Abstract 4188: CD4+ T cells mediate non-canonical rejection of major histocompatibility class-I deficient pancreatic tumors independently of CD8+ T cells

Identifying and interrogating novel immune-mediated mechanisms of tumor rejection is a major clinical opportunity. Immune checkpoint blockade (ICB) induces tumor rejection primarily via cytolytic CD8+ T cells, but many patients have primary or acquired resistance to CD8+ T cell immunity. Reduced major histocompatibility complex I (MHC I) expression is a contributing factor to resistance, and pancreatic ductal adenocarcinoma (PDAC) is historically treatment-refractory due to, among other features, low MHC expression and a dearth of T cells in the tumor. Interrogating interventions in the context of the PDAC tumor microenvironment will reveal novel mechanisms regulating sensitivity to immune-mediated destruction. Using a genetically engineered mouse model of PDAC, we previously reported that combination immunotherapy (agonistic anti-CD40 and dual ICB, anti-PD-1/CTLA-4) induced both a CD8+ and a CD4+ T cell response sufficient to mediate tumor rejection and long-lived protection. Here, we utilized CRISPR-Cas9 to disrupt the dominant MHC I (H-2Kb allele) expression in PDAC cell lines, precluding direct antigen presentation to CD8+ T cells via this molecule. Subcutaneous injection of MHC I-deficient tumor clones into wild-type mice revealed normal tumor growth at baseline, and significantly delayed tumor growth kinetics in response to combination immunotherapy (CD40/ICB) in a CD4+ T cell-dependent manner (p < 0.0001 vs. vehicle-treated mice). These findings were observed in parental PDAC clones lacking H-2Kb expression, as well as in an ovalbumin (OVA)-expressing PDAC clone. CD4+ T cells mediated tumor rejection including in OVA-tolerized mice, excluding the role of tumor-neoantigens as a major determinant of response. There was no direct contribution of tumor rejection by CD8+ T cells, despite the generation of an activated, tumor-specific CD8+ T cell response in the tumor site (24.5% vs. 1.98% in vehicle-treated mice, p < 0.003). CD4+ T cells upregulated IFN-γ production (52.3% vs. 11.9% in vehicle-treated mice, p < 0.0001), and host IFN-γ and IFN-γ receptor expression were required for response. No changes were observed in the expression of cytotoxic molecules (Granzyme A or B) by CD4+ T cells after CD40/ICB, and perforin was not required for tumor rejection. Furthermore, depletion of macrophage and myeloid cells or genetic knockout of inducible nitric oxide synthase did not dampen CD4+ T cell immunity against established PDAC tumors. Thus, CD4+ T cells mediated PDAC tumor rejection via non-canonical mechanisms, excluding cytotoxic molecules and re-direction of anti-tumor macrophage effector functions after treatment with anti-CD40/ICB. This study provides critical insight into novel mechanisms of tumor clearance beyond direct CD8+ T cell:tumor cell interactions, and opportunities to exploit CD4 T+ cells as regulators of tumor immunity. Citation Format: Samuel I. Kim, Charu Arora, Ioannis I. Verginadis, Christopher R. Cassella, Nune Markosyan, Constantinos Koumenis, Robert H. Vonderheide, Katelyn T. Byrne. CD4+ T cells mediate non-canonical rejection of major histocompatibility class-I deficient pancreatic tumors independently of CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4188.