MacLachlan et al. used structure, mutagenesis, and T cell stimulation to examine a 20mer 5T4 epitope (associated with colorectal cancer) and to distinguish the contributions of the core nonamer and peptide flanking residues (PFRs) on HLA binding and TCR stimulation. While 5T4 had weak binding affinity (determined primarily by the nonamer core), PFRs increased TCR recognition of the HLA-bound nonamer and subsequent activation of anti-5T4 CD4+ T cells. The C-terminal PFR exhibited high flexibility and could form an ordered secondary structure resembling the native antigen. Mutations in PFRs may contribute to neoantigen recognition.
CD4(+) T-cells recognize peptide antigens, in the context of human leukocyte antigen (HLA) class II molecules (HLA-II), which through peptide flanking residues (PFRs) can extend beyond the limits of the HLA-binding. The role of the PFRs during antigen recognition is not fully understood; however, recent studies have indicated that these regions can influence TCR affinity and pHLA-II stability. Here, using various biochemical approaches including peptide sensitivity ELISA and ELISpot assays, peptide binding assays and HLA-II tetramer staining, we focused on CD4(+) T-cell responses against a tumor antigen, 5T4 oncofetal trophoblast glycoprotein (5T4), which have been associated with improved control of colorectal cancer. Despite their weak T-cell receptor (TCR) binding affinity, we found that anti-5T4 CD4(+) T-cells are polyfunctional and that their PFRs are essential for TCR recognition of the core bound nonamer. The high-resolution (1.95 A) crystal structure of HLA-DR1 presenting the immunodominant 20-mer peptide 5T4111-130, combined with molecular dynamic simulations, revealed how PFRs explore the HLA-proximal space to contribute to antigen reactivity. These findings advance our understanding of what constitutes an HLA-II epitope and indicate that PFRs can tune weak-affinity TCR-pHLA-II interactions.