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
NKG2D is an important immunosurveillance receptor that triggers a unique signal transduction pathway resulting in a variety of functional outcomes. NKG2D couples to the non-ITAM-containing DAP10 and initiates at least two signaling branches that are both required for cytotoxicity. Transformed, infected, or healthy cells can express NKG2D ligands, and NKG2D(+) lymphocytes can be unaffected, costimulated, or fully activated by the NKG2D-ligand interaction. The NKG2D-mediated response can be modulated by factors such as cytokine milieu and possibly the particular ligand expressed. Thus, this nontraditional NKG2D-DAP10 initiated signal pathway enables lymphocytes to differentially respond to ligands based on the cellular environment.
Assuntos
Receptores Imunológicos/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Humanos , Ativação Linfocitária , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptores de Células Matadoras Naturais , Transdução de Sinais/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.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Proteína Adaptadora GRB2/metabolismo , Células Matadoras Naturais/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptores Imunológicos/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Humanos , Modelos Biológicos , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Fosforilação , Receptores de Células Matadoras Naturais , Transdução de Sinais/imunologiaRESUMO
The two isoforms of phospholipase C (PLC)-gamma couple immune recognition receptors to important calcium- and protein kinase C-dependent cellular functions. It has been assumed that PLC-gamma1 and PLC-gamma2 have redundant functions and that the receptors can use whichever PLC-gamma isoform is preferentially expressed in a cell of a given hemopoietic lineage. In this study, we demonstrate that ITAM-containing immune recognition receptors can use either PLC-gamma1 or PLC-gamma2, whereas the novel NK cell-activating receptor NKG2D preferentially couples to PLC-gamma2. Experimental models evaluating signals from either endogenous receptors (FcR vs NKG2D-DAP10) or ectopically expressed chimeric receptors (with ITAM-containing cytoplasmic tails vs DAP10-containing cytoplasmic tails) demonstrate that PLC-gamma1 and PLC-gamma2 both regulate the functions of ITAM-containing receptors, whereas only PLC-gamma2 regulates the function of DAP10-coupled receptors. These data suggest that specific immune recognition receptors can differentially couple to the two isoforms of PLC-gamma. More broadly, these observations reveal a basis for selectively targeting the functions initiated by distinct immune recognition receptors.
Assuntos
Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/imunologia , Receptores Imunológicos/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular Tumoral , Células Clonais , Citotoxicidade Imunológica , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Fosfolipase C gama , Fosforilação , Receptores Fc/metabolismo , Receptores Imunológicos/química , Receptores de Células Matadoras Naturais , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T Citotóxicos/imunologia , Fosfolipases Tipo C/química , Tirosina/químicaRESUMO
The immune recognition receptor complex NKG2D-DAP10 on natural killer cells is stimulated by specific ligands carried on virus-infected and malignant cells. Because DAP10 does not have an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic tail, its ability to trigger killing has been debated. Here we show that a crucial Tyr-Ile-Asn-Met amino acid motif in the cytoplasmic tail of DAP10 couples receptor stimulation to the downstream activation of phosphatidylinositol 3-kinase, Vav1, Rho family GTPases and phospholipase C. Unlike that of ITAM-containing receptors, the activation of NKG2D-DAP10 proceeds independently of Syk family protein tyrosine kinases. Yet the signals initiated by NKG2D-DAP10 are fully capable of inducing killing. Our findings identify a previously unknown mechanism by which receptor complexes that lack ITAM motifs can trigger lymphocyte activation.