Eomesodermin and T-bet mark developmentally distinct human natural killer cells

Abstract

Immaturity of the immune system of human fetuses and neonates is often invoked to explain their increased susceptibility to infection; however, the development of the fetal innate immune system in early life remains incompletely explored. We now show that the most mature NK cells found in adult (or postnatal) human circulation (CD94^-CD16⁺) are absent during ontogeny. Human fetal NK cells were found to express the 2 signature T-box transcription factors essential for the development of all murine NK and NK-like cells, eomesodermin (Eomes) and T-bet. The single-cell pattern of Eomes and T-bet expression during ontogeny, however, revealed a stereotyped pattern of reciprocal dominance, with immature NK cells expressing higher amounts of Eomes and more mature NK cells marked by greater abundance of T-bet. We also observed a stereotyped pattern of tissue-specific NK cell maturation during human ontogeny, with fetal liver being more restrictive to NK cell maturity than fetal bone barrow, spleen, or lung. These results support the hypothesis that maturation of human NK cells has a discrete restriction until postnatal life, and provide a framework to better understand the increased susceptibility of fetuses and newborns to infection.

Figure 1. Fetal tissues contain fewer mature NK cells than umbilical cord blood and adult samples.

(A) Representative flow cytometry plots of CD94 and CD16 expression on lineage-negative (Lin^neg: depleted of T cells, B cells, DCs, monocytes, granulocytes, erythroid cells, and CD34⁺ precursor cells) CD161⁺ lymphocytes from indicated tissues. Fetal samples are from an 18-week gestational age specimen. (B) Mean frequencies ± SEM of Lin^negCD161⁺ cells of indicated stages from indicated tissues. Fetal bone marrow, n = 20; fetal liver, n = 23; fetal lung, n = 11; fetal spleen, n = 15; umbilical cord blood (UCB), n = 6; adult peripheral blood mononuclear cells (PBMCs), n = 4; adult spleen, 6 independent measurements made on n = 2 specimens. Significance determined using 2-way ANOVA with Bonferroni post-hoc tests. *P < 0.001, **P < 0.05 compared with adult PBMCs; ***P < 0.001 compared with all other tissues.

Figure 2. Both Eomes- and T-bet–expressing NK cells are found in fetal tissues and Eomes^hi cells are enriched in stage 4 and fetal liver.

(A) Representative flow cytometry plots of intracellular Eomes and T-bet expression on lineage-negative (Lin^neg: depleted of T cells, B cells, DCs, monocytes, granulocytes, erythroid cells, and CD34⁺ precursor cells) CD161⁺ cells gated on indicated stages based on CD94 and CD16 expression from indicated tissues. (B) Mean frequencies ± SEM of stage 3 (top), stage 4 (middle), and stage 5a (bottom) Eomes^loT-bet^lo (L), Eomes^hi (E) and T-bet^hi (T) NK cells in indicated samples. Fetal bone marrow, n = 20; fetal liver, n = 23; fetal lung, n = 11; fetal spleen, n = 15; umbilical cord blood (UCB), n = 6; adult peripheral blood mononuclear cells (PBMCs), n = 3; adult spleen, 5 independent measurements made on n = 2 specimens. Significance determined using 2-way ANOVA with Bonferroni post-hoc tests. **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant).

Figure 3. Eomes^hi cells are CXCR6⁺CD69⁺.

Representative flow cytometry plots of Eomes^hi and T-bet^hi NK cells showing CXCR6 and CD69 staining. Experiment was performed 3 times; n = 4 for fetal specimens and n = 2 for adult spleen.

Figure 4. Eomes^hi cells are less mature than T-bet^hi cells based on killer immunoglobulin receptor (KIR) and NKG2A expression.

(A) Mean frequencies ± SEM of KIR expression (based on pan-KIR staining for KIR2DL1, KIR2DS1, KIR2DL2, KIR2DS2, KIR2DL3, KIR2DS3, KIR2DS4, and KIR2DS5) on lineage-negative (Lin^neg: depleted of T cells, B cells, DCs, monocytes, granulocytes, erythroid cells, and CD34⁺ precursor cells) CD161⁺ stage 4 and 5 Eomes^hi (open circles) and T-bet^hi (closed circles) cells from indicated tissues. Fetal bone marrow (BM), n = 7; fetal liver, n = 8; fetal lung, n = 9; fetal spleen, n = 8; umbilical cord blood (UCB), n = 6; adult spleen, 5 independent measurements made on n = 2 specimens. (B) Median fluorescence intensity (MFI) ± SEM of NKG2A on KIR-negative and KIR-positive Eomes^hi (open circles) and T-bet^hi (closed circles) stage 5a (CD94⁺CD16⁺) cells from indicated tissues. Fetal BM, n = 7; fetal liver, n = 8; fetal lung, n = 9; fetal spleen, n = 8; UCB, n = 2; adult spleen (Adult S.), 5 independent measurements made on n = 2 specimens. Significance determined using 2-way ANOVA with Bonferroni post-hoc tests. *P < 0.05, ***P < 0.001, ****P < 0.0001. ns, not significant.

Figure 5. Eomes^hi cells are less mature than T-bet^hi cells based on granzyme B and perforin expression.

(A) Representative flow cytometry histogram showing median fluorescence intensity (MFI) of granzyme B and perforin expression on 15-week gestational age lung specimen stage 5a cells gated on Eomes^hi (blue line) and T-bet^hi (red line) cells compared to Eomes^lo T-bet^lo (L) stage 3 cells (shaded gray histogram). (B) Mean frequencies ± SEM of granzyme B or perforin MFI on Eomes^hi (open circles) and T-bet^hi (closed circles) cells from indicated tissues and stages. Fetal bone marrow (BM), n = 5; fetal liver, n = 6; fetal lung, n = 7; fetal spleen, n = 6; umbilical cord blood (UCB), n = 6; adult spleen (Adult S.), 4 independent measurements made on n = 2 specimens. Significance determined using 2-way ANOVA with Bonferroni post-hoc tests. *P < 0.05, ***P < 0.001, ****P < 0.0001. ns, not significant.

Figure 6. Eomes^hi cells appear before T-bet^hi cells in an in vitro NK cell development culture system.

(A) Mean frequencies ± SEM of lineage-negative (Lin^neg: depleted of T cells, B cells, DCs, monocytes, granulocytes, erythroid cells, and CD34⁺ precursor cells) CD161⁺ cells on days 14, 21, and 28 of culture on EL08.ID2 cells. Each data point represents cells derived from a unique hematopoietic stem cell (HSC) source performed in 2 independent experiments. (B) Mean frequencies ± SEM of indicated stages of NK cell development on days 14, 21, and 28 of culture on EL08.ID2 cells; n = 4, representative of 2 independent experiments each performed with unique HSC sources. (C) Representative flow cytometry plot of intracellular Eomes and T-bet staining on indicated stages from day 14 of culture on EL08.ID2 cells. (D) Mean frequencies ± SEM of Eomes^hi (open circles) and T-bet^hi (closed circles) cells from indicated stages on days 14, 21, and 28 of culture on EL08.ID2 cells; n = 4, representative of 2 independent experiments each performed with unique HSC sources.