Simona Sivori

NKp46 is the major triggering receptor involved in the natural cytotoxicity of fresh or cultured human NK cells. Correlation between surface density of NKp46 and natural cytotoxicity against autologous, allogeneic or xenogeneic target cells

NKp46 is a novel triggering receptor expressed by all human NK cells that is involved in natural cytotoxicity. In this study we show that the surface density of NKp46 may vary in different NK cells and that a precise correlation exists between the NKp46 phenotype of NK clones and their natural cytotoxicity against HLA‐class I‐unprotected allogeneic or xenogeneic cells. Thus, NKp46bright clones efficiently lysed human and murine tumor cells while NKp46dull clones were poorly cytolytic against both types of target cells. We also show that the NKp46 phenotype of NK clones correlates with their ability to lyse HLA‐class I‐unprotected autologous cells. Finally, NKp46 was found to be deeply involved in the natural cytotoxicity mediated by freshly derived NK cells. This was indicated both by the inhibition of cytolysis after monoclonal antibody‐mediated masking of NKp46 and by the correlation existing between the natural cytotoxicity of fresh NK cells derived from different donors and their NKp46 phenotype. In conclusion, these studies strongly support the concept that NKp46 plays a central role in the physiological triggering of NK cells and, as a consequence (in concert with killer inhibitory receptors), in the NK‐mediated clearance of abnormal cells expressing inadequate amounts of HLA‐class I molecules.

Human natural killer cell receptors and co‐receptors

Summary: 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 either HLA class I negative or deficient. This indicates that the NK receptors responsible for the induction of cytotoxicity recognize ligands on target cells different from HLA class I molecules. On this basis, the process of NK‐cell triggering can be considered as a mainly non‐MHC‐restricted mechanism. The recent identification of a group of NK‐specific triggering surface molecules has allowed a first series of pioneering studies on the functional/molecular characteristics of such receptors. The first three members of a receptor family that has been termed natural cytotoxicity receptors (NCR) are represented by NKp46, NKp44 and NKp30. These receptors are strictly confined to NK cells, and their engagement induces a strong activation of NK‐mediated cytolysis. A direct correlation exists between the surface density of NCR and the ability of NK cells to kill various target cells. 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. Notably, a dysfunction of 2B4 has been associated with a severe form of immunodeficiency termed X‐linked lymphoproliferative disease. Future studies will clarify whether also the altered expression and/or function of other NK‐triggering molecules may represent a possible cause of immunological disorders.

2B4 functions as a co‐receptor in human NK cell activation

Natural cytotoxicity receptors (NKp46, NKp44 and NKp30) play a predominant role in human NK cell triggering during natural cytotoxicity. Human 2B4 also induced NK cell activation in redirected killing assays using anti‐2B4 monoclonal antibodies (mAb) and murine targets. Since this effect was confined to a fraction of NK cells, this suggested a functional heterogeneity of 2B4 molecules. Here we show that activation via 2B4 in redirected killing against murine targets is strictly dependent upon the engagement of NKp46 by murine ligand (s) on target cells. Thus, NK cell clones expressing high surface density of NKp46 (NKp46bright) were triggered by anti‐2B4 mAb, whereas NKp46dull clones were not although they expressed a comparable surface density of 2B4. mAb‐mediated modulation of NKp46 molecules in NKp46bright clones had no effect on the expression of 2B4 while it rendered cells unresponsive to anti‐2B4 mAb. Finally, anti‐2B4 mAb could induce NK cell triggering in NKp46dull clones provided that suboptimal doses of anti‐NKp44 or anti‐CD16 mAb were added to the redirected killing assay. These results indicate that differences in responses do not reflect a functional heterogeneity of 2B4 but rather depend on the co‐engagement of triggering receptors.

Molecular and functional characterization of IRp60, a member of the immunoglobulin superfamily that functions as an inhibitory receptor in human NK cells.

In this study we describe the functional and molecular characterization of IRp60 (inhibitory receptor protein 60), an inhibitory receptor expressed on all human NK cells. The IRp60 molecule has been identified by the generation of three novel monoclonal antibodies (mAb). Cross‐linking of IRp60 by specific mAb strongly inhibits the spontaneous cytotoxicity of NK cells as well as the NK‐mediated cytolytic activity induced via different non‐HLA‐specific or HLA‐specific activating receptors. IRp60 is a 60‐kDa glycoprotein that, upon sodium pervanadate treatment, becomes tyrosine phosphorylated and associates with the SH2‐containing phosphatases SHP‐1 and SHP‐2. The IRp60 gene is located on human chromosome 17 and encodes a molecule belonging to the immunoglobulin (Ig) superfamily characterized by a single V‐type Ig‐like domain in the extracellular portion. The cytoplasmic tail contains three classical immunoreceptor tyrosine‐based inhibitory motifs. Southern blot analysis revealed cross‐hybridization with monkey and mouse genomic DNA, thus suggesting that IRp60 may be conserved among different species. Moreover, based on the use of different anti‐IRp60 mAb, we could identify two IRp60 allelic variants. Since IRp60 is also expressed by other cell types, including T cell subsets, monocytes and granulocytes, it may play a more general role in the negative regulation of different leukocyte populations.

