RESUMO
We studied the factors that control IL-17 production in human Mycobacterium tuberculosis infection. CD4(+) cells from healthy tuberculin reactors produced IL-17 in response to autologous M. tuberculosis-stimulated monocytes, and most IL-17(+) cells were Ag experienced, CD4(+)CD62L(-). IL-17 production by CD4(+) cells was inhibited by anti-IL-23, but not by Abs to IL-1, IL-6, or TGF-beta. Anti-NKG2D reduced IL-17 production and the frequency of CD4(+)CD62(-) IL-17(+) cells, suggesting that NKG2D stimulates IL-17 production. CD4(+)NKG2D(+) cells did not produce IL-17. Monocytes and alveolar macrophages from healthy donors produced IL-23 in response to M. tuberculosis. Addition of CD4(+) cells markedly enhanced IL-23 production by M. tuberculosis-stimulated monocytes, and this was inhibited by anti-NKG2D and by Abs to UL-16 binding protein (ULB)1, a ligand for NKG2D on APCs. We conclude that binding of NKG2D to UL-16 binding protein (ULB)1 contributes to IL-23-dependent IL-17 production by CD4(+) cells in human M. tuberculosis infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Interleucina-17/biossíntese , Interleucina-23/biossíntese , Subfamília K de Receptores Semelhantes a Lectina de Células NK/fisiologia , Tuberculose/imunologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/microbiologia , Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/microbiologia , Estudos de Casos e Controles , Células Cultivadas , Proteínas Ligadas por GPI , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos Alveolares/microbiologia , Proteínas de Membrana/metabolismo , Monócitos/microbiologia , Mycobacterium tuberculosis/imunologiaRESUMO
The activating receptor, NKG2D, is expressed on a variety of immune effector cells and recognizes divergent families of major histocompatibility complex (MHC) class I-related ligands, including the MIC and ULBP proteins. Infection, stress, or transformation can induce NKG2D ligand expression, resulting in effector cell activation and killing of the ligand-expressing target cell. The human cytomegalovirus (HCMV) membrane glycoprotein, UL16, binds to three of the five known ligands for human NKG2D. UL16 is retained in the endoplasmic reticulum and cis-Golgi apparatus of cells and causes MICB to be similarly retained and stabilized within cells. Coexpression of UL16 markedly reduces cell surface levels of MICB, ULBP1, and ULBP2, and decreases susceptibility to natural killer cell-mediated cytotoxicity. Domain swapping experiments demonstrate that the transmembrane and cytoplasmic domains of UL16 are important for intracellular retention of UL16, whereas the ectodomain of UL16 participates in down-regulation of NKG2D ligands. The intracellular sequestration of NKG2D ligands by UL16 represents a novel HCMV immune evasion mechanism to add to the well-documented viral strategies directed against antigen presentation by classical MHC molecules.
Assuntos
Citomegalovirus/imunologia , Células Matadoras Naturais/imunologia , Receptores Imunológicos/metabolismo , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Ligação Competitiva , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/virologia , Reações Cruzadas , Citomegalovirus/genética , Citomegalovirus/patogenicidade , Citomegalovirus/fisiologia , Citotoxicidade Imunológica , Regulação para Baixo , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Ligantes , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Células Matadoras Naturais , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Cell surface proteins major histocompatibility complex (MHC) class I-related chain A (MICA) and UL16-binding proteins (ULBP) 1, 2, and 3 are up-regulated upon infection or tumor transformation and can activate human natural killer (NK) cells. Patches of cross-linked raft resident ganglioside GM1 colocalized with ULBP1, 2, 3, or MICA, but not CD45. Thus, ULBPs and MICA are expressed in lipid rafts at the cell surface. Western blotting revealed that glycosylphosphatidylinositol (GPI)-anchored ULBP3 but not transmembrane MICA, MHC class I protein, or transferrin receptor, accumulated in detergent-resistant membranes containing GM1. Thus, MICA may have a weaker association with lipid rafts than ULBP3, yet both proteins accumulate at an activating human NK cell immune synapse. Target cell lipid rafts marked by green fluorescent protein-tagged GPI also accumulate with ULBP3 at some synapses. Electron microscopy reveals constitutive clusters of ULBP at the cell surface. Regarding a specific molecular basis for the organization of these proteins, ULBP1, 2, and 3 and MICA are lipid modified. ULBP1, 2, and 3 are GPI anchored, and we demonstrate here that MICA is S-acylated. Finally, expression of a truncated form of MICA that lacks the putative site for S-acylation and the cytoplasmic tail can be expressed at the cell surface, but is unable to activate NK cells.
