Raffaella Augugliaro

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.

Analysis of the molecular mechanism involved in 2B4-mediated NK cell activation: evidence that human 2B4 is physically and functionally associated with the linker for activation of T cells.

While 2B4 is a well‐known surface receptor involved in NK cell triggering and induction of cytotoxicity against CD48‐positive target cells, little is known about the downstream events which lead to NK cell activation. In this study we show that, in normal human NK cells, 2B4 constitutively associates with the linker for activation of T cells (LAT). Antibody‐mediated engagement of 2B4 resulted in tyrosine phosphorylation not only of 2B4 but also of the associated LAT molecules. Moreover, tyrosine phosphorylation of LAT led to the recruitment of intracytoplasmic signaling molecules including phospholipase Cγ and Grb2. These data support the concept that 2B4 may mediate NK cell triggering via a LAT‐dependent signaling pathway.

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.

Evidence that the KIR2DS5 gene codes for a surface receptor triggering natural killer cell function

In this study, after immunization with NK cells from a KIR2DS5+ donor and screening on cell transfectants expressing different members of the killer immunoglobulin‐like receptor (KIR) family, we generated a mAb, DF200, reacting with several KIR2D receptors including KIR2DL1/L2/L3, KIR2DS1/S2 and KIR2DS5. By the analysis of peripheral blood NK cells and in vitro derived NK cell clones, we have demonstrated for the first time that KIR2DS5 is expressed at the cell surface in discrete subsets of NK cells and, after DF200 mAb‐mediated engagement, can induce both cytotoxicity and cytokine release. Using co‐transfection and co‐immunoprecipitation, we found that KIR2DS5 associates with the DAP12 signaling polypeptide. Finally, soluble KIR2DS5‐Fc fusion protein does not bind to cell transfectants expressing different HLA‐C alleles, suggesting that, if KIR2DS5 does recognize HLA‐C molecules, this may only occur in the presence of certain peptides.

Melanoma cells become resistant to NK-cell-mediated killing when exposed to NK-cell numbers compatible with NK-cell infiltration in the tumor

During the past few years, a number of studies reported that different melanoma cell lines could be extensively lysed in vitro by IL‐2‐activated NK cells at appropriate effector/target ratios. Here, we show, by histological evaluation of different melanoma lesions, that NK/target‐cell ratios compatible with those allowing efficient melanoma cell killing in vitro are hardly reached at the tumor site. We then investigated the outcome of cocultures established at low NK/melanoma cell ratios. After initial NK‐mediated lysis, residual melanoma cells acquired resistance to IL‐2‐activated NK cells. This reflected primarily an increased expression, on melanoma cells, of classical and nonclassical HLA class I molecules, accompanied by a partial downregulation of NKG2D‐ligands, and was dependent on NK‐mediated IFN‐γ release. Consistently, melanoma lesions showed a higher HLA class I expression on tumor cells that were proximal to infiltrating NK cells. In long‐term cocultures, the “protective phenotype” acquired by melanoma cells was lost over time. However, this phenomenon was counteracted by downregulation of relevant activating receptors in cocultured NK cells. Analysis of different NK‐cell‐activating cytokines indicated that IL‐15 can partially overcome this novel tumor escape mechanism suggesting that IL‐15, rather than IL‐2, may be eligible for NK‐cell‐based immunotherapy.