Interleukin-4 regulates eomesodermin in CD8+ T cell development and differentiation.
ABSTRACT: Interleukin (IL)-4 is a cytokine classically associated with CD4(+) T helper type 2 differentiation, but has been recently shown to also be required for the development of CD8(+) innate-like lymphocytes. CD8(+) innate-like lymphocytes are non-conventional lymphocytes that exhibit characteristics typically associated with memory CD8(+) T cells, including expression of the T-box transcription factor Eomesodermin (Eomes). Here we investigate the signaling pathways required for IL-4 induction of Eomes and CD8(+) innate-like lymphocyte markers in murine CD8SP thymocytes and peripheral CD8(+) T cells. We demonstrate that IL-4 is sufficient to drive Eomes expression and the CD8(+) innate-like lymphocyte phenotype through cooperation between STAT6- and Akt-dependent pathways. Furthermore, we show that while IL-4 has little effect on the induction of Eomes in the setting of robust T cell receptor (TCR) activation, this cytokine promotes Eomes in the setting of attenuated TCR stimulation in mature CD8(+) T cells suggesting that cytokine signaling pathways may direct cell fate when TCR signals are limiting.
Project description:T cell development in the thymus produces multiple lineages of cells, including innate T cells such as ?? TCR(+) cells, invariant NKT cells, mucosal-associated invariant T cells, and H2-M3-specific cells. Although innate cells are generally a minor subset of thymocytes, in several strains of mice harboring mutations in T cell signaling proteins or transcriptional regulators, conventional CD8(+) T cells develop as innate cells with characteristics of memory T cells. Thus, in Itk-deficient mice, mature CD4(-)CD8(+) (CD8 single-positive [SP]) thymocytes express high levels of the transcription factor eomesodermin (Eomes) and are dependent on IL-4 being produced in the thymic environment by a poorly characterized subset of CD4(+) thymocytes expressing the transcriptional regulator promyelocytic leukemia zinc finger. In this study, we show that a sizeable proportion of mature CD4(+)CD8(-) (CD4SP) thymocytes in itk(-/-) mice also develop as innate Eomes-expressing T cells. These cells are dependent on MHC class II and IL-4 signaling for their development, indicating that they are conventional CD4(+) T cells that have been converted to an innate phenotype. Surprisingly, neither CD4SP nor CD8SP innate Eomes(+) thymocytes in itk(-/-) or SLP-76(Y145F) mice are dependent on ?? T cells for their development. Instead, we find that the predominant population of Eomes(+) innate itk(-/-) CD4SP thymocytes is largely absent in mice lacking CD1d-specific invariant NKT cells, with no effect on innate itk(-/-) CD8SP thymocytes. In contrast, both subsets of innate Eomes(+)itk(-/-) T cells require the presence of a novel promyelocytic leukemia zinc finger-expressing, SLAM family receptor adapter protein-dependent thymocyte population that is essential for the conversion of conventional CD4(+) and CD8(+) T cells into innate T cells with a memory phenotype.
Project description:CD8+ T cell exhaustion commonly occurs in chronic infections and cancers. During T cell exhaustion there is a progressive and hierarchical loss of effector cytokine production, up-regulation of inhibitory co-stimulatory molecules, and eventual deletion of antigen specific cells by apoptosis. A key factor that regulates T cell exhaustion is persistent TCR stimulation. Loss of this interaction results in restoration of CD8+ T cell effector functions in previously exhausted CD8+ T cells. TCR stimulation is also important for the differentiation of Eomeshi anti-viral CD8+ effector T cells from T-bethi precursors, both of which are required for optimal viral control. However, the molecular mechanisms regulating the differentiation of these two cell subsets and the relative ratios required for viral clearance have not been described. We show that TCR signal strength regulates the relative expression of T-bet and Eomes in antigen-specific CD8+ T cells by modulating levels of IRF4. Reduced IRF4 expression results in skewing of this ratio in the favor of Eomes, leading to lower proportions and numbers of T-bet+ Eomes- precursors and poor control of LCMV-clone 13 infection. Manipulation of this ratio in the favor of T-bet restores the differentiation of T-bet+ Eomes- precursors and the protective balance of T-bet to Eomes required for efficient viral control. These data highlight a critical role for IRF4 in regulating protective anti-viral CD8+ T cell responses by ensuring a balanced ratio of T-bet to Eomes, leading to the ultimate control of this chronic viral infection.
