RESUMEN
Endothelial function and integrity are compromised after allogeneic bone marrow transplantation (BMT), but how this affects immune responses broadly remains unknown. Using a preclinical model of CMV reactivation after BMT, we found compromised antiviral humoral responses induced by IL-6 signaling. IL-6 signaling in T cells maintained Th1 cells, resulting in sustained IFN-γ secretion, which promoted endothelial cell (EC) injury, loss of the neonatal Fc receptor (FcRn) responsible for IgG recycling, and rapid IgG loss. T cell-specific deletion of IL-6R led to persistence of recipient-derived, CMV-specific IgG and inhibited CMV reactivation. Deletion of IFN-γ in donor T cells also eliminated EC injury and FcRn loss. In a phase III clinical trial, blockade of IL-6R with tocilizumab promoted CMV-specific IgG persistence and significantly attenuated early HCMV reactivation. In sum, IL-6 invoked IFN-γ-dependent EC injury and consequent IgG loss, leading to CMV reactivation. Hence, cytokine inhibition represents a logical strategy to prevent endothelial injury, thereby preserving humoral immunity after immunotherapy.
Asunto(s)
Trasplante de Médula Ósea , Infecciones por Citomegalovirus , Inmunidad Humoral , Interleucina-6 , Antivirales , Trasplante de Médula Ósea/efectos adversos , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/metabolismo , Inmunoglobulina G , Interleucina-6/metabolismo , Animales , RatonesRESUMEN
Tissue health is dictated by the capacity to respond to perturbations and then return to homeostasis. Mechanisms that initiate, maintain, and regulate immune responses in tissues are therefore essential. Adaptive immunity plays a key role in these responses, with memory and tissue residency being cardinal features. A corresponding role for innate cells is unknown. Here, we have identified a population of innate lymphocytes that we term tissue-resident memory-like natural killer (NKRM) cells. In response to murine cytomegalovirus infection, we show that circulating NK cells were recruited in a CX3CR1-dependent manner to the salivary glands where they formed NKRM cells, a long-lived, tissue-resident population that prevented autoimmunity via TRAIL-dependent elimination of CD4+ T cells. Thus, NK cells develop adaptive-like features, including long-term residency in non-lymphoid tissues, to modulate inflammation, restore immune equilibrium, and preserve tissue health. Modulating the functions of NKRM cells may provide additional strategies to treat inflammatory and autoimmune diseases.
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Infecciones por Citomegalovirus , Muromegalovirus , Humanos , Animales , Ratones , Células Asesinas Naturales , Inmunidad Adaptativa , Linfocitos T , Inmunidad InnataRESUMEN
Cone Dystrophy with Supernormal Rod Response (CDSRR) is a rare autosomal recessive disorder leading to severe visual impairment in humans, but little is known about its unique pathophysiology. We have previously shown that CDSRR is caused by mutations in the KCNV2 (Potassium Voltage-Gated Channel Modifier Subfamily V Member 2) gene encoding the Kv8.2 subunit, a modulatory subunit of voltage-gated potassium (Kv) channels. In a recent study, we validated a novel mouse model of Kv8.2 deficiency at a late stage of the disease and showed that it replicates the human electroretinogram (ERG) phenotype. In this current study, we focused our investigation on young adult retinas to look for early markers of disease and evaluate their effect on retinal morphology, electrophysiology and immune response in both the Kv8.2 knockout (KO) mouse and in the Kv2.1 KO mouse, the obligate partner of Kv8.2 in functional retinal Kv channels. By evaluating the severity of retinal dystrophy in these KO models, we demonstrated that retinas of Kv KO mice have significantly higher apoptotic cells, a thinner outer nuclear cell layer and increased activated microglia cells in the subretinal space. Our results indicate that in the murine retina, the loss of Kv8.2 subunits contributes to early cellular and physiological changes leading to retinal dysfunction. These results could have potential implications in the early management of CDSRR despite its relatively nonprogressive nature in humans.
