CD45-Csk Phosphatase-Kinase Titration Uncouples Basal and Inducible T Cell Receptor Signaling during Thymic Development

Division of Rheumatology, University of California San Francisco, San Francisco, CA 94143, USA.
Immunity (Impact Factor: 21.56). 03/2010; 32(3):342-54. DOI: 10.1016/j.immuni.2010.03.006
Source: PubMed


The kinase-phosphatase pair Csk and CD45 reciprocally regulate phosphorylation of the inhibitory tyrosine of the Src family kinases Lck and Fyn. T cell receptor (TCR) signaling and thymic development require CD45 expression but proceed constitutively in the absence of Csk. Here, we show that relative titration of CD45 and Csk expression reveals distinct regulation of basal and inducible TCR signaling during thymic development. Low CD45 expression is sufficient to rescue inducible TCR signaling and positive selection, whereas high expression is required to reconstitute basal TCR signaling and beta selection. CD45 has a dual positive and negative regulatory role during inducible but not basal TCR signaling. By contrast, Csk titration regulates basal but not inducible signaling. High physiologic expression of CD45 is thus required for two reasons-to downmodulate inducible TCR signaling during positive selection and to counteract Csk during basal TCR signaling.

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Available from: Arthur Weiss, Jun 30, 2014
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    • "Since no studies to date have uncovered profound effects of LYP-W620 on thymic selection, an alternative explanation is that TCR signaling in thymocytes might be controlled by multiple redundant phosphatases. LYP-dependent effects might not be detectable in the presence of a dominant regulator such as CD45, which plays a major role in both positive and negative regulation of TCR signaling in double positive and single positive thymocytes [53]. "
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    ABSTRACT: A C1858T (R620W) variation in the PTPN22 gene encoding the tyrosine phosphatase LYP is a major risk factor for human autoimmunity. LYP is a known negative regulator of signaling through the T cell receptor (TCR), and murine Ptpn22 plays a role in thymic selection. However, the mechanism of action of the R620W variant in autoimmunity remains unclear. One model holds that LYP-W620 is a gain-of-function phosphatase that causes alterations in thymic negative selection and/or thymic output of regulatory T cells (Treg) through inhibition of thymic TCR signaling. To test this model, we generated mice in which the human LYP-W620 variant or its phosphatase-inactive mutant are expressed in developing thymocytes under control of the proximal Lck promoter. We found that LYP-W620 expression results in diminished thymocyte TCR signaling, thus modeling a "gain-of-function" of LYP at the signaling level. However, LYP-W620 transgenic mice display no alterations of thymic negative selection and no anomalies in thymic output of CD4(+)Foxp3(+) Treg were detected in these mice. Lck promoter-directed expression of the human transgene also causes no alteration in thymic repertoire or increase in disease severity in a model of rheumatoid arthritis, which depends on skewed thymic selection of CD4(+) T cells. Our data suggest that a gain-of-function of LYP is unlikely to increase risk of autoimmunity through alterations of thymic selection and that LYP likely acts in the periphery perhaps selectively in regulatory T cells or in another cell type to increase risk of autoimmunity.
    PLoS ONE 02/2014; 9(2):e86677. DOI:10.1371/journal.pone.0086677 · 3.23 Impact Factor
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    • "How the engaged TCR couples to Src kinase-mediated ITAM phosphorylation is an important question that has led to various models mainly based on clustering or conformational changes of the TCR/CD3 complex [37], [38]. In agreement with very recent data [17], [39], we found that unstimulated T cells are equipped with a pool of catalytically active Lck and Fyn that appears to be substantial and not upregulated in response to TCR stimulation in naïve CD4+ T cells. For the first time, we provide evidence for a positive feedback loop between LAT and the TCR/Src kinase signaling modules in which LAT promotes TCR signal initiation through active Lck and Fyn. "
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    ABSTRACT: One of the earliest activation events following stimulation of the T cell receptor (TCR) is the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD3-associated complex by the Src family kinase Lck. There is accumulating evidence that a large pool of Lck is constitutively active in T cells but how the TCR is connected to Lck and to the downstream signaling cascade remains elusive. We have analyzed the phosphorylation state of Lck and Fyn and TCR signaling in human naïve CD4+ T cells and in the transformed T cell line, Hut-78. The latter has been shown to be similar to primary T cells in TCR-inducible phosphorylations and can be highly knocked down by RNA interference. In both T cell types, basal phosphorylation of Lck and Fyn on their activatory tyrosine was observed, although this was much less pronounced in Hut-78 cells. TCR stimulation led to the co-precipitation of Lck with the transmembrane adaptor protein LAT (linker for activation of T cells), Erk-mediated phosphorylation of Lck and no detectable dephosphorylation of Lck inhibitory tyrosine. Strikingly, upon LAT knockdown in Hut-78 cells, we found that LAT promoted TCR-induced phosphorylation of Lck and Fyn activatory tyrosines, TCRζ chain phosphorylation and Zap-70 activation. Notably, LAT regulated these events at low strength of TCR engagement. Our results indicate for the first time that LAT promotes TCR signal initiation and suggest that this adaptor may contribute to maintain active Lck in proximity of their substrates.
    PLoS ONE 11/2010; 5(11):e15114. DOI:10.1371/journal.pone.0015114 · 3.23 Impact Factor
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    ABSTRACT: For over three decades now, the T cell receptor (TCR) for antigen has not ceased to challenge the imaginations of cellular and molecular immunologists alike. T cell antigen recognition transcends every aspect of adaptive immunity: it shapes the T cell repertoire in the thymus and directs T cell-mediated effector functions in the periphery, where it is also central to the induction of peripheral tolerance. Yet, despite its central position, there remain many questions unresolved: how can one TCR be specific for one particular peptide-major histocompatibility complex (pMHC) ligand while also binding other pMHC ligands with an immunologically relevant affinity? And how can a T cell's extreme specificity (alterations of single methyl groups in their ligand can abrogate a response) and sensitivity (single agonist ligands on a cell surface are sufficient to trigger a measurable response) emerge from TCR-ligand interactions that are so low in affinity? Solving these questions is intimately tied to a fundamental understanding of molecular recognition dynamics within the many different contexts of various T cell-antigen presenting cell (APC) contacts: from the thymic APCs that shape the TCR repertoire and guide functional differentiation of developing T cells to the peripheral APCs that support homeostasis and provoke antigen responses in naïve, effector, memory, and regulatory T cells. Here, we discuss our recent findings relating to T cell antigen recognition and how this leads to the thymic development of foreign-antigen-responsive alphabetaT cells.
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