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  Methods for regulating t cell subsets by modulating transcription factor activityUSPTO Application #: 20080038235Title: Methods for regulating t cell subsets by modulating transcription factor activity Abstract: Methods for modulating production of a T helper type 2 (Th2)-associated cytokine, in particular interleukin-4, by modulating the activity of one or more transcription factors that cooperate with NF-AT family proteins to regulate expression of a Th2-associated cytokine gene are disclosed. In one embodiment, the activity of a maf family protein (e.g., c-Maf or a small maf protein, such as p18) is modulated. In another embodiment, the activity of a protein that interacts with an NF-AT family protein (e.g., NIP45) is modulated. Combination methods, for example wherein the activities of a maf family protein and an NF-AT protein are modulated or the activities of a maf protein and NF-AT-interacting protein are modulated, are also encompassed by the invention. Methods for modulating development of T helper type 1 (Th1) or T helper type 2 (Th2) subsets in a subject using agents that modulate transcription factor activity are also disclosed. (end of abstract)
Agent: Lahive & Cockfield, LLP - Boston, MA, US Inventors: Laurie H. Glimcher, I-Cheng Ho USPTO Applicaton #: 20080038235 - Class: 424093210 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Whole Live Micro-organism, Cell, Or Virus Containing, Genetically Modified Micro-organism, Cell, Or Virus (e.g., Transformed, Fused, Hybrid, Etc.), Eukaryotic Cell The Patent Description & Claims data below is from USPTO Patent Application 20080038235. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This application is a continuation application of U.S. Ser. No. 10/236,629, entitled "Methods and Compositions for Regulating T cell Subsets by Modulating Transcription Factor Activity", filed Sep. 5, 2002, which is a continuation application of U.S. Ser. No. 08/755,592, entitled "Methods and Compositions for Regulating T cell Subsets by Modulating Transcription Factor Activity", filed Nov. 25, 1996, which is a continuation-in-part of U.S. Ser. No. 08/636,602, entitled "Methods and Compositions for Regulating T cell Subsets by Modulating Transcription Factor Activity", filed Apr. 23, 1996, the entire contents of which are expressly incorporated herein by reference. This application is also related to U.S. Ser. No. 08/755,584, entitled "NF-AT Interacting Protein NIP45 and Methods of Use Therefor", filed Nov. 25, 1996 (Attorney Docket No. HUI-026), the entire contents of which are expressly incorporated herein by reference. BACKGROUND OF THE INVENTION [0003] CD4+ T helper cells are not a homogeneous population but can be divided on the basis of cytokine secretion into at least two subsets termed T helper type 1 (Th1) and T helper type 2 (Th2) (see e.g., Mosmann, T. R. et al. (1986) J. Immunol. 136:2348-2357; Paul, W. E. and Seder, R. A. (1994) Cell 76:241-251; Seder, R. A. and Paul, W. E. (1994) Ann. Rev. Immunol. 12:635-673). Th1 cells secrete interleukin-2 (L-2) and interferon-.gamma. (IFN-.gamma.) while Th2 cells produce interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10) and interleukin-13 (IL-13). Both subsets produce cytokines such as tumor necrosis factor (TNF) and granulocyte/macrophage-colony stimulating factor (GM-CSF). In addition to their different pattern of cytokine expression, Th1 and Th2 cells are thought to have differing functional activities. For example, Th1 cells are involved in inducing delayed type hypersensitivity responses, whereas Th2 cells are involved in providing efficient "help" to B lymphocytes and stimulating production of IgG1 and IgE antibodies. [0004] There is now abundant evidence that the ratio of Th1 to Th2 cells is highly relevant to the outcome of a wide array of immunologically-mediated clinical diseases including autoimmune, allergic and infectious diseases. For example, in experimental leishmania infections in mice, animals that are resistant to infection mount predominantly a Th1 response, whereas animals that are susceptible to progressive infection mount predominantly a Th2 response (Heinzel, F. P., et al. (1989) J. Exp. Med. 169:59-72; Locksley, R. M. and Scott, P. (1992) Immunoparasitology Today 1:A58-A61). In murine schistosomiasis, a Th1 to Th2 switch is observed coincident with the release of eggs into the tissues by female parasites and is associated with a worsening of the disease condition (Pearce, E. J., et al. (1991)J. Exp. Med. 173:159-166; Grzych, J-M., et al. (1991)J. Immunol. 141:1322-1327; Kullberg, M. C., et al. (1992) J. Immunol. 