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

Abstract

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)2D3, enhances CAR-T-cell functionality and improves therapeutic outcomes. We demonstrate that 1α,25(OH)2D3 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)2D3 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)2D3 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.

1. Introduction

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), accounting for approximately 30%–40% of newly diagnosed cases worldwide [1,2]. DLBCL is a biologically and clinically heterogeneous disease that is often characterized by rapid progression and early relapse [3,4]. The current standard treatment for DLBCL is immunochemotherapy, which typically consists of rituximab, a monoclonal antibody targeting CD20, combined with the CHOP regimen (cyclophosphamide, doxorubicin, vincristine, and prednisone) [5]. While this treatment induces remission in many patients, approximately 30%–40% ultimately experience relapse or develop refractory disease, posing significant therapeutic challenges [6].

CD19-directed chimeric antigen receptor (CAR) T cell therapy has emerged as a transformative treatment for patients with relapsed or refractory (R/R) DLBCL by redirecting patient-derived T cells to selectively recognize and eliminate CD19-positive lymphoma cells [6–8]. Multiple clinical trials have demonstrated durable responses and complete remission rates of 40%–58% in heavily pretreated patients, leading to the approval of several commercial CD19 CAR-T products for DLBCL treatment [9–12]. Despite these remarkable initial responses, the long-term efficacy of CAR-T therapy remains limited, as most relapses occur within the first year, and fewer than half of responders maintain ongoing responses after five years [13,14]. Notably, DLBCL patients who relapsed after CD19 CAR-T-cell therapy face poor outcomes, with a median overall survival (OS) shorter than 6 months [15,16]. This variability in the clinical outcomes of CD19 CAR-T treatment may be attributable to several factors, including intrinsic tumor resistance, immune evasion mechanisms within the tumor microenvironment, and, most critically, dysfunction of the infused CAR-T cells [17].