Endogenous Neoantigen-Specific CD8 T Cells Identified in Two Glioblastoma Models Using a Cancer Immunogenomics Approach

The "cancer immunogenomics" paradigm has facilitated the search for tumor-specific antigens over the last 4 years by applying comprehensive cancer genomics to tumor antigen discovery. We applied this methodology to identify tumor-specific "neoantigens" in the C57BL/6-derived GL261 and VM/Dk-derived SMA-560 tumor models. Following DNA whole-exome and RNA sequencing, high-affinity candidate neoepitopes were predicted and screened for immunogenicity by ELISPOT and tetramer analyses. GL261 and SMA-560 harbored 4,932 and 2,171 non-synonymous exome mutations, respectively, of which less than half were expressed. To establish the immunogenicities of H-2K(b) and H-2D(b) candidate neoantigens, we assessed the ability of the epitopes predicted in silico to be the highest affinity binders to activate tumor-infiltrating T cells harvested from GL261 and SMA-560 tumors. Using IFN gamma ELISPOT, we confirmed H-2D(b)-restricted Imp3(D81N) (GL261) and Odc1(Q129L) (SMA-560) along with H-2K(b)-restricted E2f8(K272R) (SMA-560) as endogenous tumor-specific neoantigens that are functionally immunogenic. Furthermore, neoantigen-specific T cells to Imp3(D81N) and Odc1(Q129L) were detected within intracranial tumors as well as cervical draining lymph nodes by tetramer analysis. By establishing the immunogenicities of predicted high-affinity neoepitopes in these models, we extend the immunogenomics-based neoantigen discovery pipeline to glioblastoma models and provide a tractable system to further study the mechanism of action of T cell-activating immunotherapeutic approaches in preclinical models of glioblastoma. (C) 2016 AACR.