• natural killer cell receptors;
  • natural killer cell differentiation;
  • natural killer cell cytokine production;
  • cytolytic activity;
  • haploidentical hematopoietic stem cell transplantation


  1. Top of page
  2. Abstract
  3. Introduction
  8. Literature Cited

Natural killer (NK) cells are important components of the innate immunity and play a key role in host defense by virtue of their ability to release cytokines and to mediate cytolytic activity against tumor cells and virus-infected cells. NK cells were first described more than 30 years ago on the basis of their peculiar functional capabilities. Subsequently, thanks to the production of a variety of monoclonal antibodies, it became possible to identify surface receptors and markers expressed by NK cells as well as to characterize their functional properties. Here, we provide a brief historical overview about the discovery of human NK cell receptors and we delineate the main phenotypic features of differentiating and mature NK cells in healthy donors as well as their alterations in certain pathologic conditions. © 2013 International Society for Advancement of Cytometry


  1. Top of page
  2. Abstract
  3. Introduction
  8. Literature Cited

Natural killer (NK) cells were originally described on a functional basis. Thus, Herberman and his group reported that certain lymphocytes from healthy donors had the ability to recognize and kill a variety of tumor cells in a spontaneous fashion, that is, without previous activation ([1]). Subsequent morphological analysis revealed that most of these “naturally” cytotoxic cells display a typical and homogeneous morphology. Accordingly, they were defined large granular lymphocytes (LGL). Only at the beginning of the 1980s Lanier, Perussia and their groups selected two different monoclonal antibodies (mAbs; NK-15 and B73.1, respectively) directed against an antigen expressed by LGL ([2-4]). These mAbs, similarly to another mAb (HNK-1, CD57), appeared to specifically recognize NK cells, although they reacted only with a fraction of LGL. The targeted antigen was named CD16 (FcγRIIIa). It is a low affinity receptor for the Fc fragments of IgG. CD16 was found to activate the antibody-dependent cell-mediated cytotoxicity (ADCC) leading to NK-mediated killing of antibody-coated target cells.

CD56 (neural cell adhesion molecule, N-CAM) is another important NK cell antigen belonging to the Ig superfamily. Its functional role was confined to the ability to mediate homophilic adhesion ([5]). More recently, a second functional capability has been described, namely the ability to bind fibroblast growth factor 1 (FGFR1) ([6]). Based on the differential expression of CD56 levels, two major subsets of NK cells were defined and termed CD56dim cells and CD56bright. Differently from CD56dim cells, CD56bright also express CD117 (or c-kit), which binds to stem cell factor (SCF), a stromal cell-derived cytokine synthesized by fibroblasts and other cell types. Another molecule expressed by CD56bright NK cell subset is the high affinity IL-2 receptor α chain (IL-2Rα, CD25). CD56dim cells only express the heterodimer IL-2Rβ/IL-2Rγ (CD122/132), representing an intermediate affinity receptor, which binds both IL-2 and IL-15. These differences in IL-2R expression are reflected in the different proliferative capacity of CD56bright versus CD56dim cells ([7]). NK cells were also found to express adhesion molecules including lymphocyte-associated molecule-2 (LFA-2 or CD2), LFA-3 (CD58), and LFA-1 (CD11a/CD18) playing an important role in NK cell cytolytic function.

Despite the identification of several NK cell markers, the mechanism by which NK cells could kill tumor while sparing normal cells remained unclear. A pioneering hypothesis was proposed by Ljunggren and Karre in the late 1980. Based on the finding that murine NK cells efficiently killed mutant lymphoma cells that had lost surface MHC-class-I (MHC-I) molecules, but failed to lyse parental MHC-I+ cells, the authors suggested that NK cells preferentially kill target cells “missing” MHC-I (self) expression ([8]). The missing self hypothesis provided a clue for the subsequent molecular definition of the mechanisms involved in NK-mediated tumor cell killing. Parallel experiments in mice and humans revealed the existence of MHC-I-specific inhibitory receptors. In particular, human receptors were originally identified thanks to the production of mAbs (EB6 and GL183), specific for two surface molecules (named p58.1 and p58.2) expressed by subsets of NK cells and capable of modulating NK cell functions. The NK cell subsets identified on the basis of the expression (or lack) of these molecules displayed different capabilities of killing allogeneic cells, including normal phytohemagglutinin (PHA)-induced lymphoblast. Subsequent studies revealed that p58 molecules were indeed HLA-I-specific inhibitory receptors with the unique capability of recognizing allotypic determinants expressed by different groups of HLA-C alleles ([9-12]). p58 molecules, belonging to the Ig superfamily, were the prototypes of Killer Ig-like receptors (KIRs). Remarkably also activating forms of KIR were identified (see Table 1). Molecular analyses revealed major differences in their transmembrane and intracytoplasmic portions. Thus, while inhibitory KIR were characterized, in their (long) intracytoplasmic tail, by immune-receptor tyrosine-based inhibition motifs, capable of recruiting and activating the phosphatases SHP1 and SHP2, the activating KIRs were found to display a short intracytoplasmic tail and to be coupled to ITAM-bearing signaling polypeptides (DAP12) ([13-15]). Subsequently a wide array of inhibitory NK receptors was identified including additional KIRs with two or three extracellular domains, the HLA-E-specific CD94/NKG2A heterodimer, LIR/ILT2 (CD85j), and a number of non-HLA-specific receptors including p75/AIRM1 (Siglec7) and IRp60 ([16-19]).

Table 1. Human NK cell markers and receptors
  1. aInhibits cytotoxicity and stimulate IFNγ.

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The selection of mAbs capable of interfering with NK-mediated killing allowed to identify the prototypes (and major) activating receptors involved in tumor cell lysis, that is, NKp46, NKp44, and NKp30 ([20-24]). These receptors were collectively named natural cytotoxicity receptors (NCR). Further studies revealed that NKp46 is also expressed in mice. In addition, being strictly NK specific, NKp46 represents a reliable marker for human (and murine) NK cell identification. Notably, while both NKp46 and NKp30 are present in both resting and activated NK cells, NKp44 is expressed only in cultured, cytokine-activated, NK cells. Conversely, NKp44 was found to be constitutively expressed on pDC and on NCR+ innate lymphoid cell-3 (ILC3) in peripheral tissue ([25, 26]) while NKp30 on subsets of T lymphocytes ([27]). Other activating receptors are not restricted to NK cells. These include NKG2D, NKp80, DNAM-1, and 2B4 that are expressed also on T cell subsets, while NTB-A is present also on T and B cells. It is of note that 2B4 and NTB-A primarily function as coreceptors amplifying NK cell responses induced by NCR or cytokines ([24]). Finally, NK cells have been shown to express various toll-like receptors (TLR), including TLR 2, 3, 7, 8, and 9. It should be noted that the presently used techniques for TLR detection are usually represented by transcript detection (mRNA) or stimulation with agonist ligands while they do not include the use of flow cytometry and specific mAbs. Notably, the engagement of TLRs by their specific ligands strongly upregulates NK cell cytotoxicity and cytokine production ([28-33]). These findings revealed that NK cells can rapidly sense and respond to various microbial products, thus suggesting that they can play a role also in early defenses against various pathogens.


  1. Top of page
  2. Abstract
  3. Introduction
  8. Literature Cited

NK cells originate from CD34+ hematopoietic stem cells (HSCs) through discrete stages of development, by the sequential acquisition of receptors and functions, and they share developmental relationships with cells of other lineages. NK cell commitment requires the expression of transcription factors such as nuclear factor IL-3 regulated (NFIL3 also termed E4BP4) and thymocyte selection-associated HMG box factor (TOX) ([34-37]). Moreover, they derive from an inhibitor of DNA binding 2 (ID2) positive precursor similar to other innate lymphoid cells, T lymphocytes, and dendritic cells ([26]). It has also been shown that common myeloid and granulocytes-monocytes precursors retain the ability to differentiate in vitro into NK cells ([38-41]).

