Inhibition of RBPJ transcription complex promotes IL-17 and IFN-γ secretion by CD4⁺ T cells in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is characterized by an immunosuppressive tumor microenvironment (TME), indicating that immune cell activation is a promising approach. The use of γ-secretase inhibitors (GSIs) to control high Notch signaling activity is currently one of the traditional methods for clinical immunotherapy of HCC. However, the lack of substrate specificity in GSIs often leads to serious side effects. In contrast, a novel small molecule compound, RBPJ inhibitor-1 (RIN1), which selectively blocks the functional interaction between RBPJ and Notch intracellular domain (NICD), has been found to inhibit CD8+ T cells exhaustion in HCC effectively. However, its impact on CD4+ T cells is still unknown. This study found that RIN1 stimulated T cell IL-17 and IFN-γ secretion, and drove more T cell differentiation towards Th17.1 (CD161+, CD183+, CD191-). Furthermore, RIN1 upregulated T cell STAT3, STAT4, TBX21 protein levels, enhanced STAT3 and RORγt binding to the IL-17 promoter, and facilitated STAT4 and TBX21 enrichment on IFNG promoter. RIN1 also boosted T cell-mediated antitumor immunity and inhibited HCC cells' epithelial-mesenchymal transition. Notably, IL-17R knockdown in HCC cells partially reverted RIN1-enhanced T cell antitumor effects. In vivo, RIN1 promoted the expression of IL-17 and IFN-γ in CD4+ TILs while suppressing PD-1 expression and reducing the frequency of Treg cells, exhibiting tumor growth inhibition. These findings suggested that RIN1 enhances CD4+ T cell-mediated antitumor immunity in HCC by modulating gene transcription and cell subset differentiation, highlighting its potential as an immunostimulatory agent (Graphical abstract).