Themis controls thymocyte selection through regulation of T cell receptor-mediated signaling

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA.
Nature Immunology (Impact Factor: 20). 09/2009; 10(8):848-56. DOI: 10.1038/ni.1766
Source: PubMed


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.

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    • "We have shown that soon after TCR stimulation, THEMIS associates to LAT, via the adapter protein GRB2, leading to THEMIS phosphorylation , and that this recruitment is required for THEMIS to function in T conv development (Paster et al, 2013). Despite the severe phenotype of THEMIS-deficient mice and initial hints that THEMIS was implicated in regulating TCR signalling (Fu et al, 2009; Brockmeyer et al, 2011), its precise molecular and functional role has remained obscure. "
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    ABSTRACT: THEMIS is critical for conventional T-cell development, but its precise molecular function remains elusive. Here, we show that THEMIS constitutively associates with the phosphatases SHP1 and SHP2. This complex requires the adapter GRB2, which bridges SHP to THEMIS in a Tyr-phosphorylation-independent fashion. Rather, SHP1 and THEMIS engage with the N-SH3 and C-SH3 domains of GRB2, respectively, a configuration that allows GRB2-SH2 to recruit the complex onto LAT. Consistent with THEMIS-mediated recruitment of SHP to the TCR signalosome, THEMIS knock-down increased TCR-induced CD3-ζ phosphorylation, Erk activation and CD69 expression, but not LCK phosphorylation. This generalized TCR signalling increase led to augmented apoptosis, a phenotype mirrored by SHP1 knock-down. Remarkably, a KI mutation of LCK Ser59, previously suggested to be key in ERK-mediated resistance towards SHP1 negative feedback, did not affect TCR signalling nor ligand discrimination in vivo. Thus, the THEMIS:SHP complex dampens early TCR signalling by a previously unknown molecular mechanism that favours T-cell survival. We discuss possible implications of this mechanism in modulating TCR output signals towards conventional T-cell development and differentiation. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.
    The EMBO Journal 12/2014; 34(3). DOI:10.15252/embj.201387725 · 10.43 Impact Factor
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    • "In the Themis-knockout mice, however, results were different. We and other groups observed unaltered activation of ERK and calcium influx in Themis deficient immature DP thymocyte upon anti-CD3 antibody stimulation [2-3-4], although one group reported impaired activation of these signaling events [5]. Moreover, recent study showed that TCR-dependent activation of ERK, p38 and Vav1 were reduced in Themis deficient CD4SP and CD8SP thymocytes [9]. "
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    ABSTRACT: 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-β 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 ΔCore1- but not ΔCore2-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.
    PLoS ONE 02/2014; 9(2):e89115. DOI:10.1371/journal.pone.0089115 · 3.23 Impact Factor
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    • "For example, the Rag1, Rag2 and Themis mRNAs, which are involved in the differentiation and maturation of thymocytes, were regulated in both strains (Figure 6). Themis is expressed in a tightly regulated manner during T cell development in late double-negative (DN) and especially in double-positive (DP) thymocytes [46]. It is down-regulated after positive selection and is expressed at low levels in mature T cells. "
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    ABSTRACT: Due to recent studies indicating that the deregulation of microRNAs (miRNAs) in T cells contributes to increased severity of rheumatoid arthritis, we hypothesized that deregulated miRNAs may interact with key mRNA targets controlling the function or differentiation of these cells in this disease. To test our hypothesis, we used microarrays to survey, for the first time, the expression of all known mouse miRNAs in parallel with genome-wide mRNAs in thymocytes and naïve and activated peripheral CD3(+) T cells from two mouse strains the DBA-1/J strain (MHC-H2q), which is susceptible to collagen induced arthritis (CIA), and the DBA-2/J strain (MHC-H2d), which is resistant. Hierarchical clustering of data showed the several T cell miRNAs and mRNAs differentially expressed between the mouse strains in different stages of immunization with collagen. Bayesian statistics using the GenMir(++) algorithm allowed reconstruction of post-transcriptional miRNA-mRNA interaction networks for target prediction. We revealed the participation of miR-500, miR-202-3p and miR-30b*, which established interactions with at least one of the following mRNAs: Rorc, Fas, Fasl, Il-10 and Foxo3. Among the interactions that were validated by calculating the minimal free-energy of base pairing between the miRNA and the 3'UTR of the mRNA target and luciferase assay, we highlight the interaction of miR-30b*-Rorc mRNA because the mRNA encodes a protein implicated in pro-inflammatory Th17 cell differentiation (Rorγt). FACS analysis revealed that Rorγt protein levels and Th17 cell counts were comparatively reduced in the DBA-2/J strain. This result showed that the miRNAs and mRNAs identified in this study represent new candidates regulating T cell function and controlling susceptibility and resistance to CIA.
    PLoS ONE 06/2013; 8(1):e54803. DOI:10.1371/journal.pone.0054803 · 3.23 Impact Factor
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