Parsing digital or analogue TCR performance through piconewton forces
T lymphocytes leverage T-cell receptor (TCR) recognition of aberrant peptides bound to major histocompatibility complex molecules (pMHCs) on altered cells to protect the mammalian host from infectious pathogens and cancerous transformations. While adaptive immunity requires efficient TCR performance, comparison metrics are widely limited to force-free assays. Recent data reveals, however, that mechanical load on the TCR-pMHC bond resulting from cellular motions between a T cell-antigen presenting cell conjugate formed during immune surveillance tunes TCR specificity and sensitivity. Here, we cloned TCRs from lung resident CD8 T cells following influenza A virus (IAV) infection directed against two immunodominant epitopes, the luminous nucleoprotein NP366-374 /Db and sparse acid polymerase PA224-233/Db pMHCs, arrayed at >1000 vs <10 copies/cell, respectively. Using optical tweezers-based methods to apply biologically relevant loads, we observe that certain TCRs belonging to the NP repertoire require many ligands for activation while others only few, functioning in analogue or digital modes, correspondingly. In contrast, prevalent TCRs tested in the PA repertoire all perform digitally. When held at a critical force, moreover, even digital TCRs are distinguishable, exhibiting dramatic increases in bond lifetime and performance relating to their structural transition rate and distance. Digital performance is best for both specificities in vitro and in vivo, but undiscernible using conventional functional avidity or TCR sequence distance metrics. Correlates of optimal biophysical parameters include magnitudes of ERK phosphorylation, CD3 surface loss, and activation marker upregulation.  Our findings underscore the requirement to match TCR performance quality with pMHC copy number, as nature does. Given tumor antigen array paucity, engaging choice digital TCRs appears critical for T-cell adoptive cancer therapies and vaccines. Overall design: To determine response of specific TCR-bearing T cells to influenza virus, CD8+ retrogenic T cells bearing unique TCR were adoptively transferred into C57BL/6 mice hosts that were then infected with influenza A virus(IAV). The transferred retrogenic T cells expressed unique fluorophores permitting their isolation by FACs from dissociated lymph node and lung of mice at 7 days post-IAV infection. We then performed RNA-Seq and gene expression analysis of the lymph node- and lung-isolated retrogenic T cells for 3 unique TCR on 3 individual mice for each TCR (i.e. 3 replicates of lymph node, and of lung, for 3 TCR: 18 samples).