148:3264-3270). Many human diseases, including chronic infections (such as with human immunodeficiency virus (HIV) and tuberculosis) and certain metastatic carcinomas, also are characterized by a Th1 to Th2 switch (see e.g., Shearer, G. M. and Clerici, M. (1992) Prog. Chem. Immunol. 54:21-43; Clerici, M and Shearer, G. M. (1993) Immunology Today 14:107-111; Yamamura, M., et al. (1993) J. Clin. Invest. 91:1005-1010; Pisa, P., et al. (1992) Proc. Natl. Acad. Sci. USA 89:7708-7712; Fauci, A. S. (1988) Science 239:617-623). Furthermore, certain autoimmune diseases have been shown to be associated with a predominant Th1 response. For example, patients with rheumatoid arthritis have predominantly Th1 cells in synovial tissue (Simon, A. K., et al. (1994) Proc. Natl. Acad. Sci. USA 91:8562-8566) and experimental autoimmune encephalomyelitis (EAE) can be induced by autoreactive Th1 cells (Kuchroo, V. K., et al. (1993) J. Immunol. 151:4371-4381). [0005] The ability to alter or manipulate ratios of Th1 and Th2 subsets requires an understanding of the mechanisms by which the differentiation of CD4 T helper precursor cells (Thp), which secrete only IL-2, choose to become Th1 or Th2 effector cells. It is clear that the cytokines themselves are potent Th cell inducers and form an autoregulatory loop (see e.g., Paul, W. E. and Seder, R. A. (1994) Cell 76:241-251; Seder, R. A. and Paul, W. E. (1994) Ann. Rev. Immunol. 12:635-673). Thus, IL-4 promotes the differentiation of Th2 cells while preventing the differentiation of precursors into Th1 cells, while IL-12 and IFN-.gamma. have the opposite effect. One possible means therefore to alter Th1:Th2 ratios is to increase or decrease the level of selected cytokines. Direct administration of cytokines or antibodies to cytokines has been shown to have an effect on certain diseases mediated by either Th1 or Th2 cells. For example, administration of recombinant IL-4 or antibodies to IL-12 ameliorate EAE, a Th1-driven autoimmune disease (see Racke; M. K. et al. (1994) J. Exp. Med. 180:1961-1966; and Leonard, J. P. et al. (1995) J. Exp. Med. 181:381-386), while anti-IL-4 antibodies cure the Th2-mediated parasitic disease, Leishmania major (Sadick, M. D. et al. (1990) J. Exp. Med. 171:115-127). However, as therapeutic options, systemic administration of cytokines or antibodies may have unwanted side effects and, accordingly, alternative approaches to manipulating Th1/Th2 subsets are still needed. [0006] The molecular basis for the tissue-specific expression of IL-4 in Th2 cells, or any T cell cytokine, has remained elusive. One possibility is the presence of repressor proteins that selectively silence cytokines. Transcriptional silencing has been well documented for bacteria, yeast and mammalian genes. Examples include E. coli thermoregulation genes (Goransson, M. et al. (1990) Nature 344:682-685), yeast .alpha.2 mating type genes (Keleher, C. A. et al. (1988) Cell 53:927-936) and mammalian MHC class I and TcR.alpha. genes (Weisman, J. D. and Singer, D. S. (1991) Mol. Cell. Biol. 11:4228-4234; Winoto, A. and Baltimore, D. (1989) Cell 59:649-655). Indeed, early experiments involving injection of IL-2 genomic DNA into Xenopus oocytes suggested the existence of a repressor protein for IL-2 in resting versus activated T cell extracts (Mouzaki, A. et al. (1991) EMBO J. 10: 1399-1406). These studies suggested that the absence of IL-2 production in resting T cells was due to proteins that silenced the transcription of IL-2 by interacting with negative elements in the IL-2 promoter. [0007] A second possibility is the existence of Th selective transactivators. A family of four related transcription factors called Nuclear Factor of Activated T cells (NF-AT), plays a key role in the regulation of cytokine gene expression (see e.g., Emmel, E. A. et al. (1989) Science 246:1617-1620; Flanagan, W. M. et al. (1991) Nature 352:803-807; Jain, J. et al. (1993) Nature 365:352-355; McCaffrey, P. G. et al. (1993) Science 262:750-754; Rao, A. (1994) Immunol. Today 15:274-281; Northrop, J. P. et al. (1994) Nature 369:497). However, NF-AT family members can bind to and transactivate the promoters of multiple cytokine genes including IL-2 and IL-4 (Rooney, J. et al. (1995) Immunity 2:545-553; Szabo, S. J. et al. (1993) Mol. Cell. Biol. 13:4793-4805; Flanagan, W. M. et al. (1991) Nature 352:803-807; Northrop, J. P. et al. (1994) Nature 369:497). Thus, they are not likely to be responsible for directing Th1- or Th2-specific cytokine transcription. Most, if not all, NF-AT binding sites in cytokine promoter regulatory regions are accompanied by nearby