INTRODUCTION: Our understanding of how immune-based strategies designed to enhance T-cell activation might effectively control glioblastoma progression has been limited by our ability to identify and monitor tumor-specific T cells. The "cancer immunogenomics" approach has facilitated the search for tumor-specific antigens over the past 4 years by applying comprehensive cancer genomics to tumor antigen discovery. We applied this methodology to identify tumor-specific "neoantigens" in preclinical brain tumor models susceptible to checkpoint immunotherapy.
The C57BL/6-derived GL-261 and VM/Dk-derived SMA-560 H-2b haplotype brain tumors underwent DNA whole exome and RNA sequencing. High-affinity candidate neoepitopes were predicted by using in silico computational approaches. Tumor-specific T-cell recognition was assessed using enzyme-linked immunospot (ELISPOT) and mutant-specific tetramer analyses of tumor-infiltrating lymphocytes from tumors injected subcutaneously and intracranially.
The GL-261 and SMA-560 tumors harbored 4644 and 2066 nonsynonymoous exome mutations, respectively, of which half were expressed. We applied H-2Db- and H-2Kb-specific neoantigen prediction algorithms to identify candidate tumor antigens. To validate the immunogenicity of these candidate neoantigens, we assessed tumor-specific T-cell responses of tumor-infiltrating lymphocytes harvested from GL-261 and SMA-560 tumors transplanted heterotopically and orthotopically into their respective syngeneic hosts. Using interferon-? ELISPOT and tetramer analysis, we confirmed H2-Db-restricted IMP3-D81N (GL-261) and ODC1-Q129L (SMA-560) as endogenous neoantigens that were functionally immunogenic. Neoantigen-specific T cells were detected within intracranial tumors as well as draining lymph nodes.
We credentialed the immunogenicity of 2 predicted high-affinity neoepitopes in well-studied orthotopic syngeneic preclinical brain tumor models, GL-261 and SMA-560. Furthermore, we identified tetramer-positive, antigen-specific T-cell populations within transplanted brain tumors and draining lymph nodes. We therefore extend cancer immunogenomics-based neoantigen discovery to glioblastoma models and establish a foundation to expand studies of the tumor-specific mutanome and explore fundamental mechanisms of T-cell activation in central nervous system immunobiology.