Biology and structure of pMHC receptors functioning as mechanosensors in the [alpha][beta] T-cell lineage

ABSTRACT – OVERALL T lymphocytes utilize  T cell receptors (TCRs) to distinguish self from non-self through recognition of sparse antigenic peptides bound to MHC molecules (pMHC) arrayed on antigen presenting cells (APC). With remarkable specificity and sensitivity,  T lymphocytes can destroy host cells altered by viruses, other infectious pathogens or cancerous transformations while leaving normal cellular counterparts intact. Until recently, it was unclear how such TCR discrimination was achieved, given a lack of somatic mutations of TCR genes to boost receptor-ligand affinity unlike with B cell receptors. Contrary to conventional ligand associations exemplified by antigen-antibody interactions, moreover, it is now evident that physical force plays a crucial role in non- equilibrium TCR-based T cell activation. Here we investigate the overarching hypothesis that the  T-cell lineage receptors that recognize pMHC ligands, namely TCRs and their thymocyte-specific precursor, preTCRs, function as mechanosensors, transducing biomechanical forces to impact thymocyte development as well as T- cell antigen recognition, activation, and memory specification. Both TCRs and preTCRs utilize force to induce reversible structural transitions involving clonotypic immunoglobulin (Ig)-like domains. Project 1 shall elucidate various structural and biophysical features driving TCR mechanosensing using transformative optical tweezers (OT)-based single molecule and single cell measurements to determine non-equilibrium dynamics and energy landscapes under force linked to digital vs analog TCR performance. In turn, CD8 memory repertoires against influenza A virus (IAV) epitopes arrayed at sparse to abundant pMHC ligand densities shall be compared along with their variable domain centricities at the pMHC interface association with memory subtypes. Spatial transcriptomics in lung tissue operating in compliant vs. non-compliant locales will be assessed using TCR B6 and retrogenic mice to understand how TCR biomechanics are optimized for selective cellular niches during IAV infection. Project 2 shall map IAV-related preTCR and TCR structures and bond properties to thymocyte signaling, development and repertoire diversification in thymic epithelial cultures using bulk and 10X single cell RNAseq for complete tracking and analysis. Structural and biophysical insights will be used in conjunction with peptide variants to sculpt the T cell repertoire to generate thymocytes expressing mature TCR IAV specificities. Project 3 shall perform multifaceted computational analysis of TCR load propagation and conformational dynamics to calculate stress and strain distributions across TCR-pMHC molecules during molecular dynamics simulations under force (MDf), determine the propagation pathway and examine signal relay to CD3 subunits and the transmembrane helices based on holoreceptor cryoelectron microscopy structures. An Administrative Core (A), a Protein Production Core (B) and an NMR Core (C) will assist Projects to discern how force and dynamic structures empower  T lineage recognition of pMHC with basic and translational importance.