RESUMEN
In humans, natural killer (NK) cell function is regulated by a series of receptors and coreceptors with either triggering or inhibitory activity. Here we describe a novel 60-kD glycoprotein, termed NTB-A, that is expressed by all human NK, T, and B lymphocytes. Monoclonal antibody (mAb)-mediated cross-linking of NTB-A results in the induction of NK-mediated cytotoxicity. Similar to 2B4 (CD244) functioning as a coreceptor in the NK cell activation, NTB-A also triggers cytolytic activity only in NK cells expressing high surface densities of natural cytotoxicity receptors. This suggests that also NTB-A may function as a coreceptor in the process of NK cell activation. Molecular cloning of the cDNA coding for NTB-A molecule revealed a novel member of the immunoglobulin superfamily belonging to the CD2 subfamily. NTB-A is characterized, in its extracellular portion, by a distal V-type and a proximal C2-type domain and by a cytoplasmic portion containing three tyrosine-based motifs. NTB-A undergoes tyrosine phosphorylation and associates with the Src homology 2 domain-containing protein (SH2D1A) as well as with SH2 domain-containing phosphatases (SHPs). Importantly, analysis of NK cells derived from patients with X-linked lymphoproliferative disease (XLP) showed that the lack of SH2D1A protein profoundly affects the function not only of 2B4 but also of NTB-A. Thus, in XLP-NK cells, NTB-A mediates inhibitory rather than activating signals. These inhibitory signals are induced by the interaction of NTB-A with still undefined ligands expressed on Epstein-Barr virus (EBV)-infected target cells. Moreover, mAb-mediated masking of NTB-A can partially revert this inhibitory effect while a maximal recovery of target cell lysis can be obtained when both 2B4 and NTB-A are simultaneously masked. Thus, the altered function of NTB-A appears to play an important role in the inability of XLP-NK cells to kill EBV-infected target cells.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/virología , Herpesvirus Humano 4/inmunología , Células Asesinas Naturales/inmunología , Trastornos Linfoproliferativos/inmunología , Glicoproteínas de Membrana/inmunología , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Citotoxicidad Inmunológica , Cartilla de ADN/genética , Humanos , Técnicas In Vitro , Trastornos Linfoproliferativos/genética , Masculino , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Ratones , Mutación , Receptores Inmunológicos/química , Receptores Inmunológicos/genética , Dominios Homologos srcRESUMEN
Natural Killer (NK) lymphocytes were initially described as potent effector cells that, unlike T lymphocytes, were able to kill targets in the absence of a priori stimulation and without specific recognition mechanisms. Over the past ten years however, it has been clearly demonstrated that NK cell function is regulated by a number of surface receptors that bind specific ligands expressed by target cells. Some of these receptors display inhibitory functions and recognize MHC class I molecules expressed by normal autologous cells that, as a consequence, are spared from indiscriminate NK-mediated killing. Other receptors are involved in NK cell activation against allogeneic cells or cells that, upon viral infection or tumor transformation, down-regulate MHC Class I expression. Altogether these data provide important advances toward the understanding of the complexity of the molecular mechanisms that regulate NK-mediated functions.
Asunto(s)
Células Asesinas Naturales/inmunología , Citotoxicidad Inmunológica , Humanos , Células Asesinas Naturales/citología , Receptores Inmunológicos/inmunologíaRESUMEN
2B4 is a surface molecule involved in activation of the natural killer (NK) cell-mediated cytotoxicity. It binds a protein termed Src homology 2 domain-containing protein (SH2D1A) or signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), which in turn has been proposed to function as a regulator of the 2B4-associated signal transduction pathway. In this study, we analyzed patients with X-linked lymphoproliferative disease (XLP), a severe inherited immunodeficiency characterized by critical mutations in the SH2D1A gene and by the inability to control Epstein-Barr virus (EBV) infections. We show that, in these patients, 2B4 not only fails to transduce triggering signals, but also mediates a sharp inhibition of the NK-mediated cytolysis. Other receptors involved in NK cell triggering, including CD16, NKp46, NKp44, and NKp30, displayed a normal functional capability. However, their activating function was inhibited upon engagement of 2B4 molecules. CD48, the natural ligand of 2B4, is highly expressed on the surface of EBV(+) B cell lines. Remarkably, NK cells from XLP patients could not kill EBV(+) B cell lines. This failure was found to be the consequence of inhibitory signals generated by the interaction between 2B4 and CD48, as the antibody-mediated disruption of the 2B4-CD48 interaction restored lysis of EBV(+) target cells lacking human histocompatibility leukocyte antigen (HLA) class I molecules. In the case of autologous or allogeneic (HLA class I(+)) EBV(+) lymphoblastoid cell lines, restoration of lysis was achieved only by the simultaneous disruption of 2B4-CD48 and NK receptor-HLA class I interactions. Molecular analysis revealed that 2B4 molecules isolated from either XLP or normal NK cells were identical. As expected, in XLP-NK cells, 2B4 did not associate with SH2D1A, whereas similar to 2B4 molecules isolated from normal NK cells, it did associate with Src homology 2 domain-containing phosphatase 1.