Assuntos
Proteínas de Transporte/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores Imunológicos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Sequência de Bases , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Primers do DNA/genética , Proteínas Ligadas por GPI , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Células Matadoras Naturais/ultraestrutura , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Proteínas de Membrana , Microscopia Eletrônica , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptores de Células Matadoras Naturais , Linfócitos T/ultraestruturaRESUMO
Human cytomegalovirus (HCMV) encodes UL18, a major histocompatibility complex (MHC) class I homologue that binds to the leukocyte immunoglobulin-like receptor (LIR)-1 (also called ILT2/CD85j/LILRB1), an inhibitory receptor expressed on myeloid and lymphoid immune cells. The molecular basis underlying the high affinity binding of UL18 to LIR-1, compared to MHC class I molecules (MHC-I), is unclear. Based on a comparative structural analysis of a molecular model of UL18 with the crystal structure of the HLA-A2/LIR-1 complex, we identified three regions in UL18 influencing interaction with LIR-1. Comparison of the relative binding affinities of mutated UL18 proteins to LIR-1 demonstrated the importance of specific residues in each region. Substitution of residues K42/A43 and Q202, localized in the alpha1 and alpha3 domains, respectively, reduced binding affinity to LIR-1 nearly by half. The model also suggested the formation of an additional disulfide bridge in the alpha3 domain of UL18 between residues C240 and C255, not present in MHC-I. Substitution of either cysteine residue prevented association of UL18 to beta2m, abolishing binding to LIR-1. All observed differences in binding affinities translated directly into functional consequences in terms of inhibition of IFN-gamma production by T cells, mediated through the UL18-LIR-1 interaction. The larger amount of interacting regions, combined with an increased stability of the alpha3 and beta2m domains allow a higher recognition affinity of UL18 by LIR-1.
Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Western Blotting , Complexo CD3/metabolismo , Proteínas do Capsídeo/genética , Células Cultivadas , Dicroísmo Circular , Cristalografia por Raios X , Dissulfetos/química , Dissulfetos/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Ativação Linfocitária , Conformação Proteica , Linfócitos T/metabolismo , Microglobulina beta-2/metabolismoRESUMO
The MHC class I chain-related molecules (MICs) have previously been shown to be induced on most epithelial tumor cells. Engagement of MIC by the activating immune receptor NKG2D triggers NK cells and augments antigen-specific CTL anti-tumor immunity. The MIC-NKG2D system was proposed to participate in epithelial tumor immune surveillance. Paradoxically, studies suggest that tumors may evade MIC-NKG2D-mediated immunity by MIC shedding-induced impairment of effector cell function. Here we demonstrate the first evidence to our knowledge of a significant correlation of MIC shedding and deficiency in NK cell function with the grade of disease in prostate cancer. MIC is widely expressed in prostate carcinoma. The presence of surface target MIC, however, is counteracted by shedding. A significant increase in serum levels of soluble MIC (sMIC) and deficiency in NK cell function was shown in patients with advanced cancer. Finally, the deficiency in NK cell function can be overcome by treatment with IL-2 or IL-15 in vitro. Our results suggest that (a) deficiency in MIC-NKG2D immune surveillance may contribute to prostate cancer progression, (b) sMIC may be a novel biomarker for prostate cancer, and (c) using cytokines to restore MIC-NKG2D-mediated immunity may have clinical significance for prostate cancer in cell-based adaptive immunotherapy.
