WO2009052561A1 - Compositions and methods for manipulating an immune response - Google Patents

Abstract

A method for reducing antigen presentation by one or more antigen presenting cells is described. The method comprises the steps of introducing an exogenous apoptosis-inducing agent into the cytosol of the one or more antigen presenting cells, and triggering apoptosis of the one or more antigen presenting cells by an apopto sis-triggering complex that includes the exogenous apoptosis-inducing agent and an endogenous apoptosis-inducing agent. Reduction in antigen presentation by antigen presenting cells in accordance with the method described, can result in a reduced immune response, a reduction in cross-presentation of antigens to one or more CD8+ T- cells, a reduction in interleukin 12 expression, and/or a reduction in toll-like receptor 3 expression.

Description

COMPOSITIONS AND METHODS FOR MANIPULATING AN IMMUNE

RESPONSE

FIELD OF THE INVENTION

The present invention relates to compositions and methods for manipulating an immune response.

In particular, the present invention relates to compositions and methods for reducing cross-presentation of an antigen by antigen-presenting cells (APCs) to T-cells. The invention has been developed primarily as a composition and a method for manipulating an immune response and will be described hereinafter with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use.

BACKGROUND OF THE INVENTION

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Cross-presentation (or cross-priming) denotes the ability of certain antigen presenting cells (APCs) to internalise, process, and present extracellular antigens with major histocompatibility complex (MHC) class I receptors to CD8⁺ T-cells (cytotoxic T- cells, CTLs) in contrast to direct priming, which results from the presentation of endogenous antigens. Cross-presentation is necessary for induction of immunity against most tumours and against viruses that do not infect antigen presenting cells. It is also required for induction of cytotoxic immunity by vaccination with protein antigens, for example, tumour vaccinations. Cross-presentation of antigens is an important process for generating cytotoxic T-cell responses (Heath et al., Immunol. Rev. 199:9-26, 2004). APCs capable of cross- presentation are predominantly CD8⁺ dendritic cells (DCs). However, macrophages, B- cells, and liver sinusoidal endothelial cells have also been shown to be capable of cross- presentation. The reasons for this cell activity and the exact mechanisms involved in cross-presentation remain contentious although several mechanisms have been proposed (Rodriguez et al., Nat. Cell Biol. 1:362-368, 1999; Shen et al., Immunity 21:155-165, 2004; and Ackerman et al., Immunity 25:607-617, 2006). There is evidence to suggest that the specialised ability of dendritic cells for cross-presentation may be explained, at least in part, by the presence of a unique, size-selective intracellular pathway for export of an antigen from endosomal compartments into the cytosol for access to the conventional MHC class I processing pathway (Rodriguez et al., Nat. Cell Biol. 1:362- 368, 1999), although this has not specifically been shown for the CD8⁺ dendritic cell subset. Recent data also suggest that the cytosolic cross-presentation pathway is the major relevant mechanism in vivo (Palmowski et al., J. Immunol. 177:983-990, 2006) and that the machinery that confers cross-presentation specialisation is located downstream from antigen internalisation (Schnorrer et al., Proc. Natl. Acad. Sci. U.S.A. 103:10729-10734, 2006).

The mitochondrial intrinsic pathway is one of the most characterised pathways of apoptosis. One initiator of this pathway in mammals is cytochrome c, a soluble 13 kDa mitochondrial protein that also functions as a component of the electron transport chain. Upon receiving an apoptotic stimulus, cytochrome c is released from the intermembrane space of the mitochondrion into the cytosol of the cell, where it binds to its partner protein, apoptotic-protease-activating-factor 1 (Apaf-1) to form a pro-caspase-9- activating heptametric protein complex known as the apoptosome (Schafer et al., Developmental Cell 10:549-561, 2006). Apaf-1 -mediated caspase activation is accomplished through a series of downstream effector caspases eventually resulting in cell death. Specific ablation of the apoptotic function of cytochrome c using a mouse model has shown that the only major activator of Apaf-1 is cytochrome c and that the only significant protein that cytochrome c can activate during apoptosis is Apaf-1 (Hao et al., Cell 121 :579-591, 2005). Previously, despite the progress in understanding the mechanism of cross- presentation, in vivo application of cross-presentation to manipulate (either by enhancing or reducing) a CD8⁺ T-cell response remains to be exploited. This is in part due to the inability to promote selective enhancement (adjuvanting) or selective reduction (e.g., deletion) of the cross-presenting cell or its function. As described above, cross- presenting cells possess a specific cytosolic export feature. This feature has not previously been used to export an apoptosis-inducing agent, for example, cytochrome c, into the cytosol of the antigen-presenting cell to trigger apoptosis and therefore permit the manipulation of an immune response.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION

Accordingly, in one aspect the present invention provides a method for reducing antigen presentation by one or more antigen presenting cells, the method comprising introducing an exogenous apoptosis-inducing agent that enters into the cytosol of the one or more antigen presenting cells, wherein the introduction of the exogenous apoptosis-inducing agent results in the triggering of apoptosis of the one or more antigen presenting cells by an apopto sis-triggering complex comprising the exogenous apoptosis-inducing agent and an endogenous apoptosis-inducing agent.

The exogenous apoptosis-inducing agent may directly form an apoptosis-triggering complex with an endogenous apoptosis-inducing agent. In other examples, the exogenous apoptosis-inducing agent may be modified or activated prior to the formation of an apoptosis-triggering complex. Thus, the exogenous apoptosis-inducing agent, when part of an apoptosis-triggering complex, may be in the same form as introduced into the cell or may be in a modified or activated form.

Preferably, the exogenous apoptosis-inducing agent is a purified protein, or a portion or an active fragment thereof. In one example, the protein is cytochrome c, or a portion or an active fragment thereof. Alternatively, the exogenous apoptosis-inducing agent is a toxin (e.g., streptolysin), or a portion thereof, or another cytotoxic agent (e.g., granzyme B) that can elicit cell death or inactivation (such that the host cell can no longer cross- present) after being transported to the cytosol.

Cytochrome c from any vertebrate can be used in the invention. Specific, non- limiting examples of cytochrome c that can be used in the invention include human cytochrome c (e.g., NP 061820: mgdvekgkki fimkcsqcht vekggkhktg pnlhglfgrk tgqapgysyt aanknkgiiw gedtlmeyle npkkyipgtk mifvgikkke eradliaylk katne (SEQ ID NO:2) and NM Ol 8947 (SEQ ID NO: I)), horse cytochrome c (e.g., P00004 and XM 001498822: mgdvekgkki fvqkcaqcht vekggkhktg pnlhglfgrk tgqapgftyt danknkgitw keetlmeyle npkkyipgtk mifagikkkt eredliaylk katne (SEQ ID NOs:3 and 4)), and murine cytochrome c (e.g., XP 980234 and XM 975140: mgdvekgkki fvqkcaqcht vekggkhktg pnlhglfgrk tgqaagfsyt danknkgitw gedtlmeyle npkkyipgtk mifagikkkg eradliaylk katne (SEQ ID NOs:5 and 6)). As the sequence of cytochrome c is highly conserved, cytochrome c proteins from different species can be used in the methods of the invention. Any derivative of cytochrome c that also binds to Apaf-1 can also be used in the methods of the invention. For example, a derivative of cytochrome c having, for example, at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to a naturally occurring cytochrome c sequence (e.g., SEQ ID NOs:2, 4, and 6) can be used in the invention.

"Percent (%) amino acid sequence identity" with respect to the sequences described herein is defined as the percentage of amino acid residues in a candidate sequence (i.e., a derivative) that are identical with the amino acid residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purposes of determining percent amino acid sequence identity can be achieved using methods known in the art, and those skilled in the art can determine appropriate parameters for measuring alignment, including assigning algorithms needed to achieve maximal alignment over the full- length sequences being compared (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705).

