Background: The GL261 and CT2A syngeneic tumor lines are frequently used as immunocompetent orthotopic mouse models of human glioblastoma (huGBM) but demonstrate distinct differences in their responses to immunotherapy., Methods: To decipher the cell-intrinsic mechanisms that drive immunotherapy resistance in CT2A-luc and to define the aspects of human cancer biology that these lines can best model, we systematically compared their characteristics using whole exome and transcriptome sequencing, and protein analysis through immunohistochemistry, Western blot, flow cytometry, immunopeptidomics, and phosphopeptidomics., Results: The transcriptional profiles of GL261-luc2 and CT2A-luc tumors resembled those of some huGBMs, despite neither line sharing the essential genetic or histologic features of huGBM. Both models exhibited striking hypermutation, with clonal hotspot mutations in RAS genes ( Kras p.G12C in GL261-luc2 and Nras p.Q61L in CT2A-luc). CT2A-luc distinctly displayed mesenchymal differentiation, upregulated angiogenesis, and multiple defects in antigen presentation machinery (e.g. Tap1 p.Y488C and Psmb8 p.A275P mutations) and interferon response pathways (e.g. copy number losses of loci including IFN genes and reduced phosphorylation of JAK/STAT pathway members). The defect in MHC class I expression could be overcome in CT2A-luc by interferon-γ treatment, which may underlie the modest efficacy of some immunotherapy combinations. Additionally, CT2A-luc demonstrated substantial baseline secretion of the CCL-2, CCL-5, and CCL-22 chemokines, which play important roles as myeloid chemoattractants., Conclusion: Although the clinical contexts that can be modeled by GL261 and CT2A for huGBM are limited, CT2A may be an informative model of immunotherapy resistance due to its deficits in antigen presentation machinery and interferon response pathways., Competing Interests: MLS is equity holder, scientific co-founder, and advisory board member of Immunitas Therapeutics. EAC is an advisor to Amacathera, Bionaut Labs, Genenta, Inc., Insightec, Inc., DNAtrix Inc., Seneca Therapeutics, Synthetic Biologics. EAC has equity options in Bionaut Laboratories, DNAtrix, Immunomic Therapeutics, Seneca Therapeutics, Synthetic Biologics, Ternalys Therapeutics. EAC is co-founder and on the Board of Directors of Ternalys Therapeutics. EAC also is a named inventor on patents related to oncolytic HSV1 and noncoding RNAs. MCS has equity options and is a current employee of GSK. MN is a current employee of AbbVie. DN has stock ownership in Madrigal Pharmaceuticals. RJ received consultant fees from Cur, Elpis, Innocoll, SPARC, and SynDevRx,; owns equity in Accurius, Enlight, and SynDevRx; is on the Board of Trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund, and Tekla World Healthcare Fund; and received a research grant from Boehringer Ingelheim. AS currently has funding from Quark, Merck, AbbVie, Moderna and Vertex unrelated to the submitted work. AS serves on advisory boards for Surface Oncology, SQZ Biotechnologies, Selecta, Elpiscience, Monopteros, Bicara, Fibrogen, and Alixis. AS also is on scientific advisory boards for the Massachusetts General Cancer Center, Program in Cellular and Molecular Medicine at Boston Children’s Hospital, the Human Oncology and Pathogenesis Program at Memorial Sloan Kettering Cancer Center, Glaxo Smith Kline and Janssen. AS is an academic editor for the Journal of Experimental Medicine. AS has patents/pending royalties on the PD-1 pathway from Roche and Novartis. GF has patents/pending royalties on the PD-L1/PD-1 pathway from Roche, Merck MSD, Bristol-Myers-Squibb, Merck KGA, Boehringer-Ingelheim, AstraZeneca, Dako, Leica, Mayo Clinic, Eli Lilly, and Novartis. GF has served on advisory boards for Roche, Bristol-Myers-Squibb, Origimed, Triursus, iTeos, NextPoint, IgM, Jubilant, Trillium, GV20, IOME, and Geode. GF has equity in Nextpoint, Triursus, Xios, iTeos, IgM, Trillium, Invaria, GV20, and Geode. CW is an equity holder of BioNTech, Inc. DR receives institutional support through Dana-Farber Cancer Institute from Acerta Phamaceuticals, Agenus, Bristol-Myers Squibb, Celldex, EMD Serono, Enterome, Epitopoietic Research Corporation, Incyte, Inovio, Insightec, Novartis, Omniox, and Tragara; and is an advisor/consultant for Abbvie, Advantagene, Agenus, Agios, Amgen, AnHeart Therapeutics, Avita Biomedical, Bayer, Boston Biomedical, Boehringer Ingelheim, Bristol-Myers Squibb, Celldex, Deciphera, Del Mar Pharma, DNAtrix, Ellipses Pharma, EMD Serono, Genenta, Genentech/Roche, Hoffman-LaRoche, Imvax, Inovio, Kintara, Kiyatec, Medicenna Biopharma, Merck, Merck KGaA, Monteris, Neuvogen, Novartis, Novocure, Oncorus, Oxigene, Regeneron, Stemline, Sumitono Dainippon Pharma, Pyramid, Taiho Oncology, Vivacitas Oncology, and Y-mabs Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Iorgulescu, Ruthen, Ahn, Panagioti, Gokhale, Neagu, Speranza, Eschle, Soroko, Piranlioglu, Datta, Krishnan, Yates, Baker, Jain, Suvà, Neuberg, White, Chiocca, Freeman, Sharpe, Wu and Reardon.)