T cells. However, in earlier studies of oral tolerance to guinea pig myelin basic protein (MBP) in the Lewis rat model of experimental autoimmune encephalomyelitis (EAE) (1), both CD4 and CD8 regulatory T cells that secrete IL-4, IL-10 and TGF-β were described. There is a need to develop effective administration strategies for delivery of antigens to induce suppression of cell-mediated autoimmune conditions. The administration strategies must not only to be immunologically effective but also convenient, direct and safe. In work leading up to the present invention, the inventors investigated the aerosol inhalation and intranasal administration of insulin and its precursor (proinsulin) in an animal model of spontaneous IDDM, the non-obese diabetic (NOD) mouse, and showed that this was effective in reducing pancreatic islet pathology and incidence of diabetes. Furthermore, aerosol insulin induced regulatory CDδγδ T cells which contributed to the prevention of diabetes. As an alternative strategy or a strategy which is capable of being uses in combination with the aforesaid approach, the present invention further provides for blocking or otherwise delaying CTL induction and/or maturation by, for example, blocking interaction between CD40 and CD40 ligand (CD40L). SUMMARY OF THE INVENTION Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. One aspect of the present invention contemplates a method of inducing immune tolerance, including cytotoxic T-lymphocyte (CTL) tolerance, while substantially avoiding CTL immunity in response to a mucosal antigen, said method comprising selecting said mucosal antigen or modifying a mucosal antigen to disable the function of an MHC class I restricted epitope and then administering said selected or modified antigen for a time and under conditions sufficient to prevent or reduce CTL immunity to said mucosal antigen. Another aspect of the present invention contemplates a method of suppressing a cell- mediated autoimmune disease while substantially avoiding CTL immunity in response to a mucosal autoantigen, said method comprising selecting an autoantigen or modifying said mucosal autoantigen to disable the function of an MHC class I restricted epitope and then administering said selected or modified autoantigen for a time and under conditions sufficient to induce tolerance but prevent or reduce CTL immunity to said autoantigen. A further aspect of the present invention contemplates a method of suppressing a cell- mediated autoimmune disease in a subject, said method comprising the administration as an aerosol of an effective amount of an antigen associated with said autoimmune disease for a time and under conditions sufficient to prevent, reduce or otherwise ameliorate autoimmune pathology wherein said antigen substantially lacks a functional MHC class I interacting region. Yet another aspect of the present invention provides a method of preventing, reducing or otherwise ameliorating an autoimmune disease condition in a subject, said method comprising the aerosol administration to said subject of an effective amount of an antigen associated with said autoimmune disease for a time and under conditions sufficient to induce or stimulate immunoregulatory mechanisms which are protective against cell- mediated autoimmune pathology wherein said antigen substantially lacks a MHC class I interacting region. Even yet another aspect of the present invention contemplates a method of preventing, reducing or otherwise ameliorating IDDM, slowly progressive (SP) IDDM or gestational type 1 diabetes in a subject, said method comprising the administration, as an aerosol or other functionally equivalent means, to said subject of an effective amount of an autoantigen associated with IDDM for a time and under conditions sufficient for induction of regulatory T cells and/or other suitable mechanisms sufficient to suppress cell-mediated autoimmune pathology associated with IDDM wherein said autoantigen substantially lacks a functional MHC class I interacting epitope. Still another aspect of the present invention contemplates a method of inducing, suppressing or otherwise ameliorating or preventing IDDM in a subject, said method comprising administering proinsulin peptide truncated at its C-terminal antigen end to disable the function of any MHC class I restricted epitope for a time and under conditions sufficient to prevent or reduce CTL immunity and otherwise induce immune tolerance, including CTL tolerance. Still yet another aspect of the present invention provides a composition comprising an antigen associated with an autoimmune disease in an aerosol formulation including one or more pharmaceutically acceptable carriers and/or diluents. Another aspect of the present invention contemplates a method of inducing CTL tolerance while substantially avoiding CTL immunity in response to a mucosal antigen, said method comprising administering to a subject a nucleic acid molecule or analogue thereof encoding said mucosal antigen but wherein said antigen substantially lacks a functional MHC class I restricted epitope for a time and under conditions sufficient to prevent or reduce induction of CTL immunity. A further aspect of the present invention contemplates a method of preventing or suppressing IDDM while substantially avoiding CTL immunity, said method comprising administering to a subject a nucleic acid molecule or analogue thereof encoding an IDDM- associated mucosal antigen which substantially lacks a functional MHC class I restricted epitope for a time and under conditions sufficient to prevent or reduce the effects of IDDM. Another aspect of the present invention, therefore, contemplates a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering said mucosal antigen or a nucleic acid encoding same for a time and under conditions sufficient to prevent or reduce CTL immunity, before, simultaneously or sequentially with the administration of an antagonist of CTL induction and/or maturation. More particularly, the present invention contemplates a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering said mucosal antigen or a nucleic acid encoding same for a time and under conditions sufficient to prevent or reduce CTL immunity, before, simultaneously or sequentially with the administration of an antagonist of CD40L-CD4 interaction. Even another aspect of the present invention provides a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering an agent for a time and under conditions sufficient to block or otherwise delay CTL induction and/or maturation. More particularly, the present invention provides a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering an agent for a time and under conditions sufficient to block or otherwise delay CTL induction and/or maturation wherein said agent blocks or otherwise disrupts CD40-CD40L interaction. Another aspect of the present invention contemplates a use of a mucosal antigen with an inactive MHC class I epitope in the manufacture of a medicament for the treatment or prophylaxis of a disease condition in a subject. Yet another aspect of the present invention contemplates the use of an agent in the manufacture of a medicament for the treatment or prophylaxis of a disease condition, said agent capable of blocking CTL induction and/or maturation. The present invention further provides for the use in combination of any of the methodologies contemplated above. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graphical representation showing that intranasal human insulin (80 μg) at 56 days of age delays diabetes onset in female NOD mice. Figure 2 is a graphical representation showing that intranasal human proinsulin (40 μg) at 56 days of age delays diabetes onset. Figure 3 is a graphical representation showing that intranasal human proinsulin aa24-36 (40 μg at 56 days of age) delays diabetes onset. Figure 4 is a graphical representation showing that aerosol insulin induces CD8 T cells that suppress transfer of diabetes. NOD male mice (n=16/group) aged 6-9 weeks were injected with pooled splenocytes from recently-diabetic 14-19 week old females, together with either unfractionated (A) or fractionated (B-E) splenocytes from aerosol insulin- or ovalbumin-treated NOD females, and their incidence of diabetes subsequently monitored. In the experiment shown, aerosol donor mice had been treated for 10 consecutive days and then weekly from 49 days of age and were normoglycemic when sacrificed at 156 days of age. Figure 5 is a graphical representation showing that aerosol insulin induces CD8 γδ T cells that suppress transfer of diabetes. Young male NOD mice were co-injected with "diabetic" splenocytes (2 x 10 7 ) and total or fractionated splenic T cells from aerosol-treated mice, as in the legend to Figure 4. The numbers of fractionated cells injected were, in A) ~10 7 total T cells and, from aerosol insulin mice, ~10 7 γδ-depleted T cells or 1.4 x 10 5 γδT cells and, in B), from aerosol insulin mice, ~10 7 total T cells, 2 x 10 6 CD8 T cells, 2 x 10 6 γδ- depleted CD8 T cells or 1.5 x 10 5 CD8 γδ+ve T cells. Figure 6 is a diagrammatical representation showing that adoptive transfer of diabetes is suppressed by CD8 γδ T cells induced by aerosol insulin: summary of 11 experiments. Figure 7 is a graphical representation showing that intranasal human proinsulin aa24-36 (40 μg at 56 days of age) induces CD4 T cells that suppress adoptive transfer of diabetes, in NOD mice. Figure 8 is a graphical representation showing that mouse proinsulin aa26-34 and to a greater extent mouse proinsulin aa25-34 bind to the MHC class I molecule K d . In this assay, expression of K d on the surface of RMA-S cells is monitored by binding of a monoclonal anti-K d antibody detected by fluorescence in a flow cytometer. Addition of a K d -binding peptide to the cells stabilizes K and increases its expression on the cell surface, indicated by a right shift in the signal response. Isotype control = control monoclonal antibody; no peptide = constitutive K d expression; HAP and LLO = peptides from 'flu hemagglutinin and Listeria known to bind to K d and used as positive controls. Figure 9 is a graphical representation showing that mouse proinsulin aa25-34 induces K d - restricted cytotoxic T lymphocytes (CTL) in NOD mice. Six week old female mice were immunised subcutaneously with 50 μg of peptide in Complete Freund's Adjuvant. After 14 days their spleens were removed and splenocytes re-stimulated in vitro with 10 μg/ml peptide for 6 days. Splenocytes were then tested for CTL activity against 51 Cr and pep tide- loaded RMA-S target cells. Figures 10A and , 10B are diagrammatic representations showing that C-terminal truncations enhance the effect of intranasal proinsulin B-C peptide to suppress diabetes. Figure 11 is a diagrammatic representation showing postulated mechanisms of β-cell destruction in type 1 diabetes, including the role of CD40L-CD40 interaction in activating CD8 T cells to become CTLs. Figure 12 is a graphical representation showing (A) systemic and (B) oral priming of CTL require CD40L signalling. (A) A single i.p. injection of 250 μg of control mAb 