Abstract NG04: Agonistic anti-CD40 converts regulatory T cells in to Type 1 effector cells within the tumor microenvironment

Regulatory T cells (Tregs) are a pillar of the tumor microenvironment (TME), contributing to the restraint and dysfunction of adaptive immune responses against the tumor. Even small proportions of Tregs in the TME can subvert effector T cell responses, revealing the potent suppressive role of Tregs in both primary and acquired resistance to immune interventions. Therapeutically targeting Tregs to render the TME sensitive to immune destruction has been most successful using anti-CTLA-4, which is reported to bind intratumoral Tregs and mediate antibody dependent cellular cytotoxicity (ADCC). However, this therapy results in clinical responses in only a subset of patients and many tumor types are resistant to anti-CTLA-4, highlighting the need for improved options in targeting Tregs in the TME.As with most immune cells, Tregs exist on a spectrum, ranging from highly immunosuppressive to those with T helper (Th) effector functions, including production of interferon (IFN)-gamma. In studies of intestinal Treg subsets, conversion from a FoxP3+ suppressor conventional (cTreg) phenotype to a Th1-like ‘ExTreg’ phenotype has been reported. Given our recent findings that immunotherapy-induced CD4 T cells contribute to tumor rejection1,2, the impact of this switch from cTreg to ExTreg could be substantial in the context of the developing anti-tumor immune response. Here, we report for the first time the conversion of cTregs to ExTregs in the TME as a result of treatment with agonistic anti-CD40 monoclonal antibody. We employed anti-CD40 in the genetically engineered LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model of pancreatic ductal adenocarcinoma (PDAC) as a means of priming an adaptive T cell response against established, highly immunosuppressive tumors3. We have previously reported the positive impact of anti-CD40 combined with dual immune checkpoint blockade (anti-PD-1 and anti-CTLA-4; ICB) on the generation of protective CD4 and CD8 T cell responses in the context of PDAC, concurrent with a decline of Tregs in the PDAC TME1. Leveraging a PDAC tumor clone with a relatively high T cell infiltrate to assess the impact of ‘successful’ immunotherapy on the TME4, we observed significant alterations in the Treg compartment after combination anti-CD40 and ICB administration, including a global reduction in Tregs and a pattern of Treg localization at the tumor edge. Interrogation of the mechanisms underlying Treg loss after therapy revealed the effect was entirely dependent on CD40 stimulation, as anti-CD40 alone mediated this effect and mice lackingCD40 expression were resistant to Treg reduction. Given that Tregs do not express CD40, excluding the role of ADCC in mediating Treg loss in the TME, we hypothesized that CD40-expressing antigen presenting cells may mediate intratumoral Treg reduction. We found that dendritic cells (DCs) and the IL-12/IFN-gamma cytokine signaling pathway regulated Treg loss, but that Tregs were not dying at increased rates, as FoxP3+ cells did not express increased levels of cleaved caspase 3 after treatment with anti-CD40. Using FoxP3eGFP-Cre-ERT2 xGt(ROSA)26Sortm(CAGtdTomato)/Hze (R26tdTomato) mice to conduct lineage tracing experiments, we determined that Tregs in the PDAC TME downregulated FoxP3 expression, converting from a cTreg to ExTreg phenotype. Concurrently, all Treg subsets upregulated expression of the Th1transcription factor Tbet, and ExTregs increased production of IFN-gamma. This effect was completely lost in mice treated with antibodies blocking either IL-12p40 or IFN-gamma, highlighting the role of this cytokine axis in regulating ExTreg generation and acquisition of Th1effector functions. Furthermore, nuclear translocation of the nuclear factor of activated T cells (NFAT1), a reliable marker of extremely recent cognate antigen stimulation of T cells ( Citation Format: Vivien Maltez, Charu Arora, Rina Sor, Ronald N. Germain, Robert H. Vonderheide, Katelyn T. Byrne. Agonistic anti-CD40 converts regulatory T cells in to Type 1 effector cells within the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr NG04.