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

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 in vitro. Importantly, reinfusion of CD39⁺CD8⁺ TILs significantly inhibited tumor growth and demonstrated favorable safety in vivo. At the patient level, we further demonstrated that CD39⁺CD8⁺ TILs are enriched for effector programs and pathways linked to T-cell activation and cytotoxicity, and exhibit reduced TCR clonal diversity with pronounced clonal expansion. The intratumoral abundance of CD39⁺CD8⁺ TILs also correlated with earlier tumor stage and improved overall survival, and a CD39⁺CD8⁺ TIL-derived gene signature predicted ICI response and prognosis, supporting CD39 as a practical biomarker to enrich tumor-reactive CD8⁺ TILs and to improve adoptive cell transfer strategies in future clinical practice.