Themis, a T cell-specific protein important for late thymocyte development.
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ABSTRACT:
SUBMITTER: Lesourne R
PROVIDER: S-EPMC2848698 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Thymocyte-expressed molecule involved in selection (THEMIS) is a recently identified regulator of thymocyte positive selection. THEMIS's mechanism of action is unknown, and whether it has a role in TCR-proximal signaling is controversial. In this article, we show that THEMIS and the adapter molecule growth factor receptor-bound protein 2 (GRB2) associate constitutively through binding of a conserved PxRPxK motif within the proline-rich region 1 of THEMIS to the C-terminal SH3-domain of GRB2. This association is indispensable for THEMIS recruitment to the immunological synapse via the transmembrane adapter linker for activation of T cells (LAT) and for THEMIS phosphorylation by Lck and ZAP-70. Two major sites of tyrosine phosphorylation were mapped to a YY-motif close to proline-rich region 1. The YY-motif was crucial for GRB2 binding, suggesting that this region of THEMIS might control local phosphorylation-dependent conformational changes important for THEMIS function. Finally, THEMIS binding to GRB2 was required for thymocyte development. Our data firmly assign THEMIS to the TCR-proximal signaling cascade as a participant in the LAT signalosome and suggest that the THEMIS-GRB2 complex might be involved in shaping the nature of Ras signaling, thereby governing thymic selection.
| S-EPMC3607403 | biostudies-literature
Project description:Themis (thymocyte-expressed molecule involved in selection), a member of a family of proteins with unknown functions, is highly conserved among vertebrates. Here we found that Themis had high expression in thymocytes between the pre-T cell antigen receptor (pre-TCR) and positive-selection checkpoints and low expression in mature T cells. Themis-deficient thymocytes showed defective positive selection, which resulted in fewer mature thymocytes. Negative selection was also impaired in Themis-deficient mice. A greater percentage of Themis-deficient T cells had CD4(+)CD25(+)Foxp3(+) regulatory and CD62L(lo)CD44(hi) memory phenotypes than did wild-type T cells. In support of the idea that Themis is involved in TCR signaling, this protein was phosphorylated quickly after TCR stimulation and was needed for optimal TCR-driven calcium mobilization and activation of the kinase Erk.
| S-EPMC2757056 | biostudies-literature
Project description:Basigin is a highly glycosylated transmembrane protein that is expressed in a broad range of tissues and is involved in a number of physiological and pathological processes. However, the in vivo role of basigin remains unknown. To better understand the physiological and pathological functions of basigin in vivo, we generated a conditional null allele by introducing two loxP sites flanking exons 2 and 7 of the basigin gene (Bsg). Bsg(fl/fl) mice were born at the expected Mendelian ratio and showed a similar growth rate compared with wildtype mice. After crossing these mice with Lck-Cre transgenic mice, basigin expression was specifically inactivated in T cells in the resulting Lck-Cre; Bsg(fl/fl) mice. Although the birth and growth rate of Lck-Cre; Bsg(fl/fl) mice were similar to control mice, thymus development was partially arrested in Lck-Cre; Bsg(fl/fl) mice, specifically at the CD4(+)CD8(+) double-positive (DP) and CD4 single-positive (CD4(+)CD8(-), CD4SP) stages. In addition, CD4(+) T cell activation was enhanced upon Concanavalin A (Con A) or anti-CD3/anti-CD28 stimulation but not upon PMA/Ionomycin stimulation in the absence of basigin. Overall, this study provided the first in vivo evidence for the function of basigin in thymus development. Moreover, the successful generation of the conditional null basigin allele provides a useful tool for the study of distinct physiological or pathological functions of basigin in different tissues at different development stages.
| S-EPMC3879590 | biostudies-literature
Project description:Thymocyte-expressed molecule involved in selection (Themis) has been shown to be important for T cell selection by setting the threshold for positive versus negative selection. Themis interacts with the protein tyrosine phosphatase (PTP) Src-homology domain containing phosphatase-1 (Shp1), a negative regulator of the T cell receptor (TCR) signaling cascade. However, how Themis regulates Shp1 is still not clear. Here, using a very sensitive phosphatase assay on ex vivo thymocytes, we have found that Themis enhances Shp1 phosphatase activity by increasing its phosphorylation. This positive regulation of Shp1 activity by Themis is found in thymocytes, but not in peripheral T cells. Shp1 activity is modulated by different affinity peptide MHC ligand binding in thymocytes. Themis is also associated with phosphatase activity, due to its constitutive interaction with Shp1. In the absence of Shp1 in thymocytes, Themis interacts with Shp2, which leads to almost normal thymic development in Shp1 conditional knockout (cKO) mice. Double deletion of both Themis and Shp1 leads to a thymic phenotype similar to that of Themis KO. These findings demonstrate unequivocally that Themis positively regulates Shp1 phosphatase activity in TCR-mediated signaling in developing thymocytes.