sites that bind auxiliary transcription factors, usually members of the AP-1 family. It has been shown that NF-AT and AP-1 proteins bind coordinately and cooperatively and are required for full activity of the IL-2 and IL-4 promoters. Different AP-1 proteins, specifically c-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D, have been shown to bind to these sites (Rao, A. et al. (1994) Immunol. Today 15:274-281; Jain, J. et al. (1993) Nature 365:352-355; Boise, L. H. et al. (1993) Mol. Cell. Biol. 13:1911-1919; Rooney, J. et al. (1995) Immunity 2:545-553; Rooney, J. et al. (1995) Mol. Cell. Biol. 15:6299-6310). However, none of these AP-1 proteins is expressed in a Th1- or Th2-specific manner and there is no evidence for the differential recruitment of AP-1 family members to the IL-2 or IL-4 composite sites (Rooney, J. et al. (1995) Mol. Cell. Biol. 15:6299-6310). Thus, neither NF-AT proteins nor the AP-1 family members c-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D can account for the tissue-specific transcription of IL-4 in Th2 cells. SUMMARY OF THE INVENTION [0008] This invention pertains to methods for regulating production of Th2-associated cytokines and for regulating Th1 or Th2 subsets by modulating the activity of one or more transcription factors that regulate expression of Th2-specific cytokine genes. As described further herein, it has now been discovered that the tissue-specific expression of IL-4 in Th2 cells is not due to a repressor protein but rather to a Th2-specific transactivator protein. The proto-oncogene c-Maf has now been demonstrated to be responsible for the tissue-specific expression of the Th2-associated cytokine interleukin-4. Moreover, ectopic expression of c-Maf in cells other than Th2 cells (e.g., Th1 cells, B cells and non-lymphoid cells) leads to activation of the IL-4 promoter and, under appropriate conditions, production of endogenous IL-4. It further has been discovered that c-Maf and NF-AT synergize to activate Th2-associated cytokine gene expression. It still further has been discovered that a third protein that interacts with NF-AT, termed NIP45 (for NF-AT Interacting Protein 45), potentiates gene expression mediated by c-Maf and NF-AT such that when all three factors (c-Maf, NF-AT and NIP45) are active in a cell, high levels of endogenous IL-4 production is stimulated. It still further has been discovered that a small maf protein lacking a transactivation domain, such as p18, can repress Th2-associated cytokine gene expression, e.g., expression mediated by c-Maf. [0009] Accordingly, this invention pertains to methods for modulating Th2-associated cytokines expression by modulating the expression or activity of one or more transcription factors that cooperate with an NF-AT family protein to regulate the expression of Th2-associated cytokine genes. In one embodiment, the transcription factor that cooperates with an NF-AT family protein to regulate the expression of a Th2-associated cytokine gene, and thus whose expression or activity is modulated, is a Th2-specific transcription factor (e.g., a Th2-specific maf family protein). In one embodiment, the transcription factor that cooperates with an NF-AT family protein to regulate the expression of a Th2-associated cytokine gene, and thus whose expression or activity is modulated, is a maf family protein, such as c-Maf. In yet another embodiment, the transcription factor that cooperates with an NF-AT family protein to regulate the expression of a Th2-associated cytokine gene, and thus whose expression or activity is modulated, is a protein that interacts with an NF-AT family protein, such as NIP45. In yet another embodiment, the expression or activity of a small maf protein, such as p18, is modulated. The methods of the invention may involve modulating the expression or activity of one transcription factor (e.g., c-Maf or NIP45 or p18) or a combination of transcription factors (e.g., c-Maf+NF-AT, or NF-AT+NIP45, or c-Maf+NF-AT+NIP45). [0010] The modulatory methods of the invention generally involve contacting a cell with an agent that modulates the expression or activity of a transcription factor(s) such that production of the Th2-associated cytokine by a cell is modulated. In particular, preferred agents of the invention act intracellularly to modulate the activity of the transcription factor. In one embodiment, the modulatory method of the invention stimulates production of a Th2-associated cytokine. For example, Th2-associated cytokine production can be stimulated in Th1 cells, B cells or non-lymphoid cells. In another embodiment, the modulatory method of the invention