Takeshi Egawa

IL-6 programs T H -17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways

T helper cells that produce interleukin 17 (IL-17; TH-17 cells) are a distinct subset of proinflammatory cells whose in vivo function requires IL-23 but whose in vitro differentiation requires only IL-6 and transforming growth factor-β (TGF-β). We demonstrate here that IL-6 induced expression of IL-21 that amplified an autocrine loop to induce more IL-21 and IL-23 receptor in naive CD4+ T cells. Both IL-21 and IL-23, along with TGF-β, induced IL-17 expression independently of IL-6. The effects of IL-6 and IL-21 depended on STAT3, a transcription factor required for the differentiation of TH-17 cells in vivo. IL-21 and IL-23 induced the orphan nuclear receptor RORγt, which in synergy with STAT3 promoted IL-17 expression. IL-6 therefore orchestrates a series of downstream cytokine-dependent signaling pathways that, in concert with TGF-β, amplify RORγt-dependent differentiation of TH-17 cells.

Differential Requirements for Runx Proteins in CD4 Repression and Epigenetic Silencing during T Lymphocyte Development

T lymphocytes differentiate in discrete stages within the thymus. Immature thymocytes lacking CD4 and CD8 coreceptors differentiate into double-positive cells (CD4(+)CD8(+)), which are selected to become either CD4(+)CD8(-)helper cells or CD4(-)CD8(+) cytotoxic cells. A stage-specific transcriptional silencer regulates expression of CD4 in both immature and CD4(-)CD8(+) thymocytes. We show here that binding sites for Runt domain transcription factors are essential for CD4 silencer function at both stages, and that different Runx family members are required to fulfill unique functions at each stage. Runx1 is required for active repression in CD4(-)CD8(-) thymocytes whereas Runx3 is required for establishing epigenetic silencing in cytotoxic lineage thymocytes. Runx3-deficient cytotoxic T cells, but not helper cells, have defective responses to antigen, suggesting that Runx proteins have critical functions in lineage specification and homeostasis of CD8-lineage T lymphocytes.

Requirement for CARMA1 in Antigen Receptor-Induced NF-κB Activation and Lymphocyte Proliferation

Ligation of antigen receptors (TCR, BCR) on T and B lymphocytes leads to the activation of new transcriptional programs and cell cycle progression. Antigen receptor-mediated activation of NF-kappa B, required for proliferation of B and T cells, is disrupted in T cells lacking PKC theta and in B and T cells lacking Bcl10, a caspase recruitment domain (CARD)-containing adaptor protein. CARMA1 (also called CARD11 and Bimp3), the only lymphocyte-specific member in a family of membrane-associated guanylate kinase (MAGUK) scaffolding proteins that interact with Bcl10 by way of CARD-CARD interactions, is required for TCR-induced NF-kappa B activation in Jurkat T lymphoma cells. Here we show that T cells from mice lacking CARMA1 expression were defective in recruitment of Bcl10 to clustered TCR complexes and lipid rafts, in activation of NF-kappa B, and in induction of IL-2 production. Development of CD5(+) peritoneal B cells was disrupted in these mice, as was B cell proliferation in response to both BCR and CD40 ligation. Serum immunoglobulin levels were also markedly reduced in the mutant mice. Together, these results show that CARMA1 has a central role in antigen receptor signaling that results in activation and proliferation of both B and T lymphocytes.

Runx-CBFβ complexes control expression of the transcription factor Foxp3 in regulatory T cells

The transcription factor Foxp3 has an indispensable role in establishing stable transcriptional and functional programs of regulatory T cells (Treg cells). Loss of Foxp3 expression in mature Treg cells results in a failure of suppressor function, yet the molecular mechanisms that ensure steady, heritable Foxp3 expression in the Treg cell lineage remain unknown. Using Treg cell–specific gene targeting, we found that complexes of the transcription factors Runx and CBFβ were required for maintenance of Foxp3 mRNA and protein expression in Treg cells. Consequently, mice lacking CBFβb exclusively in the Treg cell lineage had a moderate lymphoproliferative syndrome. Thus, Runx-CBFβ complexes maintain stable high expression of Foxp3 and serve as an essential determinant of Treg cell lineage stability.