Project description:Conventional and nonconventional T cell development occur in the thymus. Nonconventional thymocytes that bear characteristics typically associated with innate immune cells are termed innate-like lymphocytes (ILLs). Mice harboring a tyrosine to phenylalanine mutation in the adaptor protein Src homology 2 domain-containing leukocyte protein of 76 kDa at residue 145 (Y145F mice) develop an expanded population of CD8(+)CD122(+)CD44(+) ILLs, typified by expression of the T-box transcription factor eomesodermin. Y145F mice also have an expanded population of ?? T cells that produce copious amounts of IL-4 via a mechanism that is dependent on the BTB-ZF transcription factor promyelocytic leukemia zinc finger. Using mice with T cell-specific deletion of Eomes, we demonstrate that this transcription factor is required for CD8(+) ILL development in Y145F as well as wild-type mice. Moreover, we show that promyelocytic leukemia zinc finger and IL-4 are also required for the generation of this ILL population. Taken together, these data shed light on the cell-intrinsic and cell-extrinsic factors that drive CD8(+) ILL differentiation.
Project description:T-cell memory is critical for long-term immunity. However, the factors involved in maintaining the persistence, function, and phenotype of the memory pool are undefined. Eomesodermin (Eomes) is required for the establishment of the memory pool. Here, we show that in T cells transitioning to memory, the expression of high levels of Eomes is not constitutive but rather requires a continuum of cell-intrinsic NFκB signaling. Failure to maintain NFκB signals after the peak of the response led to impaired Eomes expression and a defect in the maintenance of CD8 T-cell memory. Strikingly, we found that antigen receptor [T-cell receptor (TCR)] signaling regulates this process through expression of the NFκB-dependent kinase proviral integration site for Moloney murine leukemia virus-1 (PIM-1), which in turn regulates NFκB and Eomes. T cells defective in TCR-dependent NFκB signaling were impaired in late expression of Pim-1, Eomes, and CD8 memory. These defects were rescued when TCR-dependent NFκB signaling was restored. We also found that NFκB-Pim-1 signals were required at memory to maintain memory CD8 T-cell longevity, effector function, and Eomes expression. Hence, an NFκB-Pim-1-Eomes axis regulates Eomes levels to maintain memory fitness.
Project description:Memory CD8+ T cells have the ability to provide lifelong immunity against pathogens. Although memory features generally arise after challenge with a foreign antigen, naïve CD8 single positive (SP) thymocytes may acquire phenotypic and functional characteristics of memory cells in response to cytokines such as interleukin-4. This process is associated with the induction of the T-box transcription factor Eomesodermin (EOMES). However, the underlying molecular mechanisms remain ill-defined. Using epigenomic profiling, we show that these innate memory CD8SP cells acquire only a portion of the active enhancer repertoire of conventional memory cells. This reprograming is secondary to EOMES recruitment, mostly to RUNX3-bound enhancers. Furthermore, EOMES is found within chromatin-associated complexes containing BRG1 and promotes the recruitment of this chromatin remodelling factor. Also, the in vivo acquisition of EOMES-dependent program is BRG1-dependent. In conclusion, our results support a strong epigenetic basis for the EOMES-driven establishment of CD8+ T cell innate memory program.
Project description:CD8(+) T-cell development in the thymus generates a predominant population of conventional naive cells, along with minor populations of "innate" T cells that resemble memory cells. Recent studies analyzing a variety of KO or knock-in mice have indicated that impairments in the T-cell receptor (TCR) signaling pathway produce increased numbers of innate CD8(+) T cells, characterized by their high expression of CD44, CD122, CXCR3, and the transcription factor, Eomesodermin (Eomes). One component of this altered development is a non-CD8(+) T cell-intrinsic role for IL-4. To determine whether reduced TCR signaling within the CD8(+) T cells might also contribute to this pathway, we investigated the role of the transcription factor, IFN regulatory factor 4 (IRF4). IRF4 is up-regulated following TCR stimulation in WT T cells; further, this up-regulation is impaired in T cells treated with a small-molecule inhibitor of the Tec family tyrosine kinase, IL-2 inducible T-cell kinase (ITK). In contrast to WT cells, activation of IRF4-deficient CD8(+) T cells leads to rapid and robust expression of Eomes, which is further enhanced by IL-4 stimulation. In addition, inhibition of ITK together with IL-4 increases Eomeso up-regulation. These data indicate that ITK signaling promotes IRF4 up-regulation following CD8(+) T-cell activation and that this signaling pathway normally suppresses Eomes expression, thereby regulating the differentiation pathway of CD8(+) T cells.