The activating form of CD94 receptor complex: CD94 covalently associates with the Kp39 protein that represents the product of the NKG2-C gene

Inhibitory receptor complexes formed by CD94 and NKG2‐A (Kp43) molecules have been implicated in HLA class I recognition by human natural killer (NK) cells. Additional forms of CD94 receptors have recently been described in NK cells characterized by the lack of NKG2‐A expression. These CD94 receptors were shown to display activating functions. Immunoprecipitation with anti‐CD94 monoclonal antibodies (mAb) led to the identification, in these cells, of a 39‐kDa (Kp39) molecule that was originally believed to represent an activating isoform of the CD94 molecules. In the present study we show that the Kp39 molecule is covalently associated with CD94 and displays a protein backbone (26 kDa) similar to that of NKG2‐A (Kp43) glycoproteins. Peptide mapping analysis indicates that Kp39 and NKG2‐A glycoproteins belong to the same molecular family. A novel NKG2‐specific mAb (termed P25) has been generated that specifically reacts with both NKG2‐A and NKG2‐C molecules, but fails to recognize NKG2‐E molecules. Analysis of polyclonal and clonal NK cells shows that P25 mAb reacts with all NKG2‐A+ cells and with a fraction of CD94+ cells lacking the expression of NKG2‐A. These data indicate that NKG2‐C molecules are indeed expressed only in a subset of cells lacking the expression of NKG2‐A. The CD94‐associated Kp39 molecule can be detected only in NKG2‐A− P25+ cells, i.e. cells expressing NKG2‐C molecules. Indeed, reverse transcription‐polymerase chain reaction analysis performed on a large panel of NK clones indicates that NKG2‐A− P25+ NK clones express the NKG2‐C transcript. Notably, the cytolytic activity of these clones can be triggered by the P25 mAb in redirected killing analysis. Finally, biochemical analysis of COS7 cells cotransfected with CD94 and NKG2‐C demonstrates the identity between Kp39 and NKG2‐C molecules. Altogether, our data demonstrate that NKG2‐C molecules associate with CD94 to form an activating receptor complex in a subset of human NK cells.

Involvement of natural cytotoxicity receptors in human natural killer cell-mediated lysis of neuroblastoma and glioblastoma cell lines

The surface receptors involved in natural killer (NK) cell triggering during the process of target cell lysis have been at least in part identified. These are members of a novel family of receptors that has been termed natural cytotoxicity receptors (NCR). The first three members of this emerging group of receptors are the NKp46, NKp44 and NKp30 molecules that all belong to the immunoglobulin superfamily. Blocking of these receptors inhibits NK-mediated cytotoxicity against a wide variety of tumor target cells. In the present study, we show that these NCR are also involved in NK-mediated killing of tumor cells of neural origin. Glioblastoma and neuroblastoma target cells were efficiently killed by all NK clones analyzed since little protection from NK lysis was mediated by HLA class I molecules. Blocking of one or another NCR inhibited cytotoxicity; however, optimal inhibition was only observed when the three receptors were blocked simultaneously. A sharp difference in cytotoxicity against neural tumors was demonstrated between NCR(bright) and NCR(dull) NK clones, further supporting the notion that NCR play a critical role in the induction of cytotoxicity against tumor target cells of different histotype. Finally, our data also indicate that CD16 does not function as a triggering receptor involved in lysis of neural tumors since no difference in cytotoxicity could be substantiated between CD16(+) and CD16(-) NK clones and no correlation could be detected between the NCR(bright)/NCR(dull) phenotype and CD16 expression.

Natural killer cells expressing the KIR2DS1-activating receptor efficiently kill T-cell blasts and dendritic cells: implications in haploidentical HSCT

