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1.
J Leukoc Biol ; 78(2): 359-71, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15937142

RESUMO

The major histocompatibility complex nonrestricted cytotoxic leukemic T cell line T acute lymphoblastic leukemia (TALL)-104 is being pursued as a therapeutic agent for cancer. However, the receptors and effector mechanisms responsible for its broad tumoricidal function remain undefined. Here, we examined the roles played by natural cytotoxicity receptors (NCR), killer cell immunoglobulin-like receptors, cytolytic granule components, and tumor necrosis factor (TNF) family members in tumor recognition and lysis by TALL-104 cells. The perforin-granzyme pathway, TNF-related apoptosis-inducing ligand (TRAIL), and Fas were each involved in the lysis of particular tumor targets by TALL-104. Furthermore, phorbol 12-myristate 13-acetate/ionomycin treatment induced surface expression of Fas-L and TRAIL. In addition, supernatants from CD3-stimulated TALL-104 cultures exhibited antiproliferative activity, which was blocked 50-90% by anti-TNF-alpha monoclonal antibody (mAb). Although negative for the NCR natural killer (NK)p44, this cell line was found to express NKp46. An anti-NKp46 antibody strongly blocked TALL-104-mediated lysis of certain targets and directly induced cytokine production, granule release, and redirected lysis responses. Anti-NKG2D and anti-2B4 also stimulated redirected cytotoxicity by TALL-104. By contrast, anti-NKG2A mAb did not stain the cells or inhibit killing responses. Alternatively, KIR3DL2 was detected on TALL-104, and expression of its reported ligand, human leukocyte antigen (HLA)-A, on target cells provided protection from cytotoxicity. Thus, NKp46, NKG2D, and 2B4 are activating receptors, and KIR3DL2 is an inhibitory receptor on TALL-104. The data demonstrate the ability of TALL-104 cells to recognize a wide variety of tumors with NK cell receptors and kill them with a broad arsenal of cytolytic effector mechanisms, including cytolytic granules and TNF family ligands.


Assuntos
Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/imunologia , Transferência Adotiva , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD/imunologia , Proteínas Reguladoras de Apoptose , Complexo CD3/imunologia , Carcinógenos/farmacologia , Degranulação Celular/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Proteína Ligante Fas , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Proteínas Imediatamente Precoces/imunologia , Ionomicina/farmacologia , Ionóforos/farmacologia , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Glicoproteínas de Membrana/imunologia , Proteínas Monoméricas de Ligação ao GTP/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptor 1 Desencadeador da Citotoxicidade Natural , Neoplasias/imunologia , Neoplasias/terapia , Receptores Imunológicos/imunologia , Receptores KIR , Receptores KIR3DL2 , Receptores de Células Matadoras Naturais , Transdução de Sinais/efeitos dos fármacos , Família de Moléculas de Sinalização da Ativação Linfocitária , Linfócitos T Citotóxicos/citologia , Ligante Indutor de Apoptose Relacionado a TNF , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/imunologia , Fatores de Necrose Tumoral/imunologia , Células U937
2.
J Immunol ; 174(7): 3859-63, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15778339

RESUMO

KIR2DL4 (2DL4, CD158d), a member of the human killer cell Ig-like receptor (KIR) family, triggers potent IFN-gamma responses but weak cytotoxicity in resting NK cells. 2DL4 mRNA has been detected in most NK cell clones from most humans examined, but surface protein expression is detectable only on CD56(high) NK cells from certain donors. The receptor possesses a transmembrane arginine residue, suggesting association with a signaling accessory protein that has remained elusive. We provide biochemical and functional evidence that FcepsilonRI-gamma (gamma) associates with 2DL4 to promote surface expression and provide signal transducing function. Weak cytolytic responses triggered through 2DL4 may result from low stoichiometric association with gamma. Selective association with gamma distinguishes 2DL4 from all other activating forms of the KIR family, which alternatively associate with DNAX-activating protein (DAP)12.


Assuntos
Células Matadoras Naturais/fisiologia , Receptores de IgE/fisiologia , Receptores Imunológicos/fisiologia , Transdução de Sinais/imunologia , Antígenos de Superfície , Células Cultivadas , Citotoxicidade Imunológica , Humanos , Células Jurkat , Células Matadoras Naturais/citologia , Ligação Proteica , Receptores de IgE/imunologia , Receptores de IgE/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Receptores KIR , Receptores KIR2DL4 , Regulação para Cima
3.
J Immunol ; 172(12): 7385-92, 2004 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-15187115

