RESUMEN
BACKGROUND: Advanced gastric cancer (GC) has a poor prognosis. This study aimed to identify novel GC-related genes as potential therapeutic targets. METHODS: Killer cell lectin-like receptor G2 (KLRG2) was identified as a candidate gene by transcriptome analysis of metastatic GC tissues. Small interfering RNA-mediated KLRG2 knockdown in human GC cell lines was used to investigate KLRG2 involvement in signaling pathways and functional behaviors in vitro and in vivo. Clinicopathological data were analyzed in patients stratified according to tumor KLRG2 mRNA expression. RESULTS: KLRG2 knockdown in GC cells decreased cell proliferation, migration, and invasion; caused cell cycle arrest in G2/M phase; induced apoptosis via caspase activation; suppressed JAK/STAT and MAPK-ERK1/2 pathway activities; and upregulated p53 and p38 MAPK activities. In mouse xenograft models of peritoneal metastasis, the number and weight of disseminated GC nodules were decreased by KLRG2 knockdown. High tumor levels of KLRG2 mRNA were significantly associated with lower 5-year overall survival (OS) and relapse-free survival (RFS) rates in patients with Stage I-III GC (5-year OS rate: 64.4% vs. 80.0%, P = 0.009; 5-year RFS rate: 62.8% vs. 78.1%, P = 0.030). CONCLUSIONS: KLRG2 knockdown attenuated the malignant phenotypes of GC cells via downregulation of JAK/STAT and MAPK-ERK1/2 pathway activity and upregulation of p38 MAPK and p53. Targeted suppression of KLRG2 may serve as a new treatment approach for GC.
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Quinasas Janus , Neoplasias Gástricas , Humanos , Animales , Ratones , Quinasas Janus/genética , Quinasas Janus/metabolismo , Transducción de Señal , Neoplasias Gástricas/patología , Sistema de Señalización de MAP Quinasas , Proteína p53 Supresora de Tumor/genética , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismo , Proliferación Celular/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , ARN Mensajero/metabolismo , Receptores Similares a Lectina de Células NK/genética , Receptores Similares a Lectina de Células NK/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión GénicaRESUMEN
Natural killer (NK) cells recognize virally infected cells and tumors. NK cell function depends on balanced signaling from activating receptors, recognizing products from tumors or viruses, and inhibitory receptors (such as KIR/Ly49), which recognize major histocompatibility complex class I (MHC-I) molecules. KIR/Ly49 signaling preserves tolerance to self but also conveys reactivity toward MHC-I-low target cells in a process known as NK cell education. Here, we found that NK cell tolerance and education were determined by the subcellular localization of the tyrosine phosphatase SHP-1. In mice lacking MHC-I molecules, uneducated, self-tolerant Ly49A+ NK cells showed accumulation of SHP-1 in the activating immune synapse, where it colocalized with F-actin and the signaling adaptor protein SLP-76. Education of Ly49A+ NK cells by the MHC-I molecule H2Dd led to reduced synaptic accumulation of SHP-1, accompanied by augmented signaling from activating receptors. Education was also linked to reduced transcription of Ptpn6, which encodes SHP-1. Moreover, synaptic SHP-1 accumulation was reduced in NK cells carrying the H2Dd-educated receptor Ly49G2 but not in those carrying the noneducating receptor Ly49I. Colocalization of Ly49A and SHP-1 outside of the synapse was more frequent in educated compared with uneducated NK cells, suggesting a role for Ly49A in preventing synaptic SHP-1 accumulation in NK cell education. Thus, distinct patterning of SHP-1 in the activating NK cell synapse may determine NK cell tolerance.
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Antígenos Ly , Células Asesinas Naturales , Ratones , Animales , Receptores Similares a Lectina de Células NK/metabolismo , Antígenos Ly/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Sinapsis/metabolismoRESUMEN
Effector CD8+ T cells are crucial players in adaptive immunity for effective protection against invading pathogens. The regulatory mechanisms underlying CD8+ T cell effector differentiation are incompletely understood. In this study, we defined a critical role of mediator complex subunit 1 (Med1) in controlling effector CD8+ T cell differentiation and survival during acute bacterial infection. Mice with Med1-deficient CD8+ T cells exhibited significantly impaired expansion with evidently reduced killer cell lectin-like receptor G1+ terminally differentiated and Ly6c+ effector cell populations. Moreover, Med1 deficiency led to enhanced cell apoptosis and expression of multiple inhibitory receptors (programmed cell death 1, T cell Ig and mucin domain-containing-3, and T cell immunoreceptor with Ig and ITIM domains). RNA-sequencing analysis revealed that T-bet- and Zeb2-mediated transcriptional programs were impaired in Med1-deficient CD8+ T cells. Overexpression of T-bet could rescue the differentiation and survival of Med1-deficient CD8+ effector T cells. Mechanistically, the transcription factor C/EBPß promoted T-bet expression through interacting with Med1 in effector T cells. Collectively, our findings revealed a novel role of Med1 in regulating effector CD8+ T cell differentiation and survival in response to bacterial infection.
