Exercise-mobilized lymphocytes enhance the function of cytokine-induced memory-like NK cells against myeloid leukemia

Short-term activation of natural killer (NK) cells with interleukin-12 (IL-12), IL-15, and IL-18 (IL-12/15/18) gives rise to cytokine-induced memory-like (CIML) NK cells after adoptive transfer, which exhibit enhanced antitumor 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 threefold to fourfold increase in circulating NK cells enriched for gene programs and surface proteins linked to antitumor activity. Here, we tested whether exercise-mobilized NK cells could improve the function of IL-12/15/18-activated NK (aNK) cells in vitro and CIML NK cells in vivo. Eighteen healthy donors performed 20 minutes of graded cycling up to 80% maximal oxygen uptake, with blood collected at rest and during exercise. NK cells purified from rest and exercise (NK-X) were cultured overnight with IL-15 (NK or NK-X) or IL-12/15/18 (aNK or aNK-X). End points included in vitro cytotoxicity and tumor control in leukemia-bearing xenogeneic mice. aNK-X cells exhibited stronger cytotoxicity against 2 myeloid leukemia cell lines than aNK cells from the same donors, accompanied by increased interferon gamma production, enhanced degranulation, and an enriched phenotype (higher NKG2A-/NKG2D+, CD57+, CD16+). Notably, NK-Xs displayed greater cytotoxic activity than NKs and were comparable with aNK cells, although aNK-X cells outperformed both. In mice, CIML NK-X cells combined with exercise-mobilized donor lymphocyte infusion (DLI-X) prolonged engraftment, delayed tumor progression, and extended survival relative to CIML NK cells combined with standard DLI. These findings demonstrate that exercise-induced NK cell mobilization combined with cytokine preactivation yields an adoptive cell therapy product with superior antileukemic activity. This trial was registered at www.ClinicalTrials.gov as NCT06643221.