Potently Cytotoxic Natural Killer Cells Initially Emerge from Erythro-Myeloid Progenitors during Mammalian Development

Abstract

Natural killer (NK) cells are a critical component of the innate immune system. However, their ontogenic origin has remained unclear. Here, we report that NK cell potential first arises from Hoxa^neg/low Kit⁺CD41⁺CD16/32⁺ hematopoietic-stem-cell (HSC)-independent erythro-myeloid progenitors (EMPs) present in the murine yolk sac. EMP-derived NK cells and primary fetal NK cells, unlike their adult counterparts, exhibit robust degranulation in response to stimulation. Parallel studies using human pluripotent stem cells (hPSCs) revealed that HOXA^neg/low CD34⁺ progenitors give rise to NK cells that, similar to murine EMP-derived NK cells, harbor a potent cytotoxic degranulation bias. In contrast, hPSC-derived HOXA⁺ CD34⁺ progenitors, as well as human cord blood CD34⁺ cells, give rise to NK cells that exhibit an attenuated degranulation response but robustly produce inflammatory cytokines. Collectively, our studies identify an extra-embryonic origin of potently cytotoxic NK cells, suggesting that ontogenic origin is a relevant factor in designing hPSC-derived adoptive immunotherapies.

Keywords: EMP; HSC-independent; adoptive immunotherapy; definitive hematopoiesis; erythro-myeloid progenitor; human pluripotent stem cells; natural killer cells; ontogeny; primitive hematopoiesis; yolk sac.

Figure 1:. NK cell potential first arises from erythro-myeloid progenitors (EMP) in the yolk sac.

(A) Representative flow cytometric analyses of the immunophenotypic identification of NK cells cultured from adult mouse bone marrow common lymphoid progenitors (Adult CLP; Lin⁻Kit⁺CD127⁺) or from E9.5 yolk sac (E9.5 YS; see S1A) on OP9 stromal cells. (B) NK cell potential arises from cultured E8.5, E9.5, and E10.5 yolk sac tissues. YS-whole yolk sac, Prim-primitive hematopoietic progenitors, EMP (E9.5 YS, Kit⁺CD16/32⁺CD41⁺), Not-EMP-Kit+ yolk sac cells excluding CD16/32⁺CD41⁺ cells. (C) Limiting dilution assay of NK cell potential from sorted E9.5 EMPs. (D) NK cells are present in fetal liver as early as E13.5 and increase in numbers over the next two days (NK1.1⁺CD49b⁺Ter119⁻CD117⁻CD3ε⁻CD19⁻CD127⁻, see S1C), n=3. (E) Representative flow cytometric analyses of higher scatter properties of primary fetal liver and EMP-derived NK cells (purple) compared to their adult counterparts (black). Images of Wright Giemsa-stained NK cells derived from E9.5 EMP (left) or adult CLP (right; scale bar 10 μm, 100X magnification). (F) Quantification of side scatter (SSC) of NK cells isolated from E15.5 fetal liver or from adult spleen (in vivo), and derived from the in vitro culture of E9.5 EMP or of adult CLP, n=3. (G) Representative flow cytometric analyses of degranulation, measured by CD107a expression, of PMA-activated primary murine NK cells isolated from E15.5 fetal liver or from adult spleen (in vivo), and derived from E9.5 EMP or Adult-CLP (in vitro). (H) Quantification of NK-cells that express CD107a after PMA/ionomycin-activation. n≥3. (I) Median fluorescence intensity (MFI) of CD107a on CD107a⁺ PMA-activated NK cells isolated from E15.5 fetal liver (in vivo) or derived from E9.5 EMP (in vitro), relative to MFI of CD107a on CD107a⁺ PMA/ionomycin-activated NK cells isolated from adult spleen or derived from adult CLP (black line=1), n=3. Mean ± SEM. *p <0.01, **p<0.001, ***p<0.0001. Paired student’s t-test. See also Figure S1.

Figure 2:. Lineage tracing supports an HSC-independent source of fetal NK cells.

(A) Experimental design for lineage tracing of hematopoietic cell populations in fetuses of Csf1r^MerCreMer X Rosa26^YFP mice following a single dose of tamoxifen at E9.5. (B) Gating strategy for detection of YFP+ NK cells (CD3ε⁻CD19⁻CD127⁻CD122⁺NK1.1⁺CD49b⁺) in the livers of E15.5 fetuses. Examples of fluorescence minus one (FMO) controls for NK1.1 and CD49b are also shown. (C) Quantification of lineage labeling of NK cells (as in B), as well as long-term hematopoietic stem cells (LT-HSCs-lineage⁻Kit⁺Sca1⁺CD150⁺CD48⁻), erythroid (Ter119+) cells, granulocytes (Ly6G+), monocytes (Ly6C+), and macrophages (F4/80+) (as in S2A,B). Statistical differences between %YFP in experimental (n=5) compared to littermate controls lacking Csf1r^MerCreMer n=3. Mean ± SEM. (D) Gating strategy detecting YFP+ NK cells (CD3ε⁻CD19⁻CD127⁻CD122⁺NKp46⁺) in the livers of E14.5 fetuses. (E) Quantification of lineage labeling of NK cells (as in D), and other hematopoietic lineage cells (as in S2 A,B) in the E14.5 liver. Statistical difference between %YFP in experimental (n=6) compared to littermate controls (n=4). Mean ± SEM. Unpaired two tail student’s t-test, *p<0.05, **p<0.01, ***p<0.001. See also Figure S2.