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Linfocitos T CD8-positivos , Subunidad 1 del Complejo Mediador , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular , Subunidad 1 del Complejo Mediador/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mucinas/metabolismo , ARN/metabolismo , Receptores Similares a Lectina de Células NK/metabolismo , Proteínas de Dominio T Box/metabolismoRESUMEN
Inclusion body myositis (IBM) is an inflammatory myopathy of aged people with poor response to therapy. To characterize muscle-invading inflammatory cells, we performed immunohistochemical and ultrastructural studies on muscle biopsies from 10 patients with IBM with durations of illness from 3 to 84 months. At the surface of muscle fibers, 79% and 48% of CD8+ cells were positive for killer cell lectin-like receptor subfamily G, member 1 (KLRG1) and CD57, respectively. CD8+KLRG1+ cells are highly differentiated cytotoxic cells. On an average, 27% of CD8-CD57+KLRG1+ cells at the surface were CD4+. Proportions of CD28+ cells among KLRG1+ cells showed a negative correlation with duration of illness (r = -0.68). These changes indicated progressive differentiation of CD8+ T cells. Moreover, PD-1 expression on CD57+ and CD8+ cells increased early, then fluctuated, and reincreased in later stages. PD ligand-1 (PD-L1) and PD-L2 were expressed on adjacent cells including muscle fibers. T cell large granular lymphocytes (LGLs) are potent effector cells and cells with ultrastructure indistinguishable from LGLs were seen in the sarcoplasm along with lymphocytes undergoing degeneration. Together, along the course of IBM, some inflammatory cells retained the potential for cytotoxicity whereas others indicated suppression by exhaustion, senescence, or through the PD-1 pathway.
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Miositis por Cuerpos de Inclusión , Antígeno B7-H1/metabolismo , Antígenos CD28/metabolismo , Linfocitos T CD8-positivos/metabolismo , Humanos , Ligandos , Fibras Musculares Esqueléticas/patología , Miositis por Cuerpos de Inclusión/patología , Fenotipo , Receptor de Muerte Celular Programada 1/metabolismo , Receptores Similares a Lectina de Células NK/metabolismoRESUMEN
MHC class I (MHC I) expression in the host influences NK cells in a process termed education. The result of this education is reflected in the responsiveness of NK cells at the level of individual cells as well as in the repertoire of inhibitory MHC I-specific receptors at the NK cell system level. The presence of MHC I molecules in the host environment gives rise to a skewed receptor repertoire in spleen NK cells where subsets expressing few (one or two) inhibitory receptors are expanded whereas subsets with many (three or more) receptors are contracted. It is not known whether this MHC I-dependent skewing is imposed during development or after maturation of NK cells. In this study, we tested the hypothesis that the NK cell receptor repertoire is shaped already early during NK cell development in the bone marrow. We used mice with a repertoire imposed by a single MHC I allele, as well as a C57BL/6 mutant strain with exaggerated repertoire skewing, to investigate Ly49 receptor repertoires at different stages of NK cell differentiation. Our results show that NK cell inhibitory receptor repertoire skewing can indeed be observed in the bone marrow, even during the earliest developmental steps where Ly49 receptors are expressed. This may partly be accounted for by selective proliferation of certain NK cell subsets, but other mechanisms must also be involved. We propose a model for how repertoire skewing is established during a developmental phase in the bone marrow, based on sequential receptor expression as well as selective proliferation.
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Médula Ósea , Subfamilia A de Receptores Similares a Lectina de Células NK , Animales , Antígenos Ly/metabolismo , Médula Ósea/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Subfamilia A de Receptores Similares a Lectina de Células NK/genética , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Receptores Similares a Lectina de Células NK/metabolismo , Receptores de Células Asesinas Naturales/metabolismoRESUMEN
Natural killer (NK) cell responses depend on the balance of signals from inhibitory and activating receptors. However, how the integration of antagonistic signals occurs upon NK cell-target cell interaction is not fully understood. Here we provide evidence that NK cell inhibition via the inhibitory receptor Ly49A is dependent on its relative colocalization at the nanometer scale with the activating receptor NKG2D upon immune synapse (IS) formation. NKG2D and Ly49A signal integration and colocalization were studied using NKG2D-GFP and Ly49A-RFP-expressing primary NK cells, forming ISs with NIH3T3 target cells, with or without the expression of single-chain trimer (SCT) H2-Dd and an extended form of SCT H2-Dd-CD4 MHC-I molecules. Nanoscale colocalization was assessed by Förster resonance energy transfer between NKG2D-GFP and Ly49A-RFP and measured for each synapse. In the presence of their respective cognate ligands, NKG2D and Ly49A colocalize at the nanometer scale, leading to NK cell inhibition. However, increasing the size of the Ly49A ligand reduced the nanoscale colocalization with NKG2D, consequently impairing Ly49A-mediated inhibition. Thus, our data shows that NK cell signal integration is critically dependent on the dimensions of NK cell ligand-receptor pairs by affecting their relative nanometer-scale colocalization at the IS. Our results together suggest that the balance of NK cell signals and NK cell responses is determined by the relative nanoscale colocalization of activating and inhibitory receptors in the immune synapse.
