Unbiased functional identification and therapeutic targeting of T cell neoantigens in a spontaneous murine squamous cell carcinoma [Single cell]

The comparative resistance of some cancers including head and neck squamous cell carcinoma to checkpoint blockade is speculated to derive from the low frequency of expressed somatic mutations targeted by T cells as neoantigens. SCCVII, a spontaneously arising murine squamous carcinoma resembling human HNSCC in several key features, is likewise poorly immunogenic as irradiated tumor cells alone fail to induce protective immunity within syngeneic hosts. Justifying use of this model to identify NeoAgs, we confirm activated CD4+ and CD8+ T cells are detectable and essential for vaccine efficacy of SCC VII and polyI:C co-administration. Whole-exome sequencing tumor versus normal genome identified 39 nonsynonymous missense mutations that were synthesized into 81 representative 20-mers. NeoAg-specific CD4+ T cell IFN-? responses were found against mutations of Pik3ca, Ctnnd1, and Otud5 while both CD4+ and CD8+ T cells produced IFN-? when stimulated by a single Cltc mutation during in vitro recall assays. Prophylactic immunization with a mixture of all stimulatory peptides or the Cltc NeoAg alone protected hosts from subsequent tumor challenge. Further, the Cltc NeoAg, eliciting both CD4+ and CD8+ T cell responses, was also therapeutically beneficial in vivo. Anti-PD-1 combinatorial blockade resulted in synergistic tumor rejection via boosting Cltc-specific responses and increasing response diversification via epitope spreading. These data show that a functional NeoAg identification platform can be used to select immunotherapeutically relevant targets and filtration of neoepitopes that co-prime both CD4+ and CD8+ T cell responses is superior for practical intervention of poorly immunogenic tumors. Overall design: For identifcation of TCRs from cells responding to NeoAg in vitro, TCR clonality was assessed in IFN-?-captured CD4+ and CD8+ T cells via analysis of single-cell RNA-Seq data. FASTQ files were first generated using Bcl2fastq (v2.17.0). Quality control reports that were generated by FastQC (v0.11.2) (14) showed Nextera contamination, therefore cutadapt (v1.13) (15) was run with an adapter mask of 35 to exclude the contaminated regions. The FASTQ files were then edited by the Seqtk (v1.3) package to increase read quality. The trimfq function within Seqtk was used with an error rate threshold of 0.01 to trim the ends of each read and increase the data quality. The trimmed files were analyzed using TraCeR (v0.6.0) (16) and the reference genome GRCm38. TCR a and ß chains detected by TraCeR were completed to full-length sequences by adding corresponding leader peptide sequences retrieved from the international ImMunoGeneTics information system (IMGT). Productive TCR a and ß chains were considered if clonal, which was defined as a and ß chains identified together in more than one cell.