Background Host T-cell response is limited to only a small fraction of nonsynonymous mutations; however, the molecular properties of those immunogenic neoantigens remain elusive.

Methods Here, we interrogated the HLA class I ligandome of a microsatellite instability (MSI)-type cancer cell line using a proteogenomic approach, and found an immunogenic 9-mer neoantigen, AKF9. The AKF9 was a non-anchor type neoantigen that harbored a single amino-acid substitution (Asn > Lys) at position 8, which did not affect the HLA-binding affinity.

Results In order to assess a determinant of the immunogenicity, we prepared a panel of AKF9 variants with substitutions at position 8, and found that CD8+ T-cell responses were biased toward residues with structural difference from the wild-type. Interestingly, a substitution with moderate structural change (Asp) also induced reactive T cells; however, in contrast to the others, induced T cells frequently cross-reacted to the wild type HLA ligand. To validate these findings, we used in silico prediction of accessible surface areas and scored the difference between neoantigens and wild types (ΔASA). Evaluation of reported clinical datasets demonstrated that patient T-cell induction was positively correlated with ΔASA values, while cross-reactivity of induced T cells was inversely correlated.

Conclusions Our results indicate that dissimilarity is key for both T-cell induction and discrimination from self. ΔASA may help predict immunogenic non-anchor type neoantigens inducing specific T-cell response from a variety of cancer mutation pools.