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Keywords:

  • Hematopoietic stem cells;
  • Liver NK cells;
  • NK-cell development

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Hepatic NK cells constitute ∼40% of hepatic lymphocytes and are phenotypically and functionally distinct from blood NK cells. Whether hepatic NK cells derive from precursors in the BM or develop locally from hepatic progenitors is still unknown. Here, we identify all five known sequential stages of NK-cell development in the adult human liver and demonstrate that CD34+ hepatic progenitors can generate functional NK cells. While early NK-cell precursors (NKPs) were similar in liver and blood, hepatic stage 3 NKPs displayed immunophenotypical differences, suggesting the onset of a liver-specific NK-cell development. Hepatic stage 3 NKPs were RORCneg and did not produce IL-17 or IL-22, excluding them from the lymphoid tissue-inducer (LTi) subset. In vitro culture of hepatic NKPs gave rise to functional NK cells exhibiting strong cytotoxicity against K562 targets. To determine whether hepatic NKPs are stably residing in the liver, we analyzed donor and recipient-derived cells in transplanted livers. Shortly after liver transplantation all donor NKPs in liver grafts were replaced by recipient-derived ones, indicating that hepatic NKPs are recruited from the bloodstream. Together, our results show that NKPs are continuously recruited from peripheral blood into the liver and can potentially differentiate into liver-specific NK cells.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Natural killer (NK) cells are an important component of the innate immune system. Without prior activation, NK cells can kill infected or malignant cells and produce cytokines that contribute to shaping both innate and adaptive immune responses. Recent studies have defined five main stages of human NK-cell development 1, 2. NK cells start from stage 1 (also named pro-NK: CD34+CD117CD94), and proceed through stage 2 (or pre-NK: CD34+CD117+CD94) and stage 3 (or iNK: CD34CD117+CD94), into stage 4 corresponding to the CD56bright NK cells (CD3CD56brightCD16−/dim), and stage 5 corresponding to the CD56dim NK cells (CD3CD56dimCD16+) NK cells 1–3. These last two stages, which constitute the mature NK-cell subsets, are both fully differentiated but their plasticity enables CD56bright to convert into CD56dim cells 4–6.

Originally, NK cells were thought to develop from CD34+ hematopoietic stem cells (HSCs) in BM. However, recent data suggest that very early phases of development, such as the generation of stage 1 NK-cell precursors (NKPs) from HSCs, occurs in the BM 1 whereas later phases may occur in peripheral organs, as shown for secondary lymphoid tissues 7. Accordingly, a complete pathway of NK-cell differentiation comprising all five stages of NK-cell development has been found in BM, peripheral blood, LNs and tonsils 1–2, 7, while the three final stages of NK-cell development were detected in human uterus 8. In addition, in vitro studies have demonstrated that functional NK cells can be generated by stimulation of CD34+ cells isolated from human cord blood, BM, fetal liver, thymocytes 1, 9–13, secondary lymphoid tissues 1–2, 7, intestine 14, 15 and uterus 8, 16.

The liver contains large numbers of leukocytes, with a peculiar lymphocyte distribution characterized by the enrichment of CD8+ T cells, NKT cells and NK cells when compared with that of peripheral blood 17, 18. Hepatic NK cells, additionally, are phenotypically and functionally distinct from blood NK cells 17, 19 and have important functions in defense against viral infections 20, 21, tumor metastases 22, hepatocellular carcinoma 23 and prevention of liver fibrosis 24, 25. It is not known whether hepatic NK cells develop from local precursors or are of systemic origin. Similar to secondary lymphoid tissues, hepatic NK cells are enriched for the CD56bright subset 17, 19, suggesting that liver NK cells may also develop locally. During fetal life the liver is the main site of hematopoiesis 26, 27. Shortly after birth, however, the liver drastically reduces its hematopoietic activity and the BM becomes the main site of hematopoiesis throughout normal adult life 26, 27. Nevertheless, the adult liver maintains the potential to reconstitute hematopoiesis, as observed in pathological conditions resulting in BM dysfunction in which the liver rehabilitates its hematopoietic activity 27, 28. Additional evidence of this potential hematopoietic role of the adult liver derives from animal studies, in which isolated hepatic HSCs have been shown to successfully reconstitute hematopoiesis in lethally irradiated animals 26, 29.