Our present knowledge on human NK cell differentiation comes mostly from in vitro studies. Upon culture in the presence of appropriate cytokines, NK cells can be generated in vitro from HSCs or more committed progenitors ([42]). For long, the bone marrow (BM) has been considered the only site of human NK cell differentiation. However, there is evidence that NK cell development from HSCs may occur in tissues other than BM. When properly cultured, CD34+ cells isolated from different sites (peripheral blood—PB, umbilical cord blood—UCB, thymus, secondary lymphoid organs, foetal, or adult liver and decidua) give rise to mature NK cells ([43-47]). Moreover, flow cytometric analysis of cells isolated from various peripheral tissues helped to identify NK cells at different stages of differentiation, suggesting that CD34+ cells or NK-lineage-committed precursors may recirculate from the BM to the periphery where they eventually undergo differentiation towards NK cells. Notably, in this context, CD117+CD56+/− and CD117+/−CD56+/−CD94+ immature NK cells were identified in tonsils and lymph nodes (LN). Moreover, NK-committed CD34+CD127+CD122+ (ID2+E4BP4+) cells were identified in decidual tissue and CD34+CD117CD94 and CD34CD117+/−CD94+/− in liver and CD34CD117+ in intestinal lamina propria. All these immature precursors could differentiate in vitro towards NK cells ([42, 45-47]).

Protocols for in vitro NK cell differentiation from CD34+ HSCs may differ in the cytokines added at the beginning of cultures (IL-3) or in the presence or absence of stromal cells as feeder cells. The minimal requirements for in vitro NK cell differentiation include cytokines that promote CD34+ cell survival, proliferation (SCF and Flt3-L), and lymphoid commitment (IL-7) and also cytokines that induce the generation of NK cell precursors and subsequently of mature NK cells (IL-15 or IL-2). It has been demonstrated that Flt3-L and SCF induce the expression of IL-2/IL-15 receptor beta-chain (CD122), thus generating cells that can respond to IL-15 and further differentiate towards NK cells ([42]).

The sequential acquisition of receptors and functional capabilities during in vitro NK cell differentiation can be monitored by flow cytometry. Three main stages can be identified, namely, NK cell precursors (NKP), immature NK cells (iNK), and mature NK cells (mNK). Although at the beginning of cultures mature NK cells are absent, at later stages, all three cell subsets can coexist in the same culture as differentiation is not fully synchronized in cultures. Figure 1 recapitulates the kinetic of acquisition of cell surface markers during in vitro NK cell development. Figure 2 shows a representative gating strategy useful to analyze UCB-derived CD34+ differentiation towards NK cells.


Figure 1. Schematic representation of NK cell differentiation. CD34+ cells, cultured in the presence of SCF, Flt3-L, IL-7, IL-15 (+ o - IL-21), differentiate towards NK cells through the sequential acquisition of receptors/markers and functional properties. Three main subsets can be identified: NK cell precursors, immature NK cells, and mature NK cells.

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Figure 2. Flow cytometric analysis of in vitro differentiating NK cells. UCB-derived CD34+ cells were cultured in the presence of SCF, Flt3-L, IL-7, IL-15, and IL-21. After 20 days of culture, cells were harvested and either analyzed for the surface expression of markers informative of NK cell differentiation or stimulated 4 h with PMA/Ionomycin and IL-23 in the presence of monensin and then analyzed for cytokine production. Data were acquired with a Gallios flow-cytometer (Beckman Coulter) and analyzed with the FlowJo 8.8.6 software. Sequential gates were applied to exclude doublets, dead cells (detected using fixable viability dyes), and Lin+ cells (CD3, CD19, and CD14). By the analysis of gated Lin CD33+ cells as CD161 versus CD56 it is possible to further gate on CD161+CD56 cells, that is, NK precursor cells (light blue line), and CD161+/dimCD56+ cells. CD161+/dimCD56+ cells can be further divided into CD56+LFA-1 immature NK cells (dark blue line) and CD56+LFA-1+ mature NK cells (red line). Histograms show the expression of the indicated surface markers or cytokines in the three cell subsets. Fixable viability dye-eFluor450; CD3-eFluor450; CD19-eFluor450; CD14-APC-eFluor780; CD33-APC; CD56-PC7; CD161-PerCPCy5.5; LFA-1-FITC; all other markers PE.

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Figure 3. Peripheral NK cell subsets. Schematic representation of NK cell differentiation from CD56bright to CD56dim.

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CD122 expression, that indicates NK cell commitment, is hardly detectable by flow cytometry ([42]). However, NK cell precursors can be identified as CD34(CD33+)CD117+CD244+CD161+CD56 cells. Although CD117 and CD244 can be expressed already on CD34+ cells, the first appearance of CD161 during in vitro NK cell differentiation is characteristic of the NKP stage ([48]). Subsequently, NK cell precursors acquire the expression of CD56. It is of note that, at this and at later differentiation stages, NK cells may coexpress CD33, a marker typical of the myeloid lineage. Different reports showed that CD33+(CD13+CD115+/−) myeloid progenitors could switch towards the NK cell lineage, particularly in the presence of corticosteroid or CXCL8 ([39, 41, 49]). Interestingly, the activating receptor NKp44, which is absent in most of freshly isolated PB NK cells from healthy donors, appears early in NKP/iNK CD161+CD56+/− cells. As previously mentioned, NKp44 expression by in vitro-derived NK cell precursors may be a consequence of the exposure to IL-15. Indeed, mature PB NK cells up-regulate NKp44 expression when cultured in the presence of IL-2 or IL-15 ([21]). NKp44+ iNK cells do not coexpress IL-3R (CD123), ILT3 (CD85k), or BDCA2 (CD303). Therefore, they can easily be distinguished from pDC. In addition, pDC do not grow under these culture conditions and express NKp44 functioning as an inhibitory receptor ([25, 50, 51]). Subsequently, CD117+CD161+CD56+NKp44+ immature (non cytolytic) NK cells acquire other activating receptors such as NKp46, NKG2D, and DNAM-1. The first inhibitory receptor to be expressed during in vitro (and in vivo) NK cell differentiation is CD94/NKG2A ([42]). Concomitantly to CD94 acquisition, iNK cells down-regulate CD117. These phenotypic changes shortly precede the expression of CD11a/CD18 (LFA-1) adhesion molecules ([49, 52, 53]). These events mark the achievement of a mature phenotype reminiscent of that detected in PB CD56bright NK cells. In vitro-derived mature NK cells have acquired cytolytic activity and express high levels of CD94/NKG2A, LFA-1, and activating receptors, while they are CD117low/+ and they can undergo partial down-regulation of CD161 (expressed at lower levels than in NKP or iNK cells). CD16 and KIR expression represent the last steps of NK cell differentiation; however, these receptors are not always clearly detectable in in vitro-derived NK cells. Different from mature NK cells, in NKP and iNK cells CD244 acts as an inhibitory receptor, as, at these stages of maturation, cells do not express SAP adaptor molecule, which is responsible for the transduction of coactivating signals in mature NK cells ([54]). Although a small population of CD161+56 NKP can kill target cells via a TRAIL-dependent mechanism ([55]), the expression of inhibitory CD244, together with the absence or low expression of perforin and granzymes and of LFA-1, explains the weak (if any) cytolytic activity of immature NK cells.

NKP and iNK cells differ from mature NK cells also in their peculiar cytokine secretion profile. NKPs produce GM-CSF, IL-5, and IL-13, while iNK cells secrete IL-22 and large amounts of CXCL8 ([49, 56]). Interestingly, in iNK cells CD161 and NKp44 cross-linking can induce CXCL8 secretion. It is of note that CD161 stimulation does neither induce cytokine release nor degranulation (detectable by the assessment of CD107a expression) in both in vitro-derived or PB-derived mature NK cells. Thus, an activating function mediated by CD161 seems to be restricted to iNK cells ([49]). Immature NK cells also express cytoplasmic TNF-α. This cytokine, together with IFN-γ, is also secreted by in vitro-derived and ex vivo isolated mature NK cells. It is of note that in vitro-derived CD117+CD161+CD56+NKp44+CD94(LFA-1) cells are a heterogeneous cell population and may include not only iNK cells but also NCR+ ILC3 (also known as NK22 cells) ([26, 57]). These cells, which are characterized by the expression of RORγt, have been identified in vivo within tonsils and intestinal mucosa. It is possible that in vitro generated CD161+CD56+LFA-1+IL-22+ cells may belong to ILC3 rather than to NK cells. However, further analysis of human NCR+ ILC3 and NK cells differentiation pathways is needed to clarify whether NCR+ ILC3 cells represent a distinct cell lineage or an intermediate step of NK cell development.


