RESUMEN
BACKGROUND: Although most individuals effectively control herpesvirus infections, some suffer from severe and/or recurrent infections. A subset of these patients possess defects in natural killer (NK) cells, lymphocytes that recognize and lyse herpesvirus-infected cells; however, the genetic etiology is rarely diagnosed. PLCG2 encodes a signaling protein in NK-cell and B-cell signaling. Dominant-negative or gain-of-function variants in PLCG2 cause cold urticaria, antibody deficiency, and autoinflammation. However, loss-of-function variants and haploinsufficiency have not been reported to date. OBJECTIVES: The investigators aimed to identify the genetic cause of NK-cell immunodeficiency in 2 families and herein describe the functional consequences of 2 novel loss-of-function variants in PLCG2. METHODS: The investigators employed whole-exome sequencing in conjunction with mass cytometry, microscopy, functional assays, and a mouse model of PLCG2 haploinsufficiency to investigate 2 families with NK-cell immunodeficiency. RESULTS: The investigators identified novel heterozygous variants in PLCG2 in 2 families with severe and/or recurrent herpesvirus infections. In vitro studies demonstrated that these variants were loss of function due to haploinsufficiency with impaired NK-cell calcium flux and cytotoxicity. In contrast to previous PLCG2 variants, B-cell function remained intact. Plcg2+/- mice also displayed impaired NK-cell function with preserved B-cell function, phenocopying human disease. CONCLUSIONS: PLCG2 haploinsufficiency represents a distinct syndrome from previous variants characterized by NK-cell immunodeficiency with herpesvirus susceptibility, expanding the spectrum of PLCG2-related disease.
Asunto(s)
Haploinsuficiencia , Síndromes de Inmunodeficiencia , Fosfolipasa C gamma , Animales , Humanos , Ratones , Infecciones por Herpesviridae , Síndromes de Inmunodeficiencia/genética , Células Asesinas Naturales , Transducción de Señal , Fosfolipasa C gamma/genéticaRESUMEN
CD28 is the major costimulatory receptor on T cells regulating proliferation, survival and effector function. Acquired mutations in the extracellular domain of CD28 have been identified in patients with cutaneous T cell lymphoma, angioimmunoblastic T cell lymphoma and other T cell neoplasms, suggesting it may contribute to disease pathogenesis. We used a heterologous system in which mutant human CD28 was expressed on primary murine T cells deficient in CD28 to ascertain how specific mutations identified in a genetic screen of patients with cutaneous T cell lymphoma affected normal T cell function. All three mutant CD28 proteins examined enhanced CD28-dependent T cell proliferation and effector function. These data suggest that the mutant CD28 isoforms could accelerate tumor cell growth and increase tumor burden in affected patients. Interruption of CD28:ligand interactions may be an effective, targeted therapy for a subset of patients whose tumors bear the mutant CD28 receptor.
Asunto(s)
Antígenos CD28/genética , Antígeno CTLA-4/genética , Linfoma Cutáneo de Células T/genética , Mutación , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígenos CD28/inmunología , Antígeno CTLA-4/inmunología , Proliferación Celular , Supervivencia Celular , ADN Complementario/genética , ADN Complementario/inmunología , Expresión Génica , Humanos , Activación de Linfocitos , Linfoma Cutáneo de Células T/inmunología , Linfoma Cutáneo de Células T/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/inmunología , Cultivo Primario de Células , Linfocitos T/patología , Transfección , TransgenesRESUMEN
Juvenile dermatomyositis (JDM) is a pediatric autoimmune disease associated with characteristic rash and proximal muscle weakness. To gain insight into differential lymphocyte gene expression in JDM, peripheral blood mononuclear cells from 4 new-onset JDM patients and 4 healthy controls were sorted into highly enriched lymphocyte populations for RNAseq analysis. NK cells from JDM patients had substantially greater differentially expressed genes (273) than T (57) and B (33) cells. Upregulated genes were associated with the innate immune response and cell cycle, while downregulated genes were associated with decreased ribosomal RNA. Suppressed ribosomal RNA in JDM NK cells was validated by measuring transcription and phosphorylation levels. We confirmed a population of low ribosome expressing NK cells in healthy adults and children. This population of low ribosome NK cells was substantially expanded in 6 treatment-naïve JDM patients and was associated with decreased NK cell degranulation. The enrichment of this NK low ribosome population was completely abrogated in JDM patients with quiescent disease. Together, these data suggest NK cells are highly activated in new-onset JDM patients with an increased population of low ribosome expressing NK cells, which correlates with decreased NK cell function and resolved with control of active disease.