Previous research has shown the presence of CD34+ lymphoid progenitors in the adult human liver, nevertheless none of these studies has indicated whether these progenitors are stably resident in the liver or has investigated the local presence of intermediate NK-cell developmental stages or the potential to derive functional NK cells from hepatic CD34+ progenitors 26, 28. In this study, we investigated whether the liver is a possible site of NK-cell development by identifying hepatic NKPs, characterizing their origin and assessing their capacity to mature into functional NK cells in vitro.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Identification of NK-cell precursors within the adult human liver

To identify hepatic NKPs by flow cytometry we isolated mononuclear cells (MNCs) from fresh liver perfusates (n=15) and fresh liver biopsies (n=4) collected during liver transplantation (LTX) procedures. The first three developmental stages were initially gated from viable cells, negative for the lineage markers CD3, CD14, CD19 and for CD94 (Fig. 1A, 1–3), and were then identified according to their expression of CD34 and CD117 (Fig. 1A, 4). CD56bright and CD56dim NK cells were identified on the basis of CD56 and CD16 expression within viable CD3CD56+ NK cells (Fig. 1A, 5).

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Figure 1. NKPs in liver graft perfusates, liver graft biopsies and peripheral blood. (A) Gating strategy for the identification of NKPs and NK cells. Stage 1–3 NKPs were identified among CD45+ viable (7AAD) cells within the lymphocyte gate. Lineage+ (CD3, CD14, CD19) cells and CD94+ cells were excluded (1–3). Gating of NKPs (4) was based on isotype controls (for CD94, CD34 and CD117) and was obtained by recording at least 2×106 events. CD56bright and CD56dim NK cells were gated within CD3CD56+ cells on the basis of their CD56/CD16 expression (5). Dot plots represent liver graft perfusates, but similar profiles were observed for liver biopsies and peripheral blood. (B) Numbers of NKPs and NK cells in (1) liver perfusates (n=15), (2) liver biopsies (n=4) and (3) peripheral blood of healthy volunteers (n=5). NK cells were enumerated in additional samples of liver perfusates (total n=21) and blood from healthy volunteers (total n=13).

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We identified all these sequential developmental stages in liver perfusate and liver biopsies (Fig. 1B, 1 and 2) and we confirmed their presence in peripheral blood (Fig. 1BA, 3). As expected, the number of cells in the first three stages of development (expressed as percentage of viable lymphocytes) was uniformly low, (Fig. 1B). Additional control experiments (n=6) proved that stage 1–3 NKPs were CD45+, thus excluding contamination by endothelial cells (data not shown). Expression of CD45RA, characterizing a subset of CD34+ cells in BM, blood and especially LNs that can develop into CD56bright NK cells 7, was observed on 57±10% of stage 1 cells, 41±10% of stage 2 and 55±7% of stage 3 cells in liver perfusates (n=6). In liver biopsies we detected similar numbers of stage 1 and 2 NKPs compared with liver perfusates and higher proportions of stage 3 cells (0.45% of total hepatic lymphocytes, range: 0.37–0.54, compared with 0.06%, range: 0.01–0.29, in perfusate) (Fig. 1B, 2). As expected, the frequency of hepatic NK cells was high and reflected the known 3:1 ratio between the CD56bright and CD56dim subsets 19 both in liver perfusates and biopsies (Fig. 1A and B). The number of NKPs in peripheral blood was similar to what we found in liver and the proportion of CD56bright and CD56dim reflected the known 9:1 ratio (Fig. 1B, 3).

We then analyzed CD94 expression on hepatic NK cells by gating on the following sequential subsets: CD56brightCD94high, CD56dimCD94high and CD56dimCD94low cells (Fig. 2A) 6. Our results indicated that all three subsets are present in both liver and peripheral blood, but that their distribution is different in the two compartments, possibly contributing to the hypothesis of a local hepatic differentiation into liver-specific NK cells.

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Figure 2. Expression of CD94, CD127, CD25 and CD117 by NKPs and NK cells from liver or peripheral blood. (A) Representative dot plot of the three subsets identified on the basis of CD56, CD16 and CD94 expression. Percentages indicate medians with ranges from 7 independent experiments for perfusates and 5 independent experiments for peripheral blood. p-Values of differences in subset frequencies in the two tissues were calculated with the Mann–Whitney test. (B) Expression of CD127, CD25 and CD117 on NKPs and NK cells from liver (both perfusates and biopsies, closed circles) and peripheral blood (both healthy individuals and organ donors, open triangles). p-Values of differences in the expression of these markers between liver and blood-derived cells were calculated with the Mann–Whitney test.