In allogeneic HSCT, NK-cell alloreactivity is determined by the presence in the donor of NK cells expressing inhibitory killer cell Ig-like receptors (KIRs) that recognize HLA class I allotypes present in the donor but lacking in the recipient. Dominant KIR ligands are the C1 and C2 epitopes of HLA-C. All HLA-C allotypes have either the C1 epitope, the ligand for KIR2DL2/L3, or the C2 epitope, the ligand for KIR2DL1/S1. Here, we show that, in alloreactive NK-cell responses, KIR2DS1 expression represents a remarkable advantage as it allows efficient killing of C2/C2 or C1/C2 myelomonocitic dendritic cells (DCs) and T-cell blasts. When DCs or T-cell blasts were derived from C2/C2, Bw4/Bw4 donors, the activating signals delivered by KIR2DS1 could override the inhibition generated by NKG2A or KIR2DL2/L3 expressed on the same NK-cell clone. Furthermore, substantial lysis of C2/C2, Bw4/Bw6 targets was mediated by KIR2DS1(+) NK cells coexpressing KIR3DL1. Importantly, in the case of C1/C2 targets, KIR2DS1(+) NK cells were inhibited by the coexpression of KIR2DL2/L3 but not of NKG2A. Thus, KIR2DS1 expression in HSC donors may substantially increase the size of the alloreactive NK-cell subset leading to an enhanced ability to limit GVHD and improve engrafment.

A novel KIR-associated function: Evidence that CpG DNA uptake and shuttling to early endosomes is mediated by KIR3DL2

Human natural killer (NK) cells express Toll-like receptor 9 (TLR9) transcript and, upon exposure to microbial CpG oligodeoxynucleotide (ODN), release cytokines and kill target cells. Here we show that NK cell treatment with CpG ODN results in down-modulation of KIR3DL2 inhibitory receptor from the cell surface and in its cointernalization with CpG ODN. CpG ODN-induced interferon-γ (IFN-γ) release is mostly confined to KIR3DL2(+) NK cells, thus suggesting a crucial role of KIR3DL2 in CpG ODN-mediated NK responses. Using soluble receptor molecules, we demonstrate the direct binding of KIR3DL2 to ODNs and we show that the D0 domain is involved primarily in this interaction. KIR3DL2 modulation is also induced in malignant cells of Sézary cutaneous T-cell lymphoma, a disease in which KIR3DL2 represents a typical marker of malignant T cells. Confocal microscopy analysis suggests that, in human NK cells, CpG ODN can encounter TLR9 in early endosomes after being shuttled to these sites by KIR3DL2, which functions as a CpG ODN receptor at the cell surface. This novel KIR-associated function emphasizes the antimicrobial role of NK cells in the course of infection.

Comparison of different CpG oligodeoxynucleotide classes for their capability to stimulate human NK cells.

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides (CpG ODN) mimic the immunostimulatory activity of microbial DNA via Toll‐like receptor (TLR)9. Previous studies indicated that human NK cells express functional TLR3 and TLR9, since their cytokine release and cytolytic function could be incremented by poly(I:C) or ODN A/B, respectively. We have now evaluated the capability of a novel class of CpG ODN, termed ODN C, to modulate the function of human NK cells in the presence of exogenous cytokines. We show that NK cells isolated from peripheral blood and cultured with ODN C, in the presence of either IL‐12 or IL‐8, express higher levels of CD69 as compared to those stimulated with either ODN A or ODN B. Moreover, NK cells cultured with ODN C displayed higher cytolytic activity against tumor cell lines. These effects were not confined to freshly isolated peripheral blood NK cells since polyclonal NK cell populations that had been cultured in the presence of exogenous IL‐2 for several weeks also displayed higher cytolytic activity and cytokine release after culture in the presence of ODN C. Remarkably, NK cells displaying poor responses to ODN A/B were efficiently stimulated by ODN C.

CD59 is physically and functionally associated with natural cytotoxicity receptors and activates human NK cell-mediated cytotoxicity

Triggering of cytotoxicity in human NK cells is induced by the combined engagement of several triggering receptors. These include primary receptors such as NKG2D and the natural cytotoxicity receptors (NCR) NKp30, NKp46 and NKp44, while other molecules, including 2B4, NTB‐A and NKp80, function as co‐receptors. As reported in the present study, during an attempt to identify novel NK receptors or co‐receptors, we found that CD59 functions as a co‐receptor in human NK cell activation; engagement of CD59 by specific mAb delivers triggering signals to human NK cells, resulting in enhancement of cytotoxicity. Similar to other NK co‐receptors, the triggering function of CD59, a glycosylphosphatidylinositol (GPI)‐linked protein, depends on the simultaneous engagement of primary receptors such as NCR. Accordingly, CD59‐dependent triggering was virtually restricted to NK cells expressing high surface densities of NKp46, and mAb‐mediated modulation of NKp46 resulted in markedly decreased responses to anti‐CD59 mAb. Biochemical analysis revealed that CD59 is physically associated with NKp46 and NKp30. Moreover, engagement of CD59 resulted in tyrosine phosphorylation of CD3ζ chains associated with these NCR, but not those associated with CD16. Thus, CD59‐mediated costimulation of NK cells requires direct physical interaction of this GPI‐linked protein with primary triggeringNK receptors.