Asunto(s)
Subfamilia A de Receptores Similares a Lectina de Células NK , Subfamilia K de Receptores Similares a Lectina de Células NK , Animales , Proteínas Portadoras/metabolismo , Antígenos H-2 , Antígeno de Histocompatibilidad H-2D/metabolismo , Células Asesinas Naturales , Lectinas Tipo C/metabolismo , Ligandos , Ratones , Células 3T3 NIH , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Receptores Similares a Lectina de Células NK/metabolismo , Receptores de Células Asesinas Naturales/metabolismoRESUMEN
BACKGROUND: Severe dengue, characterized by shock and organ dysfunction, is driven by an excessive host immune response. We investigated the role of hyperinflammation in dengue pathogenesis. METHODS: Patients recruited into an observational study were divided into 3 plasma leak severity grades. Hyperinflammatory biomarkers were measured at 4 time points. Frequencies, activation, and cytotoxic potential of natural killer (NK) cells were analyzed by flow cytometry. RNA was extracted from sorted CD56+ NK cells and libraries were prepared using SMART-Seq and sequenced using HiSeq3000 (Illumina). RESULTS: Sixty-nine patients were included (grade 0, 42 patients; grade 1, 19 patients; grade 2, 8 patients). Patients with grade 2 leakage had higher biomarkers than grade 0, including higher peak ferritin levels (83.3% vs 45.2%) and H-scores (median, 148.5 vs 105.5). NK cells from grade 2 patients exhibited decreased expression of perforin and granzyme B and activation markers. RNA sequencing revealed 3 single-nucleotide polymorphisms in NK cell functional genes associated with more severe leakage-NK cell lectin-like receptor K1 gene (KLRK1) and perforin 1 (PRF1). CONCLUSIONS: Features of hyperinflammation are associated with dengue severity, including higher biomarkers, impaired NK cell function, and polymorphisms in NK cell cytolytic function genes (KLRK1 and PRF1). Trials of immunomodulatory therapy in these patients is now warranted.
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Dengue Grave , Humanos , Biomarcadores/metabolismo , Ferritinas , Granzimas/genética , Granzimas/metabolismo , Células Asesinas Naturales , Perforina/genética , Perforina/metabolismo , Polimorfismo Genético , Receptores Similares a Lectina de Células NK/genética , Receptores Similares a Lectina de Células NK/metabolismo , ARNRESUMEN
Massive infiltrated and enriched decidual macrophages (dMφ) have been widely regarded as important regulators of maternal-fetal immune tolerance and trophoblast invasion, contributing to normal pregnancy. However, the characteristics of metabolic profile and the underlying mechanism of dMφ residence remain largely unknown. Here, we observe that dMφ display an active glycerophospholipid metabolism. The activation of ENPP2-lysophosphatidic acid (LPA) facilitates the adhesion and retention, and M2 differentiation of dMφ during normal pregnancy. Mechanistically, this process is mediated through activation of the LPA receptors (LPAR1 and PPARG/PPARγ)-DDIT4-macroautophagy/autophagy axis, and further upregulation of multiple adhesion factors (e.g., cadherins and selectins) in a CLDN7 (claudin 7)-dependent manner. Additionally, poor trophoblast invasion and placenta development, and a high ratio of embryo loss are observed in Enpp2±, lpar1-/- or PPARG-blocked pregnant mice. Patients with unexplained spontaneous abortion display insufficient autophagy and cell residence of dMφ. In therapeutic studies, supplementation with LPA or the autophagy inducer rapamycin significantly promotes dMφ autophagy and cell residence, and improves embryo resorption in Enpp2± and spontaneous abortion mouse models, which should be dependent on the activation of DDIT4-autophagy-CLDN7-adhesion molecules axis. This observation reveals that inactivation of ENPP2-LPA metabolism and insufficient autophagy of dMφ result in resident obstacle of dMφ and further increase the risk of spontaneous abortion, and provides potential therapeutic strategies to prevent spontaneous abortion.Abbreviations: ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; Atg5: autophagy related 5; ATG13: autophagy related 13; BECN1: beclin 1; CDH1/E-cadherin: cadherin 1; CDH5/VE-cadherin: cadherin 5; CFSE: carboxyfluorescein succinimidyl ester; CLDN7: claudin 7; CSF1/M-CSF: colony stimulating factor 1; CSF2/GM-CSF: colony stimulating factor 2; Ctrl: control; CXCL10/IP-10: chemokine (C-X-C) ligand 10; DDIT4: DNA damage inducible transcript 4; dMφ: decidual macrophage; DSC: decidual stromal cells; ENPP2/ATX: ectonucleotide pyrophosphatase/phosphodiesterase 2; Enpp2±: Enpp2 heterozygous knockout mouse; ENPP2i/PF-8380: ENPP2 inhibitor; EPCAM: epithelial cell adhesion molecule; ESC: endometrial stromal cells; FGF2/b-FGF: fibroblast