Ligand-Dependent [alpha][beta]TCR Function

ABSTRACT – PROJECT 1 The αβ T lymphocyte ligand recognition process operates under nonequilibrium conditions, in contrast to that of B cells and secreted antibodies. Physical force tunes ligand recognition sensitivity and specificity mediated by αβT cell receptors (TCRs) during their immune surveillance to detect foreign peptides bound to major histocompatibility molecules (pMHC) on diseased cells. Mechanistic elucidation requires deep understanding of single molecule biophysics and structural biology in concert with T lymphocyte biology. The strategy of Project 1 is to pair single molecule and single cell measurements using optical tweezers (OT) in Aim 1 with functional biological impact in Aim 2. We utilize a portfolio of influenza A virus (IAV)-specific CD8 clonotypes specific for pMHC at abundant, intermediate and sparse array on antigen presenting cells (APCs). Our focus is on quality of T cell performance in lymph node (LN) as well as lung along with characterization of primary and secondary memory TCR features. Spatial transcriptomics in lung will assess single cell transcriptomes and paired TCR sequencing, allowing TCR function in compliant and stiff tissue niches to be revealed and defining parameters necessary to elicit digital T cells in distinct cellular environments. These highest quality CD8 T cells can detect even a few copies of a ligand per cell, manifest long bond lifetimes, and undergo structural transitioning through a mechanism that involves cycling between conformational states on a dynamic energy landscape. Mechanistic details will be evaluated including force toggling required to drive transitioning, force magnitude comparison to cell micromechanical properties, and molecular underpinnings of conformational transitions. A focus will be on strengthening and weakening mutations that are informed by Project 3 molecular dynamics with the aspirational goal to engineer digital performance into any TCR and hence regulate T cell recognition function in the future. We shall further evolve our OT-based capabilities by merging single molecule single cell (SMSC) biophysical and single cell activation requirement (SCAR) assays into the SM-SCAR assay where input profiles to a single TCR can directly test requirements for activation. In contrast to digital cells, T cells entering primary memory in LNs show profiles with relatively weak binding and analog performance (i.e., multiple pMHC copies per APC required to activate T cells) in the context of high density pMHC array. Interestingly, differentiation into effector and central memory subsets appear to be linked to Vα- or Vβ-pMHC binding bias, respectively, with signature force bond lifetime profiles implying that selective load through TCR subunits impacts signaling and hence resultant transcriptomes. Finally, we shall define the activation threshold and interrogate properties of primary memory T cells, to investigate cross-reactivity through pMHC containing single point variations that may engender heterotypic protection and to contrast primary memory with secondary memory recall responses.