Roberto Biassoni

Molecular clones of the p58 NK cell receptor reveal immunoglobulin-related molecules with diversity in both the extra- and intracellular domains

Recognition of major histocompatibility class I molecules on target cells by natural killer (NK) cells confers selective protection from NK-mediated lysis. Cross-linking of the p58 NK receptor, involved in the recognition of HLA-C alleles, delivers a negative signal that prevents target cell lysis. Molecular cloning of the p58 NK receptor reported here revealed a new member of the immunoglobulin superfamily. Five distinct p58 receptors, with sequence diversity in the immunoglobulin-related domains, were identified in a single individual. All NK clones tested expressed at least one p58 member. Three different types of transmembrane and cytoplasmic domains exist, even among receptors with closely related extracellular domains. These data revealed a repertoire of NK cells with clonally distributed p58 receptors exhibiting diversity in both extracellular and intracellular domains.

Transforming growth factor β1 inhibits expression of NKp30 and NKG2D receptors: Consequences for the NK-mediated killing of dendritic cells

The surface density of the triggering receptors responsible for the natural killer (NK)-mediated cytotoxicity is crucial for the ability of NK cells to kill susceptible target cells. In this study, we show that transforming growth factor β1 (TGFβ1) down-regulates the surface expression of NKp30 and in part of NKG2D but not that of other triggering receptors such as NKp46. The TGFβ1-mediated inhibition of NKp30 surface expression reflects gene regulation at the transcriptional level. NKp30 has been shown to represent the major receptor involved in the NK-mediated killing of dendritic cells. Accordingly, the TGFβ1-dependent down-regulation of NKp30 expression profoundly inhibited the NK-mediated killing of dendritic cells. On the contrary, killing of different NK-susceptible tumor cell lines was variably affected, reflecting the differential usage of NKp30 and/or NKG2D in the lysis of such tumors. Our present data suggest a possible mechanism by which TGFβ1-producing dendritic cells may acquire resistance to the NK-mediated attack.

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.

Major histocompatibility complex class I-specific receptors on human natural killer and T lymphocytes

Summary: Human NK cells express several specialized inhibitory receptors that recognize major histocompatibility complex (MHC) class I molecules expressed on normal cells. The lack of expression of one or more HLA CLASS I alleles leads to NK‐mediated target cell lysis. Receptors specific for groups of HLA‐C (p58), HLA‐B (p70) and HLA‐A (p140) alleles belong to the Ig superfamily with two or three Ig‐like domains in their extracellular portion, and a long cytoplasmic tail containing ITIM motifs and associated with a non‐polar transmembrane portion. In contrast, the CD94/NKG2‐A receptor complex is composed of type II proteins with a C‐type lectin domain which displays a more broad specificity for different class I alleles. Recently, activatory forms of the HLA‐C‐specific receptors have been identified in some donors. They are virtually identical to the inhibitory forms in their extracellular portions, but display a short cytoplasmic tail lacking ITIM motifs associated with a Lys‐containing transmembrane portion (p50). A subset of activated T‐lymphocytes. primarily CD8+ and oligoclonal or monoclonal in nature, express NK‐type class I‐specific receptors. These receptors exert an inhibitory activity on T‐cell receptor‐mediated functions and may provide an important mechanism of down‐regulation of T‐cell responses.

Role of NKG2D in tumor cell lysis mediated by human NK cells: cooperation with natural cytotoxicity receptors and capability of recognizing tumors of nonepithelial origin

NKG2D is a recently described activating receptor expressed by both NK cells and CTL. In this study we investigated the role of NKG2D in the natural cytolysis mediated by NK cell clones. The role of NKG2D varied depending on the type of target cells analyzed. Lysis of various tumors appeared to be exclusively natural cytotoxicity receptors (NCR) dependent. In contrast, killing of anothergroup of target cells, including not only the epithelial cell lines HELA and IGROV‐1, but also the FO‐1 melanoma, the JA3 leukemia, the Daudi Burkitt lymphoma and even normal PHA‐induced lymphoblasts, involved both NCR and NKG2D. Notably, NK cell clones expressing low surface densities of NCR (NCRdull) could lyse these tumors in an exclusively NKG2D‐dependent fashion. Remarkably, notall of these targets expressed MICA/B, thus implying the existence of additional ligands recognized by NKG2D, possibly represented by GPI‐linked molecules. Finally, we show that the engagement of different HLA class I‐specific inhibitory receptors by either specific antibodies or the appropriate HLA class I ligand led to inhibition of NKG2D‐mediated NK cell triggering.

Natural cytotoxicity receptors that trigger human NK-cell-mediated cytolysis

Natural killer (NK) cells can detect whether cells have undergone tumour transformation or viral infection. The discovery of specific inhibitory receptors for major histocompatibility complex class I molecules clarified the basis of this discrimination. However, the receptors responsible for NK-cell triggering in the process of natural cytotoxicity remained elusive until recently. Here, Alessandro Moretta and colleagues describe the identification and characterization of several such receptors.

What is a natural killer cell

Natural killer cells have been arbitrarily defined using a number of different phenotypic and functional criteria. We asked Lorenzo Moretta if we have truly discovered the core identity of this critical player in eliciting immune responses.

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.

NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo

NK cells use a variety of receptors to detect abnormal cells, including tumors and their metastases. However, in the case of melanoma, it remains to be determined what specific molecular interactions are involved and whether NK cells control metastatic progression and/or the route of dissemination. Here we show that human melanoma cell lines derived from LN metastases express ligands for natural cytotoxicity receptors (NCRs) and DNAX accessory molecule-1 (DNAM-1), two emerging NK cell receptors key for cancer cell recognition, but not NK group 2 member D (NKG2D). Compared with cell lines derived from metastases taken from other anatomical sites, LN metastases were more susceptible to NK cell lysis and preferentially targeted by adoptively transferred NK cells in a xenogeneic model of cell therapy. In mice, DNAM-1 and NCR ligands were also found on spontaneous melanomas and melanoma cell lines. Interference with DNAM-1 and NCRs by antibody blockade or genetic disruption reduced killing of melanoma cells. Taken together, these results show that DNAM-1 and NCRs are critical for NK cell-mediated innate immunity to melanoma cells and provide a background to design NK cell-based immunotherapeutic strategies against melanoma and possibly other tumors.

Human natural killer cells: Origin, clonality, specificity, and receptors

Publisher Summary This chapter focuses on human natural killer (NK) cells. NK cells are considered as effector cells that lyse tumor cells or virally infected cells via nonspecific mechanisms. Both their cytolytic effect and the spectrum of their antitumor activity are greatly increased after culture of NK cells in interleukin- 2 (IL-2). IL-2-activated NK cells represent the most potent effector cells among the lymphokine-activated killer (LAK) cells. Several of the generally accepted theories concerning NK cells have been challenged by evidence based on a variety of new experimental approaches and technological breakthroughs. NK cells may originate from precursors present in early fetal liver and even in immature postnatal thymocytes. They specifically recognize class I molecules. Different from T cells, binding of NK cells to class I molecules appears to result in a “negative signal” leading to target cell protection. Many tumor cells that are susceptible to lysis mediated by NK/LAK cells express normal amounts of surface class I antigens. This phenomenon is explained in that (1) the tumor cells express class I antigens that do not function as specific protective elements for the allogeneic NK cells most frequently used in the cytolytic assays and (2) the tumor cells may lack the expression of single class I alleles functioning as protective elements. The ability of activated NK cells to lyse efficiently a wider spectrum of tumor cells can be interpreted as the expression of appropriate activation receptors on these cells as a consequence of cell activation.