ThPOK acts late in specification of the helper T cell lineage and suppresses Runx-mediated commitment to the cytotoxic T cell lineage

The transcription factor ThPOK has been shown to be required and sufficient for CD4+CD8− thymocyte generation, yet the mechanism through which ThPOK orchestrates CD4 helper T cell lineage differentiation remains unclear. Here we utilized reporter mice to track expression of transcription factors in developing thymocytes. Distal promoter-driven Runx3 (Runx3d) expression was restricted to MHC class I-selected thymocytes. In ThPOK-deficient mice, Runx3d expression was de-repressed in MHCII-selected thymocytes, contributing to their redirection to the CD8 T cell lineage. In the absence of both ThPOK and Runx, redirection was prevented and cells potentially belonging to the CD4 lineage, presumably specified independently of ThPOK, were generated. Our results suggest that MHCII-selected thymocytes are directed towards the CD4 lineage independently of ThPOK, but require ThPOK to prevent Runx-dependent differentiation towards the CD8 lineage.The transcription factor ThPOK is required and sufficient for the generation of CD4+CD8− thymocytes, yet the mechanism by which ThPOK orchestrates differentiation into the CD4+ helper T cell lineage remains unclear. Here we used reporter mice to track the expression of transcription factors in developing thymocytes. Distal promoter–driven expression of the gene encoding the transcription factor Runx3 was restricted to major histocompatibility complex (MHC) class I–selected thymocytes. In ThPOK-deficient mice, such expression was derepressed in MHC class II–selected thymocytes, which contributed to their redirection to the CD8+ T cell lineage. In the absence of both ThPOK and Runx, redirection was prevented and cells potentially belonging to the CD4+ lineage, presumably specified independently of ThPOK, were generated. Our results suggest that MHC class II–selected thymocytes are directed toward the CD4+ lineage independently of ThPOK but require ThPOK to prevent Runx-dependent differentiation toward the CD8+ lineage.

Development of Promyelocytic Zinc Finger and ThPOK-Expressing Innate γδ T Cells Is Controlled by Strength of TCR Signaling and Id3

The broad-complex tramtrack and bric a brac-zinc finger transcriptional regulator(BTB-ZF), promyelocytic leukemia zinc finger (PLZF), was recently shown to control the development of the characteristic innate T cell phenotype and effector functions of NK T cells. Interestingly, the ectopic expression of PLZF was shown to push conventional T cells into an activated state that seems to be proinflammatory. The factors that control the normal expression of PLZF in lymphocytes are unknown. In this study, we show that PLZF expression is not restricted to NK T cells but is also expressed by a subset of γδ T cells, functionally defining distinct subsets of this innate T cell population. A second BTB-ZF gene, ThPOK, is important for the phenotype of the PLZF-expressing γδ T cells. Most importantly, TCR signal strength and expression of inhibitor of differentiation gene 3 control the frequency of PLZF-expressing γδ T cells. This study defines the factors that control the propensity of the immune system to produce potentially disease-causing T cell subsets.