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Since NKPs are present in peripheral blood 1, we next aimed at detecting possible phenotypical differences between liver and blood-derived NKPs. To exclude that detected phenotypical differences between the tissues analyzed (liver from deceased organ donors and peripheral blood from healthy individuals) were related to the pre-morbid state of the organ donor, we herein included also peripheral blood samples from organ donors obtained at the time of perfusate collection (n=4; Fig. 1 Supporting Information). We compared the expression of CD127, CD25 and CD117 on liver and blood-derived NKPs (Fig. 2B). Given the low frequency of NKPs, we only considered as reliable those experiments in which we measured at least 50 events for each developmental stage. Stage 1 and 2 cells showed similar immunophenotypes in the two tissues. However, a high percentage of liver-derived stage 3 cells were positive for CD127 (73.2±5.1%), while only 31.4±7.7% blood-derived stage 3 cells expressed CD127 (p=0.001). Moreover, CD25 expression was substantially higher on blood-derived stage 3 cells compared with their hepatic counterparts (61.0±7.6% of CD25+ stage 3 cells in blood and 34.2±7.0% in liver, p=0.02). With regard to NK cells, only 5.0±2.5% of hepatic CD56bright NK cells was CD127+, while 18.8±5.4% of blood-derived CD56bright NK cells expressed the same marker (p=0.007). Similarly, CD117 was expressed only by 0.8±0.2% of hepatic CD56bright NK cells while was present on 29.1±4.7% of CD56bright cells derived from peripheral blood (p=0.0001). Conversely, CD117 was completely absent from CD56dim NK cells of both tissues.

Overall, these immunophenotypical differences indicate that hepatic NKPs differ from their blood counterparts from stage 3 onwards. Altogether, our results show that all NK-cell developmental stages, from multipotent CD34+ hematopoietic progenitor cells to mature NK cells, are present in the adult human liver.

Hepatic stage 3 cells express CD127 but are not lymphoid tissue-inducer cells

To assess if NKPs in liver perfusates have characteristics of LTi cells (CD127+RORC+, IL-22 and IL-17 production 30–32) we FACS sorted stages 1, 2 and 3 NKPs from fresh perfusates and measured by qPCR the mRNA levels of the transcription factors RORC and ID2, as well as the cytokines IL-22, IL-17 and IFN-γ. RORC mRNA was neither detected in NKPs nor NK cells from perfusates (Fig. 3). Intracellular staining confirmed the absence of RORC in stage 1 and 2 cells, while only 6±3% of stage 3 cells were RORCpos (n=3; data not shown). Adding to this, IL-17 (data not shown) and IL-22 mRNA were undetectable in NKPs and NK cells, while ID2 mRNA, essential for NK-cell development beyond stage 3 cells 33, was detected in all hepatic NKPs (Fig. 3). These data indicate that hepatic NKPs, including stage 3 cells, are real NK-cell precursors and are not committed to the LTi-lineage.

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Figure 3. Expression of transcription factors and cytokines in hepatic NKPs and NK cells. qPCR analysis of ID2, RORC, IFN-γ and IL-22 in NKPs from fresh liver perfusates. mRNA of NK cells isolated from liver perfusate and LTi cells from tonsils were used as controls. Relative gene expression levels were normalized to GAPDH. Data shown are from one experiment representative of 2 independent qPCR analyses performed on mRNA extracted from: pools of NKPs isolated from 11 perfusates, NK cells from 3 perfusates and tonsil LTi cells from 3 tonsils.

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Hepatic NK-cell precursors develop into functional NK cells in vitro

Sorted stages 1, 2 and 3 from fresh liver perfusates were cultured, in the presence of cytokines that support NK-cell differentiation in vitro 1, 11–13, 34, to assess their capacity to differentiate into NK cells. After cell sorting, stage 1 cells reached only 50–70% of purity and the large majority died during the first weeks of culture, most probably indicating the need for a layer of feeder cells for appropriate expansion 2, 7, 31. Conversely, cell sorting of stage 2 and 3 cells yielded highly pure populations (average purity of >97%; Fig. 4A). After the first 2 or 3 weeks the rate of expansion of stage 2 and 3 cells required redistribution of the cells into additional wells. After 4 weeks of culture, all stage 2 cells had developed into stage 3 NKPs (median of 21%; range: 8–58%) or NK cells (75%; range: 47–99%; n=8; Fig. 4B). Of these latter NK cells, 76±9% belonged to the CD56bright subset and 21±8% to the CD56dim subset (Fig. 4B). Importantly, in these cultures we did not detect stage 1 or stage 2 cells.