Asunto(s)
Antígenos CD , Herpesvirus Humano 4/inmunología , Péptidos y Proteínas de Señalización Intracelular , Células Asesinas Naturales/inmunología , Trastornos Linfoproliferativos/inmunología , Glicoproteínas de Membrana/inmunología , Receptores Inmunológicos/inmunología , Transducción de Señal , Cromosoma X , Secuencia de Bases , Proteínas Portadoras/genética , Línea Celular , Membrana Celular/metabolismo , Preescolar , ADN Complementario , Ligamiento Genético , Humanos , Células Asesinas Naturales/virología , Activación de Linfocitos/inmunología , Trastornos Linfoproliferativos/sangre , Trastornos Linfoproliferativos/genética , Masculino , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Receptor 1 Gatillante de la Citotoxidad Natural , Receptores Inmunológicos/metabolismo , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Células Tumorales CultivadasRESUMEN
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 Cgamma and Grb2. These data support the concept that 2B4 may mediate NK cell triggering via a LAT-dependent signaling pathway.
Asunto(s)
Antígenos CD , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Glicoproteínas de Membrana/fisiología , Receptores Inmunológicos , Humanos , Fosforilación , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Fosfolipasas de Tipo C/fisiología , Tirosina/metabolismoRESUMEN
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
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.
Asunto(s)
Inmunoglobulinas/química , Células Asesinas Naturales/metabolismo , Receptores Inmunológicos/química , Secuencia de Aminoácidos , Animales , Antígenos CD , Secuencia de Bases , Southern Blotting , Mapeo Cromosómico , Cromosomas Humanos Par 17 , Clonación Molecular , Sondas de ADN , ADN Complementario/análisis , Haplorrinos , Humanos , Inmunoglobulinas/genética , Ratones , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores KIR , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
In this study, by the generation of a specific monoclonal antibody, we identified p75/AIRM1 (for adhesion inhibitory receptor molecule 1), a novel inhibitory receptor that is mostly confined to human natural killer cells. p75/AIRM1 is a 75-kD glycoprotein that, upon sodium pervanadate treatment, becomes tyrosine phosphorylated and associates to src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-1. The p75/AIRM1 gene is located on human chromosome 19 and encodes a novel member of the sialoadhesin family characterized by three immunoglobulin-like extracellular domains (one NH(2)-terminal V-type and two C2-type) and a classical immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic portion. The highest amino acid sequence similarity has been found with the myeloid-specific CD33 molecule and the placental CD33L1 protein. Similar to other sialoadhesin molecules, p75/AIRM1 appears to mediate sialic acid-dependent ligand recognition.
