Front. Immunol. Frontiers in Immunology Front. Immunol. 1664-3224 Frontiers Media S.A. 10.3389/fimmu.2016.00262 Immunology Review Human Circulating and Tissue-Resident CD56bright Natural Killer Cell Populations Melsen Janine E. 1 * Lugthart Gertjan 1 Lankester Arjan C. 1 Schilham Marco W. 1 1Department of Pediatrics, Leiden University Medical Center, Leiden, Netherlands

Edited by: Eric Vivier, Centre d’Immunologie de Marseille-Luminy, France

Reviewed by: Francisco Borrego, Cruces University Hospital, Spain; Francesco Colucci, University of Cambridge, UK

 *Correspondence: Janine E. Melsen, j.e.melsen@lumc.nl

Specialty section: This article was submitted to NK Cell Biology, a section of the journal Frontiers in Immunology

 30 06 2016 2016 7 262 02 05 2016 21 06 2016 Copyright © 2016 Melsen, Lugthart, Lankester and Schilham. 2016 Melsen, Lugthart, Lankester and Schilham

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Two human natural killer (NK) cell subsets are usually distinguished, displaying the CD56dimCD16+ and the CD56brightCD16−/+ phenotype. This distinction is based on NK cells present in blood, where the CD56dim NK cells predominate. However, CD56bright NK cells outnumber CD56dim NK cells in the human body due to the fact that they are predominant in peripheral and lymphoid tissues. Interestingly, within the total CD56bright NK cell compartment, a major phenotypical and functional diversity is observed, as demonstrated by the discovery of tissue-resident CD56bright NK cells in the uterus, liver, and lymphoid tissues. Uterus-resident CD56bright NK cells express CD49a while the liver- and lymphoid tissue-resident CD56bright NK cells are characterized by co-expression of CD69 and CXCR6. Tissue-resident CD56bright NK cells have a low natural cytotoxicity and produce little interferon-γ upon monokine stimulation. Their distribution and specific phenotype suggest that the tissue-resident CD56bright NK cells exert tissue-specific functions. In this review, we examine the CD56bright NK cell diversity by discussing the distribution, phenotype, and function of circulating and tissue-resident CD56bright NK cells. In addition, we address the ongoing debate concerning the developmental relationship between circulating CD56bright and CD56dim NK cells and speculate on the position of tissue-resident CD56bright NK cells. We conclude that distinguishing tissue-resident CD56bright NK cells from circulating CD56bright NK cells is a prerequisite for the better understanding of the specific role of CD56bright NK cells in the complex process of human immune regulation.

 CD56bright NK cell populations tissue resident lymphoid tissues liver uterus NK cell development UL 2011-5133 KWF Kankerbestrijding10.13039/501100004622 Introduction

Since the discovery of natural killer (NK) cells in 1975 (1, 2), major advances were made in deciphering the role of NK cells in health and disease. It is currently accepted that NK cells are not just “killers” that lyse infected or transformed cells but can also play an important role in modulation of immune responses due to the secretion of immunoregulatory cytokines (e.g., IFN-γ and TNF-α) and chemokines (e.g., CCL3 and CCL4). Based on this cytokine secretion profile, NK cells are classified into group 1 of the large family of innate lymphoid cells (ILCs). Developmentally, NK cells are not related to the other (non-cytotoxic) ILCs, and can be distinguished from the remaining ILCs by the expression of the transcription factor Eomesodermin (EOMES) and the cytolytic protein perforin (3, 4).

In humans, two conventional NK cell subsets have been phenotypically defined based on CD56 and CD16 (FCRγIII) surface expression: CD56brightCD16−/+ and CD56dimCD16+. While the function of CD56 [neural cell adhesion molecule (NCAM)] on NK cells is not completely understood yet, CD16 can mediate antibody-dependent cellular cytotoxicity (5). Since most research in human NK cell biology is based on peripheral blood, the herein predominant CD56dim NK cell population is most extensively investigated. Based on circulating NK cells, CD56bright and CD56dim NK cells have usually been described as two functionally distinct subsets, cytokine producing and cytolytic, respectively. However, several observations challenge this strict difference, as that both subsets can be cytotoxic or produce cytokines, after appropriate in vitro stimulation. Upon target cell recognition, resting CD56dim NK cells are highly cytotoxic, but can produce cytokines as well (6–8). In contrast, CD56bright NK cells require monokine activation (combinations of IL2/IL12/IL15/IL18) to acquire cytolytic activity and produce cytokines (6, 9–11).

Although the CD56dim NK cells predominate in blood, the CD56bright NK cells are far more abundant in the human body due to their enrichment in lymphoid and non-lymphoid tissues (12–18). In addition, CD56bright NK cells comprise the major NK cell population in inflamed and cancer tissues (12, 14, 19). Recently, tissue-resident CD56bright NK cells were identified in liver, uterus, and lymphoid tissues, which points toward a tissue-specific function of CD56bright NK cells (13, 15–17, 20–22). In order to understand the NK cell diversity, it is essential to focus on how CD56bright NK cells develop, distribute, and acquire or alter their phenotype and function specifically in a particular organ. The first four developmental stages (i.e., from hematopoietic stem cell to CD56bright NK cell) were already reviewed extensively elsewhere and will not be discussed here (23, 24). This review attempts to improve the understanding of human circulating and tissue-resident CD56bright NK cells by reappraising their distribution and developmental, functional, and phenotypical characteristics. In addition, we will address to the developmental relationship between CD56bright (stage 4) and CD56dim NK cells (stage 5) and speculate on the position of tissue-resident CD56bright NK cells within the NK cell developmental pathway.

 Distribution, Phenotype and Function

CD56bright NK cells are widely distributed throughout the human body. When compared with blood, CD56bright NK cells are enriched in most human tissues. They represent the majority of NK cells in lymph nodes, tonsil, stomach, gut, liver, uterus, adrenal gland, and visceral adipose tissue (12–18). Although CD56bright NK cells seem to be outnumbered by CD56dim NK cells in lung, kidney, mammillary tissue, bone marrow and spleen, this is probably a reflection of the high blood perfusion of these organs (12, 13, 18, 25). Most knowledge on the phenotype and function of CD56bright NK cells is derived from blood, but it is important to realize that unique subsets of tissue-resident CD56bright NK cells have been described in lymphoid tissues, liver and uterus (13, 15, 22, 26). Conceivably, more organs contain tissue-resident CD56bright NK cell populations. To the best of our knowledge, no tissue-resident CD56dim NK cells have been described to date. Although residency is often used as a term for organ-infiltrating NK cells, it is generally not discussed whether these NK cells are just trafficking through the organ, or truly tissue resident. In this review, we only apply the term “resident” if there is substantial evidence, which allows to distinguish the tissue-resident CD56bright NK cells from circulating CD56bright NK cells. The lack of CD56 expression on murine NK cells hampers the one to one comparison of CD56bright NK cells to their murine counterpart. Due to limitations in obtaining human tissue samples, important findings in mice will be included in this review to cover the lack of human data.