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Figure 4. Differentiation of sorted hepatic NKPs into cytotoxic NK cells. (A) Representative example of FACS sorting of perfusate MNCs into stages 2 and 3 NKPs (purity>97%). Stages 2 and 3 NKPs were initially gated from CD45+ viable (7AAD) cells within the lymphocyte gate. Lineage+ (CD3, CD14, CD15, CD19, CD56) cells and CD94+ cells were excluded and stages 2 and 3 NKPs were then sorted on the basis of their CD34 and CD117 expression. (B) Sorted stages 2 and 3 NKPs were cultured for 4 wk, then harvested and phenotypically analyzed by flow cytometry. Dot plots are representative of 8 independent experiments for stage 2 cells and 6 independent experiments for stage 3 cells. (C) Progressive induction of CD94 expression during differentiation of stage 2 NKPs. Dot plots are representative of three independent experiments. (D) Cytotoxic capacity of in vitro-derived NK cells. After 4 wk of culture, the NK cells obtained from hepatic stage 2 or 3 NKPs were co-incubated for 4 h with K562 cells at two different E:T ratios (2:1 and 5:1). The percentage of K562 cells killed by NK cells was determined as previously described 19. Results show means±SD of five independent experiments. (E) Cytotoxicity and degranulation of in vitro-derived NK cells. Representative dot plots showing the percentage of K562 targets killed upon co-incubation with NK cells (upper panel) and the amount of NK cells degranulating in the presence of K562 targets (lower panel). The plots shown depict stage 2-derived NK cells and are representative of five independent experiments; percentages of K562 killing and degranulation are expressed as mean±SD.

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After 4 weeks of culture of stage 3 cells, none of the cells converted to stage 1 or 2, a variable proportion remained in stage 3 (24%; range: 0.6–47%) and the majority had developed into mature NK cells (72%; range: 51–90%, n=6, Fig. 4B). Most of the stage 3-derived NK cells belonged to the CD56bright subset (81.1±7.6%), while 12.8±9.6% was CD56dim. NK cells that developed in vitro from stage 2 and 3 lacked Killer Ig-like Receptors (KIRs) (data not shown). In addition, a large part of the in vitro-derived CD56dim NK cells expressed CD94 (93.9±2.2%), while a smaller fraction of the CD56bright NK cells was CD94+ (59.5±9.9%). The expression of CD94 was also analyzed during the first week of culture and these data indicated that CD94 was sequentially upregulated during development (n=3; Fig. 4C). Altogether these data confirm that freshly isolated stage 2 and 3 NKPs from adult human liver are able to give rise to CD56bright and CD56dim mature NK cells.

To assess if the NK cells we obtained in vitro from stage 2 and 3 NKPs are functionally competent we performed cytotoxicity assays with the classical K562 target cell line and measured CD107a expression on NK cells to assess their degranulation capacity. Stage 2-derived NK cells exhibited potent cytotoxicity (84±6% of K562 targets were killed in a 2:1 E:T ratio and 87±6% in a 5:1 ratio; Fig. 4D and E) and an average degranulation of 43±4% of the cells (background of spontaneous degranulation was 13±2%; p<0.001; n=5; Fig. 4E). Similarly, stage 3-derived NK cells killed most of the K562 targets (63±12% in 2:1 and 86±5% in 5:1 E:T ratios; Fig. 4D) and exhibited an average degranulation of 52±10% (background of spontaneous degranulation was 18±3%; p=0.009; n=5).

Hepatic NKPs are rapidly replaced by precursors recruited from the circulation

To investigate whether the NKPs we detected in human liver are stably resident in the organ or are recruited from the circulation we collected biopsies from five liver grafts explanted during a re-transplantation (re-LTX) procedure 1 week to 2 years after the first transplantation. In these samples, we determined the presence of NKPs and NK cells from the donor. In all five explanted liver grafts we detected only recipient-derived but not donor-derived NKPs (Table 1A), indicating that hepatic NKPs are replaced by blood-derived NKPs within 1 wk after LTX. On the contrary, in all the biopsies donor-derived NK cells co-existed with recipient NK cells. To investigate whether donor-derived NK cells were also detectable in the recipient circulation late after LTX, we collected peripheral blood samples from five LTX patients between 10 and 17 months post-LTX. Donor-derived NK cells and their precursors were not found in any of these post-LTX blood samples where, instead, all detected NK cells and NKPs were derived from the recipient (Table 1B).

