HOPX is required for the generation of umbilical cord blood-derived memory-like NK cells induced by three cytokines

Background Natural killer (NK) cells represent a highly promising form of cancer immunotherapy. Recent studies have utilized umbilical cord blood (UCB) as a source of NK cells and achieved encouraging results. However, several challenges remain, including the limited number of NK cells that can be obtained from UCB, as well as the difficulty of ex vivo expansion and functional persistence, which hinder large−scale clinical applications. In addition, the absence of standardized culture systems leads to inconsistent cell purity and cytotoxic activity, thereby limiting the efficacy and translational potential of NK cell−based tumor therapy. Method We demonstrated that in vitro activation by a combination of cytokines, followed by prolonged expansion with high doses of IL-2, can induce and expand memory-like NK cells from UCB. We performed both in vivo and in vitro investigations into the unique properties of these memory-like NK cells, and analyzed their heterogeneity via single-cell sequencing. Results These memory-like NK cells displayed augmented proliferation and sustained cytotoxic efficacy. Via single-cell analysis, we detected considerable heterogeneity among UCB-derived NK cells. We identified six cell subsets with well-defined functional characteristics in expanded UCB-derived NK cells, conducted a systematic and in-depth analysis of the gene expression profile of each subset, obtained findings distinct from previous studies, and unraveled the unique transcriptional features of umbilical cord blood-derived NK cells. Moreover, memory-like NK cells exhibited a markedly higher proportion displaying a proliferative phenotype. Notably, we found the HOPX is required in the generation of memory-like NK cells derived from UCB. Conclusion In conclusion, memory-like NK cells derived from umbilical cord blood exhibit excellent ex vivo expansion capacity and sustained cytotoxicity in vitro and in vivo . HOPX protein is required for the generation and functional maintenance of these cells. Therefore, such cells hold promising clinical potential in tumor immunotherapy.