| S-EPMC6275480 | biostudies-literature
Project description:Themis (also named Gasp) is a newly identified Grb2-binding protein that is essential for thymocyte positive selection. Despite the possible involvement of Themis in TCR-mediated signal transduction, its function remains unresolved and controversial. Themis contains two functionally uncharacterized regions called CABIT (cysteine-containing, all-? in Themis) domains, a nuclear localization signal (NLS), and a proline-rich sequence (PRS). To elucidate the role of these motifs in Themis's function in vivo, we established a series of mutant Themis transgenic mice on a Themis(-/-) background. Deletion of the highly conserved Core motif of CABIT1 or CABIT2 (Core1 or Core2, respectively), the NLS, or the PRS abolished Grb2-association, as well as TCR-dependent tyrosine-phosphorylation and the ability to induce positive selection in the thymus. The NLS and Core1 motifs were required for the nuclear localization of Themis, whereas Core2 and PRS were not. Furthermore, expression of ?Core1- but not ?Core2-Themis conferred dominant negative-type inhibition on T cell development. Collectively, our current results indicate that PRS, NLS, CABIT1, and CABIT2 are all required for positive selection, and that each of the CABIT domains exerts distinct functions during positive selection.
| S-EPMC3931654 | biostudies-other
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.
| S-EPMC3545802 | biostudies-other
Project description:In vertebrates, multiple transcription factors (TFs) bind to gene regulatory elements (promoters, enhancers, and silencers) to execute developmental expression changes. ChIP experiments are often used to identify where TFs bind to regulatory elements in the genome, but the requirement of TF-specific antibodies hampers analyses of tens of TFs at multiple loci. Here we tested whether TF binding predictions using ATAC-seq can be used to infer the identity of TFs that bind to functionally validated enhancers of the Cd4, Cd8, and Gata3 genes in thymocytes. We performed ATAC-seq at four distinct stages of development in mouse thymus, probing the chromatin accessibility landscape in double negative (DN), double positive (DP), CD4 single positive (SP4) and CD8 SP (SP8) thymocytes. Integration of chromatin accessibility with TF motifs genome-wide allowed us to infer stage-specific occupied TF binding sites within known and potentially novel regulatory elements. Our results provide genome-wide stage-specific T cell open chromatin profiles, and allow the identification of candidate TFs that drive thymocyte differentiation at each developmental stage.
| S-EPMC5884796 | biostudies-literature
Project description:Considerable cross-talk exists between mechanisms controlling genome architecture and gene expression. AgR loci are excellent models for these processes because they are regulated at both conformational and transcriptional levels to facilitate their assembly by V(D)J recombination. Upon commitment to the double-negative stage of T cell development, Tcrb adopts a compact conformation that promotes long-range recombination between Vβ gene segments (Trbvs) and their DβJβ targets. Formation of a functional VβDβJβ join signals for robust proliferation of double-negative thymocytes and their differentiation into double-positive (DP) cells, where Trbv recombination is squelched (allelic exclusion). DP differentiation also is accompanied by decontraction of Tcrb, which has been thought to separate the entire Trbv cluster from DβJβ segments (spatial segregation-based model for allelic exclusion). However, DP cells also repress transcription of unrearranged Trbvs, which may contribute to allelic exclusion. We performed a more detailed study of developmental changes in Tcrb topology and found that only the most distal portion of the Trbv cluster separates from DβJβ segments in DP thymocytes, leaving most Trbvs spatially available for rearrangement. Preferential dissociation of distal Trbvs is independent of robust proliferation or changes in transcription, chromatin, or architectural factors, which are coordinately regulated across the entire Trbv cluster. Segregation of distal Trbvs also occurs on alleles harboring a functional VβDβJβ join, suggesting that this process is independent of rearrangement status and is DP intrinsic. Our finding that most Trbvs remain associated with DβJβ targets in DP cells revises allelic exclusion models from their current conformation-dominant to a transcription-dominant formulation.
| S-EPMC4506872 | biostudies-literature
Project description:T cell antigen receptor (TCR) signaling in CD4(+)CD8(+) double-positive thymocytes determines cell survival and lineage commitment, but the genetic and molecular basis of this process is poorly defined. To address this issue, we used ethylnitrosourea mutagenesis to identify a previously unknown T lineage-specific gene, Themis, which is critical for the completion of positive selection. Themis contains a tandem repeat of a unique globular domain (called 'CABIT' here) that includes a cysteine motif that defines a family of five uncharacterized vertebrate proteins with orthologs in most animal species. Themis-deficient thymocytes showed no substantial impairment in early TCR signaling but did show altered expression of genes involved in the cell cycle and survival before and during positive selection. Our data suggest a unique function for Themis in sustaining positive selection.
| S-EPMC2908989 | biostudies-literature
Project description:Intrathymic differentiation of T lymphocytes begins as early as intrauterine stage, yet the T cell lineage decisions of human fetal thymocytes at different gestational ages are not currently understood. Here, we performed integrative single-cell analyses of thymocytes across gestational ages. We identified conserved candidates underlying the selection of T cell receptor (TCR) lineages in different human fetal stages. The trajectory of early thymocyte commitment during fetal growth was also characterized. Comparisons with mouse data revealed conserved and species-specific transcriptional dynamics of thymocyte proliferation, apoptosis and selection. Genome-wide association study (GWAS) data associated with multiple autoimmune disorders were analyzed to characterize susceptibility genes that are highly expressed at specific stages during fetal thymocyte development. In summary, our integrative map describes previously underappreciated aspects of human thymocyte development, and provides a comprehensive reference for understanding T cell lymphopoiesis in a self-tolerant and functional adaptive immune system.
| S-EPMC8284395 | biostudies-literature