Exhausted CD8⁺ T Cells Promote Ovarian Cancer Immunosuppression via CCL3-Driven M2 Macrophage Polarization

High-grade serous ovarian cancer (HGSOC) is an aggressive malignancy marked by high recurrence rates, poor prognosis, and limited response to immune checkpoint inhibitors, primarily attributable to its immunologically “cold” tumor microenvironment. To profile the immunological landscape of HGSOC, we conducted single-cell RNA sequencing (scRNA-seq) on 84,065 cells from tumor tissues of eight treatment-naïve patients and one normal ovarian tissue, identifying six major cell clusters and revealing substantial immune cell infiltration. Further analysis of CD8⁺ T cells identified two key subpopulations—precursor and terminally exhausted T cells—and delineated their developmental trajectories. The accumulation of exhausted CD8⁺ T cells (Tex) suggested an immunosuppressive tumor microenvironment. Integrated trajectory inference and high-dimensional weighted gene co-expression network analysis (hdWGCNA) identified CCL3 as a novel hub gene specifically expressed in Tex cells. Communication analysis suggested that Tex cells may interact with M2 macrophages via the CCL3–CCR1 ligand–receptor axis. Functional validation confirmed that: (1) secretomes from Tex cells—but not effector T cells—significantly promoted M2 polarization in both THP-1 and bone marrow-derived macrophages (CD206⁺ THP-1: 83.8% vs. 51.4%, p < 0.001; CD206⁺ BMDM: 72.4% vs. 41.5%, p < 0.001); and (2) recombinant CCL3 acted synergistically with IL-4/IL-10 to further enhance M2 polarization (59.2% vs. 37.7%, p = 0.008). Collectively, our findings unveil a previously unrecognized immunoregulatory axis whereby exhausted CD8⁺ T cells drive immunosuppression via CCL3–CCR1–mediated communication with M2 macrophages, presenting a promising therapeutic target to reverse the immune-cold tumor microenvironment in HGSOC.