Distal colonocytes targeted by C. rodentium recruit T-cell help for barrier defence.

Interleukin 22 (IL-22) has a non-redundant role in immune defence of the intestinal barrier1-3. T cells, but not innate lymphoid cells, have an indispensable role in sustaining the IL-22 signalling that is required for the protection of colonic crypts against invasion during infection by the enteropathogen Citrobacter rodentium4 (Cr). However, the intestinal epithelial cell (IEC) subsets targeted by T cell-derived IL-22, and how T cell-derived IL-22 sustains activation in IECs, remain undefined. Here we identify a subset of absorptive IECs in the mid-distal colon that are specifically targeted by Cr and are differentially responsive to IL-22 signalling. Major histocompatibility complex class II (MHCII) expression by these colonocytes was required to elicit sustained IL-22 signalling from Cr-specific T cells, which was required to restrain Cr invasion. Our findings explain the basis for the regionalization of the host response to Cr and demonstrate that epithelial cells must elicit MHCII-dependent help from IL-22-producing T cells to orchestrate immune protection in the intestine.

IL-2-induced Stat3 Signaling is Critical for Effector Treg Cell Programming.

Maintenance of immune homeostasis to the intestinal mictrobiota is dependent on a population of effector regulatory T (eTreg) cells that develop from microbiota-reactive induced (i)Treg cells. A cardinal feature of eTreg cells is their production of IL-10, which plays a non-redundant role in immune tolerance of commensal microbes. Here, we identify an unexpected role for IL-2-induced Stat3 signaling to program iTreg cells for eTreg cell differentiation and Il10 transcriptional competency. IL-2 proved to be both necessary and sufficient for eTreg cell development - contingent on Stat3 output of the IL-2 receptor coordinate with IL-2 signaling during early Treg cell commitment. Induction of iTreg cell programming in absence of IL-2-induced Stat3 signaling resulted in impaired eTreg cell differentiation and a failure to produce IL-10. An IL-2 mutein with reduced affinity for the IL-2Rγ (γ c ) chain was found to have blunted IL-2R Stat3 output, resulting in a deficiency of Il10 transcriptional programming that could not be fully rescued by Stat3 signaling subsequent to an initial window of iTreg cell differentiation. These findings expose a heretofore unappreciated role of IL-2 signaling that acts early to program subsequent production of IL-10 by developing eTreg cells, with broad implications for IL-2-based therapeutic interventions in immune-mediated diseases.

Batf stabilizes Th17 cell development via impaired Stat5 recruitment of Ets1-Runx1 complexes.

Although the activator protein-1 (AP-1) factor Batf is required for Th17 cell development, its mechanisms of action to underpin the Th17 program are incompletely understood. Here, we find that Batf ensures Th17 cell identity in part by restricting alternative gene programs through its actions to restrain IL-2 expression and IL-2-induced Stat5 activation. This, in turn, limits Stat5-dependent recruitment of Ets1-Runx1 factors to Th1- and Treg-cell-specific gene loci. Thus, in addition to pioneering regulatory elements in Th17-specific loci, Batf acts indirectly to inhibit the assembly of a Stat5-Ets1-Runx1 complex that enhances the transcription of Th1- and Treg-cell-specific genes. These findings unveil an important role for Stat5-Ets1-Runx1 interactions in transcriptional networks that define alternate T cell fates and indicate that Batf plays an indispensable role in both inducing and maintaining the Th17 program through its actions to regulate the competing actions of Stat5-assembled enhanceosomes that promote Th1- and Treg-cell developmental programs.

A nonredundant role for T cell-derived interleukin 22 in antibacterial defense of colonic crypts.

