Unbiased testing of all single nucleotide variants specific to a tumor for anti-tumor CD8+ T cell immune response

Abstract

Abstract Neoepitopes presented in the context of MHC render tumors immunogenic. However, not all potential neoepitopes elicit immunity, and the attributes of immunogenic neoepitopes are yet to be fully defined. Based on the premise that regressing tumors must elicit T cells that recognize the true anti-tumor neoepitopes, we have developed and used here an ELISPOT based high-throughput assay that allows testing of the immunogenicity of a large number of candidate neoepitopes. Naïve C57BL/6 mice were challenged with the colorectal tumor line MC38 and treated, starting day 5 post-tumor challenge, with a checkpoint-blocking antibody, leading to the beginning of tumor regression. MC38-specific CD8+ T cell response was observed in tumor-bearing hosts with regressing tumors. Exome and transcriptome sequencing of MC38 and accompanying bioinformatic analyses have revealed >800 single nucleotide variations (SNVs) in MC38. To identify the SNVs contributing to the immunogenicity of MC38, reactivity of the anti-MC38 CD8+ T cells isolated from mice with regressing tumors, towards individual peptides corresponding to each identified SNV, was tested. Indeed, CD8+ T cells against a number of mutant peptides were detected. These immunogenic mutant peptides are potential MHC I-restricted neoepitopes of MC38 and their role in tumor rejection is being tested.