  1. Top of page
  2. Abstract
  3. Introduction
  8. Literature Cited

As mentioned above, different stages of NK cell differentiation have been identified also in fresh NK cells from peripheral tissues and PB. For example, although the majority of NK cells, in LN and tonsils, are CD56bright, also CD34CD117+CD56+CD94 as well as CD34CD117CD56+CD94+ cells can be detected and isolated. Moreover, at least in some instances, NK cells at these differentiation stages may derive from CD34+ cells present in these tissues. This strongly suggests that CD34+ cells, rather than committed precursors, are likely to migrate from BM to peripheral tissues.

PB NK cells are not homogeneous but are composed of different subsets. These subsets can represent different stages of differentiation and/or identify NK cells with different functional capabilities. As mentioned above, it is possible to distinguish two main NK subpopulations, on the basis of the expression of different levels of CD56: (CD3)CD56bright cells and (CD3)CD56dimcells. These NK cell subsets differ for a variety of receptors and functional activities. CD56bright were mainly considered as cytokine producers, while CD56dim as cytotoxic effector cells. Recently, it has clearly been established that “cytotoxic” CD56dim cells can also release large amounts of cytokines upon receptor-mediated triggering. In addition, they are phenotypically and functionally heterogeneous (see below).

CD56bright cells, which account only for 10% of PB and spleen NK cells, are mainly confined within secondary lymphoid organs where the ratio between CD56bright and CD56dim is inverted as compared to PB. The CD56bright subset is characterized by the absence or low expression of lytic granules and by a relevant, but relatively late, cytokine production that may influence immune responses. Within the CD56bright subset isolated from different districts, slight differences exist both in phenotypic characteristics and in the response to cytokine stimulation. Thus, LN CD56bright cells are KIRNKp44NKp30NKp46lowCD16CD94/NKG2A+ while PB CD56bright cells are usually characterized by the KIRNKp44NKp30lowNKp46+CD16+/−CD94/NKG2A+CCR7+CD62L+ phenotype. KIR expression may be detected in a minor fraction of PB CD56bright cells (<10%) and could reflect an initial progression towards further NK cell differentiation ([58, 59]). Upon stimulation with IL-2 or IL-12 ([60]), CD56bright NK cells produce IFN-γ. In addition, both PB and LN CD56bright KIR NK cells may de novo express CD16, KIRs, and perforin ([61]).

CD56dimCD16+ cells represent the large majority of PB NK cells. The developmental relationship between CD56bright and CD56dim was controversial; however, recent reports suggested that CD56dim derive from CD56bright cells. Thus, it has been shown that in CD56brightCD16 cells telomeres are significantly longer than in CD56dimCD16bright cells ([62]). In addition, analysis of human NK cell development in humanized immune system (HIS) mice demonstrated that NK cell development from CD34+ cells progressed from CD56brightCD16KIR to CD56dimCD16+KIR and finally to CD56dimCD16+KIR+([63]). When PB CD56bright NK cells were infused in NOD/SCID or NOD/SCID/γc−/− mice, they progressed towards CD56dim cells and acquired CD16 and KIRs expression ([6, 63, 64]). Moreover, independent studies identified cells with phenotypic and functional properties common to both CD56bright and CD56dim cells, suggesting the existence of intermediate stages in the progression from CD56bright towards CD56dim cells. Indeed, CD56brightCD16CD62L+CD27+KIRCD94/NKG2Abrightperforinlow NK cells would gradually acquire CD16, KIR, cytotoxic granules, CX3CR1, and CD57. In parallel, maturing NK cells down-modulate CD62L, CD27, CCR7, CD56, and CD94/NKG2A. For example, while CD27 and CD62L are expressed by all CD56bright cells, in CD56dim NK cells they are expressed by a subset that retains a high proliferating potential ([64, 65]). Conversely, the expression of CX3CR1 identifies more mature cells characterized by a low proliferating capacity ([66]). As mentioned above, differences in the proliferating capacity can be explained at least in part, by different patterns of cytokine receptor expression (see receptors for IL-2, IL-15, and IL-7) (Figure 3).

The expression of CD57 has been associated with late stages of CD8 T and NK cells maturation. This molecule is only expressed by a fraction of CD56dim NK cells, but not by CD56bright NK cells. CD57 expression on CD56dim cells increases concomitantly with that of KIRs and with the down-regulation of NKG2A. As a matter of fact, CD56dim CD57 NK cells display higher proliferative capabilities than CD57+ cells ([67]). It is of note that during the differentiation from CD56brigth to CD56dim, NK cells modulate adhesion molecules and chemokine receptors (including CCR7, CXCR1, CX3CR1, and ChemR23), thus changing their homing capability (i.e., from secondary lymphoid organs to inflamed tissues) ([68]).

Until recently, the role of CD56dimCD16+ cells was mainly confined to their cytolytic activity against virus-infected and tumor cells and to the CD16-mediated ADCC. However, recent studies revealed different CD56dimCD16+ subpopulations characterized not only by a peculiar phenotype but also by their different proliferating capacity and susceptibility to activating stimuli. More importantly, CD56dim NK cells can rapidly release cytokines upon NCR cross-linking. In addition, the secretion of chemokines/cytokines required the engagement of different number/density of activating receptors ([69, 70]). Chemokines such as MIP-1α (CCL3) and RANTES (CCL5) are released within 1 h, while IFN-γ and TNFα require longer time intervals, but still compatible with the time of innate responses (i.e., few hours). These data indicate that CD56dim cells can assure a rapid NK cell intervention during the early phases of innate immune responses.

HLA-I-specific receptors, in particular KIRs, revealed to be important to understand the mechanism of NK cells activation/inactivation and for defining peripheral mature NK cell subsets. They are even more important because they allowed the identification of alloreactive NK cells that play a fundamental role in the therapy of high risk leukemias in the haploidentical HSC transplantation (haplo-HSCT). While in an autologous setting, in most instances, NK cells do not attack normal self cells because they express receptor specific for self HLA-class I molecules, in an allogeneic setting, donor cells may express KIRs that do not recognize HLA-class I alleles of the recipient. These “alloreactive” NK subsets can be identified by appropriate combination of mAbs specific for different KIRs and NKG2A ([71]). This, together with appropriate genetic analysis performed on potential donors and recipient, allows identifying the most suitable donor of HSCs. In addition, it permits to monitor the generation of alloreactive NK cells deriving from donor HSCs and their persistence in patients as well as to establish correlations between the size of the alloreactive NK subset and the clinical outcome. It is of note that alloreactive NK cells not only kill leukemic blasts residual after the conditioning regimen but also eliminate patients DCs and T lymphocytes, thus preventing both Graft versus Host Disease (GvHD) and Host versus Graft reaction ([72, 73]).


