Abstract 6474: Comprehensive immunophenotypic profiling in anti-PD-1-responsive and -resistant mouse models reveals perforin-expressing CD4+ T cells as key effectors of cytolytic activity

Abstract Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have dramatically advanced cancer therapy, but resistance mechanisms remain a significant hurdle. This study explores immune changes in syngeneic mouse models sensitive (MC-38) or resistant (LLC1) to anti-PD-1 monoclonal antibody (mAb) treatment. Multicolor flow cytometry was employed to assess immune cell profiles in the tumor microenvironment (TME) and tumor-draining lymph nodes (TdLNs). In the MC-38 model, anti-PD-1 mAb therapy significantly altered immune landscapes, including enhanced dendritic cell (DC) and macrophage populations and decreased myeloid-derived suppressor cells (MDSCs). Upregulated antigen presentation molecules (MHC I/II) and functional markers (CD80, CD86, CCR7, PD-L1) were observed in tumor-associated DCs and macrophages. Notably, there was an increase in tumor-infiltrating CD4+ T cells, CD8+ T cells, including central memory (TCM) and effector memory (TEM) subsets, regulatory T cells (Tregs), natural killer (NK) cells, and natural killer T (NKT) cells. A key finding was the enhanced cytotoxic potential across various lymphocyte subsets, with perforin identified as a pivotal marker among 3 different cytolytic markers, correlating with therapeutic efficacy. Tumor volume inversely correlated with levels of perforin-expressing CD4+ T cells and NKT cells, highlighting their critical roles in antitumor responses. Conversely, the LLC1 model displayed limited immune alterations following anti-PD-1 mAb treatment, emphasizing distinct resistance mechanisms. This study delineates the dynamic immunophenotypic changes induced by anti-PD-1 therapy and identifies perforin, particularly in CD4+ T cells and NKT cells, as a promising biomarker for treatment response. Furthermore, these findings suggest potential combinatorial strategies targeting DC/MDSC ratios and perforin pathways to overcome resistance in refractory tumors. Citation Format: Hiroyuki Inoue, Takayuki Hamasaki, Kazuhiko Inoue, Akira Nakao, Noriyuki Ebi, Hirofumi Minomo, Ichiro Nagata, Naoto Horai, Masaki Fujita. Comprehensive immunophenotypic profiling in anti-PD-1-responsive and -resistant mouse models reveals perforin-expressing CD4+ T cells as key effectors of cytolytic activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6474.