Background: Glioblastoma is a fatal disease despite aggressive multimodal therapy. PD-1 blockade, a therapy that reinvigorates hypofunctional exhausted CD8 T cells (Tex) in many malignancies, has not shown efficacy in glioblastoma. Loss of CD4 T cells can lead to an exhausted CD8 T-cell phenotype, and terminally exhausted CD8 T cells (Tex term) do not respond to PD-1 blockade. GL261 and CT2A are complementary orthotopic models of glioblastoma. GL261 has a functional CD4 T-cell compartment and is responsive to PD-1 blockade; notably, CD4 depletion abrogates this survival benefit. CT2A is composed of dysfunctional CD4 T cells and is PD-1 blockade unresponsive. We leverage these models to understand the impact of CD4 T cells on CD8 T-cell exhaustion and PD-1 blockade sensitivity in glioblastoma. Methods: Single-cell RNA sequencing was performed on flow sorted tumor-infiltrating lymphocytes from female C57/BL6 mice implanted with each model, with and without PD-1 blockade therapy. CD8+ and CD4+ T cells were identified and separately analyzed. Survival analyses were performed comparing PD-1 blockade therapy, CD40 agonist or combinatorial therapy. Results: The CD8 T-cell compartment of the models is composed of heterogenous CD8 Tex subsets, including progenitor exhausted CD8 T cells (Tex prog), intermediate Tex, proliferating Tex, and Tex term. GL261 is enriched with the PD-1 responsive Tex prog subset relative to the CT2A and CD4-depleted GL261 models, which are composed predominantly of the PD-1 blockade refractory Tex term subset. Analysis of the CD4 T-cell compartments revealed that the CT2A microenvironment is enriched with a suppressive Treg subset and an effector CD4 T-cell subset that expresses an inhibitory interferon-stimulated (Isc) signature. Finally, we demonstrate that addition of CD40 agonist to PD-1 blockade therapy improves survival in CT2A tumor-bearing mice. Conclusions: Here, we describe that dysfunctional CD4 T cells are associated with terminal CD8 T-cell exhaustion, suggesting CD4 T cells impact PD-1 blockade efficacy by controlling the severity of exhaustion. Given that CD4 lymphopenia is frequently observed in patients with glioblastoma, this may represent a basis for resistance to PD-1 blockade. We demonstrate that CD40 agonism may circumvent a dysfunctional CD4 compartment to improve PD-1 blockade responsiveness, supporting a novel synergistic immunotherapeutic approach. Keywords: CD4-positive T-lymphocytes; CD8-positive T-lymphocytes; brain neoplasms; immunotherapy; lymphocytes, tumor-infiltrating.

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