Differentiation and functional regulation of human fetal NK cells (original) (raw)
NK cell differentiation and KIR acquisition start early in fetal development. NK cells, defined as CD45+CD3–CD56+ cells, have been reported to exist in the human fetal liver as early as gestational week 6 as well as in fetal spleen from gestational week 15 (17). Here we used a more stringent definition of NK cells (CD7+CD3–CD14–CD19–CD34– cells expressing CD56, NKG2A, or CD16) to identify NK cells (Figure 1A and Supplemental Figure 1, A and B; supplemental material available online with this article; doi:10.1172/JCI68989DS1). CD7 is expressed by B cells, T cells, and NK cells (28) and as such is a useful marker of lymphocytes. To verify that staining with CD7 did not exclude a large population of CD7–CD161+ immature stage III pre-NK cells (29), we analyzed the expression of CD7 on CD161+CD3–CD14–CD19–CD45+ lymphocytes. Virtually all of the CD161+ cells expressed CD7 in bone marrow, liver, lung, lymph nodes, and spleen (Supplemental Figure 1D), demonstrating that gating on CD7 did not exclude a significant number of immature NK cells. NK cells were most frequent in fetal liver and lung, in which they made up 50%–60% of the CD7+ cells on average. Relatively high frequencies of NK cells were also detected in fetal spleen, whereas the frequency was low in fetal bone marrow and mesenteric lymph nodes (mLNs) (Figure 1B).
NK cells are present in human fetal tissues. (A) Representative staining and gating strategy for identification of NK cells in fetal lung. NK cells were defined using the Boolean algorithm (“CD56+ or NKG2A+ or CD16+” ) after gating on live CD3–CD14–CD19–CD34–CD7+ cells. For detailed information on the identification of fetal NK cells, see Supplemental Figure 1. (B) Frequency of NK cells, as percentages of CD7+ cells in fetal liver (n = 10), fetal lung (n = 15), fetal spleen (n = 14), fetal bone marrow (n = 11), fetal mLNs (n = 11), and adult PBMCs (n = 7). Horizontal bars indicate the mean percentage, and dots indicate the number of samples tested. (C) Staining of NKp46+ cells in fetal lung. Arrows indicate NKp46+ cells. Two NKp46+ cells are shown in the top and bottom left corners. The staining is representative of sections from 4 individual fetal lungs at gestational week 18–22. Representative staining of EpCAM expression (epithelial marker, top right) and CD31 expression (endothelial marker, bottom right) in fetal lung sections. Scale bar: 20 μm.
Although our flow cytometry data demonstrated that NK cells were the dominant lymphocyte population in fetal lung and liver, they did not reveal where the NK cells were located in these tissues. NKp46 is expressed by virtually all fetal NK cells, but by very few other cells (Supplemental Figure 1B), and is thus specific for fetal NK cells in the tissues analyzed. The fetal lungs analyzed in this study (gestational week 15–22) correspond to the canalicular stage of lung development, where clearly visible terminal buds and acinar tubules are lined with EpCAM+ epithelial cells (30). NKp46+ cells were scattered throughout the mesenchyme of the lung, between the terminal buds and acinar tubules, and were only rarely intraepithelial in their location (Figure 1C). Also in fetal liver, NKp46+ cells were scattered and had no apparent association with endothelial (CD31+) or epithelial (EpCAM+) structures (data not shown).
