Massimo Vitale

Characterization of an Antigen That Is Recognized on a Melanoma Showing Partial HLA Loss by CTL Expressing an NK Inhibitory Receptor

Melanoma lines MEL.A and MEL.B were derived from metastases removed from patient LB33 in 1988 and 1993, respectively. The MEL.A cells express several antigens recognized by autologous cytolytic T lymphocytes (CTL) on HLA class I molecules. The MEL.B cells have lost expression of all class I molecules except for HLA-A24. By stimulating autologous lymphocytes with MEL.B, we obtained an HLA-A24-restricted CTL clone that lysed these cells. A novel gene, PRAME, encodes the antigen. It is expressed in a large proportion of tumors and also in some normal tissues, albeit at a lower level. Surprisingly, the CTL failed to lyse MEL.A, even though these cells expressed the gene PRAME. The CTL expresses an NK inhibitory receptor that inhibits its lytic activity upon interaction with HLA-Cw7 molecules, which are present on MEL.A cells and not on MEL.B. Such CTL, active against tumor cells showing partial HLA loss, may constitute an intermediate line of anti-tumor defense between the CTL, which recognize highly specific tumor antigens, and the NK cells, which recognize HLA loss variants.

Identification of PVR (CD155) and Nectin-2 (CD112) as Cell Surface Ligands for the Human DNAM-1 (CD226) Activating Molecule

Human natural killer (NK) cells express a series of activating receptors and coreceptors that are involved in recognition and killing of target cells. In this study, in an attempt to identify the cellular ligands for such triggering surface molecules, mice were immunized with NK-susceptible target cells. On the basis of a functional screening, four mAbs were selected that induced a partial down-regulation of the NK-mediated cytotoxicity against the immunizing target cells. As revealed by biochemical analysis, three of such mAbs recognized molecules of ∼70 kD. The other mAb reacted with two distinct molecules of ∼65 and 60 kD, respectively. Protein purification followed by tryptic digestion and mass spectra analysis, allowed the identification of the 70 kD and the 65/60 kD molecules as PVR (CD155) and Nectin-2 δ/α (CD112), respectively. PVR-Fc and Nectin-2-Fc soluble hybrid molecules brightly stained COS-7 cells transfected with the DNAM-1 (CD226) construct, thus providing direct evidence that both PVR and Nectin-2 represent specific ligands for the DNAM-1 triggering receptor. Finally, the surface expression of PVR or Nectin-2 in cell transfectants resulted in DNAM-1–dependent enhancement of NK-mediated lysis of these target cells. This lysis was inhibited or even virtually abrogated upon mAb-mediated masking of DNAM-1 (on NK cells) or PVR or Nectin-2 ligands (on cell transfectants).

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.

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.

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.

Effector and regulatory events during natural killer–dendritic cell interactions

Summary:  The different cell types of the innate immune system can interact with each other and influence the quality and strength of an immune response. The cross talk between natural killer (NK) cells and myeloid dendritic cells (DCs) leads to NK cell activation and DC maturation. Activated NK cells are capable of killing DCs that fail to undergo proper maturation (‘DC editing’). Encounters between NK cells and DCs occur in both inflamed peripheral tissues and lymph nodes, where both cell types are recruited by chemokines released in the early phases of inflammatory responses. Different NK cell subsets (CD56brightCD16− versus CD56+CD16+) differ in their homing capabilities. In particular, CD56brightCD16− NK cells largely predominate the lymph nodes. In addition, these two subsets display major functional differences in their cytolytic activity, cytokine production, and ability to undergo proliferation. NK cell functions are also greatly influenced by the presence of polarizing cytokines such as interleukin (IL)‐12 and IL‐4. The cytokine microenvironment reflects the presence of different cell types that secrete such cytokines in response to microbial products acting on different Toll‐like receptors (TLRs). Moreover, NK cells themselves can respond directly to microbial products by means of TLR3 and TLR9. Thus, it appears that the final outcome of a response to microbial infection may greatly vary as a result of the interactions occurring between different pathogen‐derived products and different cell types of the innate immunity system. These interactions also determine the quality and strength of the subsequent adaptive responses. Remarkably, NK cells appear to play a key role in this complex network.

The natural killer cell-mediated killing of autologous dendritic cells is confined to a cell subset expressing CD94/NKG2A, but lacking inhibitory killer Ig-like receptors

The cognate NK–DC interaction in inflamed tissues results in NK cell activation and acquisition of cytotoxicity against immature DC (iDC). This may represent a mechanism of DC selection required for the control of downstream adaptive immune responses. Here we show that killing of monocyte‐derived iDC is confined to the NK cell subset that expresses CD94/NKG2A, but not killer Ig‐like receptors (KIR). Consistent with these data, the expression of HLA‐E (i.e. the cellular ligand of CD94/NKG2A) was down‐regulated in iDC. On the other hand, HLA‐B and HLA‐C down‐regulation in iDC was not sufficient to induce cytotoxicity in NK cells expressing KIR3DL1 or KIR2DL. Remarkably, CD94/NKG2A+KIR– NK cells were heterogeneous in their ability to kill iDC and an inverse correlation existed between their CD94/NKG2A surface density and the magnitude of their cytolytic activity. It is conceivable that the reduced CD94/NKG2A surface density enables these cells to efficiently sense the decrease of HLA‐E surface expression in iDC. Finally, most NK cells that lysed iDC did not kill mature DC that express higher amounts of HLA class I molecules (including HLA‐E)as compared with iDC. However, a small NK cell subset was capable of killing not only iDC but also mature DC.

The small subset of CD56brightCD16- natural killer cells is selectively responsible for both cell proliferation and interferon-γ production upon interaction with dendritic cells

The encounter of NK cells with dendritic cells (DC) undergoing maturation may result in the induction of NK cell proliferation. Whether such proliferation involves most NK cells or just a subset has yet to be determined. In the present study we analyzed the nature of such proliferating NK cells by combining carboxyfluorescein succinimidyl ester staining and double‐fluorescence cytofluorimetric analysis. Freshly isolated peripheral blood NK cells cultured with LPS and immature DC underwent proliferation; however, proliferating cells were confined to a minor NK cell subset. This subset is characterized by the CD56brightCD16–NKG2A+KIR– surface phenotype (KIR, killer Ig‐like receptor). This was further confirmed by the fact that, after cell sorting, only the CD56bright NK cells were able to proliferate in response to the DC stimulus, whereas the CD56dull were not. We also provide evidence that the CD56bright subset is the main source of IFN‐γ‐producing NK cells, upon interaction with DC. The CD56brightCD16– NK cells express a panel of surface molecules including CD62L, CCR7 and CXCR3 that may allow their homing either to secondary lymphoid compartments or to inflamed tissues. This implies that, in vivo, the interactions between DC undergoing maturation and CD56bright NK cells may occur in different tissues and have different functional implications.