Characterization of CD8⁺ T-cell responses to non-anchor-type HLA class I neoantigens with single amino-acid substitutions

CD8⁺ T cells are capable of recognizing mutation-derived neoantigens displayed by HLA class I molecules, thereby exhibiting the ability to distinguish between cancer and normal cells. However, accumulating evidence has shown that only a small fraction of nonsynonymous somatic mutations give rise to clinically relevant neoantigens. The properties of such neoantigens, which must be presented by HLA and immunogenic to induce a T-cell response, remain elusive. In this study, we explored the HLA class I ligandome of a human cancer cell line with microsatellite instability using a proteogenomic approach. The results demonstrated that neoantigens accounted for only 0.34% of the HLA class I ligandome, and most neoantigens were encoded by genes with abundant expression. Thereafter, T-cell responses were prioritized, and immunodominant neoantigens were defined using naive CD8⁺ T cells derived from healthy donors. AKF9, an immunogenic neoantigen with a mutation at a non-anchor position, formed a stable peptide-HLA complex. T-cell responses were analyzed against a panel of AKF9 variants with single amino-acid substitutions, in which mutations did not alter the high HLA-binding affinity and stability. The responses varied across individuals, demonstrating the impact of heterogeneous T-cell repertoires in this human cancer model. Moreover, responses were biased toward a variant group with large structural changes compared to the wild-type peptide. Thus, naive T-cell induction can be attributed to multiple determinants. Combining structural dissimilarity with gene-expression levels, HLA-binding affinity, and stability may further help prioritize the immunogenicity of non-anchor-type neoantigens.