Citations

AbstractStudying signal transduction in skin‐resident T cells (sr‐T cells) can be limited by the small size of clinical biopsies. Here, we isolated sr‐T cells from clinical samples and analysed signalling protein complexes by multiplex immunoprecipitation detected by flow cytometry (mIP‐FCM). In samples from two independent donors, antigenic stimulation induced signalling proteins to join shared complexes that were observed in seven pairwise combinations among five proteins. This demonstrates that sr‐T cells isolated from small clinical samples provide sufficient material for mIP‐FCM‐based analysis of signalling‐induced protein complexes. We propose that this strategy may be useful for gaining improved mechanistic insight of sr‐T cell signal transduction associated with dermatological disease.

Abstract Unlike BCR and secreted Ig, TCR expression is not thought to occur in a bivalent form. The conventional monovalent model of TCR/CD3 is supported by published studies of complexes solubilized in the detergent digitonin, in which bivalency was not observed. We revisited the issue of TCR valency by examining complexes isolated from primary αβ T cells after solubilization in digitonin. Using immunoprecipitation followed by flow cytometry, we unexpectedly observed TCR/CD3 complexes that contained two TCRs per complex. Standard anti-TCR Abs, being bivalent themselves, tended to bind with double occupancy to bivalent TCRs; this property masked the presence of the second TCR per complex in certain Ab binding assays, which may partially explain why previous data did not reveal these bivalent complexes. We also found that the prevalence of bivalency among fully assembled, mature TCR/CD3 complexes was sufficient to impact the functional performance of immunoprecipitated TCRs in binding antigenic peptide/MHC-Ig fusion proteins. Both TCR positions per bivalent complex required an Ag-specific TCR to effect optimal binding to these soluble ligands. Therefore, we conclude that in primary T cells, TCR/CD3 complexes can be found that are physically and functionally bivalent. The expression of bivalent TCR/CD3 complexes has implications regarding potential mechanisms by which Ag may trigger signaling. It also suggests the possibility that the potential for bivalent expression could represent a general feature of Ag receptors.

Abstract The CD3ε cytoplasmic tail contains a conserved proline-rich sequence (PRS) that influences TCR–CD3 expression and signaling. Although the PRS can bind the SH3.1 domain of the cytosolic adapter Nck, whether the PRS is constitutively available for Nck binding or instead represents a cryptic motif that is exposed via conformational change upon TCR–CD3 engagement (CD3Δc) is currently unresolved. Furthermore, the extent to which a cis-acting CD3ε basic amino acid-rich stretch (BRS), with its unique phosphoinositide-binding capability, might impact PRS accessibility is not clear. In this study, we found that freshly harvested primary thymocytes expressed low to moderate basal levels of Nck-accessible PRS (“open-CD3”), although most TCR–CD3 complexes were inaccessible to Nck (“closed-CD3”). Ag presentation in vivo induced open-CD3, accounting for half of the basal level found in thymocytes from MHC+ mice. Additional stimulation with either anti-CD3 Abs or peptide–MHC ligands further elevated open-CD3 above basal levels, consistent with a model wherein antigenic engagement induces maximum PRS exposure. We also found that the open-CD3 conformation induced by APCs outlasted the time of ligand occupancy, marking receptors that had been engaged. Finally, CD3ε BRS–phosphoinositide interactions played no role in either adoption of the initial closed-CD3 conformation or induction of open-CD3 by Ab stimulation. Thus, a basal level of open-CD3 is succeeded by a higher, induced level upon TCR–CD3 engagement, involving CD3Δc and prolonged accessibility of the CD3ε PRS to Nck.

Most mechanisms of cell development, physiology, and signal transduction are controlled by protein-protein interactions. Immunoprecipitation of multiprotein complexes detected by flow cytometry (IP-FCM) is a means to quantitatively measure these interactions. The high sensitivity of this method makes it useful even when very little biomaterial is available for analysis, as in the case of rare primary cell subsets or patient samples. Detection of the T cell antigen receptor associated with the CD3 multiprotein complex from as few as 300 primary murine T cells is presented as an example. The method is compatible with quantitative flow cytometry techniques, making it possible to estimate the number of coimmunoprecipitated molecules. Both constitutive and inducible protein-protein interactions can be analyzed, as illustrated in related methodology using glutathione S -transferase–fusion protein pull-down experiments. IP-FCM represents a robust, quantitative, biochemical technique to assess native protein-protein interactions, without requiring genetic engineering or large sample sizes.