Anna Pessino

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.

The murine homologue of the human NKp46, a triggering receptor involved in the induction of natural cytotoxicity

The NKp46 molecule has been proposed to play the role of triggering receptor in the natural cytotoxicity mediated by human NK cells. In this study we have identified the gene encoding the murine NKp46 homologue that we termed MAR‐1. The MAR‐1 gene is localized on chromosome 7 that is synthenic to the human chromosome 19 where the NKp46 gene is located. MAR‐1 encodes a type I transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily that, like human NKp46, is characterized by two C2‐type Ig‐like domains, a transmembrane portion containing a positively charged residue and a cytoplasmic tail lacking the immunoreceptor tyrosine‐based activation motif (ITAM). The MAR‐1 protein is expressed on the surface of cell transfectants and displays a molecular mass of approximately 46 kDa similar to that of its human counterpart. Semiquantitative RT‐PCR analysis showed that MAR‐1, similar to the human NKp46, is selectively expressed by NK cells. The MAR‐1 protein displays 58 % identity with the human NKp46 receptor. This high homology together with the presence of a charged amino acid (Arg) in the transmembrane portion suggest that MAR‐1 may associate at the cell membrane into a multimeric complex with ITAM containing polypeptides.

p49, a putative HLA class I-specific inhibitory NK receptor belonging to the immunoglobulin superfamily

NK cells display several killer inhibitory receptors (KIR) specific for different alleles of MHC class I molecules. A family of KIR are represented by type I transmembrane proteins belonging to the immunoglobulin superfamily (Ig‐SF). Besides cDNA encoding for these KIR, additional cDNA have been identified which encode for Ig‐SF receptors with still undefined specificity. Here we analyze one of these cDNA, termed cl.15.212, which encodes a type I transmembrane protein characterized by two extracellular Ig‐like domains and a 115‐amino acid cytoplasmic tail containing a single immuno‐receptor tyrosine‐based inhibitory motif (ITIM) which is typical of KIR. cl.15.212 cDNA displays approximately 50 % sequence homology with other Ig‐SF members. Different from the other KIR, cl.15.212 mRNA is expressed by all NK cells and by a fraction of KIR+ T cell clones. cl.15.212 cDNA codes for a membrane‐bound receptor displaying an apparent molecular mass of 49 kDa, thus termed p49. To determine the specificity of the cl.15.212‐encoded receptor, we generated soluble fusion proteins consisting of the ectodomain of p49 and the Fc portion of human IgG1. Soluble molecules bound efficiently to 221 cells transfected with HLA‐G1, ‐A3, ‐B46 alleles and weakly to ‐B7 allele. On the other hand, they did not bind to 221 cells either untransfected or transfected with HLA‐A2, ‐B51, ‐Cw3 or ‐Cw4. The binding specificity of soluble p49‐Fc was confirmed by competition experiments using an anti‐HLA class I‐specific monoclonal antibody. Finally, different cDNA encoding for molecules homologous to cl.15.212 cDNA have been isolated, two of which lack the sequence encoding the transmembrane portion, thus suggesting they may encode soluble molecules.

TLR activation of tumor-associated macrophages from ovarian cancer patients triggers cytolytic activity of NK cells

We analyzed the functional outcome of the interaction between tumor‐associated macrophages (TAMs) and natural killer (NK) cells. TAMs from ascites of ovarian cancer patients displayed an alternatively activated functional phenotype (M2) characterized by a remarkably high frequency and surface density of membrane‐bound IL‐18. Upon TLR engagement, TAMs acquired a classically activated functional phenotype (M1), released immunostimulatory cytokines (IL‐12, soluble IL‐18), and efficiently triggered the cytolytic activity of NK cells. TAMs also induced the release of IFN‐γ from NK cells, which however was significantly lower compared with that induced by in vitro‐polarized M2 cells. Most tumor‐associated NK cells displayed a CD56bright, CD16neg or CD56bright, CD16dim phenotype, and very poor cytolytic activities, despite an increased expression of the activation marker CD69. They also showed downregulation of DNAM‐1, 2B4, and NTB‐A activating receptors, and an altered chemokine receptor repertoire. Importantly however, when appropriately stimulated, NK cells from the patients, including those cells isolated from ascites, efficiently killed autologous TAMs that expressed low, “nonprotective” levels of HLA class I molecules. Overall, our data show the existence of a complex tumor microenvironment in which poorly cytolytic/immature NK cells deal with immunosuppressive tumor‐educated macrophages.

Extracellular release of the 'differentiation enhancing factor', a HMG1 protein type, is an early step in murine erythroleukemia cell differentiation.