Asunto(s)
Cromosomas Humanos Par 19 , Células Asesinas Naturales/inmunología , Receptores Inmunológicos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Glicoproteínas/genética , Glicoproteínas/inmunología , Humanos , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ligandos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Receptores Inmunológicos/inmunología , Dominios Homologos srcRESUMEN
Surface receptors involved in natural killer (NK) cell triggering during the process of tumor cell lysis have recently been identified. Of these receptors, NKp44 is selectively expressed by IL-2- activated NK cells and may contribute to the increased efficiency of activated NK cells to mediate tumor cell lysis. Here we describe the molecular cloning of NKp44. Analysis of the cloned cDNA indicated that NKp44 is a novel transmembrane glycoprotein belonging to the Immunoglobulin superfamily characterized by a single extracellular V-type domain. The charged amino acid lysine in the transmembrane region may be involved in the association of NKp44 with the signal transducing molecule killer activating receptor-associated polypeptide (KARAP)/DAP12. These molecules were found to be crucial for the surface expression of NKp44. In agreement with data of NKp44 surface expression, the NKp44 transcripts were strictly confined to activated NK cells and to a minor subset of TCR-gamma/delta+ T lymphocytes. Unlike genes coding for other receptors involved in NK cell triggering or inhibition, the NKp44 gene is on human chromosome 6.
Asunto(s)
Citotoxicidad Inmunológica , Inmunoglobulinas/genética , Células Asesinas Naturales/inmunología , Receptores Inmunológicos/genética , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Células COS , Compartimento Celular , Cromosomas Humanos Par 6 , Clonación Molecular , Secuencia Conservada , ADN Complementario/genética , Humanos , Inmunoglobulinas/clasificación , Inmunoglobulinas/inmunología , Inmunoglobulinas/metabolismo , Subgrupos Linfocitarios/inmunología , Proteínas de la Membrana , Ratones , Datos de Secuencia Molecular , Receptor 2 Gatillante de la Citotoxidad Natural , Unión Proteica , ARN Mensajero/aislamiento & purificación , Receptores Inmunológicos/clasificación , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Especificidad de la EspecieRESUMEN
After culture in interleukin (IL)-2, natural killer (NK) cells acquire an increased capability of mediating non-major histocompatibility complex (MHC)-restricted tumor cell lysis. This may reflect, at least in part, the de novo expression by NK cells of triggering receptors involved in cytolysis. In this study we identified a novel 44-kD surface molecule (NKp44) that is absent in freshly isolated peripheral blood lymphocytes but is progressively expressed by all NK cells in vitro after culture in IL-2. Different from other markers of cell activation such as CD69 or VLA.2, NKp44 is absent in activated T lymphocytes or T cell clones. Since NKp44 was not detected in any of the other cell lineages analyzed, it appears as the first marker specific for activated human NK cells. Monoclonal antibody (mAb)-mediated cross-linking of NKp44 in cloned NK cells resulted in strong activation of target cell lysis in a redirected killing assay. This data indicated that NKp44 can mediate triggering of NK cell cytotoxicity. mAb-mediated masking of NKp44 resulted in partial inhibition of cytolytic activity against certain (FcgammaR-negative) NK-susceptible target cells. This inhibition was greatly increased by the simultaneous masking of p46, another recently identified NK-specific triggering surface molecule. These data strongly suggest that NKp44 functions as a triggering receptor selectively expressed by activated NK cells that, together with p46, may be involved in the process of non-MHC-restricted lysis. Finally, we show that p46 and NKp44 are coupled to the intracytoplasmic transduction machinery via the association with CD3zeta or KARAP/DAP12, respectively; these associated molecules are tyrosine phosphorylated upon NK cell stimulation.
Asunto(s)
Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Antígenos de Histocompatibilidad Menor/inmunología , Receptores Inmunológicos/aislamiento & purificación , Biomarcadores , Células Cultivadas , Citometría de Flujo , Humanos , Interleucina-2/farmacología , Células Asesinas Naturales/efectos de los fármacos , Receptor 1 Gatillante de la Citotoxidad Natural , Receptor 2 Gatillante de la Citotoxidad Natural , Receptores Inmunológicos/inmunología , Transducción de SeñalRESUMEN
Limited information is available on the surface molecules that are involved in natural killer (NK) cell triggering. In this study, we selected the BAB281 monoclonal antibody (mAb) on the basis of its ability to trigger NK-mediated target cell lysis. BAB281 identified a novel NK cell-specific surface molecule of 46 kD (p46) that is expressed by all resting or activated NK cells. Importantly, unlike the NK cell antigens identified so far, the expression of p46 was strictly confined to NK cells. Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones. The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A. Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex-restricted cytolysis mediated by human NK cells.