growth factor 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPCPD1: glycerophosphocholine phosphodiesterase 1; HE: heterozygote; HIF1A: hypoxia inducible factor 1 subunit alpha; HNF4A: hepatocyte nuclear factor 4 alpha; HO: homozygote; ICAM2: intercellular adhesion molecule 2; IL: interleukin; ITGAV/CD51: integrin subunit alpha V; ITGAM/CD11b: integrin subunit alpha M; ITGAX/CD11b: integrin subunit alpha X; ITGB3/CD61: integrin subunit beta 3; KLRB1/NK1.1: killer cell lectin like receptor B1; KRT7/cytokeratin 7: keratin 7; LPA: lysophosphatidic acid; LPAR: lysophosphatidic acid receptor; lpar1-/-: lpar1 homozygous knockout mouse; LPAR1i/AM966: LPAR1 inhibitor; LY6C: lymphocyte antigen 6 complex, locus C1; LYPLA1: lysophospholipase 1; LYPLA2: lysophospholipase 2; Lyz2: lysozyme 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MARVELD2: MARVEL domain containing 2; 3-MA: 3-methyladenine; MBOAT2: membrane bound O-acyltransferase domain containing 2; MGLL: monoglyceride lipase; MRC1/CD206: mannose receptor C-type 1; MTOR: mechanistic target of rapamycin kinase; NP: normal pregnancy; PDGF: platelet derived growth factor; PLA1A: phospholipase A1 member A; PLA2G4A: phospholipase A2 group IVA; PLPP1: phospholipid phosphatase 1; pMo: peripheral blood monocytes; p-MTOR: phosphorylated MTOR; PPAR: peroxisome proliferator activated receptor; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; PPARGi/GW9662: PPARG inhibitor; PTPRC/CD45: protein tyrosine phosphatase receptor type, C; Rapa: rapamycin; RHEB: Ras homolog, mTORC1 binding; SA: spontaneous abortion; SELE: selectin E; SELL: selectin L; siCLDN7: CLDN7-silenced; STAT: signal transducer and activator of transcription; SQSTM1: sequestosome 1; TJP1: tight junction protein 1; VCAM1: vascular cell adhesion molecule 1; WT: wild type.
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Aborto Espontáneo , Autofagia , Aborto Espontáneo/genética , Aborto Espontáneo/metabolismo , Actinas/metabolismo , Aciltransferasas/metabolismo , Animales , Autofagia/genética , Beclina-1/metabolismo , Cadherinas/metabolismo , Quimiocina CXCL10/metabolismo , Claudinas/metabolismo , Molécula de Adhesión Celular Epitelial/metabolismo , Ésteres/metabolismo , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Glicerofosfolípidos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Fosfolipasas A2 Grupo IV/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Factor 1 Inducible por Hipoxia/metabolismo , Integrinas/metabolismo , Queratina-7/metabolismo , Ligandos , Lisofosfolipasa/metabolismo , Lisofosfolípidos/metabolismo , Proteína 2 con Dominio MARVEL , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Monoacilglicerol Lipasas/metabolismo , Muramidasa/metabolismo , PPAR gamma/metabolismo , Fosfolipasas , Fosfolipasas A1/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Embarazo , Pirofosfatasas/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores Similares a Lectina de Células NK/metabolismo , Selectinas/metabolismo , Proteína Sequestosoma-1/metabolismo , Sirolimus , Serina-Treonina Quinasas TOR/metabolismo , Tioléster HidrolasasRESUMEN
OBJECTIVE: Natural killer (NK) cell-deficient mice are useful models in biomedical research. NOD/SCID mice have been used as a model of this type in research. However, the actual status of NK cells in NOD/SCID mice and CB17/SCID mice in comparison with that in BALB/c mice has not been sufficiently evaluated. METHODS: Splenocytes from naïve or poly(I:C)-treated mice were isolated for phenotyping and analysis of cytotoxicity-related molecules and inhibitory receptors; for cytotoxicity assay, purified NK cells were also used. RESULTS: The proportion of splenic NK cells did not differ significantly between NOD/SCID and CB17/SCID mice. The perforin levels in NK cells were similar between the poly(I:C)-treated CB17/SCID and NOD/SCID mice, while the granzyme B and NKG2A/C/E levels in NK cells from NOD/SCID mice were significantly lower than those from CB17/SCID mice. Moreover, the NKG2D and Ly49A levels in NK cells from NOD/SCID mice were higher than those from CB17/SCID. The splenocytes from CB17/SCID mice showed higher cytotoxicity than those from NOD/SCID mice, while the cytotoxicity of purified NK cells basically did not differ between the two strains. After in vitro stimulation with cytokines, the splenocytes from CB17/SCID mice showed higher IFN-γ production than those from NOD/SCID mice; however, NK cells did not. CONCLUSION: There was no significant difference in the proportion of splenic NK cells between CB17/SCID and NOD/SCID mice, and the function of NK cells was only partially compromised in NOD/SCID mice. Caution should be taken when considering the use of NOD/SCID mice as an NK-deficient model.
