Preconditioning determines engraftment and therapeutic efficacy of anti-CD19 CAR-T cells in murine lupus

Anti-CD19 chimeric antigen receptor (CAR) T cells have emerged as a promising therapeutic strategy for autoimmune diseases, including systemic lupus erythematosus. An unresolved question is how immunosuppressive preconditioning regimens influence CAR-T cell engraftment and therapeutic efficacy. In murine models, whole-body irradiation is required for optimal CAR-T cell engraftment. However, irradiation alone can ameliorate lupus disease, confounding attribution of CAR-T-specific effects. To delineate the contribution of CAR-T cells beyond irradiation, we investigated how conditioning intensity shapes immunologic and physiological outcomes in lupus-prone New Zealand Black/New Zealand White (NZB/W) F1 mice. We generated murine anti-CD19 CAR-T cells incorporating a CD28 costimulatory domain and compared their efficacy following low-, intermediate- and high-dose irradiation. High-dose irradiation induced disease improvement that limited mechanistic discrimination between irradiation- and CAR-T-mediated effects, whereas low-dose irradiation failed to support durable CAR-T cell engraftment. In contrast, an intermediate-dose irradiation regimen in 22-week-old mice achieved consistent CAR-T cell engraftment in lymphoid organs while minimizing irradiation-driven disease modulation. Under these conditions, CAR-T cell treatment induced significant depletion of B cells in blood and spleen, including naïve B cells, marginal zone B cells, and plasma cells, accompanied by reduced anti-double-stranded DNA antibody levels and prevention of renal disease progression. Collectively, these findings demonstrate that intermediate-intensity conditioning enables mechanistic separation of CAR-T-mediated immune remodeling from irradiation effects in murine lupus and provide a preclinical framework that aligns with the pressing need for reduced-intensity lymphodepletion strategies in human CAR-T therapy for autoimmune diseases.