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Background Glioblastoma multiforme (GBM) is a devastating primary CNS malignancy, and current treatments are ineffective and inevitably result in tumor relapse. Given the potential for T cells to target treatment-resistant tumor cells, we seek to understand and improve T cell priming in the context of GBM. While cDC1 are required for CD8 + T cell priming in a murine model of GBM,1 there remains a major gap in knowledge regarding the functional limitations of cDC1 and other APCs and how these limitations can be overcome in a CNS tumor setting. Given the established synergy between CD40 and TLR agonism in amplifying antigen-specific T cell responses in vaccine and peripheral tumor settings,2 3 we hypothesized that that combination CD40 and TLR3 agonism would improve the immunostimulatory capacity of APCs, and cDC1 in particular, to drive T cell priming and control GBM growth.Methods Using a syngeneic, orthotopic CT2A-ZsGreen model of GBM, we performed magnetic resonance imaging to size match tumors before treating immune-competent mice with anti-CD40, polyI:C (TLR3 agonist), and anti-PD-1 or control IgG2a. Mice were monitored daily for survival or harvested for flow cytometry 24 hours after anti-CD40 and polyI:C administration.Results This treatment regimen drastically improves survival, with therapeutic efficacy completely dependent upon the presence of T cells and largely driven by the infiltration of T cells primed in the periphery, indicative of a need for tumor antigen drainage ( figure 1B). Many mice eradicate their tumors, as evidenced by magnetic resonance imaging and durable survival. These ‘long-term survivors’ are also protected from rechallenge, which coincides with the emergence of a tissue resident memory T cell population (figures 1C and D). Further studies reveal that CD40 agonism drives the survival benefit (figure 2A). Despite increases in the frequency of intratumoral CCR7+ cDC1 with migratory capacity, there are no detectable changes in tumor antigen drainage to the meninges or draining lymph nodes. While both polyI:C and anti-CD40 activate cDC1 as measured by CD86 expression, expression of the costimulatory molecule CD70 is uniquely induced by CD40 agonism and may be the critical aspect of cDC1 activation that drives tumor control (figure 2B-I).Conclusions This work demonstrates the potential for CD40 agonism in improving anti-GBM T cell responses, particularly regarding the formation of immune memory to protect patients from tumor recurrence. Moreover, our data suggest a critical role for the CD40/CD70/CD27 signaling axis, which we are currently testing the necessity and sufficiency of.Acknowledgements We thank the National Cancer Institute and Focused Ultrasound Foundation for financial support and the UVA Flow Cytometry Core for exceptional technical assistance. Figure 1A was made using Biorender.References Bowman-Kirigin JA, Desai R, Saunders BT, Wang AZ, Schaettler MO, Liu CJ, Livingstone AJ, Kobayashi DK, Durai V, Kretzer NM, Zipfel GJ, Leuthardt EC, Osbun JW, Chicoine MR, Kim AH, Murphy KM, Johanns TM, Zinselmeyer BH, Dunn GP. The conventional dendritic cell 1 subset primes CD8+ T cells and traffics tumor antigen to drive antitumor immunity in the brain. Cancer Immunol Res. 2023;11(1):20–37.Ahonen CL, Doxsee CL, McGurran SM, Riter TR, Wade WF, Barth RJ, Vasilakos JP, Noelle RJ, Kedl RM. Combined TLR and CD40 triggering induces potent CD8+ T cell expansion with variable dependence on type I IFN. J Exp Med. 2004;199(6):775–784.Sanchez PJ, McWilliams JA, Haluszczak C, Yagita H, Kedl RM. Combined TLR/CD40 stimulation mediates potent cellular immunity by regulating dendritic cell expression of CD70 in vivo. J Immunol. 2007;178(3):1564–1572.Abstract 899 Figure 1Agonistic CD40 and TLR3 agonism promotes T cell dependent survival and functional immune memory on an immune checkpoint inhibitor backgroundAbstract 899 Figure 2Survival benefit is driven by CD40 agonism and results in robust cDC1 activation and CD70 expression