Gretta L. Stritesky

Stat3 and Stat4 Direct Development of IL-17-Secreting Th Cells

IL-17-secreting CD4+ T cells are critically involved in inflammatory immune responses. Development of these cells is promoted in vivo and in vitro by IL-23 or TGFβ1 plus IL-6. Despite growing interest in this inflammatory Th subset, little is known about the transcription factors that are required for their development. We demonstrate that Stat3 is required for programming the TGFβ1 plus IL-6 and IL-23-stimulated IL-17-secreting phenotype, as well as for RORγt expression in TGFβ1 plus IL-6-primed cells. Moreover, retroviral transduction of a constitutively active Stat3 into differentiating T cell cultures enhances IL-17 production from these cells. We further show that Stat4 is partially required for the development of IL-23-, but not TGFβ1 plus IL-6-primed IL-17-secreting cells, and is absolutely required for IL-17 production in response to IL-23 plus IL-18. The requirements for Stat3 and Stat4 in the development of these IL-17-secreting subsets reveal additional mechanisms in Th cell fate decisions during the generation of proinflammatory cell types.

The transcription factor PU.1 is required for the development of IL-9-producing T cells and allergic inflammation

CD4+ helper T cells acquire effector phenotypes that promote specialized inflammatory responses. We show that the ETS-family transcription factor PU.1 was required for the development of an interleukin 9 (IL-9)-secreting subset of helper T cells. Decreasing PU.1 expression either by conditional deletion in mouse T cells or the use of small interfering RNA in human T cells impaired IL-9 production, whereas ectopic PU.1 expression promoted IL-9 production. Mice with PU.1-deficient T cells developed normal T helper type 2 (TH2) responses in vivo but showed attenuated allergic pulmonary inflammation that corresponded to lower expression of Il9 and chemokines in peripheral T cells and in lungs than that of wild-type mice. Together our data suggest a critical role for PU.1 in generating the IL-9-producing (TH9) phenotype and in the development of allergic inflammation.

IL-6 controls Th17 immunity in vivo by inhibiting the conversion of conventional T cells into Foxp3 regulatory T cells

The conditions leading to the induction of adaptive Foxp3+ regulatory T cells (T-regs) from peripheral T cells in vivo are incompletely understood. Here, we show that unresponsiveness of T cells to IL-6 by T cell-selective deletion of gp130 or immunization of wild-type mice with antigen in incomplete Freunds adjuvant (IFA), which fails to induce IL-6, promotes the conversion of peripheral CD4+ T cells into adaptive Foxp3+ T-regs. Thus, both T cell-conditional gp130 knockout (KO) mice immunized with MOG35-55 in complete Freunds adjuvant (CFA) and wild-type mice immunized with MOG35-55 in IFA develop overwhelming antigen-specific T-reg responses and are protected from experimental autoimmune encephalomyelitis (EAE). Depletion of T-regs restores T helper (Th)17 responses and clinical EAE in MOG/CFA-immunized T cell-conditional gp130 KO mice, but not in MOG/IFA-immunized wild-type mice. We conclude that in the absence of T-regs, IL-6 signaling is dispensable for the induction of Th17 cells, and alternative pathways exist to induce Th17 cells and EAE in the absence of IL-6 signaling. However, IL-6 signaling is dominant in inhibiting the conversion of conventional T cells into Foxp3+ T-regs in vivo, and in the absence of IL-6 signaling, no other cytokine can substitute in inhibiting T-reg conversion. These data identify IL-6 as an important target to modulate autoimmune responses and chronic inflammation.

IL-23 Promotes Maintenance but Not Commitment to the Th17 Lineage

IL-23 plays a critical role establishing inflammatory immunity and enhancing IL-17 production in vivo. However, an understanding of how it performs those functions has been elusive. In this report, using an IL-17-capture technique, we demonstrate that IL-23 maintains the IL-17-secreting phenotype of purified IL-17+ cells without affecting cell expansion or survival. IL-23 maintains the Th17 phenotype over multiple rounds of in vitro stimulation most efficiently in conjunction with IL-1β. However, in contrast to Th1 and Th2 cells, the Th17 phenotype is not stable and when long-term IL-23-stimulated Th17 cultures are exposed to Th1- or Th2-inducing cytokines, the Th17 genetic program is repressed and cells that previously secreted IL-17 assume the cytokine secreting profile of other Th subsets. Thus, while IL-23 can maintain the Th17 phenotype, it does not promote commitment to an IL-17-secreting lineage.

Selection of Self-Reactive T Cells in the Thymus

On the whole, the healthy adaptive immune system is responsive to foreign antigens and tolerant to self. However, many individual lymphocytes have, and even require, substantial self-reactivity for their particular functions in immunity. In this review, we discuss several populations of lymphocytes that are thought to experience agonist stimulation through the T cell receptor during selection: nTreg cells, iNKT cells, nIELs, and nTh17s. We discuss the nature of this self-reactivity, how it compares with conventional T cells, and why it is important for overall immune health. We also outline molecular pathways unique to each lineage and consider possible commonalities to their development and survival.