Lineage Diversion of T Cell Receptor Transgenic Thymocytes Revealed by Lineage Fate Mapping

Background The binding of the T cell receptor (TCR) to major histocompatibility complex (MHC) molecules in the thymus determines fates of TCRαβ lymphocytes that subsequently home to secondary lymphoid tissue. TCR transgenic models have been used to study thymic selection and lineage commitment. Most TCR transgenic mice express the rearranged TCRαβ prematurely at the double negative stage and abnormal TCRαβ populations of T cells that are not easily detected in non-transgenic mice have been found in secondary lymphoid tissue of TCR transgenic mice. Methodology and Principal Findings To determine developmental pathways of TCR-transgenic thymocytes, we used Cre-LoxP-mediated fate mapping and show here that premature expression of a transgenic TCRαβ diverts some developing thymocytes to a developmental pathway which resembles that of gamma delta cells. We found that most peripheral T cells with the HY-TCR in male mice have bypassed the RORγt-positive CD4+8+ (double positive, DP) stage to accumulate either as CD4−8− (double negative, DN) or as CD8α+ T cells in lymph nodes or gut epithelium. Likewise, DN TCRαβ cells in lymphoid tissue of female mice were not derived from DP thymocytes. Conclusion The results further support the hypothesis that the premature expression of the TCRαβ can divert DN thymocytes into gamma delta lineage cells.

Runx and ThPOK: A balancing act to regulate thymocyte lineage commitment

CD4‐positive helper T cells and CD8‐positive cytotoxic T cells comprise the majority of T lymphocytes present in secondary lymphoid organs and are essential for acquired immunity. These two populations are derived from common precursors in the thymus and selected through interaction between their clonal T‐cell receptors and major histocompatibility complex molecules. Although intensely studied as a model system for binary cell fate decisions, the mechanisms underlying the helper versus cytotoxic lineage choice in the thymus has been elusive. In the past few years, it has been demonstrated that the Runx family of transcription factors, particularly Runx3, is essential for the generation of cytotoxic lineage T cells, whereas the ThPOK zinc finger transcription factor that plays a crucial role in the differentiation of the helper lineage. Recent works have implied that a cross‐regulation between Runx and ThPOK contributes to appropriate thymocyte lineage commitment. In this article, recent findings on the transcription factor networks governing thymocyte lineage decisions are discussed, focusing on the two factors, and provide insights into mechanisms of lineage‐specific gene regulation in the process of T‐cell commitments. J. Cell. Biochem. 107: 1037–1045, 2009.

Regulation of Fas-mediated immune homeostasis by an activation-induced protein, Cyclon

Activation-induced cell death (AICD) plays an essential role in the contraction of activated T cells after eradication of pathogen. Fas (APO-1/CD95) is one of the key cell surface proteins that mediate AICD in CD4(+) and CD8(+) T cells. Despite its prime importance in cell death, regulation of Fas expression in T cells is poorly understood. Here we show that Cyclon, a newly identified cytokine-inducible protein, is induced in T cells on T-cell receptor ligation and important for immune homeostasis. Transgenic expression of Cyclon ameliorated autoimmune phenotype in mice lacking subunits of IL-2R. Transgenic expression of Cyclon markedly enhanced AICD through increased expression of Fas whose expression is essential for Cyclon action. Finally, we demonstrated that activated but not resting CD4(+) T cells with targeted deletion of a Cyclon allele show reduced AICD and expression of Fas, indicating a critical role of Cyclon in Fas expression in activated T cells. We think that our data provide insight into expression regulation of Fas in T cells.

Antagonistic interplay between ThPOK and Runx in lineage choice of thymocytes

Differentiation of CD4(+)CD8(+) double-positive (DP) thymocytes into either CD4(+)-helper or CD8(+)-cytotoxic lineages involves several phases. It has been suggested that, following initial specification to one of the lineages by a set of lineage-specific genes during positive selection, stable cell identity is established during the commitment process by eliminating differentiation potential toward the other lineage. While the Runx3 transcription factor fixes the Cd4 gene into a silenced state during cytotoxic-lineage cell differentiation, the ThPOK transcription factor is both necessary and sufficient to generate a CD4(+)CD8(-) phenotype in post-selection thymocytes, regardless of the MHC specificity of the TCRs. Recent studies have revealed that a reciprocal antagonistic interplay between Runx3 and ThPOK is a central component in the transcription factor network governing the helper versus cytotoxic-lineage decision.