Table 1. Proportions of donor-derived NKPs and NK cells in transplanted livers and recipient peripheral blood after liver transplantation
NKPs and NK cells as % of lymphocytes in liver grafts after LTX (% of donor-derived within each subset)
Time post-LTXPatient-1 7 daysPatient-2 16 daysPatient-3 1 yearPatient-4 2 yearsPatient-5 2 years
  1. a

    Proportions of NKPs and NK cells in (A) first liver grafts or (B) peripheral blood of recipients, at different time points after LTX. Numbers of cells in each stage of NK-cell development are expressed as percentage of total viable lymphocytes. Numbers in parentheses indicate the percentage of donor-derived cells within each stage.

A
Stage 10.4 (0)0.2 (0)0.1 (0)0.2 (0)0.1 (0)
Stage 20.07 (0)0.01 (0)0.04 (0)0.05 (0)0.01 (0)
Stage 30.3 (0)0.06 (0)0.1 (0)0.1 (0)0.1 (0)
NK10.1 (6.8)11.6 (1.5)42.8 (1.7)7.9 (1.7)32.2 (4.8)
NKPs and NK cells as % of total lymphocytes in peripheral blood after LTX (% of donor-derived within each subset)
Time post-LTXPatient-1 10 monthsPatient-2 14 monthsPatient-3 15 monthsPatient-4 15 monthsPatient-5 17 months
B
Stage 10.3 (0)0.2 (0)0.2 (0)0.2 (0)0.5 (0)
Stage 20.05 (0)0.03 (0)0.09 (0)0.02 (0)0.07 (0)
Stage 30.01 (0)0.01 (0)0.05 (0)0.03 (0)0.06 (0)
NK7.8 (0)10.5 (0)14.1 (0)6.6 (0)11.3 (0)

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study, we demonstrate for the first time that the adult human liver contains precursors of NK cells that can develop into functional CD56bright and CD56dim mature NK cells. We have shown that all five stages of NK-cell development are detectable in the adult liver (both in biopsies and perfusates) that stage 2 and 3 NKPs can generate cytotoxic NK cells in vitro and that early NKPs are recruited from peripheral blood. As proposed by Freud and Caligiuri 1, the selective enrichment of CD56bright NK cells in a specific organ (as was shown for the human liver in our previous study 19), together with the presence of the full range of developmental intermediates, here shown as spanning the continuum of differentiation from CD34+ HSCs to CD3CD56+ NK cells, suggest that the specific organ may be a site of NK-cell development in vivo. Altogether our results support a model in which hepatic NKPs originate from circulating precursors (most probably derived from the BM), relocate into the liver and hence can differentiate into a population of NK cells with liver-specific characteristics. Tissue-specific NK-cell development is possible since, in contrast to T-cell development (which requires a stage of selection within the thymus for the removal of auto-reactive T cells) NK-cell education ensuring self-tolerance can potentially take place in any tissue, as long as the local microenvironment is supportive 1. Importantly IL-15, a key cytokine for NK-cell development, is present in the liver 26, 27, 35 (produced by Kupffer cells 36) and IL-7, known to increase NK-cell proliferation, is produced by hepatocytes 27.

Our immunophenotypical definition of NKPs is derived from the model proposed by Caligiuri et al. that is based on the lack of lineage markers and CD94 and on the combined expression of CD34 and CD117 2. While stage 1 and 2 cells are still multipotent, full commitment to the NK-cell lineage occurs at stage 3 of development 1, 2, 37, 38. Therefore, the actual NKPs may be only a subset of stage 1 and 2 cells 2, as already suggested by studies in human lymph nodes which identified specific precursors of CD56bright NK cells within stages 1 and 2 as expressing CD45RA 7. We found that around 60% of hepatic stage 1 cells and 40% of stage 2 cells expressed CD45RA, suggesting that these cell populations contain specific precursors of CD56bright NK cells.