Hallmarks of Tissue-Resident CD56<sup>bright</sup> NK Cells

In order to be retained within the tissue, tissue-resident CD56bright NK cells should possess characteristics, which prevent egress from the tissue. One of the mechanisms involved in residency is attributed to CD69, which is absent from blood-derived NK cells (13). Originally, CD69 was identified as an early activation marker, but today CD69 is known to be associated with tissue residency by suppressing sphingosine-1-phospate receptor 1 (S1PR1) surface expression (27–29). Although initially identified in the context of T- and B-cell migration, S1PRs have also been proposed to mediate the egress of NK cells from tissues into blood and lymph in mice, driven by a S1P gradient (30–35). It has not been confirmed whether S1PRs are expressed as protein on the cell surface of human NK cell subsets in blood and tissues. At transcriptional level, however, both S1PR1 and S1PR5 are expressed in circulating human NK cells, with the latter being selective for CD56dim NK cells (33, 36, 37). In contrast to S1PR1, S1PR5 is not inhibited by CD69 (32). Another potential mechanism for tissue homing and/or residency is the engagement of chemokine receptors. For instance, CXCR6 and CCR5 are both highly expressed on tissue-resident CD56bright NK cells in lymphoid tissues and liver, but have a low expression on blood-derived CD56bright NK cells, which instead express CCR7 (13, 15, 38). A third mechanism of tissue retention is driven by the expression or absence of adhesion molecules. For instance, the integrin CD49a is highly expressed on uterine CD56bright NK cells, but absent from blood NK cells (20). Furthermore, tissue-resident CD56bright NK cells lack CD62L (L-selectin), which is like CCR7 involved in recruitment of circulating NK cells to lymphoid tissues via high endothelial venules (HEVs) (15, 17, 18, 38, 39). Altogether, based on the expression of CD69, chemokine receptors, and adhesion molecules, tissue-resident CD56bright NK cells can be distinguished from circulating CD56bright NK cells. In addition to the phenotypical differences, tissue-resident CD56bright NK cells are functionally different from their circulating counterparts as will be discussed in the next sections.

 Lymphoid Tissues Lymph Node

Lymph nodes contain 40% of the lymphocytes in the human body, of which 2–5% consist of NK cells (Figure 1A) (12, 13, 18, 40). More than 75% of the NK cells in lymph nodes have a CD56bright phenotype (Figure 1B) (13, 18, 25). Accumulating evidence suggests a model in which CD56bright NK cells circulate from the blood to tissues, enter the lymphatic system, and eventually migrate back to the periphery via the efferent lymph (41, 42). The mechanisms governing the migration to and infiltration of lymphoid tissues by CD56bright NK cells are mainly deduced from chemokine receptor expression on circulating CD56bright NK cells. As discussed earlier, circulating CD56bright NK cells express CCR7 and CD62L (38, 39). The chemokines engaging CCR7, CCL19, and CCL21 are both highly expressed in lymph nodes (12). HEVs might not be the only route for circulating CD56bright NK cells to enter the lymph node. NK cells in seroma fluid, which represents an accumulation of afferent lymph, resemble circulating CD56bright NK cells regarding low expression of CCR5, killer-cell immunoglobulin-like receptor (KIR), and CD16 and high expression of CCR7 and CD62L (12, 43). This suggests that circulating CD56bright NK cells enter the lymph node both via HEVs and afferent lymph vessels.

Distribution of NK cell populations in blood and tissues. The distribution of CD56dim, non-resident CD56bright, and tissue-resident CD56bright NK cells is depicted as percentage of (A) total lymphocytes and (B) total NK cells within blood (13), lymph node (13, 18, 25), spleen (13, 18, 25), bone marrow (13, 18, 25), tonsil (18, 44), liver (15, 45, 46), endometrium (16, 20), and decidua (20, 47). Tissue-resident CD56bright NK cells were defined as CD69+CXCR6+ (lymph node, spleen, and bone marrow), NKp44+CD103+ (tonsil), CD69+CXCR6+ (liver), and CD49a+ (endometrium and decidua). For phenotypical details, see Figure 2 and Table 1.

Recently, we identified a major lymphoid tissue-resident NK cell subset in lymph node, spleen, and bone marrow based on co-expression of CD69 and CXCR6 (Figure 2; Table 1) (13). In the lymph node, lymphoid tissue NK (ltNK) cells account for 60% of all NK cells and cover the majority of the CD56bright NK cell compartment (Figure 1B). LtNK cells display a slightly less intense CD56 and more intense NKp46 expression compared with circulating CD56bright NK cells. In addition, the majority of ltNK cells is CD16−, CD49a−, and CD27+ (13). Interestingly, most ltNK cells do not express DNAX accessory molecule 1 (DNAM1), an activating receptor which is uniformly expressed on circulating CD56bright NK cells (13). The remaining CD56brightCD69− NK cells closely resemble circulating CD56bright NK cells, suggesting that these cells are blood-derived CD56bright NK cells transiently circulating through the lymph node (13).

Phenotype of circulating and tissue-resident CD56bright NK cells. The cell surface markers on NK cells that are discriminative between circulating and tissue-resident NK cells in lymphoid tissue, liver, and uterus are shown (see references in text and Table 1). Circulating CD56bright NK cells typically express the lymphoid tissue homing makers CD62L (L-selectin) and CCR7. In addition, CD117 (c-kit) and CD127 (IL-7Rα) are expressed by a fraction of circulating CD56bright NK cells. Lymphoid tissue-resident NK cells express CD69 and CXCR6. Tonsil-resident NK cells (defined as NKp44+CD103+) express in addition ITGβ7, CD49a, and partly CD9. The majority of CD69+CXCR6+ liver-resident NK cells express CCR5. In contrast to circulating CD56bright NK cells, only a fraction of lymphoid tissue-, tonsil-, and liver-resident NK cells express DNAM1. A subset of CD49a+ uterus-resident NK cells (endometrium and decidua) expresses CD69, ITGβ7, CD103, and NKp44. The reported DNAM1 expression in the uterus is contradicting in the literature and therefore indicated with −*. % indicates that only a fraction of the NK cell population is positive for the marker.

Phenotype of circulating and tissue-resident CD56bright NK cells.

Reference Blood (13, 15, 17, 38) Lymph node, spleen, marrow (13, 18) Tonsil (18, 44) Liver (15, 45, 46) Uterus (16, 17, 20, 48, 49)
Definition CD69+CXCR6+ NKp44+CD103+ CD69+CXCR6+ CD49a+CD103− CD49a+CD103+
CD56 ++ + +/++ + +++ +++
CD69 – + + + – +
Cytokine receptors
CD117 (c-kit) % – N.A. N.A. – –
CD127 (IL7-Rα) % – – N.A. – –
Chemokine receptors
CCR7 + – – – – –
CCR5 – % N.A. % – –
CXCR6 – + + + N.A. N.A.
NK cell receptors
DNAM1 + % % % –a –a
KIR – – % – % %
NKp44 – – + – – %
NKp46 + ++ ++ ++ + +
Adhesion molecules
CD9 – N.A. % N.A. % +
CD49a (ITGα1) – – + – + +
CD62L (L-selectin) + – – – – –
CD103 (ITGαE) – – + N.A. – +
ITGβ7 N.A. N.A. + N.A. – +

The cell surface markers which are discriminative for circulating CD56bright NK cells, lymph node-, spleen-, bone marrow-, tonsil-, liver-, and uterus-resident NK cells are summarized. Uterus-resident NK cells can be subdivided based on CD103 expression.

