Patent Application
20200261576
In compliance with 37 C.F.R. § 1.52, a computer readable form of the sequence listing is submitted herewith, file name: 84243WO_ST25.txt; size 43 KB; created on: Jul. 10, 2014; using PatentIn-3.5, which is hereby incorporated by reference in its entirety.
Regulatory T cells (Treg cells) hold promise for autoimmune disease therapy. However, a challenge remains as to how to induce antigen-specific Treg cells that only target inflammatory immune cells without compromising the entire immune response. Peripheral immune tolerance is key to preventing overreactivity of the immune system to various antigens. CD4+CD25+Foxp3+ regulatory T (Treg) cells are critical for maintaining immune tolerance, and deficiency of Treg cells causes severe autoimmune diseases and chronic inflammation. Indeed, the emergence and characterization of CD4+ CD25+ Foxp3+ Treg cells have offered the hope of developing novel immunotherapy for human autoimmune diseases and chronic inflammation.” However, the lack of knowledge of antigen specificity and the difficulty of expanding thymus-derived Treg cells (tTreg cells) have limited their potential clinical application. The discovery of TGFβ induction of Treg cells (iTreg cells) from peripheral naive CD4+ T cells has brought new hope of inducing antigen-specific Treg cells for autoimmune disease therapy5,6. However, published studies have been limited to the prevention of experimental diseases by pre-injection of in vitro induced antigen-specific iTreg cells into unmanipulated mice or in vivo by induction of antigen-specific Treg cells in naive mice before the disease is established. There is a considerable difference in the immune status of an unmanipulated, naive mouse and a mouse with an established disease. The immune tolerance toward self-tissues in naive mice is broken in mice with autoimmunity, where the autoantigen-responsive immune cells are uncontrollably activated and proinflammatory cytokines are produced. Treg cells that fully exhibit immunosuppressive capacity in the immune quiescent state in naïve mice may lose their suppressive activity or even convert to effector cells under the dysregulated inflammation in mice with autoimmune diseases. This problem is particularly salient in clinical settings, in which patients with autoimmune disease present with an already dysregulated immune response. Therefore, a challenge is to make cells in the inflammatory, dysregulated immune system in animals with established autoimmune diseases, and ultimately in patients, that can specifically inhibit inflammation in the organs/tissues affected and treat the diseases, i.e. to reprogram the dysregulated immune system in animals and patients so that it is restored, or to direct it to an immune-tolerant state to the target auto-antigens in the tissues affected with autoimmunity.
Accordingly, there is a need in the art for novel autoimmune disease therapies.
The present invention provides a therapeutic method for the treatment of autoimmune or autoinflammatory diseases by first breaking down the dysregulated immune system and then reprogramming the immune system to restore tolerance to the patient's self-antigens by induction of antigen specific regulatory T cells. It has been shown here that only with the combination of apoptosis, phagocytes, and antigen can antigen-specific Treg cells be optimally generated and long-term immune tolerance developed, i.e., the proper antigenic peptide needs to be introduced in a timely manner into subjects in which an immunoregulatory milieu was created by apoptosis-triggered phagocytes.
Exemplary tolerizing and/or treatment methods of the invention involve a) identifying a subject as sufficing from an autoimmune disease or disorder; performing at least one of the following steps: b) administering an effective amount of an anti-CD4 antibody, anti-CD8 antibody, or both to the subject to induce apoptosis in T cells of the subject suffering from the autoimmune disease or disorder; b) administering an effective amount of low-dose irradiation to the subject suffering from the autoimmune disease or disorder to induce apoptotic cells with adoptive transfer of said macrophage; and/or b) administering an effective amount of an anti-CD8 antibody and/or an anti-CD-20 antibody to the subject to induce depletion and apoptosis of B cells and T cells of the subject suffering from the autoimmune disease or disorder; and c) administering an autoantigen specific to the autoimmune disease or disorder that the subject is suffering from, whereby the subject is tolerized to the antigen of the autoimmune or autoinflammatory disease and the disease or disorder is treated. The invention also features kits for carrying out the methods of the invention.
In one aspect, this invention provides a method of tolerizing a subject suffering from an autoimmune or autoinflammatory disease or disorder to an antigen associated with the autoimmune disease or disorder comprising steps a to c in order: a) identifying a subject as suffering from an autoimmune disease or disorder; b) administering an effective. amount of an anti-CD4 antibody, anti-CD8 antibody, or both to the subject to induce apoptosis in T cells of the subject suffering from the autoimmune disease or disorder; and c) administering an autoantigen specific to the autoimmune disease or disorder that the subject is suffering from, whereby the subject is tolerized to the antigen of the autoimmune or autoinflammatory disease.
In another aspect, the invention provides a method of treating a subject suffering from an autoimmune or autoinflammatory disease or disorder comprising steps a to c in order: a) identifying a subject as suffering from an autoimmune disease or disorder; b) administering an effective amount of an anti-CD4 antibody, anti-CDS antibody, or both to the subject to induce apoptosis in T cells of the subject suffering from the autoimmune disease or disorder; and c) administering an autoantigen specific to the autoimmune disease or disorder that. the subject is suffering from, whereby the subject is tolerized to the autoantigen, thereby treating the autoimmune or autoinflammatory disease or disorder.
In one embodiment, the autoantigen is one or more autoantigens selected from the group consisting of: the myelin basic protein (MBP), the myelin proteolipid protein (PLP), insulin, GAD65 (glutamic acid decarboxylase), DiaPep277, heal-shock proteins (Hsp65, Hsp90, DnaJ), immunoglobulin binding protein (BiP), heterogeneous nuclear RNPs, annexin V, calpastatin, type II collagen, glucose-6-phosphate isomerase (GPI), elongation factor human cartilage gp39, and mannose binding lectin (MBL).
In another embodiment of the above aspects, step b is performed more than once prior to the performance of step c. In a further embodiment of the above aspects, the time for performance of step b and the time of performance of step c are separated by 3 to 14 days. In still another embodiment of the above aspects, step b induces apoptosis in a subset of T cells. In another embodiment of the above aspects, performance of steps a, b, and c is more effective than the performance of either steps a and b or steps a and c alone.
In one embodiment of the above aspects, the method further comprises monitoring the subject for amelioration of at least one sign or symptom of an autoimmune disease or disorder.
In another embodiment of the above aspects, the autoimmune disease or disorder is selected from the group consisting of multiple sclerosis, diabetes mellitus and rheumatoid arthritis, Sjogren's syndrome, and systemic sclerosis.
In certain embodiments, the monitoring comprises a diagnostic test or assessment. In another embodiment, the diagnostic test or assessment is selected from the expanded Disability Status Scale, the timed 25-foot walk test or the nine-hole peg test. In another related embodiment, the diagnostic test or assessment is selected from an oral glucose tolerance test (OGTT) glycosylated hemoglobin test or fasting plasma glucose test. In a further related embodiment, the diagnostic test or assessment is selected from the American College of Rheumatology (ACR) response, the Simplified Disease Activity Index (SDAI), the Clinical Disease Activity Index (CDAI) or the Global Arthritis Score (GAS).
In certain embodiments, the diagnostic test or assessment comprises determining the amount of inflammatory cell infiltration.
In another embodiment, the subject suffering from an autoimmune disease or disorder is at a late stage of disease.
In another embodiment, the method further comprises administration of an additional agent.
In one embodiment, the autoimmune disease or disorder is type I diabetes mellitus.
In certain embodiments, the methods of the invention also include a step of administering an anti-CD20 antibody to the subject suffering from an autoimmune or autoinflammatory disease or disorder.
Another aspect of the invention provides a method of treating a subject suffering from an autoimmune or autoinflammatory disease or disorder that includes performing the following steps in order: a) identifying a subject as suffering from an autoimmune disease or disorder; b) administering an effective amount of low-dose irradiation and macrophage to the subject sufficing from the autoimmune disease or disorder to induce apoptotic cells together with adoptive transfer of the macrophage; and c) administering an autoantigen specific to the autoimmune disease or disorder that the subject is suffering from, where the subject is tolerized to the autoantigen, effecting treatment of the autoimmune or autoinflammatory disease or disorder.
A further aspect of the invention provides a method of treating a subject suffering from an autoimmune or autoinflammatory disease or disorder that involves performing the following steps in order: a) identifying a subject as suffering from an autoimmune disease or disorder; b) administering an amount of an anti-CD8 antibody and/or an anti-CD-20 antibody to the subject effective to induce depletion and/or apoptosis of B cells and T cells (e.g., CD8− T cells) of the subject suffering from the autoimmune disease or disorder; and c) administering an autoantigen specific to the autoimmune disease or disorder that the subject is suffering from, where the subject is tolerized to the autoantigen, thereby treating the autoimmune or autoinflammatory disease or disorder.
In another aspect, the invention features a kit comprising an effective amount of an anti-CD4 antibody, anti-CD8 antibody in a pharmaceutical carrier; an autoantigen specific to an autoimmune or autoinflammatory disease or disorder; and instructions for use in treating the autoimmune or autoinflammatory disease or disorder.
