NK cell function is finely regulated by a series of inhibitory or activating receptors. The inhibitory receptors, specific for major histocompatibility complex (MHC) class I molecules, allow NK cells to discriminate between normal cells and cells that have lost the expression of MHC class I (e.g., tumor cells). In the absence of sufficient signaling by their HLA class I-specific inhibitory receptors, human natural killer (NK) cells become activated and display potent cytotoxicity against cells that are HLA class I negative. This indicates that the NK receptors responsible for the induction of cytotoxicity recognize ligands on target cells different from HLA class I molecules. These receptors have been termed natural cytotoxicity receptors (NCR) and include NKp46, together with NKp44 and NKp30 (Bottino et al. 2000). A direct correlation exists between the surface density of NCR and the ability of NK cells to kill various target cells (Sivori et al. 1999). Importantly, mAb-mediated blocking of these receptors has been shown to suppress cytotoxicity against most NK-susceptible target cells. However, the process of NK-cell triggering during target cell lysis may also depend on the concerted action of NCR and other triggering receptors, such as NKG2D, or surface molecules, including 2B4 and NKp80, that appear to function as co-receptors rather than as true receptors.
Distribution and Function of NKp46 on NK Cells
NKp46 is a surface molecule of 46 kDa expressed by all human NK cells (including the CD56bright CD16− subset) irrespective of their state of activation. It is mostly confined to NK cells and its engagement induces a strong activation of NK-mediated cytolysis, Ca++ mobilization, and cytokine release (Moretta et al. 2001; Sivori et al. 1997).
In healthy donors, within the CD56dim NK cells, two distinct NK cell subsets (termed NCRdull and NCRbright NK cell subsets) can be often distinguished on the basis of the different surface densities of NKp46 and NKp30 molecules. The NCR surface density directly correlates with the magnitude of the NK-mediated natural cytotoxicity and provides a rational explanation for the clonal heterogeneity of NK cells in killing target cells (Sivori et al. 1999). In particular, in healthy donors, most of the terminally differentiated CD56dim NK cells (characterized by the CD56dimKIR+NKG2A−CD57+ phenotype) display a lower surface expression of NCRs (Bjorkstrom et al. 2010; Lopez-Verges et al. 2010).
On the other hand, CD56bright NK cells are characterized by higher NKp46 surface expression as compared to CD56dim NK cells.
More recently, in HCMV+ individuals a further small reduction of the NKp46 and NKp30 expression has been described in memory-like NK cells that are characterized by a highly differentiated surface phenotype, CD56dimCD16brightLIR-1+KIR+NKG2A−NKG2C+CD57+, and by acquisition of some hallmarks of adaptive immunity, that is, clonal expansion, enhanced effector function (i.e., a functional enhancement in terms of ADCC and IFN-γ production), longevity, as well as given epigenetic modifications (Muntasell et al. 2013; Rölle and Brodin 2016).
More recently, in HCMV+ individuals, a novel NK cell subset has been identified within the fully mature NK cells. This NK cell subset is characterized by the expression of the inhibitory PD-1 receptor and marked downregulation of NKp30 and NKp46. Indeed, the NCRs expression of PD-1+ NK cells is maximally reduced as compared to that of PD-1− NK cells contained within the highly differentiated KIR+NKG2A−CD57+ NK cell subset (Pesce et al. 2016).
Molecular/Biochemical Characterization of NKp46
NKp46 is encoded by the NCR1 gene, which maps in the telomeric region of “leukocyte receptor complex” (LRC) on human chromosome 19q13.42 (Kelley et al. 2005; Pessino et al. 1998). The NCR1 gene is conserved during speciation, at least in rodents, bovine, and primates (Biassoni 2008).
The crystal structure of NKp46 indicates a certain degree of structural conservation with KIR2DL2, CD89, glycoprotein VI platelet collagen receptor, LILRB1 (LIR-1/ILT2/CD85j), and LILRB2 (LIR2/ILT4/CD85d) molecules, which are encoded by genes that map close to each other on chromosome 19q13.42 (Ponassi et al. 2003).
The identification of the NKp46 ligand(s) on cancer cells is still being investigated. At this regard, it has been suggested that membrane-associated heparan sulfate proteoglycans (HSPGs) might be involved in the recognition of tumor cells by NKp46 and NKp30 (Bloushtain et al. 2004). These results have been not confirmed by other studies, at least for NKp30-mediated killing (Warren et al. 2005). Moreover, since heparan sulfate is also found on normal cells that are not killed by NK cells, it is likely that it does not represent a specific ligand of NKp46 or NKp30. Indeed, heparan sulfate is already known as a co-ligand for different growth factors, chemokines, lipid-binding proteins, and adhesion proteins (Capila and Linhardt 2002).
NKp46 is also involved in the killing of virus-infected cells, and it has been described to recognize the hemagglutinin of influenza virus (IV-HA) and the hemagglutinin-neuraminidase of Sendai virus (SV-HN) (Arnon et al. 2004; Gazit et al. 2006; Mandelboim et al. 2001). This recognition seems to depend on the sialylation of NKp46 receptor, in particular on α2,6-linked sialic acid carried by Thr-225 residue (located into the membrane proximal domain of NKp46) (Arnon et al. 2004). The Thr-225 of NKp46 plays a critical role in interaction not only with viral hemagglutinins, but also with unknown tumor cellular ligands, probably via different mechanisms not involving its sialylation (Arnon et al. 2004).