The immunophenotypical definition of stage 3 cells can include also LTi cells 31. LTi cells, required for the formation of lymph nodes during embryogenesis, exhibit the same phenotype as stage 3 cells but, in addition, express CD127 and RORC, and produce IL-22 and IL-17 30–32. Although a large proportion of hepatic stage 3 cells expressed CD127 we here have shown that they are real NKPs, since these cells had low or absent RORC expression, at both protein and mRNA level, and did not produce IL-22 or IL-17. The potential of hepatic NKPs to develop into NK cells was further supported by their expression of ID2. Altogether, our phenotypical analysis and qPCR data have shown that the liver contains specific stage 1, 2 and 3 NKPs.

When hepatic NKPs and NK cells from organ donors were compared with their counterparts in peripheral blood from healthy individuals, a number of differences emerged in terms of expression of CD25, CD127 and CD117. Confirmation that these differences were related to the tissue of origin rather than the pre-morbid state of the organ donor was obtained by comparing paired blood and liver samples from the same organ donors (Fig. 1, Supporting Information). Interestingly, differences in expression of the abovementioned molecules were detected only from stage 3 of differentiation, suggesting that stage 3 cells are influenced by the hepatic environment and may constitute the first step toward the differentiation of liver-specific NK cells. Furthermore, hepatic CD56bright NK cells differed from their blood counterparts in that they exhibited lower expression of CD127 and complete absence of CD117. Additional differences between blood and liver NK cells were detected in terms of CD94 expression. The level of CD94 expression has been described to identify a functional intermediary between the classical CD56bright and CD56dim NK cells 6 that defines three sequential subsets: CD56brightCD94high, CD56dimCD94high and CD56dimCD94low cells (Fig. 2A). Comparison of these three subsets in liver and blood shows a clear difference in their proportions, suggesting here again, as already postulated for decidua 39 and secondary lymphoid tissue 2, that specific types of NK cells may develop locally in different tissues.

We here show that stage 2 and 3 cells isolated from liver develop into functional NK cells in vitro. Our experiments showed that stage 2 cells progressed into stage 3 and, similar to stage 3 cultures, ultimately differentiated into NK cells, largely belonging to the CD56bright subset. In vitro-derived NK cells progressively upregulated CD94 during the culture (Fig. 4C). CD94 expression is known to be highly correlated with NKG2A levels 40, and the expression of the inhibitory dimeric receptor CD94/NKG2A, which occurs prior to KIRs expression, is generally linked to the acquisition of cytotoxic properties 41, 42. In our experiments none of the in vitro-derived NK cells expressed KIRs after 4 weeks of culture, confirming previous in vitro observations 11, 12, 43, 44. Despite the lack of KIRs, our in vitro-derived NK cells were highly functional: the cells degranulated in response to K562 targets, exhibiting a 1.5- to 2-fold higher cytotoxicity than our previously analyzed hepatic NK cells 19. Besides demonstrating the cytotoxic potential of our in vitro-derived NK cells, these observations confirm that the lack of engagement of inhibitory KIRs is not the only mechanism triggering NK cell responses against MHC class Inegative targets 45, 46.

Our observation that hepatic, donor NKPs are rapidly replaced after LTX by recipient-derived NKPs that infiltrate the graft, suggests that stage 1 and 2 NKPs from the BM migrate into (or traffic through) the liver. This concept is supported by experiments in adult mice indicating that part of the HSCs from BM enter the bloodstream and traffic to multiple peripheral organs (including the liver), where they reside shortly before entering draining lymphatics to return to the blood and, eventually, the BM 47. Importantly, a study by Massberg et al. 47 describes the liver as one of the peripheral organs in which tissue-resident HSCs are constantly replenished by blood-derived HSCs and are turned over within a matter of days. Our results indicating the presence of donor-derived NK cells in the grafted organ long time after LTX suggest at least two possible interpretations. First, hepatic NKPs transferred by LTX may displace to other sites, such as the BM, and hence constitute a long-term source of donor cells which may relocate again into the liver. This hypothesis is supported by data showing the regular release of HSCs from BM to blood in humans 48. Alternatively, the liver may contain a population of long-lived NK cells as demonstrated by recent experimental animal data showing that long-lived NK cells reside not only in lymphoid tissues, but also in non-lymphoid tissues and particularly in the liver 49, 50. Observations in mice indicate that NK cells, activated specifically by a chemical hapten 51 or a virus 49, 52 or non-specifically by a state of lymphopenia 53 or inflammatory cytokines 54, acquire classical characteristics of immune memory that include an extended lifespan, the ability to self-renew, and the capacity to mount robust and protective recall responses 52.

