Ryoji Yagi

GATA3 controls Foxp3+ regulatory T cell fate during inflammation in mice

Tregs not only keep immune responses to autoantigens in check, but also restrain those directed toward pathogens and the commensal microbiota. Control of peripheral immune homeostasis by Tregs relies on their capacity to accumulate at inflamed sites and appropriately adapt to their local environment. To date, the factors involved in the control of these aspects of Treg physiology remain poorly understood. Here, we show that the canonical Th2 transcription factor GATA3 is selectively expressed in Tregs residing in barrier sites including the gastrointestinal tract and the skin. GATA3 expression in both murine and human Tregs was induced upon TCR and IL-2 stimulation. Although GATA3 was not required to sustain Treg homeostasis and function at steady state, GATA3 played a cardinal role in Treg physiology during inflammation. Indeed, the intrinsic expression of GATA3 by Tregs was required for their ability to accumulate at inflamed sites and to maintain high levels of Foxp3 expression in various polarized or inflammatory settings. Furthermore, our data indicate that GATA3 limits Treg polarization toward an effector T cell phenotype and acquisition of effector cytokines in inflamed tissues. Overall, our work reveals what we believe to be a new facet in the complex role of GATA3 in T cells and highlights what may be a fundamental role in controlling Treg physiology during inflammation.

The Transcription Factor GATA3 Is Critical for the Development of All IL-7Rα-Expressing Innate Lymphoid Cells

Innate lymphoid cells (ILCs) are critical in innate immune responses to pathogens and lymphoid organ development. Similar to CD4(+) T helper (Th) cell subsets, ILC subsets positive for interleukin-7 receptor α (IL-7Rα) produce distinct sets of effector cytokines. However, the molecular control of IL-7Rα(+) ILC development and maintenance is unclear. Here, we report that GATA3 was indispensable for the development of all IL-7Rα(+) ILC subsets and T cells but was not required for the development of classical natural killer cells. Conditionally Gata3-deficient mice had no lymph nodes and were susceptible to Citrobactor rodentium infection. After the ILCs had fully developed, GATA3 remained important for the maintenance and functions of ILC2s. Genome-wide gene expression analyses indicated that GATA3 regulated a similar set of cytokines and receptors in Th2 cells and ILC2s, but not in ILC3s. Thus, GATA3 plays parallel roles in regulating the development and functions of CD4(+) T cells and IL-7Rα(+) ILCs.

STAT6-Dependent Regulation of Th9 Development

Th cell effector subsets develop in response to specific cytokine environments. The development of a particular cytokine-secreting pattern requires an integration of signals that may promote the development of opposing pathways. A recent example of this paradigm is the IL-9–secreting Th9 cell that develops in response to TGF-β and IL-4, cytokines that, in isolation, promote the development of inducible regulatory T cells and Th2 cells, respectively. To determine how the balance of these factors results in priming for IL-9 secretion, we examined the effects of each pathway on transcription factors that regulate Th cell differentiation. We demonstrated that TGF-β induces the PU.1-encoding Sfpi1 locus and that this is independent of IL-4–induced STAT6 activation. IL-4–activated STAT6 is required for repressing the expression of T-bet and Foxp3 in Th9 cells, transcription factors that inhibit IL-9 production, and STAT6 is required for the induction of IRF4, which promotes Th9 development. These data established a transcription factor network that regulates IL-9 and demonstrated how combinations of cytokine signals generate cytokine-secreting potential by altering the expression of a panel of transcription factors.

The Transcription Factor T-bet Is Induced by Multiple Pathways and Prevents an Endogenous Th2 Cell Program during Th1 Cell Responses

T-bet is a critical transcription factor for T helper 1 (Th1) cell differentiation. To study the regulation and functions of T-bet, we developed a T-bet-ZsGreen reporter mouse strain. We determined that interleukin-12 (IL-12) and interferon-γ (IFN-γ) were redundant in inducing T-bet in mice infected with Toxoplasma gondii and that T-bet did not contribute to its own expression when induced by IL-12 and IFN-γ. By contrast, T-bet and the transcription factor Stat4 were critical for IFN-γ production whereas IFN-γ signaling was dispensable for inducing IFN-γ. Loss of T-bet resulted in activation of an endogenous program driving Th2 cell differentiation in cells expressing T-bet-ZsGreen. Genome-wide analyses indicated that T-bet directly induced many Th1 cell-related genes but indirectly suppressed Th2 cell-related genes. Our study revealed redundancy and synergy among several Th1 cell-inducing pathways in regulating the expression of T-bet and IFN-γ, and a critical role of T-bet in suppressing an endogenous Th2 cell-associated program.