Interleukin (IL)-22 is central to immune defense at barrier sites. We examined the contributions of innate lymphoid cell (ILC) and T cell-derived IL-22 during Citrobacter rodentium (C.r) infection using mice that both report Il22 expression and allow lineage-specific deletion. ILC-derived IL-22 activated STAT3 in C.r-colonized surface intestinal epithelial cells (IECs) but only temporally restrained bacterial growth. T cell-derived IL-22 induced a more robust and extensive activation of STAT3 in IECs, including IECs lining colonic crypts, and T cell-specific deficiency of IL-22 led to pathogen invasion of the crypts and increased mortality. This reflected a requirement for T cell-derived IL-22 for the expression of a host-protective transcriptomic program that included AMPs, neutrophil-recruiting chemokines, and mucin-related molecules, and it restricted IFNγ-induced proinflammatory genes. Our findings demonstrate spatiotemporal differences in the production and action of IL-22 by ILCs and T cells during infection and reveal an indispensable role for IL-22-producing T cells in the protection of the intestinal crypts.

TH17 cells require ongoing classic IL-6 receptor signaling to retain transcriptional and functional identity.

Acting in concert with TGF-ß, interleukin-6 (IL-6) signaling induces T helper 17 (TH17) cell development by programming TH17-related genes via signal transducers and activators of transcription 3 (STAT3). A role for IL-6 signaling beyond the inductive phase of TH17 cell development has not been defined because IL-23 signaling downstream of TH17 cell induction also activates STAT3 and is thought responsible for TH17 cell maintenance. Here, we find that IL-6 signaling is required for both induction and maintenance of mouse TH17 cells; IL-6Rα-deficient TH17 cells rapidly lost their TH17 phenotype and did not cause disease in two models of colitis. Cotransfer of wild-type TH17 cells with IL-6Rα-deficient TH17 cells induced colitis but was unable to rescue phenotype loss of the latter. High IL-6 expression in the colon promoted classic, or cis, rather than transreceptor signaling that was required for maintenance of TH17 cells. Thus, ongoing classic IL-6 signaling underpins the TH17 program and is required for TH17 cell maintenance and function.

Insulin-Like Growth Factors Are Key Regulators of T Helper 17 Regulatory T Cell Balance in Autoimmunity.

Appropriate balance of T helper 17 (Th17) and regulatory T (Treg) cells maintains immune tolerance and host defense. Disruption of Th17-Treg cell balance is implicated in a number of immune-mediated diseases, many of which display dysregulation of the insulin-like growth factor (IGF) system. Here, we show that, among effector T cell subsets, Th17 and Treg cells selectively expressed multiple components of the IGF system. Signaling through IGF receptor (IGF1R) activated the protein kinase B-mammalian target of rapamycin (AKT-mTOR) pathway, increased aerobic glycolysis, favored Th17 cell differentiation over that of Treg cells, and promoted a heightened pro-inflammatory gene expression signature. Group 3 innate lymphoid cells (ILC3s), but not ILC1s or ILC2s, were similarly responsive to IGF signaling. Mice with deficiency of IGF1R targeted to T cells failed to fully develop disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Thus, the IGF system represents a previously unappreciated pathway by which type 3 immunity is modulated and immune-mediated pathogenesis controlled.

Batf Pioneers the Reorganization of Chromatin in Developing Effector T Cells via Ets1-Dependent Recruitment of Ctcf.

The basic leucine zipper transcription factor activating transcription factor-like (Batf) contributes to transcriptional programming of multiple effector T cells and is required for T helper 17 (Th17) and T follicular helper (Tfh) cell development. Here, we examine mechanisms by which Batf initiates gene transcription in developing effector CD4 T cells. We find that, in addition to its pioneering function, Batf controls developmentally regulated recruitment of the architectural factor Ctcf to promote chromatin looping that is associated with lineage-specific gene transcription. The chromatin-organizing actions of Batf are largely dependent on Ets1, which appears to be indispensable for the Batf-dependent recruitment of Ctcf. Moreover, most of the Batf-dependent sites to which Ctcf is recruited lie outside of activating protein-1-interferon regulatory factor (Ap-1-Irf) composite elements (AICEs), indicating that direct involvement of Batf-Irf complexes is not required. These results identify a cooperative role for Batf, Ets1, and Ctcf in chromatin reorganization that underpins the transcriptional programming of effector T cells.

Differential IL-2 expression defines developmental fates of follicular versus nonfollicular helper T cells.