In light of the evidence we here report, we propose the following model for the NK-cell development in the adult liver: early NKPs (stages 1 and 2) are continuously recruited from peripheral blood into the liver, where they differentiate, under the influence of the local microenvironment, into the so-called “liver-specific” NK cells. This hepatic population, enriched in CD56bright cells and highly cytotoxic 19, has important local functions such as the defense against viral infections and malignant cells and the protection from liver fibrosis. Besides contributing to our understanding of NK-cell biology, our results shed more light onto the mechanisms determining prolonged intragraft NK-cell chimerism after LTX, which are possibly involved in induction of immunological tolerance to liver grafts 26, 55.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Isolation of mononuclear cells from liver graft perfusate, blood and liver biopsy

Perfusates were collected from human liver grafts during the back table procedure and MNCs were isolated as previously described 19. MNCs from peripheral blood samples of either healthy controls or LTX patients were isolated by Ficoll density gradient centrifugation under standard conditions.

Liver graft biopsies were obtained from healthy grafts just before transplantation or from explanted liver grafts in patients who underwent re-LTX and were collected in University of Wisconsin (UW) preservation solution. Fresh biopsies were cut into small pieces and incubated with digestion medium: 1640 RPMI (Cambrex Bio Science, Verviers, Belgium) with collagenase type IV (0.5 mg/mL; Gibco, Breda, The Netherlands) and DNase type I (0.02 mg/mL; Roche Diagnostics, Manheim, Germany) for 40 min at 37°C. Subsequently, the tissue pieces were mashed over a nylon mesh filter (200 μm pore diameter) to obtain a single-cell suspension. The suspension was centrifuged at 360 rpm for 2 min to allow sedimentation of hepatocytes. After hepatocyte removal, cells were washed and prepared for standard Ficoll gradient centrifugation to obtain MNCs.

Flow cytometry, cell sorting and qPCR

The following monoclonal Abs were used in this study: CD3-AmCyan, CD14-FITC, CD15-FITC, CD16-PacificBlue, CD34-allophycocyanin, CD45-AmCyan, CD107a-PE, anti-HLA-A2-FITC, IgG1-PE-Cy7, anti-RORC-PE, IgG1-PE (BD Biosciences, San Jose, CA, USA); CD3-FITC, CD56-allophycocyanin, CD94-PE, CD117-PE-Cy7, CD158a-allophycocyanin, CD158b-allophycocyanin (Beckman Coulter Immunotech, Marseille, France); CD25-PacificBlue, CD19-FITC (BioLegend, San Diego, CA, USA); CD127-allophycocyanin-Cy7 and IgG1-allophycocyanin (eBioscence, San Diego, CA, USA); anti-HLA-Bw4-FITC (from One Lambda, Canoga Park, USA). NKPs were gated from viable cells by exclusion of CD3+, CD14+ and CD19+ cells and according to the following combinations: stage 1: CD34+CD117CD94; stage 2: CD34+CD117+CD94; stage 3: CD34CD117+CD94. Mature NK cells were gated as CD56bright cells: CD3CD56brightCD16−/dim; CD56dim cells: CD3CD56lowCD16+. All analyses were performed using FACS Canto II flow cytometer (BD Bioscences) equipped with BD FACS-Diva software, version 6.1.1. Non-viable cells were excluded using 7-AAD (BD Biosciences).

Flow cytometric cell sorting was used to freshly isolate NK-cell developmental stages for either qPCR analysis or in vitro cell culture. For gene-expression analysis, stage 1, 2 and 3 cells were sorted from 11 different fresh liver perfusates according to the immunophenotypical definition described in the Introduction. Mature NK cells were isolated from fresh perfusates by negative selection, using the NK-Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer's instructions. Cell sorting was preceded by enrichment of NKPs from perfusate-derived MNCs by magnetic column depletion (Miltenyi Biotec) of lin+ cells (CD3, CD14, CD15 and CD56 cells). Upon labeling with CD34, CD117 and CD94 mAb, NKPs were sorted with BD FACSAria II Cell Sorter (BD Biosciences) following the scheme reported in the Introduction section and depicted in Fig. 1. During FACS sorting, additional labeling with mAbs against lineage markers (CD3, CD56, CD14, CD15 and CD19) allowed maximal exclusion of committed lineage+ cells and contaminating NK cells. Tonsils were collected during routine tonsillectomies contingent on informed consent. Tissue was cut into small pieces and cell suspensions were prepared by disrupting the tissue with a GentleMacs (Miltenyi Biotech) in the presence of 0.5 mg/mL collagenase type IV (Sigma, St. Louis, MO, USA). MNCs were isolated from Ficoll gradients. Prior to sorting, tonsil MNCs were enriched for LTis by labeling with anti-CD117 biotin (eBioscience) and positive selection of the CD117+ cells using streptavidin microbeads and VarioMacs (Miltenyi Biotech). LTi cells were sorted as defined by the expression of the following markers: CD45+CD3CD14CD19CD34CD117+CD127+NKp44+.

