Introduction/Background Despite evidence suggesting a potential role for immunotherapy in Epithelial Ovarian Cancer (EOC), initial attempts had limited efficacy. A better characterization of tumor infiltrating lymphocytes’ (TIL) immunophenotype appears crucial to deeply understand their role in anti-tumor immunity and to set the basis for their potential modulation to optimize adoptive cell therapies approaches. We extensively characterized the composition and phenotype of immune cells in EOC to identify pathways involved in limiting anti-tumor immunity.

Methodology Immune infiltrate was investigated for phenotype in 48 EOC specimens by immunohistochemistry (IHC) and flow cytometry (FC). Furthermore, the gene expression of tumor samples was evaluated with a panel of 799 immune and cancer-related genes by the Nanostring platform. FC was also used to compare T cells isolated from tumor, ascites and peripheral blood of 19 patients for memory phenotype and for the expression of multiple inhibitory receptors (IRs) and of activation markers.

Results Both IHC and FC revealed a high infiltration by T lymphocytes and myeloid cells, while B cells were scanty. High-dimensional analysis of FC data identified 2 metaclusters of CD4⁺ and CD8⁺ T cells exclusively present in tumors, characterized by a CD137⁺CD39⁺PD-1⁺TIM-3⁺CD45RA^-CD62L^-CD95⁺ phenotype. Gene expression profile revealed a peculiar microenvironment in samples characterized by high TIL content, with increased expression of immunity- and myeloid-related genes. Accordingly, the ligands for IRs and co-stimulatory molecules were mainly provided by myeloid rather than neoplastic cells.

Conclusion Our data suggest that EOC is infiltrated by antigen-experienced T lymphocytes displaying features of both activation and partial exhaustion, possibly driven by IRs ligands expression by infiltrating myeloid cells.