Asunto(s)
Antígenos de Superficie/inmunología , Células Asesinas Naturales/inmunología , Lectinas Tipo C , Activación de Linfocitos , Amidohidrolasas/metabolismo , Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos de Superficie/análisis , Calcio/metabolismo , Células Clonales , Reactivos de Enlaces Cruzados/metabolismo , Citocinas/biosíntesis , Pruebas Inmunológicas de Citotoxicidad/métodos , Citotoxicidad Inmunológica/inmunología , Regulación hacia Abajo , Glicosilación , Hexosaminidasas/metabolismo , Humanos , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Subfamília D de Receptores Similares a Lectina de las Células NK , Receptor 1 Gatillante de la Citotoxidad Natural , Neuraminidasa/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Receptores KIR , Receptores KIR2DL3RESUMEN
Human natural killer (NK) cells express inhibitory (p58) or activatory (p50) receptors for HLA-C alleles. Here, we describe a novel member of the p58/p50 family that is expressed by a subset of NK cells in about one third of donors. This molecule, termed p50.3, mediates NK cell triggering as revealed by the induction of intracellular free calcium mobilization, cytokine release and cytotoxicity. In addition, anti-p50.3 monoclonal antibody (mAb) induced a selective, strong proliferation of p50.3+ NK cells in peripheral blood lymphocytes. Although p50.3 molecules do not appear to display an obvious HLA class I specificity, they are usually coexpressed with known inhibitory receptors for HLA class I alleles, mAb-mediated cross-linking of these receptors leads to inhibition of the anti-p50.3 mAb-induced NK cell activation and proliferation. Surface p50.3 molecules are glycoproteins of approximately 55-58 kDa which, upon deglycosylation, display a relative molecular mass of 36 kDa, similar to that of deglycosylated (activatory) p50 receptors. Analysis of the two-dimensional peptide maps of the 50.3 molecules revealed a high homology with the other HLA-C-specific p58/p50 receptors. The use of a set of oligodeoxynucleotide primers, previously shown to amplify the activatory (p50) forms of HLA-C-specific receptors, consistently amplified in p50.3+ clones a cDNA sequence termed KKA3. This sequence belongs to the p58/p50 multigene family, that encodes for a transmembrane protein specifically stained by anti-p50.3 mAb in cell transfectants. Similar to p50 molecules, the KKA3-encoded molecules are characterized by two extracellular immunoglobulin-like domains, by the presence of a lysine in the transmembrane region and a short (39 amino acids) cytoplasmic tail which does not contain immune receptor tyrosine-based activation motifs (ITAM)-like sequences.
Asunto(s)
Células Asesinas Naturales/inmunología , Activación de Linfocitos/efectos de los fármacos , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/farmacología , Secuencia de Bases , Clonación Molecular , Citotoxicidad Inmunológica , Epítopos/inmunología , Humanos , Células Asesinas Naturales/química , Células Asesinas Naturales/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Receptores Inmunológicos/química , Receptores KIR , Receptores KIR2DL3 , Homología de Secuencia de Aminoácido , Transcripción Genética/inmunologíaRESUMEN
Natural killer (NK) cells express clonally distributed receptors for different groups of HLA class I alleles. The Z27 monoclonal antibody described in this study recognizes a p70 receptor specific for HLA-B alleles belonging to the Bw4 supertypic specificity. Single amino acid substitutions in the peptide-binding groove of HLA-B2705 molecules influenced the recognition by some, but not all, p7O/Z27+ clones. This suggests the existence of a limited polymorphism within the p7O family of receptors. The pattern of reactivity of monoclonal antibody Z27 revealed that Bw4-specific receptors may be expressed alone or in combination with different (GL183 and/or EB6) p58 molecules. Analysis of NK clones coexpressing p58 and p7O receptors allowed us to demonstrate that the two molecules represent physically and functionally independent receptors. The expression of p7O molecules either alone or in combination with EB6 molecules provided the molecular basis for understanding the cytolytic pattern of two previously defined groups of "alloreactive" NK cell clones ("group 3" and "group 5").
