Atomistic TCR-ligand interactions instruct memory T-cell differentiation and crossreactivity

While memory T-cells provide protective immunity against specific pathogens, their post-encounter differentiation into central (TCM) and/or effector (TEM) subpopulations remains enigmatic. We thus explored the CD8 T-cell receptor (TCR) repertoire of 242 murine TCR?? clonotypes directed against an immunodominant influenza A virus (IAV) peptide/major histocompatibility complex (pMHC) ligand, the nucleoprotein NP366-374/Db, leveraging single-cell transcriptomics with paired TCR sequencing, mechanosensing metrics, and in vivo memory development plus TCR-pMHC structural analyses. Polar TEM and more variegated TCM repertoires as well as bipotential “bipolar” clonotypes (TBP) revealed weak force-dependent bonding parameters associated with heterosubtypic IAV crossreactivities. TCM and TEM polarities manifest pMHC binding skewed to TCR?- versus TCR?-subunit mechanotransduction, respectively, unlike the more signaling-balanced TBP TCRs. In sum, TCR diversity anticipates pathogen evolution whereas divergent signaling regulates memory fate. Overall design: NP1_GEX, NP1_TCR, NP2_GEX, and NP2_TCR were derived from NP366-374-Db tetramer+ tetramer? Zombie Aqua? CD8ß? CD44? T cells sorted from pooled mesenteric lymph nodes (mLNs) of 10 C57BL/6 (B6) mice at day 34 following primary infection with the PR8 strain of influenza A virus (IAV). NP1 and NP2 originated from the same pooled sample but were separated during barcoding. DN2_LN_GEX and DN2_LN_TCR were obtained from Zombie Aqua? CD8ß? CD44? T cells isolated from pooled mLNs of 2 B6 mice undergoing a memory recall response, 2 days after heterologous challenge with the X31 strain. DN4_LN_GEX and DN4_LN_TCR were similarly derived from pooled mLNs of 2 B6 mice, 4 days after X31 challenge. NS_LN_GEX and NS_LN_TCR were isolated from Zombie Aqua? CD44? CD8ß? T cells sorted from pooled mLNs of four B6 mice at day 33 post-infection.