6C8 (open squares) or anti-CD40L mAb MRl (open circles) was given to C57B1/6 mice one day before challenge with 20 x 10 6 i.v. OVA-coated H-2K bm" ' splenocytes to prime CTL. After 14 days mice were killed and their splenocytes tested for CTL activity, expressed as OVA- specific lysis for representative individual mice. (B) The same doses of 6C8 or MRl were given to mice that were then fed 20 mg OVA on three alternate days. After 14 days, without further priming, mice were killed and their splenocytes tested for CTL activity, this time expressed as lytic units per spleen for individual mice (n=12/group). Figure 13 is a graphical representation showing activation and expansion of OVA-specific CTL by oral OVA requires CD40L. C57B1/6 recipient mice congenic for Ly5.1 were adoptively transferred with 3 x 10 6 transgenic OT-I cells (Ly5.2) and then given control mAb 6C8 or anti-CD40L mAb MRl, 250 μg i.p. Mice from each treatment group were then divided into two groups and fed either PBS or 20 mg OVA in PBS on three alternate days. mAb treatment was repeated before the third feeding. Mice were killed 14 days from the start of feeding and the numbers and phenotype of OT-I cells in their spleens analyzed by flow cytometry. (A) Dot-plots of individual mice show CD44 (left) and CD62L (L- selectin) (right) expression on OT-I cells. The % of cells expressing a high level of CD44 or a low level of CD62L is shown in the corresponding quadrant. The number of OT-I cells per spleen (B) CD44 expression (C) and % CD62L 10 (D) OT-I cells in individual recipient mice treated with 6C8 or MRl and then fed PBS or OVA are shown for a single experiment, but similar results were obtained three experiments. Figure 14 is a graphical representation showing anti-CD40L treatment prevents induction of diabetes by oral OVA in RIP-OVA 10 mice. PJP-OVA 10 mice bearing OT-I and OT-II cells were injected with control mAb 6C8 or anti-CD40L mAb MRl, 250 μg i.p. To mimic low-dose oral tolerance regimens, mice were then fed OVA, 0.5 mg on five alternate days. Blood glucose was measured 12 days after the start of feeding and values above 13 mmol/1 were considered diagnostic of diabetes. Data are pooled from two experiments. Figure 15 is a graphical representation showing anti-CD40L treatment does not limit oral tolerance to systemic priming of CTL. CTL activity in response to i.v. priming with OVA- coated splenocytes (A) or to s.c. priming with OVA in CFA (B) is similarly attenuated by oral OVA in mice treated with control mAb 6C8 and anti-CD40L mAb MRl . Mice were injected with 6C8 or MRl, 250 μg i.p. and then fed either PBS (black squares) or 20 mg OVA in PBS (open circles) on three alternate days. After 14 days or 21 days (not shown), mice were primed systemically and seven days later killed and their splenocytes recovered for a standard in vitro 51 Cr release assay of CTL activity. Primed splenocytes as effectors (E) were tested against 51 Cr-loaded cells as targets (T). Each plot represents an individual mouse. CTL activity plots for individual mice were converted into lytic units from four experiments (C) after priming as in (A) and from two experiments (D) after priming as in (B), in which mice received either PBS or 20 mg oral OVA on three alternate days (C) or PBS or 0.5 mg oral OVA on five alternate days (D). Figure 16 is a graphical representation showing anti-CD40L treatment does not limit oral tolerance to systemic priming of T-cell proliferation (A) and IFN-γ (B) responses, or antibody production (C). Mice (n=3/group) were injected with control mAb 6C8 or anti- CD40L mAb MRl 250 μg i.p. They were then fed either PBS or 20 mg OVA in PBS on three alternate days. After seven days, mice were immunized s.c. with OVA (0.1 mg) in CFA in the base of tail. Ten days later, spleens and inguinal lymph nodes and sera were harvested for measurement of T-cell proliferation and cytokine production in the absence (solid) or presence (hatched) of O.lmg/ml OVA (mean and standard deviation shown for spleen), and anti-OVA antibodies, as described in Methods. Figure 17 is a graphical representation showing the effect of treatment with anti-CD40L monoclonal antibody (MR-1) on diabetes incidence in NOD mice given aerosol insulin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is predicated on the surprising discovery that insulin or its precursor could be used to induce immune tolerance. Accordingly, one aspect of the present invention contemplates a method of inducing immune tolerance while substantially avoiding CTL immunity in response to a mucosal antigen in a subject, said method comprising selecting said mucosal antigen or modifying a mucosal antigen to disable the function of an MHC class I restricted epitope and then administering said selected or modified antigen for a time and under conditions sufficient to prevent or reduce CTL immunity to said mucosal antigen. Generally, the mucosal antigen is used for preventing a CTL-mediated autoimmune disease such as but not limited to diabetes and in particular, IDDM. Accordingly, another aspect of the present invention contemplates a method of suppressing a cell-mediated autoimmune disease while substantially avoiding CTL immunity in response to a mucosal autoantigen, said method comprising selecting an autoantigen or modifying said mucosal autoantigen to disable the function of an MHC class I-restricted epitope and then administering said selected or modified autoantigen for a time and under conditions sufficient to induce tolerance but prevent or reduce CTL immunity to said autoantigen. Administration of the autoantigen may be by DNA or polypeptide/peptide delivery and may be by any appropriate means but the preferred route of administration is via mucosal surfaces including via oral, nasal, pharyngeal, or bronchial passages and via aerosol including intranasal aerosol. Yet another aspect of the present invention contemplates a method of suppressing a cell- mediated autoimmune disease in a subject, said method comprising the administration as an aerosol of an effective amount of an antigen associated with said autoimmune disease for a time and under conditions sufficient to prevent, reduce or otherwise ameliorate autoimmune pathology wherein said antigen substantially lacks a functional MHC class I interacting region. More particularly, the present invention provides a method of preventing, reducing or otherwise ameliorating an autoimmune disease condition in a subject, said method comprising the aerosol administration to said subject of an effective amount of an antigen associated with said autoimmune disease for a time and under conditions sufficient to induce or stimulate immunoregulatory mechanisms which are protective against cell- mediated autoimmune pathology wherein said antigen substantially lacks a MHC class I interacting region. Reference hereinafter to "immunoregulatory mechanisms" should be understood as a reference to all mechanisms which regulate cell-mediated immune responses including, but not limited to, regulation of T-cell functional activity, for example, regulation by one or more of suppressor CD4 T cells, Thl, Th2 or CD8 T cells including γδ T cells (referred to herein as "regulatory T cells"), or via regulation of cytokine production by lymphoid, myeloid or stromal cells. The present invention is predicated in part on the recognition by the inventors that some mucosal autoantigens contain epitopes for MHC class I-restricted CTLs. As a result, administration of these antigens may result in CTL immunity and CTL tolerance. Accordingly, the present invention requires the selection of- antigens which lack or to modify the antigens to remove functional MHC class I interacting epitopes. In a particularly preferred embodiment, the MHC class I epitope is an MHC class I (K )- restricted epitope. The present invention is hereinafter described with respect to preventing, reducing or otherwise ameliorating IDDM, slowly progressive IDDM (SPIDDM) also referred to as latent autoimmune diabetes in adults [LADN] and gestational diabetes due to underlying IDDM. This is done, however, with the understanding that the present invention extends to a range of cell-mediated autoimmune conditions. Accordingly, another aspect of the present invention contemplates a method of preventing, reducing or otherwise ameliorating IDDM, SPIDDM or gestational diabetes in a subject, said method comprising the administration, as an aerosol or other functionally equivalent means, to said subject of an effective amount of an autoantigen associated with IDDM for a time and under conditions sufficient for induction of regulatory T cells and/or other suitable mechanisms sufficient to suppress cell-mediated autoimmune pathology associated with IDDM wherein said autoantigen substantially lacks a functional MHC class I interacting epitope. Reference hereinafter to "IDDM" includes IDDM, SPIDDM and gestational IDDM. The regulatory T cells induced will depend on the form of antigen and its route of administration. For example, when an undegraded, conformationally-intact polypeptide or whole protein molecule is administered (e.g. insulin), CD8 T cells and, more particularly, CD8γδ T cells are induced. Smaller peptides such as proinsulin peptides (e.g. proinsulin peptide 24-36) generally induce CD4 T cells and, more particularly, CD4αβ T cells. Whole proteins may be degraded to peptides to generate predominantly CD4 regulatory T cells, particularly if administration is via the oral route. The absence of a functional MHC class I interacting epitope includes a single or multiple amino acid deletion encompassing all or part of the epitope region. Alternatively, the epitope may be blocked by other means such as by an antibody or other molecular interaction. A particularly preferred form of administration is intranasal administration via an aerosol spray, drip or vapour. The preferred antigen associated with IDDM used for intranasal administration or other route of administration is preproinsulin or proinsulin as well as insulin and their immune response stimulatory derivatives thereof such as but not limited to peptide fragments of proinsulin, preproinsulin or insulin, provided that such antigens lack or substantially lack a functional MHC class I-associating region which is involved in inducing CTL immunity. Immune response stimulation preferably includes regulatory T cell stimulation. However, any islet antigen may be employed such as, but not limited to, glutamic acid decarboxylase (GAD) in its various isoforms (for example, GAD 65 and GAD 67) or derivatives thereof and tyrosine phosphatase IN-2 or derivatives thereof. The antigens may be from human or any non-human species such as mouse. The most preferred antigen is a proinsulin peptide modified to inactivate the MHC class I interacting region, defined by amino acids 24 to 33. The interacting region may be modified by generating peptides lacking one or more key MHC class I anchor residues or comprising modified residues such that MHC class I binding is reduced. Preferably, the proinsulin peptide undergoes a C-terminal truncation to inactivate