Asunto(s)
Dermatomiositis , Adulto , Humanos , Niño , Leucocitos Mononucleares/metabolismo , Células Asesinas Naturales/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , ARN Ribosómico/genéticaRESUMEN
While large DNA viruses are thought to have low mutation rates, only a small fraction of their genomes have been analyzed at the single-nucleotide level. Here, we defined the genetic stability of murine cytomegalovirus (MCMV) by whole-genome sequencing. Independently assembled sequences of three sister plaques showed only two single-base-pair substitutions after in vitro passage. In vivo-passaged MCMV likewise demonstrated low mutation rates, comparable to those after in vitro passage, indicating high genome stability of MCMV at the single-nucleotide level in the absence of obvious selection pressure.
Asunto(s)
Genoma Viral , Inestabilidad Genómica , Muromegalovirus/genética , Animales , Secuencia de Bases , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Muromegalovirus/metabolismo , Mutación , Sistemas de Lectura Abierta , Análisis de Secuencia de ADNRESUMEN
Juvenile dermatomyositis (JDM) is a debilitating pediatric autoimmune disease manifesting with characteristic rash and muscle weakness. To delineate signaling abnormalities in JDM, mass cytometry was performed with PBMCs from treatment-naive JDM patients and controls. NK cell percentages were lower while frequencies of naive B cells and naive CD4+ T cells were higher in JDM patients than in controls. These cell frequency differences were attenuated with cessation of active disease. A large number of signaling differences were identified in treatment-naive JDM patients compared with controls. Classification models incorporating feature selection demonstrated that differences in phospholipase Cγ2 (PLCγ2) phosphorylation comprised 10 of 12 features (i.e., phosphoprotein in a specific immune cell subset) distinguishing the 2 groups. Because NK cells represented 5 of these 12 features, further studies focused on the PLCγ2 pathway in NK cells, which is responsible for stimulating calcium flux and cytotoxic granule movement. No differences were detected in upstream signaling or total PLCγ2 protein levels. Hypophosphorylation of PLCγ2 and downstream mitogen-activated protein kinase-activated protein kinase 2 were partially attenuated with cessation of active disease. PLCγ2 hypophosphorylation in treatment-naive JDM patients resulted in decreased calcium flux. The identification of dysregulation of PLCγ2 phosphorylation and decreased calcium flux in NK cells provides potential mechanistic insight into JDM pathogenesis.
Asunto(s)
Dermatomiositis/metabolismo , Células Asesinas Naturales/metabolismo , Fosfolipasa C gamma/metabolismo , Transducción de Señal , Adolescente , Niño , Preescolar , Dermatomiositis/inmunología , Femenino , Humanos , Leucocitos Mononucleares , Masculino , FosforilaciónRESUMEN
Polyarticular juvenile idiopathic arthritis (JIA) is among the most challenging of the JIA subtypes to treat. Even with current biologic therapies, the disease remains difficult to control in a substantial subset of patients, highlighting the need for new therapies. The aim of this study was to use the high dimensionality afforded by mass cytometry with phospho-specific antibodies to delineate signaling abnormalities in immune cells from treatment-naive polyarticular JIA patients. Peripheral blood mononuclear cells were isolated from 17 treatment-naive polyarticular JIA patients, 10 of the patients after achieving clinical remission, and 19 healthy controls. Samples were stimulated for 15 minutes with IL-6 or IFN-γ and analyzed by mass cytometry. Following IFN-γ stimulation, increased STAT1 and/or STAT3 phosphorylation was observed in subsets of CD4 T cells and classical monocytes from treatment-naive patients. The enhanced IFN-γ signaling was associated with increased expression of JAK1 and SOCS1 in CD4 T cells. Furthermore, substantial heterogeneity in surface marker expression was observed among the subsets of CD4 T cells and classical monocytes with increased IFN-γ responsiveness. The identification of enhanced IFN-γ signaling in CD4 T cells and classical monocytes from treatment-naive polyarticular JIA patients provides mechanistic support for investigations into therapies that attenuate IFN-γ signaling in this disease.