The term "antigen" is used herein to refer to any substance (e.g., a polypeptide, protein, peptide, polysaccharide, or fragment thereof) that can stimulate an immune response. In certain examples, an antigen according to the invention is processed by antigen presenting cells, leading to induction of an immune response, as described herein. In the case of polypeptide, protein, or peptide antigens, the term "antigen" is to be considered as encompassing full-length proteins, as well as fragments including epitopes to which immunity is induced. Practice of the invention does not require the use or identity of any particular antigen. Rather, the methods of the invention result in reduction of antigen presentation in general, and are not limited to any specific antigen.

The methods in accordance with the invention provide a differential induction of apoptosis or inactivation of cross-presenting cells. Advantageously, the methods provide a correlation between cross-presenting function and apoptosis; as apoptosis is only triggered when exogenous cytochrome c, or a portion or an active fragment thereof, is delivered to the cytosol. Furthermore, in certain examples, the methods can be completely performed in vivo. Thus, the processing/presentation of the antigen or portion thereof does not need to contain any ex vivo steps and avoids any potential artifacts during the isolation and culture of DCs. Most advantageously, the methods not only provide a tool for further study of the in vivo contribution of cross-presentation of an antigen, or a portion thereof, for induction of a CD8⁺ T-cell response, but permits specific targeting of cross-presenting DCs to manipulate CD8⁺ T-cell responses.

It is a further advantage of the present invention that the methods may be beneficial to subjects (such as human patients) in minimizing transplant rejection, by reducing an immune response to an allograft or xenograft. The present invention can apply to either donor grafts or the recipient by inactivating either donor cross-presenting cells or host cross-presenting cells. Thus, the invention includes treatment of candidate live organ, tissue, or cell donors; donated organs, tissues, or cells themselves; and recipients of donated organs, tissues, or cells; or a combination thereof. Examples of contexts in which the methods of the invention can be used thus include transplantation of heart, kidney, lung, liver, pancreas, bones, tendons, cornea, heart valves, veins, skin, bone marrow cells, pancreatic islet cells, and blood transfusions.

It is a further advantage of the present invention that the methods may also be beneficial in reducing acute immune responses in certain subjects (such as human patients), thereby reducing tissue damage.

It is a further advantage of the present invention that the methods may also be beneficial in the prevention or treatment of autoimmune disease, inflammatory diseases or conditions, neoplastic disease (for example, cancer-associated immunopathology), viral disease (for example, influenza or respiratory syncytial disease, as well as viral infection-associated immunopathology), bacterial infection (for example, bacteria infection-associated immunopathology), and any disease where there is T-cell-mediated pathology. Subjects treated using the methods of the invention include, for example, humans, as well as other mammals, such as livestock and domestic pets.

The terms "prevention" or "preventing" as used herein are intended to encompass blocking, inhibiting, or decreasing the likelihood of occurrence of a disease or condition, while the terms "treatment" or "treating" are intended to encompass providing a therapeutic benefit to an existing disease or condition, such as by reducing one or more symptoms (temporarily or permanently) or providing a cure.

Whilst not wishing to be bound to any particular theory of how the invention works, Applicant believes that the exogenous apoptosis-inducing agent interacts with an endogenous apoptosis-inducing agent to form an apoptosis-triggering complex in the one or more antigen presenting cells.

Preferably, the immune response is an adaptive immune response.

Preferably, the exogenous apoptosis-inducing agent is admitted to the one or more APCs via their extracellular environment. Alternatively, the exogenous apoptosis- inducing agent is admitted intracellularly to the one or more APCs. Preferably, the apoptosis-inducing agent is internalised by the one or more APCs by pinocytosis, macropinocytosis, phagocytosis, receptor uptake (for example, mannose receptor or Fc receptor for immune complexes), or other pathway.

Preferably, the exogenous apoptosis-inducing agent is produced in an expression system, and thus can be in the form of a nucleotide sequence located in an expression cassette for ligation into a vector. Preferably, the vector is selected from a cloning vector or an expression vector. The expression vector can be selected from the group consisting of mammalian (for example, pCI), yeast (for example, Pichia vectors), or bacterial expression vectors (for example, pJC40). Most preferably, the vector is used in an expression system to produce cytochrome c or a portion thereof for subsequent administration to a patient.

Preferably, the endogenous apoptosis-inducing agent is Apaf-1.

Preferably, the apopto sis-triggering complex is the apoptosome.

Preferably, the one or more APCs are selected from the group consisting of dendritic cells, macrophages, B-lymphocytes, and liver sinusoidal endothelial cells. More preferably, the one or more APCs are dendritic cells. Most preferably, the one or more APCs are CD8⁺ dendritic cells.

Preferably, the antigen, or a portion thereof, is cross-presented on the surface of one or more APCs in association with a MHC receptor. Most preferably, the antigen, or a portion thereof, is bound to a MHC class I receptor.

Preferably, cytochrome c, or a portion or an active fragment thereof, is selectively internalised by dendritic cells in vivo and in vitro.

Reduction in antigen presentation by antigen presenting cells, according to the invention, can result in a reduced immune response, for example, by a reduction in cross-presentation of antigens to one or more CD8⁺ T-cells. Treatment according to the methods of the invention can optionally further result in a reduction in inter leukin 12 expression, and/or a reduction in toll-like receptor 3 expression.

In yet a further aspect, the present invention provides pharmaceutical compositions comprising cytochrome c, or a portion or an active fragment thereof, and a pharmaceutically acceptable diluent (e.g., sterile water or saline), excipient, solvent, adjuvant, or carrier. In another example, the pharmaceutical composition can comprise cytochrome c in lyophilized form, and such a composition can be reconstituted with a pharmaceutically acceptable diluent prior to administration. Optionally, the pharmaceutical compositions can also include one or more additional pharmaceutically active agents.

The pharmaceutical composition can be administered parenterally, e.g., intravenously, intraperitoneally, subcutaneously, transcutaneously, intradermally, or intramuscularly, or can be administered mucosally (e.g., orally) or topically (e.g., by inhalation). Typically, protein-based therapeutics are administered by injection, which and may require frequent administration, because of the half-life of proteins. To reduce the need for multiple injections, the pharmaceutical composition can be formulated for administration by inhalation or transdermal means. In other examples, PEGylation can be used to increase circulation half-life, thereby reducing frequency of injections. PEGylation can be achieved by nonspecific conjugation of the protein with linear poly(ethylene glycol) chains or branched poly(ethylene glycol), or by site-specific PEGylation achieved using mutagenesis techniques (Chaubal et al, Pharmaceutical Technology 30(10), 2006).

Acceptable carriers for use in the invention include saline or buffers such as phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 residues) polypeptides; proteins, such as serum albumin (e.g., human serum albumin or bovine serum albumin), gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagines, arginine, and lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, maltose, and dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, PLURONICS™, and PEG. Optionally, the formulations include a pharmaceutically acceptable salt, such as sodium chloride at about physiological concentrations. Optionally, the formulations of the invention can contain a pharmaceutically acceptable preservative (e.g., benzyl alcohol, phenol, m-cresol, methylparaben, or propylparaben). Further, the active ingredients can be entrapped in microcapsule or sustained-release preparations can be prepared.

Procedures for the preparation of dosage unit forms are readily available to those skilled in the art from texts such as Pharmaceutical Handbook. A Martindale Companion Volume, ed. Ainley Wade, 19^th edition, The Pharmaceutical Press London; CRC Handbook of Chemistry and Physics, ed. Robert C. Weast, Ph.D., CRC Press Inc.; Goodman and Gilman 's, The Pharmacological basis of Therapeutics, 9^th edition, McGraw Hill; Remington, The Science and Practice of Pharmacy, 19^th edition, ed. Alfonso R. Gennaro, Mack Publishing Co., Easton, PA; and Remington 's Pharmaceutical Sciences, 20^th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA.

Depending on the condition treated, administration can be short or long-term. For example, in the case of certain autoimmune diseases, it may be desirable to administer the composition of the invention to a patient experiencing an exacerbation of symptoms, to assist in achieving remission, upon which treatment may optionally be terminated (or reduced). Similarly, in the case of organ or tissue transplantation, it may be desirable to administer the compositions of the invention to the recipient just prior to, during, or after transplantation (or a combination thereof).