Differentiation enhancing factor (DEF) is a 29 kDa protein expressed in murine erythroleukemia (MEL) cells and active in promoting a significant increase in the rate of hexamethylenebisacetamide induced differentiation of these cells. The factor was recently shown to possess an amino acid sequence identical to that reported for one of the HMG1 proteins, designated as ‘amphoterin’ on the basis of its highly dipolar sequence. In the present study, we have expressed DEF cDNA in an E. coli strain and found that the recombinant protein has functional properties identical to those observed with native DEF. Furthermore, we demonstrate that, following MEL cell stimulation with the chemical inducer, DEF is secreted in large amounts in the extracellular medium. In fact, the N‐terminal sequence and the partial amino acid sequence of tryptic peptides from the secreted protein correspond to those of DEF isolated from the soluble fraction of resting MEL cells. These results are indicative for an extracellular localization as the site of action of DEF and suggest a novel function for proteins belonging to the HMG1 family. Finally, the early decay of DEF mRNA, in chemical induced MEL cells, support the hypothesis that the involvement of the enhancing factor occurs and is completed in the early phases of cell differentiation.

p49, A putative HLA-G1-specific inhibitory NK receptor belonging to the Immunoglobulin Superfamily

NK cells display several killer inhibitory receptors (KIRs) specific for different alleles of major histocompatibility complex (MHC) class I molecules. A family of KIRs are represented by type I transmembrane proteins belonging to the Immunoglobulin Superfamily (Ig-SF). In the present study we describe a cDNA, termed cl.15.212, that encodes for a type I transmembrane protein displaying approximately 50% sequence homology with other Ig-SF members. The protein encoded by cl.15.212 (termed p49 according to its apparent molecular weight of 49 kDa) is characterized by two extracellular Ig-like domains, a 115-amino acid cytoplasmic tail containing a single immuno-receptor tyrosine-based inhibitory motif (ITIM) typical of KIR. Different from the other KIRs, the cl.15.212 transcript is expressed by all NK cells and by a fraction of T-cell clones expressing KIR. To determine the specificity of the cl.15.212-encoded receptor, we generated a chimeric protein, formed by the ectodomain of p49 and the Fc portion of human IgG1 (p49-Fc). Soluble molecules bound efficiently to LCL721.221 (221) cells transfected with HLA-G1, -A3, -B46 alleles and weakly to the -B7 allele. On the other hand, they did not bind to 221 cells either untransfected or transfected with HLA-A2, -B51, -Cw3, or-Cw4.

Protein kinase C isoforms in murine erythroleukemia cells and their involvement in the differentiation process

In addition to α, δ and ε‐protein kinase C, murine erythroleukemia cells contain ζ‐PKC and also a c‐PKC isoform, named α1, which shows cross‐reactivity with an anti‐α‐PKC antipeptide antibody. In a C44 MEL cell clone, characterized by a high rate of differentiation, both c‐PKC forms are expressed at a level higher than that of the N23 MEL cell clone which differentiates at a low rate and contains higher levels of ε‐PKC and particularly of the δ‐PKC isozyme. In the course of MEL cell differentiation, δ‐PKC in N23 cells and α1‐PKC in C44 cells are rapidly down‐regulated and the overall process is almost completed before cell commitment. Of the other three PKC isozymes present in both clones, only α‐PKC is down‐regulated to a significant extent. It is proposed that modulation of the signal delivered by each PKC isozyme is one of the biochemical mechanisms involved in MEL cell differentiation.

Differentiation of HL60 promyelocytic cells is promoted by a 'differentiation enhancing factor' produced by erythroleukemia cells

A differentiation enhancing factor isolated from murine erythroleukemia cells is also a potent enhancer of the differentiation of HL60 human promyelocytic leukemia cells, induced by retinoic acid and by phorbol ester. This stimulating effect is the result of a large increase in the sensitivity of HL60 cells for retinoic acid and for phorbol 12‐myristate 13‐acetate (20‐fold and 40‐fold, respectively). Accelerated differentiation induced by the protein factor, and monitored by the appearance of marker enzymes, is accompanied by a large increase in the fluctuation of the levels of protein kinase C (PKC) isozymes in HL60 cells. These results provide further support for the role of this new protein factor in cell differentiation and indicate that other cell types are susceptible to its biological effect.

p49, a putative HLA classs I-specific inhibitory NK receptor belonging to the immunoglobulin super-family (Vol 28(6) 1998, pp 1980-1990)

Page 1980 Due to a printing error the affiliation given for Michele Cilli was wrongly indicated; the correct affiliation is: Istituto Scientifico Tumori and Centro Biotecnologie Avanzate, Genova, Italy