In a further aspect, the invention provides a kit comprising an effective amount of an autoantigen specific to an autoimmune or autoinflammatory disease or disorder; and instructions for use in treating or preventing the autoimmune or autoinflammatory disease or disorder in a subject, optionally when used in combination with administration of one or more of the following: administering an effective amount of an anti-CD4 antibody, anti-CD-8 antibody, or both to the subject to induce apoptosis in T cells of the subject suffering from the autoimmune disease or disorder; administering an effective amount of low-dose irradiation and macrophage to the subject suffering from the autoimmune disease or disorder to induce apoptotic cells with adoptive transfer of the macrophage; and/or administering an amount of an anti-CDS antibody and an anti-CD-20 antibody to the subject effective to induce depletion and/or apoptosis of B cells and T cells (e.g., CD81 T cells) of the subject suffering from the autoimmune disease or disorder.
To facilitate an understanding of the present invention, a number of terms and phrases are defined below.
As used herein, the singular forms “a”, “an”, and “the” include plural forms unless the context clearly dictates otherwise. Thus, for example, reference to “a biomarker” includes reference to more than one biomarker.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive.
As used herein, the terms “comprises,” “comprising,” “containing,” “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like: “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
“Absence” of an autoantibody means that an autoantibody which is indicative fair at least one autoimmune disorder is not immunologically detectable. Accordingly, the autoantibody is not. hound to an autoantigen. Any immunoassay known in the art can be used, including an exemplary assay such as ELISA performed upon blood obtained from a subject (initially) identified as having an autoimmune disease or disorder and optionally undergoing a treatment as described herein.
As used herein, the term “antibody” is meant to refer to a full-length (i.e., naturally occurring or thrilled by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecule or an immunologically active (i.e., antigen-binding) portion of an immunoglobulin molecule, like an antibody fragment. In certain embodiments, the antibody is anti-CD4 or anti-CD8.
As used herein, the term “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
As used herein, the term “autoantigen” is meant to refer to any antigen that stimulates autoantibodies in the organism that produced it.
As used herein, the term “autoimmune disease or disorder” is meant to refer to a disease state caused by an inappropriate immune response that is directed to a self-encoded entity which is known as an autoantigen. Examples of autoimmune diseases include vasculitis, arthritis, autoimmune diseases of the connective tissue, inflammatory bowel diseases, autoimmune diseases of the liver and the bile duct, autoimmune disease of the thyroid gland, dermatologic autoimmune diseases, neurologic immune diseases, Diabetes type 1. Exemplary vasculitis can be selected from medium to small vessel vasculitis or large vessel vasculitis, exemplary arthritis can be selected from seronegative and seropositive rheumatoid arthritis, psoriatic arthritis, Bechterew's disease, juvenile idiopathic arthritis; exemplary inflammatory bowel diseases can be selected from Crohn's disease or ulcerative colitis; exemplary diseases of the liver and the bile duet can be selected from autoimmuno-hepatitis, primary biliary cirrhosis and primary sclerosing Cholangitis; exemplary autoimmune diseases of the thyroid gland can be selected from Hashimoto's thyreoiditis and Grave's disease; exemplary autoimmune diseases of the connective tissue can be selected from systemic lupus erythematosus (SLE) disease, Sjogren's syndrome (SS), scleroderma, dermato- and poly-myositis, Sharp syndrome, systemic sclerosis and CREST syndrome; exemplary neurologic autoimmune diseases can be selected from multiple sclerosis (MS), chronic inflammatory demyelating polyneuropathy (CIDP) and myasthenia gravis. Medium to small vasculitis can optionally be selected from classical panarteritis nodosa, granulomatosis with polyangiitis, microscopic panarteritis, Churg-Strauss syndrome Behcet's disease and the large vessel vasculitis can optionally be selected from giant cell arteritis, polymyalgia rheumatic and Takayasu's arteritis. An autoimmune disease or disorder in a subject can be identified by any art-recognized method, including by assessment of symptoms or by evaluation of marker levels (e.g., autoantibody levels).
As used herein, the term “autoinflammatory disease or disorder” is meant to refer to a group of disorders characterized by seemingly unprovoked inflammation.
As used herein, the terms “determining”, “assessing”, “assaying”, “measuring” and “detecting” refer to both quantitative anti qualitative determinations, and as such, the term “determining” is used interchangeably herein with “assaying,” “measuring,” and the like.
The term “subject” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.
As used herein, the term “tolerizing” is meant to refer to a failure to attack the body's own proteins and other antigens. The term “tolerizing” may also include inducing tolerance, inducing immunological tolerance, or rendering nonimmunogenic.
As used herein, the term “treating” is meant to include alleviating, preventing and/or eliminating one or mom symptoms associated with inflammatory responses or an autoimmune disease. It will be appreciated that, although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated.
This invention is based, at least in part, on the discovery that tolerization to antigens by T cell depletion using anti-CD4 and/ or anti-CD8 antibodies or other apoptotic cell induction methods to produce apoptosis, followed by antigen administration could be used for the tolerization of a dysfunctional immune system.
Featured in the present invention are methods of tolerizing a subject suffering from an autoimmune or autoinflammatory disease or disorder to an antigen associated with the autoimmune disease or disorder comprising steps a to c in order: a) identifying a subject as suffering from an autoimmune disease or disorder; b) administering an effective amount of an anti-CD4 antibody, anti-CD8 antibody, or both to the subject to induce apoptosis in T cells of the subject suffering from the autoimmune disease or disorder; and c) administering an autoantigen specific to the autoimmune disease or disorder that the subject is suffering from, whereby the subject is tolerized to the antigen of the autoimmune or autoinflammatory disease. Also featured are methods of treating a subject suffering from an autoimmune or autoinflammatory disease or disorder comprising steps a to c in order: a) identifying a subject. as sufficing from an autoimmune disease or disorder; b) administering an effective. amount of an anti-CD4 antibody, anti-CD8 antibody, or both to the subject to induce apoptosis in T cells of the subject suffering from the autoimmune disease or disorder; and c) administering an autoantigen specific to the autoimmune disease or disorder that the subject is suffering from, whereby the subject is tolerized to the autoantigen, thereby treating the autoimmune or autoinflammatory disease or disorder.
It has further been discovered and is disclosed herein that step b) of the above method can optionally be substituted with or supplemented by a step of b) administering an effective amount of low-dose irradiation and macrophage to the subject sufficing from the autoimmune disease or disorder to induce apoptotic cells with adoptive transfer of the macrophage or b) administering an effective amount of an anti-CD8 antibody and/or an anti-CD20 antibody to the subject to induce depletion and/or apoptosis of B cells and/or cells of the subject. suffering from the autoimmune disease or disorder. Following such alternate administering steps, an autoantigen specific to the autoimmune disease or disorder that the subject is suffering from can then be administered, with the effect of tolerizing the subject to the autoantigen, thereby treating the autoimmune or autoinflammatory disease or disorder in the subject.
In certain embodiments, step b is performed more than once prior to the performance of step c. In other further embodiments, the Lime fair performance of step b and the time of performance of step c are separated by 1 to 21 days, more preferably 3 to 14 days.
The immune system develops tolerance to self-antigens early in life, primarily through the process of deleting self-reactive cell clones in the thymus. This means that in order to impose tolerance in the adult to new antigens, such as those on an allograft, it is necessary either to ablate the entire immune system and attempt to recapitulate development. with presentation of the new antigens in the thymus with a fresh source of haemopoietic stem cells, or to find a means to reprogramme the peripheral T cell repertoire in situ. The development of monoclonal antibodies that can deplete or modulate cell function in vivo have made both of these routes to tolerance a practical possibility. Monoclonal antibodies that could deplete either CD4+ or CD8+ cells in mice became available in the 1980s and were found to be able to suppress the rejection of allogeneic skin or hone marrow grafts.35 While T cell depletion strategies of immunosuppression are still practically useful in clinical bone marrow36 and organ transplantation to this day37, it was the discovery that a brief treatment. with non-depleting CD4 antibodies could induce a permanent state of antigen specific tolerance in mice38 that has provided a potential route to true therapeutic reprogramming of the adult immune system.
Cluster of differentiation 4 (hereinafter, referred to as “CD4”) is a glycoprotein having a molecular weight of about 55 kDa, which is expressed on the cell surface of most of thymic about ⅔ of peripheral blood T cells, monocytes, and macrophage. CD4 is a type I transmembrane protein in which four immunoglobulin superfamily domains (designated in order as D1 to D4 from the N terminal to the cell membrane side) are present on the outside of the cells, and two N-linked sugar chains in total are hound to the domains D3 to D4. CD4 binds to a major histocompatibility complex (MHC) class II molecule through D1 and D2 domains, and then activates the T cells. Further, it is also known that CD4 polymerizes through D3 and D4 domains. CD4 is also known as T4, and the gene has been cloned in 1985, and the DNA sequence, the amino acid sequence and the three-dimensional structure of CD4 are publicly available from a known database. For example, these can be obtained by reference to Accession Nos. P01730 (SWISSPROT), MI2807 (EMBL).
Although antibodies against CD4 were the first to be found capable of inducing tolerance to protein antigens, it has become clear that other antibody specificities are capable, either when used alone or in combinations, of reprogramming the immune system39. While non-depleting CD4 antibody used alone is sufficient to achieve tolerance to long-lived protein antigens, such as foreign IgG, it was found to be essential to combine this with anti-CD8 antibodies to achieve reliable tolerance to skin grafts38.