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Células Asesinas Naturales/inmunología , Ratones Endogámicos NOD/inmunología , Ratones SCID/inmunología , Animales , Antígenos Ly/metabolismo , Citocinas/biosíntesis , Citotoxicidad Inmunológica , Inmunidad Innata , Técnicas In Vitro , Células Asesinas Naturales/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Animales , Modelos Inmunológicos , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismo , Receptores Similares a Lectina de Células NK/metabolismo , Especificidad de la EspecieRESUMEN
The ability of cancer cells to invade and disseminate can be affected by components of the surrounding microenvironment. To identify dermal components that regulate the growth of epidermal carcinomas, we studied the genetic disease called xeroderma pigmentosum that bears mutations in genes involved in the nucleotide excision repair of DNA. Patients with xeroderma pigmentosum are more prone to develop cutaneous tumors than the general population and their dermal fibroblasts display the features of dermal cancer-associated fibroblasts, which promote the invasion of keratinocytes. Here, we report that 3-dimensional dermal cultures of fibroblasts from healthy donors but not from patients with xeroderma pigmentosum complementation group C express CLEC2A, which is the ligand of the activating NK cell receptor NKp65. A similar loss of CLEC2A was observed in sporadic dermal cancer-associated fibroblasts and upon the culture of fibroblasts with cutaneous squamous cell carcinoma-conditioned medium. Using an innovative 3-dimensional organotypic skin culture model that contain NK cells in addition to fibroblasts and squamous cell carcinoma cells, we unveiled a key role of CLEC2A that orchestrates a crosstalk between fibroblasts and NK cells, thereby leading to the control of squamous cell carcinoma invasion. These findings indicate that CLEC2A-expressing dermal fibroblasts play a major role in immune surveillance of the skin.
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Fibroblastos Asociados al Cáncer/patología , Carcinoma de Células Escamosas/inmunología , Lectinas Tipo C/deficiencia , Neoplasias Cutáneas/inmunología , Xerodermia Pigmentosa/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Fibroblastos Asociados al Cáncer/inmunología , Carcinoma de Células Escamosas/patología , Comunicación Celular/inmunología , Células Cultivadas , Niño , Preescolar , Técnicas de Cocultivo , Proteínas de Unión al ADN/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Vigilancia Inmunológica , Lactante , Recién Nacido , Células Asesinas Naturales/inmunología , Masculino , Invasividad Neoplásica/inmunología , Invasividad Neoplásica/patología , Cultivo Primario de Células , Receptores Similares a Lectina de Células NK/metabolismo , Piel/inmunología , Piel/patología , Neoplasias Cutáneas/patología , Microambiente Tumoral/inmunología , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/inmunología , Adulto JovenRESUMEN
Human gnathostomiasis is mainly caused by third-stage larvae of Gnathostoma spinigerum (G. spinigerum L3). Excretory-secretory products (ES) released from infective helminthic larvae are associated with larval migration and host immunity modulation. Natural killer (NK) cells have important immune functions against helminth infection. Currently, the effects of ES from G. spinigerum L3 (G. spinigerum ES) on NK cell activity are unclear. This study investigated whether G. spinigerum ES affected human NK cells. Human normal peripheral blood mononuclear cell (PBMC) cultures were used to mimic immune cells within the circulation. PBMC were co-cultured with G. spinigerum ES (0.01-0.05 µg/ml) for 5 or 7 days. Levels of IFN-γ in cultured supernatants were measured by enzyme-linked immunosorbent assay. The expressions of mRNA encoding NK cell receptors, especially the C type killer cell lectin-like family (KLR; NKG2A, NKG2C, and NKG2D) and IFN-γ in ES induced PBMC were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). ES induced PBMC markedly decreased the levels of IFN-γ and increased the expressions of NKG2A and NKG2D on NK cells. In conclusion, low amounts of G. spinigerum ES modulated NK cells by downregulating the transcription of IFN-γ and upregulating the expressions of KLR (NKG2A and NKG2D receptors) during the 7-day observation period. These findings indicate more in-depth studies of NK cell function are required to better understand the mechanism involved in immune evasive strategies of human gnathostomiasis.