the MHC class I epitope. As a result, induction of CTL immunity is disassociated from induction of tolerance, including CTL tolerance. Accordingly, another aspect of the present invention contemplates a method of inducing, suppressing or otherwise ameliorating or preventing IDDM in a subject, said method comprising administering proinsulin peptide truncated at its C-terminal antigen end to disable the function of any MHC class I restricted epitope for a time and under conditions sufficient to prevent or reduce CTL immunity and otherwise induce immune tolerance, including CTL tolerance. As is discussed further below, the methods of the present invention may also be used in combination with a strategy to block CTL induction and/or maturation. In one approach, for example, the CD40-CD40 ligand (CD40L) interaction is blocked. The term "derivatives" includes fragments, parts, portions, chemical equivalents, mutants, homologues and analogues of the antigens. Analogues may be derived from natural synthetic or recombinant sources and include fusion proteins. Chemical equivalents of an antigen can act as a functional analog of an antigen. Chemical equivalents may not necessarily be derived from an antigen but may share certain conformational similarities. Alternatively, chemical equivalents may be specifically designed to mimic certain physiochemical properties of an antigen. Chemical equivalents may be chemically synthesised or may be detected following, for example, natural product screenings. A homologue of an antigen contemplated herein includes but is not necessarily limited to antigens derived from human or any non-human species such as mouse. Derivatives include one or more insertions, deletions or substitutions of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in said peptide although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences include fusions with other peptides or polypeptides. It is possible, for example, that the subject preferred peptides may be substituted by other peptides or functional homologues or analogues. A hybrid peptide may comprise a combination of peptides. The term "aerosol" is used in its most general sense to include any formulation capable of administration via nasal, pharyngeal, bronchial or oral passages. Aerosols generally comprise particles of liquid or solid suspended in a gas or vapour. Conveniently, the aerosol is a colloidal system such as a mist in which the dispersion medium is a gas. The method of administering the aerosol formulation may be by any means and may be achieved using a hand pump, electric pump, pressurized dispenser, nasal drip or other convenient means. Furthermore, drop size may determine lung penetration and the size of the droplets may need to be manipulated to maximize efficacy of administration. It should be understood that the method of the present invention extends to direct application of said formulations to intranasal surfaces. In a particularly preferred embodiment, the aerosol is delivered at a rate of from about 1 to about 20 litres/min and preferably from about 2 to about 15 litres/min at a droplet size of from about 0.1 to about 10 μm and more preferably from about 0.1 to about 6 μm. Conveniently, a stock solution of antigen is prepared at a concentration of from about 0.5 to about 20 mg/ml or more preferably from about 1.0 to about 10 mg/ml of carrier solution. Commercially available insulin is particularly useful which is about 4 mg/ml. A useful dose is from about 50 1 to 1000 μl and preferably 100 μl to 500 μl from the stock solution. The antigen may be administered alone or by formulation in or with an adjuvant. The adjuvant is selected from a range of adjuvants which enhance an immunoregulatory response including cholera toxin B, heat labile toxin of E. coli, saponin, Quill A extracts and other derivatives of saponin, DΕAΕ-dextran, dextran sulphate, aluminium salts, and non-ionic block co-polymers. The adjuvant may include other immunomodulators, such as cytokines (for example, IL-4 or IL-13), muramyl-dipeptide and derivatives, and cell wall components, for example, cell wall lipoprotein from Gram-ve bacteria such as E.coli, from species of Mycobacteria or Corynebacteria. The adjuvant formulation may include a combination of two or more of the adjuvants listed. These lists are not to be taken as exhaustive. The selection of adjuvant is in part dependent on the species being targeted and is based on the level and duration of the immune response required and on the lack of reactogenicity (i.e. tissue compatibility). The level of active component and adjuvant are chosen to achieve the desired level and duration of immune response. The antigen is administered in a therapeutically effective amount. A therapeutically effective amount means that amount necessary at least partly to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular conditions being treated, the severity of the condition and individual patient parameters including age, physical conditions, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgement. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reasons. Generally, daily oral doses of antigen will be from about 0.01 mg/ per dose per subject per day to 1000 mg/per dose per subject per day. Small doses (0.01-1 mg) may be administered initially, followed by increasing doses up to about 1000 mg/kg per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent patient tolerance permits. A single dose may be administered or multiple doses may be required on an hourly, daily, weekly or monthly basis. Effective amounts of antigen vary depending on the individual but may range from about 0.1 μg to about 100 mg, preferably from about 1 μg to about 10 mg and more preferably from about 5 μg to 20 mg per dose per subject. In particular, lower doses may be contemplated for aerosol or intranasal administration, for example, ng-μg doses may be optimal. In a related aspect of the present invention the subject undergoing treatment may be any human or animal in need of therapeutic or prophylactic treatment. The immune status generally, and specifically levels of regulatory T cells and cytokine profiles, may be readily determined throughout any treatment regime using conventional methods known to those skilled in the art. For example, regulatory T cell levels may be monitored by cytometric analysis following labelling with commercially available antibodies specific to T-cell subsets. Other examples of methods suitable for determining the status of the subject include purification of peripheral blood mononuclear cells by density centrifugation followed by stimulation by incubation with well known antigens such GAD, IA-2 family members, preproinsulin, proinsulin or insulin or peptide sequences from these antigens. Resulting proliferation may be quantified by assaying for inco oration of H 3 thymidine. The cytokine profile can be determined approximately 24- 72 hours after stimulation by antigen. Said cytokines can be detected using, for example, specific cytokine antibodies. Within about 24 hours after stimulation with antigen, stimulated cells can be phenotypically characterized by, for example, flow cytometric analysis of activation marker expression (for example, CD69, CD44, CTLA4, CD25). Following cell surface labelling of activated cells, said cells may be further fixed and incubated with fluorochrome labelled antibodies to specific cytokines to determine intracellular cytokine levels. In particular, for example, cells may be further assessed by double labelling assays. The double labelled cells may be analysed utilizing flow cytometric analysis or fluorescence microscopy. Another aspect of the present invention provides a composition comprising an antigen associated with an autoimmune disease in an aerosol formulation including one or more pharmaceutically acceptable carriers and/or diluents. Preferably, the autoimmune disease is IDDM. Preferably, the antigen is an islet antigen such as modified forms of insulin, or a precursor thereof such as preproinsulin, proinsulin or their derivatives (e.g. proinsulin peptide 24-36) or GAD or tyrosine phosphatases IA-2 or derivatives thereof wherein said antigens are modified to prevent an MHC class I epitope from functioning. Preferably, the antigen and route of administration induce regulatory T cells, such as in relation to whole molecules such as insulin CD8 T cells and most preferably CD8γδ T cells or, in relation to smaller molecules such as proinsulin peptide 24-36, CD4 T cells and most preferably CD4αβ T cells. In an alternative embodiment, a nucleic acid molecule encoding an IDDM-associated autoantigen is administered. Generally, according to this embodiment, intranasal or other suitable administration of a nucleic acid molecule such as DNA encoding proinsulin or a modified form thereof induces a population of CD4 T cells which suppresses development of diabetes. Preferably, the nucleic acid molecule encodes a peptide lacking a functional MHC class I interacting molecule. The nucleic acid molecule is preferably DNA such as cDNA or genomic DNA or is a DNA:RNA hybrid. It is particularly preferred to have the nucleic acid molecule in the form of a plasmid or vector. The nucleic acid molecule may also contain additional or substitution analogues of nucleotide bases in order to enhance stability. Accordingly, another aspect of the present invention contemplates a method of inducing immune tolerance, including CTL tolerance, while substantially avoiding CTL immunity in response to a mucosal antigen, said method comprising administering to a subject a nucleic acid molecule or analogue thereof encoding said mucosal antigen but wherein said antigen substantially lacks a functional MHC class I-restricted epitope for a time and under conditions sufficient to prevent or reduce induction of CTL immunity. More particularly, the present invention contemplates a method of preventing or suppressing IDDM while substantially avoiding CTL immunity, said method comprising administering to a subject a nucleic acid molecule or analogue thereof encoding an IDDM- associated autoantigen which substantially lacks a functional MHC class I-restricted epitope for a time and under conditions sufficient to prevent or reduce the effects of IDDM. Still yet another aspect of the present invention contemplates other methods for dissociating CTL immunity from CTL tolerance. In particular, the maturation of CTL to effector "killer" cells requires priming by antigen-presenting cells such as dendritic cells. The dendritic cells present the antigenic peptide (i.e. epitope) as a complex with MHC class I molecules to the T-cell receptor of CD8 CTL. The dendritic cell itself is primed to perform this function by prior interaction with a "helper" CD4 T cell through the interaction between CD40 ligand (CD40L) on the T cell and CD40 on the dendritic cell (see Figure 11). CD8 T cells themselves have also been shown to express CD40L. It is proposed, therefore, in another embodiment, in conjunction with the administration of a mucosal antigen to administer an antagonist of CD40L-CD40 interaction. An