Tumor rejection in Cblb(−/−) mice depends on IL-9 and Th9 cells

BACKGROUND: Casitas B lymphoma-b (Cbl-b) is a central negative regulator of cytotoxic T and natural killer (NK) cells and functions as an intracellular checkpoint in cancer. In particular, Th9 cells support mast cell activation, promote dendritic cell recruitment, enhance the cytolytic function of cytotoxic T lymphocytes and NK cells, and directly kill tumor cells, thereby contributing to tumor immunity. However, the role of Cbl-b in the differentiation and antitumor function of Th9 cells is not sufficiently resolved. METHODS: Using Cblb(−/−) mice, we investigated the effect of knocking out Cblb on the differentiation process and function of different T helper cell subsets, focusing on regulatory T cell (Treg) and Th9 cells. We applied single-cell RNA (scRNA) sequencing of in vitro differentiated Th9 cells to understand how Cbl-b shapes the transcriptome and regulates the differentiation and function of Th9 cells. We transferred tumor-model antigen-specific Cblb(−/−) Th9 cells into melanoma-bearing mice and assessed tumor control in vivo. In addition, we blocked interleukin (IL)-9 in melanoma cell-exposed Cblb(−/−) mice to investigate the role of IL-9 in tumor immunity. RESULTS: Here, we provide experimental evidence that Cbl-b acts as a rheostat favoring Tregs at the expense of Th9 cell differentiation. Cblb(−/−) Th9 cells exert superior antitumor activity leading to improved melanoma control in vivo. Accordingly, blocking IL-9 in melanoma cell-exposed Cblb(−/−) mice reversed their tumor rejection phenotype. Furthermore, scRNA sequencing of in vitro differentiated Th9 cells from naïve T cells isolated from wildtype and Cblb(−/−) animals revealed a transcriptomic basis for increased Th9 cell differentiation. CONCLUSION: We established IL-9 and Th9 cells as key antitumor executers in Cblb(−/−) animals. This knowledge may be helpful for the future improvement of adoptive T cell therapies in cancer.