In response to infection, naïve CD4+ T cells differentiate into two subpopulations: T follicular helper (TFH) cells, which support B cell antibody production, and non-TFH cells, which enhance innate immune cell functions. Interleukin-2 (IL-2), the major cytokine produced by naïve T cells, plays an important role in the developmental divergence of these populations. However, the relationship between IL-2 production and fate determination remains unclear. Using reporter mice, we found that differential production of IL-2 by naïve CD4+ T cells defined precursors fated for different immune functions. IL-2 producers, which were fated to become TFH cells, delivered IL-2 to nonproducers destined to become non-TFH cells. Because IL-2 production was limited to cells receiving the strongest T cell receptor (TCR) signals, a direct link between TCR-signal strength, IL-2 production, and T cell fate determination has been established.

IL-1R signaling promotes STAT3 and NF-κB factor recruitment to distal cis-regulatory elements that regulate Il17a/f transcription.

Interleukin (IL)-1ß plays a critical role in IL-6ß- and transforming growth factor ß (TGFß)-initiated Th17 differentiation and induction of Th17-mediated autoimmunity. However, the means by which IL-1 regulates various aspects of Th17 development remain poorly understood. We recently reported that IL-1ß enhances STAT3 phosphorylation via NF-κB-mediated repression of SOCS3 to facilitate Il17 transcription and Th17 differentiation, identifying an effect of IL-1 signaling on proximal events of STAT3 signaling. Here, we show that IL-1ß promotes STAT3 binding to key cis-elements that control IL-17 expression. Additionally, we demonstrate that the IL-1-induced NF-κB factor RelA directly regulates the Il17a/f loci in cooperation with STAT3. Our findings reveal that IL-1 impacts both proximal signaling events and downstream interactions between transcription factors and cis-regulatory elements to promote Il17a/f transcription and Th17 differentiation.

IL-1 signaling modulates activation of STAT transcription factors to antagonize retinoic acid signaling and control the TH17 cell-iTreg cell balance.

Interleukin 17 (IL-17)-producing helper T cells (TH17 cells) and CD4(+) inducible regulatory T cells (iTreg cells) emerge from an overlapping developmental program. In the intestines, the vitamin A metabolite retinoic acid (RA) is produced at steady state and acts as an important cofactor to induce iTreg cell development while potently inhibiting TH17 cell development. Here we found that IL-1 was needed to fully override RA-mediated expression of the transcription factor Foxp3 and induce protective TH17 cell responses. By repressing expression of the negative regulator SOCS3 dependent on the transcription factor NF-κB, IL-1 increased the amplitude and duration of phosphorylation of the transcription factor STAT3 induced by TH17-polarizing cytokines, which led to an altered balance in the binding of STAT3 and STAT5 to shared consensus sequences in developing T cells. Thus, IL-1 signaling modulated STAT activation downstream of cytokine receptors differently to control the TH17 cell-iTreg cell developmental fate.

Deletion of a conserved cis-element in the Ifng locus highlights the role of acute histone acetylation in modulating inducible gene transcription.

Differentiation-dependent regulation of the Ifng cytokine gene locus in T helper (Th) cells has emerged as an excellent model for functional study of distal elements that control lineage-specific gene expression. We previously identified a cis-regulatory element located 22 kb upstream of the Ifng gene (Conserved Non-coding Sequence -22, or CNS-22) that is a site for recruitment of the transcription factors T-bet, Runx3, NF-κB and STAT4, which act to regulate transcription of the Ifng gene in Th1 cells. Here, we report the generation of mice with a conditional deletion of CNS-22 that has enabled us to define the epigenetic and functional consequences of its absence. Deletion of CNS-22 led to a defect in induction of Ifng by the cytokines IL-12 and IL-18, with a more modest effect on induction via T-cell receptor activation. To better understand how CNS-22 and other Ifng CNSs regulated Ifng transcription in response to these distinct stimuli, we examined activation-dependent changes in epigenetic modifications across the extended Ifng locus in CNS-22-deficient T cells. We demonstrate that in response to both cytokine and TCR driven activation signals, CNS-22 and other Ifng CNSs recruit increased activity of histone acetyl transferases (HATs) that transiently enhance levels of histones H3 and H4 acetylation across the extended Ifng locus. We also demonstrate that activation-responsive increases in histone acetylation levels are directly linked to the ability of Ifng CNSs to acutely enhance Pol II recruitment to the Ifng promoter. Finally, we show that impairment in IL-12+IL-18 dependent induction of Ifng stems from the importance of CNS-22 in coordinating locus-wide levels of histone acetylation in response to these cytokines. These findings identify a role for acute histone acetylation in the enhancer function of distal conserved cis-elements that regulate of Ifng gene expression.

