Strong spontaneous tumor neoantigen responses induced by a natural human carcinogen

Abstract

A key to improving cancer immunotherapy will be the identification of tumor-specific "neoantigens" that arise from mutations and augment the resultant host immune response. In this study we identified single nucleotide variants (SNVs) by RNA sequencing of asbestos-induced murine mesothelioma cell lines AB1 and AB1-HA. Using the NetMHCpan 2.8 algorithm, the theoretical binding affinity of predicted peptides arising from high-confidence, exonic, non-synonymous SNVs was determined for the BALB/c strain. The immunoreactivity to 20 candidate mutation-carrying peptides of increased affinity and the corresponding wild-type peptides was determined using interferon-γ ELISPOT assays and lymphoid organs of non-manipulated tumor-bearing mice. A strong endogenous immune response was demonstrated to one of the candidate neoantigens, Uqcrc2; this response was detected in the draining lymph node and spleen. Antigen reactive cells were not detected in non-tumor bearing mice. The magnitude of the response to the Uqcrc2 neoantigen was similar to that of the strong influenza hemagglutinin antigen, a model tumor neoantigen. This work confirms that the approach of RNAseq plus peptide prediction and ELISPOT testing is sufficient to identify natural tumor neoantigens.

Keywords: HA, hemagglutinin; MCA, methylcholanthrene; NGS, next-generation sequencing; RNAseq; SNV, single nucleotide variant.; dLN, lymph node draining the tumor; epitope prediction; mesothelioma; neoantigen.

Figure 1.

Flow diagram showing the selection process to identify candidate neoantigens for testing.

Figure 2.

Endogenous immune responses to candidate neoantigens in mesothelioma tumor-bearing mice. (A) Representative duplicate wells from interferon-γ ELISPOT analysis of total dLN cell preparation from non-treated AB1 tumor-bearing mice against pools of peptides. (B) Summary of ELISPOT data showing mean ± SD for the deconvolution of peptide pool B. ELISPOT assays were performed in triplicate; **P < 0.01.

Figure 3.

(A) Endogenous immune responses to wild-type and mutant Urcqc2 in the dLN and spleen of 7 mesothelioma-bearing mice. ELISPOT assays were performed in triplicate; * P < 0.05. (B) Comparison of endogenous immune responses to wild-type and mutant Urcqc2, and the hemagglutinin peptide CL4 in the dLN of 15 AB1-HA tumor-bearing mice. ELISPOT assays were performed in triplicate; ns, not significant. (C) Representative triplicate wells from interferon-γ ELISPOT analysis of total dLN cell preparation from 4 non-treated AB1-HA tumor-bearing mice.