TIPE2 knockdown enhances the anti-tumor efficacy of NKG2D CAR-T cells against pancreatic cancer via activating NF-κb signaling pathway

Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) is an intracellular immune checkpoint protein known to suppress T cell activation and effector function. Despite its role in limiting T cell responses, CAR-T cells are prone to TIPE2-mediated inhibitory signaling. We therefore hypothesized that inhibiting this immune checkpoint would enhance CAR-T cell anti-tumor function. To overcome TIPE2-mediated negative regulation, we engineered a novel second-generation NKG2D-based CAR-T cell by incorporating TIPE2-targeting shRNA sequences directly into the CAR construct. TIPE2 knockdown efficiency in the CAR constructs was measured by qPCR and western blot analysis. The functional and mechanistic properties of TIPE2-downregulated CAR-T cells were evaluated in vitro by flow cytometry, including analysis of activation, cytotoxicity, exhaustion, apoptosis, proliferation, and differentiation. Antitumor efficacy was further validated in vivo using a preclinical pancreatic cancer mouse model. Flow cytometry analysis revealed that TIPE2-deficient CAR-T cells exhibited significantly higher expression of activation (CD69), degranulation (CD107a), cytotoxic (GzmB), and cytokine (IFN-γ) markers, resulting in more efficient tumor cell elimination compared to conventional CAR-T cells. TIPE2 silencing also reduced T cell exhaustion, lowered susceptibility to apoptosis, and enhanced proliferation when co-cultured with Panc-28 pancreatic cancer cells. Moreover, TIPE2 inhibition skewed CAR-T cells differentiation towards an effector phenotype (T EFF ), characterized by higher T-bet expression and reduced Eomes production. Mechanistically, these functional enhancements were mediated by increased NF-κB signaling, as confirmed by elevated p-p65 expression and functional reversal upon NF-κB inhibition. Consistently, TIPE2-deficient CAR-T cells exhibited significantly improved anti-tumor efficacy in vivo compared to wild-type CAR-T cells. We successfully developed TIPE2-downregulated NKG2D-CAR-T cells that exhibited enhanced activation and cytotoxicity while limiting apoptosis and exhaustion against NKG2D ligand-expressing pancreatic tumors, highlighting TIPE2 as a promising intracellular immune checkpoint target for optimizing CAR-T cell therapy in solid tumors.