Notch simultaneously orchestrates multiple helper T cell programs independently of cytokine signals.

Two models are proposed to explain Notch function during helper T (Th) cell differentiation. One argues that Notch instructs one Th cell fate over the other, whereas the other posits that Notch function is dictated by cytokines. Here we provide a detailed mechanistic study investigating the role of Notch in orchestrating Th cell differentiation. Notch neither instructed Th cell differentiation nor did cytokines direct Notch activity, but instead, Notch simultaneously regulated the Th1, Th2, and Th17 cell genetic programs independently of cytokine signals. In addition to regulating these programs in both polarized and nonpolarized Th cells, we identified Ifng as a direct Notch target. Notch bound the Ifng CNS-22 enhancer, where it synergized with Tbet at the promoter. Thus, Notch acts as an unbiased amplifier of Th cell differentiation. Our data provide a paradigm for Notch in hematopoiesis, with Notch simultaneously orchestrating multiple lineage programs, rather than restricting alternate outcomes.

The Th17 family: flexibility follows function.

Discovery of the T-helper 17 (Th17) subset heralded a major shift in T-cell biology and immune regulation. In addition to defining a new arm of the adaptive immune response, studies of the Th17 pathway have led to a greater appreciation of the developmental flexibility, or plasticity, that is a feature of T-cell developmental programs. Since the initial finding that differentiation of Th17 cells is promoted by transforming growth factor-ß (TGFß), it became clear that Th17 cell development overlapped that of induced regulatory T (iTreg) cells. Subsequent findings established that Th17 cells are also unusually flexible in their late developmental programming, demonstrating substantial overlap with conventional Th1 cells through mechanisms that are just beginning to be understood but would appear to have important implications for immunoregulation at homeostasis and in immune-mediated diseases. Herein we examine the developmental and functional features of Th17 cells in relation to iTreg cells, Th1 cells, and Th22 cells, as a basis for understanding the contributions of this pathway to host defense, immune homeostasis, and immune-mediated disease.

Th22 cells are an important source of IL-22 for host protection against enteropathogenic bacteria.

Interleukin-22 (IL-22) is central to host protection against bacterial infections at barrier sites. Both innate lymphoid cells (ILCs) and T cells produce IL-22. However, the specific contributions of CD4(+) T cells and their developmental origins are unclear. We found that the enteric pathogen Citrobacter rodentium induced sequential waves of IL-22-producing ILCs and CD4(+) T cells that were each critical to host defense during a primary infection. Whereas IL-22 production by ILCs was strictly IL-23 dependent, development of IL-22-producing CD4(+) T cells occurred via an IL-6-dependent mechanism that was augmented by, but not dependent on, IL-23 and was dependent on both transcription factors T-bet and AhR. Transfer of CD4(+) T cells differentiated with IL-6 in the absence of TGF-ß ("Th22" cells) conferred complete protection of infected IL-22-deficient mice whereas transferred Th17 cells did not. These findings establish Th22 cells as an important component of mucosal antimicrobial host defense.

Reciprocal interactions of the intestinal microbiota and immune system.

The emergence of the adaptive immune system in vertebrates set the stage for evolution of an advanced symbiotic relationship with the intestinal microbiota. The defining features of specificity and memory that characterize adaptive immunity have afforded vertebrates the mechanisms for efficiently tailoring immune responses to diverse types of microbes, whether to promote mutualism or host defence. These same attributes can put the host at risk of immune-mediated diseases that are increasingly linked to the intestinal microbiota. Understanding how the adaptive immune system copes with the remarkable number and diversity of microbes that colonize the digestive tract, and how the system integrates with more primitive innate immune mechanisms to maintain immune homeostasis, holds considerable promise for new approaches to modulate immune networks to treat and prevent disease.