Asunto(s)
Antígenos HLA-B/metabolismo , Células Asesinas Naturales/metabolismo , Receptores Inmunológicos/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Línea Celular , Células Clonales/metabolismo , Citotoxicidad Inmunológica , Femenino , Haplotipos/genética , Humanos , Tolerancia Inmunológica , Isoantígenos/inmunología , Células Asesinas Naturales/clasificación , Masculino , Ratones , Ratones Endogámicos BALB C , Mutagénesis Sitio-Dirigida , Linaje , Unión Proteica , Receptores KIR , Receptores KIR2DL3RESUMEN
The natural killer (NK) cell-specific p58 molecules EB6 and GL183 have been shown to represent the putative surface receptors for two distinct groups of human histocompatibility leukocyte antigen (HLA) C alleles. Interaction between p58 receptors and class I molecules expressed on target cells results in inhibition of the NK-mediated cytolytic activity and thus in target cell protection. In the present study, we show that EB6 molecules may also act as receptors mediating NK cell triggering. Activatory EB6 molecules were found to be confined only to certain donors. Moreover, in these donors, only a fraction of EB6+ NK clones expressed the activatory form of EB6 molecules, while the remaining clones expressed the conventional inhibitory form. Biochemical analysis of the activatory EB6 molecules revealed a molecular mass of approximately 50 kD (p50), thus differing from the 58-kD inhibitory form. This difference was not due to differential glycosylation of the same protein, as revealed by deglycosylation experiments of isolated EB6 molecules. Treatment of purified p58 or p50/EB6 molecules with proteolytic enzymes, including V8-protease, chymotrypsin, and papain, showed only minor differences in the resulting peptides. Treatment with pepsin followed by two-dimensional peptide mapping demonstrated that, although the majority of peptides migrated in identical positions, differences between the two forms could be detected for at least one major peptide. Anti-EB6 monoclonal antibody (mAb)-mediated cross-linking of p50 molecules was required to trigger the cytolytic activity and the intracellular calcium ([Ca+2]i) increases in appropriate NK clones. Likewise, mAb-mediated cross linking of the p58 EB6 molecules was needed to inhibit the cytolytic activity; however, in this case, no [Ca+2]i increases could be detected. In NK clones expressing the inhibitory p58 EB6 receptors, soluble anti-EB6 mAb prevented recognition of protective Cw4 molecules and reconstituted target cell lysis. In contrast, in clones expressing the activatory p50/EB6 receptor, EB6 masking frequently resulted in partial inhibition of the cytolytic activity against Cw4+ target cells. Therefore, it appears that NK clones expressing the p50/EB6 receptors are induced to lyse Cw4+ target cells upon specific interaction with Cw4 molecules. This concept was further substantiated by experiments in which target cells were represented by the HLA-negative LCL721.221 cell line transfected with the Cw4 allele. Phenotypic and functional analysis of a large number of NK clones showed that clones expressing the activatory p50/EB6 molecules consistently coexpressed inhibitory receptors for other HLA class I alleles.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Antígenos HLA-C/metabolismo , Células Asesinas Naturales/inmunología , Activación de Linfocitos/fisiología , Receptores Inmunológicos/fisiología , Alelos , Anticuerpos Monoclonales/inmunología , Calcio/metabolismo , Citotoxicidad Inmunológica , Glicosilación , Antígenos HLA-C/genética , Antígenos HLA-C/inmunología , Humanos , Peso Molecular , Mapeo Peptídico , Receptores KIR , Receptores KIR2DL3 , Transducción de Señal/fisiologíaRESUMEN
Four monoclonal antibodies (mAb) termed NKTA255, NKTA72, 1F1 and 1B1 were selected on the basis of their ability to inhibit the cytolytic activity of natural killer (NK) cell clones against P815 target cells. These mAb selectively reacted with normal or tumor cells of hematopoietic origin and displayed a cellular distribution similar to that of CD45 or CD11a/CD18 antigens. Immunoprecipitation experiments showed that they reacted with molecules with an apparent molecular mass of 40 kDa under both reducing and nonreducing conditions ("p40" molecules), thus differing from CD45 or CD11a/CD18 antigens as well as from the "inhibitory" receptors for HLA class I molecules (i.e. p58, CD94 and NKB1 molecules). Double-immunofluorescence analysis of peripheral blood mononuclear cells allowed the identification of three distinct populations on the basis of the fluorescence intensity of cells stained with anti-p40 mAb. p40bright cells were homogeneously HLA-DR-positive, p40medium cells were HLA-DR-negative but co-expressed CD56 antigens, while p40dull cells were all CD3+. Anti-p40 mAb strongly inhibited the lysis of K562 target cells, mediated by fresh NK cells, as well as the lysis of P815 target cells by NK or T cell clones. In addition, in redirected killing assays, anti-p40 mAb strongly reduced the anti-CD16 mAb-induced cytolytic activity of NK cell clones. On the contrary, they did not inhibit either the anti-CD3 or anti-T cell receptor mAb-mediated cytolytic activity of T cell clones or the lysis of allogeneic phytohemagglutinin blasts mediated by specific cytolytic T cell clones. The p40-induced inhibition of the NK cytotoxicity required optimal cross-linking, as anti-p40 mAb could inhibit the lysis of Fc gamma receptor (Fc gamma R)-positive but not of Fc gamma R-negative target cells. In addition, (Fab')2 fragments of anti-p40 mAb failed to inhibit the lysis of Fc gamma R-positive target cells. In conclusion, p40 molecules represent a new type of inhibitory surface molecule that appears to play a general regulatory role in the NK-mediated cytolysis.
