Assuntos
Psoríase/genética , Receptores KIR3DL1/genética , Adulto , Idade de Início , Estudos de Casos e Controles , Epitopos/genética , Epitopos de Linfócito T/metabolismo , Feminino , Antígenos HLA-B/metabolismo , Humanos , Masculino , Receptores KIR3DL1/antagonistas & inibidores , Fatores de RiscoRESUMO
Stable surface expression of human inhibitory killer cell Ig-like receptors (KIRs) is critical for controlling NK cell function and maintaining NK cell tolerance toward normal MHC class I(+) cells. Our recent experiments, however, have found that Ab-bound KIR3DL1 (3DL1) readily leaves the cell surface and undergoes endocytosis to early/recycling endosomes and subsequently to late endosomes. We found that 3DL1 internalization is at least partially mediated by an interaction between the µ2 subunit of the AP-2 clathrin adaptor complex and ITIM tyrosine residues in the cytoplasmic domain of 3DL1. Disruption of the 3DL1/µ2 interaction, either by mutation of the ITIM tyrosines in 3DL1 or mutation of µ2, significantly diminished endocytosis and increased surface expression of 3DL1 in human primary NK cells and cell lines. Furthermore, we found that the 3DL1/AP-2 interaction is diminished upon Ab engagement with the receptor, as compared with untreated cells. Thus, we have identified AP-2-mediated endocytosis as a mechanism regulating the surface levels of inhibitory KIRs through their ITIM domains. Based on our results, we propose a model in which nonengaged KIRs are internalized by this mechanism, whereas engagement with MHC class I ligand would diminish AP-2 binding, thereby prolonging stable receptor surface expression and promoting inhibitory function. Furthermore, this ITIM-mediated mechanism may similarly regulate the surface expression of other inhibitory immune receptors.
Assuntos
Complexo 2 de Proteínas Adaptadoras/metabolismo , Endocitose/fisiologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores KIR/metabolismo , Complexo 2 de Proteínas Adaptadoras/química , Complexo 2 de Proteínas Adaptadoras/genética , Anticorpos/metabolismo , Linhagem Celular , Citotoxicidade Imunológica , Endossomos/metabolismo , Expressão Gênica , Antígenos de Histocompatibilidade Classe I , Humanos , Ligação Proteica/imunologia , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/metabolismo , Transporte Proteico , Receptores KIR3DL1/antagonistas & inibidores , Receptores KIR3DL1/metabolismoRESUMO
OBJECTIVES: The aim of this study was to develop a small interfering RNA (siRNA) against the expression of KIR3DL1 receptor on natural killer (NK) cells, in order to promote the ability of NK cells to destroy human immunodeficiency virus (HIV)-infected cells and thus prevent failure of siRNA therapy targeting human immunodeficiency virus type 1 (HIV-1) virus among HIV-1 infected patients in vitro. METHODS: A siRNA targeting KIR3DL1 was synthesized and then modified with cholesterol, methylene, and sulfate. The inhibitory action of the siRNAs on primary cultured NK cells was detected. The amount of IFN-γ and TNF-α secretions in NK cells was measured. The intended functions of NK cells in vitro were analyzed by CFSE and PI methods. RESULTS: There were no significant differences in inhibiting the expression of KIR3DL1 on NK cells between the modified and unmodified siRNAs, while inhibition by each of them differed significantly from controls. The amount of IFN-γ and TNF-α secretions in the NK cells was abundant due to unsuccessful expression of KIR3DL1 on NK cells, which further promoted function of the NK cells. CONCLUSION: The siRNA against KIR3DL1 could enhance the ability of the NK cells to kill the HIV-1 infected cells in vitro and successfully prevented the failure of siRNA therapy targeting the HIV-1 virus. Therefore, it can act as a potential gene therapeutic agent among HIV-1 infected people.
Assuntos
Fatores Imunológicos/farmacologia , Células Matadoras Naturais/imunologia , RNA Interferente Pequeno/farmacologia , Receptores KIR3DL1/antagonistas & inibidores , Adulto , Células Cultivadas , Feminino , Terapia Genética/métodos , Infecções por HIV/terapia , Humanos , Fatores Imunológicos/genética , Interferon gama/metabolismo , Masculino , Pessoa de Meia-Idade , RNA Interferente Pequeno/genética , Receptores KIR3DL1/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The inhibitory killer cell Ig-like receptors (KIR) negatively regulate NK cell cytotoxicity by activating the Src homology 2 domain-containing protein tyrosine phosphatases 1 and 2 following ligation with MHC class I molecules expressed on normal cells. This requires tyrosine phosphorylation of KIR on ITIMs in the cytoplasmic domain. Surprisingly, we have found that KIR3DL1 is strongly and constitutively phosphorylated on serine and weakly on threonine residues. In this study, we have mapped constitutive phosphorylation sites for casein kinases, protein kinase C, and an unidentified kinase on the KIR cytoplasmic domain. Three of these phosphorylation sites are highly conserved in human inhibitory KIR. Functional studies of the wild-type receptor and serine/threonine mutants indicated that phosphorylation of Ser(394) by protein kinase C slightly suppresses KIR3DL1 inhibitory function, and reduces receptor internalization and turnover. Our results provide evidence that serine/threonine phosphorylation is an important regulatory mechanism of KIR function.