++ and +++ indicate relatively higher levels of expression.

% indicates that only a fraction of the NK cell population is positive for the marker.

aContradicting literature exist on the DNAM expression on uterine NK cells.

N.A., not assessed.

Lymphoid tissue NK cells were tested in the functional assays classically used for NK cells. The ltNK cells were less potent IFN-γ producers compared with circulating CD56bright NK cells nor did they lyse K562 target cells as efficient as CD56dim NK cells (13). However, the expression of EOMES and perforin distinguishes ltNK cells from the helper-ILC1 group (13). These phenotypical and functional characteristics, combined with their specific location in lymphoid tissues where immune responses are initiated and shaped, point to a distinct yet undefined role of ltNK cells (50).

Notably, without the use of tissue-resident markers, CD69 and CXCR6, ltNK cells could previously not be distinguished from circulating CD56bright NK cells. Therefore, a re-examination of the function of CD56bright NK cells in lymphoid tissue is necessary, in particular in the lymph node where a large population of ltNK cells co-exists next to the non-resident or circulating CD56bright NK cells.

 Spleen and Marrow

The spleen and bone marrow contain 14 and 10% of the total lymphocyte pool. NK cells constitute 5–20% and 4% of lymphocytes in spleen and marrow, respectively (Figure 1A) (13, 18, 40, 43). The CD56bright and CD56dim NK cells are equally distributed in the spleen, but 90% of CD56bright NK cells consist of ltNK cells (Figure 1B) (13, 25, 40). Similar to the spleen, the bone marrow is enriched for CD56bright NK cells, of which the majority consists of ltNK cells (13, 25). The phenotype of ltNK cells in spleen and marrow resembles the ltNK cell population in lymph node. As mentioned before, the non-resident CD56brightCD69− and CD56dim NK cells in marrow and spleen closely resemble the circulating CD56bright and CD56dim NK cells and are probably circulating NK cells contained in the tissue at time of isolation. Previously, the spleen has been reported to be enriched in CD27+ and NKp46bright NK cells, which could be a reflection of ltNK cells. These findings further illustrate the importance of using tissue-resident markers to distinguish circulating from tissue-resident CD56bright NK cells (51, 52). The manner in which NK cells enter the spleen differs from lymph node entrance, because the spleen does not contain afferent lymphatic vessels or HEVs (53). In mice, NK cells enter the spleen via arterioles in the marginal zone, rather than via arterioles directly connected to the red pulp, where most NK cells reside (54). Unfortunately, there is a lack of human studies focusing on how NK cells migrate to the spleen and bone marrow.

 Tonsil

In tonsil, although the CD56bright subset is predominant, only 0.4% of the total lymphocytes consist of NK cells (Figure 1A) (18). Seventeen percent of the total NK cell population in the tonsil co-expresses CD69 and CXCR6 (Figure 1B) (44). In contrast to the ltNK cells in lymph node, marrow, and spleen, these tonsil-resident NK cells also express NKp44, CD103, CD49a, Integrinβ7, and partly CD9 (Figure 2; Table 1) (44). Of note, tonsil-resident NK cells should be distinguished from NKp44+ ILC3s, which are located in the mucosa surrounding the lymphoid follicles and secrete preferentially IL-22 (55, 56). Similar to circulating CD56bright NK cells, the total pool of CD56bright NK cells was shown to produce high levels of IFN-γ and to become cytolytic upon IL-2 and/or IL-12 stimulation (18). Tonsils do not have afferent lymph vessels but HEVs are present, which might support the trafficking of NK cells. Similarly, CCL19 and CCL21 are secreted to attract circulating CD56bright NK cells, which might explain the high content of CD56bright NK cells which lack a tissue-resident phenotype (57).

 Liver

Hepatic NK cells comprise 40% of all hepatic lymphocytes (Figure 1A) (45). Recently, a major liver-resident EOMES+CD56bright NK cell population has been described, which comprises 45% of the hepatic NK cells and closely resembles ltNK cells phenotypically and functionally (Figure 1B) (15, 45, 46). Liver-resident CD56bright NK cells are characterized by a simultaneous expression of CD69 and CXCR6 (46). They have a high expression of CCR5 and NKp46, and low expression of DNAM1, as indirectly concluded from phenotypical analysis on total hepatic CD56bright NK cells (Figure 2; Table 1) (15, 45).

An independent report demonstrated the presence of a distinct minor liver-resident cell population characterized by CD49a expression (21). Those CD56brightCD49a+ cells make up 2% of the total NK cell compartment in the liver but are not present in every individual (41% of donors) (13, 15, 21). The expression of CXCR6 has not been described. However, these cells do not express EOMES, suggesting that they do not belong to the NK cell lineage (21). Due to the low prevalence of this CD49a+EOMES− cell population, we can indirectly conclude that the major CD69+CXCR6+ liver-resident NK cell population is negative for CD49a. The IFN-γ production of liver-resident CD56bright NK cells after 4-h stimulation with IL12 and IL18 was lower compared with the non-resident hepatic NK cells (46). Similar to ltNK cells, liver-resident CD56bright NK cells express perforin and granzyme B at a low level, further supporting a non-cytotoxic function (46).

Several studies in mice demonstrated the existence of hapten and virus-specific hepatic NK cell memory, mediated by cells expressing CD49a and CXCR6 (21, 58, 59). In contrast, splenic CXCR6+ NK cells, which potentially resemble the human ltNK cells, were not able to mediate a memory response (58). Thus, although CXCR6 expression is not restricted to the liver, only hepatic NK cells were found to mediate a memory response in mice. Nevertheless, it would be interesting to further study the memory capacities of the highly prevalent CXCR6+ liver- and lymphoid tissue-resident CD56bright NK cells in humans.