Asunto(s)
Antígenos de Superficie/fisiología , Citotoxicidad Inmunológica , Anticuerpos Monoclonales/inmunología , Antígenos CD11/fisiología , Antígenos CD18/fisiología , Humanos , Células Asesinas Naturales/inmunología , Antígenos Comunes de Leucocito/fisiología , Complejo Receptor-CD3 del Antígeno de Linfocito T/fisiología , Receptores de IgG/fisiología , Linfocitos T/inmunologíaRESUMEN
The natural killer cell (NK)-specific p58 surface molecules, recognized by the GL183 and EB6 monoclonal antibodies (mAb), have been shown to represent the putative NK receptor for HLA-C molecules. The interaction between p58 receptors and HLA-C results in inhibition of the NK-mediated target cell lysis. In this study, GL183-EB6+ clones (Cw4-specific), after mAb-induced surface modulation of EB6 molecules, acquired the ability to lyse the Cw4+ C1R cells. In NK clones co-expressing both GL183 and EB6 molecules and unable to kill Cw3-protected target cells, the mAb-induced modulation of EB6 molecules resulted both in selective co-modulation of GL183 molecules and in the lysis of Cw3-transfected P815 murine cells. In line with the co-modulation experiments we also show that the GL183 and EB6 molecules can be co-immunoprecipitated from GL183+/EB6+ clones after cell lysis in the presence of digitonin. The p58 receptor also revealed an association with molecules belonging to the zeta family (i.e. CD3 zeta and Fc epsilon RI gamma chains). Two-dimensional diagonal gel analysis of the p58 complex immunoprecipitated from polyclonally activated p58+ NK cells indicated a preferential association with CD3 zeta chains either in the form of covalently linked zeta-zeta homodimers or in the form of zeta-gamma heterodimers, while gamma-gamma homodimers were detectable in low amounts. However, p58+ clones displaying a unique association with gamma-gamma homodimers could also be isolated. Probing the immunoprecipitated p58 complex with anti-p56lck antibody also revealed an association with this member of the src family. In addition, mAb-mediated signaling of NK clones via p58 molecules induced increments of p58/p56lck association. However, under the same experimental conditions that induced optimal in vivo tyrosine phosphorylation of the CD16-associated CD3 zeta chains, no tyrosine phosphorylation was detected in the p58-associated CD3 zeta chains. In these in vivo experiments neither anti-CD16 nor anti-p58 mAb could induce tyrosine phosphorylation of the gamma chains. Finally, the anti-p58-mediated inhibition of the NK cell triggering via CD16 molecules was not accompanied by a down-regulation of the tyrosine phosphorylation of the CD16-associated CD3 zeta chains.