Figure 3:. Human WNT-independent hematopoietic progenitors have NK cell potential.

(A) Representative flow cytometric analyses of CD56⁺ NK cell and CD15⁺ granulocyte potential of WNTi CD34⁺43⁺, WNTd CD34⁺ and cord blood CD34⁺ progenitors, n=12. (B) Quantification of geometric mean fluorescence intensity (MFI) of SSC from CD56⁺ WNTi-derived NK, WNTd-derived NK, and CB-DERIVED NK cells. n=55 (WNTi-NK), n=56 (WNTd-NK) and n=52 (CB-NK). Mean ± SEM. (C) Morphology of WNTi-, WNTd-, and CB-NK CD56⁺ and WNTi-CD15⁺ cells, obtained as in Supplemental Figure 3A, under transmission electron microscopy (scale bar=1 μm, 5,000X magnification). (D) Representative flow cytometric analyses of CD56 and CD3ε expression on Week 4 of OP9-DL4 co-cultures, gated on Live (DAPI⁻) singlets. (E) Representative flow cytometric analyses of CD94 and CD16 expression of CD56⁺ hPSC-derived WNTi-NK, WNTd-NK and CB-NK cells, over time. (F) Quantification of CD94 and CD16 expression on each CD56⁺ cell population, as shown in E. Week 2, n=5, Week 3, n=11, Week 4 n=58 (WNTi-derived NK and WNTd-derived NK) and n=54 (CB-NK), Week 5, n=4. Mean ± SEM. Gated on Live (DAPI⁻) singlet, CD56⁺ cells. (G) Representative histogram of CD16 expression on WNTi-derived NK (purple), WNTd-derived NK (blue), and CB-derived NK (black). Gated on CD56⁺ cells. Mean ± SEM. Paired and unpaired student’s t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Figure S3,S4.

Figure 4:. Whole transcriptome sequencing of WNTd-derived NK, WNTi-derived NK, and CB-DERIVED NK cells.

(A-C) Heat maps showing gene expression within WNTi and WNTd CD34⁺ progenitors, and CD56⁺ WNTi-derived NK, WNTd-derived NK, and CB-derived NK cells, of (A) Heat map of genes (RPKM >1 in each group) enriched within WNTi-derived NK cells (279), WNTd-derived NK (100), common to hPSC-NK cells (274), CB-derived NK genes (236), genes shared by either WNTd -and CB-NK (100) or WNTi- and CB-NK (70), and all NK cell populations (1043). Dendrogram was constructed using one minus Pearson correlation with averaged linkage. Color scale is row dependent, with highest RPKM set to red and lowest RPKM set to blue. (B) Heat map of transcription factors associated with NK cell specification and development, NK cell “signature” genes, and Killer-cell immunoglobulin-like receptors (KIRs). (C) Heat map of common NK cell surface receptors and cytolytic enzymes and proteins required for NK cell-mediated effector functions (upper) and differential gene expression (lower) between NK cell populations. Each square represents the average RPKM of each gene per sample group, n=3. (D-E) Gene Set Enrichment Analysis (GSEA) of WNTi-derived NK cells compared to WNTd-derived NK cells. (D) Quantification of False Discover Rate (FDR) q-value correlate transcriptional and functional differences observed in WNTi-derived NK and WNTd-derived NK cells. (E) Select enrichment plots from gene sets significantly enriched in WNTi-derived NK (purple border) or WNTd-derived NK cells (blue border). See also Figure S4.

Figure 5:. WNTi-derived NK and WNTd-derived NK cells are functionally distinct.

CD56⁺ NK cells from each ontogenic origin were isolated by FACS and assessed for IFN-[γ] and CD107a by flow cytometry. (A, B) Response of CD56⁺ cells to K562 tumor targets (effector:target (E:T) ratio 1.25:1). (A) Representative flow cytometric analyses of CD107a and IFN-[γ] expression within CD56⁺ NK cells. B) Quantification of CD107a⁺IFN-γ⁻ and CD107a⁻IFN-[γ]⁺ populations, as in A. n≥7. Mean ± SEM. (C, D) Response of CD56⁺ cells to ADCC. (C) Representative flow cytometric analyses of CD107a and IFN-[γ] expression within CD56⁺ NK cells. (D) Quantification of CD107a⁺IFN-[γ]⁻ and CD107a⁻IFN-[γ]⁺ populations, as in C. n=6. (E, F) Response of CD56⁺ cells to PMA/ionomycin stimulation. (E) Representative flow cytometric analyses of CD107a and IFN-[γ] expression within CD56⁺ NK cells. (F) Quantification of CD107a⁺IFN-[γ]⁻ and CD107a⁻IFN-[γ]⁺ populations, as in E. n=4. Mean ± SEM. (G). Quantification of specific killing by 7-AAD uptake of Rituximab-coated Raji cells. n>4. Mean ± SEM. Two-way ANOVA with multiple comparisons. ****p<0.0001. (H, I) Response of CD56⁺CD16⁺ WNTi-derived NK, WNTd-derived NK, CB-DERIVED NK, and PB-NK cells to K562 tumor targets. E:T ratio of 1.25:1. (H) Representative flow cytometric analyses s of CD107a and IFN-[γ] within CD56⁺CD16⁺ NK cells. (I) Quantification of CD107a⁺IFN-[γ]⁻ and CD107a⁻IFN-[γ]⁺ populations, as in H. n = 4. Mean ± SEM. Paired and unpaired student’s t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Figure S5.