RESUMO

Human NK cells use class I MHC-binding inhibitory receptors, such as the killer cell Ig-like receptor (KIR) family, to discriminate between normal and abnormal cells. Some tumors and virus-infected cells down-regulate class I MHC and thereby become targets of NK cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatases, Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) and SHP-2, to two phosphorylated cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). KIR2DL5 is a type II member of the KIR2D family with an atypical extracellular domain and an intracytoplasmic domain containing one typical ITIM and one atypical ITIM sequence. Although KIR2DL5 structure is expressed by approximately 50% of humans and is conserved among primate species, its function has not been determined. In the present study, we directly compared functional and biochemical properties of KIR2DL5, KIR3DL1 (a type I KIR with two ITIMs), and KIR2DL4 (the only other type II KIR, which has a single ITIM) in a human NK-like cell line. Our results show that KIR2DL5 is an inhibitory receptor that can recruit both SHP-1 and SHP-2, and its inhibitory capacity is more similar to that of the cytoplasmic domain of KIR2DL4 than KIR3DL1. Interestingly, inhibition of NK cell cytotoxicity by KIR2DL5 was blocked by dominant-negative SHP-2, but not dominant-negative SHP-1, whereas both dominant-negative phosphatases can block inhibition by KIR3DL1. Therefore, the cytoplasmic domains of type II KIRs (2DL4 and 2DL5) exhibit distinct inhibitory capacities when compared with type I KIRs (3DL1), due to alterations in the canonical ITIM sequences.


Assuntos
Células Matadoras Naturais/imunologia , Ativação Linfocitária , Proteínas Tirosina Fosfatases/metabolismo , Receptores Imunológicos/fisiologia , Sequência de Aminoácidos , Linhagem Celular , Citoplasma/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Testes de Precipitina , Ligação Proteica , Proteína Fosfatase 1 , Proteína Fosfatase 2 , Estrutura Terciária de Proteína , Transporte Proteico , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores KIR , Receptores KIR2DL4 , Receptores KIR3DL1 , Transfecção
4.
J Immunol ; 172(2): 899-906, 2004 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-14707061

RESUMO

NKp44 (NCR2) is a member of the natural cytotoxicity receptor (NCR) family that is expressed on activated human NK cells. We dissected structural attributes of NKp44 to determine their contributions to receptor function. Our results demonstrate that surface expression and NK cell activation by NKp44 is mediated through noncovalent association with the immunoreceptor tyrosine-based activation motif-containing protein, DAP12. Physical linkage to DAP12 requires lysine-183 in the NKp44 transmembrane domain. Intriguingly, the cytoplasmic domain of NKp44 also contains a sequence that matches the immunoreceptor tyrosine-based inhibitory motif (ITIM) consensus. By expressing a chimeric receptor in an NK-like cell line, we found that this ITIM-like motif from NKp44 lacks inhibitory capacity in a redirected cytotoxicity assay. The NKp44 cytoplasmic tyrosine was efficiently phosphorylated in the chimeric receptor upon treating the cells with pervanadate, but it was unable to recruit ITIM-binding negative effector phosphatases. We also generated NK-like cell lines expressing epitope-tagged wild-type or tyrosine to phenylalanine mutant (Y238F) versions of NKp44 and compared their capacities to induce activation marker expression, promote IFN-gamma production, or stimulate target cell cytotoxicity. We did not detect any tyrosine-dependent reduction or enhancement of NK cell activation through wild-type vs. Y238F mutant NKp44. Finally, the cytoplasmic tyrosine-based sequence did not provide a docking site for the AP-2 clathrin adaptor, nor did it potentiate receptor internalization. In summary, all activating properties and surface expression of NKp44 are mediated through its association with DAP12, and the putative ITIM in the NKp44 cytoplasmic domain does not appear to attenuate activating function.


Assuntos
Citoplasma/imunologia , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Fragmentos de Peptídeos/fisiologia , Receptores Imunológicos/metabolismo , Receptores Imunológicos/fisiologia , Complexo 2 de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Linhagem Celular Tumoral , Membrana Celular/imunologia , Membrana Celular/metabolismo , Citoplasma/genética , Humanos , Células Jurkat , Ativação Linfocitária/genética , Lisina/fisiologia , Proteínas de Membrana , Dados de Sequência Molecular , Receptor 2 Desencadeador da Citotoxicidade Natural , Fenilalanina/genética , Ligação Proteica/genética , Ligação Proteica/imunologia , Estrutura Terciária de Proteína/genética , Receptores Imunológicos/biossíntese , Tirosina/genética
5.
J Immunol ; 171(7): 3415-25, 2003 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-14500636