 Uterus

The uterine mucosa is populated by EOMES+CD56bright NK cells (16, 17, 23). In contrast to blood, there are hardly any CD56dim NK cells detectable in endometrium (no pregnancy) and decidua (pregnancy) (16, 17, 20, 60). Independent of the stage of the menstrual cycle, NK cells make up 30% of the endometrial lymphocytes (Figure 1A), although the absolute number of lymphocytes and NK cells increases robustly in the secretory stage (16). During early pregnancy, however, more than 70% of the lymphocytes in the uterine decidua is represented by CD56bright NK cells (Figure 1A) (47). Phenotypically, endometrial and decidual CD56bright NK cells closely resemble each other, and will be further referred to as uterine CD56bright NK cells. The CD56 expression of the uterine NK cells is even more intense than their circulating CD56bright counterparts (Table 1) (17, 61). All uterine NK cells display CD49a but not CCR5, discriminating them from the circulating, lymphoid tissue, and liver-resident NK cell populations (Figure 2) (16, 20). DNAM1 has been reported to be absent on uterine NK cells, although a contradicting report on this observation exists (20, 48, 62). Recently, it was shown that a fraction of uterine NK cells expresses the heterodimer CD103/ITGβ7, NKp44, as well as CD69 (20, 62). Conversely, an earlier study reported that all decidual NK cells express CD69 (17). Despite this discrepancy concerning the CD69 expression, both the CD56brightCD103− and CD56brightCD103+ NK cells are likely to represent a tissue-resident CD56bright NK cell population, as demonstrated by the expression of KIRs, CD9, and poor IFN-γ production and cytotoxicity (16, 17, 20, 49, 63, 64). Moreover, transcriptome analysis of decidual NK cells and circulating NK cells highlighted the uniqueness of the uterine NK cells (17, 48). To the best of our knowledge, the presence of chemokine receptors, such as CXCR6 and CCR5, has not been reported. Compared with circulating CD56bright NK cells, decidual CD56bright NK cells highly express the activating receptors NKG2C and NKG2E at RNA level; however, <30% is NKG2C+ on protein level (17, 62, 65).

Initially, a suppressive function of decidual NK cells was thought to be essential to provide maternal–fetal tolerance (17, 64). However, accumulating evidence points toward a more active role of decidual CD56bright NK cells in regulating placentation, as reviewed elsewhere (66). Decidual NK cells are considered to stimulate trophoblast invasion and spinal artery remodeling via the production of various chemokines and angiogenic factors (including angiopoietins and GM-CSF) (67–69). Mice lacking decidual NK cells exhibit abnormalities in pregnancy, including abnormal vascular remodeling of decidual arteries (70). Although the process of placentation in humans is different, specific combinations of fetal HLA-C alleles, presented by trophoblasts, and maternal KIR expression were shown to be associated with successful placentation (69, 71). The similarities between endometrial and decidual CD56bright NK cells suggest that decidual CD56bright NK cells are a direct reflection of endometrial CD56bright NK cells in a pregnant tissue microenvironment. Taken together, the phenotypical and functional profile of the uterine CD56bright NK cell compartment supports their unique functional role during pregnancy.

 Developmental Relationship Between Circulating CD56<sup>dim</sup> and CD56<sup>bright</sup> NK Cells

Thus far, we discussed the tissue-resident and circulating/non-resident CD56bright NK cells within the tissues. Still, the origin of the different CD56bright NK cell populations and their relation to the CD56dim NK cell subset remains unclear. The circulating CD56bright NK cells have been extensively investigated and are generally considered to be the precursors of the CD56dim NK cells. In the last section of this review, we will summarize the current evidence in favor and against the linear relationship between the circulating CD56bright and CD56dim NK cells, and speculate on the position of tissue-resident NK cells in this developmental pathway.

Several studies provided clues about the developmental relationship between CD56bright NK cells and CD56dim NK cells. First, it was shown that CD56bright, but not CD56dim NK cells, constitutively express the high-affinity IL-2Rα (CD25) and display a high proliferative response in the presence of picomolar concentrations of IL-2 (72, 73). Because CD56bright NK cells have significantly longer telomeres compared with CD56dim NK cells, they have been assumed to have a shorter proliferative history (74). A commonly used marker for immaturity, the tyrosine kinase c-kit (receptor for stem cell factor, CD117) is expressed on a fraction of CD56bright NK cells, but is absent on CD56dim NK cells (75, 76). In addition, the recovery of CD56bright NK cells in the first weeks after hematopoietic stem cell transplantation (HSCT) precedes the reconstitution of CD56dim NK, a sequential occurrence potentially pointing toward a developmental relationship (74, 77). Together, these findings resulted in the hypothesis that CD56dim NK cells are derived from CD56bright NK cells.

Differentiation from CD56<sup>bright</sup> to CD56<sup>dim</sup> NK Cells <italic>In Vitro</italic>

In efforts to provide evidence for this hypothesis, numerous studies aimed to recapitulate the differentiation from CD56bright to CD56dim NK cells in vitro. CD56bright NK cells were shown to acquire a CD56dim-like phenotype upon in vitro activation with IL-2, IL-15, and/or co-culture with T cells. This resulted in the upregulation of CD16 and KIRs and the downregulation of IL-7Rα (CD127), CD117, CXCR3, and CCR7 (10, 74). However, the intensity of CD56 expression was not reduced on monokine-activated CD56bright NK cells. The presence of fibroblast growth factor receptor 1 (FGFR1) was demonstrated to be critical for the in vitro differentiation of CD56bright NK cells to cytotoxic CD56dim NK cells in a contact-dependent manner (78). FGFR1 is a ligand for CD56 and is constitutively expressed on fibroblasts (79, 80). The high density of CD56 on CD56bright NK cells may thus be of importance in the interaction with fibroblasts and differentiation toward CD56dim NK cells.

 Differentiation from CD56<sup>bright</sup> to CD56<sup>dim</sup> NK Cells <italic>In Vivo</italic>

The in vivo evaluation of the relationship between CD56bright and CD56dim NK cells is hampered by the lack of CD56 expression on murine NK cells. The vast majority of human CD56bright NK cells displayed a reduction of CD56 expression intensity after infusion into immune-deficient mice (78). Whether these in vivo differentiated CD56dim NK cells were phenotypically and functionally similar to human blood-derived CD56dim NK cells were not addressed in this study, leaving the possibility that the bright CD56 expression is not sustained in mice lacking human fibroblasts expressing FGFR1. An alternative for murine experiments can be provided by the study of rhesus macaques. Gene tracking data in rhesus macaques transplanted with lentiviral barcoded hematopoietic stem cells demonstrated that the lineage origin of the macaque NK cell homologs of CD56bright (CD56+CD16−) and CD56dim (CD56−CD16+) NK cells is different (81). While the CD56bright homolog was derived from the same progenitors as T-cell, B-cell, and myeloid cells, the CD56dim homolog displayed a unique clonal pattern, suggesting that these cells do not develop from the CD56bright population but may belong to an independent lineage (81).

In addition to mice and macaques studies, human NK cell deficiencies can provide clues about the developmental relationship between CD56bright and CD56dim NK cells. Mutations in the transcription factor gene GATA2 result in the absence of CD56bright NK cells while CD56dim NK cells are still present (82). This observation argues against the theory that CD56dim NK cells are derived from CD56bright NK cells. On the other hand, humans with a partial minichromosome maintenance complex 4 (MCM4) deficiency, a molecule involved in proliferation, have reduced numbers of circulating CD56dim NK cells but normal numbers of CD56bright NK cells (83). This could indicate that maintenance of the CD56dim NK cell subset requires proliferation, which might be dependent or independent of the CD56bright NK cells. To the best of our knowledge, there are no mutations in transcription factors described, which cause a lack of CD56dim NK cells while the CD56bright NK cells are spared. Recently, Zeb2 was identified as the essential regulator of terminal NK cell maturation in mice and shown to be higher expressed in circulating CD56dim compared with CD56bright NK cells (84). Together, these studies emphasize the need for additional experimental evidence on the transcriptional regulation of human NK cell development.