Having identified NK cells in different fetal organs, we next asked whether fetal NK cells were predominantly immature or whether we could find evidence of NK cell differentiation. Based on current models for adult NK cell differentiation (25, 31), we examined 3 subsets of NK cells: NKG2A+CD16– (corresponding to CD56bright), NKG2A+CD16+, and NKG2A–CD16+ (corresponding to CD56dim NK cells with an increasingly differentiated phenotype). All 3 subsets were present in all fetal organs studied, but the distribution varied substantially among organs, with most differentiated NK cells in fetal lung and the least differentiated NK cells in lymph nodes and liver (Figure 2A). The expression of CD56 was in general high on fetal NK cells, and we could not readily discriminate discrete CD56bright from CD56dim NK cell populations in fetal tissues. Nevertheless, in line with adult NK cell differentiation, the mean fluorescence intensity of CD56 decreased with increasing differentiation of fetal NK cells (Supplemental Figure 2). However, fetal NK cells were less differentiated compared with adult peripheral blood NK (PBNK) cells, and approximately 90% of the fetal NK cells expressed NKG2A, irrespective of organ (Figure 2A). Interestingly, there was no change in the distribution of the NKG2A+CD16–, NKG2A+CD16+, and NKG2A–CD16+ subsets during gestational weeks 15–22 (Figure 2B and data not shown), suggesting that NK cell differentiation in the human fetus starts before gestational week 15 and is stable from gestational week 15–22.
Fetal NK cell differentiation and KIR acquisition starts early in the developing fetus and varies among organs. (A) Frequency of NKG2A+CD16–, NKG2A+CD16+, and NKG2A–CD16+ subsets of total NK cells in fetal organs (black circles), full-term umbilical cord blood (UCB; white circles), and adult PBMCs (aPBMC; white circles) (fetal mLN, n = 8; liver, n = 13; spleen, n = 11; bone marrow, n = 11; lung, n = 24; umbilical cord blood, n = 4; adult PBMCs, n = 31). (B) Frequency of NKG2A+CD16–, NKG2A+CD16+, and NKG2A–CD16+ subsets of NK cells in fetal lung stratified over gestational weeks (gw) 15–18 (black, n = 9), 18–20 (gray, n = 9), and 20–22 (white, n = 6). Error bars represent SD, and bars represent mean. (C) KIR2DL1, KIR2DL3, and KIR3DL1 expression on fetal lung NK cells. (D) Frequency of NK cells expressing KIR2DL1, KIR2DL3, and/or KIR3DL1 (defined by the Boolean gate “KIR2DL1+ and/or KIR2DL3+ and/or KIR3DL1+”) (lung, n = 21; spleen, n = 10; bone marrow, n = 8; mLN, n = 8; liver, n = 12; umbilical cord blood, n = 14; adult PBMCs, n = 26). Horizontal bars indicate the mean percentage, and dots indicate the number of samples tested.
Similar to NKG2A, KIR expression is also associated with differentiation of CD56dim NK cells in adults (25). To our knowledge, only one previous report has studied KIR expression on fetal NK cells, showing that they are expressed by a small subset of fetal liver NK cells at gestational week 14 (32). However, it is currently not known whether KIRs are expressed on NK cells in other fetal organs or whether KIR expression is associated with fetal NK cell differentiation, as is the case for adult PBNK cells. All the fetal organs studied here contained NK cells that expressed KIR2DL1, KIR2DL3, and KIR3DL1, the 3 major inhibitory KIRs, but the frequency varied considerably among the organs studied (Figure 2, C and D). Fetal lung contained the highest frequency of KIR+ NK cells (≈30%), followed by spleen and bone marrow (≈20%). In contrast, expression of KIRs on NK cells from fetal mLNs and liver was low (<10%) (Figure 2D). Similar to adult PBNK cells (25), KIR expression was associated with differentiation and was increasingly more common on fetal NKG2A+CD16+ and NKG2A–CD16+ NK cells, compared with that on NKG2A+CD16– NK cells (Figure 3A). Surprisingly, the frequency of KIR+ cells on NKG2A+CD16+ and NKG2A–CD16+ fetal lung NK cells was at least as high as that on adult PBNK cells, with an average of ≈70% of NKG2A–CD16+ fetal lung NK cells expressing KIR (Figure 3A). In contrast, the frequency of KIR-expressing NK cells in fetal liver and full-term umbilical cord blood was lower than in fetal lung and adult PBNK cells, irrespective of which subset was analyzed (Figure 3A). The frequency of KIR-expressing fetal NK cells did not increase between gestational weeks 15 and 22 (Figure 3B and data not shown), in line with the stable distribution of NKG2A+CD16–, NKG2A+CD16+, and NKG2A–CD16+ fetal NK cell subsets during this period of time (Figure 2B).
