High dimensional flow cytometric analysis reveals distinct NK cell subsets but conserved response to cytokine stimulation in umbilical cord blood and adult peripheral blood

Abstract

Adoptive cellular therapies using Natural Killer (NK) cells are of growing interest. NK cells can be obtained from various sources, including umbilical cord blood (UCB) and adult peripheral blood (APB). Understanding the diversity of NK cell populations and their receptor expression in both UCB and APB will guide future therapeutic designs. In this study, we used a 20-colour flow cytometry panel to compare unstimulated and cytokine-activated UCB and APB NK cells. Our analysis showed that UCB NK cells express slightly higher levels of the immune checkpoints PD-1, TIGIT and CD96 compared to their APB counterparts. Unsupervised hierarchical clustering and principal component analyses revealed previously unappreciated differences in NK cell populations from UCB and APB. UCB was characterised by an enrichment in CD56neg as well as mature NKp46neg and CD56+CD16+ NK cell populations whereas CD57+ terminally differentiated NK cells with variable expression of KIRs and CD16 were found in APB. These populations were conserved following two-days of IL-15 culture as well as overnight stimulation with IL-12, IL-15, and IL-18. Interestingly, cytokine stimulation was associated with the up-regulation of LAG-3 and DNAM-1 together with the downregulation of NKG2D, TIGIT and CD16 on multiple NK cell subsets in both UCB and APB. TIM-3 was also up-regulated with activation, but only in UCB. Overall, our data indicate that NK cells in UCB have a more immature phenotype than APB NK cells and UCB NK cells might be more amenable to immune checkpoint therapy.