In these examples, the compositions can be administered as sole therapeutic agents or in combination with other therapeutic agents, such as immunosuppressive agents (e.g., tacrolimus, LEA29Y, anti-CD25, and anti-thymocyte globulin).

In the case of short-term administration to patients who are hospitalized for their treatment, it may be advantageous to administer the compositions by intravenous infusion via an intravenous line, either continually or intermittently. In out-patient situations, it may be preferred to use subcutaneous, intramuscular, or mucosal (e.g., oral) routes.

In the case of organ, tissue, or cell transplants, as noted above, the organ, tissue, or cells may be treated with the compositions of the invention. For solid organs or tissues, the composition can be applied to the surface of the organ or tissue, or infused into the organ or tissue, while for cell-based grafts (and also for solid organs or tissues), the organ, tissue, or cell-based graft can be placed in a liquid medium including the composition.

In yet a further aspect, the present invention provides use of the pharmaceutical compositions in accordance with the invention (including an apoptosis-inducing agent, such as cytochrome c or a portion or active fragment thereof) for the prevention or treatment of autoimmune disease, inflammation, transplant rejection, neoplastic disease (for example, cancer-associated immunopathology), viral disease (for example, influenza or respiratory syncytial disease, as well viral infection-associated immunopathology), bacterial infection (for example, bacteria infection-associated immunopathology), and any diseases where there is T-cell-mediated pathology.

The compositions of the present invention can be administered simultaneously or consecutively with one or more other pharmaceutically active agents effective in the prevention or treatment of autoimmune disease, inflammatory diseases or conditions, transplant rejection, neoplastic disease (for example, cancer-associated immunopathology), viral disease (for example, influenza or respiratory syncytial disease, as well as viral infection-associated immunopathology), bacterial infection (for example, bacteria infection-associated immunopathology), and any disease where there is T-cell- mediated pathology.

In yet a further aspect, the present invention provides use of an apoptosis- inducing agent (e.g., cytochrome c, or a portion or an active fragment thereof) in the manufacture or preparation of a medicament for the prevention or treatment of autoimmune disease, inflammatory diseases or conditions, transplant rejection, neoplastic disease (for example, cancer-associated immunopathology), viral disease (for example, influenza or respiratory syncytial disease, as well as viral infection-associated immunopathology), bacterial infection (for example, bacteria infection-associated immunopathology), and any diseases where there is T-cell-mediated pathology.

In yet a further aspect, the present invention provides a method of preventing or treating autoimmune disease, inflammatory diseases or conditions, transplant rejection, neoplastic disease (for example, cancer-associated immunopathology), viral disease (for example, influenza or respiratory syncytial disease, as well as viral infection-associated immunopathology), bacterial infection (for example, bacteria infection-associated immunopathology), and any diseases where there is T-cell-mediated pathology, comprising administering a prophylactically or therapeutically effective dose of cytochrome c, or a portion or an active fragment thereof, to a patient in need thereof.

Preferably, the prophylactically or therapeutically effective amount of cytochrome c, or a portion or an active fragment thereof, is from 0.5 mg/kg to 1250 mg/kg; more preferably from 5 mg/kg to 750 mg/kg; more preferably from 50 to 500 mg/kg; more preferably from 50 mg/kg to 250 mg/kg; and most preferably 250 mg/kg.

In yet a further aspect, the present invention provides a method of preventing or treating a disease selected from autoimmune disease, inflammatory diseases or conditions, transplant rejection, neoplastic disease (for example, cancer-associated immunopathology), viral disease (for example, influenza or respiratory syncytial disease, as well as viral infection-associated immunopathology), bacterial infection (for example, bacteria infection-associated immunopathology), and any disease where there is T-cell- mediated pathology, comprising administering a prophylactically or therapeutically effective dose of cytochrome c, or a portion or an active fragment thereof, in combination with a known treatment of the selected diseases.

In yet a further aspect, the present invention provides a use of a prophylactically or therapeutically effective dose of cytochrome c, or a portion or an active fragment thereof, in combination with another pharmaceutically active agent effective in the prevention or treatment of autoimmune disease, inflammatory diseases or conditions, transplant rejection, neoplastic disease (for example, cancer-associated immunopathology), viral disease (for example, influenza or respiratory syncytial disease, as well as viral infection-associated immunopathology), bacterial infection (for example, bacteria infection-associated immunopathology), and any disease where there is T-cell- mediated pathology in the preparation or manufacture of a medicament for the prevention or treatment of autoimmune disease, inflammatory diseases or conditions, transplant rejection, neoplastic disease, viral disease, for example, influenza or respiratory syncytial disease and any disease where there is T-cell-mediated pathology.

The term "T-cell mediated pathology" as used in the context of the present invention is intended to encompass, but is not limited to, any pathological state or condition that is initiated and maintained by a T-cell subtype; initiated by a T-cell subtype and maintained by another cell subtype, for example a B-cell, or any pathological state or condition that may in other ways involve a T-cell.

The term "inflammatory disease or condition" as used in the context of the present application is intended to encompass, but is not limited to, any disease or condition characterized by a localized protective reaction of tissue to irritation, injury, or infection, resulting in pain, redness, swelling, and/or loss of function. Non-limiting examples of inflammatory conditions that can be prevented or treated according to the invention include arthritis, gastritis, pericarditis, ileitis, osteitis, and encephalitis.

Chronic inflammation can be related to persistent infection, ulceration, mechanical or chemical irritation, or autoimmune disease.

The term "autoimmune disease" as used in the context of the present invention is intended to encompass, but is not limited to, any disease state or condition that involves one or more components of cellular or humoral immunity. In general, autoimmune diseases are characterized by an immune response in an individual that is directed against a tissue, cells, or organs of the individual. Examples of autoimmune diseases that can be prevented or treated according to the methods of the invention include the following: acute disseminated encephalomyelititis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), anemia (e.g., autoimmune hemolytic anemia), autoimmne hepatitis, autoimmune inner ear disease, pemphigoid (e.g., bullous pemphigoid), celiac disease, dermatomyositis, diabetes mellitus (type I and type II), insulin resistance, metabolic syndrome, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Raynaud's syndrome, Reiter's disease, nephritis, neuropathy, inflammatory bowel disease or colitis (e.g., Crohn's disease, ulcerative colitis, Behcet's disease, collagenous colitis, and lymphocytic colitis), thyroiditis (e.g., Hashimoto's disease), thrombocytopenia (e.g., idiopathic thrombocytopenic purpura), lupus (e.g., systemic lupus erythematosis), mixed connective tissue disease, sclerosis (e.g., multiple sclerosis, systemic sclerosis, primary progressive multiple sclerosis, relapsing remitting multiple sclerosis, progressive systemic sclerosis, amyotrophic lateral sclerosis, atherosclerosis, and arteriosclerosis), amyloidosis, rheumatic fever, respiratory distress syndrome, meningitis, autoimmune haemato logical disorders, inflammation of the uvea, iritis, choroiditis, sudden hearing loss, encephalitis, uveitis, glomerulonephritis, allergy, eczema, myasthenia gravis, pemphigus vulgaris, pernicious anaemia, polymyositis, primary biliary cirrhosis, psoriasis, dermatitis, urticaria, scleroderma, pulmonary fibrosis, chronic pulmonary inflammatory disease, autoimmune myocarditis, arthritis (e.g., rheumatoid arthritis), sarcoidosis, Sjόgen's syndrome, vasculitis, arteritis, and vitiligo.

The term "neoplastic disease" as used in the context of the present invention is intended to encompass, but is not limited to, any abnormal proliferation of a cell in a tissue, an organ or a biological fluid. Such abnormal proliferation may be benign or malignant. The neoplastic disease may be a solid tumour or a liquid tumour, such as a leukaemia or the like. The methods of the invention are particularly useful in the treatment of neoplastic disease in the context of preventing or treating immunopathology associated with cancer.

