The CXCR3 ligands CXCL9, 10, and 11 play critical roles in the amplification of immune responses by recruiting CXCR3⁺ immune effector cells to the tumor site. Taking advantage of this property of CXCR3 ligands, we aimed to establish a novel approach to identify immunogenic mutated-antigens. We examined the feasibility of using CXCR3 ligand mRNAs as sensors for detection of specific immune responses in human and murine systems. We further investigated whether this approach is applicable for the identification of immunogenic mutated-antigens by using murine sarcoma lines. Rapid synthesis of CXCR3 ligand mRNAs occurred shortly after specific immune responses in both human and murine immune systems. Particularly, in CMS5 tumor-bearing mice, we detected specific immune responses to mutated mitogen-activated protein kinase 2 (ERK2), which has previously been identified as an immunogenic mutated-antigen. Furthermore, by combining this approach with whole-exome and transcriptome sequencing analyses, we identified an immunogenic neo-epitope derived from mutated staphylococcal nuclease domain-containing protein 1 (Snd1) in CMS7 tumor-bearing mice. Most importantly, we successfully detected the specific immune response to this neo-epitope even without co-administration of anti-cytotoxic T-lymphocyte protein-4 (CTLA-4), anti-programmed cell death-1 (PD-1) and anti-glucocorticoid-induced TNFR-related protein (GITR) antibodies, which vigorously augmented the immune response and consequently enabled us to detect the specific immune response to this neo-epitope by conventional IFNγ intracellular staining method.

Our data indicate the potential usefulness of this strategy for the identification of immunogenic mutated-antigens. We propose that this approach would be of great help for the development of personalized cancer vaccine therapies in future.