  1. Top of page
  2. Abstract
  3. Introduction
  8. Literature Cited

The surface phenotypic and functional properties of NK cells can be significantly altered under different pathologic conditions. For example, the down-regulation of NCR expression has been described in both hematologic malignancies and viral infections, where it is likely to represent a mechanism by which virus-infected or tumor cells become resistant to NK cell-mediated lysis. In most acute myeloid leukemia (AML) patients, PB-NK cells are characterized by low levels of relevant activating receptors, including NKp46, NKp30, and DNAM-1, which play a major role in leukemia cell lysis ([74, 75]). These phenotypic and functional alterations could be induced by direct NK cell-tumor interactions, by soluble ligands of activating receptors or inhibitory factors released by leukemic blasts. Release of inhibitory factors appears to be a common mechanism in both hematological malignancies, including AML, acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) ([76, 77]) and in various solid tumors, including melanoma ([78, 79]), glioblastoma ([80]), breast cancer ([81]), ovarian carcinoma ([82]), and gastrointestinal stromal tumors (GIST) ([83, 84]). The inhibitory factors more commonly detected include cytokines such as TGF-β, metabolites such as the IDO-derived L-kynurenin and the prostaglandin E2. It is of note that also tumor-associated cells, for example fibroblasts, may secrete inhibitory factors, thus contributing to the establishment of a microenvironment capable of dampening antitumor immune responses ([85, 86]). The inhibitory effect may be further amplified by tumor-induced Tregs or myeloid suppressor cells. In all these cases, the down-regulation of relevant activating NK receptors is associated with functional impairment that has been observed in vitro as well as ex vivo in NK cells infiltrating tumor tissues.

With regard to infectious conditions, relevant reduction of NCR expression (NKp46 and NKp30) and impairment of cytolytic activity have been described in viremic HIV-infected individuals ([87, 88]). In addition, peripheral NK cells express relevant proportions of activation markers including HLA-DR (20–35% of NK cells) and CD69 not only in viremic patients but also after antiretroviral treatment ([89, 90]). In these patients, NK cell subsets showing HLA-DR expression have more relevant decreases in NCR expression and cytolytic activity, and reduced CD16 expression also in CD56dim cells. Interestingly, in HIV-infected patients, the expansion of an unusual CD56CD16+ NK cell subset was described for the first time ([88]). This subset displays an impaired effector function and typically shows the NKG2A, NKG2C+, KIR+, siglec-7, and NCRlow surface phenotype ([91, 92]). Both the presence of CD56 NK cells and the expansion of NKG2C+ NK cells have been detected in subjects who experienced Hantavirus ([93]), Chikungunya virus ([94]), HBV, or HCV infection ([95]) (5–75% NKG2C+ NK cells in Hantavirus, 15–90% in Chikungunya, 5–40% in HCV, and 5–80% in HBV). Along this line, it is now well established that CMV infection can play an important role in shaping the NK cell receptor repertoire, favoring the preferential expansion of NKG2C+ KIR+ NK cells and their persistence over time ([96, 97]). Since, in most instances, CMV coinfections/reactivations occur in HCV− and HIV−chronically infected patients, it is conceivable that CMV, rather than other viruses, may be responsible for the KIR+ NKG2C+ CD56 signature of NK cells found in these patients.

The imprinting on NK cell phenotype induced by CMV infection results particularly evident in immunocompromised hosts where T-cell immunity is impaired, such as chronically infected HIV patients ([98, 99]), congenital immunodeficiencies ([100, 101]), and patients undergoing HSCT. In this context, it has recently been shown that CMV reactivation can promote a rapid NK cell development after UCBT ([59, 102]). As shown in Figure 4, NK cells achieve more rapidly a full maturational stage in CMV-reactivating patients (right plots) as compared to noninfected ones (left plots). In particular, NK cells isolated from CMV-reactivating patients show low percentages of CD56bright NK cells and high proportions of mature NKG2AKIR+ NK cells, at variance with nonreactivating patients that display a more immature NK cell phenotype. Remarkably, in CMV-reactivating patients an unusual CD56CD16+ Siglec-7 NK cell subset was detected and was reminiscent of that described in viremic HIV-patients. The expansion of highly differentiated NK cells characterized by the NKG2C+CD57+KIR+ phenotype has been described not only after HSCT but also in some solid organ-transplanted patients undergoing acute CMV infection. As suggested by a recent study, NKG2C+ CD57+ NK cells may contain “memory” or “long-lived” NK cells ([103]).


Figure 4. Maturing NK cell phenotype is altered in patients undergoing umbilical cord blood transplantation (UBCT) by CMV infection/reactivation. (A) Freshly isolated NK cells were analyzed for the expression of the indicated surface markers in a cohort of patients undergoing UCBT. The phenotypic features of maturing NK cells are shown in two representative patients reactivating (right plots) or nonreactivating CMV (left plots), at 6 months after transplant. Data were acquired with a FacsVerse cytometer (Becton Dickinson) and analyzed with FacsSuite 1.0.2 software. CD56-PC7, CD16-PerCPCy5.5, CD57-Vioblue, NKG2A-FITC, Siglec-7, and KIR PE-labeled by isotype-specific secondary reagents have been used. (B) Schematic representation of NK cell maturation from CD34+ HSC in patients reactivating CMV. Note that the final stage is represented by CD16+ NK cells in which CD56 and Siglec-7 are down-modulated.

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HCV infection represents, in addition to HIV-1, another chronic viral infection in which relevant NK cell surface molecule perturbations have been described both during acute and during chronic phases of the disease. Differently from HIV, impairment of NK cells may be less evident by phenotypic analysis. In vitro exposure to cell-free HCV does not result in impairment of neither NK cell phenotype (as assessed by flow cytometry), nor their function, while reduced expression of NKp30 and NKG2D has been observed after contact of NK cells with HCV-JFH-infected hepatoma cell lines ([104]). On the contrary, analysis of peripheral NK cells in vivo during acute human HCV infection showed that NK cells up-regulate NKG2D expression as well as IFN-γ production both in those patients who develop chronic infection and in those who eventually clear the virus. In subjects acutely exposed to HCV, who did not develop acute or chronic disease (i.e., exposed-uninfected), increased expression of NKp30 but not of NKG2D has been reported ([105]). Thus, upon HCV challenge, acute infection may be associated to different NK cell responses.

The KIR genotype of infected patients has been shown to be associated to the course of HCV disease. Thus, it has been shown that patients with homozygous carriage of KIR2DL3:HLA-C1 genes ([106]) had a 2.5-fold higher probability to clear the virus as compared to those who did not carry this genetic background. The weaker interaction of KIR2DL3 with HLA-C1 molecules, compared to KIR2DL2:HLA-C1 or -C2 ([107]) would explain a lower degree of NK cell functional inhibition, resulting into more efficient NK cell function leading to increased clearance of the virus. Notably, this association cannot be approached by flow cytometry, and has not been so far reproduced by cytofluorimetric analysis of KIRs expression on NK cells. Finally, it should be remembered that KIRs are expressed also by some CD8+ T-cells, particularly those specific for HLA-E ([108]) and that their expression may prevent CTL function, including specific lysis of infected target cell, and that their relative contribution to HCV clearance has not been explored so far.

With regard to NCR expression during chronic HCV infection, conflicting results were initially reported with either unchanged ([109]), or significantly reduced ([110]) NKp46 and NKp30 expression. Recent observations in a prospective study of chronically HCV genotype 1 infected patients who underwent standard peg-IFN-α+ribavirin (PR) treatment showed that low NKp30 and NKp46 expression and low CD85j before treatment initiation are associated with sustained responses ([111]). On the contrary, higher NKp46 and NKp30 expressions were detected in nonresponders to PR.

Patients affected by mycobacterium tuberculosis hominis (mtb) infection may also display alterations in NK cell receptor expression. In fact, in patients affected by recent onset pulmonary tuberculosis, confirmed by clinical parameters and by direct identification of mtb, expression of NKp46, NKp30, and DNAM-1 on peripheral NK cells was significantly reduced and was associated with a marked reduction in IFN-γ production and in cytotoxic activity ([112]).


  1. Top of page
  2. Abstract
  3. Introduction
  8. Literature Cited

During the past decades, it became clear that cells of the innate immunity play a fundamental role in immune responses, not only as first barrier against pathogens but also for their ability to profoundly influence downstream adaptive immune responses. These progresses have been possible also thanks to the phenotypic identification of an array of surface molecules/receptors that allow to dissect different stages of cell maturation as well as to identify cell subsets endowed with different functional capabilities. In addition, thanks to the production and selection of a series of mAbs capable of interfering with/or inducing different cell function, major innate receptors have been identified that allowed to unravel the main mechanism involved in immune and inflammatory responses. In this context, the discovery of receptor regulating NK cell activation and the possibility to identify functional subsets by cytofluorimetric analysis has been pivotal not only for understanding NK cell biology but also, more importantly, for exploiting NK cells and their receptors in the successful cure of otherwise fatal leukemias ([71, 73]).