Asunto(s)
Artritis Juvenil/inmunología , Interferón gamma/metabolismo , Adolescente , Antirreumáticos/farmacología , Antirreumáticos/uso terapéutico , Artritis Juvenil/sangre , Artritis Juvenil/tratamiento farmacológico , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Citometría de Flujo , Humanos , Lactante , Interferón gamma/inmunología , Masculino , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: We have previously established that the gene for neutrophil cytosolic factor 2 (NCF-2) predisposes to lupus, and we have identified lupus patients with point mutations that are predicted to cause reduced NADPH oxidase activity. We undertook this study to investigate the relationship between reduced leukocyte NADPH oxidase activity and immune dysregulation associated with systemic lupus erythematosus (SLE). METHODS: We generated NCF-2-null mice, in which NADPH oxidase activity is absent, on the nonautoimmune C57BL/6 (B6) mouse background and on the NZM 2328 mouse background, a polygenic model in which mice spontaneously develop lupus. Clinical disease, serology, and immunopathology were evaluated. RESULTS: NCF-2-null mice on the B6 background were susceptible to Aspergillus fumigatus pneumonia characteristic of chronic granulomatous disease, but did not develop systemic lupus disease. In contrast, NCF-2-null and even NCF-2-haploinsufficient mice on the NZM 2328 background developed accelerated full-blown lupus with significantly accelerated lupus kidney disease. This was characterized by more rapid development of hyperactive B cell and T cell immune compartments, increased expression of type I interferon-responsive genes, and generation of neutrophil extracellular traps, which were observed even in the absence of NADPH oxidase activity. CONCLUSION: Just as patients with chronic granulomatous disease who lack NADPH oxidase rarely develop SLE, NCF-2-null mice on a nonautoimmune background were susceptible to a chronic granulomatous disease-like opportunistic infection but did not develop lupus. In contrast, on a lupus-prone background, even haploinsufficiency of NCF-2 accelerated the development of full-blown lupus disease. This establishes an interaction between reduced oxidase activity and other lupus-predisposing genes, paralleling human SLE-associated variants predicted to have only reduced NADPH oxidase activity.
Asunto(s)
Haploinsuficiencia/genética , Lupus Eritematoso Sistémico/genética , Nefritis Lúpica/genética , NADPH Oxidasas/genética , Animales , Péptidos Catiónicos Antimicrobianos , Aspergillus fumigatus , Linfocitos B/inmunología , Catelicidinas/inmunología , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Trampas Extracelulares/inmunología , Regulación de la Expresión Génica/inmunología , Predisposición Genética a la Enfermedad , Enfermedad Granulomatosa Crónica/genética , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Riñón/inmunología , Riñón/patología , Lupus Eritematoso Sistémico/inmunología , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Aspergilosis Pulmonar/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Linfocitos T/inmunologíaRESUMEN
Results from studies comparing the diversity and specificity of the TCR repertoires expressed by conventional (Tconv) and regulatory (Treg) CD4+ T cell have varied depending on the experimental system employed. We developed a new model in which T cells express a single fixed TCRα chain, randomly rearranged endogenous TCRß chains, and a Foxp3-GFP reporter. We purified CD4+Foxp3- and CD4+Foxp3+ cells, then performed biased controlled multiplex PCR and high throughput sequencing of endogenous TCRß chains. We identified >7,000 different TCRß sequences in the periphery of 5 individual mice. On average, ~12% of TCR sequences were expressed by both conventional and regulatory populations within individual mice. The CD4+ T cells that expressed shared TCR sequences were present at higher frequencies compared to T cells expressing non-shared TCRs. Furthermore, nearly all (>90%) of the TCR sequences that were shared within mice were identical at the DNA sequence level, indicating that conventional and regulatory T cells that express shared TCRs are derived from common clones. Analysis of TCR repertoire overlap in the thymus reveals that a large proportion of Tconv and Treg sharing observed in the periphery is due to clonal expansion in the thymus. Together these data show that there are a limited number of TCR sequences shared between Tconv and Tregs. Also, Tconv and Tregs sharing identical TCRs are found at relatively high frequencies and are derived from common progenitors, of which a large portion are generated in the thymus.