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Elucidating the mechanisms that controlled T cell activation requires visualization of the spatial organization of multiple proteins on the submicron scale. Here, we use stoichiometrically accurate, multiplexed, single-molecule super-resolution microscopy (DNA-PAINT) to image the nanoscale spatial architecture of the primary inhibitor of the T cell signaling pathway, Csk, and two binding partners implicated in its membrane association, PAG and TRAF3. Combined with a newly developed co-clustering analysis framework, we find that Csk forms nanoscale clusters proximal to the plasma membrane that are lost post-stimulation and are re-recruited at later time points. Unexpectedly, these clusters do not co-localize with PAG at the membrane but instead provide a ready pool of monomers to downregulate signaling. By generating CRISPR-Cas9 knockout T cells, our data also identify that a major risk factor for autoimmune diseases, the protein tyrosine phosphatase non-receptor type 22 (PTPN22) locus, is essential for Csk nanocluster re-recruitment and for maintenance of the synaptic PAG population., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations in the adaptor molecule LAT can lead to autoimmunity. In this issue of Immunity, Mingueneau et al. (2009) describe how this may not be a failure of central tolerance.

The balance between kinases and phosphatases is crucial for regulating lymphocyte signaling. In this issue, McNeill et al. (2007) show that the transmembrane phosphatase CD45 has a role as both positive and negative regulator of T cell signaling.

CD4 and CD8 genes are integral indicators of T-cell lineage commitment. New insights into the control of expression of these genes have come from recent reports implicating chromatin-remodelling factors in their regulation.

The T lymphocyte-specific protein tyrosine kinase p56lck (Lck) is an essential component of the TCR-mediated signal transduction complex. Lck knockout mice have reduced numbers of double-positive thymocytes and very few mature single-positive cells, particularly of the CD4 lineage. Here we demonstrate the ability of a tetracycline-based tissue-specific inducible Lck transgene to restore expansion of early thymocytes and maturation of single-positive cells in Lckneg mice upon induction with doxycycline. Restoration of Lck expression is particularly important for positive selection to the CD4+ lineage but has a lesser impact on selection to the CD8+ lineage, suggesting activation of Lck is an important component of the signals involved in lineage choice during thymic differentiation.

The T cell repertoire is shaped by positive and negative selection of thymocytes that express low levels of T cell receptor (TCR) and both CD4 and CD8. TCR-mediated signals that determine these selection processes are only partly understood. Vav, a GDP-GTP exchange factor for Rho-family proteins, is tyrosine phosphorylated following TCR stimulation, suggesting that it may transduce TCR signals. We now demonstrate that mice lacking Vav are viable and display a profound defect in the positive selection of both class I- and class II-restricted T cells. In contrast, Vav is not essential for negative selection, though in its absence negative selection is much less effective. Vav may influence the efficiency of TCR-induced selection events by regulating the intracellular calcium flux of thymocytes.

The T cell receptor alpha and beta chain genes donated by an H-2 class I-specific, CD8-dependent cytotoxic T cell clone were transferred, alone or in combination with the Lyt-2 gene, into a class II-restricted, CD4+ T cell hybridoma. Two important points emerged. First, the alpha and beta T cell receptor genes endowed the recipient with the H-2 class I specificity of the donor only if the same cell had also been transfected with the Lyt-2 gene. Second, the functional Lyt-2 molecule was expressed on the transfected cells in the absence of the Lyt-3 polypeptide. These results demonstrate that, besides the T cell receptor, the Lyt-2 polypeptide is the only subset-specific molecule required to retarget a class II-reactive, CD4+ T cell line toward H-2 class I molecules.

The Lyt-2/3 molecule is a glycoprotein expressed on T lymphocytes and has classically been considered a marker for the cytotoxic/suppressor T cell subset. It has been postulated to be a receptor for class I major histocompatibility complex proteins. We have used a cDNA clone encoding the analogous human protein, Leu-2/T8, to isolate mouse cDNA clones, which were used as probes to isolate mouse genomic clones. By transfection we have shown that the mouse homologue of Leu-2/T8 is Lyt-2 and not Lyt-3. We have further demonstrated that two Lyt-2 polypeptide chains are encoded by a single gene and result from alternative modes of mRNA splicing. The nucleotide sequence of cDNA clones encoding each of these polypeptide chains has been determined and shows the difference between the two Lyt-2 polypeptide chains to be in the lengths of their cytoplasmic tails.