example of such an antagonist is a CD40L antibody, such as a monoclonal antibody. The antagonist of CD40L-CD40 interaction may be administered before, simultaneously with or sequentially with the administration of the mucosal antigen. Sequential administration includes within seconds, minutes, hours, days or weeks. Simultaneous includes substantially simultaneously. This extra treatment may be in conjunction with the administration of a mucosal antigen or a nucleic acid molecule encoding a mucosal antigen. Another aspect of the present invention, therefore, contemplates a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering said mucosal antigen or a nucleic acid encoding same for a time and under conditions sufficient to prevent or reduce CTL immunity, before, simultaneously or sequentially with the administration of an antagonist of CTL induction and/or maturation. More particularly, the present invention contemplates a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering said mucosal antigen or a nucleic acid encoding same for a time and under conditions sufficient to prevent or reduce CTL immunity, before, simultaneously or sequentially with the administration of an antagonist of CD40L-CD4 interaction. Even another aspect of the present invention provides a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering an agent for a time and under conditions sufficient to block or otherwise delay CTL induction and/or maturation. More particularly, the present invention provides a method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering an agent for a time and under conditions sufficient to block or otherwise delay CTL induction and/or maturation wherein said agent blocks or otherwise disrupts CD40-CD40L interaction. Still another aspect of the present invention contemplates the use of an agent in the manufacture of a medicament for the treatment or prophylaxis of a disease condition, said agent capable of blocking CTL induction and/or maturation. The present invention is further described by the following non-limiting Examples. EXAMPLE 1 Aerosol treatment and diabetes assessment Semi-sealed boxes of eight female NOD mice were each aerosolized by connection to a standard, patient electric pump (Maymed Aerosol MKV, Anaesthetic Supplies, Sydney, Australia) and Aeroflo nebulizer (Waite & Co., Sydney). Recombinant human insulin (Humulin R, Eli Lilly) or control ovalbumin protein, at 4 mg/ml, was delivered over 10 min at an air flow rate of 12 litres/min. in a rated droplet size of <5.8 μm, to groups of 24- 32 mice. All treatments were given between 0900 and 1100 hours. Protocols and mouse care were approved and supervised by the institutional Animal Ethic Committee. Retro- orbital venous blood was sampled at least every 28 days from 100 days of age and mice considered to be diabetic if their blood glucose, confirmed by a repeat test, was >11 mM. Glucose was measured with BM-Test Glycemie (registered trademark) strips and a Reflolux (registered trademark) II meter (Boehringer-Mannheim), on a drop of blood aspirated via a glass capillary tube from the retro-orbital venous plexus of unanesthetized mice. EXAMPLE 2 Histology Mice were killed by CO 2 inhalation and the pancreas and salivary glands immediately removed into Bouin's fixative and embedded in paraffin. The insulitis score, a measure of the severity of islet infiltration, was determined blindly by two independent investigators by grading and then averaging a minimum of 15 separate islets in serial 6 μm pancreas sections stained with haematoxylin and eosin. The grading scale was: 0, no filtration, islet intact; 1, <10 peri-islet lymphoid cells, islet intact; 2, 10-20 peri-and intra-islet lymphoid cells, islet intact; 3, >20 peri- and intra-islet lymphoid cells, <50% of islet replaced or destroyed; 4, massive lymphoid infiltrate with >50% of islet replaced or destroyed. Infiltration of the salivary glands was graded by the number of lymphoid cells in clusters: 0, no cells; 1, <10 cells; 2, 10-50 cells; 3, >50 cells. EXAMPLE 3 Immune responses Spleen cells from individual normoglycemic mice were treated with a red cell lysis buffer, resuspended and incubated in quadruplicate at 2 x 10 5 /200 μl of serum-free HL-1 medium (Hycor, Irvine, CA) containing 50 μm 2-mercaptoethanol, in round-bottom wells with the indicated concentrations of antigen. After 3 days at 37 °C in 5% v/v CO 2 /air, 100 μl aliquots from each replicate supernatant were collected and stored at -70 °C for cytokine assays; the cells were then pulsed with 3H-thymidine, harvested 16 hours later and counted on a Topcount (trademark) micro-scintillation counter (Packard, Meriden, CT). Insulin was recombinant human (Humulin R, Eli Lilly), as used for aerosol treatments. Insulin B-chain peptide corresponding to amino acids 9-23 of mouse insulin II (Peptide Express, Fort Collins, CO) was more than 90% pure by HPLC analysis. GAD65 was the recombinant human form expressed with a C-terminal hexahistidine in a baculovirus system and purified by Ni 2+ chelation affinity chromatography. It was resolved as a single band in SDS-PAGE and was endotoxin-free by the quantitative Limulus lysate assay (BioWhittaker, Walkersville, MD). IL-2, -4, -10 and IFN-γ were measured by ELISAs with monoclonal antibody pairs (Pharmingen); the lower limits of detection were 62, 16, 16 and 55 pg/ml, respectively. TGF-/31 was measured with an ELISA kit (Promega) with a lower limit of detection of 16 pg/ml. To detect insulin antibodies, 125 I-labelled human insulin (approximately 100,000 cpm: specific activity 120 μCi/μg) was incubated with or without excess unlabelled insulin (10 μg/ml) in phosphate-buffered saline containing a mixture of protease inhibitors and serial log dilutions of mouse serum, for 5 days at 4°C. Complexes were then precipitated with rabbit anti-mouse globulin anti-serum, washed and counted in a gamma counter. Positive control sera (guinea pig anti-porcine insulin serum, human IDDM sera) maximally precipitated 37-54% of the total radioactivity. Non-specific binding, in the presence of excess unlabelled insulin, was ^.3%. EXAMPLE 4 Adoptive transfer of diabetes Male NOD mice aged 6-9 weeks (16/group) were irradiated (800R) from a Cobalt source and 3-6 hours later received 2 x 10 7 pooled splenocytes from recently-diabetic 14-19 week- old female NOD mice, together with 2 x 10 7 splenocytes (or cells fractionated from this number) from either aerosol insulin- or ovalbumin-treated mice, in 200 μl via the tail vein. The onset of diabetes was then monitored by measuring blood glucose starting two weeks after transfer. EXAMPLE 5 Fractionation of spleen cell populations Spleen cells were treated with red cell lysis buffer and resuspended in mouse tonicity phosphate buffered saline. Total T cells were purified by non-adherence to nylon wool. CD4 and CD8 cells were positively selected/depleted magnetically with monoclonal antibodies directly bound to MACS MicroBeads (Milteny Biotec, GmbH, Germany) according to the manufacturer's protocols, and counted as viable cells (trypan blue stain negative). Flow cytometry revealed 95% depletion of CD4 or CD8 cells, with recoveries -80% and -50% respectively. γδ T cells were positively selected/depleted by incubating T cells from aerosol-treated mice first with biotinylated GL3-1A antibody (Pharmingen, San Diego, CA) and then with streptavidin-MACS MicroBeads, followed by magnetic separation. By flow cytometry, γδ cells comprised 1-2% of NOD splenocytes and were totally depleted with GL3-1A antibody. To purify CD8 γδ T cells, CD8 T cells were first magnetically selected from total T cells with anti-CD8-FITC conjugate and anti-FITC MicroBeads. The MicroBeads were then released according to the Miltenyi Biotec protocol, and the CD8 cells magnetically separated into γδ positive and depleted fractions. Double staining and FACS analysis demonstrated total depletion of γδ cells and their recovery as a GL3-1 A high and low expressing CD8 population. EXAMPLE 6 Diabetes and insulitis Aerosol human insulin or ovalbumin were administered in different schedules to female NOD mice from 28 days of age, the earliest time at which insulitis is detectable in the colony of mice, and their incidence of diabetes and severity of insulitis subsequently measured. The incidence of diabetes was only marginally affected by a single aerosol insulin treatment at 28 days of age, being 75% by 240 days of age compared to 88% after aerosol ovalbumin. However, treatment for 3 or 10 consecutive days and then weekly significantly delayed the onset and reduced the incidence of diabetes. In five separate experiments, diabetes incidence at 156 days of age was reduced from a median of 47% in ovalbumin- treated mice to 23% in insulin-treated mice; at 240 days of age, when the cumulative incidence of diabetes approaches a maximum, the values were 79% and 49%, respectively (p=0.005, Kaplan-Meier survival statistic). There was no difference if the initial treatment was for 3 or 10 days. In another experiment, in which treatment was given for 10 consecutive days and then weekly, but not started until 49 days of age when insulitis was well-established, aerosol insulin still significantly reduced diabetes incidence at 156 days from 58% to 25% (p=0.001). Insulin treatment was associated with a significant reduction in the severity of the islet lesion, as judged by the "insulitis score", which parallelled the decrease in diabetes incidence (Table 1). Infiltration of the salivary glands by lymphoid cells (sialitis), which also occurs in NOD mice, was unaffected by aerosol insulin. In the absence of absorption-promoting agents, systemic uptake of insulin from the naso- pharyngeal mucosa in humans is insignificant (22). In NOD mice, blood glucose was not altered in the short-term by aerosol insulin. Insulin solutions labelled with 10% Evan's Blue dye were observed to be deposited in the naso-pharynx, trachea and main bronchial divisions, as well as the oesophagus. While it may be difficult, if not impossible, to avoid some gastrointestinal exposure after aerosol or intranasal delivery of soluble protein, delivery into the naso-pharynx alone is sufficient to induce tolerance (7,23,24). TABLE 1 Severity of insulitis and frequency of diabetes in NOD mice treated with aerosol protein Mice (32/group) were given either aerosol insulin or ovalbumin for 10 consecutive days and then weekly from 28 days of age. At 105 days of age, five non-diabetic mice from each group were killed for pancreas histology. The insulitis score is expressed as mean ± SD. l The insulitis score in insulin-treated mice was significantly reduced (p<0.01, Mann- Whitney U test). The diabetes frequency in insulin-treated mice significantly reduced (p=0.04, Fisher's exact test). EXAMPLE 7 Immune responses The inventors investigated if aerosol insulin treatment had altered immune responses to insulin. Unprimed T-cell proliferative responses to islet antigens, including insulin, have been reported in NOD mice (25) but have not always been reproducible (26). Proliferative responses of spleen cells (0.5-2.5 x 10 6 /ml) from