Total mRNA was extracted using the NucleoSpin RNA XS kit for small amounts of cells (≤5×105 cells) or the NucleoSpin RNA II kit (both from Macherey-Nagel, Düren, Germany) for larger amounts of cells (up to 5×106 cells), according to the manufacturer's instructions. Isolated mRNA was treated with rDNase to remove any contaminating genomic DNA. Quantification and purity of extracted mRNA was measured by Nanodrop (Thermo, Wilmington, USA). Complementary DNA (cDNA) was synthesized using the iScript cDNA Synthesis kit (Bio-rad) according to the manufacturer's instructions. QPCR was performed in duplicates using SYBRGreen (Quantace). The primer sequences used in this study are here reported. Id2 forw, 5′-CTCGCATCC CACTATTGT-3′; Id2 rev 5′-TGCTTTGCTGTCATTTGA-3′; IL-17 forw 5′-GAAGGCAGGAATCACAATC-3′; IL-17 rev 5′-GCCTCCCAGATCACAGA-3′; IL-22 forw 5′-CCCATCAGCTCCCACTGC-3′; IL-22 rev 5′-GGCACCACCTCCTGCATATA-3′; RORC forw 5′-CCCGTCAGCAGAACTG-3′; RORC rev 5′-AGCCCCAAGGTGTAGG-3′; IFN-γ forw 5′-CCAGGACCCATATGTAAAAG-3′; IFN-γ rev 5′-TGGCTCTGCATTATTTTTC-3′; GAPDH forw 5′-TGCACCACCAACTGCTTAGC-3′; GAPDH rev 5′-GGCATGGACTGTGGTCATGAG-3′. The thermocycling conditions used are here reported: 10 min at 95°C, followed by 40 cycles of 10 s at 95°C, 10 s at 58°C and 15 s at 72°C. Data were analyzed with the Bio-rad IQ-5 to establish the Ct value for each sample. Only Ct values≤35 were considered for the analysis and relative gene expression was calculated by normalization with the housekeeping gene GAPDH.

In vitro differentiation of NK cells

After sorting, cultures were initiated with 1×104 cells seeded in 96-well round-bottomed plates with 200 μL of medium consisting of 1640 RPMI, 10% heat-inactivated human AB serum, 10% FBS (Hyclone, Logan, UT, USA), penicillin (100 U/mL) and streptomycin (100 U/mL; both from Gibco). The following cytokines were added to the culture medium: stem cell factor (SCF) (20 ng/mL), FMS-like tyrosine kinase (FLT3-L) (20 ng/mL), interleukin-7 (IL-7) (20 ng/mL), IL-15 (20 ng/mL) and IL-21 (20 ng/mL) (all from PeproTech, NJ, USA). Half of the culture medium was replaced every 3–4 days. After 4 wk of culture, cells were harvested and used for phenotypical or functional analysis as previously described by our group 19.

Identification of donor-derived NK cells and their precursors after LTX

We selected donor/recipient pairs in which only one of the two parties had an HLA-A2 or HLA-Bw4 epitope. FACS analysis with a monoclonal Ab recognizing either HLA-A2 or HLA-Bw4 was used to distinguish between cells of donor and recipient origin in peripheral blood of LTX recipients or in biopsies from explanted first liver grafts in patients who underwent re-LTX. Multiple biopsies were obtained from different sites of the explanted graft. Biopsies were pooled together and processed as described in Flow cytometry, cell sorting and qPCR to isolate MNCs for FACS analysis.

The Ethical Committee of the Erasmus MC approved the study and all volunteers gave written informed consent. This project was funded by the Erasmus MC (Rotterdam, The Netherlands).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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