Other authors have suggested that vimentin, expressed on the surface of Mycobacterium tuberculosis–infected monocyte cell lines, mediates NKp46 binding to these cells, and contributes to their lysis (Garg et al. 2006).
It has been suggested that the positively charged amino acids K133, R136, H139, R142, and K146 present in the membrane proximal domain may be involved in ligand interaction. Indeed, mutagenesis of these amino acids on NKp46 to noncharged amino acids decreases the affinity of interaction with tumor cells at least 10–100 times, without affecting the interaction with viral ligands (Zilka et al. 2005).
NKp46 on T Cells
Although the cell-surface expression of NKp46 is the most selective marker for human and mouse NK cells described so far, there are non-NK populations that also express NKp46, including discrete subsets of γδ T cells in mice and subsets of intraepithelial lymphocytes in patients with celiac disease. In various chronic infectious and inflammatory diseases (such as celiac disease or CMV infection), human CTLs can aberrantly express NKp46 as well as other activating NK receptors, such as NKG2C or NKp44. These activating NK receptors, thanks to their association with ITAM-bearing molecules (DAP12, CD3ζ, or FcεRIγ), could interfere with normal T cell tolerance mechanisms by both inducing the expansion and unleashing the effector function of CTL, independently of TCR signaling (Meresse et al. 2006).
NKp46 surface expression has been shown also on transformed Sezary T cells. Surprisingly, in these cells, mAb-mediated engagement of NKp46 (that in NK cells functions as a triggering receptor) has been shown to result in inhibition of the CD3-mediated activation process (Bensussan et al. 2011).
NKp46 and ILCs
NK cells are one of the components of a broad family of innate lymphoid cells (ILCs). Different from T-cells and B-cells, ILCs are a group of lymphocytes lacking recombination activating gene (RAG)-dependent rearranged antigen receptors (Diefenbach et al. 2014). ILCs represent a heterogeneous family of cells characterized by distinct patterns of cytokine production and lineage-specific transcription factors (Vacca et al. 2016). Cytotoxic-ILC population is represented by NK cells (Seillet et al. 2016), whereas helper-ILC populations are constituted by ILC1, ILC2, and ILC3 (Diefenbach et al. 2014).
ILC3 and NK cells share some phenotypic characteristics, primarily in term of NKp46 or other NCRs (Vacca et al. 2016). In particular, inside ILC3 cells, which are characterized by the expression of the transcription factor RORγt, both NCR− and NCR+ subsets can be distinguished. Indeed, in mice a subset of CCR6− ILC3 cells is characterized by the expression of NKp46 and in humans a consistent fraction of ILC3 cells derived from tonsils and gut lamina propria expresses NKp44, NKp46, and NKp30, although to lower levels compared to human NK cells (Killig et al. 2014). Human NCR+ ILC3 cells homogenously express CCR6 (Cella et al. 2009) and are mainly located in tonsils and intestinal lamina propria, where they contribute to epithelial homoeostasis and host defense against extracellular pathogens (Montaldo et al. 2014). When human NCR+ ILC3 were identificated, they were named NK22, because of their expression of NCRs and production of IL22 (Satoh-Takayama et al. 2008; Cella et al. 2009; Vivier et al. 2009), but more recently, according to the ILC nomenclature, their name was changed to NCR+ ILC3 (Spits et al. 2013). Importantly, IL22 production by human NCR+ ILC3 cells can occur upon engagement of NKp44 (Hoorweg et al. 2012; Glatzer et al. 2013). IL22 regulates the crosstalk between epithelial cells, immune cells, and the commensal microflora, and it instructs epithelial cell functions both in the maintenance of the barrier function and in inducing antimicrobial resistance (Dudakov et al. 2015).
Differently from murine CCR6− NCR+ ILC3 cells, human NCR+ ILC3 do not express T-bet or IFN-γ ex vivo, although expression can be induced after in vitro culture (Cella et al. 2009; Glatzer et al. 2013; Killig et al. 2014).
Recently, the biology of innate producers of IL-22 in mouse conditional deletion models targeting NCR+ ILC3 but preserving B and T cells has been deeply analyzed. These data revealed the redundant role of murine NCR+ ILC3 in the control of C. rodentium infection in immunocompetent hosts and a selective role of murine NCR+ ILC3 in cecum homeostasis (Rankin et al. 2016).
NKp46 is a surface molecule of 46 kDa expressed by all human NK cells irrespective of their state of activation. It is mostly confined to NK cells and its engagement induces a strong activation of NK function. In particular, NKp46 plays an important role in the killing of virus-infected and tumor cells. The identification of the NKp46 ligand(s) on cancer cells is still being investigated. Indeed, it is important to understand whether NKp46 ligand(s) downmodulation or loss can be involved in tumor resistance to NK-mediated cytolysis. Moreover, this identification will allow designing strategies to increase tumor susceptibility to NK-mediated killing by manipulating the NKp46 ligand(s) expression at the tumor cell surface. Although NKp46 is the most selective marker for human and mouse NK cells described so far, there are non-NK populations that also express NKp46, including discrete subsets of γδ T cells in mice, human CTLs in various chronic infectious and inflammatory diseases, human transformed Sezary T cells, and mouse/human RORγt+ ILC3 cell subsets.
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