Assuntos
Antígenos HLA-A/metabolismo , Antígenos HLA-A/fisiologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/fisiologia , Neoplasias da Próstata/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antígeno CD56/metabolismo , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Humanos , Vigilância Imunológica , Interleucina-15/farmacologia , Interleucina-2/farmacologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/patologia , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores de Células Matadoras Naturais , Linfócitos T Citotóxicos/imunologia , Evasão TumoralRESUMO
NKG2D, together with NKp46 and NKp30, represents a major triggering receptor involved in the induction of cytotoxicity by both resting and activated human natural killer cells. In this study, we analyzed the expression and the functional relevance of MHC class I-related chain A (MICA) and UL16 binding protein (ULBP), the major cellular ligands for human NKG2D, in human tumor cell lines of different histological origin. We show that MICA and ULBP are frequently coexpressed by carcinoma cell lines, whereas MICA is expressed more frequently than ULBP by melanoma cell lines. Interestingly, the MICA(-) ULBP(+) phenotype was detected in most T cell leukemia cell lines, whereas the MICA(-) ULBP(-) phenotype characterized all acute myeloid leukemia and most B-cell lymphoma cell lines analyzed. These results, together with functional experiments, based on monoclonal antibody-mediated blocking of either NKG2D or its ligands, showed that killing of certain MICA(-) cell tumors is at least in part NKG2D dependent. Indeed, leukemic T cells as well as certain B-cell lymphomas were killed in a NKG2D-dependent fashion upon recognition of ULBP molecules. Moreover, ULBP could induce NKG2D-mediated NK cell triggering also in tumors coexpressing MICA. Our data suggest that the involvement of NKG2D in natural killer cell-mediated cytotoxicity strictly correlates with the expression and the surface density of MICA and ULBP on target cell tumors of different histotypes.
Assuntos
Proteínas de Transporte/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Proteínas de Transporte/biossíntese , Citotoxicidade Imunológica/imunologia , Proteínas Ligadas por GPI , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia/imunologia , Leucemia/metabolismo , Melanoma/imunologia , Melanoma/metabolismo , Proteínas de Membrana , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Neoplasias/metabolismo , Neuroblastoma/imunologia , Neuroblastoma/metabolismo , Receptores Imunológicos/imunologia , Receptores de Células Matadoras Naturais , Células Tumorais CultivadasRESUMO
OBJECTIVE: To replicate a previous study of nonmusculoskeletal responses to chiropractic intervention and to establish whether such responses are influenced by the country of study, chiropractors' attitudes, and information to patients, patients' demographic profiles, and treatment regimens. METHODS: Information obtained through questionnaires by chiropractors and patients on return visit within 2 weeks of previous treatment from chiropractic practices in Canada, United States, Mexico, Hong-Kong, Japan, Australia, and South Africa. In all, 385 chiropractors collected valid data on 5607 patients. Spinal manipulation with or without additional therapy was the intervention provided by chiropractors. Outcome measures included self-reported improved nonmusculoskeletal reactions (allergy, asthma, breathing, circulation, digestion, hearing, heart function, ringing in the ears, sinus problems, urination, and others). RESULTS: The results from the previous study were largely reproduced. Positive reactions were reported by 2% to 10% of all patients and by 3% to 27% of those who reported to have such problems. Most common were improved breathing (27%), digestion (26%), and circulation (21%). Some variables were identified that somewhat influenced the outcome: patients informed that such reactions may occur (odds ratio [OR] 1.5), treatment to the upper cervical spine (OR 1.4), treatment to lower thoracic spine (OR 1.3), and female sex (OR 1.3). However, these had a very small "explanatory" value (pseudo R2 3%). CONCLUSION: A minority of patients with self-reported nonmusculoskeletal symptoms report definite improvement after chiropractic care, and very few report definite worsening. Future studies should use stringent criteria to investigate a possible treatment effect and concentrate on specific diagnostic subgroups such as digestive problems and tinnitus.