Literature Cited

  1. Top of page
  2. Abstract
  3. Introduction
  8. Literature Cited
  • 1
    Rosenberg EB, Herberman RB, Levine PH, Halterman RH, McCoy JL, Wunderlich JR. Lymphocyte cytotoxicity reactions to leukemia-associated antigens in identical twins. Int J Cancer 1972;9:648658.
  • 2
    Lanier LL, Le AM, Phillips JH, Warner NL, Babcock GF. Subpopulations of human natural killer cells defined by expression of the Leu-7 (HNK-1) and Leu-11 (NK-15) antigens. J Immunol 1983;131:17891796.
  • 3
    Perussia B, Starr S, Abraham S, Fanning V, Trinchieri G. Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. I. Characterization of the lymphocyte subset reactive with B73.1. J Immunol 1983;130:21332141.
  • 4
    Perussia B, Acuto O, Terhorst C, Faust J, Lazarus R, Fanning V, Trinchieri G. Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. II. Studies of B73.1 antibody-antigen interaction on the lymphocyte membrane. JImmunol 1983;130:21422148.
  • 5
    Lanier LL, Chang C, Azuma M, Ruitenberg JJ, Hemperly JJ, Phillips JH. Molecular and functional analysis of human natural killer cell-associated neural cell adhesion molecule (N-CAM/CD56). J Immunol 1991;146:44214426.
  • 6
    Chan A, Hong DL, Atzberger A, Kollnberger S, Filer AD, Buckley CD, McMichael A, Enver T, Bowness P. CD56bright human NK cells differentiate into CD56dim cells:Role of contact with peripheral fibroblasts. JImmunol 2007;179:8994.
  • 7
    Caligiuri MA, Zmuidzinas A, Manley TJ, Levine H, Smith KA, Ritz J. Functional consequences of interleukin 2 receptor expression on resting human lymphocytes. Identification of a novel natural killer cell subset with high affinity receptors. J Exp Med 1990;171:15091526.
  • 8
    Karre K, Ljunggren HG, Piontek G, Kiessling R. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 1986;319:675678.
  • 9
    Moretta A, Tambussi G, Bottino C, Tripodi G, Merli A, Ciccone E, Pantaleo G, Moretta L. A novel surface antigen expressed by a subset of human CD3- CD16+ natural killer cells. Role in cell activation and regulation of cytolytic function. J Exp Med 1990;171:695714.
  • 10
    Moretta A, Bottino C, Pende D, Tripodi G, Tambussi G, Viale O, Orengo A, Barbaresi M, Merli A, Ciccone E, et al. Identification of four subsets of human CD3-CD16+ natural killer (NK) cells by the expression of clonally distributed functional surface molecules: Correlation between subset assignment of NK clones and ability to mediate specific alloantigen recognition. J Exp Med 1990;172:15891598.
  • 11
    Ciccone E, Pende D, Viale O, Di Donato C, Tripodi G, Orengo AM, Guardiola J, Moretta A, Moretta L. Evidence of a natural killer (NK) cell repertoire for (allo) antigen recognition: Definition of five distinct NK-determined allospecificities in humans. J Exp Med 1992;175:709718.
  • 12
    Moretta A, Vitale M, Bottino C, Orengo AM, Morelli L, Augugliaro R, Barbaresi M, Ciccone E, Moretta L. P58 molecules as putative receptors for major histocompatibility complex (MHC) class I molecules in human natural killer (NK) cells. Anti-p58 antibodies reconstitute lysis of MHC class I-protected cells in NK clones displaying different specificities. J Exp Med 1993;178:597604.
  • 13
    Olcese L, Lang P, Vely F, Cambiaggi A, Marguet D, Blery M, Hippen KL, Biassoni R, Moretta A, Moretta L, et al. Human and mouse killer-cell inhibitory receptors recruit PTP1C and PTP1D protein tyrosine phosphatases. JImmunol 1996;156:45314534.
  • 14
    Moretta L, Moretta A. Killer immunoglobulin-like receptors. Curr Opin Immunol 2004;16:626633.
  • 15
    Parham P. MHC class I molecules and KIRs in human history, health and survival. Nat Rev Immunol 2005;5:201214.
  • 16
    Lopez-Botet M, Perez-Villar JJ, Carretero M, Rodriguez A, Melero I, Bellon T, Llano M, Navarro F. Structure and function of the CD94 C-type lectin receptor complex involved in recognition of HLA class I molecules. Immunol Rev 1997;155:165174.
  • 17
    Cantoni C, Bottino C, Augugliaro R, Morelli L, Marcenaro E, Castriconi R, Vitale M, Pende D, Sivori S, Millo R, et al. Molecular and functional characterization of IRp60, a member of the immunoglobulin superfamily that functions as an inhibitory receptor in human NK cells. Eur J Immunol 1999;29:31483159.
  • 18
    Colonna M, Nakajima H, Cella M. Inhibitory and activating receptors involved in immune surveillance by human NK and myeloid cells. J Leukoc Biol 1999;66:718722.
  • 19
    Falco M, Biassoni R, Bottino C, Vitale M, Sivori S, Augugliaro R, Moretta L, Moretta A. Identification and molecular cloning of p75/AIRM1, a novel member of the sialoadhesin family that functions as an inhibitory receptor in human natural killer cells. J Exp Med 1999;190:793802.
  • 20
    Sivori S, Vitale M, Morelli L, Sanseverino L, Augugliaro R, Bottino C, Moretta L, Moretta A. p46, a novel natural killer cell-specific surface molecule that mediates cell activation. J Exp Med 1997;186:11291136.
  • 21
    Vitale M, Bottino C, Sivori S, Sanseverino L, Castriconi R, Marcenaro E, Augugliaro R, Moretta L, Moretta A. NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells, is involved in non-major histocompatibility complex-restricted tumor cell lysis. J Exp Med 1998;187:20652072.
  • 22
    Pende D, Parolini S, Pessino A, Sivori S, Augugliaro R, Morelli L, Marcenaro E, Accame L, Malaspina A, Biassoni R, et al. Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells. J Exp Med 1999;190:15051516.
  • 23
    Moretta A, Bottino C, Vitale M, Pende D, Cantoni C, Mingari MC, Biassoni R, Moretta L. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu Rev Immunol 2001;19:197223.
  • 24
    Marcenaro E, Carlomagno S, Pesce S, Della Chiesa M, Parolini S, Moretta A, Sivori S. NK cells and their receptors during viral infections. Immunotherapy 2011;3:10751086.
  • 25
    Bonaccorsi I, Cantoni C, Carrega P, Oliveri D, Lui G, Conte R, Navarra M, Cavaliere R, Traggiai E, Gattorno M, et al. The immune inhibitory receptor LAIR-1 is highly expressed by plasmacytoid dendritic cells and acts complementary with NKp44 to control IFNalpha production. PLoS One 2010;5:e15080.
  • 26
    Spits H, Di Santo JP. The expanding family of innate lymphoid cells: Regulators and effectors of immunity and tissue remodeling. Nat Immunol 2011;12:2127.
  • 27
    Tang Q, Grzywacz B, Wang H, Kataria N, Cao Q, Wagner JE, Blazar BR, Miller JS, Verneris MR. Umbilical cord blood T cells express multiple natural cytotoxicity receptors after IL-15 stimulation, but only NKp30 is functional. J Immunol 2008;181:45074515.
  • 28
    Sivori S, Falco M, Della Chiesa M, Carlomagno S, Vitale M, Moretta L, Moretta A. CpG and double-stranded RNA trigger human NK cells by Toll-like receptors: Induction of cytokine release and cytotoxicity against tumors and dendritic cells. Proc Natl Acad Sci USA 2004;101:1011610121.
