Fig. 6: Human high-affinity TCR engineering impairs antigen specificity.

a Structural overview of MAG-IC3-TCR (light blue of TCRα and misty rose of TCRβ) bound to the MAGE-A3-HLA-A*01:01 or Titin-HLA-A*01:01 complex. The components of MAGE-A3-derived peptide (EVDPIGHLY), Titin-derived peptide (ESDPIVAQY), HLA-A*01:01 and β2m are depicted in blue, red, purple and yellow, respectively. b NFAT-GFP population of MAG-IC3 T cells expressing CD8 or not, stimulated by a series of concentrations of MAGE-A3-HLA-A*01:01 or Titin-HLA-A*01:01 protein. The presented data represent one of three independent experiments. The statistical analyses were performed using the Mann–Whitney test. c, d Force-dependent mean lifetime curves of MAG-IC3-TCR interactions with MAGE-A3 (c) or Titin (d) when CD8 is absent or present. 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 ± SEM and summarized in Supplementary information, Tables S3, S4. The statistical analyses were performed using the Mann–Whitney U-test. e, f The analysis of TCR bond lifetime ratio and CD8 enhancement power in the medium force regime reveals low specificity and weak enhancement for engineered high-affinity TCRs. Bond lifetime ratios and CD8 enhancement power for 2C-, m33-, and m67-TCRs from Fig. 3b were replotted for comparison. All data points are presented as mean ± SEM and summarized in Supplementary information, Tables S6, S8. The statistical analyses for the panel e were conducted using unpaired studen's t-tests, and those for the panel f were performed using the Mann–Whitney U-test, with 2C-TCR–L4-MHC as a control. Statistical significance was indicated as follows: ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.