Tumor-specific CXCR6 positive precursor CD8⁺ T cells mediate tumor control in metastatic melanoma

Adoptive cell therapy (ACT) mediates durable and complete regression of various cancers. However, its efficacy is limited by the long-term persistence of cytotoxic T lymphocytes, given their irreversible dysfunction within the tumor microenvironment. Herein, we aimed to establish an artificial lung metastasis model to examine T-lymphocyte subsets, in order to identify potential effective cell subsets for ACT.

A metastatic lung melanoma mouse model was established using OVA-expressing melanoma B16 cells. Flow cytometry analysis was conducted to examine the surface markers, transcription factors, and secreted cytokines of tumor-specific CD8⁺ T cells within metastatic tissues. The infiltrated cells were sorted by flow cytometry for in vitro tumor cell killing assays or in vivo cell infusion therapy combined with chemotherapeutic drugs and immune checkpoint blockade antibodies.

Exhausted CD8⁺ T cells (Tex) exhibited high heterogeneity in metastatic tissues. Among Tex cells, the CXCR6^- precursor cell showed certain memory characteristics, including phenotype, transcription factors, and maintenance, whereas the CXCR6⁺ subpopulation partially lost these traits. Moreover, CXCR6⁺ precursor cells effectively replenished effector-like Tex cells in metastatic tissues and exerted direct cytotoxicity against tumor cells. Notably, transferring these tumor-specific CXCR6⁺ precursor-exhausted T (Texp) cells into recipients induced a substantial regression of metastasis. In addition, these cells could respond to immune checkpoint blockade, which could better control tumor metastasis.

In our study, a subset of antigen-specific CXCR6-expressing Texp cells was observed within the metastatic tissue. The cells served as a crucial source of effector-like Tex cells and exerted direct cytotoxic effects on tumor cells. Adoptive transfer of CXCR6⁺ Texp cells effectively mitigated lung metastasis in mice. This study helps elucidate the role of Texp cells in metastasis, thereby offering novel insights into enhancing the efficacy and durability of immunotherapy.