The term "another pharmaceutically effective agent" as used in the context of the present invention is intended to encompass, but is not limited to, those compounds or compositions that are useful in the prevention or treatment of the selected disease or conditions contemplated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

A preferred embodiment of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

Figure 1 (a) shows flow cytometry of splenic dendritic cells 24 hours following treatment with 5 mg of either horse or yeast cytochrome c; (b) shows absolute DC counts per spleen after horse or yeast cytochrome c treatment; (c) shows total cell numbers after horse or yeast cytochrome c treatment; (d) shows DC subset counts, and (e) shows an apoptosis assay of purified CDl Ic⁺ cells cultured in the indicated doses of cytochrome c and stained with CD8, annexin V, and PI for analysis by flow cytometry.

Figure 2 graphically shows B6 mice left untreated or given 3 daily doses of iv cytochrome c starting the day prior to injection of OVA-coated splenocytes (OCS) (a) or PK15-OVA (b); CFSE-labeled OT-I cells were adoptively transferred into all mice (a), (b, left panel). Some mice were co-injected with equal numbers of CFSE-labeled OT-II (b, right panel).

Figure 3 graphically shows the effect of cytochrome c on the abrogation of cross- presentation of OVA by CD8⁺ DCs to OT-I cells.

Figure 4 graphically shows flow cytometry plots of CFSE-labeled OT-I cells that were adoptively transferred into WT- or Apaf-l^'^-reconstituted bml mice. Mice were untreated or given 3 daily doses of cytochrome c starting the day prior to receiving OCS. Proliferation was assessed 60 h later with (a) representative and (b) quantitation of proliferating cells shown.

Figure 5 (a) shows endogenous in vivo CTL responses in B6 mice primed in vivo with OCS and treated or untreated with cytochrome c determined after 7 days; (b) shows endogenous CD8⁺ T-cell proliferation; (c) shows total numbers of CFSE^dimCD8⁺ cells from cytochrome c treated immunized, untreated immunized, or unimmunised mice, and (d) shows endogenous CD4⁺ T-cell proliferation.

Figure 6 graphically shows total tumour numbers per spleen (a) and (c), and representative FACS plots (b) from B6 mice given three daily injections of cytochrome c starting the day prior to in vivo priming with OCS and inoculated after 7 days with Eμ- wyc-GFP or Eμ-wyc-OVA cells.

Figure 7 graphically shows T cell proliferation in response to OVA presented by residual splenic CDl Ic⁺ DCs from pooled cytochrome c treated (after 24 hours) or untreated mice sorted into CD8⁺ or CD8^" subsets. Each subset was cultured continuously in duplicate for 60 hours with the indicated doses of OVA together with CFSE-labelled (a) OT-I or (b) OT-II cells.

Figure 8 shows (a) IL-12p70 production as measured by ELISA of culture supernatants and (b) TLR3 expression as measured by quantitative real-time PCR of DCs as prepared for Figure 6. ** indicates that no IL-12p70 was detected.

Figure 9 graphically shows pancreatic islet allotransplant survival as measured by normalization of blood glucose in diabetic mice. Mice were injected daily with 5 mg horse cytochrome c in phosphate buffered saline intravenously on the day prior to transplantation, then intraperitoneally for the following 7 (Long treatment) or 2 (Short treatment) days. Vehicle treated controls received phosphate buffered saline alone according to the long treatment schedule. The figure shows data from two experiments represented by solid and dashed lines respectively. Each line represents an individual mouse.

Figure 10 graphically shows that mouse islets coated with cytochrome c are not rejected in fully allogenic, immune competent recipients. Islets were isolated from Fl (C57BL/6 X Balb/c) donors and cultured overnight to recover. The following day, islets were handpicked into 200 islet aliquots and cultured for a further 24 h in either media with or without 5 mg/ml cytochrome c. After treatment, islets were recovered and 400 islets were transplanted under the kidney capsule of non-auto imune diabetic CBA recipients (RIP-Kb CBA). Blood glucose was measured via tail vein bleed at 1, 3, and 5 days post transplantation, and then weekly intervals to monitor initial diabetes reversal and then graft rejection. All grafts were surgically successful as determined by normoglycaemia within 4 days post transplantation. Islet grafts coated with cytochrome c were protected against allogenic rejection (Log-rank test: p=0.0124).

Figure 11 shows that mouse islets exposed to cytochrome c are protected against immune destruction in fully allogenic, immune competent recipients. Islet grafts treated with 5 mg/ml cytochrome c in vitro (cytc) or derived from donors treated 24 h prior to islet isolation with 5 mg intravenous cytochrome c (donor) or untreated grafts were recovered from non-autoimmune diabetic CBA recipients (RIP-Kb CBA) 100 d post transplant. Frozen sections were stained with antibodies against insulin (Ins), CD4, or CD8. Donor tissue exposed to cytochrome c (either ex vivo or in vivo) was found to have less CD4⁺ and CD8⁺ infiltrating cells and more insulin positive islet mass when compared to untreated control grafts.

PREFERRED EMBODIMENT OF THE INVENTION

A mouse model that allowed functional ablation of cross-presenting cells was designed. Dendritic cells are highly macropinocytic cells and efficient cross- presentation involves specialized machinery for endosome-to-cytosol transport. Release of endogenous cytochrome c from the mitrochondrion of higher eukaryotic organisms into the cytosol permits engagement with Apaf-1 and subsequent initiation of apoptosis within mammalian cells. In contrast, cytochrome c from lower eukaryotic organisms, for example yeast, has a low affinity for Apaf-1, and does not trigger activation of caspases and fails to initiate apoptosis (partly due to trimethylation of lysine 72). Thus, the introduction of exogenous horse cytochrome c into mice would selectively induce apoptosis in cells that possess this unique capacity for cytosolic transfer, namely, professional cross-presenting cells. Mice and reagents

C57BL/6 (B6), bml (Nikolic-Zugic et al., Eur. J. Immunol. 20:2431-2437, 1990), OT-I (Hogquist et al., Cell 76:17-27, 1994), OT-II (Barnden et al., Immunol. Cell Biol. 76:34-40, 1998), and CDl lc-mOVA (Steptoe et al., J. Immunol 178: 2094-2103, 2007) mice have been described. Lethal irradiation of bml mice consisted of two doses of 550 cGy spaced 2 hours apart using a ⁶⁰Co source on an Eldorado 8 instrument. Reconstitution of irradiated hosts was performed with E14.5 Apaf-l^"7" fetal liver cells. Neomycin sulphate (0.2% w/v) (Sigma) was added prophylactically to the drinking water of these mice for the first 3 weeks following irradiation. For haematopoietic reconstitution with Apaf-1-/- or Apaf-1+/+ stem cells, single cell suspensions were prepared from individual mouse foetal livers by repetitively pipetting the tissue through a wide-bore pipette tip to facilitate dispersion. After washing, 2-5 x 10⁶ foetal liver cells were injected Lv. into irradiated hosts. OVA (ovalbumin) protein, cytochrome c from horse heart and Saccharomyces cerevisiae were obtained from Sigma (St Louis, MO). Mice received, intravenously, 5 mg horse or yeast cytochrome c dissolved in PBS.

Splenic DC isolation and analysis

Splenic DCs were isolated as previously described (Vremec et al., J. Immunol. 164:2978-2986, 2000). To generate a single cell suspension, up to 4 spleens were cut into fragments and resuspended in 6 ml RPMI/2%FCS containing 7 mg collagenase (Type II, Worthington Biochemicals) and 1 mg DNase (Type I, Boehringer-Mannheim). Single cell suspensions were made by gentle pipetting through a wide-bore transfer pipette repetitively for 25 min at room temperature, followed by the addition of 0.6 ml of 0.1 M EDTA (pH 7.2) for 5 min to dissociate T cell-DC complexes and to inhibit further enzymatic activity of collagenase. Undigested fragments were removed by filtration through a steel sieve. This procedure was performed at 4°C. The splenocyte suspension was washed once with RPMI/2%FCS and pelleted. This was resuspended in 5 ml of cold Nycodenz medium (Nycomed Pharma) and gently overlayed onto 5 ml of Nycodenz. A further 0.5 ml of 0.01 M EDTA in FCS was layered above the splenocyte suspension. Gradient density centrifugation (Beckman) was performed at 3000 g for 15 min at 4°C. The supernatant, containing light density cells (<1.077 g/cm³ at mouse osmolarity) was collected. The splenic DCs at this stage were usually about 50% pure. This preparation was used for (i) high-speed sorting (MoFIo, Cytomation, Fort Collins, CO), (ii) immunomagnetic bead purification (AutoMACS, Miltenyi Biotec, Sydney, Australia), or (iii) immuno fluorescent labeling before analysis.