KIR expression on fetal NK cells increases with differentiation, and coexpression of several KIRs is more common on differentiated fetal lung NK cells than on adult PBNK cells. (A) Frequency of NK cells expressing KIR2DL1, KIR2DL3, and/or KIR3DL1 (defined by the Boolean gate “KIR2DL1+ and/or KIR2DL3+ and/or KIR3DL1+”) within each subset (liver, n = 12; lung, n = 21; full-term umbilical cord blood, n = 4; adult PBMCs, n = 26). (B) Frequency of fetal lung NK cells expressing KIR2DL1, KIR2DL3, and/or KIR3DL1, stratified over gestational weeks 15–18 (black, n = 4), 18–20 (gray, n = 8), and 20–22 (white, n = 6). (C) Frequency of NK cells coexpressing 0, 1, 2, or 3 KIRs (KIR2DL1, KIR2DL3, and/or KIR3DL1) in NKG2A+CD16–, NKG2A+CD16+, and NKG2A–CD16+ subsets of NK cells from fetal lung (black circles, n = 11) and adult PBMCs (white circles, n = 11). *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed Mann-Whitney test performed. ns, not significant. (D) Frequency of NKG2A+ NK cells in subsets with 0, 1, 2, or 3 KIR (KIR2DL1, KIR2DL3, and/or KIR3DL1) in fetal lung (n = 19), spleen (n = 9), umbilical cord blood (n = 11), and adult PBMCs (n = 25). Error bars represent SD, and bars represent mean. Horizontal bars indicate the mean percentage, and dots indicate the number of samples tested.
We recently showed that expression of multiple KIRs on single adult CD56dim NK cells is associated with differentiation (25). Similar to adult NK cells, expression of multiple KIRs on single fetal lung NK cells was increasingly more frequent on NKG2A+CD16+ and NKG2A–CD16+ NK cells, compared with that on NKG2A+CD16– NK cells (Figure 3C). Interestingly, the simultaneous expression of ≥2 KIRs was more frequent on NKG2A+CD16+ and NKG2A–CD16+ fetal lung NK cells compared with that on adult NKG2A+CD16+ and NKG2A–CD16+ PNBK cells, respectively (Figure 3C). The acquisition of multiple KIRs was also associated with a lower frequency of NKG2A expression, although not as pronounced as that observed in adult PBNK cells (Figure 3D), indicating that downregulation of NKG2A occurs at a later stage of NK cell differentiation, subsequent to acquisition of KIRs.
Terminal differentiation of adult NK cells is also associated with expression of CD57 (25, 33). A small percentage of fetal lung NK cells expressed low levels of CD57, compared with the abundant and high expression found on PBNK cells (Figure 4, A and B). Although the frequency of CD57-expressing NK cells was higher in NKG2A–CD16+ fetal NK cells compared with that in NKG2A+CD16+ and NKG2A+CD16– fetal NK cells, the frequency was still much lower compared with that in adult NKG2A–CD16+ NK cells (data not shown). This indicated that the observed differentiation of fetal NK cells, as evidenced by the loss of NKG2A and acquisition of multiple KIRs, was in itself not sufficient to drive any substantial upregulation of CD57. Finally, we also analyzed the intracellular expression of perforin and granzyme B in fetal NK cells. A majority of the fetal NK cells in the lung expressed both perforin and granzyme B (Figure 4C), albeit at a lower level than that observed in adult PBNK cells (Figure 4D). The expression of perforin and granzyme B was higher in the more differentiated NKG2A+CD16+ and NKG2A–CD16+ fetal NK cells, compared with that in NKG2A+CD16– NK cells, indicating that the differentiation was also accompanied with functional maturation (Figure 4C).