An updated view on transcription factor GATA3-mediated regulation of Th1 and Th2 cell differentiation

CD4 T(h) are critical for orchestrating adaptive immune responses. The expression of the transcription factor GATA3 (GATA-binding protein 3) is up-regulated or down-regulated during T(h)2 or T(h)1 cell differentiation, respectively. Furthermore, GATA3 is responsible for induction of T(h)2 differentiation and represses T(h)1 differentiation. In this review, we present an updated view on the molecular mechanisms through which GATA3 regulates T(h)1/T(h)2 differentiation. During T(h)2 cell differentiation, GATA3 directly binds to the T(h)2 cytokine gene locus at several regions and regulates expression. On the other hand, GATA3 inhibits T(h)1 cell differentiation by preventing up-regulation of IL-12 receptor β2 and STAT4 (signal transducer and activator of transcription 4) and neutralization of Runx3 (runt-related transcription factor 3) function through protein-protein interaction. GATA3 may also directly act on the Ifng gene. In summary, GATA3 serves as a transcriptional activator or repressor through direct action on transcriptional machinery and/or affecting chromatin remodeling at many critical loci encoding cytokines, cytokine receptors, signaling molecules as well as transcription factors that are involved in the regulation of T(h)1 and T(h)2 differentiation.

The Transcription Factors T-bet and Runx Are Required for the Ontogeny of Pathogenic Interferon-γ-Producing T Helper 17 Cells

T helper 17 (Th17) cells can give rise to interleukin-17A (IL-17A)- and interferon (IFN)-γ-double-producing cells that are implicated in development of autoimmune diseases. However, the molecular mechanisms that govern generation of IFN-γ-producing Th17 cells are unclear. We found that coexpression of the Th1 and Th17 cell master transcription factors, T-bet and retinoid-related orphan receptor gamma-t (RORγt), respectively, did not generate Th cells with robust IL-17 and IFN-γ expression. Instead, development of IFN-γ-producing Th17 cells required T-bet and Runx1 or Runx3. IL-12-stimulated Th17 cells upregulated Runx1, which bound to the Ifng locus in a T-bet-dependent manner. Reciprocally, T-bet or Runx1 deficiency or inhibition of Runx transcriptional activity impaired the development of IFN-γ-producing Th17 cells during experimental autoimmune encephalomyelitis, which correlated with substantially ameliorated disease course. Thus, our studies identify a critical role for T-bet and Runx transcription factors in the generation of pathogenic IFN-γ-producing Th17 cells.

Efficient cytokine-induced IL-13 production by mast cells requires both IL-33 and IL-3

BACKGROUND IL-13 is a critical effector cytokine for allergic inflammation. It is produced by several cell types, including mast cells, basophils, and TH2 cells. In mast cells and basophils its induction can be stimulated by cross-linkage of immunoglobulin receptors or cytokines. The IL-1 family members IL-33 and IL-18 have been linked to induction of IL-13 production by mast cells and basophils. In CD4 TH2 cells IL-33-mediated production of IL-13 requires simultaneous signal transducer and activator of transcription (STAT) 5 activation. OBJECTIVE Here we have addressed whether cytokine-induced IL-13 production in mast cells and basophils follows the same logic as in TH2 cells: requirement of 2 separate signals. METHODS By generating a bacterial artificial chromosome (BAC) transgenic IL-13 reporter mouse, we measured IL-13 production in mast cells and basophils. RESULTS In mast cells harvested from peritoneal cavities, 2 cytokine signals are required for IL-13 production: IL-33 and IL-3. In bone marrow mast cells IL-13 production requires IL-33, but the requirement for a STAT5 inducer is difficult to evaluate because these cells require the continuous presence of IL-3 (a STAT5 activator) for survival. Poorer STAT5 inducers in culture (IL-4 or stem cell factor) result in less IL-13 production on IL-33 challenge, but the addition of exogenous IL-3 enhances IL-13 production. This implies that bone marrow-derived mast cells, like peritoneal mast cells and TH2 cells, require stimulation both by an IL-1 family member and a STAT5 inducer to secrete IL-13. Basophils follow the same rule; splenic basophils produce IL-13 in response to IL-18 or IL-33 plus IL-3. CONCLUSION Optimal IL-13 production from mast cells and basophils requires 2 cytokine signals.