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Gnathostoma/inmunología , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Receptores Similares a Lectina de Células NK/metabolismo , Animales , Técnicas de Cocultivo , Regulación hacia Abajo , Gnathostoma/crecimiento & desarrollo , Gnathostomiasis/inmunología , Humanos , Células Asesinas Naturales/metabolismo , Larva/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Regulación hacia ArribaRESUMEN
Unrelated cord blood (CB) is an excellent alternative as an allogeneic donor source for stem cell transplantation. CB transplantation is associated with lower incidence of severe acute graft versus host disease (GVHD) and chronic GVHD but similar rates of malignant relapse compared with other unrelated donor cell transplants. NK cells are critical innate immune components and the comparison of CB vs. peripheral blood (PB) NK cells is relatively unknown. NK cell receptor expression, cell function, and maturation may play a role in the risk of relapse after CB transplant. We investigated CB vs. PB NK cell subset cytotoxicity, function, receptor expression, and genomic and proteomic signatures. The CB CD56dim compared with PB CD56dim demonstrated significantly increased expression of NKG2A and NKG2D, respectively. CB vs. PB CD56dim NK cells had significantly decreased in vitro cytotoxicity against a variety of non-Hodgkin lymphoma targets. Various proteins were significantly under- and over-expressed in CB vs. PB CD56dim NK cells. Microarray analyses and qRT-PCR in CB vs. PB CD56dim demonstrated significantly increased expression of genes in cell regulation and development of apoptosis, respectively. In summary, CB vs. PB CD56dim NK cells appear to be earlier in development, have decreased functional activity, and increased capacity for programmed cell death, suggesting that CB NK cells require functional and maturational stimulation to achieve similar functional levels as PB CD56dim NK cells.
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Antígeno CD56/sangre , Sangre Fetal/inmunología , Células Asesinas Naturales/inmunología , Adulto , Apoptosis/genética , Apoptosis/inmunología , Antígeno CD56/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Sangre Fetal/citología , Sangre Fetal/metabolismo , Genómica , Humanos , Inmunofenotipificación , Células Asesinas Naturales/metabolismo , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismo , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Proteómica , Receptores Similares a Lectina de Células NK/metabolismo , Transducción de Señal , Transcriptoma/inmunologíaRESUMEN
All memory T cells mount an accelerated response on antigen reencounter, but significant functional heterogeneity is present within the respective memory T-cell subsets as defined by CCR7 and CD45RA expression, thereby warranting further stratification. Here we show that several surface markers, including KLRB1, KLRG1, GPR56, and KLRF1, help define low, high, or exhausted cytokine producers within human peripheral and intrahepatic CD4+ memory T-cell populations. Highest simultaneous production of TNF and IFN-γ is observed in KLRB1+KLRG1+GPR56+ CD4 T cells. By contrast, KLRF1 expression is associated with T-cell exhaustion and reduced TNF/IFN-γ production. Lastly, TCRß repertoire analysis and in vitro differentiation support a regulated, progressive expression for these markers during CD4+ memory T-cell differentiation. Our results thus help refine the classification of human memory T cells to provide insights on inflammatory disease progression and immunotherapy development.
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Linfocitos T CD4-Positivos/inmunología , Citocinas/metabolismo , Hepatopatías/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Receptores Similares a Lectina de Células NK/metabolismo , Anciano , Anciano de 80 o más Años , Linfocitos T CD4-Positivos/metabolismo , Citocinas/inmunología , Humanos , Memoria Inmunológica , Hígado/patología , Hepatopatías/sangre , Hepatopatías/patología , Persona de Mediana Edad , Receptores Acoplados a Proteínas G/inmunología , Receptores Similares a Lectina de Células NK/inmunologíaRESUMEN
Objective: To study the functional effects of killer cell lectin-like receptor subfamily G member 1 (KLRG1) expression on natural killer cells (NK cell) in chronic hepatitis B virus infection (HBV). Methods: Peripheral blood mononuclear cells (PBMC) were extracted from 120 patients with chronic hepatitis B virus infection and 19 healthy persons. The frequency of NK cells and KLRG1+ NK cells in peripheral blood was detected by flow cytometry. Interferon-γ levels secreted by NK cells were detected in peripheral blood. Statistical analysis of experimental data was performed using GraphPad Prism 6.03 software. Results: The frequency of NK cells in HBV-infected group (16.92% ± 7.9%) was not significantly different from that in healthy controls (10.57% ± 6.5%). The frequency of KLRG1+NK cells in HBV-infected group was significantly higher (49.43% ± 21.2%) than that to healthy control group (31.60% ± 17.9%), (t = 7.347 6, P < 0.001). IFN-γ secretion of KLRG1 + NK cells in HBV-infected patients (2.59% ± 1.0%) were significantly lower than healthy controls (5.96% ± 2.4%), (P = 0.009). Conclusion: HBV infection can increase the expression of KLRG1 in NK cells and further reduce the secretion of IFN-γ in NK cells, which may be an important cause for chronic HBV infection.