The frequency of CD57+ NK cells in fetal lung is low. (A) Representative staining of CD16 and CD57 on NK cells from fetal lung and adult PBNK. (B) Frequency and mean fluorescence intensity of CD57 expression on NK cells from fetal lung (black circles) and adult PBNK (white circles). Bars represent mean. Representative stainings of perforin and granzyme B expression in NKG2A+CD16– (long dashed lines), NKG2A+CD16+ (dotted line), and NKG2A–CD16+ (solid line) subsets of NK cells in (C) fetal lung and (D) adult PBMC. The non-NK cell Boolean gate was used as a reference (filled gray). The stainings are representative of 3 independent stainings in 3 individual donors.
In summary, our analysis revealed that several fetal organs contain a high frequency of KIR-expressing, differentiated NK cells, which suggests that they may be functional and that NKG2A and KIRs may be involved in the regulation of fetal NK cell responses.
Fetal NK cells display decreased responses to HLA class I–negative target cells but are highly responsive to cytokine stimulation. It was reported previously that NK cells in fetal liver are able to kill HLA class I–negative cells and anti-CD16–coated P815 cells, but not as efficiently as adult PBNK cells (17). To date, it is neither known whether fetal NK cells in other organs are functional nor whether fetal NK cells respond to cytokine stimulation. Therefore, we tested fetal and adult NK cell responses in parallel with several different stimuli, including HLA class I–negative cells, antibody-coated cells, and cytokines. For practical reasons, we chose to compare fetal tissue NK cells with adult blood NK cells, as it was logistically very hard to coordinate simultaneous collection of adult and fetal tissues on the same day. However, a separate comparison of matched adult blood and adult lung NK cells revealed that they had a similar degree of differentiation, as measured by expression of CD16, NKG2A, and CD57 (data not shown). A much larger fraction of adult PBNK cells compared with fetal NK cells expressed high levels of CD57 (Figure 4, A and B), a molecule associated with late NK cell differentiation and altered NK cell function (25, 33). In order to compare adult and fetal NK cells with a similar degree of differentiation, we therefore analyzed CD57– NK cells in all subsequent functional analysis.
NK cells from fetal lung, spleen, and liver degranulated in response to stimulation with HLA class I–negative K562 cells, as measured by CD107a upregulation, but to a lower degree than adult PBNK cells (Figure 5, A and B). Adult PBNK cells responded at a similar level as that of matched adult lung NK cells (Supplemental Figure 3A), verifying that the use of adult PBNK cells in the assays is representative for adult lung NK cells also. When lung and liver NK cells from the same fetus could be directly compared, fetal NK cells from the liver responded better than those from the lung (data not shown). Fetal lung NK cells also killed K562 cells (Figure 5C), indicating that the observed degranulation correlated with killing of target cells. However, fetal NK cells killed K562 cells to a lower extent than adult PBNK cells, in line with the reduced degranulation by fetal NK cells after stimulation with K562 cells. In addition to K562 cells, fetal NK cells also degranulated in response to HLA class I–negative 721.221 cells (Figure 5D). To test to what extent fetal NK cells could mediate ADCC, we stimulated them with 721.221 cells coated with rituximab (a therapeutic humanized antibody against CD20). Fetal lung NK cells responded strongly to rituximab-coated 721.221 cells, both with respect to degranulation (Figure 5D) and cytotoxicity (Figure 5E), indicating that stimulation via CD16 at least partially overrides the fetal NK cell hyporesponsiveness observed against HLA class I–negative target cells.
