BPS2026 – Interrogating T cell receptor interactions with peptide-MHC through structure-guided mutational analysis

Studies have shown that germline contacts may underlie MHC restriction, supporting the idea that TCR-MHC specificity is partially encoded in the germline (Garcia et al., 1996; Marrack & Scott-Browne, 2010). To investigate how germline-encoded TCR contacts influence TCR-pMHC interactions and T cell activation. Nine MART-1 peptide variants (a melanoma-associated antigen), generated by single alanine substitutions, were pulsed onto T2 cells (1×10 5 /well) and co-cultured with TCR-transduced Jurkat T cells at a 1:1 ratio for 14 h. Activation was assessed by CD69 staining and NFAT-GFP reporter signal via flow cytometry. Four α and two β chain TCR variants were generated by single alanine substitution at predicted polar contacts within germline-encoded CD1 and CD2 regions. Jurkat T cells were co-transduced with the α and β chain lentiviruses, then co-cultured for 14 h with T2 cells pulsed with peptides that had retained activity in the initial screen. Activation was assessed by CD69 and NFAT-GFP expression via flow cytometry. Two MART-1 variants, MART1-1A (E1A, 15.6%) and MART1-9A (T9A, 11.5%), induced dose-dependent activation. Another peptide, MART-91, derived from Mycobacterium and previously reported to share a similar motif with MART-1, induced the strongest activation (28.6%). Based on these results, MART1-1A and MART-91 were selected for testing TCR variants. While substitutions in the β chain yielded inconclusive results, disrupting polar contacts in the α chain abolished activation in response to both peptides. In contrast, the germline TCR (germline-TCR156) maintained robust activation, particularly in response to MART-91. These findings support a role for germline-encoded TCR residues in modulating TCR-MHC recognition and specificity, enabling tolerance of variation at peptide contact sites. Future studies will aim to repeat β chain experiments to clarify the ambiguous results, which may have been due to a failed transfection.