  • 29
    Chalifour A, Jeannin P, Gauchat JF, Blaecke A, Malissard M, N'Guyen T, Thieblemont N, Delneste Y. Direct bacterial protein PAMP recognition by human NK cells involves TLRs and triggers alpha-defensin production. Blood 2004;104:17781783.
  • 30
    Hart OM, Athie-Morales V, O'Connor GM, Gardiner CM. TLR7/8-mediated activation of human NK cells results in accessory cell-dependent IFN-gamma production. J Immunol 2005;175:16361642.
  • 31
    Sivori S, Carlomagno S, Moretta L, Moretta A. Comparison of different CpG oligodeoxynucleotide classes for their capability to stimulate human NK cells. Eur J Immunol 2006;36:961967.
  • 32
    Marcenaro E, Ferranti B, Falco M, Moretta L, Moretta A. Human NK cells directly recognize Mycobacterium bovis via TLR2 and acquire the ability to kill monocyte-derived DC. Int Immunol 2008;20:11551167.
  • 33
    Sivori S, Falco M, Moretta L, Moretta A. Extending killer Ig-like receptor function: From HLA class I recognition to sensors of microbial products. Trends Immunol 2010;31:289294.
  • 34
    Gascoyne DM, Long E, Veiga-Fernandes H, de Boer J, Williams O, Seddon B, Coles M, Kioussis D, Brady HJ. The basic leucine zipper transcription factor E4BP4 is essential for natural killer cell development. Nat Immunol 2009;10:11181124.
  • 35
    Kamizono S, Duncan GS, Seidel MG, Morimoto A, Hamada K, Grosveld G, Akashi K, Lind EF, Haight JP, Ohashi PS, et al. Nfil3/E4bp4 is required for the development and maturation of NK cells in vivo. J Exp Med 2009;206:29772986.
  • 36
    Aliahmad P, de la Torre B, Kaye J. Shared dependence on the DNA-binding factor TOX for the development of lymphoid tissue-inducer cell and NK cell lineages. Nat Immunol 2010;11:945952.
  • 37
    Yun S, Lee SH, Yoon SR, Kim MS, Piao ZH, Myung PK, Kim TD, Jung H, Choi I. TOX regulates the differentiation of human natural killer cells from hematopoietic stem cells in vitro. Immunol Lett 2011;136:2936.
  • 38
    Perez SA, Sotiropoulou PA, Gkika DG, Mahaira LG, Niarchos DK, Gritzapis AD, Kavalakis YG, Antsaklis AI, Baxevanis CN, Papamichail M. A novel myeloid-like NK cell progenitor in human umbilical cord blood. Blood 2003;101:34443450.
  • 39
    Vitale C, Cottalasso F, Montaldo E, Moretta L, Mingari MC. Methylprednisolone induces preferential and rapid differentiation of CD34+ cord blood precursors toward NK cells. Int Immunol 2008;20:565575.
  • 40
    Grzywacz B, Kataria N, Blazar BR, Miller JS, Verneris MR. Natural killer-cell differentiation by myeloid progenitors. Blood 2011;117:35483558.
  • 41
    Vitale C. Plasticity of NK-cell differentiation. Blood 2011;117:34823483.
  • 42
    Freud AG, Caligiuri MA. Human natural killer cell development. Immunol Rev 2006;214:5672.
  • 43
    Mingari MC, Vitale C, Cantoni C, Bellomo R, Ponte M, Schiavetti F, Bertone S, Moretta A, Moretta L. Interleukin-15-induced maturation of human natural killer cells from early thymic precursors: Selective expression of CD94/NKG2-A as the only HLA class I-specific inhibitory receptor. Eur J Immunol 1997;27:13741380.
  • 44
    Freud AG, Becknell B, Roychowdhury S, Mao HC, Ferketich AK, Nuovo GJ, Hughes TL, Marburger TB, Sung J, Baiocchi RA, et al. A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells. Immunity 2005;22:295304.
  • 45
    Moroso V, Famili F, Papazian N, Cupedo T, van der Laan LJ, Kazemier G, Metselaar HJ, Kwekkeboom J. NK cells can generate from precursors in the adult human liver. Eur J Immunol 2011;41:33403350.
  • 46
    Vacca P, Vitale C, Montaldo E, Conte R, Cantoni C, Fulcheri E, Darretta V, Moretta L, Mingari MC. CD34+ hematopoietic precursors are present in human decidua and differentiate into natural killer cells upon interaction with stromal cells. Proc Natl Acad Sci USA 2011;108:24022407.
  • 47
    Chinen H, Matsuoka K, Sato T, Kamada N, Okamoto S, Hisamatsu T, Kobayashi T, Hasegawa H, Sugita A, Kinjo F, et al. Lamina propria c-kit+ immune precursors reside in human adult intestine and differentiate into natural killer cells. Gastroenterology 2007;133:559573.
  • 48
    Bennett IM, Zatsepina O, Zamai L, Azzoni L, Mikheeva T, Perussia B. Definition of a natural killer NKR-P1A+/CD56-/CD16- functionally immature human NK cell subset that differentiates in vitro in the presence of interleukin 12. J Exp Med 1996;184:18451856.
  • 49
    Montaldo E, Vitale C, Cottalasso F, Conte R, Glatzer T, Ambrosini P, Moretta L, Mingari MC. Human NK cells at early stages of differentiation produce CXCL8 and express CD161 molecule that functions as an activating receptor. Blood 2012;119:39873996.
  • 50
    Fuchs A, Cella M, Kondo T, Colonna M. Paradoxic inhibition of human natural interferon-producing cells by the activating receptor NKp44. Blood 2005;106:20762082.
  • 51
    Swiecki M, Colonna M. Unraveling the functions of plasmacytoid dendritic cells during viral infections, autoimmunity, and tolerance. Immunol Rev 2010;234:142162.
  • 52
    Zamai L, Del Zotto G, Buccella F, Galeotti L, Canonico B, Luchetti F, Papa S. Cytotoxic functions and susceptibility to apoptosis of human CD56(bright) NK cells differentiated in vitro from CD34(+) hematopoietic progenitors. Cytometry A 2012;81A:294302.
  • 53
    Zamai L, Galeotti L, Del Zotto G, Canonico B, Mirandola P, Papa S. Identification of a NCR+/NKG2D+/LFA-1(low)/CD94(-) immature human NK cell subset. Cytometry A 2009;75A:893901.
  • 54
    Sivori S, Falco M, Marcenaro E, Parolini S, Biassoni R, Bottino C, Moretta L, Moretta A. Early expression of triggering receptors and regulatory role of 2B4 in human natural killer cell precursors undergoing in vitro differentiation. Proc Natl Acad Sci USA 2002;99:45264531.
  • 55
    Zamai L, Ahmad M, Bennett IM, Azzoni L, Alnemri ES, Perussia B. Natural killer (NK) cell-mediated cytotoxicity: Differential use of TRAIL and Fas ligand by immature and mature primary human NK cells. J Exp Med 1998;188:23752380.
  • 56
    Loza MJ, Zamai L, Azzoni L, Rosati E, Perussia B. Expression of type 1 (interferon gamma) and type 2 (interleukin-13, interleukin-5) cytokines at distinct stages of natural killer cell differentiation from progenitor cells. Blood 2002;99:12731281.
  • 57
    Spits H, Artis D, Colonna M, Diefenbach A, Di Santo JP, Eberl G, Koyasu S, Locksley RM, McKenzie AN, Mebius RE, et al. Innate lymphoid cells—A proposal for uniform nomenclature. Nat Rev Immunol 2013;13:145149.
  • 58
    Andre P, Spertini O, Guia S, Rihet P, Dignat-George F, Brailly H, Sampol J, Anderson PJ, Vivier E. Modification of P-selectin glycoprotein ligand-1 with a natural killer cell-restricted sulfated lactosamine creates an alternate ligand for L-selectin. Proc Natl Acad Sci USA 2000;97:34003405.
  • 59