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Hepatitis B Crónica/complicaciones , Células Asesinas Naturales/metabolismo , Leucocitos Mononucleares , Receptores Similares a Lectina de Células NK/metabolismo , Estudios de Casos y Controles , Virus de la Hepatitis B , Hepatitis B Crónica/diagnóstico , Hepatitis B Crónica/virología , Humanos , Interferón gamma , Células Asesinas Naturales/inmunología , Receptores Similares a Lectina de Células NK/genéticaRESUMEN
Natural killer (NK) cells express MHC class I (MHC-I)-specific receptors, such as Ly49A, that inhibit killing of cells expressing self-MHC-I. Self-MHC-I also "licenses" NK cells to become responsive to activating stimuli and regulates the surface level of NK-cell inhibitory receptors. However, the mechanisms of action resulting from these interactions of the Ly49s with their MHC-I ligands, particularly in vivo, have been controversial. Definitive studies could be derived from mice with targeted mutations in inhibitory Ly49s, but there are inherent challenges in specifically altering a single gene within a multigene family. Herein, we generated a knock-in mouse with a targeted mutation in the immunoreceptor tyrosine-based inhibitory motif (ITIM) of Ly49A that abolished the inhibitory function of Ly49A in cytotoxicity assays. This mutant Ly49A caused a licensing defect in NK cells, but the surface expression of Ly49A was unaltered. Moreover, NK cells that expressed this mutant Ly49A exhibited an altered inhibitory receptor repertoire. These results demonstrate that Ly49A ITIM signaling is critical for NK-cell effector inhibition, licensing, and receptor repertoire development.
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Citotoxicidad Inmunológica/inmunología , Genes MHC Clase I/inmunología , Motivo de Inhibición del Inmunorreceptor Basado en Tirosina , Células Asesinas Naturales/inmunología , Subfamilia A de Receptores Similares a Lectina de Células NK/fisiología , Receptores Similares a Lectina de Células NK/metabolismo , Animales , Células Cultivadas , Células Asesinas Naturales/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores Similares a Lectina de Células NK/genética , Tirosina/metabolismoRESUMEN
Natural killer (NK) cells play an important role in preventing cancer development. NK group 2 member D (NKG2D) is an activating receptor expressed in the membrane of NK cells. Tumour cells expressing NKG2DL become susceptible to an immune-dependent rejection mainly mediated by NK cells. The paradoxical roles of transforming growth factor beta (TGF-ß) in regulation of NKG2DL are presented in many studies, but the mechanism is unclear. In this study, we showed that TGF-ß up-regulated the expression of NKG2DLs in both PC3 and HepG2 cells. The up-regulation of NKG2DLs was characterized by increasing the expression of UL16-binding proteins (ULBPs) 1 and 2. TGF-ß treatment also increased the expression of transcription factor SP1. Knockdown of SP1 significantly attenuated TGF-ß-induced up-regulation of NKG2DLs in PC3 and HepG2 cells, suggesting that SP1 plays a key role in TGF-ß-induced up-regulation of NKG2DLs. TGF-ß treatment rapidly increased SP1 protein expression while not mRNA level. It might be due to that TGF-ß can elevate SP1 stability by activating PI3K/AKT signalling pathway, subsequently inhibiting GSK-3ß activity and decreasing the association between SP1 and GSK-3ß. Knockdown of GSK-3ß further verified our findings. Taken together, these results revealed that AKT/GSK-3ß-mediated stabilization of SP1 is required for TGF-ß induced up-regulation of NKG2DLs. Our study provided valuable evidence for exploring the tumour immune modulation function of TGF-ß.