Fetal NK cells degranulate in response to target cells and are highly responsive to cytokine stimulation. (A) Representative plots of degranulation (measured as CD107a cell surface expression) and IFN-γ production by fetal lung NK cells (top row) and adult PBNK cells (bottom row), following stimulation with target cells (left) or cytokines (right). (B) NK cell degranulation (fetal lung, n = 14; fetal spleen, n = 5; fetal liver, n = 8; fetal mLN, n = 2; adult PBMCs, n = 7) and (C) cytotoxicity in response to K562 cells (fetal lung, n = 7; adult PBMCs, n = 9). ***P < 0.001. (D) NK cell degranulation in response to 721.221 cells and rituximab-coated 721.221 cells and (E) mean cytotoxicity against rituximab-coated 721.221 cells (fetal lung, n = 6; adult PBMCs, n = 10). (F) Frequency of IFN-γ–positive NK cells after stimulation with IL-12 and IL-18 or (G) IL-12 alone. (H) Frequency of NK cells degranulating after overnight priming with IL-12 and IL-18, followed by coculture with K562 cells. Bars represent mean, and error bars represent SD. Horizontal bars indicate the mean percentage, and dots indicate the number of samples tested. ***P < 0.001, paired t test. The frequency of responding (CD107a+ or IFN-γ+) cells cultured in the control (R10 medium alone) was subtracted in B, D, F, and G. The degranulation response from control wells stimulated with medium alone (left) or IL-12/18 (right), without K562 coculture, was subtracted in H. (C and E) Linear regression analysis was performed, testing whether slopes were significantly different.
In contrast to the decreased responsiveness to HLA class I–negative target cells, fetal NK cells responded strongly to stimulation with IL-12 and IL-18. A majority (≈70%–80%) of the NK cells from all fetal organs tested (lung, spleen, liver, and mLN) produced IFN-γ after stimulation with IL-12 and IL-18 (Figure 5, A and F). The frequency of IFN-γ–producing fetal NK cells was at least as high as that in adult CD56dim and CD56bright PBNK cells (Figure 5F). In addition, fetal lung NK cells also responded strongly to stimulation with IL-12 alone (Figure 5G), whereas adult PBNK cells responded poorly, suggesting that fetal NK cells have a lower threshold of activation with respect to cytokine stimulation. In addition to direct activation of NK cells, stimulation with IL-12 and IL-18 can prime target cell responses by adult NK cells. Similarly, overnight culture with IL-12 and IL-18 significantly increased the functional responsiveness to K562 cells by fetal lung NK cells (Figure 5H). Taken together, our results demonstrate that fetal NK cells are highly responsive to cytokine stimulation but are hyporesponsive to HLA class I–negative target cells.
NKG2A educates fetal NK cells. The finding that fetal NK cells had a decreased response to HLA class I–negative target cells indicated that there are differences in the regulation of fetal and adult NK cell function. Education via NKG2A and KIRs is critical for optimal NK cell responses to HLA class I–negative or allogeneic cells in adults (24), but it is currently unknown whether these receptors educate fetal NK cells. We have previously shown that fetal tissue lymphocytes express normal levels of HLA class I (19), and we also verified the expression of HLA class I molecules on fetal cells in this study (Supplemental Figure 4), demonstrating that ligands for both NKG2A and KIRs are expressed in the fetal tissues. NKG2A has a strong educational effect on NK cells in adults, and NKG2A+ NK cells in adults respond well to stimulation with HLA class I–negative target cells (23, 34). In addition, NKG2A confers an educational signal to NK cells during the early phases of the reconstitution of the immune system after hematopoietic stem cell transplantation (35). Given the high frequency of NKG2A+ fetal NK cells (Figure 2A), we asked whether NKG2A conferred an educational signal in the developing fetus. To compare NK cell education via NKG2A in fetal and adult NK cells, we analyzed degranulation after stimulation, and gated on CD57–KIR– NK cells, thus normalizing for the effects of differentiation coupled to expression of CD57 (25, 33) and KIR-mediated education (21). NKG2A+CD57–KIR– fetal lung NK cells (as well as fetal liver and spleen NK cells; data not shown) responded better than NKG2A–CD57–KIR– NK cells after stimulation with K562 cells (Figure 6A) as well as after stimulation with 721.221 cells (Figure 6B) and rituximab-coated 721.221 cells (Figure 6C). Confirming previous studies, adult NKG2A+CD57–KIR– PBNK cells also responded better than NKG2A–CD57–KIR– PBNK cells after stimulation with K562 cells (Figure 6A), 721.221 cells (Figure 6B), or rituximab-coated 721.221 cells (Figure 6C). Similarly, adult lung NKG2A+ NK cells responded better than adult lung NKG2A– NK cells to stimulation with K562 cells (Supplemental Figure 3B). Thus, NKG2A educates fetal and adult NK cells, thereby rendering them reactive to target cells lacking HLA-E, while potentially remaining tolerant to HLA-E–expressing cells.
