Background Lung metastasis remains the primary cause of tumor-related mortality, with limited treatment options and unsatisfactory efficacy. In preclinical studies, T helper 9 (T H 9) cells have shown promise in treating solid tumors. However, it is unclear whether T H 9 cells can tackle more challenging situations, such as established lung metastases. Moreover, comprehensive exploration into the nuanced biological attributes of T H 9 cells is imperative to further unravel their therapeutic potential. Methods We adoptively transferred T H 1, T H 9, and T H 17 cells into subcutaneous, in situ , and established lung metastases models of osteosarcoma and triple-negative breast cancer, respectively, comparing their therapeutic efficacy within each distinct model. We employed flow cytometry and an in vivo imaging system to evaluate the accumulation patterns of T H 1, T H 9, and T H 17 cells in the lungs after transfusion. We conducted bulk RNA sequencing on in vitro differentiated T H 9 cells to elucidate the chemokine receptor CXCR4, which governs their heightened pulmonary tropism relative to T H 1 and T H 17 cell counterparts. Using Cd4 cre Cxcr4 flox/flox mice, we investigate the effects of CXCR4 on the lung tropism of T H 9 cells. We performed mass spectrometry to identify the E3 ligase responsible for CXCR4 ubiquitination and elucidated the mechanism governing CXCR4 expression within T H 9 cellular milieu. Ultimately, we analyzed the tumor immune composition after T H 9 cell transfusion and evaluated the therapeutic efficacy of adjunctive anti-programmed cell death protein-1 (PD-1) therapy in conjunction with T H 9 cells. Results In this study, we provide evidence that T H 9 cells exhibit higher lung tropism than T H 1 and T H 17 cells, thereby exhibiting exceptional efficacy in combating established lung metastases. CXCR4-CXCL12 axis is responsible for lung tropism of T H 9 cells as ablating CXCR4 in CD4 + T cells reverses their lung accumulation. Mechanistically, tumor necrosis factor receptor-associated factor 6 (TRAF6)-driven hyperactivation of NF-κB signaling in T H 9 cells inhibited ITCH-mediated ubiquitination of CXCR4, resulting in increased CXCR4 accumulation and enhanced lung tropism of T H 9 cells. Besides, T H 9 cells’ transfusion significantly improved the immunosuppressed microenvironment. T H 9 cells and anti-PD-1 exhibit synergistic effects in tumor control. Conclusions Our findings emphasized the innate lung tropism of T H 9 cells driven by the activation of TRAF6, which supports the potential of T H 9 cells as a promising therapy for established lung metastases.

Natural lung-tropic TH9 cells: a sharp weapon for established lung metastases