Cytokines are secreted proteins that regulate cellular growth and differentiation. These factors are especially important in regulating immune and inflammatory responses, and are critical in the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, psoriasis, allergy and asthma. Targeting cytokines and cytokine signaling are successful new strategies in treating these diseases, underscoring the need to better understand the molecular basis of cytokine action as it relates to the pathogenesis of immune-mediated disease. A critical means through which cytokines exert their effect is activation of receptor-associated Janus kinases, or JAKs, and the activation of a family of transcription factors called STATs (signal transducers and activators of transcription);this has been the focus of our work for nearly two decades. One important action of cytokines in which STAT proteins are key is the differentiation of different subsets of T cells to attain distinct fates. An important population of T cells responsible for preventing autoimmune disease is regulatory T cells, or Treg cells. In this FY, we have examined factors that can destabilize Treg cells. We were particularly interested in the role of Treg cells in acute graft-versus-host disease (GvHD), which is a major cause of mortality in allogeneic bone marrow transplantation. We found STAT3 limits FoxP3+ Treg cell numbers following allogeneic bone marrow transplant (BMT) by promoting instability of natural Treg (nTreg) cells and inhibiting formation of induced Treg (iTreg) cells. Previous work has established that microRNAs control T cell stability and plasticity. During this reporting period we found that Treg cells had high expression of the microRNA miR-10a and that miR-10a was induced by retinoic acid and transforming growth factor beta (TGFb). We previously established that IL-2, acting via STAT5, attenuates differentiation of another important T cell subset, Th17 cells. This year, we extend these findings to show that IL-15 also tunes IL-17 production. Using a mouse model of multiple sclerosis, experimental autoimmune encephalitis (EAE), we found that Il15-deficient mice displayed worse autoimmune disease, whereas IL-15 administration and IL-17A neutralization reduced severity. IL-17 is not made by T cells exclusively;in fact, our previous work revealed the existence of innate lymphoid cells (ILCs) that produce IL-17 and IL-22. This year we reported that a subset of ILCs (NKp46+ ILC22 or ILC3s) express T-bet (encoded by Tbx21) and the presence of these cells in the intestinal lamina propria is dependent upon this factor. During this FY we also gained insight into the actions of the immunosuppressive cytokine IL-27. We found that IL-27 priming of naive T cells upregulated expression of programmed death ligand 1 (PD-L1) in STAT1-dependent manner. We further found that IL-27-primed T cells inhibited the differentiation of Th17 cells in trans through PD-1-PD-L1 interactions. We found this limited immune-mediated pathology in EAE. A major focus of the laboratorys efforts over the past several years has been to approach the issue of helper T cell specification using new tools that allow genomic views of differentiating cells. A powerful technique has been chromatin immunoprecipitation and massive parallel sequencing (ChIP-seq). This technique can be used to understand genome-wide actions of transcription factors and also to understand epigenetic changes associated with cellular specification. In this FY, we collaborated with NCI investigators to show that the transcription factors Thpok and LRF are necessary and partly redundant for T helper cell differentiation. We employed Chip-seq to define the actions of these transcription factors. We also used Chip-seq technology to identify specific genes in Th17 cells to better understand how they might contribute to autoimmune disease. We showed that tissue inhibitor of metalloproteinase 1 (TIMP1), a secreted protein with pleiotropic effects on cellular growth, survival and integrity of the extracellular matrix, is preferentially produced by Th17 and Th1 cells. We also showed that Th1 and Th17 cell TIMP1 regulation follows separate mechanisms, with a requirement for STAT4 in the former and STAT3 in the latter. Finally, we demonstrated that, when restricted to T cells, expression of TIMP1 promotes neuropathology in EAE. Thus, production of TIMP1 by Th1 and Th2 cells appears to contribute to immunopathology associated with autoimmune disease. Genetic polymorphisms within in the BACH2 gene are associated with numerous autoimmune and allergic diseases including asthma, Crohn's disease, celiac disease, vitiligo, multiple sclerosis and type 1 diabetes. This year we showed that BACH2 is required for efficient formation of regulatory (Treg) cells and that absence of Bach2 in mice is associated with lethal autoimmunity. Assessment of the genome-wide function of BACH2, using Chip-seq, revealed that it represses genes associated with effector cell differentiation. Consequently, its absence during Treg polarization resulted in inappropriate diversion to effector lineages. In addition, BACH2 constrained full effector differentiation within Th1, Th2 and Th17 cell lineages. These findings identify BACH2 as a key regulator of CD4 helper T-cell differentiation that prevents inflammatory disease by controlling the balance between tolerance and immunity. A major factor governing the stability of cellular phenotype is the epigenetic landscape. Genes comprise just a small fraction of the genome (2%);most of the genome appears to be switches that regulate gene expression. This year we reported our efforts to map a particular type of switch, namely active enhancer elements in T helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the activation of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. Transcriptome analysis further supports a functional role for enhancers regulated by STATs. Importantly, expression of lineage-defining master regulators in STAT-deficient cells fails to fully recover the chromatin signature of STAT-dependent enhancers. Thus, these findings point to a critical role of STATs as environmental sensors in dynamically molding the specialized enhancer architecture of differentiating cells.

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National Institute of Arthritis and Musculoskeletal and Skin Diseases
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Shih, Han-Yu; Sciumè, Giuseppe; Mikami, Yohei et al. (2016) Developmental Acquisition of Regulomes Underlies Innate Lymphoid Cell Functionality. Cell 165:1120-33
Schwartz, Daniella M; Bonelli, Michael; Gadina, Massimo et al. (2016) Type I/II cytokines, JAKs, and new strategies for treating autoimmune diseases. Nat Rev Rheumatol 12:25-36
Gadina, Massimo; Schwartz, Daniella M; O'Shea, John J (2016) Decernotinib: A Next-Generation Jakinib. Arthritis Rheumatol 68:31-4
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Yang, Xiang-Ping; Jiang, Kan; Hirahara, Kiyoshi et al. (2015) EZH2 is crucial for both differentiation of regulatory T cells and T effector cell expansion. Sci Rep 5:10643
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Hirahara, Kiyoshi; Onodera, Atsushi; Villarino, Alejandro V et al. (2015) Asymmetric Action of STAT Transcription Factors Drives Transcriptional Outputs and Cytokine Specificity. Immunity 42:877-89
Witte, Steven; O'Shea, John J; Vahedi, Golnaz (2015) Super-enhancers: Asset management in immune cell genomes. Trends Immunol 36:519-26

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