Cytokines regulate cellular growth and differentiation, along with immune and inflammatory responses. They are critical in the pathogenesis of autoimmune diseases such as rheumatoid arthritis, SLE, IBD, psoriasis, allergy and asthma. Targeting cytokines and cytokine signaling has led to successful new strategies for 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 by 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; this has been the focus of our work for the last two decades. One important action of cytokines in which STATs play a key role is the differentiation of different subsets of lymphocytes to attain distinct fates, and this too has been a longstanding interest of the lab. The transcription factor STAT5 is fundamental to the mammalian immune system. However, the relationship between its two paralogs, STAT5A and STAT5B, and the extent to which they are functionally distinct in various tissues, has not been fully ascertained. We used mouse models of paralog deficiency to demonstrate that they are not equivalent for CD4+ helper T cells. We found that STAT5B is dominant for both effector and regulatory responses and, therefore, uniquely necessary for immunological tolerance. Comparative analysis of genomic distribution and transcriptomic output confirms that STAT5B has far greater impact but, surprisingly, the data point towards asymmetric expression (i.e. paralog dose), rather than distinct functional properties, as the key distinguishing feature. Thus, relative abundance of STAT5 imposes functional specificity (or dominance) in the face of widespread structural homology. In related collaborative work, we found that the Stat5a/b locus is subject to additional lineage-specific transcriptional control. CRISPR/Cas9-mediated genome editing was used to delete these sites in mice and determine their biological function. Mutant animals exhibited an 80% reduction of Stat5 levels and a concomitant reduction of STAT5-dependent gene expression. We have previously shown that STATs, especially STAT1 and STAT4, are important drivers of expression of a lineage defining transcription factor (LDTF) in T helper 1 (Th1) cells termed T-bet encoded by Tbx21). IFN-g, a member of a large family of anti-pathogenic and anti-tumor IFNs, acting through STAT1 induces T-bet, which in turn supports IFN-g production in a feed-forward manner. In a study published this year, we showed that a cell-intrinsic role of T-bet determines how T cells perceive their secreted product in the environment. In the absence of T-bet, IFN-g aberrantly induced a type I IFN transcriptomic program in T cells. Specifically, T-bet preferentially repressed genes and pathways ordinarily activated by type I IFNs to ensure that its transcriptional response did not evoke an aberrant amplification of type I IFN signaling circuitry, otherwise triggered by its own product. Thus, this work identified an unanticipated function of a well-known LDTF. In addition to promoting Th1 effector commitment and production of the signature cytokine IFN-g, T-bet acts as a repressor in differentiated Th1 cells to prevent aberrant autocrine type I IFN and downstream signaling. Blimp-1 (encoded by Prdm1) is an LDTF expressed in differentiated effector T cells that extinguishes the fate of T follicular helper cells and limits autoimmunity. We found unexpectedly that STAT3 and not STAT6 plays a critical role in regulating Blimp-1 in TH2 cells. Furthermore, we found that the cytokine interleukin-10 (IL-10) acted directly on TH2 cells and was necessary and sufficient to induce optimal Blimp-1 expression through STAT3. Moreover, we found that Blimp-1 and STAT3 amplified IL-10 production in TH2 cells, creating a strong autoregulatory loop that enhanced Blimp-1 expression. Increased Blimp-1 in T cells antagonized STAT5-regulated cell cycle and antiapoptotic genes to limit cell expansion. By contrast, we found a very distinct mode of regulation of Blimp-1 in Th17 cells compared to Th2 cells. Interleukin-23 (IL-23) is a pro-inflammatory cytokine required for the pathogenicity of Th17 cells. We found that IL-23 induces Blimp-1 and peripheral deletion of this transcription factor resulted in reduced Th17 activation and reduced severity of autoimmune encephalomyelitis. Furthermore, genome-wide occupancy and overexpression studies in Th17 cells revealed that Blimp-1 co-localized with transcription factors RORt, STAT-3, and p300 at the Il23r, Il17a/f, and Csf2 cytokine loci to enhance their expression. Blimp-1 also directly bound to and repressed cytokine loci Il2 and Bcl6. An important transcription factor that antagonizes effector differentiation of T cells is Bach2. In collaboration, we extended our previous work on Bach2 in CD4 T cells to study its role on the differentiation state of CD8 T cells. We found that the BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 sites to Jun family signal-dependent transcription factors. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation, but in effector cells reduced expression enabling unrestrained induction of TCR-driven programs. Polymorphisms of the BACH2 locus are associated with multiple autoimmune diseases including rheumatoid arthritis, lupus, diabetes, IBD, asthma, multiple sclerosis and other diseases. In related work, we found that BACH2 mutations underlie a previously unrecognized Mendelian monogenic primary immunodeficiency manifested by immunodeficiency and autoimmunity. Innate lymphoid cells (ILCs) represent a subset of cells that play key roles in host defense, barrier integrity, and homeostasis and mirror adaptive CD4 T helper (Th) cell subtypes in both usage of effector molecules and transcription factors. To better understand the relationship between ILC subsets and their Th cell counterparts, we measured genome-wide chromatin accessibility. We found that chromatin in proximity to effector genes is selectively accessible in ILCs prior to high-level transcription upon activation. Accessibility of these regions is acquired in a stepwise manner during development and changes little after in vitro or in vivo activation. Conversely, dramatic chromatin remodeling occurs in naive CD4 T cells during Th cell differentiation during parasite infection. This alteration results in a substantial convergence of Th2 cells toward ILC2 regulomes. Our data indicate extensive sharing of regulatory circuitry across the innate and adaptive compartments of the immune system, in spite of their divergent developing pathways. During this year, we have investigated the role of STAT5 in ILCs and the relationship between ILC poised epigenomes and acute activation in terms of rapid regulation of gene expression. In related epigenomic work, we investigated the role of histone variants in regulation of transcription. We found that mH2A1.2 is required for gene expression and cellular differentiation. Moreover, H3K27 acetylation was found to be dependent upon mH2A1.2, indicating a role for this factor in enhancer activation.
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