RESUMO

Killer cell Ig-like receptor (KIR)2DL4 (2DL4, CD158d) was previously described as the only KIR expressed by every human NK cell. It is also structurally atypical among KIRs because it possesses a basic transmembrane residue, which is characteristic of many activating receptors, but also contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). We expressed epitope-tagged 2DL4 in an NK-like cell line to study receptor function. Three distinct 2DL4 cDNA clones were analyzed: one encoding the "conventional" 2DL4 with the cytoplasmic ITIM (2DL4.1) and two encoding different cytoplasmic truncated forms lacking the ITIM (2DL4.2 and 2DL4(*)). Surprisingly, one truncated receptor (2DL4.2), which is the product of a prevalent human 2DL4 allele, was not expressed on the cell surface, indicating that some individuals may lack functional 2DL4 protein expression. Conversely, both 2DL4.1 and 2DL4(*) were expressed on the cell surface and up-regulated by IL-2. Analysis of primary NK cells with anti-2DL4 mAb confirmed the lack of surface expression in a donor with the 2DL4.2 genotype. Donors with the 2DL4.1 genotype occasionally expressed receptor only on CD56(high) NK cells, although their expression was up-regulated by IL-2. Interestingly, Ab engagement of epitope-tagged 2DL4 triggered rapid and robust IFN-gamma production, but weak redirected cytotoxicity in an NK-like cell line, which was the opposite pattern to that observed upon engagement of another NK cell activating receptor, NKp44. Importantly, both 2DL4.1 and 2DL4(*) exhibited similar activation potential, indicating that the ITIM does not influence 2DL4.1 activating function. The unique activation properties of 2DL4 suggest linkage to a distinct signaling pathway.


Assuntos
Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/fisiologia , Interferon gama/biossíntese , Interleucina-2/fisiologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores Imunológicos/biossíntese , Receptores Imunológicos/fisiologia , Adjuvantes Imunológicos/genética , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/genética , Membrana Celular/imunologia , Membrana Celular/metabolismo , Citoplasma/imunologia , Citoplasma/metabolismo , Fragmentação do DNA/imunologia , Regulação para Baixo/imunologia , Genótipo , Humanos , Interleucina-2/farmacologia , Células Jurkat , Ativação Linfocitária/imunologia , Dados de Sequência Molecular , Receptor 2 Desencadeador da Citotoxicidade Natural , Receptores Imunológicos/genética , Receptores KIR , Receptores KIR2DL4 , Retroviridae/genética , Retroviridae/imunologia , Transdução Genética , Tirosina/metabolismo , Regulação para Cima/imunologia , Receptor fas/fisiologia
6.
J Immunol ; 168(10): 5047-57, 2002 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-11994457

RESUMO

Killer cell Ig-like receptors (KIR) are MHC class I-binding immunoreceptors that can suppress activation of human NK cells through recruitment of the Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) to two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains. KIR2DL4 (2DL4; CD158d) is a structurally distinct member of the KIR family, which is expressed on most, if not all, human NK cells. 2DL4 contains only one ITIM in its cytoplasmic domain and an arginine in its transmembrane region, suggesting both inhibitory and activating functions. While 2DL4 can activate IFN-gamma production, dependent upon the transmembrane arginine, the function of the single ITIM of 2DL4 remains unknown. In this study, tandem ITIMs of KIR3DL1 (3DL1) and the single ITIM of 2DL4 were directly compared in functional and biochemical assays. Using a retroviral transduction method, we show in human NK cell lines that 1) the single ITIM of 2DL4 efficiently inhibits natural cytotoxicity responses; 2) the phosphorylated single ITIM recruits SHP-2 protein tyrosine phosphatase, but not SHP-1 in NK cells; 3) expression of dominant-negative SHP-1 does not block the ability of 2DL4 to inhibit natural cytotoxicity; 4) surprisingly, mutation of the tyrosine within the single ITIM does not completely abolish inhibitory function; and 5) this correlates with weak SHP-2 binding to the mutant ITIM of 2DL4 in NK cells and a corresponding nonphosphorylated ITIM peptide in vitro. These results reveal new aspects of the KIR-inhibitory pathway in human NK cells, which are SHP-1 and phosphotyrosine independent.


Assuntos
Citoplasma/imunologia , Citotoxicidade Imunológica , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/imunologia , Fosfotirosina/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Receptores Imunológicos/fisiologia , Transdução de Sinais/imunologia , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Citoplasma/enzimologia , Citoplasma/metabolismo , Testes Imunológicos de Citotoxicidade , Citotoxicidade Imunológica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células Matadoras Naturais/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica/genética , Ligação Proteica/imunologia , Proteína Fosfatase 1 , Estrutura Terciária de Proteína/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Receptores Imunológicos/biossíntese , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores KIR , Receptores KIR2DL4 , Receptores KIR3DL1 , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Células Tumorais Cultivadas , Vaccinia virus/enzimologia , Vaccinia virus/genética , Domínios de Homologia de src/imunologia
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