Abstract NG04: Agonistic anti-CD40 converts regulatory T cells in to Type 1 effector cells within the tumor microenvironment
Authors: Maltez Vivien , Arora Charu , Sor Rina , Germain Ronald N. , Vonderheide Robert H. , Byrne Katelyn T. 
Published: April 2023

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 (&lt;45minutes), was observed at increased levels within the ExTreg compartment, an effect that was lost in mice treated with MHC II blocking antibody. Coupled with increased expression of phosphorylated STAT1 expression within the ExTreg compartment, these findings suggest a role for DCs in presenting antigen directly to ExTregs in the context of IL-12/IFN-gamma stimulation to promote Th1 effector functions in the PDAC TME. Importantly, the generation of Th1-likeExTregs was not observed in mice treated with anti-CTLA-4, emphasizing the unique contribution of CD40 in driving this conversion and acquisition of anti-tumor functions within the ExTreg compartment. The use of agonistic CD40 antibody to reprogram Tregs in the suppressive PDAC TME reveals a previously underappreciated potential of this immunotherapeutic approach. Here our findings suggest that Tregs in the TME may not be a harbinger of poor prognostic outcomes and instead find that Treg plasticity may be leveraged for improved patient outcomes. Simultaneously alleviating T cell suppression while promoting effector functions may provide early modifications in the TME during a crucial time point of the nascent anti-tumor immune response that could ultimately dictate therapeutic responsiveness. This work was supported in part by the Intramural Research Program of NIAID, the Postdoctoral Research Associate Training (PRAT) Program, and NCI at the NIH, in addition to the Parker Institute for Cancer Immunotherapy.1. Morrison, A. H., Diamond, M. S., Hay, C. A., Byrne, K. T. & Vonderheide, R. H. Sufficiency ofCD40 activation and immune checkpoint blockade for T cell priming and tumor immunity. Proc National Acad Sci 201918971 (2020). doi:10.1073/pnas.19189711172. Huffman, A. P., Lin, J. H., Kim, S. I., Byrne, K. T. & Vonderheide, R. H. CCL5 mediatesCD40-driven CD4+ T-cell tumor infiltration and immunity. Jci Insight (2020).doi:10.1172/jci.insight.1372633. Byrne, K. T. & Vonderheide, R. H. CD40 Stimulation Obviates Innate Sensors and Drives T Cell Immunity in Cancer. Cell Reports 15, 2719-2732 (2016).4. Li, J., Byrne, K. T., Yan, F., Yamazoe, T., Chen, Z., Baslan, T., Richman, L. P., Lin, J. H., Sun, Y. H., Rech, A. J., Balli, D., Hay, C. A., Sela, Y., Merrell, A. J., Liudahl, S. M., Gordon, N., Norgard, R. J., Yuan, S., Yu, S., Chao, T., Ye, S., Eisinger-Mathason, T. S. K., Faryabi, R. B., Tobias, J. W., Lowe, S. W., Coussens, L. M., Wherry, E. J., Vonderheide, R. H. & Stanger, B. Z. Tumor Cell-Intrinsic Factors Underlie Heterogeneity of Immune Cell Infiltration and Response to Immunotherapy. Immunity 49, 178-193.e7 (2018).

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.