Fig. 1: Strengthening TCR–pMHC 3D binding affinity by TCR CDR3α mutagenesis impairs the TCR specificity in the presence of CD8.

a Structural overview of 2C-TCR (cornflower blue), m33-TCR (medium purple), and m67-TCR (cyan) in complex with R4-MHC are depicted. The R4 (SIYRYYGL) peptide, H-2K^b and β2-microglobulin within R4–MHC complex are highlighted in gold, purple, and yellow, respectively. A zoomed-in view of the interactions between the R4 peptide (SIYRYYGL, upper panel) or the L4 peptide (SIYLYYGL, bottom panel) and CDR3s of 2C-TCR is shown in dashed boxes on the left; the peptide is represented in yellow ribbon with the hotspot residue shown in stick, and CDR3s are represented in surface colored according to electrostatic properties (positive charge in red and negative charge in blue) on the right. b 3D binding affinity \(({K}_{{{{\rm{d}}}}})\) of 2C-, m33-, and m67-TCRs binding to R4- or L4-MHCs, respectively. c–f IL-2 production of 2C, m33 and m67 hybridoma T cells expressing CD8 or not, when stimulated by RMA-S cells pulsed with different concentrations of R4 (c, d) or L4 (e, f) peptide. The data for both the presence and absence of CD8 were obtained from the same experimental batch. The TCR sensitivity is determined by the lowest antigen concentration that started to elicit the IL-2 production from hybridoma T cells (5% potency, or P₅). The presented data represent one of three independent experiments. The analyses were conducted using the Mann–Whitney U-test. g, h The comparison of TCR specificity of 2C-, m33-, or m67-TCRs in the absence (g) or presence (h) of CD8. Error bars are ± SEMs. The analyses were conducted using unpaired student's t-tests. Statistical significance was indicated as follows: ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.