either insulin or ovalbumin-treated mice, aged 56-105 days, to human insulin or ovalbumin (0.2, 2.0, 20 and 40 μg/ml), in different serum-supplemented or serum-free media varied by less than two-fold above basal and were usually depressed below basal at the highest concentration of insulin. Insulin at high concentrations has been reported to inhibit T-cell responses (27). In contrast, in the ovalbumin-treated control mice but not the insulin-treated mice, responses to insulin B- chain peptide a 9-23, a dominant epitope for NOD mouse islet-derived T-cell clones (19), were significant (Table 2). Furthermore, ovalbumin mice had significantly higher responses than insulin mice to human glutamic acid decarboxylase 65 (GAD65), previously reported to stimulate splenic T cells in NOD mice (25). In mice from both treatment groups, proliferative responses to non-antigen-specific stimulation by concanavalin A or T-cell receptor CD3 monoclonal antibody, 145-2C11, were similar (Table 2) and no different to untreated mice, indicating that aerosol treatment did not cause general immunosuppression. IL-2, IFN-γ and TGF-βl secretion in response to insulin B chain 9-23 were not significantly different between insulin- and ovalbumin-treated mice; however, the levels of IL-4 and particularly IL-10 were higher from cells of insulin-treated mice (Table 3). TABLE 2 Proliferative responses of splenocytes from aerosol-treated mice Splenocytes from three mice per group were assayed in quadruplicate in HL-1 serum-free medium. Statistical comparisons (Mann-Whitney U tests) were between the 12 results for each group. TABLE 3 Cytokine secretion by splenocytes from aerosol-treated mice to 40 μg/ml of mouse insulin II B chain peptide (a 9-23) Supernatants from replicate culture wells (Table 2) were sampled after three days incubation and assayed for cytokines. Insulin antibodies were measured by a standard immunoprecipitation assay with sera (n=12/group) from insulin- and ovalbumin-treated mice aged 70-105 days. Precipitation of I-insulm radioactivity by antibodies in sera from insulin-treated mice (12.7 ± 3.6%; mean precipitated cpm ± SD) was significantly higher (p<0.01, Mann Whitney U test) than in ovalbumin-treated mice (6.9 ± 2.5%). This increase in the "level" of insulin antibodies after aerosol insulin, together with the suppression of T cell proliferation and the increase in IL-4 and IL-10 responses to insulin B-chain peptide, is consistent with the phenomenon of immune deviation, as described after oral MBP in Lewis rats (1) and intranasal GAD peptides in NOD mice (28). β-cell destruction within the DTH lesion of IDDM is an example of Thl -mediated process (10,11), whose inhibition by aerosol insulin might be expected to shift the Thl/Th2 balance towards Th2 in response to key islet antigens. Defective suppressor T-cell function has been postulated to shift the balance towards Thl in IDDM (11). It seems unlikely that the reduced T cell proliferative response to GAD could reflect "bystander" suppression due to the secretion of the Th2 cytokines IL4 and IL- 10 (1) by insulin aerosol-induced regulatory cells because, apart from an absence of added insulin in the cultures with GAD, responses to conA and anti-CD3 were not impaired. A direct explanation is that the reduced response to GAD reflects the protective effect of aerosol insulin on insulitis and β-cell destruction. This implies that at least some GAD immunity is secondary and that immunity to (pro)insulin may have a more proximal role in β-cell destruction. Although NOD mouse T-cell responses to human GAD65 have been reported to be stronger and to appear earlier than those to native human insulin (25), it was found that transgenic expression of mouse proinsulin II in NOD mouse antigen presenting cells completely prevents insulitis and diabetes (29). EXAMPLE 8 Regulatory CD8 γδ T cells The inventors investigated whether aerosol insulin induced regulatory cells that could inhibit the adoptive transfer of diabetes by pathogenic, effector T cells. In the classic adoptive transfer model (30) (see Figure 6), spleen cells from diabetic NOD female mice transferred intravenously to young, irradiated non-diabetic syngeneic male or female recipients cause clinical diabetes in the majority within 4 weeks. When 2 x 10 7 spleen cells were co-injected from older, diabetic mice with an equal number of spleen cells from aerosol ovalbumin mice, the majority of young recipients developed diabetes within 4-5 weeks; in contrast, after co-injection with spleen cells from aerosol insulin mice, only a minority developed diabetes (Figure 4A). Diabetes incidence was suppressed by >75% in six separate experiments with either splenocytes or nylon wool-non-adherent splenocytes (enriched for T cells) from aerosol insulin mice. Spleen cells were then fractionated to identify the regulatory cells responsible for the suppression of diabetes transfer. Depletion and positive selection of CD4 and CD8 cells clearly showed that CD8 cells were wholly responsible for the suppression of transfer (Figure 4B). Depletion of CD4 cells did not alter the ability of residual spleen cells from aerosol insulin mice to suppress transfer (Figure 4B), and positively selected CD4 cells did not suppress transfer (Figure 4C). On the other hand, there was no suppression by CD8- depleted spleen cells from aerosol insulin mice (Figure 4D), whereas positively-selected CD8 cells suppressed transfer (Figure 4E). The partial suppression by positively-selected CD8 cells, in contrast to the rapid development of diabetes after their depletion, is probably due to the inefficient recovery of CD8 cells; in this experiment, 7 x 10 5 purified CD8 cells were co-injected into each recipient with 2 x 10 7 spleen cells from diabetic mice. T cells bearing γδ receptors have been shown to have an immunoregulatory role (31-36). Interestingly, it has been reported that total peripheral blood γδ cells decrease concomitantly with loss of β-cell function in humans with sub-clinical IDDM (37). To determine if the suppression of diabetes transfer that was observed was due to γδ T cells, the inventors fractionated spleen cells with the anti-γδ T-cell monoclonal antibody, GL3- 1A (38). Depletion of γδ T cells, like that of CD8 cells, completely abrogated the ability of nylon wool non-adherent spleen cells from insulin aerosol-treated mice to suppress adoptive transfer of diabetes (Figure 5A). Conversely, relatively small numbers of γδ T cells from insulin aerosol-treated mice could suppress transfer. Diabetes incidence after transfer was decreased by 50% for at least 70 days when 1.4 x 10 5 γδ T cells were co- injected with 2 x 10 7 spleen cells from diabetic mice (Figure 5A). The splenic CD8 and γδ T cells that suppressed diabetes transfer were one and the same, and not two interdependent populations. Thus, the ability of CD8 cells from insulin aerosol-treated mice to suppress transfer was abolished if they were first depleted of γδ T cells, whereas small numbers of γδ cells purified from the CD8 cells prevented transfer (Figure 5B). A summary of the results from 11 different co-transfer experiments is presented in Figure 6. FACS analysis revealed that γδ cells reactive with GL3 antibody constitute 1.6-2.4% of total and ~ 1% of CD8 + cells in the spleens of 12-16 week-old female NOD mice. These values were no different between groups of mice treated with insulin or ovalbumin aerosol. However, because of their low abundance distinct sub-populations of antigen-specific CD8 γδ T cells would be difficult to distinguish this way. The higher protection with fractionated cells, for example, sequentially-purified CD8 γδ cells (Figure 6), is quantitative and reflects their higher absolute number relative to that in unfractionated cells. EXAMPLE 9 Aerosolinization of insulin Aerosol inhalation as a mode of insulin delivery to the mucosa was as effective as oral insulin (22,23) in reducing diabetes incidence in the NOD mouse. The fact that it was therapeutic after the onset of insulitis is especially relevant to the prevention of IDDM in at-risk humans with sub-clinical disease in whom the presence of circulating islet-antigen reactive antibodies and T cells is taken to reflect underlying insulitis. Indeed, compared to humans with recently-diagnosed IDDM, NOD mice have more intense insulitis and the majority of females to progress to diabetes (10,11,24). Aerosol insulin had no obvious metabolic effect but induced a population of regulatory CD8 γδ T cells, small numbers of which suppressed the ability of pathogenic effector T cells to adoptively transfer diabetes. These antigen-induced "suppressor" T cells protective against cell-mediated autoimmune pathology have not been previously described. Oral tolerance has been associated with a decrease in cellular and sometimes an increase in humoral antigen-specific immunity, and with either CD8 or CD4 T cells that secrete, respectively, TGF-β or IL-4, IL-10 and TGF-βl (8). However, these regulatory cells have not been identified as bearing γδ receptors. In NOD mice, oral tolerance to insulin was attributed to regulatory CD4 T cells (21). In accordance with the present invention CD8 γδ T cells account for the regulatory cells induced by aerosol insulin. EXAMPLE 10 Intranasal insulin (Figure 1), proinsulin (Figure 2) or proinsulin peptide 24-36 (Figure 3) Commercially available insulin at 4 mg/ml, or proinsulin or proinsulin peptide 24-36 at 1-4 mg/ml in either insulin carrier solution or mouse tonicity-phosphate buffered saline, was applied in a volume of 10-20 μl to the nostrils of unanaesthetized, restrained NOD female mice at either 28 or 56 days of age. Note that by 56 days of age all mice exhibit underlying islet inflammation (insulitis). -*Single doses of insulin, proinsulin or proinsulin peptide 24-36 at either 28 or 56 days of age each significantly delayed the onset of diabetes in NOD female mice, compared to control proteins ovalbumin or hen egg lysozyme. On a comparative dose basis, proinsulin and proinsulin peptide 24-36 were more effective than insulin. These effects are greater with repeated doses of these proteins or peptide. In female mice pretreated with a single intranasal dose (40 μg) of proinsulin 24-36, whole splenocytes, and whole splenocytes depleted of CD8 but not CD4 T cells, significantly suppressed the adoptive transfer of diabetes by splenocytes from diabetic mice (Figure 7). Female mice were treated at 28 days of age, and then killed and their splenocytes taken for adoptive co-transfers at 56 days of age. EXAMPLE 11 Clinical trial of intranasal insulin in at-risk individuals The intranasal insulin trial (INIT) involves administration of intranasal insulin to at-risk but otherwise healthy first-degree relatives with immune markers of IDDM, including circulating antibodies and T cells reactive with islet autoantigens. Our subjects have at least two antibodies, to insulin, GAD or tyrosine phosphatase IA-2, and peripheral blood T cell responses to insulin or proinsulin peptide 24-36, and sometimes to GAD and IA-2 peptides. The rationale is to induce mucosa-mediated immune