TSPAN32 enhances CAR-T cell potency by assembling IL-2 receptor complex and amplifying its intracellular signal transduction

Background Chimeric antigen receptor-engineered T cells (CAR-T) have shown substantial therapeutic potential in hematologic malignancies, but further improvement in T-cell functional capacity is needed to optimize efficacy. We observed reduced TSPAN32 expression in T cells from peripheral blood of patients with B-cell lymphoma. This study aimed to investigate whether TSPAN32 enhances CAR-T cell antitumor activity and to explore the underlying mechanism.Methods TSPAN32 expression was assessed in T cells isolated from patients with B-cell lymphoma. T cells were engineered to co-express TSPAN32 and CD19-CAR, and their antitumor efficacy and cytokine secretion were evaluated in vitro and in subcutaneous tumor models in vivo. Gene expression profiling was performed to identify signaling pathways associated with TSPAN32 overexpression. Mechanistic studies examined the interaction between TSPAN32 and CD25 and its effect on IL-2 signaling. In addition, a transgenic mouse model with endogenous TSPAN32 overexpression and a TSPAN32-specific antibody (FF-37) were used to assess therapeutic potential.Results TSPAN32 expression was reduced in T cells from B-cell lymphoma patients. Co-expression of TSPAN32 with CD19-CAR significantly enhanced antitumor activity and cytokine production compared with CD19-CAR alone in vitro. In vivo, T cells engineered with both CAR and TSPAN32 showed superior therapeutic efficacy in subcutaneous tumor models. Gene expression profiling indicated increased IL-2 signaling activation in TSPAN32-high CAR-T cells. Mechanistically, TSPAN32 interacted with CD25, promoting its aggregation on the T-cell surface and enhancing IL-2 signal transduction. Endogenous TSPAN32 overexpression in transgenic mice increased resistance to subcutaneous tumor growth. Furthermore, the TSPAN32-specific antibody FF-37 increased TSPAN32 expression and improved CAR-T antitumor efficacy.Conclusions TSPAN32 enhances CAR-T cell antitumor function by promoting CD25 aggregation and IL-2 signaling activation. Increasing TSPAN32 expression, either through genetic engineering or the TSPAN32-specific antibody FF-37, may represent a promising strategy to improve CAR-T cell therapy.