Assuntos
Internacionalidade , Manipulação Quiroprática , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Inquéritos e Questionários , Resultado do TratamentoRESUMO
We evaluated the capacity of NK cells to influence expansion of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) in response to microbial Ags, using Mycobacterium tuberculosis as a model. We previously found that Tregs expand when CD4(+) cells and monocytes are exposed to M. tuberculosis. Addition of NK cells that were activated by monokines (IL-12, IL-15, and IL-18) or by exposure to M. tuberculosis-stimulated monocytes reduced Treg expansion in response to M. tuberculosis. NK cell inhibition of Treg expansion was not mediated through IFN-gamma. Activated NK cells lysed expanded, but not freshly isolated Tregs. Although monokines increased NK cell expression of the activating receptors NKp46, NKG2D, 2B4, CD16, and DNAM-1, only anti-NKG2D and anti-NKp46 inhibited NK cell lysis of expanded Tregs. Of five NKG2D ligands, only UL16-binding protein 1 (ULBP1) was up-regulated on M. tuberculosis-expanded Tregs, and anti-ULBP1 inhibited NK cell lysis of expanded Tregs. M. tuberculosis-stimulated monocytes activated NK cells to lyse expanded Tregs, and this was also inhibited by anti-NKG2D and anti-ULBP1, confirming the physiological relevance of this effect. Our study identifies a potential new role for NK cells in maintaining the delicate balance between the regulatory and effector arms of the immune response.
Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Mycobacterium tuberculosis/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/microbiologia , Antígenos CD4/análise , Células Cultivadas , Fatores de Transcrição Forkhead/análise , Proteínas Ligadas por GPI , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Interferon gama/metabolismo , Subunidade alfa de Receptor de Interleucina-2/análise , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Monocinas/farmacologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptores Imunológicos/agonistas , Receptores de Células Matadoras NaturaisRESUMO
MHC class I family members serve multiple functions beyond antigen presentation. We provide insight into the structure, expression and function of the Mill subfamily. This family includes two surface glycoproteins, Mill1 and Mill2. Protein sequences for Mill1 and Mill2 are most highly related to the NKG2D ligands, MICA and MICB, but neither of them bound to NKG2D. Computer-based protein modelling indicated that hereditary haemochromatosis protein (HFE), a molecule involved in iron uptake, was most similar. Mill1 and Mill2 were observed on cycling thymocytes, proliferating smooth muscle cells and fibroblasts. Using soluble Mill proteins, we found evidence for a soluble ligand in serum. Like HFE, the Mill family may be involved in nutrient metabolism. Skin was one of the only three organs found to express transcripts for both Mill1 and Mill2. Addition of antibodies specific for Mill2 to wounded skin enhanced healing. Our results suggest a role for the Mill proteins in cellular metabolism, with possible therapeutic significance.
Assuntos
Genes MHC Classe I/fisiologia , Glicoproteínas de Membrana/fisiologia , Cicatrização/fisiologia , Processamento Alternativo , Animais , Western Blotting , Proliferação de Células , Clonagem Molecular , Simulação por Computador , Feminino , Fibroblastos , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/química , Proteínas de Membrana/química , Camundongos , Camundongos Endogâmicos BALB C , Miócitos de Músculo Liso/metabolismo , Técnicas de Cultura de Órgãos , Ratos , Ratos Endogâmicos Lew , Pele/metabolismo , Linfócitos T , Timo/metabolismo , Útero/fisiologia , Microglobulina beta-2/metabolismoRESUMO
Natural killer (NK) cells are a major component of the host innate immune defence against various pathogens. Several viruses, including Human immunodeficiency virus 1 (HIV-1), have developed strategies to evade the NK-cell response. This study was designed to evaluate whether HIV-1 could interfere with the expression of NK cell-activating ligands, specifically the human leukocyte antigen (HLA)-I-like MICA and ULBP molecules that bind NKG2D, an activating receptor expressed by all NK cells. Results show that the HIV-1 Nef protein downmodulates cell-surface expression of MICA, ULBP1 and ULBP2, with a stronger effect on the latter molecule. The activity on MICA and ULBP2 is well conserved in Nef protein variants derived from HIV-1-infected patients. In HIV-1-infected cells, cell-surface expression of NKG2D ligands increased to a higher extent with a Nef-deficient virus compared with wild-type virus. Mutational analysis of Nef showed that NKG2D ligand downmodulation has structural requirements that differ from those of other reported Nef activities, including HLA-I downmodulation. Finally, data demonstrate that Nef expression has functional consequences on NK-cell recognition, causing a decreased susceptibility to NK cell-mediated lysis. These findings provide a novel insight into the mechanisms evolved by HIV-1 to escape from the NK-cell response.