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Factores de Transcripción Forkhead/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T Reguladores/inmunología , Timo/inmunología , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Linaje de la Célula , Células Clonales , Femenino , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Homología de Secuencia de Aminoácido , Timo/citología , Timo/metabolismoRESUMEN
Natural killer (NK) cells play critical roles in antiviral immunity. While the importance of effector mechanisms such as interferons has been demonstrated through knockout mice, specific mechanisms of how viruses are recognized and controlled by NK cells are less well defined. Previous genetic studies have mapped the resistance genes for murine cytomegalovirus (MCMV), herpes simplex virus-1 (HSV-1), and ectromelia virus to the NK gene complex on murine chromosome 6, a region containing the polymorphic Ly49 and Nkrp1 families. Genetic resistance to MCMV in C57BL/6 has been attributed to Ly49H, an activation receptor, through susceptibility of the recombinant inbred strain BXD-8 that lacks Ly49h (also known as Klra8) but derived about half of its genome from its DBA/2 progenitor. However, it remained possible that epigenetic effects could account for the MCMV phenotype in BXD-8 mice. Herein, we report the generation of a novel congenic murine strain, B6.BXD8-Klra8 ( Cmv1-del )/Wum, on the C57BL/6 genetic background to evaluate the effect of deletion of a single NK activation receptor, Ly49H. Deletion of Ly49H rendered mice much more susceptible to MCMV infection. This increase in susceptibility did not appear to be a result of a difference in NK cell expansion or interferon-gamma (IFN-gamma) production between the C57BL/6 and the B6.BXD8 strains. On the other hand, the deletion of Ly49h did not otherwise affect NK cell maturation or Ly49D expression and had no effect on susceptibility to HSV-1 or ectromelia virus. In conclusion, Ly49h is necessary for genetic resistance to MCMV, but not HSV-1 or ectromelia virus.
Asunto(s)
Infecciones por Citomegalovirus/virología , Muromegalovirus/patogenicidad , Subfamilia A de Receptores Similares a Lectina de Células NK/fisiología , Animales , Southern Blotting , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/inmunología , Femenino , Citometría de Flujo , Genotipo , Herpes Simple/genética , Herpes Simple/inmunología , Herpes Simple/virología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/patogenicidad , Células Asesinas Naturales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Repeticiones de Microsatélite , Muromegalovirus/genética , Muromegalovirus/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de SupervivenciaRESUMEN
Natural killer (NK) cell tolerance mechanisms are incompletely understood. One possibility is that they possess self-specific activation receptors that result in hyporesponsiveness unless modulated by self-major histocompatability complex (MHC)-specific inhibitory receptors. As putative self-specific activation receptors have not been well characterized, we studied a transgenic C57BL/6 mouse that ubiquitously expresses m157 (m157-Tg), which is the murine cytomegalovirus (MCMV)-encoded ligand for the Ly49H NK cell activation receptor. The transgenic mice were more susceptible to MCMV infection and were unable to reject m157-Tg bone marrow, suggesting defects in Ly49H(+) NK cells. There was a reversible hyporesponsiveness of Ly49H(+) NK cells that extended to Ly49H-independent stimuli. Continuous Ly49H-m157 interaction was necessary for the functional defects. Interestingly, functional defects occurred when mature wild-type NK cells were adoptively transferred to m157-Tg mice, suggesting that mature NK cells may acquire hyporesponsiveness. Importantly, NK cell tolerance caused by Ly49H-m157 interaction was similar in NK cells regardless of expression of Ly49C, an inhibitory receptor specific for a self-MHC allele in C57BL/6 mice. Thus, engagement of self-specific activation receptors in vivo induces an NK cell tolerance effect that is not affected by self-MHC-specific inhibitory receptors.
