Exercise-mobilized donor lymphocytes enhance blinatumomab activity against B-ALL

Abstract 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 up 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 Exercise preferentially mobilized CD8⁺ EM and EMRA T cells and increased CD8⁺ T-cell NKG7 expression (~ 70% vs. ~55%), enriching DLI-X for cytotoxic effector populations relative to DLI. 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. Clinical trial registry This trial was retrospectively registered on clinicaltrials.gov under protocol NCT06643221 , since October 8th 2024. https://clinicaltrials.gov/study/NCT0664322 1.