Assuntos
Produtos do Gene nef/farmacologia , HIV-1/química , Células Matadoras Naturais/imunologia , Receptores Imunológicos/metabolismo , Linhagem Celular , Citotoxicidade Imunológica , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , HIV-1/imunologia , Humanos , Ligantes , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptores de Células Matadoras Naturais , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
At an early phase of viral infection, contact and cooperation between dendritic cells (DCs) and NK cells activates innate immunity, and also influences recruitment, when needed, of adaptive immunity. Influenza, an adaptable fast-evolving virus, annually causes acute, widespread infections that challenge the innate and adaptive immunity of humanity. In this study, we dissect and define the molecular mechanisms by which influenza-infected, human DCs activate resting, autologous NK cells. Three events in NK cell activation showed different requirements for soluble mediators made by infected DCs and for signals arising from contact with infected DCs. IFN-alpha was mainly responsible for enhanced NK cytolysis and also important for CD69 up-regulation, whereas IL-12 was necessary for enhancing IFN-gamma production. Increased CD69 expression and IFN-gamma production, but not increased cytolysis, required recognition of influenza-infected DCs by two NK cell receptors: NKG2D and NKp46. Abs specific for these receptors or their known ligands (UL16-binding proteins 1-3 class I-like molecules for NKG2D and influenza hemagglutinin for NKp46) inhibited CD69 expression and IFN-gamma production. Activation of NK cells by influenza-infected DCs and polyinosinic:polycytidylic acid (poly(I:C))-treated DCs was distinguished. Poly(I:C)-treated DCs did not express the UL16-binding protein 3 ligand for NKG2D, and in the absence of the influenza hemagglutinin there was no involvement of NKp46.
Assuntos
Células Dendríticas/imunologia , Influenza Humana/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/imunologia , Receptores Imunológicos/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Proteínas de Transporte/imunologia , Células Dendríticas/virologia , Hemaglutininas/imunologia , Humanos , Imunidade Inata , Lectinas Tipo C , Ativação Linfocitária/efeitos dos fármacos , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptor 1 Desencadeador da Citotoxicidade Natural , Poli I-C/imunologia , Poli I-C/farmacologia , Receptores de Células Matadoras Naturais , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Proteínas Virais/imunologiaRESUMO
Human cytomegalovirus (HCMV) employs a variety of strategies to modify or evade the host immune response, and natural killer (NK) cells play a crucial role in controlling cytomegalovirus infections in mice and humans. Activation of NK cells through the receptor NKG2D/DAP10 leads to killing of NKG2D ligand-expressing cells. We have previously shown that HCMV is able to down-regulate the surface expression of some NKG2D ligands, ULBP1, ULBP2, and MICB via the viral glycoprotein UL16. Here, we show that the viral gene product UL142 is able to down-regulate another NKG2D ligand, MICA, leading to protection from NK cytotoxicity. UL142 is not able to affect surface expression of all MICA alleles, however, which may reflect selective pressure on the host to thwart viral immune evasion, further supporting an important role for the MICA-NKG2D interaction in immune surveillance.
Assuntos
Antígenos de Histocompatibilidade Classe I/biossíntese , Glicoproteínas de Membrana/fisiologia , Receptores Imunológicos/metabolismo , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Citomegalovirus/imunologia , Regulação para Baixo , Células HeLa , Humanos , Células Matadoras Naturais , Ligantes , Glicoproteínas de Membrana/biossíntese , Camundongos , Dados de Sequência Molecular , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptores de Células Matadoras Naturais , Alinhamento de Sequência , Proteínas Virais/biossínteseRESUMO
NKG2D is an activating receptor that is expressed on most natural killer (NK) cells and CD8(+) T cells. MHC class I-related chain A(MICA) and UL16-binding protein (ULBP) 1, 2, and 3 are well-known ligands for NKG2D. Human gastric cancer cell lines, SNU216 and SNU638 cells which expressed UL16-binding protein (ULBP) were susceptible to NK cells in a NKG2D-dependent manner. However, SNU484 and SNU620 cells which had no ULBP on their surface were resistant to NK cells. ULBP 1, 2, and 3 are glycosylphosphatidylinositol (GPI)-anchored proteins which are sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC). When SNU620 cells were treated with U73122, an inhibitor of PI-PLC, the surface expression of ULBP was elevated with increased NK susceptibility. Pre-incubating NK cells with culture supernatants of SNU620 or SNU638 cells, which contained soluble ULBP protein, reduced NK cell activity by decreasing surface expression of NKG2D in NK cells. Furthermore, recombinant ULBP-Fc induced the down-regulation of NKG2D expression in NK cells. Taken together, down-regulation of NKG2D by soluble ULBP provides a potential mechanism by which gastric cancer cells escape NKG2D-mediated attack by the immune cells.