NKG2A educates fetal NK cells. Degranulation by NKG2A–CD57–KIR– and NKG2A+CD57–KIR– fetal lung NK cells and NKG2A–CD57–KIR–CD56dim and NKG2A+CD57–KIR–CD56dim adult PBNK cells after stimulation with (A) K562 cells, (B) 721.221 cells, or (C) rituximab-coated 721.221 cells. The degranulation in cultures with medium alone was subtracted from the response to the indicated stimulation. *P < 0.05, **P < 0.01, ***P < 0.001, paired t test; #P < 0.05, Wilcoxon matched-pairs signed-rank test.
KIR expression on fetal NK cells results in hyporesponsiveness. In addition to the education via NKG2A, adult NK cells are educated via the interaction between KIR and self-HLA class I molecules (24). KIR-mediated education and functional inhibition are important for the regulation of NK cell allogeneic responses, in which target cells lack ligand(s) for inhibitory KIRs. As such, education of fetal NK cells via paternally inherited HLA class I could potentially result in the generation of fetal NK cells reactive toward allogeneic maternal cells. We have previously used HLA class I–negative target cells as a model to reveal potential alloreactive NK cell responses (36). The use of K562 and 721.221 cells facilitates the comparison among different donors and provides a more sensitive tool compared with primary allogeneic target cells, which normally induce very weak in vitro responses, even by freshly isolated adult NK cells (our own unpublished observations). Similar to NKG2A, education via self-HLA–specific inhibitory KIRs is evident in the early phases of immune reconstitution after hematopoietic stem cell transplantation in adults (35), indicating that education via KIRs could also be functional during the development of fetal NK cells. To investigate whether this was the case, we compared the degranulation of NKG2A+CD57–KIR– NK cells with NKG2A+CD57– NK cells expressing either only self-HLA class I–binding KIRs (self KIR) or only non-self-HLA class I–binding KIRs (non-self KIR) in response to K562 cells. Surprisingly, fetal lung NKG2A+CD57– NK cells expressing either only self or non-self KIRs responded significantly less than did fetal NKG2A+CD57– NK cells lacking KIR expression (Figure 7, A and B). Similarly, fetal lung NKG2A–CD57– NK cells expressing KIRs responded less well to stimulation than did fetal NKG2A–CD57– NK cells lacking expression of KIRs (Figure 7, C and D). Due to the low frequency of NKG2A–CD57– fetal NK cells, further division into self- and non-self KIR+ NK cells in this subset was unfortunately not possible.
KIR-expressing fetal NK cells are hyporesponsive to stimulation with K562 cells. (A) Degranulation by NKG2A+CD57– fetal lung NK cells and NKG2A+CD57–CD56dim adult PBNK cells expressing no KIR, only self KIR (S), or only non-self KIR (NS) (defined using a Boolean function of KIR2DL1, KIR2DL3, and KIR3DL1 expression and KIR-HLA ligand genotype) after stimulation with K562 cells. (B) Relative change in response to K562 cells compared with NKG2A+CD57– NK cells lacking KIR expression. (C) Degranulation by NKG2A–CD57– fetal lung NK cells and NKG2A–CD57–CD56dim adult PBNK cells expressing no KIR or any combination of KIR (as in A) after coculture with K562 cells. (D) Relative change in response to K562 cells compared with NKG2A–CD57– NK cells lacking KIR expression. (E) Degranulation by NKG2A+CD57– fetal lung NK cells and NKG2A+CD57–CD56dim adult PBNK cells expressing 0, 1, 2, or 3 KIRs (KIR2DL1, KIR2DL3, and/or KIR3DL1) after stimulation with K562 cells. (F) Relative change in degranulation compared with NK cells lacking expression of KIRs. Bars represent mean, and error bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA analysis of repeated measurements with Bonferroni’s post-test; #P < 0.001, unpaired t test. Horizontal bars indicate the mean percentage, and dots indicate the number of samples tested.
