Umbilical Cord Blood and iPSC-Derived Natural Killer Cells Demonstrate Key Differences in Cytotoxic Activity and KIR Profiles

Abstract

Natural killer (NK) cells derived or isolated from different sources have been gaining in importance for cancer therapies. In this study, we evaluate and compare key characteristics between NK cells derived or isolated from umbilical cord blood, umbilical cord blood hematopoietic stem/progenitor cells, peripheral blood, and induced pluripotent stem cells (iPSCs). Specifically, we find CD56⁺ NK cells isolated and expanded directly from umbilical cord blood (UCB56) and NK cells derived from CD34⁺ hematopoietic stem/progenitors in umbilical cord blood (UCB34) differ in their expression of markers associated with differentiation including CD16, CD2, and killer Ig-like receptors (KIRs). UCB56-NK cells also displayed a more potent cytotoxicity compared to UCB34-NK cells. NK cells derived from iPSCs (iPSC-NK cells) were found to have variable KIR expression, with certain iPSC-NK cell populations expressing high levels of KIRs and others not expressing KIRs. Notably, KIR expression on UCB56 and iPSC-NK cells had limited effect on cytotoxic activity when stimulated by tumor target cells that express high levels of cognate HLA class I, suggesting that in vitro differentiation and expansion may override the KIR-HLA class I mediated inhibition when used across HLA barriers. Together our results give a better understanding of the cell surface receptor, transcriptional, and functional differences between NK cells present in umbilical cord blood and hematopoietic progenitor-derived NK cells which may prove important in selecting the most active NK cell populations for treatment of cancer or other therapies.

Keywords: KIRs; cancer; natural killer cells; stem cells; umbilical cord blood.

Figure 1

UCB56 and UCB34 NK cell phenotypes by mass cytometry. tSNE plots demonstrating the range (positive—red, negative—blue) of expression of NK cell markers of function, activity, maturation, and identity by mass cytometry. UCB34 NK cells from Donor 2 are grouped in the top rows while UCB56 NK cells from Donor 2 for each marker are grouped in the bottom rows.

Figure 2

KIR Expression of UCB56 and UCB34 NK cells (A) KIR phenotypes by mass cytometry. tSNE plots demonstrating the range (positive—red, negative—blue) of expression of KIR markers. UCB34 NK cells from Donor 2 are grouped in the top rows while UCB56 NK cells from Donor 2 for each marker are grouped in the bottom rows. (B) NK cell KIR markers by flow cytometry of Donor 1, 2, and 3 UCB34 and UCB56 NK cells (red) compared to isotype controls (blue). Representative panels are shown from n = 3 replicates.

Figure 3

UCB56 NK and UCB34 NK cell killing activity against lymphoblastic 721.221 and myeloid K562 tumors (A) Cell death and apoptosis by caspase 3,7 activation, and 7-AAD staining of PBNK cells, UCB56 NK cells, and UCB34 NK cells with 721.221 WT, cw3, cw4, and K562 cells at Effector:Target ratios from 1.25:1 up to 20:1 in 4-h co-culture. Experiments were completed in triplicate and representative panels are shown from n = 3 replicates. All statistical analysis is of the comparisons between UCB56 and UCB34 NK cells. (B) Tumor cells alone (red) and tumor cell killing by PB-NK (blue), UCB56 (purple), and UCB34 NK cells (orange) measured by Incucyte live-imaging system over 24 h. Experiments were completed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

KIR expression and cytotoxic activity of iPSC-derived KIRPos and KIRNeg NK cells. (A) NK cell KIR markers by flow cytometry of iPSC-KIRPos and iPSC-KIRNeg NK cells (red) compared to isotype controls (blue). Representative panels are shown from n = 3 replicates. (B) NK cell markers by flow cytometry of iPSC-KIRPos and iPSC-KIRNeg NK cells (red) compared to isotype controls (blue). (C) Cell death and apoptosis by caspase 3,7 activation and 7-AAD staining of 721.221 WT, cw3, cw4, SK-N-AS, IMR32, NBLS, and K562 cells with PBNK cells (blue), iPSC-KIRPos NK cells (green), and iPSC-KIRNeg NK cells (purple) after 4-h co-culture at effector:target ratios from 1.25:1 up to 20:1 (721.221 cells) or 0.3:1 up to 5:1 (neuroblastoma cells). Representative panels are shown from n = 3 replicates. All statistical analysis is of the comparisons between KIRPos and KIRNeg iPSC-NK cells. (D) Tumor cells alone (red) and tumor cell killing by PBNK (blue), iPSC-KIRPos (green), and iPSC-KIRNeg NK cells (purple) measured by Incucyte live-imaging system over 24 h. Experiments are completed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

RNA Sequencing analysis of gene expression among UCB56 NK, iPSC NK, PB NK cells, NK 92 cells, and 3 cell lines, n = 3 biological replicates. (A) Heat maps of the correlation coefficient between each NK cell population. (B) Plot of the number of differentially expressed genes between iPSC-NK cells and the other NK cell populations. (C) Cluster analysis of differentially expressed genes. Genome-wide differentially expressed genes were analyzed among each cell population. Log10(FPKM+1) value was used for clustering. (D) Cluster analysis of genes in the NK cell mediated cytotoxicity pathway (KEGG PATHWAY: hsa04650). Hundred twenty four genes in this pathway were analyzed. (E) Cluster analysis of differentially expressed genes between UCB34 and UCB56 pre- and post-expansion with antigen presenting cells (APC) as well as iPSC NK cells post-expansion. Genome-wide differentially expressed genes were analyzed among each cell population. Log10(FPKM+1) value was used for clustering. (F) Volcano plot of differentially expressed genes between UCB34 and UCB56 cells pre-APC expansion. The x-axis indicates the fold change in gene expression between the different samples, the y-axis indicates the statistical significance (adjusted p-values) of the differences. Significantly up and down regulated genes are highlighted in red and green, respectively. Genes that were not differentially expressed between the groups are in blue. (G) Volcano plot of differentially expressed genes between UCB34 and UCB56 cells post-APC expansion. The x-axis indicates the fold change in gene expression between the different samples, the y-axis indicates the statistical significance (adjusted p-values) of the differences. Significantly up and down regulated genes are highlighted in red and green, respectively. Genes that were not differentially expressed between the groups are in blue.