TGF-ß in Th17 cell development: the truth is out there.

As Th17 cell developmental requirements continue to be studied, Gutcher et al. (2011) demonstrate in this issue of Immunity that autocrine TGF-ß cytokine promotes Th17 cell development and maintenance.

Regulation of the Ifng locus in the context of T-lineage specification and plasticity.

Study of the development of distinct CD4(+) T-cell subsets from naive precursors continues to provide excellent opportunities for dissection of mechanisms that control lineage-specific gene expression or repression. Whereas it had been thought that the induction of transcription networks that control T-lineage commitment were highly stable, reinforced by epigenetic processes that confer heritability of functional phenotypes by the progeny of mature T cells, recent findings support a more dynamic view of T-lineage commitment. Here, we highlight advances in the mapping and functional characterization of cis elements in the Ifng locus that have provided new insights into the control of the chromatin structure and transcriptional activity of this signature T-helper 1 cell gene. We also examine epigenetic features of the Ifng locus that have evolved to enable its reprogramming for expression by other T-cell subsets, particularly T-helper 17 cells, and contrast features of the Ifng locus with those of the Il17a-Il17f locus, which appears less promiscuous.

Modular utilization of distal cis-regulatory elements controls Ifng gene expression in T cells activated by distinct stimuli.

Distal cis-regulatory elements play essential roles in the T lineage-specific expression of cytokine genes. We have mapped interactions of three trans-acting factors-NF-kappaB, STAT4, and T-bet-with cis elements in the Ifng locus. We find that RelA is critical for optimal Ifng expression and is differentially recruited to multiple elements contingent upon T cell receptor (TCR) or interleukin-12 (IL-12) plus IL-18 signaling. RelA recruitment to at least four elements is dependent on T-bet-dependent remodeling of the Ifng locus and corecruitment of STAT4. STAT4 and NF-kappaB therefore cooperate at multiple cis elements to enable NF-kappaB-dependent enhancement of Ifng expression. RelA recruitment to distal elements was similar in T helper 1 (Th1) and effector CD8(+) T (Tc1) cells, although T-bet was dispensable in CD8 effectors. These results support a model of Ifng regulation in which distal cis-regulatory elements differentially recruit key transcription factors in a modular fashion to initiate gene transcription induced by distinct activation signals.

Epigenetic instability of cytokine and transcription factor gene loci underlies plasticity of the T helper 17 cell lineage.

Phenotypic plasticity of T helper 17 (Th17) cells suggests instability of chromatin structure of key genes of this lineage. We identified epigenetic modifications across the clustered Il17a and Il17f and the Ifng loci before and after differential IL-12 or TGF-beta cytokine signaling, which induce divergent fates of Th17 cell precursors. We found that Th17 cell precursors had substantial remodeling of the Ifng locus, but underwent critical additional modifications to enable high expression when stimulated by IL-12. Permissive modifications across the Il17a-Il17f locus were amplified by TGF-beta signaling in Th17 cells, but were rapidly reversed downstream of IL-12-induced silencing of the Rorc gene by the transcription factors STAT4 and T-bet. These findings reveal substantial chromatin instability of key transcription factor and cytokine genes of Th17 cells and support a model of Th17 cell lineage plasticity in which cell-extrinsic factors modulate Th17 cell fates through differential effects on the epigenetic status of Th17 cell lineage factors.

Interplay between the TH17 and TReg cell lineages: a (co-)evolutionary perspective.

The origins of the adaptive immune system and the basis for its unique association with vertebrate species have been a source of considerable speculation. In light of recent advances in our understanding of the developmental and functional links between the induced regulatory T cell and T helper 17 cell lineages, and their specialized relationship to the gut, we speculate that the co-evolution of these adaptive immune pathways might have given primitive vertebrates a means to benefit from the diversification of their commensal microbiota.