Abstract
To determine the respective contribution of the LAT transmembrane adaptor and CD5 and CD6 transmembrane receptors to early TCR signal propagation, diversification, and termination, we describe a CRISPR/Cas9-based platform that uses primary mouse T cells and permits establishment of the composition of their LAT, CD5, and CD6 signalosomes in only 4 mo using quantitative mass spectrometry. We confirmed that positive and negative functions can be solely assigned to the LAT and CD5 signalosomes, respectively. In contrast, the TCR-inducible CD6 signalosome comprised both positive (SLP-76, ZAP70, VAV1) and negative (UBASH3A/STS-2) regulators of T cell activation. Moreover, CD6 associated independently of TCR engagement to proteins that support its implication in inflammatory pathologies necessitating T cell transendothelial migration. The multifaceted role of CD6 unveiled here accounts for past difficulties in classifying it as a coinhibitor or costimulator. Congruent with our identification of UBASH3A within the CD6 signalosome and the view that CD6 constitutes a promising target for autoimmune disease treatment, single-nucleotide polymorphisms associated with human autoimmune diseases have been found in the Cd6 and Ubash3a genes.
Highlights
Following TCR triggering, the LAT transmembrane adaptor assembles a multimolecular signaling complex known as the LAT signalosome (Balagopalan et al, 2010)
Homology-directed repair (HDR) template coding for (1) a 100bp-long Lat 59 homology arm; (2) an OST tag flanked on both sides by a Gly-Ser-Gly spacer; (3) a 19-aa-long self-cleaving peptide of the porcine teschovirus-1 2A, known as P2A (Kim et al, 2011); (4) a sequence coding for CD90.1, a protein expressed at the T cell surface; and (5) a 100-bp-long Lat 39 homology arm (Fig. 1 A)
We developed a fast-track interactomics approach permitting assessment of the composition and dynamics of signalosomes assembling in primary T cells in response to TCR stimulation
Summary
Following TCR triggering, the LAT transmembrane adaptor assembles a multimolecular signaling complex known as the LAT signalosome (Balagopalan et al, 2010). The LAT signalosome ensures the propagation and diversification of TCR signals, it does not work in isolation, and other T cell surface receptors regulate early T cell activation. It remains to determine the composition of the LAT, CD5, and CD6 signalosomes in primary T cells and quantify their respective contributions to early TCR signal propagation and termination. On T cells, it colocalizes with the TCR at the immunological synapse (IS) and negatively regulates TCR signals in response to foreign peptides bound to MHC molecules (Azzam et al, 2001; Brossard et al, 2003; Peña-Rossi et al, 1999; Tarakhovsky et al, 1995). Recent data suggest that CD5 constitutes the main T cell–surface receptor capable of recruiting the E3 ubiquitin-protein ligases CBL and CBLB in response to TCR stimulation, thereby promoting ubiquitylation of colocalized signaling effectors (Voisinne et al, 2016)
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