Exercise-Mobilized Donor Lymphocytes Enhance Blinatumomab Activity Against B-ALL.

Background: Donor lymphocyte infusion (DLI) is used to prevent and treat leukemic relapse after allogeneic hematopoietic cell transplantation, although its efficacy against lymphoid malignancies remains limited. Blinatumomab (Blina), a CD3/CD19 bispecific T-cell engager, is increasingly used to enhance DLI activity against acute lymphoblastic leukemia (ALL). We previously demonstrated that exercise-mobilized DLI (DLI-X) exhibits superior anti-leukemic activity against myeloid malignancies. Here, we investigated whether DLI-X combined with Blina enhances cytotoxicity against CD19⁺ B-ALL. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from healthy participants at rest (DLI) and during the final stage of a 20-minute graded cycling protocol to 80% VO₂max (DLI-X). Cytotoxicity against CD19⁺ RS4;11 B-ALL cells was assessed in vitro. T-cell activation and effector function were evaluated by 8-color flow cytometry. In vivo efficacy was determined in NSG-IL-15 mice engrafted with RS4;11 cells and treated with DLI or DLI-X in combination with Blina or vehicle, with tumor burden monitored by bioluminescence imaging. Results: DLI and DLI-X alone exhibited limited cytotoxicity against RS4;11 cells. Blina significantly enhanced cytotoxicity of both products, with DLI-X+Blina demonstrating >20% greater tumor killing than DLI+Blina (~85% vs. ~62%). Compared with DLI, DLI-X displayed enhanced Blina-induced T-cell effector function, including increased CD107a degranulation, greater TNF-α production by CD4⁺ T cells, higher IFN-γ production by CD8⁺ T cells, and 10-15% greater upregulation of CD137 and OX40. In vivo, DLI-X+Blina achieved up to 35% greater control of tumor burden than DLI+Blina. Conclusions: Exercise mobilizes T cells with enhanced responsiveness to Blina, resulting in superior activation and cytotoxicity against CD19⁺ B-ALL cells in vitro and improved tumor control in vivo. These findings support the use of exercise-mobilized DLI as a strategy to enhance the efficacy of bispecific T-cell engager therapy following allogeneic hematopoietic cell transplantation. Trial registration: ClinicalTrials.gov, NCT06643221. Registered retrospectively on October 8, 2024.