In certain embodiments of the present invention, CD4- and CD8-depleting antibodies are used to induce T cell apoptosis. It has been shown here that only with the combination of apoptosis, phagocytes, and antigen can antigen-specific cells be optimally generated and long-term immune tolerance developed, i.e., the proper antigenic peptide needs to be introduced in a timely manner into subjects in which an immunoregulatory milieu was created by apoptosis-triggered phagocytes.
Anti-CD3 antibodies, or fragments thereof, have been employed in the treatment of autoimmune diseases, including diabetes. For example, U.S. Pat. No. 7,041,289 and published Canadian Patent Application No. 2,224,256 teach the treatment of autoimmune diseases, including diabetes, by administering an anti-CD3 antihody, or fragment thereof. However, in the present invention, use of anti-CD4 or anti-CD8 antibodies is preferable to the use of anti-CD3 antibodies.
It has previously been shown that CD3-specific antihody is able to deplete large numbers of T cells and consequently induce remission of EAE through an apoptosis-mediated mechanism14. However, CD3-specific antihody-mediated immune tolerance has two possible unwanted side effects. One is that it can transiently powerfully trigger TCR an T cells to release large amounts of pro-inflammatory cytokines including IFNγ, TNFα, and IL-6 in vivo, which may not only interfere with the generation of Treg cells, but also is a major barrier to translate the therapy into the future clinical settings. The other potential drawback of the CD3-specific antihody treatment is that the antihody engages TCR on all T cells indiscriminately, which could theoretically direct all T cells to differentiate into Treg cells or other T cell subsets depending on the environmental cytokine milieu. This might lead to Treg cells lacking antigen specificity, which would potentially render unwanted side effects to the animals and patients.
Slops of monitoring the subject suffering front an autoimmune disease or disorder may be included in the methods of the invention. In certain embodiments, the methods of tolerizing or treating a subject further comprise monitoring the subject for amelioration of at least one sign or symptom of an autoimmune disease.
According to embodiments of the present invention, monitoring can be by specific diagnostic methods with quantitative measures of disease severity. Art-recognized diagnostic methods are preferably used, for example, in multiple sclerosis, the Expanded Disability Status Scale and two quantitative tests (the timed 25-fool walk test and the nine-hole peg test) can be used separately and in combination, to detect improvement. In diabetes, an oral glucose tolerance test (OGTT) can be used to monitor how well the body handles a standard amount of glucose. HbA1C (A1C or glycosylated hemoglobin test) measures average blood glucose control for the past 2 to 3 months. Diabetes is diagnosed when the A1C is 6.5% or higher. The fasting plasma glucose test (FPG) is used to determine the amount of glucose in the plasma, as measured in mg/dL. In rheumatoid arthritis. The American College of Rheumatology (ACR) Core Data Set was developed to provide a consistent group of outcome measures for RA. ACR20, 50, and 70 responses have been used. The Disease Activity Score (DAS) and its derivatives, DAS28 (a 28-joint count) and DAS-CRP (using CRP in place of ESR), are widely used. The Simplified Disease Activity Index (SDAI) and an even further simplified version (no acute phase reactant needed), the Clinical Disease Activity Index (CDAI), have also been proposed. The Global Arthritis Score (GAS) is a sum of three measures, patient pain, the raw mHAQ score, and tender joint count, and is closely correlated with both the SDAI and DAS.
The present invention is useful for treating autoimmune and autoinflammatory diseases, and in particular embodiments, any autoimmune diseases with at least tine known specific autoantigen. The present invention is also contemplated as useful for preventing or treating allogenic transplantation rejection via depletion of the immune cells of a recipient by one or more of the methods disclosed elsewhere herein, followed by administration of the allogeneic antigens front a donor that would otherwise trigger (non-self) transplantation rejection.
In both autoimmune and inflammatory diseases the condition arises through aberrant reactions of the human adaptive or innate immune systems. Autoinflammatory diseases are a relatively new category of diseases that are different from autoimmune diseases. However, autoimmune and autoinflammatory diseases share common characteristics in that both groups of disorders result from the immune system attacking the body's own tissues, and also result in increased inflammation. The term “autoimmune disease” is meant to refer to a disease state caused by an inappropriate immune response that is directed to a self-encoded entity which is known as an autoantigen. An autoimmune disease results when a host's immune response fails to distinguish foreign antigens from sell-molecules (autoantigens) thereby eliciting an aberrant immune response. The immune response towards self-molecules in an autoimmune disease results in a deviation from the normal state of self-tolerance, which involves the destruction of T cells and B cells capable of reacting against autoantigens, which has been prevented by events that occur in the development of the immune system early in life. The cell surface proteins that play a central role in regulation of immune responses through their ability to bind and present processed peptides to T cells are the major histocompatibility complex (MHC) molecules (Rothbard, J. B. et al., 1991. Annu. Rev. Immunol. 9:527). Autoimmune diseases are further considered cell mediated or antibody mediated. Cell mediated autoimmune diseases arise from activities of lymphocytes such as T cells and natural killer cells, while antibody mediated diseases are caused by attack of antibodies produced by B cells and secreted into the circulatory system. Examples of cell mediated autoimmune conditions or diseases are diabetes, multiple sclerosis, and Hashimoto's thyroiditis. Examples of antibody mediated conditions or diseases are systemic lupus erythematosus and myasthenia gravis.
Exemplary autoimmune diseases that can be treated by the methods of the invention include, but are not limited to, autoimmune disease selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, alopecia greata, anklosing spondylitis, antiphospholipid syndrome, autoimmune addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (alps), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome immune deficiency, syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, Crest syndrome, Crohn's disease, Dego's disease, dermatomyositis, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglohulinemia, fibromyalgia-libromyositis, grave's disease, guillain-barre, hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), Iga nephropathy, insulin dependent diabetes (Type I), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglancular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-man syndrome, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.
In certain embodiments, the autoimmune disease or disorder is preferably selected from the group consisting of multiple sclerosis, diabetes mellitus and rheumatoid arthritis, graft versus host diseases (GVHD) in bone marrow transplantation, organ transplantation such as kidney, liver, heart, skin, and others, in transplantation the antigen can be simply apoptotic donor leukocytes from blood; allergy/asthma, the antigen can be whatever allergen the individual is sensitive; other autoimmune diseases such as RA and systemic sclerosis.
In further embodiment, the method is useful for the treatment of an autoimmune disease that is in a later stage. Frequently, autoimmune diseases are recognized at later stages of the disease. For example, as organ-specific autoimmune diseases do not become manifest until well-advanced, interventive therapies must inhibit late-stage disease processes. While not to be limited by a particular theory, one reason for this is because the method “resets” the immune system.
In one embodiment, the autoimmune disease or disorder is Sjogren's syndrome (“SS”). Experimental Sjogren's syndrome (“ESN”) can be induced in mice using an exemplary procedure set forth in, e.g., Lin et al. (Ann. Rheum Dis 2014; 0: 1-9). Specifically, an ESS mouse model can be induced in 8-week-old female wildtype mice (e.g., C57BL/6 mice) by introduction of salivary gland proteins as described in Lin et al. (Int Immunol 2011; 23: 613-24). For ESS induction of such mice, each mouse received subcutaneous multiinjections on the back with 0.1 ml of the emulsion on days 0 and 7, respectively. On day 14, a booster injection was carried out with a dose of 1 mg/mL salivary gland (SG) proteins emulsified in Freund's incomplete adjuvant (Sigma-Aldrich). Mice immunized with either proteins extracted from pancreas or adjuvant alone can serve as controls. Phenotypes associated with development of ESS in such mice include reduced saliva secretion, elevated serum autoantibody production and tissue destruction with lymphocytic infiltration in submandibular gland. Performance of the methods disclosed herein for treating or preventing autoimmune or autoinflammatory diseases by first breaking down the dysregulated immune system and then reprogramming the immune system to restore tolerance to the patient's self-antigens by induction of antigen specific regulatory T cells is contemplated upon both model mice such as those described above and upon subjects having or at risk of developing Sjogren's syndrome.
Autoantigens
It has been shown herein that with the combination of apoptosis, phagocytes, and antigen can antigen-specific Treg cells be optimally generated and long-term immune tolerance developed, the proper antigenic peptide needs to be introduced in a timely manner into subjects in which an immunoregulatory milieu was created by apoptosis-triggered phagocytes. In addition, the specificity of the antigenic peptide is also critical in tolerance induction.
Accordingly, certain aspects of the invention include methods and compositions concerning antigenic compositions including segments, fragments, or epitopes of polypeptides, peptides, nucleic acids, carbohydrates, lipids and other molecules that provoke or induce an antigenic response, generally referred to as antigens. As used herein, an “autoantigen” is a cellular molecule and usually is a protein. An autoantigen is typically not antigenic because the immune system is tolerized to its presence in the body under normal conditions. An autoantigen can be produced by natural cells, using recombinant methods, or through chemical synthesis, as appropriate. In particular, autoantigens, or antigenic segments or fragments of such autoantigens, which lead to the destruction of a cell via an autoimmune response, can be identified and used in the methods claimed herein.
Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS) caused by lymphocyte and macrophage infiltrations into the white matter resulting in demyelination. The disease is commonly observed in young Caucasian adults with Northern European ancestry and is associated with the HLA-DR2 haplotype. Myelin basic protein (MBP) is thought to be one of the major target antigens in the pathogenesis of MS. Particularly, T cell reactivity to the immunodominant MBP 85-99 epitope is found in subjects carrying HLA-DR2, a genetic marker for susceptibility to MS. MS has been linked to the autoimmune response of T cells to myelin self-antigens presented by HLA-DR2 with which MS is genetically associated. Myelin basic protein (MBP) is a major candidate autoantigen in this disease. Its immunodominant epitope, MBP85-99, forms a complex with HLA-DR2. Copolymer 1 (Cop1, Copaxone®, Glatiramer Acetate, poly(Y, E, A, K) n), a random amino acid copolymer [poly (Y,E,A,K)n or YEAK] as well as two new synthetic copolymers [poly (F,Y,A,K)n or FYAK, and poly (V,W,A,K)n or VWAK] also form complexes with HLA-DR2 (DRA/DRB1*1501) and compete with MBP85-99 for binding. U.S. 20070264229, incorporated by reference in its entirety herein, provides MS autoantigens that can be used in the claimed method.
The understanding of the cell-mediated pathological process leading to MS has been advanced by the development of an animal model known as experimental autoimmune encephalomyelitis (EAE). EAE in mice mimics the inflammatory infiltrate, the neurological paralytic symptoms and demyelination observed in MS. EAE is mediated by CD4 T cells and can be induced actively by immunization with myelin antigens or their immunodominant peptides emulsified in complete Freund's adjuvant in combination with pertussis toxin injections. The myelin components myelin basic protein (MBP), proteolipid protein and myelin oligodendrocyte glycoprotein are the most studied encephalitogenic self-antigens. In certain embodiments, the self-antigen is myelin proteolipid protein (PLP).
Type I diabetes is an organ-specific autoimmune disease caused by chronic inflammation (insulitis), which damages the insulin producing β-cells of the pancreatic Islets of Langerhans. Dendritic cells (DCs) are generally the first cells of the immune system to process β-cell autoantigens and, by promoting autoreactivity, play a major role in the onset of insulitis. Although no cure for diabetes presently exists, the onset of insulitis can be diminished in the non-obese diabetic (NOD) mouse type 1 diabetes model by inoculation with endogenous β-cell autoantigens. These include the single peptide vaccines insulin, GAD65 (glutamic acid decarboxylase), and DiaPep277 (an immunogenic peptide from the 60-kDa heat shock protein).
Rheumatoid arthritis (RA) is a major systemic autoimmune disease. Etiology of the disease most likely involves genetic risk factors, activation of autoimmune response as well as environmental factors. The disease is systemic at all stages, characterized by inflammatory cell infiltration, synovial cell proliferation, destruction of cartilage and aberrant post-translational modifications of self-proteins that may play a role in breaking T and B cell tolerance. However, in patients with established disease, a synovial manifestation clearly dominates.
The early clinical presentation may not be specific since RA is initially indistinguishable from other forms of arthritis. So far, there is no single biomarker for the early detection of RA. The characteristic feature of this disorder is the presence of autoantibodies in the patient serum that distinguishes it from non-autoimmune joint pathogenesis like reactive arthritis or osteoarthritis (OA).
Several autoantibodies have been descried in RA including antibodies against heat-shock proteins (Hsp65, Hsp90, DnaJ), immunoglobulin binding protein (BiP), heterogeneous nuclear RNPs, annexin V, calpastatin, type II collagen, glucose-6-phosphate isomerase (GPI), elongation factor human cartilage gp39 [7] and mannose binding lectin (MBL). There are some antigens such as citrullinated vimentin, type II collagen, fibrinogen and alpha enolase against which high titers of autoantibodies are specifically found in RA patients' sera. More recent discoveries include antibodies to carbamylated antigens (anti-CarP), to peptidyl arginine deiminase type 4 (PAD4), to BRAF (v raf murine sarcoma viral oncogene homologue B1) and to 14 autoantigens identified by phage display technology.
A pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antimicrobials such as antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, transdermal, or subcutaneous administration, and the active compound can be coated in a material to protect it from inactivation by the action of acids or other adverse natural conditions.
The methods of the invention include incorporation of administering an effective amount of an anti-CD4 antibody, anti-CD8 antibody, or both to the subject and administering an autoantigen specific to the autoimmune disease or disorder that the subject is suffering from. Accordingly, the methods of the invention include an anti-CD4 antihody, anti-CD8 antibody, or both, and an autoantigen, as provided herein into a pharmaceutical composition suitable for administration to a subject. A composition of the present invention can be administered by a variety of methods known in the art as will be appreciated by the skilled artisan. The active compound can be prepared with carriers that will protect it against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Many methods for the preparation of such formulations are patented and are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, Ed., Marcel Dekker, Inc., NY, 1978. Therapeutic compositions for delivery in a pharmaceutically acceptable carrier are sterile, and are preferably stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
Dosage regimens can be adjusted to provide the optimum desired response (e.g., tolerizing a subject and/or a therapeutic response). For example, a single bolus or oral dose can be administered, several divided doses can be administered over time, or the dose can be proportionally induced or increased as indicated by the exigencies of the disease situation.
A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective dose of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compound of the invention employed in the pharmaceutical composition at a level lower than that required in order to achieve the desired therapeutic effect, and increase the dosage with time until the desired effect is achieved.
In another embodiment, the pharmaceutical composition may also include also an additional therapeutic agent, i.e. in combination with an additional agent or agents. Examples of materials that can be used as combination therapeutics for treatment of autoimmune disease as additional therapeutic agents include: an antibody or an antibody fragment that can bind specifically to an inflammatory molecule or an unwanted cytokine such as interleukin-6, interleukin-8, granulocyte macrophage colony stimulating factor, and tumor necrosis factor-.alpha.; an enzyme inhibitor which can be a protein, such as alpha1-antitrypsin, or aprotinin; an enzyme inhibitor which can be a cyclooxygenase inhibitor; an engineered binding protein, for example, an engineered protein that is a protease inhibitor such an engineered inhibitor of a kallikrein; an antibacterial agent, which can be an antibiotic such as amoxicillin, rifampicin, erythromycin; an antiviral agent, which can be a low molecular weight chemical, such as acyclovir, a steroid, for example a corticosteroid, or a sex steroid such as progesterone; a non-steroidal anti-inflammatory agent such as aspirin, ibuprofen, or acetaminophen: an anti-cancer agent such as methotrexate, cis-platin, 5-fluomuracil, or adriamycin; a cytokine blocking agent; an adhesion molecule blocking agent; or a cytokine.
An additional therapeutic agent can be a cytokine, which as used herein includes without limitation agents which are naturally occurring proteins or variants and which function as growth factors, lymphokines, interferons particularly interferon-beta, tumor necrosis factors, angiogenic or antiangiogenic factors, orythropoietins, thrombopoietins, interleukins, maturation factors, chemotactic proteins, or the like.
An improvement in the symptoms as a result of such administration is noted by a decrease in frequency of recurrences of episodes of the autoimmune condition such as MS, by decrease in severity of symptoms, and by elimination of recurrent episodes for a period of time after the start of administration. Quantitative measures of disease severity are provided herein. A therapeutically effective dosage preferably reduces symptoms and frequency of recurrences by at least about 20%, for example, by at least about 40%, by at least about 60%, and by at least about 80% or by about 100% elimination of one or more symptoms, or elimination of recurrences of the autoimmune disease, relative to untreated subjects. The period of time can be at least about one month, at least about six months, or at least about one year.
The invention also contemplates administration of an additional agent.
Exemplary agents include, hut are not limited to non-steroidal anti-inflammatory drugs (NSAIDs), such as Aspirin, Choline and magnesium salicylates, Choline salicylate, Celecoxib, Diclofenac potassium, Diclofenac sodium, Diclofenac sodium with misoprostol, Etodolac, Fenoprofen calcium, Flurbiprofen, Ibuprofen, Indomethacin, Ketoprofen, Magnesium salicylate, Meclofenamate sodium, Mefenamic acid, Meloxicam, Nabumetone, Naproxen, Naproxen sodium, Oxaprozin, Piroxicam, Rolecoxib, Salsalate, Sodium salicylate, Sulindac Tolmetin sodium, Valdecoxib.
Other exemplary agents include disease-modifying antirheumatic drugs (DMARDs), for example, but not limited to, abatacept, adalimumab, azathioprine, chloroquine and hydroxychloroquine (antimalarials), ciclosporin (Cyclosporin A), D-penicillamine, etanercept, golimumab, gold salts (sodium aurothiomalate, auranofin), infliximab, leflunomide, methotrexate (MTX), rituximab, sulfasalazine (SSZ).
Other exemplary agents include metformin, glipizide, glyburide, glimepiride, acarbose, pioglitazone, Sitagliptin, Saxagliptin, Repaglinide, Nateglinide, Exenatide, Liraglutide.
Other exemplary agents include corticosteroids, beta-interferons, glatiramer acetate, fingolimod, natalizumab, mitoxantrone, teriflunomide.
Also provided are kits. Kits according to the present invention can contain pharmaceutical compositions for use in the methods of the Invention (e.g. anti-CD4 antibody, anti-CD8 antibody, or both, and an autoantigen specific to the autoimmune disease or disorder). In preferred embodiments, the kits contain all of the components necessary to perform the methods of the invention, including directions for performing the methods, and any necessary software for analysis and presentation of results.