Immunofluorescent staining

Anti-CD 1 Ic, CD4, CD8, CD3, CDl Ib, CD 19, Vα2, B220, F4/80, TCRβ, annexin V, and streptavidin were purchased from PharMingen (San Diego, CA) or Caltag (Burlingame, CA). Single cell suspensions were prepared for analysis or sorting by flow cytometry at at 4°C. Approximately 0.5—5 x 10⁶ cells were stained in 50 ml of suspension media containing fluorochrome-conjugated mAb or reagent for 30 min and then washed with a large volume of suspension buffer. Propidium iodide (PI) was added at 1 μg/ml just prior to analysis for exclusion of dead cells.

Cell lines

The pig kidney cell line, PKl 5 (ATCC, Manassas, VA) was stably lipofected using FuGENE6 (Roche Diagnostics Corporation) with the mammalian expression vector pCI-neo (Promega) encoding membrane OVA (designated PK15-OVA), and maintained in the presence of 1.1 mg/ml Geneticin (G418, GibcoBRL, Gaithersburg, MD) and 4 μg/ml puromycin (Sigma). Stable Eμ-myc cell lines bearing GFP (Eμ-myc- GFP) or GFP and OVA (Eμ-myc-OWA) were generated by retroviral transduction.

Retroviral expression constructs were made by subcloning into a pMIG vector (MSCV- IRES-GFP: GFP sequence is that of enhanced green fluorescent protein). Membrane OVA was inserted into the pMIG vector upstream of the IRES-GFP cassette. Pools of Em-myc cells over-expressing GFP alone (Em-myc-GFP) or both mOVA and GFP (Em- myc-OVA) were generated by retroviral infection and purified by flow cytometric sorting of GFP+ cells on a MoFIo instrument after exclusion of dead cells. Following this, the Em-myc-GFP and Em-myc-OVA cell lines were cultured in FMA medium for 5 days to obtain sufficient cells prior to in vivo passage of GFP- or OVA/GFP-expressing Em-myc cells in a B6 mouse. Tumour cells were harvested from lymph nodes of mice after 11 days and single cell suspensions of these cells served as tumour stocks for experimental use. For storage, tumour cells were frozen in FCS containing 10% DMSO (Merck) at -70°C and thawed prior to intravenous inoculation.

Proliferation & CTL assays

Usually OVA-coated bml splenocytes were used as the immunogen. In vivo OT- I and OT-II proliferation assays, and in vitro proliferation and in vivo CTL assays were performed as described (Wilson et al, 7:165-172, 2006). T cells were purified from pooled lymph nodes of OT-I or OT-II mice and stained with FITC-conjugated anti-CD4 or anti-CD8 mAb for 30 min at 4°C. After washing once with PBS/2%, cells were incubated with anti-PE microbeads (Miltenyi Biotec) for 15 min, washed and resuspended in 3 ml PBS/2%. Transgenic CD4+ (OT-II) or CD8+ T cells (OT-I) were magnetically isolated by positive selection on an AutoMACS machine (Miltenyi Biotec). The T cell preparations were routinely 98-100% pure, as determined by flow cytometry. These cells were labeled with 5,6-carboxyfluorescein-succinimidyl-ester (CFSE; Molecular Probes). Purified T cells were pelleted and suspended in 1 ml of warm PBS/1% FCS at 1 x 10⁷ cells/ml. After transfer to a new dry tube, 1 μl of 5 niM CFSE for every 1 x 10⁷ cells was added and the suspension vortexed thoroughly to ensure consistent labeling. Cells were incubated for 10 min at 37°C before three washes with 10 ml PBS/2%, cell counting and resuspension in PBS (for in yivo injections) or RPMI 1640/10% (for in vitro culture).

Endogenous proliferative responses

A modified CFSE dilution method was used (see above). B6 mice received 3 daily doses of cytochrome c starting the day prior to intravenous immunization with 2x10⁷ OCS. A week later, equivalent numbers of splenocytes were labeled with 5 μM of CFSE (Molecular Probes, Eugene, OR) for 10 min at 37°C. CFSE-labeled cells were plated at 5x10⁶AnI with OVA at 37⁰C for 4 days. Cells were enriched for viable T-cells using density centrifugation. FACS analysis was performed (FACSAria, BD Biosciences, San Jose, CA).

RNA extraction and quantitative real-time PCR

RNA extraction and quantitative real-time RT-PCR analysis was performed. Cell pellets for mRNA extraction were snap-frozen on dry ice and stored at -7O⁰C until used. Total cellular mRNA was isolated using TRIReagent (Ambion) according to the manufacturer's instructions, and then purified by phenol-chloroform extraction and ethanol precipitation. 125 μg of glycogen (Boehringer-Mannheim) was added as a carrier to each sample.

IL-12 ELISA

ELISAs for murine IL- 12 by PharMingen were used on culture supernatants of DCs stimulated with 0.5 μM CpGl 688 (Geneworks) and 20 ng/ml IFN-γ (PharMingen).

Islet isolation and transplantation

Islet donors were female (C57BL/6 x BALB/c) Fl mice. Pancreata were collagenase digested, and islets were separated from pancreatic exocrine tissue on

Histopaque-1077 (Sigma, St Louis, MO, USA) density gradients (Liu et al, Transplant Proc. 27:3208-3210, 1995). Islets were picked clean under a dissecting microscope and cultured for 2 days. Four hundred islets were transplanted under the kidney capsule of allogeneic diabetic CBA.RIP-K^b mice. CBA.RIP-K^b mice develop spontaneous and stable, non- immune diabetes due to expression of the RIP-K^b transgene in their islets and their use as allogeneic islet graft recipients has been described (Sutherland et al., Int. J. Exp. Diab. Res. 3:37-45, 2002). Recipient CBA.RIP-K^b mouse blood glucose levels were measured just prior to transplantation then periodically as indicated.

Statistical analysis A Student's t test was used to compare two sets of data.

EXAMPLES

Example 1 - Cytochrome c selectively depletes DCs in vivo and in vitro

To confirm that cytochrome c selectively depletes DCs in vivo and in vitro, C57BL/6 (B6) mice were injected with either horse or yeast cytochrome c. After 16-24 h, the relative proportion and numbers of splenic CDl Ic⁺ CD4^" CD8⁺ (CD8⁺), CDl Ic⁺ CD4⁺ CD8^" (CD4⁺), CDl Ic⁺ CD4- CD8^" (DN), and total CDl Ic⁺ cells were compared by flow cytometry. As can be seen in Figure 1, a 2-3 -fold reduction in both the percentage (a) and number (b) of CD8⁺ DCs was consistently observed after administration of horse cytochrome c (p<0.0001 compared with untreated and p=0.005 compared with yeast cytochrome c treated). A small reduction was also seen in the CD4⁺ DC subset after horse cytochrome c treatment compared with no treatment. However, when compared with yeast cytochrome c treatment, no significant difference was found in absolute CD4⁺ DC numbers after horse cytochrome c treatment (p=0.34). No reduction was observed in the CD4^" CD8^" subset (p=0.2).

To address whether other splenic populations were affected by cytochrome c treatment, the above experiments were repeated. Stained splenocytes, in parallel with CDl Ib and F4/80, CDl 9 and B220, or TCR-β and CD3, were analyzed by flow cytometry. Horse cytochrome c treatment resulted in a 15-30% decrease in total DC numbers. Both horse and yeast cytochrome c had minimal cytotoxic effects on macrophages, B and T-cells (Figure Ic). Larger cytochrome c doses of up to 15 mg did not enhance the observed reduction in CD8⁺ DCs. Thus, all subsequent experiments were performed using an intravenous dose of 5 mg.

