Fig. 5: Blocking force-enhanced interactions at the CD8–MHC binding interface impairs TCR specificity.

a Sequential conformational change of CD8–pMHC interface for 2C-TCR–R4-MHC–CD8αβ complex during cv-SMD simulations. b–d Zoomed-in snapshots of dynamic force exerted onto 2C-TCR–R4-MHC (light purple and gray surface)–CD8 (orange color, meshed surface and ribbon) complex structures at three key states: an initial state I₀ b, an unstable intermediate state I₁ c, and a stable intermediate states I₂ d. These states are characterized by the hydrophobic contact areas between CD8β-Ile2 (pink ball) and MHC. e Force-dependent mean lifetime curves for the interactions of 2C-TCR with R4-MHC, in the presence of WT CD8, or CD8 (Ile2Ala), or in the absence of WT CD8, respectively. The lifetime curves for the presence and absence of WT CD8 from Fig. 2f are replotted in Fig. 5e for comparison. The peak bond lifetimes with WT CD8, or CD8 (Ile2Ala) vs without CD8 were statistically analyzed. Single-molecule U-BFP assay ensured consistent conditions across all measurements, with each force value derived from >100 single-molecule bond lifetime measurements. The number of bond lifetime measurements per force curve for different TCR–pMHC or TCR–pMHC–CD8 pairs is summarized in Supplementary information, Table S2. All binned data points of force curves are presented as mean ± SEMs and summarized in Supplementary information, Tables S3, S4. f The analysis of TCR lifetime ratio (R4 to L4) in the presence of WT CD8, CD8 (Ile2Ala), or in the absence of WT CD8, respectively. The lifetime ratio (R4 to L4) for the presence and absence of WT CD8 from Fig. 3b was replotted in Fig. 5f for comparison. g, h IL-2 production of 2C hybridomas cells and the analysis of TCR specificity in the presence of WT CD8, CD8 (Ile2Ala), or in the absence of WT CD8, respectively. All data points are presented as mean ± SEMs and summarized in Supplementary information, Tables S6, S8. The statistical analyses for the panels e, g were conducted using the Mann–Whitney test, and the panels f, h were performed using unpaired t-tests. Statistical significance was indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001.