RESUMEN
Two major receptors involved in human natural cytotoxicity, NKp46 and NKp44, have recently been identified. However, experimental evidence suggested the existence of additional such receptor(s). In this study, by the generation of monoclonal antibodies (mAbs), we identified NKp30, a novel 30-kD triggering receptor selectively expressed by all resting and activated human natural killer (NK) cells. Although mAb-mediated cross-linking of NKp30 induces strong NK cell activation, mAb-mediated masking inhibits the NK cytotoxicity against normal or tumor target cells. NKp30 cooperates with NKp46 and/or NKp44 in the induction of NK-mediated cytotoxicity against the majority of target cells, whereas it represents the major triggering receptor in the killing of certain tumors. This novel receptor is associated with CD3zeta chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells. Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion. Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.
Asunto(s)
Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Receptores Inmunológicos/análisis , Animales , Anticuerpos Monoclonales/inmunología , Células COS , Clonación Molecular , Humanos , Receptor 1 Gatillante de la Citotoxidad Natural , Receptor 2 Gatillante de la Citotoxidad Natural , ARN Mensajero/análisis , Receptores Inmunológicos/genética , Receptores Inmunológicos/fisiología , Células Tumorales CultivadasRESUMEN
NK cells selectively lyse tumor cells which do not express one or more MHC class I alleles. The ability to discriminate between self normal or tumor cells is due to the expression of MHC class I-specific killer inhibitory receptors (KIR). In the present study we analyzed melanoma cell lines which were highly susceptible to NK cell-mediated lysis in spite of the expression of a complete set of HLA class I alleles. Quantitative analysis of the HLA class I expression using allele-specific monoclonal antibodies (mAb) revealed a down-regulation of all HLA class I molecules. Treatment of melanoma cells with IFN-gamma resulted in up-regulation of all HLA class I alleles that was paralleled by the acquisition of resistance to lysis. That resistance to lysis reflected the up-regulation of HLA class I molecules was revealed by the finding that mAb-mediated masking of either KIR or their HLA class I ligands completely restored the melanoma cell lysis. These results were obtained by the use of selected NK cell clones derived either from allogeneic or autologous donors. In addition, similar results were obtained using in vitro expanded autologous NK cell populations. Our data indicate that NK cells can lyse not only melanoma cells which have lost the expression of one or more HLA class I alleles but also cells expressing a decreased amount of class I molecules.
Asunto(s)
Genes MHC Clase I , Células Asesinas Naturales/inmunología , Melanoma/genética , Melanoma/inmunología , Alelos , Anticuerpos Monoclonales , Autoinmunidad , Linfocitos B/inmunología , Línea Celular , Transformación Celular Viral , Citotoxicidad Inmunológica , Regulación hacia Abajo , Expresión Génica , Herpesvirus Humano 4 , Humanos , Interferón gamma/farmacología , Proteínas Recombinantes , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Antígenos CD/inmunología , Células Asesinas Naturales/química , Lectinas Tipo C , Glicoproteínas de Membrana/inmunología , Receptores Inmunológicos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD/química , Células COS , Homólogo de la Proteína Chromobox 5 , ADN Complementario/genética , Dimerización , Electroforesis en Gel Bidimensional , Humanos , Células Asesinas Naturales/inmunología , Sustancias Macromoleculares , Glicoproteínas de Membrana/química , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Subfamília C de Receptores Similares a Lectina de Células NK , Subfamília D de Receptores Similares a Lectina de las Células NK , Receptores Inmunológicos/química , Receptores Inmunológicos/genética , Receptores de Células Asesinas Naturales , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The lack of classical human histocompatibility leukocyte antigen (HLA) molecules in human placenta prevents the recognition and lysis by maternal T lymphocytes but poses the problem of susceptibility to natural killer (NK) cell-mediated lysis. The nonclassical HLA class I molecule HLA-G may mediate protection from NK cells. NK cells are known to express a number of HLA class I-specific inhibitory receptors. These include members of the immunoglobulin (Ig) superfamily (p58, p70, p140), characterized by a defined allele specificity, and CD94/NKG2A with a broad specificity for different HLA class I molecules. We analyzed a series of NK cell clones derived from normal peripheral blood expressing different NK receptors (NKR). Clones were analyzed for their cytolytic activity against the HLA class I-negative 221 cell line either untransfected or transfected with HLA-G (221/G) or other informative alleles, as control. All clones expressing CD94/NKG2A [as identified by the Z199 monoclonal antibody (mAb)] displayed a markedly reduced cytolytic activity against 221/G. Moreover, mAb directed to the CD94/NKG2A complex completely restored target cell lysis. Among NKG2A-negative NK clones, different functional patterns could be detected. Clones expressing inhibitory receptors belonging to the Ig superfamily lysed 221/G target cells with equal or higher efficiency than untransfected 221 cells. These data indicated that p58, p70 and p140 do not function as HLA-G-specific inhibitory NKR, and that HLA-G-specific activating NKR also exist. Further analysis indicated that in these clones (characterized by the CD94+/NKG2A- phenotype) mAb specific for CD94, but not for the other NKR, reversed the activating effect. Infrequent clones were also isolated that, in spite of the lack of CD94/NKG2A, displayed HLA-G specificity, thus suggesting the existence of a different, still unknown NKR.
Asunto(s)
Antígenos CD/metabolismo , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/inmunología , Lectinas Tipo C , Glicoproteínas de Membrana/metabolismo , Animales , Células Clonales , Citotoxicidad Inmunológica , Femenino , Antígenos HLA-G , Humanos , Ratones , Subfamília D de Receptores Similares a Lectina de las Células NK , Embarazo , Receptores Inmunológicos/metabolismo , Trofoblastos/inmunologíaRESUMEN
Human natural killer (NK) cells express inhibitory receptors that are specific for different groups of HLA-C or HLA-B alleles. The majority of these receptors belong to the immunoglobulin (Ig) superfamily and are characterized by two or three extracellular Ig-like domains. Here we describe a novel inhibitory NK receptor that is specific for a group of HLA-A alleles. The HLA-A3-specific NK cell clone DP7 has been used for mice immunization. Two mAbs, termed Q66 and Q241, bound to the immunizing clone and stained only a subset of NK cell populations or clones. Among Q66 mAb-reactive clones, we further selected those that did not express any of the previously identified HLA-class I-specific NK receptors. These clones did not lyse HLA-A3+ (or -A11+) target cells, but lysis of these targets could be detected in the presence of Q66 or Q241 mAbs. On the other hand, target cells expressing other HLA-A alleles, including -A1, -A2, and -A24, were efficiently lysed. Moreover, none of the HLA-C or HLA-B alleles that were tested exerted a protective effect. Q66+, but not Q66- NK cell clones, expressed messenger RNA coding for a novel 3 Ig domain protein homologous to the HLA-C (p58) and HLA-B (p70) receptors. The corresponding cDNA (cl.1.1) was used to generate transient and stable transfectants in COS7 and NIH3T3 cell lines, respectively. Both types of transfectants were specifically stained by Q66 and Q241 mAbs. Since the cytoplasmic tail of Q66-reactive molecules was at least 11 amino acid longer than the other known p58/p70 molecules, we could generate an antiserum specific for the COOH-terminus of Q66-reactive molecules, termed PGP-3. PGP-3 immunoprecipitated, only from Q66+ NK cells, molecules displaying a molecular mass of 140 kD, under nonreducing conditions, which resolved, under reducing conditions, in a 70-kD band. Thus, differently from the other p58/p70 receptors, Q66-reactive molecules appear to be expressed as disulphide-linked dimers and were thus termed p140. The comparative analysis of the amino acid sequences of p58, p70, and p140 molecules revealed the existence of two cysteins proximal to the transmembrane region, only in the amino acid sequence of p140 molecules.