Abstract A069: Oncogenic EGFR signaling promotes intratumoral infiltration of antigen-specific T cells and myeloid cells while impeding T cell function in a mouse model of glioma

Abstract Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Despite the transformative impact of immunotherapies in other cancers, clinical trials in GBM have yielded only modest improvements in median overall survival. Oncogenic EGFR signaling, present in approximately 50% of GBMs, is associated with suppression of adaptive immunity, potentially mediated by tumor-associated macrophages (TAMs) that maintain a chronic interferon-response state. To investigate how EGFR signaling shapes the tumor immune microenvironment (TIME), we leveraged the MADR-mEGFRvIII (MADR) model, which expresses the constitutively active EGFR variant EGFRvIII under a tetracycline-off system. Immune checkpoint blockade is ineffective in this model due to poor intratumoral T cell infiltration, closely mirroring human GBM. Previous data demonstrated that short-term genetic EGFR ablation with doxycycline (dox) increases intratumoral CD4+ and CD8+ T cell infiltration in MADR tumors. We also previously showed that tumor-intrinsic oncogenic EGFR signaling reprograms TAMs to an inflammatory state, which, when chronically sustained, may impair T cell function. Together, these findings suggest that EGFR inhibition could be combined with T cell-based immunotherapy to recondition the TIME and enhance adaptive immunity. Thus, to study T cell trafficking and function in the context of EGFRvIII signaling, we used hgp100-specific Pmel-1 CD8+ T cells for adoptive cell transfer (ACT) in C57BL/6 mice implanted with hgp100-expressing MADR tumor cells. Mice received daily treatment with dox (20 mg/kg) or saline beginning day 9 post-implantation, followed by i.p. administration of 5.0x105 pre-activated Pmel-1 T cells on day 13. Tumor-infiltrating leukocytes (TILs) were isolated for flow cytometric analysis from tumor-bearing brain hemispheres on day 20. We hypothesized that initiating genetic EGFR ablation prior to ACT would enhance T cell infiltration, activation, and reduce TAM abundance in the TIME. However, we observed a marked increase in Pmel-1 T cell (Thy1.1+) infiltration in saline-treated tumors compared to EGFR-ablated tumors. However, in the EGFR-ablated setting, a greater proportion of infiltrating Pmel-1 T cells expressed CD25, CXCR3, and CCR2. EGFR ablation also increased the proportion of microglia (CD45loCD11b+) while decreasing the proportion of infiltrating CD45hiCD11b+ cells. These findings suggest a dual role for oncogenic EGFR signaling, where it may promote recruitment of both antigen-specific T cells and immunosuppressive myeloid cells, while dynamically shaping T-cell activation states. These data provide unique insight into the molecular mechanisms driving EGFR-influenced T cell trafficking and provide a compelling rationale for temporally optimizing EGFR inhibition and ACT. Citation Format: Marissa Li, Marissa S. Pioso, Julio C. Sanchez, Katie B. Grausam, Joshua J. Breunig, David A. Nathanson, Robert M. Prins. Oncogenic EGFR signaling promotes intratumoral infiltration of antigen-specific T cells and myeloid cells while impeding T cell function in a mouse model of glioma [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A069.