The instant application describes development of a process/pathway for generating autoantigen-specific Treg cells in vivo, which showed therapeutic effects on experimental autoimmune encephalomyelitis and nonobese diabetes in mice, and is applicable to autoimmune disease more generally. Specifically, apoptosis of immune cells was induced by systemic sublethal irradiation or depleted B and CD8+ T cells with specific antibodies and then autoantigenic peptides were administered to mice possessing established autoimmune diseases. It was mechanistically demonstrated that apoptotic cells triggered professional phagocytes (e.g., neutrophils, monocytes, macrophages, dendritic cells, and mast cells, having receptors on their surfaces which can detect harmful objects, such as bacteria) to produce transforming growth factor β, under which the autoantigenic peptides directed naïve CD4+ T cells to differentiate into Foxp3+ Treg cells, instead of into T effector cells, in vivo. These antigen-specific Treg cells specifically ameliorated autoimmunity without compromising immune responses to bacterial antigen. Thus, antigen-specific Treg cells with therapeutic activity toward autoimmunity were successfully generated. The present findings can be broadly applied to development of antigen-specific Treg cell-mediated immunotherapy for multiple sclerosis and type 1 diabetes and also other autoimmune diseases.
An antigen-specific therapy for autoimmune disease that does not compromise the overall immune response is the ultimate goal for medical researchers studying treatment of autoimmune disease. The current application provides a process/pathway to generate autoantigen-specific Treg cells in vivo in mouse autoimmunity models in which mice exhibit disease before therapeutic intervention. Herein it was identified that apoptosis-antigen therapy could suppress autoimmune T cell responses to the target tissues, without compromising the overall immune response. The dysregulaled immune system was reprogrammed and, importantly, the disease was controlled. This apoptosis-antigen-mediated immune tolerance occurred in both TH17-mediated EAE and TH1-mediated T1D. Hence, apoptosis-antigen therapy represents a new therapeutic approach that could be used in the treatment of autoimmune diseases.
Although it has been discovered that the apoptosis-antigen treatment described herein induces autoantigen-specific Treg cells, it remains possible that the already present autoantigen-specific tTreg cells may also participate in suppressing autoimmune responses in the tolerized mice. Nonetheless, adaptation of this therapy to human patients is contemplated, with dose, time, and length of the specific self-peptide injection being important factors for evaluation in clinical application to patients.
Thus, a process/pathway for reprogramming the dysregulated immune system to promote tolerance in EAE and T1D has been discovered and described herein. It is contemplated that the apoptotic induction approach described herein can be performed upon patients with autoimmunity disease or disorder. For example, anti-CD20 antibody (rituximab) has been used in patients with autoimmune diseases, and it is contemplated that such therapy can be combined with the antibody-mediated depletion (apoptosis) of B cells together with administration of known autoantigenic peptide(s) to achieve additional and better therapeutic effects for patients. Similarly, one-time low/middle dose of irradiation with the aim to induce sufficient number of apoptotic cells with adoptive transfer of autologous macrophages can also be used to induce apoptosis in patients. Total body irradiation followed by hematopoietic stem cell transplantation has been conducted previously in patients with severe autoimmune disease (Nash et al. Blood 102: 2364 2372); therefore, low-dose irradiation together with macrophage and autoantigenic peptide administration is contemplated as providing therapeutic benefits for these patients. Nonetheless, this discovery relies on the induction of autoantigen-specific Treg cells that functionally suppress autoimmunity in the target tissues without compromising the overall immune response in the host. Thus, the currently identified protocol can be applied to other types of autoimmune disease, provided that one or more self-peptides are identified.
It should be appreciated that the invention should not be construed to be limited to the examples that are now described; rather, the invention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan.
Described herein is an immunotherapy on experimental autoimmune encephalomyelitis (EAE) in mice by generating autoantigen-specific cells in vivo. Mechanistically, this was accomplished by first inducing apoptotic immune cells that trigger professional phagocytes (e.g., neutrophils, monocytes, macrophages, dendritic cells, and mast cells, having receptors on their surfaces which can detect harmful objects, such as bacteria) to produce TGFβ, and then administering auto-antigenic peptides, which directed naïve CD4+ T cells to differentiate into Foxp3+ Treg cells instead of into T effector cells in vivo. Importantly, these antigen-specific cells suppressed T cell response to the autoantigens, but not to bacterial antigen. Thus, antigen-specific cells with therapeutic activity toward EAE have been successfully generated. These findings have clinical implications for the development of therapy for various autoimmune diseases, including multiple sclerosis and diabetes.
The present invention describes, in part, the development of a novel pathway to induce antigen-specific cells in vivo that have therapeutic effects on mice with EAE. The principle of the experimental design is to first “break down” the dysregulated, autoimmune immune system, and then “reprogram” it to be immune-tolerant to self-antigens. This specific immune tolerance was accomplished by a combination of immune cell apoptosis followed by specific antigenic-peptide administration (herein apoptosis-antigen) in mice, which induced antigen-specific Treg cells (
T cell Apoptosis and Peptide Administration Leads to Long-Term Suppression of EAE
First, the hypothesis was tested of apoptosis-antigen combination to induce tolerance in a model of relapsing-remitting EAE in proteolipid protein peptide PLP139-151(pPLP)-susceptible SJL mice10,11. CD4- and Cl8-depleting antibodies were used (herein αCD4/CD8) to induce T cell apoptosis12,13. Indeed, the antibody treatment depleted 90% of CD4+ and 50% of CD8+ cells, which were recovered in about 3 weeks (data not shown). SJL mice were immunized with pPLP and complete Freud's adjuvant (CFA) to induce EAE. After mice reached the peak of disease, they were divided into five groups that were either left untreated (PBS), injected with pPLP (PLP) or received αCD4/CD8 followed by pPLP injection (αCD4/CD8+PLP), control pOVA (αCD4/CD8+IVA), or PBS (αCD4/CD8+PBS) (
However, T cells from PLP or αCD4/CD8+OVA-treated spleens showed no reduction in the above inflammatory cytokines in response to pPLP stimulation in cultures (
To exclude the possibility that the tolerance effects seen in the aforementioned therapy of EAE were due to the signaling and/or depletion of CD4+ effector T cells (and to further validate that the tolerance effects in the therapy of EAE, as described elsewhere herein, were triggered by cell apoptosis and mediated by phagocytes), B cells and CD8+ T cells were depleted with respective antibodies followed by pPLP injection in SJL mice with established EAE (
Next, the underlying mechanisms responsible for the long-term EAE remission were examined. It was hypothesized that professional phagocytes (in certain embodiments, particularly macrophages and immature DCs), by sensing and digesting apoptotic played essential roles in the tolerance induction presented here (
This question was also addressed in the prevention model of the relapsing-remitting EAE model in SJL mice. The data showed that indeed co-transfer of normal SJL splenic macrophages and iDCs with pPLP, but not with PBS, into irradiated mice before was induced significantly suppressed acute and chronic (
Since TGFβ is one of the primary cytokines produced by phagocytes upon digestion of apoptotic cells11, 15, 16, the function of TGFβ in apoptosis-antigen-mediated suppression in EAE, and T1D was determined.
TGFβ in vivo completely reversed the tolerance in NOD mice induced by apoptosis-antigen therapy (IRR+MΦ+GAD65+αTGFβ). Anti-TGFβ treated mice exhibited earlier-onset and more severe disease than untreated (PBS) NOD mice (
The role of TGFβ in apoptosis-antigen-mediated suppression of EAE was examined, using the myelin oligodendrocyte glycoprotein peptide(pMOG) induced EAE model in C57BL/6 mice. EAE mice were treated at the peak of acute with αCD4/CD8 and pMOG (herein αCD4/CD8/MOG) in the absence (αCD4/CD8MOG+Contrl Ab) and presence of anti-TGFβ neutralizing antibody (αCD4/CD8/MOG+αTGFβ) (
EAE mice were also treated at the peak of acute EAE with γ-irradiation plus phagocytes and pMOG in the absence (IRR+MΦ+MOG+contrl Ab) or presence of anti-TGFβ neutralizing antibody (IRR+MΦ+MOG+αTGFβ) (
Antigen-Specific Treg Cells were Generated in Apoptosis-Antigen Tolerized EAE Mice
Next, validation of the function of TGFβ in irradiation-phagocytes -pMOG therapeutic model of EAE in C57BL/6 mice was performed, and similar results were observed (
On the other hand, the same subpopulations of CD4+ T cells would exhibit no significant alterations of T cell responses to MT or CD3 antibody compared to untreated control mice. Indeed, non-separated CD4+ T cells from the spleens of IRR+MΦ+PLP-treated tolerized mice (
pPLP-specific Treg cell generation in other therapy models of SJL mice was then investigated with αCD20/CD8 plus pPLP (
Furthermore, the generation and function of autoantigen-specific Foxp3+ Treg cells in the tolerized mice induced by irradiation plus professional phagocytes and peptides or by αCD20/CD8 plus peptides in the prevention model of EAE in SJL, mice was also determined (
Tetramers recognizing MOG(38-49)-specific CD4+ T cells were used with Foxp3 staining to determine the specific Treg cells in C57BL/6 mice with established EAE that were suppressed with irradiation plus transfer of phagocytes plus pMOG injection (IRR+Mϕ+MOG+Control Ab). The frequency of CD4+ Foxp3+ tetramer-positive Treg cells was indeed substantially increased, and that of tetramer-positive Th17 or Th1 cells was decreased in the spinal cords of tolerized mice compared to untreated groups (
To determine the cellular sources of TGFβ in treated (tolerized) mice, cell membrane-bound TGFβ was examined in both macrophages and Treg cells (Perruche et al. Nat. Med. 14: 528-535; Nakamura et al. J. Exp. Med. 194: 629-644; Belghith et al. Nat. Med. 9: 1202-1208). C57BL/6 mice were immunized with pMOG plus FCA to develop EAE. At the peak of EAE, mice were treated with IRR+MΦ+MOG or untreated (PBS). It was observed that macrophages expressed higher amounts of latency-associated peptide (LAP) TGFβ1 than did control macrophages on the second day after apoptosis induction (day 16;
The role of Treg cells was also determined for apoptosis-antigen therapy in pMOG-induced EAE CD4+CD25+ Treg cells were depleted using anti-CD25 antibody (Sakaguchi et al. Immunol. Rev. 212: 8-27) in mice that were also treated with IRR+MΦ+MOG (
Thus, these data have provided strong evidence that autoantigen-specific Treg cells were indeed generated and functional in suppressing autoantigen-specific T effector cell responses, and this plays a major role in the therapy of induced by apoptosis-antigen combination.