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Glucógeno Sintasa Quinasa 3 beta/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Similares a Lectina de Células NK/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia Arriba/efectos de los fármacos , Células Hep G2 , Humanos , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Estabilidad Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
NK cells are the first line of defense against infected and transformed cells. Defective NK cell activity was shown to increase susceptibility for viral infections and reduce tumor immune-surveillance. With age, the incidence of infectious diseases and malignancy rises dramatically, suggesting that impaired NK cell function might contribute to disease in these individuals. We found an increased frequency of NK cells with high expression of the inhibitory killer cell lectin-like receptor G1 (KLRG1) in individuals >70 y. The role of KLRG1 in ageing is not known, and the mechanism of KLRG1-induced inhibition of NK cell function is not fully understood. We report that NK cells with high KLRG1 expression spontaneously activate the metabolic sensor AMP-activated protein kinase (AMPK) and that activation of AMPK negatively regulates NK cell function. Pre-existing AMPK activity is further amplified by ligation of KLRG1 in these cells, which leads to internalization of the receptor and allows interaction with AMPK. We show that KLRG1 activates AMPK by preventing its inhibitory dephosphorylation by protein phosphatase-2C rather than inducing de novo kinase activation. Finally, inhibition of KLRG1 or AMPK prevented KLRG1-induced activation of AMPK and reductions in NK cell cytotoxicity, cytokine secretion, proliferation, and telomerase expression. This novel signaling pathway links metabolic sensing, effector function, and cell differentiation with inhibitory receptor signaling that may be exploited to enhance NK cell activity during ageing.
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Proteínas Quinasas Activadas por AMP/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Receptores Similares a Lectina de Células NK/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Femenino , Humanos , Células Asesinas Naturales/enzimología , Masculino , Adulto JovenRESUMEN
Based on their ability to recognize and eliminate various endo- and exogenous pathogens as well as pathological alterations, Natural Killer (NK) cells represent an important part of the cellular innate immune system. Although the knowledge about their function is growing, little is known about their development and regulation on the molecular level. Research of the past decade suggests that modifications of the chromatin, which do not affect the base sequence of the DNA, also known as epigenetic alterations, are strongly involved in these processes. Here, the impact of epigenetic modifications on the development as well as the expression of important activating and inhibiting NK-cell receptors and their effector function is reviewed. Furthermore, external stimuli such as physical activity and their influence on the epigenetic level are discussed.
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Epigénesis Genética , Células Asesinas Naturales/metabolismo , Animales , Humanos , Receptores KIR/genética , Receptores KIR/metabolismo , Receptores Similares a Lectina de Células NK/genética , Receptores Similares a Lectina de Células NK/metabolismoRESUMEN
Natural killer cells express multiple receptors for major histocompatibility complex (MHC) class I, including the killer cell immunoglobulin-like receptors (KIRs) and the C-type lectin-like CD94:NKG2 receptors. The KIR locus is extremely polymorphic, paralleling the diversity of its classical MHC class I ligands. Similarly, the conservation of the NKG2 family of receptors parallels the conservation of MHC-E, the ligand for CD94:NKG2A/C/E. Binding of both CD94:NKG2 heterodimers and KIR to their respective MHC class I ligand is peptide dependent, and despite the evolution of these receptors, they have retained the property of peptide selectivity. Such peptide selectivity affects these two systems in different ways. HLA-E binding non-inhibitory peptides augment inhibition at CD94:NKG2A, while HLA-C binding non-inhibitory peptides antagonize inhibition at KIR2DL2/3, implying that KIRs are specialized to respond positively to changes in peptide repertoire. Thus, while specific KIRs, such as KIR2DL3, are associated with beneficial outcomes from viral infections, viral peptides augment inhibition at CD94:NKGA. Conversely, NKG2A-positive NK cells sense MHC class I downregulation more efficiently than KIRs. Thus, these two receptor:ligand systems appear to have complementary functions in recognizing changes in MHC class I.
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Antígenos de Histocompatibilidad Clase I/inmunología , Células Asesinas Naturales/inmunología , Péptidos/inmunología , Receptores KIR/inmunología , Receptores Similares a Lectina de Células NK/inmunología , Animales , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Células Asesinas Naturales/metabolismo , Ligandos , Modelos Inmunológicos , Péptidos/metabolismo , Unión Proteica/inmunología , Receptores KIR/metabolismo , Receptores Similares a Lectina de Células NK/metabolismoRESUMEN
Primate and rodent NK cells form highly heterogeneous lymphocyte populations owing to the differential expression of germline-encoded receptors. Many of these receptors are polymorphic and recognize equally polymorphic determinants of MHC class I. This diversity can lead to individuals carrying NK cells with different specificities. Cattle have an unusually diverse repertoire of NK cell receptor genes predicted to encode receptors that recognize MHC class I. To begin to examine whether this genetic diversity leads to a diverse NK cell population, we isolated peripheral NK cells from cattle with different MHC homozygous genotypes. Cytokine stimulation differentially influenced the transcription of five receptors at the cell population level. Using dilution cultures, we found that a further seven receptors were differentially transcribed, including five predicted to recognize MHC class I. Moreover, there was a statistically significant reduction in killer cell lectin-like receptor mRNA expression between cultures with different CD2 phenotypes and from animals with different MHC class I haplotypes. This finding confirms that cattle NK cells are a heterogeneous population and reveals that the receptors creating this diversity are influenced by the MHC. The importance of this heterogeneity will become clear as we learn more about the role of NK cells in cattle disease resistance and vaccination.