In contrast to the response patterns of fetal NK cells, adult NKG2A+CD57– PBNK cells expressing self KIRs responded better to stimulation with K562 cells than did NKG2A+CD57– PBNK cells lacking KIRs or expressing non-self KIRs (Figure 7, A and B), confirming that self KIRs educate adult NK cells. Similarly, adult NKG2A–CD57– PBNK cells expressing KIRs responded better to stimulation with K562 cells compared with NKG2A–CD57– PBNK cells lacking KIRs (Figure 7, C and D). To address whether the observed KIR-mediated hyporesponsiveness was a general feature of fetal NK cells rather than a lung-specific feature, we compared degranulation by adult NK cells from matched lung tissue and peripheral blood. Interestingly, adult lung NK cells degranulated to a similar level as adult PBNK cells from the same donor and were educated by both NKG2A and KIRs (Supplemental Figure 3, B–D). Finally, NKG2A+KIR+ fetal liver NK cells were hyporesponsive compared with NKG2A+KIR– fetal liver NK cells (Supplemental Figure 3E), whereas NK cells from adult liver have previously been demonstrated to be educated via KIRs (37). Together, this indicates that the KIR-mediated hyporesponsiveness is indeed a feature unique to fetal NK cells.
As we had observed that a large proportion of the fetal lung NK cells expressed multiple KIRs, we next asked whether the decreased responsiveness was dependent on the number of KIRs expressed. Strikingly, expression of 1 single KIR was sufficient to induce hyporesponsiveness in fetal NK cells, and coexpression of 2 or 3 KIRs further dampened the response (Figure 7, E and F). In contrast, expression of 1 or more KIRs on adult PBNK cells resulted in an increased responsiveness (Figure 7, E and F). To test whether the observed hyporesponsiveness of fetal NKG2A+CD57–KIR+ NK cells also extended to other stimuli, we analyzed NK cell responses to 721.221 cells alone as well as to 721.221 cells coated with rituximab. In all settings, KIR+ fetal NK cells responded significantly less well than did KIR– fetal NK cells (Figure 8).
KIR-expressing fetal NK cells are hyporesponsive to 721.221 cells and rituximab-coated 721.221 cells. (A) Degranulation by NKG2A+CD57– fetal lung NK cells and NKG2A+CD57–CD56dim adult PBNK cells expressing no KIR, only self KIR, or only non-self KIR (defined using Boolean function of KIR2DL1, KIR2DL3, and KIR3DL1 expression and KIR-HLA ligand genotype) after stimulation with 721.221 cells. (B) Relative change in degranulation compared with NK cells lacking expression of KIRs after stimulation with 721.221 cells. (C) Degranulation against rituximab-coated 721.221 cells by fetal and adult NK cells as in A. (D) Relative change in degranulation compared with NK cells lacking expression of KIRs after stimulation with rituximab-coated 721.221 cells. The degranulation in unstimulated cultures was subtracted from the response to the specific stimulations. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA analysis of repeated measurements with Bonferroni’s post-test; #P < 0.001, unpaired t test. Error bars represent SD.
We have previously reported that overnight priming of adult PBNK cells with cytokines is sufficient to break the hyporesponsiveness of noneducated adult NK cells to stimulation with K562 cells (25). To test whether cytokine priming was sufficient to break the hyporesponsiveness of fetal KIR+ NK cells, we cultured them overnight in IL-12 and IL-18 and subsequently measured responses to K562 cells. Although cytokine priming enhanced the fetal NK cell responses to K562 cells, the KIR+ fetal NK cells were still clearly hyporesponsive compared with KIR– fetal NK cells (Supplemental Figure 5B), indicating that the KIR-mediated hyporesponsiveness is maintained even after cytokine priming.
