Single-Cell Profiles And Prognostic Impact Of Tumor-Infiltrating Lymphocytes Coexpressing Cd39, Cd103, And Pd-1 In Ovarian Cancer

Purpose: Tumor-infiltrating lymphocytes (TIL) are strongly associated with survival in most cancers; however, the tumorreactive 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 highgrade 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: Coexpression of CD39, CD103, and PD-1 ("triplepositive" phenotype) demarcated subsets of CD8(+) TIL and CD4(+) regulatory T cells (Treg) with a highly activated/exhausted phenotype. Triple-positive CD8(+) TIL exhibited reduced T-cell receptor (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: Coexpression of CD39, CD103, and PD-1 demarcates highly activated CD8(+) and CD4(+) TIL with inferred roles in cytolytic, humoral, and regulatory immune functions. Triplepositive TIL demonstrate exceptional prognostic significance and express compelling targets for combination immunotherapy, including PD-1, CD39, and TIGIT.