TCR clonotypes intrinsically determine CD8+ T cell memory fate through atomistic differences in TCR-pMHC binding topology, where Vα-centric engagement drives TEM differentiation via enhanced CD3ζ phosphorylation, Vβ-centric engagement promotes TCM stemness and persistence, and balanced bipolar engagement generates both subsets with maximal clonal expansion and crossreactivity but increased exhaustion susceptibility, revealing that subtle structural variations in mechanotransduction pathways sculpt distinct transcriptional programs independent of signal strength or environmental factors.

Memory CD8⁺ T cells provide durable protection against recurrent infection and cancer, but how distinct memory fates are specified remains unclear. Here, we define the functional landscape of 242 murine CD8⁺ TCRαβ clonotypes specific for influenza NP366–374/H-2Dᵇ ligand (pMHC) by integrating single-cell transcriptomics and TCR sequencing with force-dependent biophysics, structural analysis, and in vivo investigation of memory development. Fate essentially tracks with TCR mechanotransduction: TCM-associated clonotypes preferentially engage pMHC through TCRβ, whereas TEM-associated clonotypes preferentially engage through TCRα. Clonotypes with balanced, bipolar signaling generate both memory subsets, optimize expansion, and display broad heterosubtypic crossreactivity but with exhaustion susceptibility. In contrast, diverse TCM clonotypes are largely clonal singlets with considerable mutant-epitope recognition in aggregate and stemness preservation, revealing complementary strategies for immunodefense coverage. Atomistically, subtle pMHC contact differences and load transmission through the TCR holoreceptor appear to tune phosphorylation, memory differentiation, and functional durability, features applicable to adoptive T-cell immunotherapies.