tolerance to insulin, based on the success of this approach in the NOD mouse, and to demonstrate safety. Commercially-available human recombinant insulin is used, which is normally given routinely by subcutaneous or intravenous injection to people with IDDM. No significant side effects have been observed in 38 high-risk subjects aged 4-30 (median 11.4 years) entered into the Trial. The possibility of mucosal irritation exists, but this has only been rare and then minor and transient. NOD mice treated with aerosol or intranasal insulin have exhibited no clinical complications, or abnormalities at autopsy. The INIT trial examines the effect of intranasal insulin on the surrogate immune markers of IDDM. The design is randomized, double-blind and placebo-controlled, with a crossover at six months. The placebo is the carrier solution normally used for insulin. The aim is to demonstrate significant effects on the levels of antibodies and T cells to insulin and other beta cell antigens. In addition, first phase insulin release (FPIR) in response to an intravenous injection of glucose, a measure of beta cell function, is monitored at the start, six months and 12 months. The crossover design gives all subjects the opportunity of treatment (an important issue for at-risk relatives), measures if any treatment effects are sustained and allows within- and between-group analyses. Treatment is administered initially daily for 10 consecutive days, then for two consecutive days weekly. After six months, treatment is crossed over (from insulin to placebo, or vice versa). The administration dose of insulin per nostril is approximately 200 μl (800 μg) of the commercial 4 mg/ml solution. The placebo is the carrier solution in which the insulin is normally dissolved. Results Intranasal insulin was associated with a significant increase (p = 0.01) in insulin antibodies overall and with a concomitant decrease in peripheral blood mononuclear (T-cell) proliferation to denatured human insulin; insulin antibody levels and T-cell proliferative responses to denatured insulin were inversely related in the first (p = 0.05) and second (p = 0.01) periods. These results are consistent with those found in the NOD mouse. There was no change in FPIR in any subject in whom this was initially measurable above the first percentile. Changes in immune parameters were not associated with changes in FPIR, e.g. an increase in insulin antibodies on insulin was not associated with deterioration of beta cell function in subjects with FPIR > 1st percentile. There were no evident side-effects of intranasal insulin and the results encourage further trials to determine the effect of intranasal insulin on FPIR long-term and diabetes incidence. EXAMPLE 12 Modification of proinsulin peptide 24-36 to remove functional MHC MHC class I-interacting eptitope Following administration of the proinsulin peptide 24-36, although CD4 regulatory T cells were induced which almost completely blocked the adoptive transfer of diabetes (when isolated and transferred with effector "diabetogenic" T cell into young irradiated NOD mice), and the onset of spontaneous diabetes was delayed but not prevented. The inventors observed that the proinsulin peptide contained predicted epitopes for MHC class I (H2- K d )-restricted CTLs, namely aa26-34 and aa25-34. Accordingly, administration of proinsulin peptide 24-36 could result in concomitant induction of CTL immunity and tolerance. First, they showed that 26-34 and especially 25-34 could bind to K d (Figure 8) and elicit CTL in NOD mice (shown for aa 26-34 in Figure 9). They then demonstrated the effect of a series of C-terminal truncations of the proinsulin aa24-36 peptide (Figure 10). Inactivation of the MHC class I-binding peptides by deletion of the position 9 anchor residue (aa34) significantly enhanced the ability of the core MHC class II (I-Ag7) binding sequence to prevent diabetes after intranasal administration. The C-terminal amino acid in position (p) 9 is a key "anchor" residue required for binding to the MHC class I molecule. EXAMPLE 13 Dissociation ofcytotoxic T-lymphocyte (CTL) immunity from oral tolerance by targeting CD40L-CD40 signalling MATERIALS AND METHODS Mice Mice were bred and maintained in The Walter and Eliza Hall Institute of Medical Research. Oral tolerance and CTL activity were determined in female C57B1/6 mice aged 6 to 8 weeks. Transgenic OT-1 / Rag 7 " mice bearing a transgenic CD8 T-cell receptor (TCR) for the MHC class I-restricted OVA 257 . 264 peptide and OT-II mice bearing a transgenic CD4 TCR for the MHC class Il-restricted OVA 23 . 3 9 peptide were used between 6 and 12 weeks as donors of OVA-reactive T cells for adoptive transfer into Ly5.1/CD45.2 congenic C57B1/6 mice (OT-I cells) and into RIP-OVA transgenic mice (OT-I and OTII cells). Induction of oral tolerance Oral tolerance was induced with two protocols corresponding to reported high and low dose OVA. OVA (Grade V, Sigma, St. Louis, MO) was administered to female C57B1/6 mice either at 20 mg on three alternate days (high dose) or at 0.5 mg on five alternate days (low dose) via intragastric intubation under light methoxyflurane (Penthrane (trademark)) anaesthesia. The endo toxin concentration of an OVA solution (lOmg/ml) measured in the Limulus lysate assay (BioWhittaker, Walkersville, MD) was < 0.5 ng/ml. CD40L signaling was blocked by administration of the hamster IgGl anti-mouse CD40L mAb MR-1 (ATCC, Rockville, MD); the control was the hamster mAb 6C8 specific for human Bcl-2. Both mAbs were purified from hybridoma cell culture medium by affinity chromatography on protein G-Sepharose (Pharmacia, Uppsala, Sweden) and injected intraperitoneally (i.p.). in a dose of 250 μg as indicated. Cytotoxic T lymphocyte (CTL) assay CTL were assayed as follows. Mice were primed i.v. with 20 x 10 6 OVA-coated H-2K bm l spleen cells (dependant on CD4 T-cell help) or subcutaneously (s.c.) in the base of tail with 200μg of OVA peptide 257-264 in CFA in 100 μl (independent of CD4 T-cell help). Depending on the experiment, mice were primed 2 or 3 weeks after receiving mAb and oral OVA. Mice were killed 7 days after priming, and their spleen cells stimulated in vitro for another 6 days before being used as effectors in a 51 Cr release assay. Lytic units were calculated by dividing the total number of effectors generated from each spleen by the total number of effectors required for 30% OVA-specific lysis. Effect of oral OVA ± anti-CD40L treatment of CTL precursors To determine the response of OVA-specific CTL precursors to oral OVA and the effect of CD40L blockade, 3 x 10 6 OT-I cells were transferred into Ly5.1 congenic female mice, which were then given either MR-1 or control 6C8 mAb on days 0 and 3. Oral OVA 20 mg was given daily on days 1-3. Mice were killed on day 14 and the numbers of splenic OT-I cells and their expression of CD44 and CD62L (L-selectin) activation markers) analyzed by FACScan using Lysys 2 (trademark) software (Becton Dickinson, San Jose, CA). Cells were incubated with FITC-conjugated anti-CD44 and anti-L-selectin mAbs (PharMingen, San Jose, CA) together with biotinylated anti-Ly5.2 mAb (PharMingen) and PE- conjugated anti-CD8 mAb (Sigma), followed by a second-step incubation with streptavidin- conjugated PerCp (PharMingen) to detect Ly5.2. Effect of oral OVA ± anti-CD40L treatment on diabetes induction in RIP-OVA to mice To examine the effect of CD40L blockade on activation of CTL in vivo be oral OVA, RIP- OVA mice which express OVA on their pancreatic β cells were adoptively transferred with 0.3 x 10 5 OT-1 cells and 0.2 x 10 6 OT-1 cells and given MR-1 or control 6C8 mAb on the day of transfer (day 0). Mice were then treated with oral OVA, 0.5 mg on five alternate days, starting from day 1. Blood glucose was measured on a drop of retro-orbital venous blood with a glucometer, on days 14 and 21 and values above 14 mmol/1 were considered to be diagnostic for diabetes. Evaluation of oral tolerance To evaluate CTL tolerance to systemic priming, 14 or 21 days after the last dose of oral OVA mice were injected i.v. with 20 x 10 6 OVA-coated H-2K bm_1 spleen cells or s.c. with 0.1 mg OVA protein in CFA and their splenic CTL activity subsequently measured as described above. To evaluate conventional indices of mucosal tolerance, 7 days after the last dose of oral OVA mice were immunized by s.c. injection with OVA (0.1 mg) in CFA in the base of tail. Ten days later, they were anesthetized, bled with a glass capillary tube from the retro-orbital venous plexus, killed by CO 2 asphyxiation, and their spleens and inguinal lymph nodes removed. Serum was harvested and stored at -20 C for assay of OVA antibodies. Cell suspensions were prepared from spleens and nodes by mechanical disruption through a stainless steel mesh, washed, counted and resuspended in RPMI-1640 medium containing 2 mM glutamine, 5 x 10 "5 2-mercaptoethanol and 5% v/v fetal calf serum for assay of proliferative and cytokine responses to OVA. IgG subclass antibodies to OVA were measured by ELISA using peroxidase-conjugated anti-mouse IgG 1, 2a, 2b or 3 antibodies (Southern Biotechnology Associates) as previously described . Proliferative responses to OVA of splenocytes (1 x 10°) or inguinal lymph node cells (5 x 10 5 ) in 200 μl medium were measured in replicates of eight in round-bottom wells of 96- well Linbro plates (Flow Labs, McLean, VA), after incubation with or without 0.1 mg/ml OVA at 37°C in 5% CO 2 / air for 96 hours. 