CD39⁺conventional CD4⁺T cells with exhaustion traits and cytotoxic potential infiltrate tumors and expand upon CTLA-4 blockade

Abstract Background Conventional CD4 + T (Tconv) lymphocytes play important roles in tumor immunity; however, their contribution to tumor elimination remains poorly understood. Methods Here we describe a subset of Tconv cells characterized by the expression of CD39. The phenotype, the effector function and transcriptional profile of tumor-infiltrating CD39 + Tconv lymphocytes from different mouse cancer models and breast cancer patients were studied by multiparametric flow cytometry and RNA sequencing. The impact of the in vivo CTLA-4 blockade on the tumor-infiltrating CD39 + Tconv population was assessed in mice grafted with the immunogenic MC38 colorectal tumor. Overall survival was evaluated in a cohort of patients from the TCGA consortium. Results In mouse cancer models, we observed that CD39 + Tconv cells accumulated in tumors as they grew but were absent in lymphoid organs. Compared to tumor CD39 − counterparts, CD39 + Tconv cells exhibited a cytotoxic and exhausted signature at the transcriptomic level, confirmed by high protein expression of inhibitory receptors and transcription factors related to the exhaustion phenotype. Additionally, CD39 + Tconv cells showed increased production of IFN gamma, granzyme B, perforin and CD107a expression, but reduced production of TNF. In vivo CTLA-4 blockade induced the expansion of tumor CD39 + Tconv cells, which maintained their cytotoxic and exhausted features. In breast cancer patients, CD39 + Tconv cells were found in tumors and in metastatic lymph nodes but were less frequent in adjacent non-tumoral mammary tissue and not detected in non-metastatic lymph nodes and blood. Human tumor CD39 + Tconv cells constituted a heterogeneous cell population with features of exhaustion, impaired TNF production, and high expression of inhibitory receptors and CD107a. We found that high CD4 and ENTPD1 (CD39) gene expression in human tumor tissues correlated with a higher overall survival rate in breast cancer patients. Conclusions We found that CD39 acts as a biomarker of Tconv cells with characteristics of both exhaustion and cytotoxic potential. CTLA-4 blockade expands CD39 + CD4 + T cells which may contribute to the reduction of tumor development. Discovering the role of CD39-expressing CD4 + T cells in the tumor microenvironment should help design new strategies to manipulate them and improve the efficacy of current immunotherapies.