Cytokines are secreted proteins that regulate cellular growth and differentiation. These factors are especially important in regulating immune and inflammatory responses, regulating lymphoid development and differentiation. Cytokines also have critical functions in regulating immune homeostasis, tolerance, and memory. Not surprisingly, cytokines are critical in the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease and psoriasis. Moreover, 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. An important action of cytokines is to drive differentiation of different subsets of T cells to attain distinct fates. This is important for efficient control of various infections, but in addition, T cells are important culprits in autoimmunity. T cells that produce IL-17, a major inflammatory cytokine, appears to contribute to the pathogenesis of many autoimmune and autoinflammatory disorders including rheumatoid arthritis, spondyloarthropathies, multiple sclerosis and inflammatory bowel diseases (IBD). Work from both animal models and genetic linkage in human disease, areas in which we have contributed in the past, point to important roles of IL-23 in driving IL-17 production and autoimmunity. A critical step through which cytokines like IL-23 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). Our work for nearly two decades has established the criticality of Jaks and Stats and the molecular basis of cytokine action. In recent years we have focused considerable effort on understanding the factors that are important for the production of IL-17 and the generation of T cells that selectively produce IL-17 (so-called Th17 cells). Specifically, we have identified a number of key roles of STAT3 in Th17 differentiation. During this FY we gained further insight into their regulation uncovering a new mechanism through the immunosuppressive cytokine IL-27 controls Th17 differentiation. 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. This limited immune-mediated pathology in a model of experimental autoimmune encephalomyelitis. Another key lymphoid cell, critical for both host defense and autoimmunity, is the follicular helper T cell. These cells are key for tuning B cell responses in germinal centers. We successfully generated a system in which we were able to generate Tfh-like cells in vitro. Upon adoptive transfer to mice that were unable to form germinal centers, these cells were capable of rescuing responses. In a separate study, we further explored the relationship between Tfh cells and other helper cell subsets. This was important as the relationship Tfh cells and other helper lineages has been incompletely understood. We found that interleukin-12 acting via STAT4 induced both the signature cytokine of Tfh cells, Il21. In addition, IL-12 also induced expression of the Tfh master regulator Bcl6. However, we found that IL-12, acting via STAT4 also induced the transcription factor T-bet, a master regulator for T cells. We found that this factor repressed features of Tfh cells. Tfh-like cells were rapidly generated after Toxoplasma gondii infection in mice, but T-bet constrained Tfh cell expansion and consequent germinal center formation and antibody production. 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 transcripton factors and also to understand epigenetic changes associated with cellular specification. We therefore took advantage of this technology to understand how T-bet was acting to curtail Tfh differentiation. To this end, we defined the genome-wide targets of T-bet and found that it directly repressed Bcl6 and other genes expressed by Tfh cells, thereby attenuating the nascent Tfh cell-like phenotype in the late phase of Th1 cell specification. This led us to conclude that Tfh and Th1 cells share a transitional stage through the signal mediated by STAT4, which promotes both phenotypes. However, T-bet represses Tfh cell functionalities, promoting full Th1 cell differentiation. The issue of whether different helper T cell subsets behave as differentiated lineages versus flexible states is important with respect to immune responses and autoimmune diathesis. Based on the above findings, we became very interested in understanding more about the inter-convertibility of helper cells. Importantly, we were able to show that both Tfh-like and in vivo-derived Tfh cells were able to produce effector cytokines in response to appropriate polarizing conditions. Conversely, we found that Th1, Th2, and Th17 cells could be reprogrammed to obtain Tfh cell characteristics. A third critical population of T cells are regulatory T cells or Treg cells. These cells are essential for preserving immune homeostasis and maintaining tolerance. 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 (BMT). The administration of FoxP3(+) regulatory T (Treg) cells has been proposed as a therapy. However, the phenotypic stability of Treg cells is an important but unresolved issue. We found STAT3 limits FoxP3(+) Treg cell numbers following allogeneic BMT by two pathways. First, STAT3 activation promotes instability of natural Treg (nTreg) cells. Second, STAT3 inhibits formation of induced Treg (iTreg) cells. A major factor governing the stability of cellular phenotype is the epigenetic landscape. Recently, there has been an explosion in our understanding of the epigenome, and we now know that junk DNA has critical regulatory functions. While we are just at the beginning of understanding the rules, we have employed what has been learned about the histone code to gain insight into helper T cell stability vs. flexibility. Through epigenetic analyses We identified epigenetic markings indicative of accessibility of the genes encoding T-bet, Gata3, and Rorc in Tfh-like and ex vivo Tfh cells and the Bcl6 locus in non-Tfh cells. This then provided a mechanism for plasticity between Tfh and other Th cell populations. Another means through which cellular responses are fine-tuned is the production of micro-RNAs. Previous work has established that microRNAs control T cell stability and plasticity. We therefore set out to find microRNAs that might influence helper cell stability. We found that regulatory T cells (T(reg) cells) had high expression of the microRNA miR-10a and that miR-10a was induced by retinoic acid and transforming growth factor-β(TGF-β) in inducible T(reg) cells. By simultaneously targeting the transcriptional repressor Bcl-6 and the corepressor Ncor2, we found that miR-10a attenuated the phenotypic conversion of inducible T(reg) cells into follicular helper T cells. We also found that miR-10a limited differentiation into the T(H)17 subset of helper T cells.
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