Asunto(s)
Envejecimiento/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Subunidades de Proteína/metabolismo , Retina/citología , Retina/metabolismo , Canales de Potasio Shab/metabolismo , Animales , Muerte Celular , Electrorretinografía , Gliosis/patología , Inmunidad , Ratones Noqueados , Microglía/patología , Visión Nocturna , Retina/fisiologíaRESUMEN
Purpose: To validate the application of a known transgenic mouse line with green fluorescent cones (Chrnb4.EGFP) to study cone photoreceptor biology and function in health and disease. Methods: Chrnb4.EGFP retinas containing GFP+ cones were compared with retinas without the GFP transgene via immunohistochemistry, quantitative real-time polymerase chain reaction, electroretinograms, and flow cytometry. The Chrnb4.EGFP line was backcrossed to the mouse models of cone degeneration, Pde6ccpfl1 and Gnat2cpfl3 , generating the new lines Gnat2.GFP and Pde6c.GFP, which were also studied as described. Results: GFP expression spanned the length of the cone cell in the Chrnb4.EGFP line, as well as in the novel Gnat2.GFP and Pde6c.GFP lines. The effect of GFP expression showed no significant changes to outer nuclear layer cell death, cone-specific gene expression, and immune response activation. A temporal decrease in GFP expression over time was observed, but GFP fluorescence was still detected through flow cytometry as late as 6 months. Furthermore, a functional analysis of photopic and scotopic electroretinogram responses of the Chrnb4 mouse showed no significant difference between GFP- and GFP+ mice, whereas electroretinogram recordings for the Pde6c.GFP and Gnat2.GFP lines matched previous reports from the original lines. Conclusions: This study demonstrates that the Chrnb4.EGFP mouse can be a powerful tool to overcome the limitations of studying cone biology, including the use of this line to study different types of cone degeneration. Translational Relevance: This work validates research tools that could potentially offer more reliable preclinical data in the development of treatments for cone-mediated vision loss conditions, shortening the gap to clinical translation.
Asunto(s)
Receptores Nicotínicos , Degeneración Retiniana , Animales , Electrorretinografía , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso , Retina , Células Fotorreceptoras Retinianas Conos , Degeneración Retiniana/genéticaRESUMEN
Mutations in the KCNV2 gene, which encodes the voltage-gated K+ channel protein Kv8.2, cause a distinctive form of cone dystrophy with a supernormal rod response (CDSRR). Kv8.2 channel subunits only form functional channels when combined in a heterotetramer with Kv2.1 subunits encoded by the KCNB1 gene. The CDSRR disease phenotype indicates that photoreceptor adaptation is disrupted. The electroretinogram (ERG) response of affected individuals shows depressed rod and cone activity, but what distinguishes this disease is the supernormal rod response to a bright flash of light. Here, we have utilized knock-out mutations of both genes in the mouse to study the pathophysiology of CDSRR. The Kv8.2 knock-out (KO) mice show many similarities to the human disorder, including a depressed a-wave and an elevated b-wave response with bright light stimulation. Optical coherence tomography (OCT) imaging and immunohistochemistry indicate that the changes in six-month-old Kv8.2 KO retinae are largely limited to the outer nuclear layer (ONL), while outer segments appear intact. In addition, there is a significant increase in TUNEL-positive cells throughout the retina. The Kv2.1 KO and double KO mice also show a severely depressed a-wave, but the elevated b-wave response is absent. Interestingly, in all three KO genotypes, the c-wave is totally absent. The differential response shown here of these KO lines, that either possess homomeric channels or lack channels completely, has provided further insights into the role of K+ channels in the generation of the a-, b-, and c-wave components of the ERG.
