CD39 Identifies the CD4⁺ Tumor-Specific T-cell Population in Human Cancer

The accumulation of tumor-specific CD4⁺ and CD8⁺ effector T cells is key to an effective antitumor response. Locally, CD4⁺ T cells promote the recruitment and effector function of tumor-specific CD8⁺ T cells and activate innate killer cells in the tumor. Here, we show that tumor-specific CD4⁺ T cells were predominantly present in the CD39⁺ subset of tumor-infiltrating lymphocytes (TIL). The CD39⁺ CD4⁺ and CD8⁺ TILs were detected in three different tumor types, and displayed an activated (PD-1⁺, HLA-DR⁺) effector memory phenotype. CD4⁺CD39⁺ single-cell RNA-sequenced TILs shared similar well-known activation, tissue residency, and effector cell-associated genes with CD8⁺CD39⁺CD103⁺ TILs. Finally, analysis of directly ex vivo cell-sorted and in vitro expanded pure populations of CD39-positive and negative CD4⁺ and CD8⁺ TILs revealed that tumor-specific antigen reactivity was almost exclusively detected among CD39⁺ cells. Immunotherapy of cancer is based on the activation of tumor-reactive CD4⁺ and CD8⁺ T cells. We show that the expression of CD39 can be used to identify, isolate, and expand tumor-reactive T-cell populations in cancers.