Introduction/Rationale: T cell receptor (TCR) functional potency is determined by membrane-proximal, two-dimensional (2D) kinetics—on-rate, off-rate, and 2D affinity—which correlate more closely with cellular responses than conventional 3D measures. The CD8 co-receptor enhances bond number and lifetime, and normalized synergy quantifies its contribution across affinity ranges. However, how individual TCR biophysical traits relate to in vivo clonal outcomes—expansion and differentiation (naïve, TCM, TEM, cytotoxic)—remains unclear in human pathogen-specific repertoires. We aim to connect quantitative 2D biophysics with single-cell phenotypes in a SARS-CoV-2 cohort to identify features predicting robust expansion and defined differentiation.
Methods: SARS-CoV-2 vaccine and infection–annotated donors were analyzed. Antigen-specific CD8⁺ T cells were identified by peptide–MHC tetramers and single-cell sequencing to recover paired TCRα/β and transcriptomic profiles. Selected TCRs were cloned into Jurkat-76 (J76) derivatives expressing or lacking CD8, including Nur77/NFAT reporter lines, to assess co-receptor dependence. 2D kinetic parameters were measured using micropipette adhesion and bio-membrane force microscopy. Single-cell phenotypes were clustered (Seurat/totalVI), linked to clonotypes, and analyzed for correlations between biophysical features, expansion, and differentiation.
Results: SARS-CoV-2–specific TCRs exhibited wide 2D affinity and CD8 dependency ranges, indicating diverse activation mechanisms. Integrating biophysical and single-cell data revealed that expansion and differentiation correlate with combined metrics of affinity, stability, and co-receptor cooperation. Highly expanded clones were not always the strongest binders; rather, normalized CD8 synergy identified low-affinity yet functionally potent clones.
Conclusion: These insights suggest that composite biophysical signatures, not affinity alone, govern clonal fate and may inform TCR-based immunotherapies and vaccine design for durable protection.