3 H-thymidine (1 μCi) was added to each well for the last 10-16 hours, the cells harvested and washed, and counted on a TopCount scintillation counter. Splenocyte or inguinal lymph node cell IFN-γ and IL-4 responses to OVA were measured by ELISPOT assay. Cells (5 x 10 5 /200 μl) were added to wells of Multiscreen Immobilon-P membrane 96-well plates (MAIPS4510; Millipore, North Ryde, Australia) that had been precoated with monoclonal rat anti-mouse IFNγ (clone R4-6A2) or IL-4 (clone 11 Bll) antibody at 5 μg/ml PBS overnight Cells were incubated with or without 0.1 mg OVA at 37°C in 5% CO 2 /air for 24 hours, then removed by washing. Membrane-bound cytokine was reacted with 4 μg/ml biotin-conjugated monoclonal rat anti-mouse IFN-γ (clone XMG1.2) or IL-4 (clone BVD6-24G2) overnight at 4°C. After washing, colour was developed with streptavidin-peroxidase followed by 3-amino-9- ethylcarbazole (NEC; Dako, Carpinteria, CN). All monoclonal antibodies were from Pharmingen, San Diego, CA. Statistics Differences between treatment groups were analyzed by the Fisher's exact test or the Mann- Whitney test RESULTS CD40L blockade impairs CTL induction by oral OVA After determining the dose (250 μg i.p.) of MR-1 mAb that blocked CTL priming by the CD4 T cell help-dependent pathway (Figure 12 A) a similar dose of either control mAb 6C8 or MR-1 mAb was given to C57B1/6 mice that were then fed 20mg OVA on three alternate days. Oral OVA induced a CTL response in 75% (9/12) of mice treated with 6C8, but in only 25% (3/12) of mice treated with MR-1 (Figure 12B). Activation and expansion of CTL by oral OVA requires CD40L To demonstrate that CTL induction by oral OVA was associated with activation and expansion of CTL precursors, the adoptively transferred OVA-specific transgenic CTL (OT-I cells) into naive Ly5.1 congenic recipients and fed them OVA. The role of CD40L in the response of OT-I cells to oral OVA was examined by pre-treating recipient mice with either control mAb 6C8 or anti-CD40L mAb MRl. In order to allow activation, proliferation, recirculation and possible cell death to occur before analyzing the final outcome, the inventors examined OT-I cells from the spleen 14 days after the last dose of oral OVA. This site and time corresponded to other protocols used, e.g. to measure OVA- induced CTL. In response to oral OVA and 6C8, OT-I cells in the spleen expanded greatly (Figure 13B) and increased CD44 (Figures 13 A, C) and decreased CD62L (Figures 13 A, D) expression, indicating that many had acquired an activated/memory phenotype. However, in the presence of MRl, the ability of oral OVA to induce expansion (Figure 13B) and activation (Figures 13 A, C, D) of OT-I cells was markedly impaired. Anti-CD40L treatment prevents induction of diabetes by oral OVA in RIP-OVA ° mice In preliminary experiments, the inventors found that transfer of a minimum of 0.3 x 10 OT-II cells and 0.2 x 10 6 OT-I cells was necessary for diabetes development after systemic priming of recipient RIP-OVA 10 mice with OVA-coated splenocytes. These numbers of OT-II and OT-I cells were, therefore, transferred into RIP-OVA 10 mice and the following day the mice were given control mAb 6C8 or anti-CD40L mAb MRl and then fed 0.5 mg OVA on five alternate days. Following oral OVA, 60% (9/15) of mice given 6C8 developed diabetes, compared to only 14% (2/14) of mice given MRl (p=0.02) (Figure 14). Anti-CD40L treatment does not prevent induction of oral tolerance Experiments in CD40L gene-targeted mice have indicated that CD40L signalling is required for induction of oral tolerance (39). However, this mutation affects the development of Peyer's patches (39) and germinal centres (40). Therefore, it was important to determine if oral tolerance could be induced in genetically unmanipulated mice treated short-term with anti-CD40L mAb. Previously, it was shown that oral OVA, while inducing CTL immunity, paradoxically suppressed the further priming of strong CTL immunity by systemic OVA (41). To determine if anti-CD40L treatment influenced this tolerogenic effect of oral OVA on CTL, C57B1/6 mice were given control mAb 6C8 or anti-CD40L mAb MRl 250 μg i.p. and then fed PBS or OVA in PBS. After 14 or 21 days they were primed in a CD4 T cell-dependent manner with i.v. OVA-coated splenocytes (Figure 15 A) or s.c. with 100 μg OVA in Complete Freund's Adjuvant (CFA) (Figure 15B). The latter method directly primes CTLs, independent of CD4 T-cell help. These experiments demonstrated that anti-CD40L treatment with MRl did not modify the tolerogenic effect of oral OVA on systemic priming of CTL by either method. This was consistent in all experiments, whether mice were challenged 14 or 21 days after high dose (20 mg on three alternate days) or low dose (0.5 mg on five alternate days) oral OVA (Figures 15C, 15D). Experiments were then performed to determine whether anti-CD40L treatment affected conventional parameters of oral tolerance. A single injection of MRl before the first dose of oral OVA (20 mg three alternate days) did not limit suppression of systemic OVA- primed T-cell proliferation or IFN-γ production (shown for splenocytes in Figures 16A and 16B, respectively) or serum anti-OVA antibodies (Figure 16C). Mean stimulation indices for proliferation decreased from 3.50 (oral PBS) to 1.96 (oral OVA) (p<0.05) after 6C8 and from 3.21 to 2.04 (ρ<0.05) after MRl (Figure 16A). Suppression of primed IFN-γ ELISPOT responses following oral OVA was more dramatic and was not affected by anti- CD40L treatment (Figure 16B). Very few IL-4 ELISPOTS (^/well) were detected under any condition. Mucosal administration of antigen can tolerize subsequent immune responses to the antigen and, in the case of autoantigens, suppress development of autoimmune disease. Mucosal administration, however, of the model protein antigen ovalbumin (OVA) also induces cytotoxic T-cell (CTL) immunity and this may cause disease. The inventors show that oral OVA-induced tolerance and CTL immunity can be dissociated by targeting the interaction between CD40L and CD40. Monoclonal antibody blockade of CD40L strengthened tolerance by preventing the simultaneous induction of CTL. This was reflected by inhibition of the activation and expansion of adoptively-transferred OVA- specific CTL (OT-1-CD8 cells) in response to oral OVA. Furthermore, in mice with transgenic expression of OVA on pancreatic β cells, CD40L blockade significantly inhibited the development of CTL (OT-1 cell)-mediated autoimmune diabetes that followed oral administration of OVA. These results show that mucosal tolerance towards CTL is induced independently of the requirements of CD40L signalling for CTL priming. Blockade of CD40L signalling could, therefore, improve the efficacy (and safety) of mucosal antigens for preventing CTL-mediated autoimmune disease. To demonstrate this, female NOD mice aged 8 weeks were treated with anti-CD40L monoclonal antibody MR- 1 or control antibody 6C8 (300 μg i.p.) just before administration of aerosol insulin (4 mg/ml, 10 minutes) or diluent control on days 1, 3, 10, 24 and 38. Anti-CD40L antibody treatment with aerosol insulin markedly reduced diabetes incidence compared to anti- CD40L antibody treatment with diluent or control antibody and either aerosol insulin or diluent (Figure 17). EXAMPLE 14 Intranasal proinsulin DNA induces CD4 T cells which prevent diabetes MATERIALS AND METHODS DNA Mouse proinsulin II cDNA or ovalbumin genomic DNA was subcloned into a plasmid vector derived from the mammalian expression vector, pCI, under the control of the CMV early promoter. The vector was modified and is designated as CIGH. Plasmids were prepared from E.coli and purified by PEG precipitation and Triton XI 14 phase partition, diluted to 2 mg DNA per ml in PBS and frozen at -20°C. Mice and treatment NOD mice were bred and maintained in The Walter and Eliza Hall Institute of Medical Research. At 3 and 5 weeks of age, 25 μl of PBS containing 50 μg DNA was given intranasally in repeat 5 μl portions to non-anaesthetized female mice. In other experiments mice were given 25 μg DNA intranasally for four consecutive weeks beginning at 3 weeks of age. Determination of diabetes Blood glucose was measured using the Advantage monitor (Boehringer Mannheim) on a drop of blood obtained via a fine glass capillary tube from the retro-orbital venous plexus. Mice were considered to be diabetic if their blood glucose was >11 mM on consecutive days. Diabetic donor mice used in adoptive transfer studies has an elevated blood glucose for <1 week. RESULTS In initial experiments, spleen cells from intranasal DNA-treated mice at 10 weeks of age were enriched for T cells (hereafter referred to as splenic T cells) by passage through nylon wool, and than either co-transferred i.v. with spleen cells from recently-diabetic NOD mice into irradiated 6 week-old male NOD mice or transferred i.v. into cyclophosphamide- treated NOD females Cyclophosphamide treatment accelerates the onset of diabetes on NOD mice. In both experimental models, a significant reduction in diabetes incidence was observed in recipient mice that received cells from proinsulin DNA-treated donors. In three experiments in which 5 x 10 6 splenic T cells were co-transferred with 2 x 10 7 "diabetic" spleen cells, the combined incidence of diabetes in recipients 4 weeks after transfer was 14% in recipients of cells from proinsulin DNA-treated donors compared to 64% in recipients of cells from ovalbumin DNA-treated donors (p=0.003) (Table 4). In parallel, 5 x 10 6 splenic T cells were transferred into 8-10 week-old female NOD mice 2 days after they had received 300 mg/kg cyclophosphamide i.p. The peak incidence of diabetes observed 17 days after cyclophosphamide was 56% in recipients of cells from ovalbumin in DNA-treated donors compared to 12.5% in recipients of cells from proinsulin DNA- treated donors (p=0.02) (Table 5). No protection was observed when 5 x 10 6 splenic T cells from mice that had received two intramuscular injections of proinsulin DNA were injected into cyclophosphamide-treated recipients (Table 5). TABLE 4 Splenic CD4 T cells from NOD mice given intranasal proinsulin II DNA suppress adoptive transfer of diabetes *p=0.003, **p=0.02 compared to ovalbumin DNA control; Fisher's exact test TABLE 5 Splenic CD4 T cells from NOD mice given intranasal proinsulin II DNA suppress cyclophosphamide-induced diabetes fc p=0.002, **p=0.04 compared to ovalbumin DNA control; Fisher's exact test The inventors next sought to identify the phenotype of the T cell responsible for protection by transferring fractional splenic T-cell population. Splenic T cells were incubated with either anti-mouse CD4 or anti -mouse CD8 monoclonal antibodies conjugated to magnetic MACS MicroBeads and purified on positive selection columns (Miltenyi). The purity of CD4 and CD8 T cells by FACS analyses was >95% and >85%, respectively. Either 4 x 10 6 CD4 T cells or 1 x 10 6 CD8 T cells were then co-transferred with 2 x 10 7 diabetic spleen cells into 6 week-old irradiated males. Diabetes incidence 4 weeks after transfer was 36% in recipients of CD4 T cells from proinsulin DNA-treated donors compared to 71% in recipients of CD4 T cells from ovalbumin DNA-treated controls (p=0.02) (Table 4). In contrast, there was no difference in diabetes incidence (94 v 83%) in recipients of co-transferred CD8 cells from either proinsulin DNA-or ovalbumin DNA-treated mice (Table 4). Similar results were obtained when either 4 x 10° CD4 T cells or 2.5 x 10 6 CD4- depleted (CD8 T cell-enriched) splenic T cells were injected into cyclophosphamide- treated 10 week-old female mice. Thus, diabetes incidence in mice that received CD4 T cells from proinsulin DNA-treated donors was significantly reduced (17%) compared to mice that received CD4 T cells from ovalbumin DNA-treated donors (56%) (p=0.04), whereas there was no difference in diabetes incidence in mice that received CD4-depleted splenic T cells from proinsulin DNA- or ovalbumin DNA-treated donors (Tables 5 and 6). TABLE 6 CD45RB hi and CD45RB 10 CD4 T cells from NOD mice given intranasal proinsulin II DNA suppress cyclophosphamide-induced diabetes "p=0.07, **p=0.4 compared to ovalbumin DNA control; Fisher's exact test In both humans and NOD mice the development of hyperglycaemia is preceded by "insulitis", which ranges form a peri-islet accumulation of lymphocytes at the vascular pole or boundary of the islet to massive infiltration into the islet associated with β-cell destruction. The inventors then examined islets of NOD mice and found that the degree of insulitis was significantly less in proinsulin DNA- compared to ovalbumin DNA-treated mice at both 70 and 100 days of age (0.89 ± 0.08 versus 1.6 ± 0.12, p < 0.01; Mann- Whitney test). Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. BIBLIOGRAPHY 1. Weiner, H.L., et al, . Annu. Rev. Immunol. 12: 809-837, 1994 2. Kikutani et al, Adv. Immunol 51: 285-322, 1992 3. Adorini et /., Springer Semin. Immunopathol 14: 18 -199, 1992 4. Muir et al, Diabet./Metab. Rev. 9: 279-287, 1993 5. Tisch et al, Proc. Natl Acad. Sci. USA 91: 437-438, 1994 6. Harrison, L.C., Mol. Med. 1: 722-727, 1995 7. Holt et al, Immunol. Today 8: 14-15, 1987 8. Wells, H.G., J. Infect. Dis. 9: 147-151, 1911 9. Chen et al, Science 265: 1237-1239. 1994 10. Honeyman et al, Springer Semin. Immunopethol. 14: 253-274, 1993 11. Bach, LF. 1994. Endocrine Rev. 15: 516-542, 1994 12. Mordes et al, Diab. Metab. Rev. 3: 725-750, 1987 13. Harrison, L.C., Immunol Today. 13: 348-352, 1992 14. Dean et al, Diabetologia 29: 339-342, 1986 15. Ziegler et al, Diabetologia 36: 402-408, 1993 16. Harrison et al, J. Clin. Invest. 89: 1161-1165, 1992 17. Rudy et al, Mol Med. 1 : 625-633, 1995 18. Ziegler et al, Diabetes 38: 358-363, 1989 19. Daniel et al, Proc. Natl Acad. Sci. USA 93: 956-958, 1986 20. Zhang et al, Proc. Natl Acad. Sci. USA 88: 10252-10256, 1991 21. Bergerot et al, J. Autoimmunity 7: 655-663, 1994 22. Moses et al, Diabetes 32: 1040-1047, 1983 23. Metzler et al, Inter. Immunol 5: 1159-1165, 1993 24. Waldo et al, Clin. Immunol. Immunopathol. 73: 30-34, 1994 25. Kaufman, D.L., Nαtwre 366: 69-72, 1993 26. Hurtenback et al, J. Autoimmunity 2: 151-161, 1989 27. Hunt et al, J. Immunol. 136: 3994-3996, 1986 28. Tian et al, J. Exp. Med. 183: 1561-1567, 1996 29. French et al, Diabetes 46: 34-39, 1997 30. Wicker et al, Diabetes 35:855-60, 1986 31. Kabelitz, D., Crit. Rev. Immunol 11: 281-303, 1992 32. Kaufman et al, Proc. Natl. Acad. Sci. USA 90: 9620-9624, 1993 33. Cardillo et al,. Eur J Immunol 23: 2597-2605, 1993 34. Gorczynski, R.M., Immunology 81: 27-35, 1994 35. Seo et al, Cancer Immunol Immunother. 40: 358-366, 1995 36. Nakata et al, Immunol Immunopathol 3: 217-222, 1995 37. Lang et al, J. Autoimmunity 6: 107-119, 1993 38. Goodman et al, J. Exp. Med. 170: 1569-1581, 1989 39. Kweon et al, J. Immunol. 162: 1904-1909, 1999 40. Xu et al, Immunity 1: 423-431, 1994 41. Blanas et al, Science 274: 1707-1709, 1996

Claims: CLAIMS 1. A method of inducing cytotoxic T-lymphocyte (CTL) tolerance while substantially avoiding CTL immunity in response to a mucosal antigen in a subject, said method comprising selecting said mucosal antigen or modifying a mucosal antigen to disable the function of an MHC class I restricted epitope and then administering said selected or modified antigen for a time and under conditions sufficient to prevent or reduce CTL immunity to said mucosal antigen. 2. A method according to Claim 1 wherein the mucosal antigen is a mucosal autoantigen. 3. A method according to Claim 2 wherein the mucosal antigen is administered as a peptide or polypeptide. 4. A method according to Claim 2 wherein the mucosal antigen is administered as DNA which encodes said mucosal antigen. 5. A method according to Claim 2 or 3 or 4 wherein the mucosal antigen or DNA encoding the mucosal antigen is administered to mucosal surfaces. 6. A method according to Claim 5 wherein administration is via one or more of the oral, nasal, pharyngeal and/or bronchial passages. 7. A method according to Claim 6 wherein administration is via aerosol administration. 8. A method of preventing, reducing or otherwise ameliorating an autoimmune disease condition in a subject, said method comprising the aerosol administration to said subject of an effective amount of an antigen associated with said autoimmune disease for a time and under conditions sufficient to induce or stimulate immunoregulatory mechanisms which are protective against cell-mediated autoimmune pathology wherein said antigen substantially lacks a MHC class I interacting region. 9. A method according to Claim 1 or 8 wherein said subject is a human. 10. A method according to Claim 1 or 9 wherein the MHC class I epitope is an MHC class I (K d )-restricted epitope. 11. A method according to Claim 1 or 8 or 9 or 10 wherein the antigen is associated with insulin dependent diabetes melitus (IDDM), slowly progressive IDDM (SPIDDM) and/or gestational diabetes. 12. A method according to any one of Claims 1 to 11 in combination with blocking or otherwise delaying CTL induction and/or maturation. 13. A method of preventing, reducing or otherwise ameliorating IDDM, SPIDDM or gestational diabetes in a subject, said method comprising the administration, as an aerosol or other functionally equivalent means, to said subject of an effective amount of an autoantigen associated with IDDM for a time and under conditions sufficient for induction of regulatory T cells and/or other suitable mechanisms sufficient to suppress cell-mediated autoimmune pathology associated with IDDM wherein said autoantigen substantially lacks a functional MHC class I interacting epitope. 14. A method according to Claim 13 wherein the subject is a human. 15. A method according to Claim 13 wherein the MHC class I epitope is an MHC class I (K d )-restricted epitope. 16. A method according to Claim 13 wherein the regulatory T cells and CD8 T cells. 17. A method according to Claim 16 wherein the CD8 T cells are CD8γδ T cells. 18. A method according to Claim 15 wherein the regulatory T cells are CD4 T cells. 19. A method according to Claim 18 wherein the CD4 T cells are CD4αβ T cells. 20. A method according to Claim 13 wherein the aerosol administration is via a spray, drip or vapour. 21. A method according to any one of Claims 13 to 20 wherein the antigen is preproinsulin or proinsulin or fragments thereof. 22. A method according to any one of Claims 13 to 20 wherein the antigen is insulin or fragments thereof. 23. A method according to Claim 21 wherein the antigen is proinsulin peptide 24-36. 24. A method according to any one of Claims 13 to 20 in combination with blocking or otherwise delaying CTL induction and/or maturation. 25. A method of inducing, suppressing or otherwise ameliorating or preventing IDDM in a subject, said method comprising administering proinsulin peptide truncated at its C-terminal antigen end to disable the function of any MHC class I restricted epitope for a time and under conditions sufficient to prevent, reduce or otherwise induce CTL tolerance. 26. A method according to Claim 25 wherein the subject is a human. 27. A method according to Claim 26 wherein the proinsulin peptide is of human, murine or porcine origin. 28. A method according to Claim 27 wherein the proinsulin peptide is of human origin. 29. A method according to Claim 25 wherein the proinsulin is administered via aerosol. 30. A method according to Claim 29 wherein the aerosol administration is via spray, drip or vapour. 31. A method according to Claim 29 or 30 wherein the proinsulin is administered at a rate of from about 1 to about 20 litres/min. 32. A method according to Claim 29 or 30 or 31 wherein the proinsulin is administered within an adjuvant. 33. A method according to Claim 32 wherein the adjuvant is selected from cholera toxin 13, heat labile toxin of E. coli, saponica or its derivative, Quill A extracts, DΕAΕ-dextran, dextran sulphate and aluminium salts. 34. A method according to Claim 32 wherein the adjuvant is a cytokine, muramyl-dipeptide or cell wall component. 35. A method according to any one of Claims 25 to 34 in combination with blocking or otherwise delaying CTL induction and/or maturation. 36. A method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering said mucosal antigen or a nucleic acid encoding same for a time and under conditions sufficient to prevent or reduce CTL immunity, simultaneously or sequentially with the administration of an antagonist of CTL induction and/or maturation. 37. A method according to Claim 36 wherein the antagonist is an antagonist of CD40 or CD40L interaction. 38. A method according to Claim 37 wherein the antagonist is anti-CD40L antibody. 39. A method according to Claim 36 wherein the subject is a human. 40. A method according to Claim 36 wherein the proinsulin peptide is of human, murine or porcine origin. 41. A method according to Claim 36 wherein the proinsulin peptide is of human origin. 42. A method according to Claim 36 wherein the proinsulin is administered via aerosol. 43. A method according to Claim 36 wherein the aerosol administration is via spray, drip or vapour. 44. A method of inducing tolerance to a mucosal antigen while substantially avoiding CTL immunity to said antigen, said method comprising administering an agent for a time and under conditions sufficient to block or otherwise delay CTL induction and/or maturation. 45. A method according to Claim 44 wherein the agent is an antagonist of CD40L-CD40 interaction. 46. A method according to Claim 45 wherein the antagonist is anti-CD40 antibody. 47. Use of a mucosal antigen with an inactive MHC class I epitope in the manufacture of a medicament for the treatment or prophylaxis of a disease condition in a subject. 48. Use according to Claim 47 wherein the subject is a human. 49. Use according to Claim 47 or 48 wherein the disease condition is IDDM, SPIDDM or gestational diabetes. 50. Use according to Claim 49 wherein the antigen is an autoantigen. 51. Use according to Claim 49 wherein the autoantigen is proinsulin. 52. Use of an agent in the manufacture of a medicament for the treatment or prophylaxis of a disease condition, said agent capable of blocking CTL induction and/or maturation. 53. Use according to Claim 52 wherein the agent blocks CD40L-CD40 interaction. 54. Use according to Claim 53 wherein the agent is anti-CD40L antibody. 55. An agent for the treatment or prophylaxis of an autoimmune disease, said agent comprising an autoantigen giving rise to said autoimmune disease, said autoantigen being modified to lack a functional MHC class I restricted epitope. 56. An agent according to Claim 54 wherein the autoimmune disease is IDDM, SPIDDM or gestational IDDM. 57. An agent according to Claim 56 wherein the autoantigen is proinsulin or insulin. 58. An agent according to any one of Claims 55 to 57 further comprising an agent which blocks CTL induction and/or maturation. 59. An agent according to Claim 58 wherein the agent blocks CD40L-CD40 interaction. 60. An agent according to Claim 59 wherein the agent is anti-CD40L antibody. 61. An agent for use in the treatment and/or prophylaxis of autoimmune conditions, said agent comprising an antagonist of CTL induction and/or maturation. 62. An agent according to Claim 61 wherein the agent blocks CD40L-CD40 interaction. 63. An agent according to Claim 62 wherein the agent is anti-CD40L antibody.

Inventors: HANNINEN ARNO, HARRISON LEONARD C, KRAMER DAVID, MARTINEZ NATHAN R, HANNINEN, ARNO, HARRISON, LEONARD, C, KRAMER, DAVID, MARTINEZ, NATHAN, R
Applicants: HANNINEN ARNO, HARRISON LEONARD C, INST MEDICAL W & E HALL, KRAMER DAVID, MARTINEZ NATHAN R, HANNINEN, ARNO, HARRISON, LEONARD, C, KRAMER, DAVID, MARTINEZ, NATHAN, R, THE WALTER AND ELIZA HALL INSTITUTE OF MEDICAL RESEARCH
Application year: 2000
Main classification: A61K38
IPC codes: A61K9, A61K39, A61K45, A61K48, A61P3, A61P37, C07K16