Gata3/Ruvbl2 complex regulates T helper 2 cell proliferation via repression of Cdkn2c expression

Significance GATA-binding protein 3 (Gata3) controls the differentiation of naive CD4 T cells into T helper 2 (Th2) cells by induction of chromatin remodeling at the Th2 cytokine gene loci. Gata3 also facilitates Th2 cell proliferation via unknown mechanisms. We have identified a functional Gata3/RuvB-like protein 2 (Ruvbl2) complex that regulates the proliferation of differentiating Th2 cells through the repression of a CDK inhibitor, cyclin-dependent kinase inhibitor 2c (Cdkn2c). Gata3 directly bound to the Cdkn2c locus in an Ruvbl2-dependent manner, and Cdkn2c-knockdown experiments indicated an important role for this molecule in the Gata3-mediated induction of Th2-cell proliferation. Ruvbl2-knockdown Th2 cells showed decreased antigen-induced expansion and caused less airway inflammation in vivo, indicating an important role for Ruvbl2 in Th2 cells in allergic reactions. GATA-binding protein 3 (Gata3) controls the differentiation of naive CD4 T cells into T helper 2 (Th2) cells by induction of chromatin remodeling of the Th2 cytokine gene loci, direct transactivation of Il5 and Il13 genes, and inhibition of Ifng. Gata3 also facilitates Th2 cell proliferation via additional mechanisms that are far less well understood. We herein found that Gata3 associates with RuvB-like protein 2 (Ruvbl2) and represses the expression of a CDK inhibitor, cyclin-dependent kinase inhibitor 2c (Cdkn2c) to facilitate the proliferation of Th2 cells. Gata3 directly bound to the Cdkn2c locus in an Ruvbl2-dependent manner. The defect in the proliferation of Gata3-deficient Th2 cells is rescued by the knockdown of Cdkn2c, indicating that Cdkn2c is a key molecule involved in the Gata3-mediated induction of Th2 cell proliferation. Ruvbl2-knockdown Th2 cells showed decreased antigen-induced expansion and caused less airway inflammation in vivo. We therefore have identified a functional Gata3/Ruvbl2 complex that regulates the proliferation of differentiating Th2 cells through the repression of a CDK inhibitor, Cdkn2c.

Thpok-independent repression of Runx3 by Gata3 during CD4+ T-cell differentiation in the thymus.

CD4+ helper T cells are essential for immune responses and differentiate in the thymus from CD4+CD8+ “double‐positive” (DP) thymocytes. The transcription factor Runx3 inhibits CD4+ T‐cell differentiation by repressing Cd4 gene expression; accordingly, Runx3 is not expressed in DP thymocytes or developing CD4+ T cells. The transcription factor Thpok is upregulated in CD4‐differentiating thymocytes and required to repress Runx3. However, how Runx3 is controlled at early stages of CD4+ T‐cell differentiation, before the onset of Thpok expression, remains unknown. Here we show that Gata3, a transcription factor preferentially and transiently upregulated by CD4+ T‐cell precursors, represses Runx3 and binds the Runx3 locus in vivo. Accordingly, we show that high‐level Gata3 expression and expression of Runx3 are mutually exclusive. Furthermore, whereas Runx3 represses Cd4, we show that Gata3 promotes Cd4 expression in Thpok‐deficient thymocytes. Thus, in addition to its previously documented role in promoting CD4‐lineage gene‐expression, Gata3 represses CD8‐lineage gene expression. These findings identify Gata3 as a critical pivot of CD4‐CD8 lineage differentiation.

Trithorax complex component Menin controls differentiation and maintenance of T helper 17 cells

Significance Epigenetic modifications, including various histone modifications, play important roles in regulating gene expression. The Trithorax group (TrxG) complex induces permissive histone modifications to activate transcription. We herein investigate the role for Menin, a component of the TrxG complex, in T helper (Th) cell differentiation, and find a critical role for Menin in differentiation and maintenance of Th17 cells. Menin is required for Th17 cell differentiation in vitro through the direct regulation of Il17a expression. Menin controls IL-17–mediated pathology in vivo. Menin is also required to maintain expression of Rorc, the gene encoding RORγt, a key transcription factor for Th17 cell function. Thus, Menin orchestrates Th17 cell differentiation and function by regulating both induction and maintenance of target gene expression. Epigenetic modifications, such as posttranslational modifications of histones, play an important role in gene expression and regulation. These modifications are in part mediated by the Trithorax group (TrxG) complex and the Polycomb group (PcG) complex, which activate and repress transcription, respectively. We herein investigate the role of Menin, a component of the TrxG complex in T helper (Th) cell differentiation and show a critical role for Menin in differentiation and maintenance of Th17 cells. Menin−/− T cells do not efficiently differentiate into Th17 cells, leaving Th1 and Th2 cell differentiation intact in in vitro cultures. Menin deficiency resulted in the attenuation of Th17-induced airway inflammation. In differentiating Th17 cells, Menin directly bound to the Il17a gene locus and was required for the deposition of permissive histone modifications and recruitment of the RNA polymerase II transcriptional complex. Interestingly, although Menin bound to the Rorc locus, Menin was dispensable for the induction of Rorc expression and permissive histone modifications in differentiating Th17 cells. In contrast, Menin was required to maintain expression of Rorc in differentiated Th17 cells, indicating that Menin is essential to stabilize expression of the Rorc gene. Thus, Menin orchestrates Th17 cell differentiation and function by regulating both the induction and maintenance of target gene expression.