    Della Chiesa M, Falco M, Podesta M, Locatelli F, Moretta L, Frassoni F, Moretta A. Phenotypic and functional heterogeneity of human NK cells developing after umbilical cord blood transplantation: A role for human cytomegalovirus?.Blood 2012;119:399410.
  • 60
    Fehniger TA, Cooper MA, Nuovo GJ, Cella M, Facchetti F, Colonna M, Caligiuri MA. 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;101:30523057.
  • 61
    Ferlazzo G, Thomas D, Lin SL, Goodman K, Morandi B, Muller WA, Moretta A, Munz C. The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic. J Immunol 2004;172:14551462.
  • 62
    Romagnani C, Juelke K, Falco M, Morandi B, D'Agostino A, Costa R, Ratto G, Forte G, Carrega P, Lui G, et al. CD56brightCD16- killer Ig-like receptor- NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation. J Immunol 2007;178:49474955.
  • 63
    Huntington ND, Legrand N, Alves NL, Jaron B, Weijer K, Plet A, Corcuff E, Mortier E, Jacques Y, Spits H, et al. IL-15 trans-presentation promotes human NK cell development and differentiation in vivo. J Exp Med 2009;206:2534.
  • 64
    Juelke K, Killig M, Luetke-Eversloh M, Parente E, Gruen J, Morandi B, Ferlazzo G, Thiel A, Schmitt-Knosalla I, Romagnani C. CD62L expression identifies a unique subset of polyfunctional CD56dim NK cells. Blood 2010;116:12991307.
  • 65
    Vossen MT, Matmati M, Hertoghs KM, Baars PA, Gent MR, Leclercq G, Hamann J, Kuijpers TW, van Lier RA. CD27 defines phenotypically and functionally different human NK cell subsets. JImmunol 2008;180:37393745.
  • 66
    Hamann I, Unterwalder N, Cardona AE, Meisel C, Zipp F, Ransohoff RM, Infante-Duarte C. Analyses of phenotypic and functional characteristics of CX3CR1-expressing natural killer cells. Immunology 2011;133:6273.
  • 67
    Bjorkstrom NK, Riese P, Heuts F, Andersson S, Fauriat C, Ivarsson MA, Bjorklund AT, Flodstrom-Tullberg M, Michaelsson J, Rottenberg ME, et al. Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education. Blood 2010;116:38533864.
  • 68
    Parolini S, Santoro A, Marcenaro E, Luini W, Massardi L, Facchetti F, Communi D, Parmentier M, Majorana A, Sironi M, et al. The role of chemerin in the colocalization of NK and dendritic cell subsets into inflamed tissues. Blood 2007;109:36253632.
  • 69
    Fauriat C, Long EO, Ljunggren HG, Bryceson YT. Regulation of human NK-cell cytokine and chemokine production by target cell recognition. Blood 2010;115:21672176.
  • 70
    De Maria A, Bozzano F, Cantoni C, Moretta L. Revisiting human natural killer cell subset function revealed cytolytic CD56(dim)CD16+ NK cells as rapid producers of abundant IFN-gamma on activation. Proc Natl Acad Sci USA 2011;108:728732.
  • 71
    Pende D, Marcenaro S, Falco M, Martini S, Bernardo ME, Montagna D, Romeo E, Cognet C, Martinetti M, Maccario R, et al. Anti-leukemia activity of alloreactive NK cells in KIR ligand-mismatched haploidentical HSCT for pediatric patients: Evaluation of the functional role of activating KIR and redefinition of inhibitory KIR specificity. Blood 2009;113:31193129.
  • 72
    Moretta A, Pende D, Locatelli F, Moretta L. Activating and inhibitory killer immunoglobulin-like receptors (KIR) in haploidentical haemopoietic stem cell transplantation to cure high-risk leukaemias. Clin Exp Immunol 2009;157:325331.
  • 73
    Moretta L, Locatelli F, Pende D, Marcenaro E, Mingari MC, Moretta A. Killer Ig-like receptor-mediated control of natural killer cell alloreactivity in haploidentical hematopoietic stem cell transplantation. Blood 2011;117:764771.
  • 74
    Costello RT, Sivori S, Marcenaro E, Lafage-Pochitaloff M, Mozziconacci MJ, Reviron D, Gastaut JA, Pende D, Olive D, Moretta A. Defective expression and function of natural killer cell-triggering receptors in patients with acute myeloid leukemia. Blood 2002;99:36613667.
  • 75
    Fauriat C, Just-Landi S, Mallet F, Arnoulet C, Sainty D, Olive D, Costello RT. Deficient expression of NCR in NK cells from acute myeloid leukemia: Evolution during leukemia treatment and impact of leukemia cells in NCRdull phenotype induction. Blood 2007;109:323330.
  • 76
    Fauriat C, Mallet F, Olive D, Costello RT. Impaired activating receptor expression pattern in natural killer cells from patients with multiple myeloma. Leukemia 2006;20:732733.
  • 77
    von Lilienfeld-Toal M, Frank S, Leyendecker C, Feyler S, Jarmin S, Morgan R, Glasmacher A, Marten A, Schmidt-Wolf IG, Brossart P, et al. Reduced immune effector cell NKG2D expression and increased levels of soluble NKG2D ligands in multiple myeloma may not be causally linked. Cancer Immunol Immunother 2010;59:829839.
  • 78
    Konjevic G, Mirjacic Martinovic K, Vuletic A, Jovic V, Jurisic V, Babovic N, Spuzic I. Low expression of CD161 and NKG2D activating NK receptor is associated with impaired NK cell cytotoxicity in metastatic melanoma patients. Clin Exp Metastasis 2007;24:111.
  • 79
    Pietra G, Manzini C, Rivara S, Vitale M, Cantoni C, Petretto A, Balsamo M, Conte R, Benelli R, Minghelli S, et al. Melanoma cells inhibit natural killer cell function by modulating the expression of activating receptors and cytolytic activity. Cancer Res 2012;72:14071415.
  • 80
    Crane CA, Han SJ, Barry JJ, Ahn BJ, Lanier LL, Parsa AT. TGF-beta downregulates the activating receptor NKG2D on NK cells and CD8+ T cells in glioma patients. Neuro Oncol 2010;12:713.
  • 81
    Mamessier E, Sylvain A, Bertucci F, Castellano R, Finetti P, Houvenaeghel G, Charaffe-Jaufret E, Birnbaum D, Moretta A, Olive D. Human breast tumor cells induce self-tolerance mechanisms to avoid NKG2D-mediated and DNAM-mediated NK cell recognition. Cancer Res 2011;71:66216632.
  • 82
    Carlsten M, Norell H, Bryceson YT, Poschke I, Schedvins K, Ljunggren HG, Kiessling R, Malmberg KJ. Primary human tumor cells expressing CD155 impair tumor targeting by down-regulating DNAM-1 on NK cells. J Immunol 2009;183:49214930.
  • 83
    Menard C, Blay JY, Borg C, Michiels S, Ghiringhelli F, Robert C, Nonn C, Chaput N, Taieb J, Delahaye NF, et al. Natural killer cell IFN-gamma levels predict long-term survival with imatinib mesylate therapy in gastrointestinal stromal tumor-bearing patients. Cancer Res 2009;69:35633569.
  • 84
    Delahaye NF, Rusakiewicz S, Martins I, Menard C, Roux S, Lyonnet L, Paul P, Sarabi M, Chaput N, Semeraro M, et al. Alternatively spliced NKp30 isoforms affect the prognosis of gastrointestinal stromal tumors. Nat Med 2011;17:700707.
  • 85
    Balsamo M, Pietra G, Vermi W, Moretta L, Mingari MC, Vitale M. Melanoma immunoediting by NK cells. Oncoimmunology 2012;1:16071609.
  • 86
    Balsamo M, Scordamaglia F, Pietra G, Manzini C, Cantoni C, Boitano M, Queirolo P, Vermi W, Facchetti F, Moretta A, et al. Melanoma-associated fibroblasts modulate NK cell phenotype and antitumor cytotoxicity. Proc Natl Acad Sci USA 2009;106:2084720852.
  • 87