Assuntos
Proteínas de Transporte/metabolismo , Regulação para Baixo , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores Imunológicos/metabolismo , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proteínas Ligadas por GPI , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Proteínas de Membrana , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptores de Células Matadoras Naturais , Solubilidade , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/metabolismoRESUMO
Immune surveillance of the airways is critical to maintain the integrity and health of the lung. We have identified a family of ligands expressed on the surface of stressed airway epithelial cells whose function is to bind the NKG2D-activating receptor found on several pulmonary lymphocytes, including natural killer cells, gammadelta(+) T cells, and CD8(+) T cells. We employed real-time PCR and flow cytometry in normal and transformed airway epithelial cell to demonstrate that major histocompatibility complex class I chain-related (MIC) B and the UL-16 binding protein (ULBP) ligands (ULBP1-4) are ubiquitously expressed at the mRNA level in all cell lines. MICA/B surface expression was present on 70% of transformed cell lines but was undetectable on primary cells. We demonstrate that MICA/B and ULBP 1, 2, 3, and 4 expression is rare or absent on the cell surface of unstimulated normal human bronchial epithelial cells although transcripts and intracellular proteins are present. Normal human bronchial epithelial cells exposed to 0.3 mM hydrogen peroxide exhibit an induction of all ligands examined on the cell surface. Surface expression is independent of changes in transcript level or total cellular protein and is mediated by the ERK family of mitogen-activated protein kinases. The induction of NKG2D ligands on stressed airway epithelial cells represents a potentially important mechanism of immune cell activation in regulation of pulmonary health and disease.
Assuntos
Células Epiteliais/metabolismo , Ligantes , Receptores Imunológicos/metabolismo , Mucosa Respiratória/citologia , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Ligadas por GPI , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Oxidantes/farmacologia , Estresse Oxidativo , Isoformas de Proteínas/metabolismo , Receptores Imunológicos/genética , Receptores de Células Matadoras Naturais , Mucosa Respiratória/metabolismoRESUMO
ULBPs are human ligands for NKG2D, an activating receptor expressed on natural killer (NK) cells, NK1.1(+) T cells, and T cells. ULBPs are expressed by a variety of leukemias, carcinomas, melanomas, and tumor cell lines. ULBP expression correlates with improved survival in cancer patients, however, the nature of the immune response that ULBPs elicit is not well understood. We report that ectopic expression of ULBP1 or ULBP2 on murine EL4 or RMA tumor cells elicits potent antitumor responses in syngeneic C57BL/6 and SCID mice. Although binding of ULBP3 to murine NKG2D could not be demonstrated in vitro, ULBP3 can also stimulate antitumor responses, suggesting that ULBP3 binds to murine NKG2D or possibly another receptor in vivo. ULBP expression was found to recruit NK cells, NK1.1(+) T cells, and T cells to the tumor. IL-15 was found to strongly enhance the immune response directed against ULBP-expressing tumors. Tumors can evade NKG2D immunity by down-regulating expression of NKG2D. Our data suggest that IL-15 may be useful for overcoming this tumor-evasion strategy. Together, these results demonstrate that ULBP expression can elicit a potent immune response and suggest that ULBPs, alone or in combination with IL-15, can be exploited for antitumor therapy.