To confirm whether cytochrome c treatment caused DC apoptosis mouse chimeras were created using mutant or wild-type (WT) fetal liver reconstitutions into bml host mice. These mice were chosen as hosts because cells from bml mice are incapable of directly presenting OVA_257-264 to OT-I CD8⁺ T-cells. After 6-12 weeks post-reconstitution, the proportion of DCs/T-cells in Apaf-l^"7" or WT-reconstituted mice was similar to that of B6 mice.

Freshly isolated splenic DCs from WT or Apaf-1^"7" mice in the presence of 0- 10mg/ml cytochrome c were cultured to determine the absolute numbers of DCs remaining in vitro after cytochrome c treatment. After 24 h, we quantified the numbers from each DC subset by calibrating against a known quantity of beads. Analysis of the absolute cell numbers revealed a 3-fold reduction in CD8⁺ DC from WT B6 mice (p=0.0001), whilst the CD8^" subset (p=0.2) and both CD8⁺ and CD8^" DCs of Apaf-1^"Λ origin was not significantly affected (Figure Id).

Apoptosis of DCs from naive B6 or Apaf-l^"'^" reconstituted mice was measured by annexin V assays of purified DCs cultured with 0-10 mg/ml cytochrome c for 6 hours. At the highest concentration used, cytochrome c induced apoptosis in almost 25% of splenic CD8⁺ DC from B6 mice, as detected by annexin V binding (Figure Ie). In contrast, cytochrome c failed to induce early apoptosis in DCs of Apaf-l^"7" origin (Figure Ie). The observed effect was dose-dependent.

Example 2 - Cytochrome c treatment impairs cross-presentation in vivo and in vitro

To verify if the cytochrome c-induced apoptosis of CD8⁺ DCs was functionally significant, impairment of cross-presentation of a cellular antigen was determined in vivo. 1x10⁶ CFSE-labeled OT-I cells were transferred into B6 mice on day -2. The mice were then injected with three daily doses of cytochrome c, starting the day prior to intravenous injection of two forms of cellular antigen: either OVA-coated bml splenocytes (OCS) (Figure 2a) or OVA expressed in a pig xenogeneic cell line, PKl 5- OVA (Figure 2b). Three injections of cytochrome c were used because of its very short circulating half-life of 4 minutes and because DCs have a rapid turnover rate. Cross- presentation was assessed 60 hours after mice had received the antigen by measuring OT-I proliferative responses in the spleen. Cross-presentation was profoundly inhibited in cytochrome c treated mice; the overall number of expanded OT-I cells was up to 27- fold lower in cytochrome c treated mice than that of untreated control B6 mice (Figure 2a, right panel).

The effect on CD4⁺ proliferative responses was then investigated. Adoptive transfer of equal numbers of CFSE- labeled OT-I and OT-II cells were injected into the same animal. MHC Class II presentation was compared to MHC Class I (cross)- presentation. Cytochrome c treatment preferentially abrogated OT-I proliferation, but resulted in a modest reduction in OT-II proliferation (Figure 2b). This effect was consistently seen when OVA-transfectants (Figure 2b) or OVA-coated cells (data not shown) were used as immunogens.

To test whether the suppression of CD8⁺ T-cell proliferation in vivo was nonspecific, we performed similar experiments in CDl lc-mOVA mice, which express membrane-bound OVA in DCs under the control of the CDl Ic promoter. Thus, OVA is constitutively synthesized and presented via the classical MHC class I pathway. When CFSE-labeled OT-I cells were adoptively transferred into these mice, equivalent proliferative responses were observed in mice treated with cytochrome c compared with mice that were not (data not shown).

Splenic CD8⁺, CD4⁺, and CD4^"CD8^' DC subsets were cultured continuously in vitro in the presence of 2 mg/ml cytochrome c and OVA. Cytochrome c selectively abrogated the ability of CD8⁺ DC to cross-present OVA to OT-I cells (data not shown) in a dose-dependent manner. The inability of CD8⁺ DCs to stimulate OT-I proliferation in the presence of cytochrome c was not related to direct toxicity of cytochrome c on T- cells in vitro, as all subsets of DCs were able to stimulate robust OT-I proliferative responses when incubated with OVA_2S7-264 peptide in vitro (data not shown). Thus, the in vitro studies confirmed the in vivo cross-presentation results.

Example 3 - Cytochrome c-mediated inhibition of cross-presentation is Apaf-1 dependent To confirm the mechanistic specificity of the observed reduction in cross- presentation, a comparison was made of how exogenous cytochrome c affected the ability to stimulate naive OT-I proliferation in Apaf-1^"'^" or WT-reconstituted mice. As expected, cross-presentation was inhibited in WT-reconstituted mice that received cytochrome c (Figure 4a, top panel). In contrast, cytochrome c treatment did not reduce the frequency (Figure 4a, bottom panel) and overall numbers (Figure 4b) of expanded OT-I cells in Apaf-1^"Λ mice. The level of cross-presentation in untreated Apaf-1^"'^"- reconstituted mice was slightly lower than that observed in WT-reconstituted mice, (this was not attributable to lower hematopoietic reconstitution as the reconstitution efficiency of the blood and DC compartments were comparable in Apaf-l^"7" and WT mice), these results cogently indicate that exogenous cytochrome c resulted in apoptosis of cross- presenting cells through an Apaf-1 -dependent pathway.

Example 4 - Cytochrome c treatment preferentially reduces CTL induction and antigen-specific proliferative response

Although cytochrome c treatment inhibited cross-presentation of cell-associated antigen to OT-I cells in vivo, it was unclear whether this phenomenon was merely idiosyncratic of transgenic T-cells. Thus, a model was designed whereby both T-cells and DCs were of endogenous origin. B6 mice were pre-treated with cytochrome c on three consecutive days, commencing a day prior to injection of OVA-coated splenocytes. In vivo CTL activity was assessed 7 days after T-cell priming. There was almost 4-fold less OVA-specific CTL activity in cytochrome c treated mice compared to untreated mice (Figure 5 a).

To assess endogenous anti-OVA proliferative responses (compared to OT-I responses), B6 mice were pre-treated with cytochrome c as described above. CFSE- labeled splenocytes were re-stimulated in vitro 7 days later with OVA. After 4 days, proliferation of endogenous CD4⁺ and CD8⁺ T-cells was assessed by CFSE dye dilution. A 2- to 3-fold impairment of CD8⁺ T-cell expansion was observed in mice treated with cytochrome c with minimal non-specific proliferation in unimmunized mice (Figure 5b,c). In contrast, there was minimal impairment of endogenous CD4⁺ T-cell responses (Figure 5d). These results indicate that cytochrome c inhibited endogenous CD8⁺ T-cell responses (both proliferative and killer responses) to cross-presentation in vivo.

Example 5 - Cytochrome c impairs immune rejection of B cell lymphoma

The effect of cytochrome c on the elimination of OVA-expressing Eμ-myc B cell lymphomas was analyzed. Mice were either left untreated or given three doses of cytochrome c at the time of priming with OCS. After 7 days, 1x10⁶ Eμ-myc-OVA or Eμ-myc-GFP cells were inoculated intravenously into primed or naive mice. Mice were left for 7 days to allow spontaneous lymphoma development. A robust antigen-specific tumour rejection response was detected in the spleens of mice that had been primed with Eμ-myc-OVA but not Eμ-myc-GFP (Figure 6a). Cytochrome c treated mice had 3-fold more tumour cells than untreated controls (Figure 6b and c), indicating that in vivo disruption of cross-presentation by cytochrome c impaired immunity to a tumour antigen.

Example 6 - Cytochrome c-resistant DCs are incapable of cross-presentation but retain a capacity for MHC Class II presentation.