Project description:Interleukin (IL)-7 is a cytokine essential for T lymphocyte development and homeostasis. However, little is known about the roles of IL-7 receptor ?-chain (IL-7R?) in late stages of T-cell development. To address this question, we established IL-7R?-floxed mice and crossed them with CD4-Cre transgenic mice. Resultant IL-7R conditional knockout (IL-7RcKO) mice exhibited marked reduction in CD8 single positive (SP) T cells, regulatory T cells (Tregs), and natural killer T (NKT) cells in thymus. The proportion and proliferation of both mature CD4SP and CD8SP thymocytes were decreased without affecting Runx expression. In addition, expression of the glucocorticoid-induced TNF receptor was reduced in CD4SP and CD8SP thymocytes, and expression of CD5 was decreased in CD8SP thymocytes. IL-7RcKO mice also showed impaired Treg and NKT cell proliferation and inhibition of NKT cell maturation. Bcl-2 expression was reduced in CD4SP and CD8SP thymocytes but not in Tregs and NKT cells, and introduction of a Bcl-2 transgene rescued frequency and CD5 expression of CD8SP thymocytes. Furthermore, IL-7RcKO mice exhibited greatly increased numbers of B cells and, to a lesser extent, ?? T and dendritic cells in thymus. Overall, this study demonstrates that IL-7R? differentially controls development and maturation of thymocyte subpopulations in late developmental stages and suggests that IL-7R expression on ?? T cells suppresses development of other cell lineages in thymus.
Project description:CD8+ T cell differentiation is controlled by the transcription factors T-bet and Eomesodermin, in concert with the cytokines IL-2, IL-10 and IL-12. Among these pathways, the mechanisms by which T-box proteins and IL-10 interact to promote a memory T cell fate remain poorly understood. Here, we show that Eomes and IL-10 drive a central memory phenotype in murine CD8+ T cells. Eomes expression led to increased IL-10 expression by the effector CD8+ T cells themselves as well as an increase in the level of the lymph node homing selectin CD62L. Furthermore, exposure of effector CD8+ T cells to IL-10 maintained CD62L expression levels in culture. Thus, Eomes promotes a step-wise transition of effector T cells towards a memory phenotype, synergizing with IL-10 to enhance the expression of CD62L. The early augmentation of lymph node homing markers by Eomes may facilitate the retention of effector T cells in the relatively low inflammatory milieu of the secondary lymphoid organs that promotes central memory development.
Project description:A hallmark of autoimmune lymphoproliferative syndrome (ALPS), caused by mutation of the Fas death receptor, is massive lymphadenopathy from aberrant expansion of CD4(-)CD8(-) (double-negative [DN]) T cells. Eomesodermin (Eomes) is a member of the T-box family of transcription factors and plays critical roles in effector cell function and memory cell fitness of CD8(+) T lymphocytes. We provide evidence in this study that DN T cells exhibit dysregulated expression of Eomes in humans and mice with ALPS. We also find that T cell-specific deletion of Eomes prevents lymphoid hypertrophy and accumulation of DN T cells in Fas-mutant mice. Although Eomes has critical physiological roles in the function and homeostasis of CD8(+) T cells, overexpression of Eomes appears to enable pathological induction or expansion of unusual CD8-related T cell subsets. Thus, antagonism of Eomes emerges as a therapeutic target for DN T cell ablation in ALPS.
Project description:CD8(+) T cells responding to intracellular infection give rise to cellular progeny that become terminally differentiated effector cells and self-renewing memory cells. T-bet and eomesodermin (Eomes) are key transcription factors of cytotoxic lymphocyte lineages. We show in this study that CD8(+) T cells lacking Eomes compete poorly in contributing to the pool of Ag-specific central memory cells. Eomes-deficient CD8(+) T cells undergo primary clonal expansion but are defective in long-term survival, populating the bone marrow niche and re-expanding postrechallenge. The phenotype of Eomes-deficient CD8(+) T cells supports the hypothesis that T-bet and Eomes can act redundantly to induce effector functions, but can also act to reciprocally promote terminal differentiation versus self-renewal of Ag-specific memory cells.