 Intermediate Stages Connecting CD56<sup>bright</sup> to CD56<sup>dim</sup> NK Cells

If CD56bright and CD56dim NK cells are successive stages in the NK cell developmental pathway, developmental intermediates should exist. Independent studies reported the existence of phenotypic and functional intermediate stages in the progression from CD56bright to CD56dim NK cells in peripheral blood of healthy donors and patients after HSCT. These studies mainly focused on CD16, CD27, or CD117, for which CD56bright NK cells have a bimodal expression profile (10, 51, 85–88). Both CD16+ and CD27− CD56bright NK cells were independently suggested to represent intermediate populations based on phenotype and functional characteristics (10, 51, 88). A relative increase of CD16+, CD27−, and CD117− CD56bright NK cells was observed early after HSCT (85, 86). Notably, the expression of CD16, CD117, and CD27 on CD56bright NK cells can also be modulated by cytokine-activation (10, 51, 74). Because the post-HSCT setting presents a cytokine-rich environment, the “intermediate” CD16+, CD27−, and CD117− CD56bright NK cells may represent cytokine-activated CD56bright NK cells instead of developmental intermediates between CD56bright and CD56dim NK cells (85, 86).

In general, the potential differentiation of circulating CD56bright NK cells to CD56dim NK cells is characterized by loss of CD27, CD117, NKG2A, and CD62L expression and gain of CD16, KIRs, and CD57 expression. Both within the CD56bright compartment (CD117↓, CD27↓, and CD16↑) as well as within the CD56dim compartment (NKG2A↓, CD62L↓, KIRs↑, and CD57↑), the sequential loss and acquisition of these surface markers do not occur in a fixed order (10, 51, 87, 89, 90). Only the extremes of these markers, for instance CD117 and CD57, are mutually exclusively expressed. Together, this illustrates that uniform intermediate stages of differentiation between CD56bright and CD56dim NK cells cannot easily be identified.

 Developmental Position of Tissue-Resident CD56<sup>bright</sup> NK Cells

Studies on the relationship between the NK cell populations have been based on blood-derived CD56bright and CD56dim NK cells. The starting point of most of these studies was a linear developmental relationship between CD56bright and CD56dim NK cells. However, the discovery of distinct tissue-resident CD56bright NK cell populations increases the number of possible relationships between the NK cell populations. Tissue-resident NK cells could be a precursor to circulating NK cells, but the absence of the immature markers CD117 and CD127 argues against this. It also seems unlikely that tissue-resident NK cells represent a transitory population between the circulating CD56bright and CD56dim NK cells. Detailed transcriptome analysis comparing uterine NK cells with both circulating CD56bright and CD56dim NK cells highlighted major differences in gene expression profile between the three NK cell populations (17). Moreover, data from transcription factor-deficient mice suggested that circulating and tissue-resident NK cells are derived from different cell lineages (91). In our opinion, the distinct phenotype and functional signature of the tissue-resident NK cell populations, together with their absence from blood, argues in favor of the hypothesis that tissue-resident CD56bright NK cells develop locally, independently of the circulating NK cells. It seems likely that the organ microenvironment is essential to induce the phenotype and retain tissue localization of tissue-resident cells. Nevertheless, additional studies are needed to shed new light on the developmental relationship between CD56bright, CD56dim and tissue-resident CD56bright NK cell populations.

 Concluding Remarks

The recent identification of tissue-resident CD56bright NK cells in the lymphoid tissues, liver, and uterus led us to reappraise the characteristics of CD56bright NK cell populations in the circulation and tissues. The function of tissue-resident CD56bright NK cells in liver and lymphoid tissues has not been elucidated, although it is very likely that these cells, such as uterine NK cells, exert tissue-specific functions.

The existence of tissue-resident NK cells raises the question whether, and if so how, all the NK cell populations are developmentally related to each other. Based on the available evidence, we conclude that it is still possible that CD56dim NK cells develop independently from the CD56bright NK cells. Tissue-resident NK cells may develop from circulating CD56bright NK cells, or follow their own developmental pathway. Current in vitro models do not sufficiently mimic the in vivo situation, especially considering the potentially important role of the tissue microenvironment in shaping the features of tissue-resident CD56bright NK cells. As mouse models do not suffice in the evaluation of human NK cell subsets, other animal models might be exploited. Studying patients with aberrations in NK cell development due to genetic mutations could provide novel insights in the origin and development of tissue-resident NK cells. Furthermore, transcriptome analysis of non-resident and resident CD56bright NK cell populations will provide tools to further decipher the role of CD56bright NK cell populations in human immune responses. In conclusion, distinguishing tissue-resident CD56bright NK cells from circulating CD56bright NK cells is a prerequisite for the better understanding of the specific role of CD56bright NK cells in the complex process of human immune regulation.

 Author Contributions

JM and GL wrote the manuscript. AL and MS critically revised the manuscript and approved it for publication.

 Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Funding

This work was financially supported with a grant from the Dutch Cancer Society (#UL 2011-5133). GL was supported by a Leiden University Medical Center MD/PhD fellowship.

 Abbreviations

DNAM1, DNAX accessory molecule 1; EOMES, eomesodermin; FGFR1, fibroblast growth factor receptor 1; HEVs, high endothelial venules; HSCT, hematopoietic stem cell transplantation; ILC, innate lymphoid cell; KIR, killer-cell immunoglobulin-like receptor; ltNK cells, lymphoid tissue natural killer cells; MCM4, minichromosome maintenance complex 4; S1PR1, sphingosine-1-phospate receptor 1.