Despite a considerable remaining population of CD8⁺DCs after cytochrome c treatment (Figure Ia), cross-presentation was still markedly impaired. Therefore, it was important to determine the antigen presentation capacity of residual DCs. To this end, B6 mice were treated with cytochrome c for 24 hours. A 50% decrease in the proportion of CD8⁺ DCs was observed (see Figure Ia). Equal numbers of sorted CD8⁺ or CD8^" DCs were cultured from untreated or cytochrome c treated mice with various concentrations of OVA protein and CFSE-labeled OT-I cells. CD8⁺ DCs from cytochrome c treated mice were far less capable of stimulating OT-I proliferation in vitro compared to CD8⁺ DCs from untreated mice (Figure 7a). The level of proliferation seen in cytochrome c treated mice approximated that of CD8^" DCs from untreated mice, which are known for their poor cross-presentation capacity. Using CFSE-labeled OT-II cells, it was observed that the remaining (cytochrome c-resistant) DCs were only defective in MHC class I but not MHC class II presentation ability as these DCs could present newly encountered OVA protein to OT-II cells equally well compared with DCs from untreated mice (Figure 7b).

Example 7 - Cytochrome c treatment preferentially reduces IL- 12 (interleukin-12) production and TLR3 (toll-like receptor 3) expression by CD8⁺ DCs

Apart from their efficiency at cross-presentation, another functional specialization of CD8⁺ DCs is their propensity for IL- 12 production in response to TLR engagement. Given that CD8⁺ DCs are the main targets of cytochrome.c-mediated depletion, the IL- 12 producing capacity of residual cytochrome c treated CD8⁺ DCs to their untreated counterparts was compared. 24 hours after treatment with cytochrome c, remaining CD8⁺ or CD8^" DC populations from untreated or cytochrome c treated B6 mice were stimulated with CpG and IFN-γ. The presence of IL-12p40 and IL-12p70 was assayed from cultured supernatants after 24 hours. On a per cell basis, CD8⁺ DC from cytochrome c treated mice produced nearly 2-fold less IL-12p40 and approximately 4-fold less IL-12p70 compared to CD8⁺ DCs from untreated mice (Figure 8a). IL-12p70 was undetectable from CD8^" DC stimulated with CpG and IFN-γ or from all unstimulated DCs (Figure 8a).

CD8⁺ DCs have also been reported to express high levels of TLR3 and this has been linked to their cross-priming ability. When residual CD8⁺ DCs from cytochrome c treated mice were quantitatively analyzed for their TLR3 expression by real-time PCR, a 1.5- to 2-fold reduction in the expression of this receptor was observed, whilst very low levels of TLR3 were expressed on CD8^" DCs (Figure 8b).

Thus, cytochrome c substantially reduced the proportion of CD8⁺ DCs that abundantly expressed IL- 12 and TLR3.

From the results above it can be seen that the CD8⁺ DC was the major DC subset reduced by cytochrome c treatment. This is in line with established evidence that this subset is the main DC mediating cross-presentation (den Haan et al., J. Exp. Med. 192:1685-1696, 2000). Whilst only half of the CD8⁺ DCs were killed by cytochrome c administration, this corresponded to a dramatic reduction in antigen-specific CD8⁺ T-cell proliferation, both for transgenic OT-I cells (Figure 2) and endogenous CD8 T-cells (Figure 5). Thus, the cytochrome c-sensitive population is the cohort of efficient cross- presenters. This was evidenced by the results in Figure 6a where the residual CD8⁺ DCs (cytochrome c-resistant) were inefficient at cross-presentation and indeed were only as effective as CD8^" DCs. Additionally, experiments at different time points when incubating DCs with cytochrome c in vitro revealed that only a proportion of CD8⁺ DCs were found to be apoptotic at any given point in time (Figures Id and e).

IL- 12 production and TLR3 expression are hallmarks of CD8⁺ DCs (Schulz et al, Nature 433:887-892, 2005; Hochrein et al., J. Immunol. 166:5448-5455, 2001). TLR3 has been shown to augment cross-priming during viral infection (Schulz et al., Nature 433:887-892, 2005) and signaling through this receptor has been reported to cause upregulation of co-stimulatory molecules and production of anti- viral cytokines (Reis et al., Semin. Immunol. 16:27-34, 2004; Sen et al., Cytokine Growth Factor Rev. 16:1-14, 2005). The fact that both IL-12 and TLR3 expression were attenuated by cytochrome c treatment indicate that the cytochrome c-sensitive DCs were the major producers of IL-12 and TLR3 and hence consistent with the observation that they are the efficient cross-presenters.

Example 8 - Cytochrome c impairs immune rejection of pancreatic islet allotransplants

The effect of cytochrome c on survival of pancreatic islet allograft survival was assessed (Figure 9). Mice were injected daily with 5 mg horse cytochrome c in phosphate buffered saline intravenously on the day prior to transplantation, then intraperitoneally for the following 7 (Long treatment) or 2 (Short treatment) days. Vehicle-treated controls received phosphate buffered saline alone according to the long treatment schedule. Initial function of all transplants was shown by normalization of blood glucose at <12 mM from pre-transplant diabetic levels of >20 mM. Blood glucose returned to pre-transplant levels in all (5/5) vehicle -treated control mice by day 55 post- transplant indicating that immune rejection of the transplants had taken place. Cytochrome c treatment protected islet transplants from immune rejection in 60% of recipients (3/6 long treatment group plus 3/4 short treatment group) such that reduced blood glucose levels were maintained in excess of 133 days for one cohort of mice (experiment terminated at this time) and in excess of 224 days for a second cohort of mice (ongoing).

Apoptosis is the mechanism by which exogenously administered cytochrome c works. This is firstly supported by the reduction in CD8⁺ DC numbers in vivo and in vitro (Figure 1). Secondly, there is an increase in annexin V staining after cytochrome c treatment (Figure Ie). As expected, this was not as dramatic as the findings measuring absolute cell numbers, presumably because annexin V staining detects early events of apoptosis and is a snapshot of cell death whereas cell number quantification is cumulative. Moreover, DCs can phagocytose dying cells that would not be accounted for during annexin V staining. Thirdly, the abrogation of cytochrome c's effect in the absence of Apaf-1 strongly argues for its mechanistic specificity via the apoptosome, namely, by exclusively inducing apoptosis in DCs that have transferred cytochrome c into the cytosol. Furthermore, yeast cytochrome c, a form that fails to initiate apoptosis, did not result in a significant in vivo reduction of DC numbers and cross-presentation.

Example 9 - Donor mouse islets exposed to cytochrome c are protected against immune rejection in fully allogenic, immune competent recipients

In Example 8, transplant hosts were treated with cytochrome c. In this Example, we assess the effect of treating graft tissue with cytochrome c. We reasoned that this may affect resident/passenger DC within tissue grafts and/or affect host DC that take up graft antigens (and hence cytochrome c that was coated on grafts). Donor islets were exposed to cytochrome c, either in vitro by addition of cytochrome c (final concentration=5 mg/ml) to the culture media after islet isolation, or in vivo by IV injection of donor mice 24 h prior to islet isolation. Following transplantation into fully allogenic, non-autoimmune diabetic recipients, islets exposed to cytochrome c stably reversed diabetes beyond 100 d post transplant, demonstrating significantly increased graft survival when compared to untreated control grafts (Figure 10). Consistent with this, when frozen sections of the islet grafts were stained for the presence insulin and CD4⁺ or CD8⁺ cells, the cytochrome c-treated grafts were found to contain less inflammatory infiltrate, and considerably more functional graft mass, when compared to untreated controls (Figure 11).

At least two routes of cross-presentation have been proposed. One pathway involves the transport of Ag from endosomes into the cytosol, whilst in the alternative pathway, Ag fails to escape from endosomes and is processed and degraded into antigenic peptides within these vesicles (Shen et al., Curr. Opin. Immunol. 18:85-91, 2006). The cytosolic route appears to be the predominant pathway used by DCs (Rodriguez et al., Nat. Cell Biol. 1 :362-368, 1999), although an alter native non- cytosolic pathway has been described (Shen et al., Immunity 21:155-165, 2004). The molecular mechanisms endowing CD8⁺ DCs with superior cross-presentation capacity are unknown. The observation that cytochrome c can induce suicide in cross-presenting DCs supports the hypothesis that cytosolic diversion is required for cross-presentation in vivo and extends it to CD8⁺ DCs as this is the only way cytochrome c induces apoptosis. This is the mechanism that confers specialization to CD8⁺ DCs as cells incapable of such transfer, even within the CD8⁺ DC subset are unable to cross present and are not depleted.