Assuntos
Proteínas de Transporte/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Interleucina-15/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Receptores Imunológicos/imunologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/uso terapêutico , Linhagem Celular Tumoral , Proteínas Ligadas por GPI , Regulação Neoplásica da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/uso terapêutico , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Células Matadoras Naturais/imunologia , Ligantes , Proteínas de Membrana , Camundongos , Camundongos SCID , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Neoplasias/genética , Neoplasias/mortalidade , Neoplasias/terapia , Receptores Imunológicos/genética , Receptores de Células Matadoras Naturais , Linfócitos T/imunologia , Evasão Tumoral/genética , Evasão Tumoral/imunologiaRESUMO
Human cytomegalovirus (HCMV) down-regulates cell surface expression of HLA class I molecules (HLA-I). UL18, an HCMV-encoded HLA-I homologue, has been proposed to protect virus-infected cells against NK cell recognition by engaging the inhibitory receptor leukocyte Ig-like receptor (LIR)-1, which also binds a broad spectrum of HLA-I alleles, including HLA-G1. Because genetic and biological differences exist among HCMV strains, we characterized laboratory (AD169) and clinical (4636, 13B, 109B) strain-derived UL18 proteins. Compared to the known AD169-derived UL18, mutations were found in clinical strain-derived UL18. They were clustered in the alpha3 domain (13B), previously shown to be critical for LIR-1 binding, or in the alpha1 domain (4636). Iotan cytotoxicity assays, pretreatment of LIR-1+ NKL with soluble 4636-UL18 completely abolished LIR-1-dependent protection from NK lysis, conferred by the expression of HLA-G1 on target cells (721.221-HLA-G1+). Similarly, flow cytometry, Biacore and ELISA experiments showed 4636-UL18 and 13B-UL18 to have the strongest binding capacity to LIR-1. Our results suggest the importance of two independent UL18 regions for LIR-1 binding, one localized on the tip of the alpha3 domain, and another composed of two loops that emerge from the alpha1 domain. Strain variations in these domains may result in different UL18-mediated effects on LIR-1+ cells during the course of HCMV infection.
Assuntos
Substituição de Aminoácidos , Antígenos CD/imunologia , Proteínas do Capsídeo/imunologia , Citomegalovirus/imunologia , Células Matadoras Naturais/imunologia , Mutação Puntual , Receptores Imunológicos/imunologia , Proteínas do Capsídeo/genética , Células Cultivadas , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Antígenos HLA/imunologia , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunidade Celular/genética , Imunidade Celular/imunologia , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Ligação Proteica/genética , Ligação Proteica/imunologia , Estrutura Terciária de Proteína/genética , Especificidade da EspécieRESUMO
NKG2D is a homodimeric C-type lectin-related receptor expressed on natural killer (NK) cells and T cells. In mice, alternative deoxyribonucleic acid (DNA) splicing generates two isoforms of NKG2D that differ in the length of their cytoplasmic domains. Their ability to induce cellular activation is mediated via association with two membrane-bound, signaling adaptor molecules, DAP10 and DAP12. It has been reported that the long form of NKG2D associates exclusively with DAP10, whereas the short variant can interact with either adaptor. The short isoform was reported to be almost undetectable in naive NK cells. Using two distinct cell types, we demonstrate that like the short isoform, the long variant of NKG2D also associates not only with DAP10 but also with DAP12. Using reporter cells (70Z/3), we demonstrate that DAP12 can compete equally with DAP10 for association with both variants of NKG2D when DAP10 and DAP12 are coexpressed. Cross-linking either isoform of NKG2D induces a calcium flux when associated exclusively with DAP10 or DAP12. Moreover, using quantitative polymerase chain reaction (PCR), we also show that the short isoform of NKG2D is expressed in naive NK cells. Our data suggest that signaling via mouse NKG2D isoforms is more complex than originally presented.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Matadoras Naturais/imunologia , Proteínas de Membrana/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Processamento Alternativo , Animais , Linhagem Celular , Células Matadoras Naturais/metabolismo , Camundongos , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Células Matadoras Naturais , Recombinação GenéticaRESUMO