Asunto(s)
Antígenos Ly/metabolismo , Tolerancia Inmunológica , Células Asesinas Naturales/inmunología , Lectinas Tipo C/metabolismo , Receptores Inmunológicos/metabolismo , Traslado Adoptivo , Animales , Trasplante de Médula Ósea/inmunología , Diferenciación Celular , Infecciones por Herpesviridae/inmunología , Células Asesinas Activadas por Linfocinas/inmunología , Células Asesinas Activadas por Linfocinas/virología , Células Asesinas Naturales/citología , Células Asesinas Naturales/virología , Ligandos , Activación de Linfocitos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Muromegalovirus/genética , Muromegalovirus/inmunología , Muromegalovirus/patogenicidad , Subfamilia A de Receptores Similares a Lectina de Células NK , Receptores Similares a Lectina de Células NK , Inmunología del Trasplante , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
A murine cytomegalovirus (MCMV)-encoded protein, m157, has a putative major histocompatibility complex class I (MHC-I) structure and is recognized by the Ly49H NK cell activation receptor. Using a monoclonal antibody against m157, in this study we directly demonstrated that m157 is a cell surface-expressed glycophosphatidylinositol-anchored protein with early viral gene kinetics. Beta-2 microglobulin and TAP1 (transporter associated with antigen processing 1) were not required for its expression. MCMV-encoded proteins that down-regulate MHC-I did not affect the expression of m157. Thus, m157 is expressed on infected cells in a manner independent of viral regulation of host MHC-I.
Asunto(s)
Infecciones por Herpesviridae/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Muromegalovirus/química , Animales , Regulación Viral de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/análisis , Ratones , Muromegalovirus/inmunologíaRESUMEN
Natural killer (NK) cells play a crucial role in the initial host defense against pathogens such as murine cytomegalovirus (MCMV). They respond rapidly and effectively control pathogen replication while the adaptive immune system is being activated. However, in the absence of an adaptive immune system, an effective initial NK cell response is not sufficient for long-term pathogen control as demonstrated by the late recrudescence of disease and mortality in immunodeficient mice infected with MCMV. In this setting, NK cells suppress the initial infection but exert enough selective pressure to drive the outgrowth of MCMV mutants that escape recognition by NK cells. Herein, we characterize the rapid emergence of escape mutants following infection with a plaque-purified MCMV isolate and demonstrate that these mutant viruses are no longer effectively controlled by NK cells. These findings suggest that late recrudescence of viral infections in certain clinical settings may also be due to viral escape from NK cells or other components of innate immunity.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Células Asesinas Naturales/inmunología , Muromegalovirus/inmunología , Traslado Adoptivo , Animales , Antígenos Ly/genética , Infecciones por Citomegalovirus/virología , ADN Viral/química , ADN Viral/genética , Citometría de Flujo , Huésped Inmunocomprometido/inmunología , Inmunohistoquímica , Células Asesinas Naturales/virología , Lectinas Tipo C , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Muromegalovirus/genética , Mutación , Reacción en Cadena de la Polimerasa , Receptores Similares a Lectina de Células NK , Análisis de Secuencia de ADN , Organismos Libres de Patógenos Específicos , Bazo/inmunología , Bazo/virologíaRESUMEN
As innate immune system components, natural killer (NK) cells respond rapidly to infections and effectively control replication of pathogens, including murine cytomegalovirus (MCMV), a double-stranded DNA beta-herpesvirus. In the absence of NK cell control, MCMV infection results in early mortality due to uncontrolled viral replication. However, here we show that even in the face of initial NK cell control, there is late recrudescence of disease and mortality in immunodeficient mice due to the outgrowth of MCMV mutants that escape recognition by innate NK cells. These data suggest that viral infections in certain clinical settings also may be due to viral escape from innate immunity.
Asunto(s)
Inmunidad Innata/inmunología , Células Asesinas Naturales/inmunología , Muromegalovirus/genética , Muromegalovirus/inmunología , Mutación/genética , Animales , Células Cultivadas , Citometría de Flujo , Infecciones por Herpesviridae/complicaciones , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/mortalidad , Infecciones por Herpesviridae/virología , Hígado/inmunología , Hígado/virología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Muromegalovirus/fisiología , Inmunodeficiencia Combinada Grave/complicaciones , Inmunodeficiencia Combinada Grave/inmunología , Bazo/inmunología , Bazo/virología , Tasa de SupervivenciaRESUMEN
Cytokines and chemokines activate and direct effector cells during infection. We previously identified a functional group of five cytokines and chemokines, namely, IFN-gamma, activation-induced T cell-derived and chemokine-related cytokine/lymphotactin, macrophage-inflammatory protein 1alpha, macrophage-inflammatory protein 1beta, and RANTES, coexpressed in individual activated NK cells, CD8(+) T cells, and CD4(+) Th1 cells in vitro and during in vivo infections. However, the stimuli during infection were not known. In murine CMV (MCMV) infection, the DAP12/KARAP-associated Ly49H NK cell activation receptor is crucial for resistance through recognition of MCMV-encoded m157 but NK cells also undergo in vivo nonspecific responses to uncharacterized stimuli. In this study, we show that Ly49H ligation by m157 resulted in a coordinated release of all five cytokines/chemokines from Ly49H(+) NK cells. Whereas other cytokines also triggered the release of these cytokines/chemokines, stimulation was not confined to the Ly49H(+) population. At the single-cell level, the production of the five mediators showed strong positive correlation with each other. Interestingly, NK cells were a major source of these five cytokines/chemokines in vitro and in vivo, whereas infected macrophages produced only limited amounts of macrophage-inflammatory protein 1alpha, macrophage-inflammatory protein1beta, and RANTES. These findings suggest that both virus-specific and nonspecific NK cells play crucial roles in activating and directing other inflammatory cells during MCMV infection.