OTHER EMBODIMENTS

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Use of singular forms herein, such as "a" and "the," does not exclude indication of the corresponding plural form, unless the context indicates to the contrary. Although the invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of the invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Other embodiments are within the following claims.

Claims

What is claimed is:

1. A method for reducing antigen presentation by one or more antigen presenting cells, the method comprising introducing an exogenous apoptosis-inducing agent that enters into the cytosol of the one or more antigen presenting cells, wherein the introduction of the exogenous apoptosis-inducing agent results in the triggering of apoptosis of the one or more antigen presenting cells by an apoptosis-triggering complex comprising the exogenous apoptosis-inducing agent and an endogenous apoptosis- inducing agent.

2. The method as claimed in claim 1, wherein the exogenous apoptosis-inducing agent is a purified protein, or a portion or an active fragment thereof.

3. The method as claimed in claim 1 or claim 2, wherein the purified protein is cytochrome c.

4. The method as claimed in claim 1 or claim 2, wherein the exogenous apoptosis- inducing agent is a toxin that elicits cell death or inactivation after being transported to the cytosol of the one or more antigen presenting cells.

5. The method as claimed in any one of claims 1 to 4, wherein the exogenous apoptosis-inducing agent is admitted to the one or more antigen presenting cells via their extracellular environment.

6. The method as claimed in any one of claims 1 to 4, wherein the exogenous apoptosis-inducing agent is admitted intracellularly to the one or more antigen presenting cells.

7. The method as claimed in any one of claims 1 to 4, wherein the exogenous apoptosis-inducing agent is internalised by the one or more antigen presenting cells by pinocytosis, macropinocytosis, phagocytosis, or receptor uptake.

8. The method as claimed in claim 7, wherein the receptor uptake is mediated by a mannose receptor or an Fc receptor.

9. The method as claimed in any one of claims 1 to 8, wherein the exogenous apoptosis-inducing agent was produced using a nucleotide sequence located in an expression cassette within an expression vector.

10. The method as claimed in any one of claims 1 to 9, wherein the endogenous apoptosis-inducing agent is Apaf-1.

11. The method as claimed in any one of claims 1 to 10, wherein the apoptosis- triggering complex is the apoptosome.

12. The method as claimed in any one of claims 1 to 11, wherein the one or more antigen presenting cells are selected from the group consisting of dendritic cells, macrophages, B-lymphocytes, and liver sinusoidal endothelial cells.

13. The method as claimed in claim 12, wherein the one or more antigen presenting cells are dendritic cells.

14. The method as claimed in claim 13, wherein, the dendritic cells are CD8⁺ dendritic cells.

15. The method as claimed in any one of claims 1 to 14, wherein the antigen is cross- presented on the surface of the one or more antigen presenting cells in association with a major histocompatibility complex (MHC) receptor.

16. The method as claimed in claim 15, wherein the MHC receptor is a MHC class I receptor.

17. The method as claimed in any one of claims 3 and 5 to 16, wherein the cytochrome c is selectively internalised by dendritic cells in vivo or in vitro.

18. The method as claimed in claim 1, wherein the reduction in antigen presentation by the one or more antigen presenting cells results in a reduced immune response.

19. The method as claimed in claim 18, wherein the reduced immune response is characterized by a reduction of a CD8⁺ T cell-mediated immune response.

20. The method as claimed in claim 1, further resulting in reducing the expression of inter leukin 12 by the one or more antigen presenting cells.

21. The method as claimed in claim 1 , further resulting in reducing the expression of toll-like receptor 3 by the one or more antigen presenting cells.

22. The method as claimed in any one of claims 1 to 21, wherein the one or more antigen presenting cells is within a subject having or at risk of developing a disease or condition characterized by T cell-mediated pathology.

23. The method as claimed in claim 22, wherein the subject is a human subject.

24. The method as claimed in claim 22 or claim 23, wherein the subject has or is at risk of developing autoimmune disease, inflammatory diseases or conditions, transplant rejection, neoplastic disease, viral disease, or bacterial disease.

25. The method as claimed in any one of claims 1 to 24, wherein the one or more antigen presenting cells is within a cell, tissue, or organ graft.

26. The method as claimed in any one of claims 22 to 24, wherein the subject has or is at risk of developing an autoimmune disease or condition selected from the group consisting of acute disseminated encephalomyelititis (ADEM), Addison's disease, ankylosing spondylitis, antiphospho lipid antibody syndrome (APS), anemia, autoimmne hepatitis, autoimmune inner ear disease, pemphigoid, celiac disease, dermatomyositis, diabetes mellitus (type I or type II), insulin resistance, metabolic syndrome, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Reynaud's syndrome, Reiter's disease, nephritis, neuropathy, inflammatory bowel disease or colitis, thyroiditis, thrombocytopenia, lupus, mixed connective tissue disease, sclerosis, amyloidosis, rheumatic fever, respiratory distress syndrome, meningitis, autoimmune haemato logical disorders, inflammation of the uvea, iritis, choroiditis, sudden hearing loss, encephalitis, uveitis, glomerulonephritis, allergy, eczema, myasthenia gravis, pemphigus vulgaris, pernicious anaemia, polymyositis, primary biliary cirrhosis, psoriasis, dermatitis, urticaria, scleroderma, pulmonary fibrosis, chronic pulmonary inflammatory disease, autoimmune myocarditis, arthritis, sarcoidosis, Sjόgen's syndrome, vasculitis, arteritis, and vitiligo.

27. The method as claimed in claim 26, wherein the sclerosis is selected from the group consisting of multiple sclerosis, systemic sclerosis, primary progressive multiple sclerosis, relapsing remitting multiple sclerosis, progressive systemic sclerosis, amyotrophic lateral sclerosis, atherosclerosis, and arteriosclerosis.

28. The method as claimed in any one of claims 22 to 24, wherein the subject has or is at risk of developing an inflammatory disease or condition selected from the group consisting of arthritis, gastritis, pericarditis, ileitis, osteitis, and encephalitis.

29. The method as claimed in claim 25, wherein the exogenous apoptosis-inducing agent is (i) administered to the recipient of the cell, tissue, or organ graft; (ii) administered to the donor of the cell, tissue, or organ graft; or (iii) contacted with the cell, tissue, or organ graft ex vivo.

30. A pharmaceutical composition comprising cytochrome c and a pharmaceutically acceptable diluent or carrier.

31. The pharmaceutical composition as claimed in claim 30, further comprising one or more additional pharmaceutically active agents.

32. Use of a pharmaceutical composition comprising an exogenous apoptosis- inducing agent for the prevention or treatment of a disease or condition characterized by T cell-mediated pathology.

33. Use of a pharmaceutical composition comprising an exogenous apoptosis- inducing agent for the preparation of a medicament for the prevention or treatment of a disease or condition characterized by T cell-mediated pathology.

34. The use as claimed in claim 32 or claim 33, wherein the exogenous apoptosis- inducing agent is cytochrome c.

35. The use as claimed in any of claims 32 to 34, wherein the disease or condition associated with T cell-mediated pathology is autoimmune disease, transplant rejection, neoplastic disease, or viral disease.

36. The use as claimed in any of claims 32 to 35, further comprising use of one or more other pharmaceutically active agents effective in the prevention or treatment of the disease or condition characterized by T cell-mediated pathology.

37. Use of cytochrome c, or a portion or an active fragment thereof, in the manufacture or preparation of a medicament for the prevention or treatment of autoimmune disease, transplant rejection, neoplastic disease, viral disease and any disease where there is T-cell-mediated pathology.

38. A method of preventing or treating autoimmune disease, transplant rejection, neoplastic disease, viral disease and any disease where there is T-cell-mediated pathology comprising administering a prophylactically or therapeutically effective dose of cytochrome c, or a portion or an active fragment thereof, to a patient in need thereof.

39. A method as claimed in any one of claims 1 to 29 or a use as claimed in any one of claims 32 to 37, substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples.

40. A pharmaceutical composition as claimed in claim 30 or 31 , substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples.