P08.22.b Systematic Comparison of Immunotherapies in Glioblastoma Reveals Distinct Immune Response Patterns

Abstract BACKGROUND Glioblastoma, the most common and lethal primary brain tumor, creates profound immunosuppression that has resisted clinical interventions. The mechanisms behind immunotherapy failures in glioblastoma remain unclear. We conducted a systematic comparison of different immunotherapeutic approaches in mouse models to better understand treatment responses. MATERIAL AND METHODS Using multiomic single-cell analysis and spatial profiling, we evaluated GL261 glioma-bearing mice treated with anti-PD-L1 antibodies, NKG2D-CAR-expressing T cells, natural killer (NK) cells, or macrophages. RESULTS All treatment models induced immune cell migration through chemotaxis. Additionally, PD-L1 and CAR T cell treatments led to an exhaustion-associated T cell signature alongside increased cytotoxic T cell presence. Further, we observed higher numbers of immunomodulatory lymphocytes and macrophages associated with these two immunotherapies. CAR macrophages and CAR NK cells exhibited enhanced cytotoxic gene signatures without increasing T cell numbers. Also, the immunomodulatory macrophage response appeared attenuated in CAR NK cells and macrophages. Notably, CAR NK cells demonstrated an enhanced antiviral response with interferon-beta expression that was not observed in other models. CONCLUSION While PD-L1 and CAR cell treatments successfully polarized the lymphoid compartment toward cytotoxic phenotypes, these approaches alone appear insufficient as they were accompanied by pronounced immunomodulatory cell responses. CAR NK cells and macrophages were characterized by attenuated immunomodulatory responses. Our findings suggest that cytotoxic lymphocytes alone fail to override tumor-associated immune suppression. This is in line with recent evidence on T helper cell roles in CAR therapy. Future immunotherapy approaches should balance cytotoxic and helper lymphocyte populations while enhancing inflammatory macrophages to overcome the immunosuppressive environment of glioblastoma