Asunto(s)
Quimiocinas/biosíntesis , Citocinas/biosíntesis , Infecciones por Herpesviridae/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/virología , Muromegalovirus/inmunología , Animales , Antígenos Ly/inmunología , Antígenos Ly/metabolismo , Antígenos Ly/fisiología , Células Cultivadas , Quimiocina CCL4 , Quimiocina CCL5/biosíntesis , Quimiocinas C , Técnicas de Cocultivo , Citocinas/fisiología , Infecciones por Herpesviridae/metabolismo , Interferón gamma/biosíntesis , Células Asesinas Naturales/metabolismo , Lectinas Tipo C , Activación de Linfocitos/inmunología , Linfocinas/biosíntesis , Proteínas Inflamatorias de Macrófagos/biosíntesis , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Subfamilia A de Receptores Similares a Lectina de Células NK , Receptores Similares a Lectina de Células NK , Sialoglicoproteínas/biosíntesisRESUMEN
Natural interferon-producing cells (IPC) respond to viruses by secreting type I interferon (IFN) and interleukin-12 (IL-12). Toll-like receptor (TLR) 9 mediates IPC recognition of some of these viruses in vitro. However, whether TLR9-induced activation of IPC is necessary for an effective antiviral response in vivo is not clear. Here, we demonstrate that IPC and dendritic cells (DC) recognize murine cytomegalovirus (MCMV) through TLR9. TLR9-mediated cytokine secretion promotes viral clearance by NK cells that express the MCMV-specific receptor Ly49H. Although depletion of IPC leads to a drastic reduction of the IFN-alpha response, this allows other cell types to secrete IL-12, ensuring normal IFN-gamma and NK cell responses to MCMV. We conclude that the TLR9/MyD88 pathway mediates antiviral cytokine responses by IPC, DC, and possibly other cell types, which are coordinated to promote effective NK cell function and MCMV clearance.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Células Dendríticas/fisiología , Células Asesinas Naturales/fisiología , Muromegalovirus/fisiología , Receptores de Superficie Celular/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígenos de Diferenciación , Citocinas/metabolismo , Interferones/metabolismo , Interleucina-12/metabolismo , Ratones , Factor 88 de Diferenciación Mieloide , Receptores Inmunológicos , Receptor Toll-Like 9 , Replicación ViralRESUMEN
Natural killer (NK) cells express inhibitory and activation receptors that recognize MHC class I-like molecules on target cells. These receptors may be involved in the critical role of NK cells in controlling initial phases of certain viral infections. Indeed, the Ly49H NK cell activation receptor confers in vivo genetic resistance to murine cytomegalovirus (MCMV) infections, but its ligand was previously unknown. Herein, we use heterologous reporter cells to demonstrate that Ly49H recognizes MCMV-infected cells and a ligand encoded by MCMV itself. Exploiting a bioinformatics approach to the MCMV genome, we find at least 11 ORFs for molecules with previously unrecognized features of predicted MHC-like folds and limited MHC sequence homology. We identify one of these, m157, as the ligand for Ly49H. m157 triggers Ly49H-mediated cytotoxicity, and cytokine and chemokine production by freshly isolated NK cells. We hypothesize that the other ORFs with predicted MHC-like folds may be involved in immune evasion or interactions with other NK cell receptors.