 References Kiessling R Klein E Wigzell H. ‘Natural’ killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur J Immunol (1975) 5:112–7.10.1002/eji.1830050208 Herberman RB Nunn ME Lavrin DH. Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic acid allogeneic tumors. I. Distribution of reactivity and specificity. Int J Cancer (1975) 16:216–29.10.1002/ijc.291016020550294 Serafini N Vosshenrich CA Di Santo JP. Transcriptional regulation of innate lymphoid cell fate. Nat Rev Immunol (2015) 15:415–28.10.1038/nri385526065585 Artis D Spits H. The biology of innate lymphoid cells. Nature (2015) 517:293–301.10.1038/nature1418925592534 Lanier LL Ruitenberg JJ Phillips JH. Functional and biochemical analysis of CD16 antigen on natural killer cells and granulocytes. J Immunol (1988) 141:3478–85.2903193 Fauriat C Long EO Ljunggren HG Bryceson YT. Regulation of human NK-cell cytokine and chemokine production by target cell recognition. Blood (2010) 115:2167–76.10.1182/blood-2009-08-23846919965656 De Maria A Bozzano F Cantoni C Moretta L. Revisiting human natural killer cell subset function revealed cytolytic CD56dimCD16+ NK cells as rapid producers of abundant IFN-γ on activation. Proc Natl Acad Sci U S A (2011) 108:728–32.10.1073/pnas.1012356108 Anfossi N André P Guia S Falk CS Roetynck S Stewart CA Human NK cell education by inhibitory receptors for MHC class I. Immunity (2006) 25:331–42.10.1016/j.immuni.2006.06.01316901727 Cooper M Fehniger TA Turner SC Chen KS Ghaheri BA Ghayur T Human natural killer cells: a unique innate immunoregulatory role for the CD56bright subset. Blood (2001) 97:3146–51.10.1182/blood.V97.10.3146 Béziat V Duffy D Quoc SN Le Garff-Tavernier M Decocq J Combadière B CD56brightCD16+ NK cells: a functional intermediate stage of NK cell differentiation. J Immunol (2011) 186:6753–61.10.4049/jimmunol.110033021555534 Takahashi E Kuranaga N Satoh K Habu Y Shinomiya N Asano T Induction of CD16+ CD56bright NK cells with antitumour cytotoxicity not only from CD16- CD56bright NK cells but also from CD16- CD56dim NK cells. Scand J Immunol (2007) 65:126–38.10.1111/j.1365-3083.2006.01883.x17257217 Carrega P Bonaccorsi I Di Carlo E Morandi B Paul P Rizzello V CD56brightPerforinlow noncytotoxic human NK cells are abundant in both healthy and neoplastic solid tissues and recirculate to secondary lymphoid organs via afferent lymph. J Immunol (2014) 192:3805–15.10.4049/jimmunol.1301889 Lugthart G Melsen JE Vervat C van Ostaijen-Ten Dam MM Corver WE Roelen DL Human lymphoid tissues harbor a distinct CD69+CXCR6+ NK cell population. J Immunol (2016) 197:78–84.10.4049/jimmunol.150260327226093 Fehniger T Cooper MA Nuovo GJ Cella M Facchetti F Colonna M CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2: a potential new link between adaptive and innate immunity. Blood (2003) 101:3052–7.10.1182/blood-2002-09-2876 Hudspeth K Donadon M Cimino M Pontarini E Tentorio P Preti M Human liver-resident CD56bright/CD16neg NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways. J Autoimmun (2016) 66:40–50.10.1016/j.jaut.2015.08.011 Manaster I Mizrahi S Goldman-Wohl D Sela HY Stern-Ginossar N Lankry D Endometrial NK cells are special immature cells that await pregnancy. J Immunol (2008) 181:1869–76.10.4049/jimmunol.181.3.186918641324 Koopman LA Kopcow HD Rybalov B Boyson JE Orange JS Schatz F Human decidual natural killer cells are a unique NK cell subset with immunomodulatory potential. J Exp Med (2003) 198:1201–12.10.1084/jem.2003030514568979 Ferlazzo G Thomas D Lin SL Goodman K Morandi B Muller WA The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic. J Immunol (2004) 172:1455–62.10.4049/jimmunol.172.3.145514734722 Dalbeth N Gundle R Davies RJ Lee YC McMichael AJ Callan MF. CD56bright NK cells are enriched at inflammatory sites and can engage with monocytes in a reciprocal program of activation. J Immunol (2004) 173:6418–26.10.4049/jimmunol.173.10.641815528382 Montaldo E Vacca P Chiossone L Croxatto D Loiacono F Martini S Unique eomes+ NK cell subsets are present in uterus and decidua during early pregnancy. Front Immunol (2016) 6:646.10.3389/fimmu.2015.00646 Marquardt N Béziat V Nyström S Hengst J Ivarsson MA Kekäläinen E Cutting edge: identification and characterization of human intrahepatic CD49a+ NK cells. J Immunol (2015) 194:2467–71.10.4049/jimmunol.140275625672754 Björkström NK Ljunggren HG Michaëlsson J. Emerging insights into natural killer cells in human peripheral tissues. Nat Rev Immunol (2016) 16:310–20.10.1038/nri.2016.3427121652 Montaldo E Vacca P Moretta L Mingari MC. Development of human natural killer cells and other innate lymphoid cells. Semin Immunol (2014) 26:107–13.10.1016/j.smim.2014.01.00624559836 Yu J Freud AG Caligiuri MA. Location and cellular stages of natural killer cell development. Trends Immunol (2013) 34:573–82.10.1016/j.it.2013.07.00524055329 Eissens DN Spanholtz J van der Meer A van Cranenbroek B Dolstra H Kwekkeboom J Defining early human NK cell developmental stages in primary and secondary lymphoid tissues. PLoS One (2012) 7:e30930.10.1371/journal.pone.003093022319595 Manaster I Mandelboim O. The unique properties of uterine NK cells. Am J Reprod Immunol (2010) 63:434–44.10.1111/j.1600-0897.2009.00794.x20055791 Bankovich AJ Shiow LR Cyster JG. CD69 suppresses sphingosine 1-phosophate receptor-1 (S1P1) function through interaction with membrane helix 4. J Biol Chem (2010) 285:22328–37.10.1074/jbc.M110.12329920463015 Shiow LR Rosen DB Brdicková N Xu Y An J Lanier LL CD69 acts downstream of interferon-alpha/beta to inhibit S1P1 and lymphocyte egress from lymphoid organs. Nature (2006) 440:540–4.10.1038/nature0460616525420 Mackay LK Braun A Macleod BL Collins N Tebartz C Bedoui S Cutting edge: CD69 interference with sphingosine-1-phosphate receptor function regulates peripheral T cell retention. J Immunol (2015) 194:2059–63.10.4049/jimmunol.140225625624457 Rivera J Proia RL Olivera A. The alliance of sphingosine-1-phosphate and its receptors in immunity. Nat Rev Immunol (2008) 8:753–63.10.1038/nri240018787560 Mayol K Biajoux V Marvel J Balabanian K Walzer T. Sequential desensitization of CXCR4 and S1P5 controls natural killer cell trafficking. Blood (2011) 118:4863–71.10.1182/blood-2011-06-36257421911833 Jenne CN Enders A Rivera R Watson SR Bankovich AJ Pereira JP T-bet-dependent S1P5 expression in NK cells promotes egress from lymph nodes and bone marrow. J Exp Med (2009) 206:2469–81.10.1084/jem.2009052519808259 Walzer T Chiossone L Chaix J Calver A Carozzo C Garrigue-Antar L Natural killer cell trafficking in vivo requires a dedicated sphingosine 1-phosphate receptor. Nat Immunol (2007) 8:1337–44.10.1038/ni152317965716 Pappu R Schwab SR Cornelissen I Pereira JP Regard JB Xu Y Promotion of lymphocyte egress into blood and lymph by distinct sources of sphingosine-1-phosphate. Science (2007) 316:295–8.10.1126/science.113922117363629 Schwab SR Pereira JP Matloubian M Xu Y Huang Y Cyster JG. Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients. Science (2005) 309:1735–9.10.1126/science.111364016151014 Hanna J Bechtel P Zhai Y Youssef F McLachlan K Mandelboim O Novel insights on human NK cells’ immunological modalities revealed by gene expression profiling. J Immunol (2004) 173:6547–63.10.4049/jimmunol.173.11.6547 Wendt K Wilk E Buyny S Buer J Schmidt RE Jacobs R. Gene and protein characteristics reflect functional diversity of CD56dim and CD56bright NK cells. J Leukoc Biol (2006) 80:1529–41.10.1189/jlb.030619116966385 Campbell JJ Qin S Unutmaz D Soler D Murphy KE Hodge MR Unique subpopulations of CD56+ NK and NK-T peripheral blood lymphocytes identified by chemokine receptor expression repertoire. J Immunol (2001) 166:6477–82.10.4049/jimmunol.166.11.647711359797 Frey M Packianathan NB Fehniger TA Ross ME Wang WC Stewart CC Differential expression and function of L-selectin on CD56bright and CD56dim natural killer cell subsets. J Immunol (1998) 161:400–8.9647249 Westermann J Pabst R. Distribution of lymphocyte subsets and natural killer cells in the human body. Clin Investig (1992) 70:539–44.1392422 Caligiuri MA. Human natural killer cells. Blood (2008) 112:461–9.10.1182/blood-2007-09-077438 Carrega P Ferlazzo G. Natural killer cell distribution and trafficking in human tissues. Front Immunol (2012) 3:347.10.3389/fimmu.2012.0034723230434 Montalto E Mangraviti S Costa G Carrega P Morandi B Pezzino G Seroma fluid subsequent to axillary lymph node dissection for breast cancer derives from an acculumulation of afferent lymph. Immunol Lett (2010) 131:67–72.10.1016/j.imlet.2010.03.002 Fuchs A Vermi W Lee JS Lonardi S Gilfillan S Newberry RD Intraepithelial type 1 innate lymphoid cells are a unique subset of IL-12- and IL-15-responsive IFN-γ-producing cells. Immunity (2013) 38:769–81.10.1016/j.immuni.2013.02.01023453631 Tanimine N Tanaka Y Abe T Piao J Chayama K Ohdan H. Functional behavior of NKp46-positive intrahepatic natural killer cells against hepatitis C virus reinfection after liver transplantation. Transplantation (2016) 100:355–64.10.1097/TP.000000000000103126714120 Stegmann KA Robertson F Hansi N Gill U Pallant C Christophides T CXCR6 marks a novel subset of T-bet(lo)Eomes(hi) natural killer cells residing in human liver. Sci Rep (2016) 6:26157.10.1038/srep26157 Bulmer JN Morrison L Longfellow M Ritson A Pace D. Granulated lymphocytes in human endometrium: histochemical and immunohistochemical studies. Hum Reprod (1991) 6:791–8.1757516 Wang F Zhou Y Fu B Wu Y Zhang R Sun R Molecular signatures and transcriptional regulatory networks of human immature decidual NK and mature peripheral NK cells. Eur J Immunol (2014) 44:2771–84.10.1002/eji.20134418324838931 Searle RF Jones RK Bulmer JN. Phenotypic analysis and proliferative responses of human endometrial granulated lymphocytes during the menstrual cycle. Biol Reprod (1999) 60:871–8.10.1095/biolreprod60.4.87110084960 Ferlazzo G Pack M Thomas D Paludan C Schmid D Strowig T. Distinct roles of IL-12 and IL-15 in human natural killer cell activation by dendritic cells from secondary lymphoid organs. Proc Natl Acad Sci U S A (2004) 101:16606–11.10.1073/pnas.040752210115536127 Vossen MT Matmati M Hertoghs KM Baars PA Gent MR Leclercq G CD27 defines phenotypically and functionally different human NK cell subsets. J Immunol (2008) 180:3739–45.10.4049/jimmunol.180.6.373918322179 Tomasello E Yessaad N Gregoire E Hudspeth K Luci C Mavilio D Mapping of NKp46(+) cells in healthy human lymphoid and non-lymphoid tissues. Front Immunol (2012) 3:344.10.3389/fimmu.2012.0034423181063 Mebius RE Kraal G. Structure and function of the spleen. Nat Rev Immunol (2005) 5:606–16.10.1038/nri166916056254 Grégoire C Cognet C Chasson L Coupet CA Dalod M Reboldi A Intrasplenic trafficking of natural killer cells is redirected by chemokines upon inflammation. Eur J Immunol (2008) 38:2076–84.10.1002/eji.20083855018624307 Cella M Fuchs A Vermi W Facchetti F Otero K Lennerz JK A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity. Nature (2008) 457:722–5.10.1038/nature07537 Spits H Artis D Colonna M Diefenbach A Di Santo JP Eberl G Innate lymphoid cells – a proposal for uniform nomenclature. Nat Rev Immunol (2013) 13:145–9.10.1038/nri3365 Page G Lebecque S Miossec P. Anatomic localization of immature and mature dendritic cells in an ectopic lymphoid organ: correlation with selective chemokine expression in rheumatoid synovium. J Immunol (2002) 168:5333–41.10.4049/jimmunol.168.10.533311994492 Paust S Gill HS Wang BZ Flynn MP Moseman EA Senman B Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses. Nat Immunol (2010) 11:1127–35.10.1038/ni.195320972432 Peng H Jiang X Chen Y Sojka DK Wei H Gao X Liver-resident NK cells confer adaptive immunity in skin-contact inflammation. J Clin Invest (2013) 123:1444–56.10.1172/JCI6638123524967 Kitaya K Yasuda J Nakayama T Fushiki S Honjo H. Effect of female sex steroids on human endometrial CD16neg CD56bright natural killer cells. Fertil Steril (2003) 79:730–4.10.1016/S0015-0282(02)04818-512620484 Lukassen HG Joosten I van Cranenbroek B van Lierop MJ Bulten J Braat DD Hormonal stimulation for IVF treatment positively affects the CD56bright/CD56dim NK cell ratio of the endometrium during the window of implantation. Mol Hum Reprod (2004) 10:513–20.10.1093/molehr/gah06715155817 Marlin R Duriez M Berkane N de Truchis C Madec Y Rey-Cuille MA Dynamic shift from CD85j/ILT-2 to NKG2D NK receptor expression pattern on human decidual NK during the first trimester of pregnancy. PLoS One (2012) 7:e30017.10.1371/journal.pone.003001722242197 Eriksson M Meadows SK Wira CR Sentman CL. Unique phenotype of human uterine