Exercise-Mobilized Lymphocytes Enhance the Function of Cytokine-Induced Memory-like NK-cells Against Myeloid Leukemia

Short-term activation of NK cells with IL-12/15/18 gives rise to cytokine-induced memory-like (CIML) NK cells after adoptive transfer, which exhibit enhanced anti-tumor activity, proliferation, and persistence. Clinical trials in high-risk leukemia have shown encouraging results, yet significant challenges remain, particularly the limited durability of cytotoxicity and the failure to achieve sustained remission. We recently demonstrated that acute exercise induces a 3-4-fold increase in circulating NK cells enriched for gene programs and surface proteins linked to anti-tumor activity. Here, we tested whether exercise-mobilized NK cells could improve the function of IL-12/15/18 activated NK-cells (aNK-cells) in vitro and CIML NK cells in vivo. Eighteen healthy donors performed 20 minutes of graded cycling up to 80% VO₂max, with blood collected at rest and during exercise. NK cells purified from rest and exercise (-X) were cultured overnight with IL-15 (NK or NK-X) or IL-12/15/18 (aNK or aNK-X). Endpoints included in vitro cytotoxicity and tumor control in leukemia-bearing xenogeneic mice. aNK-X exhibited stronger cytotoxicity against two myeloid leukemia cell lines than aNK from the same donors, accompanied by increased IFN-γ production, enhanced degranulation, and an enriched phenotype (higher NKG2A⁻/NKG2D⁺, CD57⁺, CD16⁺). Notably, NK-X displayed greater cytotoxic activity than NK and was comparable to aNK, though aNK-X outperformed both. In mice, CIML NK-X combined with exercise-mobilized donor lymphocyte infusion (DLI-X) prolonged engraftment, delayed tumor progression, and extended survival relative to CIML NK combined with standard DLI. These findings demonstrate that exercise-induced NK mobilization combined with cytokine pre-activation yields an adoptive cell therapy product with superior anti-leukemic activity. (NCT06643221).