T-Cell Receptor-CD3 Signaling Complex Extracellular Interactions Characterized by Genetic Incorporation of Unnatural Amino Acid Photo-Crosslinkers

Abstract

T-cells play a central role in the adaptive immune response to invading pathogens and cancer while largely avoiding self-reactivity. The interaction of T-cell receptors (TCRs) displayed on T-cells with peptide-major histocompatibility complexes (pMHCs) on the surface of antigen presenting cells leads to signaling through the TCR-CD3 signaling complexes, which result in the initiation of cellular immune responses. However, how molecular and structural events in TCR-recognition translate through TCR-CD3 complex and generate differences in intracellular signaling is still not clear. Previous studies have shown that the extracellular TCR-CD3 subunits interactions are important for T-cell responses to TCR engagement. The objective of this research is to understand structural organization and specific interactions among TCR and CD3 subunits, and to provide insights into how structural features and mechanisms initiating TCR-triggering determine the outcome of immune responses. To monitor interactions among TCR-CD3 subunits, we developed a system to site-specifically incorporate an unnatural amino acid photo-crosslinker p-Azido-L-phenylalanine (pAzpa) or p-benzoyl-L-phenylalanine (pBpa) into TCR expressed on cell surface. Subsequently, after activation of TCR with pMHC, photo-crosslinked TCR-CD3 complexes were immunoprecipitated and analyzed by Western Blotting. Our analysis showed that specific residues in the TCR DE, CC, FG loops and G strand play critical roles in TCR-CD3 subunit interactions. Moreover, cross-linked high molecular weight complexes suggested TCR-TCR dimerization during the signaling process. Together our results 1) reveal how TCR-CD3 signaling subunits are positioned together to perform their function, 2) suggest how the antigen specific recognition information gets transferred from outside to the signaling molecules inside cell to determine immune responses, 3) provide potential targets for modulating outcome of immune response.