Murine thymic selection quantified using a unique method to capture deleted T cells

Thymic positive and negative selection events generate a T-cell repertoire that is MHC restricted and self-tolerant. The number of T cells undergoing positive and negative selection in normal mice has never been firmly established. We generated mice that lack the proapoptotic molecule Bim (bcl2l11) together with a Nur77GFP transgene, which allowed the identification and enumeration of T cells that would normally undergo clonal deletion. Using this method, we report the striking observation that six times more cells undergo negative selection than complete positive selection. Seventy-five percent of the negatively selected cells are deleted at the double positive stage in the thymic cortex, compared with 25% at the single positive stage in the medulla. The fact that more thymocytes are highly reactive to MHC than are weakly reactive is inconsistent with a random model of recognition and suggests that T-cell recognition is MHC biased. Furthermore, Bim−/− mice had an increased number of GFPhi cells in the peripheral lymphoid tissue and a corresponding increase in antigen experienced or anergic cell phenotype. Our data also show that the CD4+ T cells that are clonally deleted experienced only slightly stronger T-cell receptor signaling than those that developed into regulatory T cells.

Temporal Induction Pattern of STAT4 Target Genes Defines Potential for Th1 Lineage-Specific Programming

STAT4 is a critical component in the development of inflammatory adaptive immune responses. It has been extensively characterized as a lineage-determining factor in Th1 development. However, the genetic program activated by STAT4 that results in an inflammatory cell type is not well defined. In this report, we use DNA isolated from STAT4-chromatin immunoprecipitation to perform chromatin immunoprecipitation-on-chip analysis of over 28,000 mouse gene promoters to identify STAT4 targets. We demonstrate that STAT4 binds multiple gene-sets that program distinct components of the Th1 lineage. Although many STAT4 target genes display STAT4-dependent IL-12-inducible expression, other genes displayed IL-12-induced histone modifications but lack induction, possibly due to high relative basal expression. In the subset of genes that STAT4 programs for expression in Th1 cells, IL-12-induced mRNA levels remain increased for a longer time than mRNA from genes that are not programmed. This suggests that STAT4 binding to target genes, while critical, is not the only determinant for STAT4-dependent gene programming during Th1 differentiation.

CD4 + T cell anergy prevents autoimmunity and generates regulatory T cell precursors

The role of anergy, an acquired state of T cell functional unresponsiveness, in natural peripheral tolerance remains unclear. In this study, we found that anergy was selectively induced in fetal antigen–specific maternal CD4+ T cells during pregnancy. A naturally occurring subpopulation of anergic polyclonal CD4+ T cells, enriched for self antigen–specific T cell antigen receptors, was also present in healthy hosts. Neuropilin-1 expression in anergic conventional CD4+ T cells was associated with hypomethylation of genes related to thymic regulatory T cells (Treg cells), and this correlated with their ability to differentiate into Foxp3+ Treg cells that suppressed immunopathology. Thus, our data suggest that not only is anergy induction important in preventing autoimmunity but also it generates the precursors for peripheral Treg cell differentiation.

Signal transducer and activator of transcription 4 limits the development of adaptive regulatory T cells

Summary T‐cell responses to a cytokine milieu instruct the development of multiple effector phenotypes. While transforming growth factor‐β1 (TGF‐β1) inhibits the development of T helper type 1 (Th1) and Th2 cells, we demonstrate that like interleukin‐6 (IL‐6) and IL‐4, IL‐12 can inhibit the development of TGF‐β1‐induced Foxp3‐expressing adaptive T regulatory (aTreg) cells. Signal transducer and activator of transcription 4 (STAT4) is critical for the response to IL‐12, although there is a parallel pathway involving T box expressed in T cells (T‐bet), and cells from mice double‐deficient in STAT4 and T‐bet are refractory to the inhibition of aTreg‐cell development by IL‐12. While the ability of these cytokines to promote Th differentiation may contribute to this effect, we observe that culture with IL‐12, or other instructive cytokines, results in an increase in repressive chromatin modifications at the Foxp3 locus that limit STAT5 binding to Foxp3, without observed effects on IL‐2 signalling pathways. In a model of allergic lung inflammation there are increased percentages of Treg cells in the lungs of Stat4−/− mice, compared with wild‐type mice, and increases in Treg cells correlate with decreased allergic inflammation. Overall, these results suggest an important role for STAT4 in regulating Treg‐cell development.

Stat4 Isoforms Differentially Regulate Inflammation and Demyelination in Experimental Allergic Encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated autoimmune disease model of multiple sclerosis. Signal transducer and activator of transcription 4 (Stat4) is a transcription factor activated by IL-12 and IL-23, two cytokines known to play important roles in the pathogenesis of EAE by inducing T cells to secrete IFN-γ and IL-17, respectively. We and others have previously shown that therapeutic intervention or targeted disruption of Stat4 was effective in ameliorating EAE. Recently, a splice variant of Stat4 termed Stat4β has been characterized that lacks 44 amino acids at the C terminus of the full-length Stat4α. In this study we examined whether T cells expressing either isoform could affect the pathogenesis of EAE. We found that transgenic mice expressing Stat4β on a Stat4-deficient background develop an exacerbated EAE compared with wild-type mice following immunization with myelin oligodendrocyte glycoprotein peptide 35–55, while Stat4α transgenic mice have greatly attenuated disease. The differential development of EAE in transgenic mice correlates with increased IFN-γ and IL-17 in Stat4β-expressing cells in situ, contrasting increased IL-10 production by Stat4α-expressing cells. This study demonstrates that Stat4 isoforms differentially regulate inflammatory cytokines in association with distinct effects on the onset and severity of EAE.