Asunto(s)
Antígenos HLA-C/metabolismo , Células Asesinas Naturales/inmunología , Proteínas Tirosina Quinasas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de IgE/metabolismo , Receptores Inmunológicos/metabolismo , Anticuerpos Monoclonales , Modulación Antigénica , Antígenos CD/análisis , Antígenos de Diferenciación de Linfocitos T/análisis , Antígenos de Superficie/metabolismo , Complejo CD3/metabolismo , Antígeno CD56 , Células Clonales , Humanos , Técnicas In Vitro , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Subgrupos Linfocitarios , Sustancias Macromoleculares , Fosforilación , Unión Proteica , Receptores de IgG/análisis , Receptor de Lamina BRESUMEN
GL183 or EB6 (p58) molecules have been shown to function as receptors for different HLA-C alleles and to deliver an inhibitory signal to natural killer (NK) cells, thus preventing lysis of target cells. In this study, we analyzed a subset of NK cells characterized by a p58-negative surface phenotype. We show that p58-negative clones, although specific for class I molecules do not recognize HLA-C alleles. In addition, by the use of appropriate target cells transfected with different HLA-class I alleles we identified HLA-B7 as the protective element recognized by a fraction of p58-negative clones. In an attempt to identify the receptor molecules expressed by HLA-B7-specific clones, monoclonal antibodies (mAbs) were selected after mice immunization with such clones. Two of these mAbs, termed XA-88 and XA-185, and their F(ab')2 fragments, were found to reconstitute lysis of B7+ target cells by B7-specific NK clones. Both mAbs were shown to be directed against the recently clustered Kp43 molecule (CD94). Thus, mAb-mediated masking of Kp43 molecules interferes with recognition of HLA-B7 and results in target cell lysis. Moreover, in a redirected killing assay, the cross-linking of Kp43 molecules mediated by the XA185 mAb strongly inhibited the cytolytic activity of HLA-B7-specific NK clones, thus mimicking the functional effect of B7 molecules. Taken together, these data strongly suggest that Kp43 molecules function as receptors for HLA-B7 and that this receptor/ligand interaction results in inhibition of the NK-mediated cytolytic activity. Indirect immunofluorescence and FACS analysis of a large number of random NK clones showed that Kp43 molecules (a) were brightly expressed on a subset of p58-negative clones, corresponding to those specific for HLA-B7; (b) displayed a medium/low fluorescence in the p58-negative clones that are not B7-specific as well as in most p58+ NK clones; and (c) were brightly expressed as in the p58+ clone ET34 (GL183-/EB6+, Cw4-specific). Functional analysis revealed that Kp43 functioned as an inhibitory receptor only in NK clones displaying bright fluorescence. These studies also indicate that some NK clones (e.g., the ET34) can coexpress two distinct receptors (p58 and Kp43) for different class I alleles (Cw4 and B7). Finally, we show that Kp43 molecules function as receptors only for some HLA-B alleles and that still undefined receptor(s) must exist for other HLA-B alleles including B27.
Asunto(s)
Antígenos CD/metabolismo , Antígenos HLA-B/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/metabolismo , Receptores de Antígenos/metabolismo , Alelos , Anticuerpos Monoclonales/inmunología , Células Clonales , Citometría de Flujo , Humanos , Subfamília D de Receptores Similares a Lectina de las Células NK , Fenotipo , Receptores de IgG/inmunología , TransfecciónRESUMEN
Human CD3-16+56+ natural killer (NK) cells have been shown to display a clonally distributed ability to recognize major histocompatibility complex (MHC) class I alleles. Opposite to T lymphocytes, in NK cells, specific recognition of MHC class I molecules appears to induce inhibition of cytolytic activity and, thus, to protect target cells. Since a precise correlation has been established between the expression of the NK-specific GL183 and EB6 surface molecules (belonging to the novel p58 molecular family) and the specificity of NK clones, we analyzed whether p58 molecules could function as receptors for MHC in human NK cells. NK clones displaying the previously defined "specificity 2" and characterized by the GL183+EB6+ phenotype, specifically recognize the Cw3 allele and thus fail to lyse the Fc gamma R+ P815 target cells transfected with Cw3. On the other hand, NK clones displaying "specificity 1" and expressing the GL183-EB6+ phenotype failed to lyse Cw4+ target cells. Addition of the F(ab')2 fragments of either GL183 or EB6 mAb as well as the XA141 mAb of IgM isotype (specific for the EB6 molecules) completely restored the lysis of Cw3-transfected P815 cells by the Cw3-specific NK clones EX2 and EX4. Similarly, both the entire EB6 mAb, its F(ab')2 fragment and the XA141 mAb reconstituted the lysis of C1R, a Fc gamma R- target cell expressing Cw4 as the only serologically detected class I antigen. Thus, it appears that masking of different members of p58 molecules prevents recognition of "protective" MHC class I alleles and thus the delivering of inhibitory signals. Further support to the concept that p58 molecules represent a NK receptor delivering a negative signal was provided by experiments in which the entire anti-p58 mAbs (of IgG isotype) could inhibit the lysis of unprotected Fc gamma R+ P815 target cells, thus mimicking the inhibitory effect of MHC class I molecules.