    De Maria A, Fogli M, Costa P, Murdaca G, Puppo F, Mavilio D, Moretta A, Moretta L. The impaired NK cell cytolytic function in viremic HIV-1 infection is associated with a reduced surface expression of natural cytotoxicity receptors (NKp46, NKp30 and NKp44). Eur J Immunol 2003;33:24102418.
  • 88
    Mavilio D, Benjamin J, Daucher M, Lombardo G, Kottilil S, Planta MA, Marcenaro E, Bottino C, Moretta L, Moretta A, et al. Natural killer cells in HIV-1 infection: Dichotomous effects of viremia on inhibitory and activating receptors and their functional correlates. Proc Natl Acad Sci USA 2003;100:1501115016.
  • 89
    Fogli M, Costa P, Murdaca G, Setti M, Mingari MC, Moretta L, Moretta A, De Maria A. Significant NK cell activation associated with decreased cytolytic function in peripheral blood of HIV-1-infected patients. Eur J Immunol 2004;34:23132321.
  • 90
    Lichtfuss GF, Cheng WJ, Farsakoglu Y, Paukovics G, Rajasuriar R, Velayudham P, Kramski M, Hearps AC, Cameron PU, Lewin SR, et al. Virologically suppressed HIV patients show activation of NK cells and persistent innate immune activation. J Immunol 2012;189:14911499.
  • 91
    Mavilio D, Lombardo G, Benjamin J, Kim D, Follman D, Marcenaro E, O'Shea MA, Kinter A, Kovacs C, Moretta A, et al. Characterization of CD56-/CD16+ natural killer (NK) cells: A highly dysfunctional NK subset expanded in HIV-infected viremic individuals. Proc Natl Acad Sci USA 2005;102:28862891.
  • 92
    Brunetta E, Fogli M, Varchetta S, Bozzo L, Hudspeth KL, Marcenaro E, Moretta A, Mavilio D. The decreased expression of Siglec-7 represents an early marker of dysfunctional natural killer-cell subsets associated with high levels of HIV-1 viremia. Blood 2009;114:38223830.
  • 93
    Bjorkstrom NK, Lindgren T, Stoltz M, Fauriat C, Braun M, Evander M, Michaelsson J, Malmberg KJ, Klingstrom J, Ahlm C, et al. Rapid expansion and long-term persistence of elevated NK cell numbers in humans infected with hantavirus. J Exp Med 2011;208:1321.
  • 94
    Petitdemange C, Becquart P, Wauquier N, Beziat V, Debre P, Leroy EM, Vieillard V. Unconventional repertoire profile is imprinted during acute chikungunya infection for natural killer cells polarization toward cytotoxicity. PLoS Pathog 2011;7:e1002268.
  • 95
    Beziat V, Dalgard O, Asselah T, Halfon P, Bedossa P, Boudifa A, Hervier B, Theodorou I, Martinot M, Debre P, et al. CMV drives clonal expansion of NKG2C+ NK cells expressing self-specific KIRs in chronic hepatitis patients. Eur J Immunol 2012;42:447457.
  • 96
    Guma M, Angulo A, Vilches C, Gomez-Lozano N, Malats N, Lopez-Botet M. Imprint of human cytomegalovirus infection on the NK cell receptor repertoire. Blood 2004;104:36643671.
  • 97
    Beziat V, Liu L, Malmberg JA, Ivarsson MA, Sohlberg E, Bjorklund AT, Retiere C, Sverremark-Ekstrom E, Traherne J, Ljungman P, et al. NK cell responses to cytomegalovirus infection lead to stable imprints in the human KIR repertoire and involve activating KIRs. Blood 2013;121:26782688.
  • 98
    Guma M, Cabrera C, Erkizia I, Bofill M, Clotet B, Ruiz L, Lopez-Botet M. Human cytomegalovirus infection is associated with increased proportions of NK cells that express the CD94/NKG2C receptor in aviremic HIV-1-positive patients. J Infect Dis 2006;194:3841.
  • 99
    Brunetta E, Fogli M, Varchetta S, Bozzo L, Hudspeth KL, Marcenaro E, Moretta A, Mavilio D. Chronic HIV-1 viremia reverses NKG2A/NKG2C ratio on natural killer cells in patients with human cytomegalovirus co-infection. AIDS 2010;24:2734.
  • 100
    Kuijpers TW, Baars PA, Dantin C, van den Burg M, van Lier RA, Roosnek E. Human NK cells can control CMV infection in the absence of T cells. Blood 2008;112:914915.
  • 101
    Farnault L, Chambost H, Michel G, Thuret I, de Saint Basile G, Fischer A, Picard C, Orlanducci F, Farnarier C, Moretta A, et al. Persistence of natural killer cells with expansion of a hypofunctional CD56(-)CD16(+) KIR(+)NKG2C(+) subset in a patient with atypical Janus kinase 3-deficient severe combined immunodeficiency. J Allergy Clin Immunol 2013;131:12301233.
  • 102
    Foley B, Cooley S, Verneris MR, Pitt M, Curtsinger J, Luo X, Lopez-Verges S, Lanier LL, Weisdorf D, Miller JS. Cytomegalovirus reactivation after allogeneic transplantation promotes a lasting increase in educated NKG2C+ natural killer cells with potent function. Blood 2012;119:26652674.
  • 103
    Lopez-Verges S, Milush JM, Schwartz BS, Pando MJ, Jarjoura J, York VA, Houchins JP, Miller S, Kang SM, Norris PJ, et al. Expansion of a unique CD57(+)NKG2Chi natural killer cell subset during acute human cytomegalovirus infection. Proc Natl Acad Sci USA 2011;108:1472514732.
  • 104
    Yoon JC, Lim JB, Park JH, Lee JM. Cell-to-cell contact with hepatitis C virus-infected cells reduces functional capacity of natural killer cells. J Virol 2011;85:1255712569.
  • 105
    Amadei B, Urbani S, Cazaly A, Fisicaro P, Zerbini A, Ahmed P, Missale G, Ferrari C, Khakoo SI. Activation of natural killer cells during acute infection with hepatitis C virus. Gastroenterology 2010;138:15361545.
  • 106
    Khakoo SI, Thio CL, Martin MP, Brooks CR, Gao X, Astemborski J, Cheng J, Goedert JJ, Vlahov D, Hilgartner M, et al. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science 2004;305:872874.
  • 107
    Winter CC, Gumperz JE, Parham P, Long EO, Wagtmann N. Direct binding and functional transfer of NK cell inhibitory receptors reveal novel patterns of HLA-C allotype recognition. JImmunol 1998;161:571577.
  • 108
    Pietra G, Romagnani C, Mazzarino P, Millo E, Moretta L, Mingari MC. Comparative analysis of NK- or NK-CTL-mediated lysis of immature or mature autologous dendritic cells. Eur J Immunol 2003;33:34273432.
  • 109
    De Maria A, Fogli M, Mazza S, Basso M, Picciotto A, Costa P, Congia S, Mingari MC, Moretta L. Increased natural cytotoxicity receptor expression and relevant IL-10 production in NK cells from chronically infected viremic HCV patients. Eur J Immunol 2007;37:445455.
  • 110
    Nattermann J, Feldmann G, Ahlenstiel G, Langhans B, Sauerbruch T, Spengler U. Surface expression and cytolytic function of natural killer cell receptors is altered in chronic hepatitis C. Gut 2006;55:869877.
  • 111
    Bozzano F, Picciotto A, Costa P, Marras F, Fazio V, Hirsch I, Olive D, Moretta L, De Maria A. Activating NK cell receptor expression/function (NKp30, NKp46, DNAM-1) during chronic viraemic HCV infection is associated with the outcome of combined treatment. Eur J Immunol 2011;41:29052914.
  • 112
    Bozzano F, Costa P, Passalacqua G, Dodi F, Ravera S, Pagano G, Canonica GW, Moretta L, De Maria A. Functionally relevant decreases in activatory receptor expression on NK cells are associated with pulmonary tuberculosis in vivo and persist after successful treatment. Int Immunol 2009;21:779791.