Single-cell profiles and prognostic impact of tumor-infiltrating lymphocytes co-expressing CD39, CD103, and PD-1 in ovarian cancer.

Purpose Tumor-infiltrating lymphocytes (TIL) are strongly associated with survival in most cancers; however, the tumor-reactive subset that drives this prognostic effect remains poorly defined. CD39, CD103, and PD-1 have been independently proposed as markers of tumor-reactive CD8+ TIL in various cancers. We evaluated the phenotype, clonality and prognostic significance of TIL expressing various combinations of these markers in high-grade serous ovarian cancer (HGSC), a malignancy in need of more effective immunotherapeutic approaches. Experimental design Expression of CD39, CD103, PD-1, and other immune markers was assessed by high-dimensional flow cytometry, single-cell sequencing, and multiplex immunofluorescence of primary and matched pre/post-chemotherapy HGSC specimens. Results Co-expression of CD39, CD103, and PD-1 ("triple-positive" phenotype) demarcated subsets of CD8+ TIL and CD4+ regulatory T cells (Tregs) with a highly activated/exhausted phenotype. Triple-positive CD8+ TIL exhibited reduced TCR diversity and expressed genes involved in both cytolytic and humoral immunity. Triple-positive Tregs exhibited higher TCR diversity and a tumor-resident phenotype. Triple-positive TIL showed superior prognostic impact relative to TIL expressing other combinations of these markers. TIGIT was uniquely upregulated on triple-positive CD8+ effector cells relative to their CD4+ Treg counterparts. Conclusions Co-expression of CD39, CD103, and PD-1 demarcates highly activated CD8+ and CD4+ TIL with inferred roles in cytolytic, humoral and regulatory immune functions. Triple-positive TIL demonstrate exceptional prognostic significance and express compelling targets for combination immunotherapy, including PD-1, CD39 and TIGIT.