To determine if antigen-specific Treg cells were indeed converted from naïve T cells in vivo in the immunosuppressive milieu triggered by apoptosis-antigen administration, TCR transgenic naïve T cells (2D2) specific to pMOG were injected into syngeneic C57BL/6 mice either treated with IRR+Mo+MOG or untreated before immunization. IRR+Mϕ+MOG-treated mice showed suppression of EAE compared to the untreated mice (
To provide unambiguous evidence that the apoptosis-antigen combination indeed promoted Foxp3 Treg cell conversion from naïve CD4+ T cells in vivo. TCR transgenic CD4+ CD25− T cells (KJ1-26+, specifically recognizing pOVA) isolated from DO11.10xRag−/− mice (which have no endogenous Foxp3+ Treg cells) were injected into syngeneic 7-week-old BALB/c mice. To avoid depletion of transferred transgenic T cells in BALB/c mice, two immune cell depletion models were used: by anti-CD8CD20 antibody injection or systemic γ-irradiation. Here, it was shown that anti-CD8/CD20 injection and pOVA administration resulted in significantly more KJ1-26+ Foxp3+ Treg cells (
The above data collectively provided clear evidence that apoptosis-antigen treatment converted naïve CD4+ T cells to antigen-specific Foxp3+ Treg cells, and this conversion required TGFβ and also phagocytes that sense and digest the apoptotic cells.
An antigen-specific therapy for autoimmune disease that does not compromise overall immune response is the ultimate goal for medical researchers and clinicians. In the above described experiments, a novel pathway was discovered for generation of autoantigen-specific Treg cells in vivo that specifically suppress autoimmune T cell responses to the target tissues without compromising the overall immune response in mice. The dysregulated immune system was reprogrammed and, importantly, the disease was controlled.
Several novel conclusions can be drawn from the current studies. First, apoptosis, rather than signaling in immune cells, is a key to initialing long-term immune tolerance. The apoptosis process requires transient yet sufficient apoptosis of cells in vivo. Supporting this conclusion is the fact that tolerance can be induced irrespective of the procedure for apoptotic cell induction or the type of apoptotic cells, as long as the phagocytes are present. Depletion of CD4+ T cells to suppress EAE was reported more than 20 years ago13,14, but the mechanisms underlying the effects were unknown. The studies here have identified that the depletion of T cells can serve as an initiator of a series of events that ultimately produces long-term immune tolerance. Indeed, depletion of non-CD4+ immune cells lead to similar therapy of EAE. The mechanisms of apoptosis-triggered tolerance reported here are also different from recent studies using non-depleting CD4-specific antibody treatment. The non-depletion CD4-specific antibody is based on the blockade of CD4 molecules on T cells and also does not involve administration of peptide21, whereas the present study relied on the transient and sufficient extent of cell apoptosis that initiated the whole tolerance process. Second, apoptosis-antigen treatment was not linked to inflammatory cytokine release by immune cells. This differs from CD3-antibody-mediated T cell depletion, which can transiently yet powerfully trigger TCR on T cells to release proinflammatory cytokines in vivo11,22,23. This lack of overt inflammatory cytokines in a TGFβ-rich immunoregulatory milieu could provide a precondition for the ensuing generation of Treg cells. Third, phagocytes24 were key in mediating the long-term immune tolerance and therapy of EAE presented here. This notion was supported by experiments of either depletion of endogenous phagocytes in tolerized mice induced by T cell depletion plus self-peptide treatment, or by adoptive transfer of syngeneic normal splenic macrophages and DCs plus self-peptide in irradiated mice. This conclusion was further supported by the data showing that depletion of immune cells alone or plus self-peptide in the absence of a sufficient number of professional phagocytes failed to generate antigen-specific cells and immune tolerance, which led to no suppression of EAE. In fact, these conditions may exacerbate by enhancing T effector cells, likely due to empty space-driven T cell expansion. Fourth, TGFβ, but not IL-10, was vital to inducing long-term immune tolerance and remission of EAE induced by apoptosis-antigen therapy through generation of antigen-specific Treg cells in vivo. Although many cells can produce TGFβ in vivo25,26, macrophages are likely the major cellular source of TGFβ in apoptosis-mediated immune tolerance immediately upon contact/digestion of apoptotic cells. Treg cells (likely antigen-specific Treg cells), however, can be another cellular source of TGFβ, especially at the later stage of the apoptosis-antigen therapy. Fifth, proper and timely antigenic peptide introduction into the transiently established immunosuppressive milieu in mice was shown to be key to induce antigen-specific cells. It has been shown here that only with the combination of apoptosis, phagocytes, and antigen can the antigen-specific Treg cells be optimally generated and long-term immune tolerance developed, the proper antigenic peptide needs to be introduced in a timely manner into mice in which an immunoregulatory milieu was created by apoptosis-triggered phagocytes. In addition, the specificity of the antigenic peptide is also critical in tolerance induction. It was found here that injecting the same amounts of an irrelevant control peptide such as pOVA instead of self-peptide (pPLP or pMOG) failed to suppress EAE in SJL or B6 mice, respectively. The administration of pOVA could theoretically induce pOVA-specific Treg cells in an apoptosis-triggered TGFβ-rich immunosuppressive microenvironment. However, these pOVA-specific cells could not survive, expand, and function sufficiently to suppress the disease, as there is no continuous pOVA stimulation in EAE mice7,18,27,31. This finding has implications in translating the study to human patients, as even if some unwanted peptide was present during the transient immunosuppressive milieu and Treg cells specific to that antigen was induced by by-product, it would not affect immune response to the antigen as long as the unwanted peptide does not stay around. Another important point to mention is that, unlike in prevention models, depletion of immune cells in the presence of phagocytes without addition of exogenous autoantigenic-peptides in the therapy models (after immunization) did result in some suppression of EAE. This phenomenon might be attributable to the fact that the mice with established likely have some endogenous self-peptide present that was derived from the immunization step. Thus, it is conceivable that dose, time, and length of the specific sell-peptide injection will be important factors to consider in future clinical application to patients.
Importantly, it was determined that the apoptosis-antigen combination treatment induced and increased antigen-specific Treg cells in vivo. TGFβ is absolutely required for this process. These antigen-specific cells could serve the major force for inducing and maintaining long-term immune tolerance, and thus inhibition of EAE and T1D. These findings were significant, at least because this appears to be the first identification that antigen-specific cells can be induced in mice with established autoimmune disease and suppress and prevent relapses of the disease, which should have clinical implication in patients with multiple sclerosis (MS) and T1D. These studies suggested that the dysregulated immune responses in patients with autoimmune diseases can be reprogrammed.
Another significant feature of this approach is that there is no obvious nonspecific overall immunosuppression or immune defect to antigens from pathogens in this induced tolerance. This conclusion is supported by the current data that the CD4+ T cells isolated from mice with long-term remission of EAE and T1D showed intact T cell responses to the pan-TCR stimulation using CD3 antibody, suggesting no overall immune defect occurred in the tolerized mice. Importantly, CD4+ T cells that exhibit tolerance to auto-antigen stimulation showed normal T cell proliferation and effector differentiation to bacterial antigens. How this occurs in vivo remains unknown, but several possible explanations can be postulated. First, immune cell depletion and the consequent suppressive immune environment are transient, and T cells recover. Second, if, in the TGFβ-immunoregulatory milieu, the body by chance also encounters bacterial or virus antigens, the pathogen-specific T cells might be unlikely to direct to Treg differentiation, but instead to T effector cells, because the pathogens could through their TLR pathways trigger inflammatory cytokines that abrogate Treg cells30, 32, 34.
In sum, described herein is the development of a novel pathway for reprogramming the dysregulated immune system and responses to EAE and T1D therapy in mice, with an ultimate use as therapy for patients with T1D, MS and other autoimmune diseases or disorders. This discovery relies on the induction of autoantigen-specific Treg cells that functionally suppress autoimmunity in the target tissues without compromising the overall immune response in the host. This protocol has applications to other types of autoimmune disease, as long as one or more self-peptides are identified.