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Distrofia del Cono/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Retina/metabolismo , Canales de Potasio Shab/metabolismo , Animales , Distrofia del Cono/diagnóstico por imagen , Distrofia del Cono/patología , Femenino , Técnicas de Inactivación de Genes , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Canales de Potasio con Entrada de Voltaje/genética , Retina/diagnóstico por imagen , Retina/patología , Canales de Potasio Shab/genética , Transmisión Sináptica , Visión Ocular/fisiologíaRESUMEN
Cytomegalovirus infection is a frequent and life-threatening complication that significantly limits positive transplantation outcomes. We developed preclinical mouse models of cytomegalovirus reactivation after transplantation and found that humoral immunity is essential for preventing viral recrudescence. Preexisting antiviral antibodies decreased after transplant in the presence of graft-versus-host disease and were not replaced, owing to poor reconstitution of donor B cells and elimination of recipient plasma cells. Viral reactivation was prevented by the transfer of immune serum, without a need to identify and target specific antigenic determinants. Notably, serotherapy afforded complete protection, provided that the serum was matched to the infecting viral strain. Thus, we define the mechanisms for cytomegalovirus reactivation after transplantation and identify a readily translatable strategy of exceptional potency, which avoids the constraints of cellular therapies.
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Anticuerpos Antivirales/sangre , Trasplante de Médula Ósea/efectos adversos , Infecciones por Citomegalovirus/prevención & control , Enfermedad Injerto contra Huésped/virología , Inmunización Pasiva , Activación Viral , Animales , Anticuerpos Neutralizantes/sangre , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/inmunología , Femenino , Inmunidad Humoral , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T/inmunología , Acondicionamiento Pretrasplante , Viremia , Latencia del VirusRESUMEN
Recent outbreaks of Ebola and Zika have highlighted the possibility that viruses may cause enduring infections in tissues like the eye, including the neural retina, which have been considered immune privileged. Whether this is a peculiarity of exotic viruses remains unclear, since the impact of more common viral infections on neural compartments has not been examined, especially in immunocompetent hosts. Cytomegalovirus is a common, universally distributed pathogen, generally innocuous in healthy individuals. Whether in immunocompetent hosts cytomegalovirus can access the eye, and reside there indefinitely, was unknown. Using the well-established murine cytomegalovirus infection model, we show that systemic infection of immunocompetent hosts results in broad ocular infection, chronic inflammation and establishment of a latent viral pool in the eye. Infection leads to infiltration and accumulation of anti-viral CD8+ T cells in the eye, and to the development of tissue resident memory T cells that localize to the eye, including the retina. These findings identify the eye as an unexpected reservoir for cytomegalovirus, and suggest that common viruses may target this organ more frequently than appreciated. Notably, they also highlight that infection triggers sustained inflammatory responses in the eye, including the neural retina.
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Infecciones por Citomegalovirus/inmunología , Citomegalovirus/fisiología , Ojo/virología , Animales , Linfocitos T CD8-positivos/inmunología , Citomegalovirus/inmunología , Citomegalovirus/patogenicidad , Modelos Animales de Enfermedad , Reservorios de Enfermedades/microbiología , Ojo/inmunología , Femenino , Memoria Inmunológica/inmunología , Inflamación/inmunología , Ratones , Ratones Endogámicos BALB C , Muromegalovirus/fisiología , Linfocitos T/inmunología , VirosisRESUMEN
Ocular antigens are sequestered behind the blood-retina barrier and the ocular environment protects ocular tissues from autoimmune attack. The signals required to activate autoreactive T cells and allow them to cause disease in the eye remain in part unclear. In particular, the consequences of peripheral presentation of ocular antigens are not fully understood. We examined peripheral expression and presentation of ocular neo-self-antigen in transgenic mice expressing hen egg lysozyme (HEL) under a retina-specific promoter. High levels of HEL were expressed in the eye compared to low expression throughout the lymphoid system. Adoptively transferred naïve HEL-specific CD4+ T cells proliferated in the eye draining lymph nodes, but did not induce uveitis. By contrast, systemic infection with a murine cytomegalovirus (MCMV) engineered to express HEL induced extensive proliferation of transferred naïve CD4+ T cells, and significant uveoretinitis. In this model, wild-type MCMV, lacking HEL, did not induce overt uveitis, suggesting that disease is mediated by antigen-specific peripherally activated CD4+ T cells that infiltrate the retina. Our results demonstrate that retinal antigen is presented to T cells in the periphery under physiological conditions. However, when the same antigen is presented during viral infection, antigen-specific T cells access the retina and autoimmune uveitis ensues.
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Autoantígenos/inmunología , Retina/inmunología , Animales , Presentación de Antígeno , Autoinmunidad , Linfocitos T CD4-Positivos/citología , Proliferación Celular , Reacciones Cruzadas , Inflamación/inmunología , Ratones , Muramidasa/inmunología , RiesgoRESUMEN
Viral infection is a common, life-threatening complication after allogeneic bone marrow transplantation (BMT), particularly in the presence of graft-versus-host disease (GVHD). Using cytomegalovirus (CMV) as the prototypic pathogen, we have delineated the mechanisms responsible for the inability to mount protective antiviral responses in this setting. Although CMV infection was self-limiting after syngeneic BMT, in the presence of GVHD after allogeneic BMT, CMV induced a striking cytopathy resulting in universal mortality in conjunction with a fulminant necrotizing hepatitis. Critically, GVHD induced a profound dendritic cell (DC) defect that led to a failure in the generation of CMV-specific CD8(+) T-cell responses. This was accompanied by a defect in antiviral CD8(+) T cells. In combination, these defects dramatically limited antiviral T-cell responses. The transfer of virus-specific cells circumvented the DC defects and provided protective immunity, despite concurrent GVHD. These data demonstrate the importance of avoiding GVHD when reconstructing antiviral immunity after BMT, and highlight the mechanisms by which the adoptive transfer of virus-specific T cells overcome the endogenous defects in priming invoked by GVHD.
Asunto(s)
Linfocitos T CD8-positivos/patología , Infecciones por Citomegalovirus/etiología , Citomegalovirus/inmunología , Células Dendríticas/patología , Enfermedad Injerto contra Huésped/complicaciones , Traslado Adoptivo , Animales , Trasplante de Médula Ósea/efectos adversos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Células Cultivadas , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/patología , Infecciones por Citomegalovirus/terapia , Células Dendríticas/inmunología , Células Dendríticas/virología , Femenino , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Enfermedad Injerto contra Huésped/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
OBJECTIVE: To use high-density genotyping to investigate the genetic associations of acute anterior uveitis (AAU) in patients with and those without ankylosing spondylitis (AS). METHODS: We genotyped samples from 1,711 patients with AAU (either primary or combined with AS), 2,339 AS patients without AAU, and 10,000 control subjects on an Illumina Immunochip Infinium microarray. We also used data for AS patients from previous genome-wide association studies to investigate the AS risk locus ANTXR2 for its putative effect in AAU. ANTXR2 expression in mouse eyes was investigated by real-time quantitative reverse transcription-polymerase chain reaction. RESULTS: A comparison between all patients with AAU and healthy control subjects showed strong association over HLA-B, corresponding to the HLA-B27 tag single-nucleotide polymorphism rs116488202. The association of 3 non-major histocompatibility complex loci, IL23R, the intergenic region 2p15, and ERAP1, reached genome-wide significance (P < 5 × 10(-8)). Five loci harboring the immune-related genes IL10-IL19, IL18R1-IL1R1, IL6R, the chromosome 1q32 locus harboring KIF21B, as well as the eye-related gene EYS, were also associated, reaching a suggestive level of significance (P < 5 × 10(-6)). Several previously confirmed AS associations demonstrated significant differences in effect size between AS patients with AAU and AS patients without AAU. ANTXR2 expression varied across eye compartments. CONCLUSION: These findings of both novel AAU-specific associations and associations shared with AS demonstrate overlapping but also distinct genetic susceptibility loci for AAU and AS. The associations in IL10 and IL18R1 are shared with inflammatory bowel disease, suggesting common etiologic pathways.