Together, these results suggest that KIR expression does not educate fetal NK cells, but instead confers hyporesponsiveness, and as such provides a potential mechanism for fetal NK cell hyporesponsiveness to HLA class I–negative target cells.
TGF-β efficiently suppresses fetal NK cells. In addition to regulation of NK cell responsiveness via NKG2A and KIRs, other mechanisms may contribute to dampening fetal NK cell responses in vivo. Several studies have shown that human NK cells are sensitive to TGF-β–mediated suppression in vitro (26, 27). It was also recently reported that TGF-β is responsible for the immaturity of neonatal NK cells in mice (15). We have previously demonstrated that TGF-β and TGF-β–related transcripts are expressed at a higher level in the developing human fetus, compared with that in adults (19), supporting the possibility that it could be involved in suppressing fetal NK cell responses in vivo. To investigate whether human fetal NK cells were affected by TGF-β, we cultured fetal cord blood mononuclear cells and adult PBMCs in the presence of either recombinant TGF-β or a TGF-β receptor kinase inhibitor (SB431542) prior to stimulation with rituximab-coated 721.221 cells (Figure 9, A and B). Upon inhibition of TGF-β receptor signaling, fetal cord blood NK cells responded significantly better compared with control cultures (Figure 9, A and B). Conversely, addition of recombinant TGF-β strongly dampened fetal NK cell responses to rituximab-coated 721.221 cells (Figure 9, A and B). In contrast, adult PBNK cells were only marginally affected by the addition of TGF-β or the TGF-β receptor kinase signaling inhibitor (Figure 9, A and B), indicating that fetal NK cells are more susceptible to TGF-β–mediated suppression than adult NK cells.
Fetal NK cells are highly susceptible to TGF-β–mediated suppression. (A) Degranulation by NKG2A+CD57– fetal cord blood NK cells and NKG2A+CD57– CD56dim adult PBNK cells after culture with TGF-β or TGF receptor kinase inhibitor (SB431542) for 48 hours and subsequent stimulation with rituximab-coated 721.221 cells for 2 hours. (B) Relative change in degranulation in response to rituximab-coated 721.221 cells after treatment with TGF-β or SB431542, compared with medium control. (C) Degranulation by NKG2A+CD57– fetal cord blood NK cells and (D) NKG2A+CD57–CD56dim adult PBNK cells expressing no KIR (KIR–) or any combination of KIR2DL1, KIR23DL3, or KIR3DL1 (KIR+) after culture with TGF-β or TGF-β receptor kinase inhibitor (SB431542) for 48 hours and subsequent stimulation with rituximab-coated 721.221 cells for 2 hours. Data are summarized from 3 individual experiments. Bars represent mean, and error bars represent SD. *P < 0.05, **P < 0.01, Mann-Whitney test; #P < 0.05, ##P < 0.01, paired t test.
Given their sensitivity to TGF-β, we next investigated the possibility that the observed KIR-mediated hyporesponsiveness was caused by an increased sensitivity of KIR+ fetal NK cells to TGF-β, compared with those lacking expression of KIRs. By analyzing NK cells in fetal cord blood, we were at the same time able to investigate whether the observed KIR-mediated hyporesponsiveness was a general feature of fetal NK cells or whether it was unique to fetal NK cells from tissues. Similar to fetal lung NK cells, NKG2A+CD57–KIR+ fetal blood NK cells responded less well to stimulation with rituximab-coated 721.221 cells than did NKG2A+CD57–KIR– fetal blood NK cells (Figure 9, C and D), corroborating that KIR-mediated hyporesponsiveness is a general feature of fetal NK cells. However, the addition of TGF-β or the TGF-β receptor kinase inhibitor (SB431542) did not reverse the KIR-mediated hyporesponsiveness (Figure 9, C and D), indicating that the differential regulation of fetal versus adult NK cell education via KIRs is independent of TGF-β signaling.