Apoptosis-antigen mediated therapy of type 1 diabetes model in NOD mice was examined. 9 wk-old NOD mice were irradiated with γ-irradiation (IRR) with dose of 200 rad. Some mice received normal splenic macrophages and DCs (MODC). Some mice were administered 5 μg of Glutamic acid decarboxylase 65 (GAD65) peptide or PBS every other day as indicated. (a), upper panel, the experimental scheme; Lower panel, the frequency of diabetes free mice. PBS (untreated control, n=3), GAD65 (GAD65 alone, n=3), IRR+MODC+GAD65 (irradiation plus MODC plus GAD65, n=5), IRR+MODC (irradiation plus MODC, n=5). (b), The frequency of Foxp3+ (left) and IFN-γ+ (right) cells within CD4+ T cells in the pancreas draining lymph nodes (DLN) are shown, as determined by flow cytometry. *P<0.05, determined by Student's t test (two-tail).
The generality of apoptosis-antigen therapy in other autoimmune settings was assessed by examining T1D in nonobese diabetic (NOD) mice (Anderson and Bluestone, Annu. Rev. Immunol. 23: 447 485). T1D develops from 12 weeks of age in female NOD mice as a result of insulitis, a leukocytic infiltrate in the pancreatic islets. GAD65 has been identified as one of the autoantigens in NOD mice and in patients with T1D (Kaufman et al. Nature 366: 69 72; Lohmann et al. Lancet 356:31 35). NOD mice were treated at the age of 9 weeks, when the mice are considered diabetic without hyperglycemia, as the inflammatory process has been initiated and is in progress, yet the levels of glucose in the blood are still within the normal range (Anderson and Bluestone). NOD mice were either untreated (PBS) or treated with GAD65 peptide (GAD65,) or with γ-irradiation followed by administration of GAD65 peptides plus phagocytes (IRR+MΦ+GAD65) (
To investigate whether the apoptosis antigen therapy was effective, in NOD mice with established T1D, hyperglycemic NOD mice with glucose levels >200 mg/dl were treated with γ-irradiation plus phagocytes and GAD65 peptide injection. Strikingly, it was found that the treatment blocked the progress of diabetes, preventing further increases in blood glucose levels, which remained at 200 to 300 mg/dl, whereas the untreated mice quickly exhibited elevated blood glucose levels (>600 mg/dl) (
To confirm that observed tolerance was mediated by induction and increase of GAD65-specific Treg cells in NOD mice, CD4+ T cells and CD4+CD25-T cells were stimulated with GAD65 peptide in the same manner as outlined in the study. Significantly decreased GAD65-driven production was observed by CD4+T cell in the IRR+MΦ+GAD65 +contrl Ab treated tolerized mice compared to untreated (PBS) mice. Levels of anti-CD3 driven CD4+ T cell production were comparable between these two groups (
An exemplary application of this approach involves administration of single-dose irradiation to a subject (e.g., 200-400 rad dosage of radiation, resulting in depletion of all types of immune cells, such as T cells, B cells, and macrophages, etc.) followed by adoptive transfer of normal macrophages and administration of autoantigenic peptides (Kasagi et al., Science Translational Medicine 6(241): 241ra78). A similar exemplary application of a different aspect of the invention involves administration of anti-CD20 and anti-CD8 antibodies to a subject (resulting in depletion of B lymphocytes and CD8+ T cells, without depleting CD4+ T cells) and administration of autoantigenic peptides (Kasagi et al., Science Translational Medicine 6(241): 241 ra78).
Despite the development of biological agents, a large portion of patients with arthritis still suffer from disability. Collagen-Induced Arthritis (CIA) is the prototype model of autoimmune arthritis, which shares many features with rheumatoid arthritis.
Mice used in the following experiments are preferably DBA/ILacJ in SPF condition however, other options include B10,RIII, B10.M-DR1 and C57BL/6, although their susceptibility varies.
Reagents for immunization are type II collagen (CII) and complete Freund's adjuvant (CFA).
Preparation is as follows:
Administration is as follows: (day 0): inject with 100 μg CII and 100 μg CFA emulsion in a total volume of 50 μl intradermally at the base of the tail.
Disease course is as follows:
Assessments are made three times a week as follows:
Treatment Groups are as follows:
Screening and grouping into comparable arms is carried out prior to the beginning of treatment. Based on the incidence and the number of groups, the estimated total number of mice in one experiment is 20.
The Endpoint is at day 56, and the following are assessed:
Systemic sclerosis (SSc) is a connective tissue disease characterized by excessive extracellular matrix deposition with an autoimmune background. Presence of autoantibodies is a central feature of SSc, antinuclear antibodies (ANAs), such as anti-DNA topoisomerase I (anti topo I) antibody, are detected of patients. Furthermore, abnormal activation of several immune cells has been identified in SSc. Prognosis is very poor in patients with diffuse type SSc (10-year survival of 55%) because of limited application of medication. Therefore, establishing treatment is urgent need for these patients.
Mice used in these experiments are C57BL/6 mice (The Jackson Laboratory) in a SPF condition.
For immunization, recombinant human topo I (TopoGEN) was dissolved in saline (500 units/ml). The topo I solution was mixed 1:1 (volume/volume) with CFA II37Ra (Sigma-Aldrich). These solutions (300 μl) were injected 4 times subcutaneously into a single location on the shaved back of the mice with a 26-gauge needle at an interval of 2 weeks. Human serum albumin (Protea Biosciences) was used as an irrelevant control human protein.
For treatment, mice were treated with either sublethal dose (200rad) of γ-Irradiation or anti-CD4/CD8 T cell depletion antibody followed by administration of professional phagocytes at the peak of disease (around day 42).
The End point for these experiments is the time point in which the differences in dermal thickness between treated mice and untreated mice are clinically apparent.
This model is chosen because TopoI is recognized as an antigen in patients with SSc. Human topo I has 93% sequence identity to mouse topo I. Further, titers of anti-topo I antibody are positively correlated with disease activity in 20% of patients with SSc. Titers of antitopo I antibody are selectively upregulated after immunization, which has correlation with lung and skin disease in this model mice. This phenomenon is similar to human disease. Finally, IL-6 and IL-17 seem to play a pathological role in this model mice. As there is evidence that irradiation plus professional phagocyte treatment induce Tregs and suppress IL-6 or IL-17 mediated inflammation in model mice, this therapy is promising and expected to fit this mouse model.
A subject having or at risk of developing Sjogren's syndrome (“SS”) is obtained or identified (exemplary subjects for Sjogren's syndrome therapy as described herein include a human subject having or at risk of developing SS, or a mouse model subject, such as mice having induced, experimental Sjogren's syndrome (“ESS”) as described, e.g., in Lin et al. (Ann. Rheum Dis 2014; 0: 1-9)). One or more of the following is administered to the subject:
(1) anti-CD4 and anti-CD8 antibodies (thereby inducing T cell apoptosis), followed by administration of auto-antigenic peptides (e.g., salivary gland peptides as described in Lin et al. (Int Immunol 2011; 23: 613-24) for mice);
(2) Anti-CD20 and anti-CD8 antibodies (thereby depleting B lymphocytes and CD8+ T cells, without depleting CD4+ T cells), followed by administration of autoantigenic peptides; and/or
(3) irradiation (optionally single dose irradiation, e.g., 200-400 rad), thereby depleting all types of immune cells such as T cells, B cells, and macrophages, etc., with adoptive transfer of normal macrophages, followed by administration of autoantigenic peptides.
The subject is then monitored for reduction, alleviation and/or therapeutic mitigation of markers and/or phenotypes of SS, and an effective anti-SS therapeutic regimen is thereby identified.
The results reported herein were obtained using the following methods and materials. Mice. D57BL/6 C57BL/6-Tg (Tera 2D2, Terb 2D2) 1Kuch (2D2), BALB/c, SJL, and CD45.1 (D57BL/6) mice were purchased from the Jackson Laboratory. DO11.10xRag1−/− mice were purchased from Taconic. Mice were maintained under specific pathogen-free conditions according to the National Institutes of Health guidelines for the use and care of live animals.
Flow Citometry. Single-cell suspension were stained with the following flourochrome-conjugated antibodies; from eBioscience: CD8 (clone 53-6.7), DO11.10 TCR (clone KJ1-26), TNF-α (clone MP6-XT22), (clone ebio17B7), CD4 (clone RM4-5), Foxp3 (clone FJK-16s), and from BD Biosciences; Vα3.2 (Clone RR3-16), vβ11 (clone RR3-15), and IFN-γ (clone XMG1.2). Foxp3 expression was examined using the eBioscience Foxp3 mouse Treg kit. MOG38-49-specific TCR tetramer (PE-labeled 1-A (b) GWYRSPESRVVII (SEQ ID NO: 1) tetramer) and I-A (b)/hCLIP taramers (negative control) were provided by NIH Tetramer Core Facility at Emory University. Cells from spinal cord or spleen were incubated with a 1:300 dilution of MOG38-49-specific TCR tetramer in DMEM plus 10%. FBS for 3 hour at 4° C. Cells were then washed and stained for cell surface markers described above. For intracellular cytokine measurement cells were incubated with PMA (5 ng/ml, Sigma), ionomyocine (250 ng/ml, Sigma) and GolgiPlug (1 μl/ml, BD Biosciences) to determine intracellular expression of IL-17, IFN-γ, and TXF-α. All samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences) and data were analyzed using Flowjo software (Treestar).