Asunto(s)
Antígenos de Superficie/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/metabolismo , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antígenos de Superficie/inmunología , Células Clonales , Antígenos HLA-C/genética , Antígenos HLA-C/metabolismo , Hemólisis , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Células Asesinas Naturales/inmunología , TransfecciónRESUMEN
Three new monoclonal antibodies (MAbs) termed 7A6, PP35 and A6/143 were isolated after mouse immunization with CD3- CD16+ NK clones. The screening procedure was based on the ability of MAbs to trigger cytolytic activity of the immunizing clones in a re-directed killing assay against the P815 murine mastocytoma cell line. The 7A6 MAb reacts with 58 kDa surface molecules that appear to belong to the same molecular family defined by the previously described NK-sub-set-specific GL183 and EB6 MAbs. However, unlike from these MAbs, the 7A6 MAb reacted with (and activated) all CD3- NK lymphocytes, independent of their sub-set assignment (based on the expression or lack of expression of EB6, GL183 and CD16). The PP35 MAb reacted with a 70 kDa surface molecule expressed on all CD3- NK cells, as well as on TCR gamma/delta + cells and on a small sub-set of TCR alpha/beta + CD8+ lymphocytes. The PP35 MAb induced activation of essentially all NK cells, although clonal analysis revealed quantitative differences in the magnitude of the cytolytic responses elicited in different clones. Finally, the A6/143 MAb reacted with a molecule of 115 kDa expressed by all human PBL. Similarly to 7A6 and PP35 MAbs, the A6/143 MAb activated all sub-sets of cloned NK cells.
Asunto(s)
Antígenos CD/inmunología , Complejo CD3/inmunología , Citotoxicidad Inmunológica , Antígenos HLA-DR/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Animales , Anticuerpos Monoclonales , Antígenos CD/análisis , Células Cultivadas , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Antígenos HLA-DR/análisis , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C/inmunología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
The effect of anti-CD69 monoclonal antibodies (mAbs) on the induction of the cytolytic activity in different types of lymphoid effector cells has been investigated. Three anti-CD69 mAbs, including the reference mAb MLR3 and two new mAbs (c227 and 31C4), have been used. All cloned CD3-CD16+ natural killer (NK) cells belonging to different subsets (as defined by the surface expression of GL183 and/or EB6 antigens) were efficiently triggered by anti-CD69 mAbs and lysed P815 mastocytoma cells in a redirected killing assay. Triggering of the cytolytic activity could also be induced in CD3-CD16- NK clones, which fail to respond to other stimuli (including anti-CD16, anti-CD2 mAbs, or phytohemagglutinin). A similar triggering effect was detected in T cell receptor (TCR) gamma/delta+ clones belonging to different subsets. On the other hand, anti-CD69 mAbs could not induce triggering of the cytolytic activity in TCR alpha/beta+ cytolytic clones. Since all thymocytes are known to express CD69 antigen after cell activation, we analyzed a series of phenotypically different cytolytic thymocyte populations and clones for their responsiveness to anti-CD69 mAb in a redirected killing assay. Again, anti-CD69 mAb triggered TCR gamma/delta+ but not TCR alpha/beta+ thymocytes. Anti-CD69 mAb efficiently triggered the cytolytic activity of "early" thymocytes lines or clones (CD3-4-8-7+), which lack all other known pathways of cell activation. Thus, it appears that CD69 molecules may initiate a pathway of activation of cytolytic functions common to a number of activated effector lymphocytes with the remarkable exception of TCR alpha/beta+ cytolytic cells.