Interrogating T-Cell Receptor Interactions with Peptide-MHC Through Structure-Guided Mutational Analysis 2219106

Abstract Introduction 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). In this study, we aimed to investigate how germline-encoded TCR contacts influence TCR—pMHC interactions and T cell activation. Methods Nine MART-1 peptide variants (a melanoma-associated antigen), generated by single alanine substitutions, were pulsed onto T2 cells (1 × 10⁵/well) and co-cultured with TCR-transduced Jurkat T cells at a 1:1 ratio for 14 hours. 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 hours 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. Results 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. Conclusion 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. Funding Source n/a Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)