Cell isolation. CD4+ T cells, CD4+CD25+ T cells, CD4+CD25− T cells, CD11b+ cells, and CD11c+ cells were isolated from spleens via either positive or negative selection using MACS isolation kits (Miltenyi Biotech) following the manufacturer's protocols. Briefly, CD4+CD25− T cells were isolated by the CD4+CD25+ regulatory T cell isolation kit (Treg kit). For isolation of CD11b+ cells (Mo) and CD11c+ cells (DCs), spleens were incubated for 20 min at 37° C. in a DMEM including 8 mg/ml collagenase. Then cells were gently meshed through a cell strainer (70 μm, BD Falcon). Mo and DCs were isolated via position selection by CD11b and CD11c microbeads, respectively. Non-CD4+ T cells were isolated via negative selection by Treg kit, and used as antigen presenting cells (APCs) after irradiation with 3000 rad of γ-irradiation (Gammacell 1000, Best Theratronics).
Cytokine assays. Splenocytes were cultured at 37° C. in 5% CO2 for 2-3 days with either soluble CD3-specific antibody (anti-CD3) (0.5 μg/ml) or MT (heat-killed M. tuberculosis, H37RA, DIFCO) (50 μg/ml) or peptides (pMOG, pPLP) (0-50 μg/ml as indicated). Cytokines were quantified in culture supernatants by ELISA; TNF-α, IL-6, and IFN-γ (BioLegend) and IL-17 (eBioscience). EAE induction, scoring, analysis and in vitro cell cultures. Peptide-induced EAE was induced in SJL mice and C57BL/6 mice as previously reported11,20. Individual mice were observed daily and clinical scores were assessed on a 0-5 scale as follows: 0, no abnormality; 1, limp tail or hind limb weakness; 2, limp tail and hind limb weakness; 3, hind limb paralysis; 4, hind limb paralysis and forelimb weakness; and 5, moribund. 7-weak-old male C57BL/6 or CD45.1 mice were immunized subcutaneously with 200 μg/mouse of pMOG emulsified in CFA (IFA supplemented with 300 μg/mouse of pPLP emulsified in CFA (MT 300 μg/mice). Mice also received 200 ng of Bordetella pertussis (List Biological Lab) i.p. on the day of immunization and 2 days later. At the end of each experiment spinal cords and brain were harvested and a part was fixed in neutral 10% formalin, extracted, embedded in paraffin and cut in 5 μm sections for H&E staining. Cells were isolated from brain and spinal cord as previously reported11. Spleen was also harvested for further staining and culture. For cell cultures, splenocytes were cultured at 37° C. in 5% CO2 for 3 days with either soluble anti-CD3 (0.5 μg/ml) or MT (50 μg/ml) or peptides (pMOG, pPLP). After 3 days culture, cells were pulsed with 1 μCi [3H] thymidine for 8-16 h. Radioactive incorporation was counted using a flatbed β-counter (Wallac). To examine the function of peptide-specific CD4+CD25+ Treg cells in the spleen of mice, CD4+, CD4+ CD25−, and CD4+CD25+ T cells were MACS sorted and cultured with irradiated APCs from peptide-immunized EAE mice in the presence of either pPLP or pMOG (10 μg/ml), or MT (50 μg/ml), or anti-CD3 (0.5 μg/ml). After 3 days of culture cells and supernatant were collected for cytokine assays and determination of T cell proliferation.
Antibodies used for in vivo. Anti-CD4 (clone Gk1.5), anti-CD8 antibody (clone 53-6.72), anti-TGFβ antibody (clone ID11) and mouse IgG1 (clone MOPC-21) were purchased from Bio X cell. Anti-CD20 antibody (clone 5D2) was a gift from Genentech. T cell depletion studies in EAE disease models. For EAE prevention studies SJL/J mice were either untreated or treated with CD4− (100 μg/mouse) and CD8− (50 μg/mouse) specific antibody (T cell depletion antibody). Some mice were immediately injected i.p. with pPLP or pOVA (25 μg/mouse) or PBS every other day for 16 days. All mice were immunized with pPLP and CFA (day 0). For EAE treatment studies, SJL mice were treated with CD4- and CD8-specific antibody and 5 μg of pPLP, pOVA or PBS i.p. every other day from day 12 to day 26 following immunization with pPLP (day 0). For EAE prevention studies, SJL mice were treated with CD4- and CD8-specific antibody at day −21, and 25 μg of pPLP, pOVA or PBS i.p. every other day from day −18 to day −2 before immunization with pPLP (day 0). In C57BL/6 mice, same T cell depletion regimen was utilized but pMOG (10 μg mouse) was administered via i.p. In some mice, anti-TGFβ or isotype control antibody (mIgG1) (200 μg/mice each day) were injected i.p. one day after T cell depletion. To examine the role of phagocytes in apoptosis-antigen combined treatment of EAE mice were treated with either 300 μl of Clodronate liposome to deplete phagocytes as reported11,15,16. B cell and CD8+ T cell depletion in EAE disease model. All mice were then immunized with pPLP and CFA (day 0) followed by twice injection of pertussis (day 0, 2). Mice were monitored for clinical score of disease and sacrificed at indicated time points. For therapy experiments, SJL mice were treated with anti-CD8/CD20 antibodies (day 9 after immunization), followed by i.p. administration of pPLP (5μg/mouse) every other day from day 10 to 23.
For prevention experiments, SJL mice were treated with anti-CD8/CD20 antibodies at day −21, and 5 μg of pPLP or PBS i.p. every other day from day −18 to day −2 before immunization with pPLP and CFA (day 0).
γ-irradiation in EAE disease model. For therapeutic experiments, mice were irradiated with 200 rad of γ-irradiation (Gammacell 40 Exactor, Best Theratronics) at the peak of acute EAE (usually day 10 after immunization) followed by normal splenic Mo/DC transfer (3 million/mice). Some mice were given 5 μg of peptides i.p. every other day from day 10-21. For prevention experiments, SJL mice were irradiated followed by normal splenic Mϕ/DC transfer (7×106/mouse). Some mice were given 25 μg of pPLP i.p. every other day from −18 to day −2 before immunization with pPLP and CFA (day 0). In certain experiments, CD4+ CD25− T cells isolated from pMOG-specific TCR transgenic mice (2D2) (CD45.2+) were adoptively transferred into CD45.1+C57BL/6 mice after the recipients were irradiated. The irradiated mice were then injected i.p. with pMOG (25 μg) every other day for 4 times. Statistical analyses. Group comparisons of parametric data were made by Student's t-test (unpaired two-tail). Data was tested for normality and variance and considered a P value of <0.05 significant.
γ-irradiation in T1D disease model. Nine-week-old female NOD mice were irradiated with 200 or 450 rads of γ-irradiation followed by transfer of MF/DCs) (2 to 3 million per mouse). Some mice were given 5 mg of GAD65 peptides intraperitoneally every other day for six times.
Conversion of pOVA-specific Treg cells by B cell and CD8+ T cell depletion antibodies. Balb/c mice were either untreated or treated with single i.p. injection of anti-CD8 (100 mg/mouse) and anti-CD20 (250 mg/mouse) at day 0. Then all mice were treated i.p. with pOVA (50 mg/day/mouse) and i.p. injection of DO11.10xRag−/− TCR-transgenic CD4+CD25 T cells (KJ1-26+, pOVA-specific). The mice treated with aCD8/20 depletion antibodies were either treated with anti-TGFβ (αTGFβ) or isotype control Ab (200 mg/day/mouse) once a day from day 2 to 4 (total 3 times, invert triangles). At day 8, all mice received i.p. injection of splenic DC (0.4 million cells/mouse). All mice were sacrificed at day 13.
Conversion of pOVA-specific Treg cells by γ-irradiation. Balb/c mice were either untreated or treated with 200 rad of γ-irradiation on day 0. All mice received i.p. injection of 5 mg of pOVA on day 1. Some mice were treated with γ-irradiation followed by i.p. injection of splenic macrophages (Mf, 1.5 million cells/mouse) on day 0, and i.p. injection of 5 mg of pOVA either in the presence of anti-TGFβ isotype control Ab treatment. Anti-TGFβ or isotype control Ab (200 mg/day/mouse) was administered once a day from day 0 to 2 (invert triangles). All mice received i.p. injection of DO11.10xRag−/− TCR transgenic CD4+CD25 T cells (KJ1-26+) day 2. At day 4, all mice were immunized with pOVA (100 mg/mouse) and (CFA (200 mg/mice) subcutaneously in the food pad. At day 11, all mice were sacrificed.
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents, publications, and CAS numbers mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
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This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/844,564, filed Jul. 10, 2013, International Patent Application Ser. No. PCT/US2014/046065 filed Jul. 10, 204, and U.S. patent application Ser. No. 14/904,054, filed Jan. 8, 2016, the entire contents of these applications are hereby incorporated by reference herein.
Research supporting this application was carried out by the United States of America as represented by the Secretary, Department of Health and Human Services. The Government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 61844564 | Jul 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14904054 | Jan 2016 | US |
| Child | 16773982 | US |
