1α,25(OH)₂D₃ prevents CD19 CAR-T cell exhaustion and differentiation via VDR-dependent transcriptional reprogramming

CD19-directed chimeric antigen receptor T (CAR-T) cell therapy is promising for treating relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL), yet its long-term efficacy remains limited by CAR-T-cell exhaustion. Given the broad immunomodulatory activities of vitamin D, we investigate whether its active form, 1α,25(OH) ₂D ₃, enhances CAR-T-cell functionality and improves therapeutic outcomes. We demonstrate that 1α,25(OH) ₂D ₃ treatment significantly mitigates exhaustion and enhances the antitumor activity of CD19 CAR-T cells derived from both healthy donors and DLBCL patients, which is further validated in xenograft mouse models. Mechanistically, we show that 1α,25(OH) ₂D ₃ upregulates the expression of the vitamin D receptor (VDR), promoting transcriptional reprogramming associated with memory-like differentiation and downregulation of exhaustion-related genes, thereby reshaping the functional heterogeneity of CAR-T cells under tumor stimulation. Our study highlights 1α,25(OH) ₂D ₃ supplementation as a safe and accessible approach to mitigate terminal differentiation and exhaustion of CAR-T cells, offering a promising strategy to enhance the clinical efficacy of CAR-T therapy in patients with R/R DLBCL.