Turka, Laurence A.

Abstract The αβ T cell antigen receptor (TCR)/CD3 complex is currently viewed as a monovalent receptor with the subunit composition αβγδε2ζ2. This model has emerged from among at least five different potential stoichiometries that were proposed in previous years. Recently, the case for the accepted stoichiometry has been supported by experiments involving co-immunoprecipitation (IP) followed by Western blot, and by blue native (BN)-PAGE. We have revisited the issue of TCR/CD3 stoichiometry by examining immunoprecipitated complexes by flow cytometry (IP-FCM), a technique which allows a quantitative estimate of the subunit constituents in native multi-protein complexes. Together with size-exclusion chromatography, the data supports the conventional stoichiometry, but provides evidence that the copy number may be two, making the TCR/CD3 a precisely bivalent complex, [αβγδε2ζ2] x 2. The expression of a bivalent TCR/CD3 complex has implications regarding possible mechanisms by which the interaction with antigen may trigger signaling. Research support: Adam G. Schrum, start-up funds from the Mayo Foundation; Laurence A. Turka, grants AI41521 and AI 43620 from NIH; Ed Palmer, grants from the Swiss National Science Foundation.

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.

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.