RESUMO
Myeloid dendritic cells (mDC) activated with a B7-DC-specific cross-linking IgM Ab (B7-DC XAb) take up and retain Ag and interact with T cell compartments to affect a number of biologic changes that together cause strong antitumor responses and blockade of inflammatory airway disease in animal models. The molecular events mediating the initial responses in mDC remain unclear. In this study we show that B7-DC XAb caused rapid phosphorylation of the adaptor protein DAP12 and intracellular kinases Syk and phospholipase C-gamma1. Pretreatment of mDC with the Syk inhibitor piceatannol blocked B7-DC XAb-induced Ag uptake with a concomitant loss of tumor protection in mice. Vaccination with tumor lysate-pulsed wild-type B7-DC XAb-activated mDC, but not TREM-2 knockout XAb-activated mDC, protected mice from lethal melanoma challenge. Multimolecular caps appeared within minutes of B7-DC XAb binding to either human or mouse mDC, and FRET analysis showed that class II, CD80, CD86, and TREM-2 are recruited in tight association on the cell surface. When TREM-2 expression was reduced in wild-type mDC using short hairpin RNA or by using mDC from TREM-2 knockout mice, in vitro DC failed to take up Ag after B7-DC XAb stimulation. These results directly link TREM-2 signaling with one change in the mDC phenotype that occurs in response to this unique Ab. The parallel signaling events observed in both human and mouse mDC support the hypothesis that B7-DC cross-linking may be useful as a therapeutic immune modulator in human patients.
Assuntos
Antígenos/imunologia , Células Dendríticas/imunologia , Glicoproteínas de Membrana/imunologia , Células Mieloides/imunologia , Receptores Imunológicos/imunologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Anticorpos/farmacologia , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Capeamento Imunológico/efeitos dos fármacos , Capeamento Imunológico/genética , Capeamento Imunológico/imunologia , Inflamação/genética , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Melanoma/genética , Melanoma/imunologia , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Fosfolipase C gama/genética , Fosfolipase C gama/imunologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Fosforilação/imunologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/genética , Transtornos Respiratórios/genética , Transtornos Respiratórios/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Estilbenos/farmacologia , Quinase Syk , Linfócitos T/imunologiaRESUMO
NK cells are innate immune cells that can eliminate their targets through granule release. In this study, we describe a specialized role for the large GTPase Dynamin 2 (Dyn2) in the regulation of these secretory events leading to cell-mediated cytotoxicity. By modulating the expression of Dyn2 using small interfering RNA or by inhibiting its activity using a pharmacological agent, we determined that Dyn2 does not regulate conjugate formation, proximal signaling, or granule polarization. In contrast, during cell-mediated killing, Dyn2 localizes with lytic granules and polarizes to the NK cell-target interface where it regulates the final fusion of lytic granules with the plasma membrane. These findings identify a novel role for Dyn2 in the exocytic events required for effective NK cell-mediated cytotoxicity.
Assuntos
Citotoxicidade Imunológica , Dinamina II/imunologia , Exocitose/imunologia , Células Matadoras Naturais/imunologia , Vesículas Secretórias/imunologia , Dinamina II/metabolismo , Imunofluorescência , Humanos , Células Matadoras Naturais/metabolismo , Lisossomos/imunologia , Lisossomos/metabolismo , Transporte Proteico/imunologia , Vesículas Secretórias/metabolismoRESUMO
Little is known about the regulatory roles of specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in cytotoxic lymphocytes. Recent information suggests that mutations in the SNARE protein syntaxin 11 result in a form of familial hemophagocytic lymphohistiocytosis (FHL). Because genetic abnormalities in key granule components (e.g., perforin) or in regulators of secretion (e.g., Munc13-4) underlie the other identified forms of FHL, we assessed whether syntaxin 11 might also serve a related regulatory role. We determined that syntaxin 11 is expressed in NK cells and activated CTLs and is located in discrete membrane-associated structures in the cytoplasm. Enhanced expression of syntaxin 11 augments the secretion and killing of tumor targets, and suppression of syntaxin 11 expression inhibits these functions. Our data identify and characterize a role for syntaxin 11 in granule exocytosis and in the generation of cell-mediated killing. These results also provide new insights on the mechanisms of hemopoietic dysregulation in FHL.
Assuntos
Testes Imunológicos de Citotoxicidade , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Proteínas Qa-SNARE/fisiologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Células Clonais , Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Exocitose/imunologia , Humanos , Células Jurkat , Células K562 , Proteínas Qa-SNARE/biossíntese , Linfócitos T Citotóxicos/citologiaRESUMO
NK cells are effector lymphocytes that can recognize and eliminate virally infected and transformed cells. NK cells express distinct activating receptors, including an ITAM-containing FcR complex that recognizes Ab-coated targets, and the DNAX-activating protein of 10 kDa-containing NKG2D receptor complex that recognizes stress-induced ligands. The regulatory role of specific tyrosine kinases in these pathways is incompletely understood. In this study, we show that, in activated human NK cells, the tyrosine kinase IL-2-inducible T cell kinase (Itk), differentially regulates distinct NK-activating receptors. Enhanced expression of Itk leads to increases in calcium mobilization, granule release, and cytotoxicity upon stimulation of the ITAM-containing FcR, suggesting that Itk positively regulates FcR-initiated cytotoxicity. In contrast, enhanced Itk expression decreases cytotoxicity and granule release downstream of the DNAX-activating protein of 10 kDa-containing NKG2D receptor, suggesting that Itk is involved in a pathway of negative regulation of NKG2D-initiated granule-mediated killing. Using a kinase mutant, we show that the catalytic activity of Itk is required for both the positive and negative regulation of these pathways. Complementary experiments where Itk expression was suppressed also showed differential regulation of the two pathways. These findings suggest that Itk plays a complex role in regulating the functions initiated by distinct NK cell-activating receptors. Moreover, understanding how these pathways may be differentially regulated has relevance in the setting of autoimmune diseases and antitumor immune responses where NK cells play key regulatory roles.
Assuntos
Sinalização do Cálcio/imunologia , Regulação Enzimológica da Expressão Gênica , Células Matadoras Naturais/imunologia , Proteínas Tirosina Quinases/imunologia , Animais , Doenças Autoimunes/enzimologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Sinalização do Cálcio/genética , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Imunidade Celular/genética , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Camundongos , Mutação , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/imunologia , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/genética , Receptores Imunológicos/biossíntese , Receptores Imunológicos/imunologiaRESUMO
NKG2D is an important immunosurveillance receptor that responds to stress-induced ligand expression on tumors and virus-infected cells. Human natural killer cells express NKG2D and require the transmembrane adaptor DAP10 to initiate their full cytotoxic activation. However, DAP10 has no immunoreceptor tyrosine-based activation motif and thus the mechanism of recruiting 'downstream' effector proteins is unclear. We show here that binding of the p85 subunit of phosphatidylinositol-3- kinase to DAP10 could not by itself trigger cell-mediated cytotoxicity and that binding of an intermediate consisting of the DAP10 binding partner Grb2 and the effector molecule Vav1 (Grb2-Vav1) to DAP10 was sufficient to initiate tyrosine-phosphorylation events. For full calcium release and cytotoxicity to occur, both Grb2-Vav1 and p85 had to bind to DAP10. These findings identify a previously unknown mechanism by which NKG2D-DAP10 mediates cytotoxicity and provides a framework for evaluating activation by other receptor complexes that lack immunoreceptor tyrosine-based activation motifs.