Asunto(s)
Espondilitis Anquilosante/genética , Uveítis Anterior/genética , Aminopeptidasas/genética , Estudios de Casos y Controles , Estudio de Asociación del Genoma Completo , Genotipo , Antígeno HLA-B27/genética , Humanos , Interleucina-10/genética , Subunidad alfa del Receptor de Interleucina-18/genética , Antígenos de Histocompatibilidad Menor , Receptores de Interleucina/genética , Receptores de Péptidos/genéticaRESUMEN
Successful replication and transmission of large DNA viruses such as the cytomegaloviruses (CMV) family of viruses depends on the ability to interfere with multiple aspects of the host immune response. Apoptosis functions as a host innate defence mechanism against viral infection, and the capacity to interfere with this process is essential for the replication of many viruses. The Bcl-2 family of proteins are the principle regulators of apoptosis, with two pro-apoptotic members, Bax and Bak, essential for apoptosis to proceed. The m38.5 protein encoded by murine CMV (MCMV) has been identified as Bax-specific inhibitor of apoptosis. Recently, m41.1, a protein product encoded by the m41 open reading frame (ORF) of MCMV, has been shown to inhibit Bak activity in vitro. Here we show that m41.1 is critical for optimal MCMV replication in vivo. Growth of a m41.1 mutant was attenuated in multiple organs, a defect that was not apparent in Bak(-/-) mice. Thus, m41.1 promotes MCMV replication by inhibiting Bak-dependent apoptosis during in vivo infection. The results show that Bax and Bak mediate non-redundant functions during MCMV infection and that the virus produces distinct inhibitors for each protein to counter the activity of these proteins.
Asunto(s)
Apoptosis/fisiología , Proteínas Inhibidoras de la Apoptosis/metabolismo , Muromegalovirus/patogenicidad , Replicación Viral/fisiología , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Inmunidad Innata , Proteínas Inhibidoras de la Apoptosis/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Muromegalovirus/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Microglial cells are the resident macrophages of the central nervous system and participate in both innate and adaptive immune responses but can also lead to exacerbation of neurodegenerative pathologies after viral infections. Microglia in the outer layers of the retina and the subretinal space are thought to be involved in retinal diseases where low-grade chronic inflammation and oxidative stress play a role. This study investigated the effect of systemic infection with murine cytomegalovirus on the distribution and dynamics of retinal microglia cells. Systemic infection with murine cytomegalovirus elicited a significant increase in the number of microglia in the subretinal space and an accumulation of iris macrophages, along with morphological signs of activation. Interferon γ (IFN-γ)-deficient mice failed to induce changes in microglia distribution. Bone marrow chimera experiments confirmed that microglial cells in the subretinal space were not recruited from the circulating monocyte pool, but rather represented an accumulation of resident microglial cells from within the retina. Our results demonstrate that a systemic viral infection can lead to IFN-γ-mediated accumulation of microglia into the outer retinal layers and offer proof of concept that systemic viral infections alter the ocular microenvironment and therefore, may influence the course of diseases such as macular degeneration, diabetic retinopathy, or autoimmune uveitis, where low-grade inflammation is implicated.
