Clonotype-Resolved Single-Cell Multi-Omics Unlocks the Profile of Tumor-Infiltrating CD39⁺CD8⁺ T Cells and Enables Adoptive Cell Therapy for Solid Tumor.

Tumor-reactive T cells are central to cancer immunotherapy, and immune checkpoint inhibitors (ICIs) have revolutionized treatment by relieving immune suppression on tumor-reactive T cells, yet response rates remain suboptimal. Adoptive T cell therapy can supplement tumor-reactive T cells, but accurately identifying tumor-reactive CD8⁺ T cells within tumor-infiltrating lymphocytes (TILs) remains challenging. CD39 (ENTPD1) is a rate-limiting enzyme in adenosine metabolism, leading to the view that CD39 is associated with immune suppression because of the inhibitory function of adenosine in tumor immunity. However, its role in tumor-reactive CD8⁺ TIL endures as controversial. In this study, we reassess the tumor-reactive potential of CD39⁺CD8⁺ TILs using clonotype-resolved single-cell multi-omics. Compared to CD39⁻CD8⁺ TILs, CD39⁺CD8⁺ TILs exhibited features of proliferation, activation, and T cell-mediated cytotoxicity, alongside reduced TCR clonal diversity and increased TCR clonal expansion, indicating tumor reactivity. TCR-T cells engineered with TCRs from CD39⁺CD8⁺ TILs mediated robust antigen-specific killing