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.

Exploring NK cell receptor dynamics in paediatric leukaemias: implications for immunotherapy and prognosis

“…Among these, clusters 3 and 5 were identified as terminally matured NK cells because of their expression of CD57. 37 Moreover, Clusters 4, 6 and 12 corresponded to terminally matured NK cells lacking CD16 and/or KIR expression, which are atypical NK cell populations identified in a previous study 38 ; they represented 19.3% of NK cells. Cluster 1 corresponded to CD56 dim CD16 + NK cells that were negative for both NKG2A and KIR and might represent maturation intermediates between CD56 br KIR À and CD56 dim KIR + NK cells.…”

“…UCB have been previously reported to express high levels of NKG2A, which has been associated with NK cells' immature development stages. 38 It is possible that the high expression of NKG2A and NKG2D in paediatric leukaemia patients reflects their young age. Interestingly, in AML, we observed significant correlations between the expression of NKG2A on NK cells and their respective ligand expression on tumor cells, suggesting that immunoediting occurred along this immunological axis.…”

“…The isolation of cells from APB and UCB samples was conducted under sterile conditions at room temperature according to previously established protocol. 38 The blood was first diluted with an equal volume of PBS, resulting in a total volume of 35 mL. A 50-mL conical tube was then prepared by underlaying 15 mL of Ficoll-Paque Plus blood cell density gradient medium (Cytivia, Massachusetts, USA).…”

“…Cells were stained with 10% mouse serum (Sigma-Aldrich), 10% rat serum (Sigma-Aldrich) and 10% MACS buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) at a 1:1:1 ratio for 10 min at 4°C to block the non-specific binding, as described in a previous study. 38 Cells were then washed with PBS and performed LiveDead staining with Fixable Viability Stain 575 V (BD Biosciences, San Jose, USA) or Zombie Aqua TM Fixable Viability Kit (Biolegend, San Deigo, USA) for 10 min at 4°C in the dark. Cells were subsequently washed with MACS buffer and stained with other cell surface antibodies for 15-20 min at 4°C.…”