inhibits production of a Th2-associated cytokine. A Th2-associated cytokine modulated in the method preferably is interleukin-4. [0011] A variety of agents can be used to stimulate the expression or activity of a transcription factor that regulates expression of a Th2-associated cytokine gene. For example, a stimulatory agent of the invention can be a nucleic acid molecule encoding the transcription factor that is introduced into and expressed in the cell. Alternatively, chemical agents that enhance the expression or activity of the transcription facto can be used as stimulatory agents. [0012] A variety of agents can be used to inhibit the expression or activity of a transcription factor that regulates expression of a Th2-associated cytokine gene. Examples of suitable inhibitory agents include antisense nucleic acid molecules that are complementary to a gene encoding the transcription factor, intracellular antibodies that bind the transcription factor (e.g., in the cell nucleus), inhibitory forms of the transcription factor (e.g., dominant-negative forms) and chemical agents that inhibit the expression or activity of the transcription factor. [0013] Combination methods, involving modulation of the expression or activity of two, three or more transcription factors that regulate Th2-associated cytokine gene expression, are also encompassed by the invention. Accordingly, in other embodiments of the invention, a cell is contacted with at least one additional agent that modulates the activity of at least one additional transcription factor that contributes to the regulation of the Th2-associated cytokine gene. Preferably, the at least one additional transcription factor whose expression or activity is modulated is selected from the group consisting of NF-AT family proteins, NF-AT-interacting proteins, maf family proteins and AP-1 family proteins. [0014] Cytokine production by a cell can be modulated in vitro or in vivo in accordance with the methods of the invention. In one embodiment, a cell is contacted with a modulating agent(s) in vitro and then the cell is administered to a subject to thereby regulate the development of Th1 and/or Th2 subsets in the subject. Accordingly, in another aspect, the invention provides methods for regulating the development of Th1 or Th2 subsets in a subject. In addition to the embodiment wherein ex vivo modified cells are administered to the subject, in another embodiment, these methods involve direct administration to the subject of an agent that modulates the activity of one or more transcription factors that regulate expression of a Th2-associated cytokine gene such that development of Th1 or Th2 cells in the subject is modulated. [0015] The modulatory methods of the invention can be used to manipulate Th1:Th2 ratios in a variety of clinical situations. For example, inhibition of Th2 formation may be useful in allergic diseases, malignancies and infectious diseases whereas enhancement of Th2 formation may be useful in autoimmune diseases and organ transplantation. BRIEF DESCRIPTION OF THE DRAWINGS [0016] FIG. 1A is a schematic of the cell fusion approach used to demonstrate that cytokine expression is not due to a repressor [0017] FIG. 1B is a reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of 1'-2 and IL-4 cytokine, and control .beta.-actin, mRNA expressed by an unfused Th1 clone (D1.1), an unfused Th2 clone (D10), Th1 and Th2 homokaryons and Th1-Th2 heterokaryons. [0018] FIG. 2A is a Northern blot analysis depicting expression of an isolated cDNA clone in Th1 cells, Th2 cells or B lymphoma cells. A control probe specific for GAPDH was used to show equal loading of RNA. [0019] FIG. 2B is a Northern blot analysis depicting upregulated expression of the isolated cDNA clone during in vitro differentiation of normal naive spleen cells into Th2 cells. Total RNA was isolated from cells harvested at the indicated time points. Culture supernatant at the appropriate dilution was measured for cytokine (IL-10) production by ELISA to determine differentiation into the Th1 or Th2 lineage. [0020] FIG. 3A is a bar graph depicting transactivation of the IL-4 promoter by c-Maf in a Th1 clone (AE7). AE7 cells were cotransfected with a wild-type IL-4 CAT reporter construct and either a control plasmid (pMEX-NeoI), a c-Maf expression plasmid (pMEX-Maf) or a c-Fos expression plasmid (pMEX-c-Fos). Half of each sample was stimulated 24 hours after transfection with antibodies to CD3. All samples were harvested 48 hours after transfection and relative CAT activities were determined. Continue reading... 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