Effector regulatory T-cell infiltration, ARID1A status, and Ex Vivo anti-PD-1 response in ovarian clear cell carcinoma: Insights from a comparative high-grade serous ovarian cancer cohort

Recent trials suggest potential benefit of immune checkpoint blockade (ICB) in ovarian clear cell carcinoma (OCCC), but the immune contexture and biomarkers that modulate ICB response remain poorly understood. We characterized tumor-infiltrating lymphocytes (TILs) in OCCC and explored associations with clinical features, ex vivo anti-PD-1-based ICB responsiveness, and biomarker expression. Peripheral blood mononuclear cells (PBMCs) and TILs were obtained from patients with OCCC (n=55). Immune checkpoint receptor and transcription factor expression were analyzed by multicolor flow cytometry. TILs were stimulated ex vivo with anti-CD3 plus anti-PD-1 with or without anti-CTLA-4, and proliferation was assessed by T-cell division. HER2, ARID1A, and L1CAM expression were evaluated by immunohistochemistry on tumor tissue. Twenty of 55 patients had stage III/IV or recurrent disease, and most tumors were grade 3. Among evaluable tumors, 9/38 exhibited HER2 2+/3+, 24/41 showed loss of ARID1A, and 29/41 showed high L1CAM expression. In flow cytometric analysis, CD8 T cells and regulatory T cells (Tregs) were enriched in tumors compared with peripheral blood. CD8⁺ TILs showed a profoundly exhausted phenotype, with higher PD-1, CTLA-4, and TOX and lower TCF-1 expression than CD8⁺ T cells in blood, while tumor-reactive CD39⁺CD103⁺ CD8 TILs were also more enriched. Tregs exhibited highly suppressive features with increased immune checkpoint receptors, CD39, and CCR8, and effector Tregs (CD45RA⁻FoxP3high) were more abundant in stage III/IV or recurrent tumors than in stage I/II disease. In ex vivo assays, CD8 TILs from a subset of patients showed robust anti-PD-1-induced proliferation, and effector Treg frequency was inversely associated with anti-PD-1-induced CD8 T-cell reinvigoration. ARID1A-deficient tumors exhibited significantly lower effector Treg infiltration, whereas HER2 and L1CAM expression were not clearly associated with T-cell phenotypes or anti-PD-1 response. When compared with our previously reported cohort of advanced-stage high-grade serous ovarian cancer (HGSOC), the percentage of tumor-infiltrating Tregs, particularly 4-1BB⁺ Tregs, was lower in advanced/recurrent OCCC than in HGSOC. In conclusion, OCCC exhibits profound exhaustion of CD8 TILs alongside infiltration of highly suppressive Tregs. Effector Treg frequency was inversely associated with anti-PD-1-induced CD8 T-cell reinvigoration, and ARID1A loss may be linked to a tumor microenvironment more permissive to anti-PD-1-based immune checkpoint blockade. This hypothesis may help explain the high ICB response rate reported in OCCC compared with HGSOC and requires confirmation in larger clinical cohorts and mechanistic studies elucidating how ARID1A loss in OCCC reprograms the tumor immune microenvironment.