NK cells and their regulation by endogenous TGF-beta. J Leukoc Biol (2004) 76:667–75.10.1189/jlb.020409015178706 Vacca P Pietra G Falco M Romeo E Bottino C Bellora F Analysis of natural killer cells isolated from human decidua: evidence that 2B4 (CD244) functions as an inhibitory receptor and blocks NK-cell function. Blood (2006) 108:4078–85.10.1182/blood-2006-04-01734316931625 El Costa H Casemayou A Aguerre-Girr M Rabot M Berrebi A Parant O Critical and differential roles of NKp46- and NKp30-activating receptors expressed by uterine NK cells in early pregnancy. J Immunol (2008) 181:3009–17.10.4049/jimmunol.181.5.300918713971 Moffett A Colucci F. Uterine NK cells: active regulators at the maternal-fetal interface. J Clin Invest (2014) 124:1872–9.10.1172/JCI6810724789879 Hanna J Goldman-Wohl D Hamani Y Avraham I Greenfield C Natanson-Yaron S Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat Med (2006) 12:1065–74.10.1038/nm145216892062 Lash GE Schiessl B Kirkley M Innes BA Cooper A Searle RF Expression of angiogenic growth factors by uterine natural killer cells during early pregnancy. J Leukoc Biol (2006) 80:572–80.10.1189/jlb.040625016816146 Xiong S Sharkey AM Kennedy PR Gardner L Farrell LE Chazara O Maternal uterine NK cell-activating receptor KIR2DS1 enhances placentation. J Clin Invest (2013) 123:4264–72.10.1172/JCI6899124091323 Ashkar AA Di Santo JP Croy BA. Interferon gamma contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation during normal murine pregnancy. J Exp Med (2000) 192:259–70.10.1084/jem.192.2.25910899912 Hiby SE Apps R Sharkey AM Farrell LE Gardner L Mulder A Maternal activating KIRs protect against human reproductive failure mediated by fetal HLA-C2. J Clin Invest (2010) 120:4102–10.10.1172/JCI4399820972337 Caligiuri MA Murray C Robertson MJ Wang E Cochran K Cameron C Selective modulation of human natural killer cells in vivo after prolonged infusion of low dose recombinant interleukin 2. J Clin Invest (1993) 91:123–32.10.1172/JCI1161617678599 Baume DM Robertson MJ Levine H Manley TJ Schow PW Ritz J. Differential responses to interleukin 2 define functionally distinct subsets of human natural killer cells. Eur J Immunol (1992) 22:1–6.10.1002/eji.18302201021370410 Romagnani C Juelke K Falco M Morandi B D’Agostino A Costa R CD56 bright CD16 − killer Ig-like receptor − NK cells display longer telomeres and acquire features of CD56 dim NK cells upon activation. J Immunol (2007) 178:4947–55.10.4049/jimmunol.178.8.4947 Nagler A Lanier LL Phillips JH. Constitutive expression of high affinity interleukin 2 receptors on human CD16-natural killer cells in vivo. J Exp Med (1990) 171:1527–33.10.1084/jem.171.5.15272139697 Matos ME Schnier GS Beecher MS Ashman LK William DE Caligiuri MA. Expression of a functional c-kit receptor on a subset of natural killer cells. J Exp Med (1993) 178:1079–84.10.1084/jem.178.3.10797688785 Nguyen S Dhedin N Vernant JP Kuentz M Al Jijakli A Rouas-Freiss N NK-cell reconstitution after haploidentical hematopoietic stem-cell transplantations: immaturity of NK cells and inhibitory effect of NKG2A override GvL effect. Blood (2005) 105:4135–43.10.1182/blood-2004-10-4113 Chan A Hong DL Atzberger A Kollnberger S Filer AD Buckley CD CD56bright human NK cells differentiate into CD56dim cells: role of contact with peripheral fibroblasts. J Immunol (2007) 179:89–94.10.4049/jimmunol.179.1.8917579025 Root LL Shipley GD. Normal human fibroblasts produce membrane-bound and soluble isoforms of FGFR-1. Mol Cell Biol Res Commun (2000) 3:87–97.10.1006/mcbr.2000.019910775505 Williams EJ Furness J Walsh FS Doherty P. Activation of the FGF receptor underlies neurite outgrowth stimulated by L1, N-CAM, and N-cadherin. Neuron (1994) 13:583–94.10.1016/0896-6273(94)90027-2 Wu C Li B Lu R Koelle SJ Yang Y Jares A Clonal tracking of rhesus macaque hematopoiesis highlights a distinct lineage origin for natural killer cells. Cell Stem Cell (2014) 14:486–99.10.1016/j.stem.2014.01.02024702997 Mace EM Hsu AP Monaco-Shawver L Makedonas G Rosen JB Dropulic L Mutations in GATA2 cause human NK cell deficiency with specific loss of the CD56bright subset. Blood (2013) 121:2669–77.10.1182/blood-2012-09-453969 Gineau L Cognet C Kara N Lach FP Dunne J Veturi U Partial MCM4 deficiency in patients with growth retardation, adrenal insufficiency, and natural killer cell deficiency. J Clin Invest (2012) 122:821–32.10.1172/JCI6101422354167 van Helden MJ Goossens S Daussy C Mathieu AL Faure F Marçais A Terminal NK cell maturation is controlled by concerted actions of T-bet and Zeb2 and is essential for melanoma rejection. J Exp Med (2015) 212:2015–25.10.1084/jem.2015080926503444 Vukicevic M Chalandon Y Helg C Matthes T Dantin C Huard B CD56 bright NK cells after hematopoietic stem cell transplantation are activated mature NK cells that expand in patients with low numbers of T cells. Eur J Immunol (2010) 40:3246–54.10.1002/eji.20094001620957748 Dulphy N Haas P Busson M Belhadj S Peffault de Latour R Robin M An unusual CD56brightCD16low NK cell subset dominates the early posttransplant period following HLA-matched hematopoietic stem cell transplantation. J Immunol (2008) 181:2227–37.10.4049/jimmunol.181.3.2227 Yu J Mao HC Wei M Hughes T Zhang J Park IK CD94 surface density identifies a functional intermediary between the CD56bright and CD56dim human NK-cell subsets. Blood (2010) 115:274–81.10.1182/blood-2009-04-21549119897577 Silva A Andrews DM Brooks AG Smyth MJ Hayakawa Y. Application of CD27 as a marker for distinguishing human NK cell subsets. Int Immunol (2008) 20:625–30.10.1093/intimm/dxn02218326863 Björkström NK Riese P Heuts F Andersson S Fauriat C Ivarsson MA Expression patterns of NKG2A, KIR, and CD57 define a process of CD56 dim NK-cell differentiation uncoupled from NK-cell education. Blood (2010) 116:3853–64.10.1182/blood-2010-04-28167520696944 Juelke K Killig M Luetke-Eversloh M Parente E Gruen J Morandi B CD62L expression identifies a unique subset of polyfunctional CD56dim NK cells. Blood (2010) 116:1299–307.10.1182/blood-2009-11-25328620505160 Sojka DK Plougastel-Douglas B Yang L Pak-Wittel MA Artyomov MN Ivanova Y Tissue-resident natural killer (NK) cells are cell lineages distinct from thymic and conventional splenic NK cells. Elife (2014) 2014:1–21.10.7554/eLife.0165924714492