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

Conventional CD4⁺ T (Tconv) lymphocytes play important roles in tumor immunity; however, their contribution to tumor elimination remains poorly understood. Here, we describe a subset of tumor-infiltrating Tconv cells characterized by the expression of CD39. In several mouse cancer models, we observed that CD39⁺ Tconv cells accumulated in tumors 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. Additionally, CD39⁺ Tconv cells showed increased production of IFN$\gamma$, granzyme B, perforin and CD107a expression, but reduced production of TNF. Around 55% of OVA-specific Tconv from B16-OVA tumor-bearing mice, expressed CD39. 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, 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. Our results identify CD39 as a biomarker of Tconv cells, with characteristics of both exhaustion and cytotoxic potential, and indicate CD39⁺ Tconv cells as players within the immune response against tumors.