Innate and adaptive immunity have not evolved separately. In this regard, the NKG2D molecule first identified on NK cells and classified as an activating NK cell receptor is also an important receptor for CD8(+) T cells. Functional analyses of human NKG2D and its ligands, i.e. UL16 binding proteins (ULBP) and MHC class I chain-related (MIC), have so far focused on immune cell-target cell situations because of the expression of NKG2D ligands on infected, stressed or transformed cells. Here, however, we address a possible function of NKG2D/ULBP-1 during the initiation of T cell responses. ULBP-1 can be detected on mature dendritic cells both in situ in the T cell areas of lymph nodes as well as in vitro after artificial maturation. FCM analysis further demonstrated that although NKG2D is expressed to some degree on all analyzed T cell subsets from peripheral blood, in vitro stimulation of T cells results in up-regulation of NKG2D on proliferating T cells. Using the sentinel lymph nodes of primary melanoma as a model for induction of defined T cell responses in vivo, we were able to demonstrate the expression of NKG2D on melanoma-associated antigen-specific T cells. Thus, our results suggest a role for NGK2D-ULBP-1 in the induction or reactivation of T cell responses.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas de Transporte/biossíntese , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/imunologia , Proteínas de Transporte/imunologia , Células Dendríticas/metabolismo , Citometria de Fluxo , Proteínas Ligadas por GPI , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Linfonodos/citologia , Linfonodos/imunologia , Melanoma/imunologia , Proteínas de Membrana , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Reação em Cadeia da Polimerase , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Receptores de Células Matadoras Naturais , Biópsia de Linfonodo SentinelaRESUMO
BALB/c thymocytes can be divided into three distinct subsets according to the expression of a ligand for the NK activation receptor NKG2D (NKG2D-L) and the expression of MHC class I (MHC-I). The first subset (MHC-Imid/NKG2D-Lhigh or "N+") is predominately CD4+CD8+ double positive (DP), comprises approximately 35% of thymocytes in a 6-8-week-old adult and contains uncommitted cells that have neither undergone selection nor are committed to death by neglect. The second subset (MHC-Ilow/NKG2D-Llow or "M-"), also mostly DP cells, comprises approximately 50% of thymocytes and consists of cells committed to death by apoptosis, likely due to neglect. By contrast, the third subset (MHC-Ihigh/NKG2D-Llow or "M+") is largely single positive (SP), represents approximately 15% of thymocytes and mostly contains more mature cells that have undergone successful positive selection. The major advantage of the analysis is that it splits DP cells into two subpopulations, one committed to death by apoptosis and the other subjected to selection. The analysis also suggests that NKG2D-L may play a role in thymocyte development.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Receptores Imunológicos/metabolismo , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Biomarcadores , Caspases/metabolismo , Regulação para Baixo , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/genética , Lectinas Tipo C , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptores de Antígenos de Linfócitos T/imunologia , Receptores Imunológicos/genética , Receptores de Células Matadoras Naturais , Subpopulações de Linfócitos T/enzimologia , Timo/citologia , Timo/metabolismo , Fatores de TempoRESUMO
Natural killer (NK) cell-mediated cytolytic activity against tumors requires the engagement of activating NK receptors by the tumor-associated ligands. Here, we have studied the role of NKG2D and natural cytotoxicity receptors (NCRs) in the recognition of human leukemia. To detect as-yet-unknown cell-surface molecules recognized by NCRs, we developed soluble forms of NKp30, NKp44, and NKp46 as staining reagents binding the putative cognate ligands. Analysis of UL16-binding protein-1 (ULBP1), ULBP2, and ULBP3 ligands for NKG2D and of potential ligands for NKp30, NKp44, and NKp46 in healthy hematopoietic cells demonstrated the ligand-negative phenotype of bone marrow-derived CD34(+) progenitor cells and the acquisition of cell-surface ligands during the course of myeloid differentiation. In acute myeloid leukemia (AML), leukemic blasts from approximately 80% of patients expressed very low levels of ULBPs and NCR-specific ligands. Treatment with differentiation-promoting myeloid growth factors, together with interferon-gamma, upregulated cell-surface levels of ULBP1 and putative NCR ligands on AML blasts, conferring an increased sensitivity to NK cell-mediated lysis. We conclude that the ligand-negative/low phenotype in AML is a consequence of cell maturation arrest on malignant transformation and that defective expression of ligands for the activating NKG2D and NCR receptors may compromise leukemia recognition by NK cells.