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Movimiento Celular , Infecciones por Citomegalovirus/patología , Interferón gamma/metabolismo , Microglía/patología , Muromegalovirus/fisiología , Retina/patología , Retina/virología , Animales , Proteínas de Unión al Calcio/metabolismo , Recuento de Células , Movimiento Celular/efectos de los fármacos , Infecciones por Citomegalovirus/virología , Femenino , Citometría de Flujo , Iris/patología , Iris/virología , Macrófagos/efectos de los fármacos , Macrófagos/patología , Macrófagos/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/metabolismo , Microglía/efectos de los fármacos , Microglía/virología , Muromegalovirus/efectos de los fármacos , Factor 88 de Diferenciación Mieloide/metabolismo , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/farmacología , Retina/efectos de los fármacos , Segmento Externo de las Células Fotorreceptoras Retinianas/efectos de los fármacos , Segmento Externo de las Células Fotorreceptoras Retinianas/patología , Segmento Externo de las Células Fotorreceptoras Retinianas/virologíaRESUMEN
Effective immunity requires the coordinated activation of innate and adaptive immune responses. Natural killer (NK) cells are central innate immune effectors, but can also affect the generation of acquired immune responses to viruses and malignancies. How NK cells influence the efficacy of adaptive immunity, however, is poorly understood. Here, we show that NK cells negatively regulate the duration and effectiveness of virus-specific CD4+ and CD8+ T cell responses by limiting exposure of T cells to infected antigen-presenting cells. This impacts the quality of T cell responses and the ability to limit viral persistence. Our studies provide unexpected insights into novel interplays between innate and adaptive immune effectors, and define the critical requirements for efficient control of viral persistence.
Asunto(s)
Antivirales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Inmunidad Innata/inmunología , Virosis/inmunología , Animales , Citotoxicidad Inmunológica/inmunología , Células Dendríticas/inmunología , Células Dendríticas/virología , Epítopos , Antígenos de Histocompatibilidad/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/virología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Subfamilia A de Receptores Similares a Lectina de Células NK/deficiencia , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Péptidos/inmunologíaRESUMEN
As with human cytomegalovirus (HCMV) infection of humans, murine CMV (MCMV) infection is widespread in its natural host, the house mouse Mus domesticus, and may consist of mixed infection with different CMV isolates. The incidence and mechanisms by which mixed infection occurs in free-living mice are unknown. This study used two approaches to determine whether mixed infection with MCMV could be established in laboratory mice. The first utilized two naturally occurring MCMV strains, N1 and G4, into which the lacZ gene was inserted by homologous recombination. The lacZ gene was used to track recombinant and parental viruses in simultaneously coinfected mice. In the second approach, a real-time quantitative PCR (qPCR) assay was used to detect viral immediate-early 1 (ie1) gene sequences in mice successively coinfected with G4 and then with the K181 MCMV strain. In both systems, mixed infection was detected in the salivary glands and lungs of experimentally infected mice. MCMV-specific antibody in sera and G4 IE1-specific cytotoxic lymphocyte responses in the spleens of twice-infected mice did not prevent reinfection. Finally, the prevalence of mixed infection in free-living mice trapped in four Australian locations was investigated using real-time qPCR to detect ie1 DNA sequences of N1, G4 and K181. Mixed infection with MCMVs containing the G4 and K181 ie1 sequences was detected in the salivary glands of 34.2 % of trapped mice. The observations that mixed infections are common in free-living M. domesticus and are acquired by immunocompetent mice through simultaneous or successive infections are important for vaccine development.
Asunto(s)
Infecciones por Herpesviridae/virología , Muromegalovirus/patogenicidad , Animales , Anticuerpos Antivirales/sangre , Especificidad de Anticuerpos , Australia , Citotoxicidad Inmunológica , Femenino , Genes Virales , Infecciones por Herpesviridae/inmunología , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/inmunología , Inmunocompetencia , Pulmón/virología , Linfocitos/inmunología , Ratones/virología , Ratones Endogámicos BALB C , Muromegalovirus/genética , Muromegalovirus/inmunología , Muromegalovirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Glándulas Salivales/virología , Bazo/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , VirulenciaRESUMEN
Signaling from the T-cell receptor (TCR) in thymocytes is negatively regulated by the RING finger-type ubiquitin ligase c-Cbl. To further investigate this regulation, we generated mice with a loss-of-function mutation in the c-Cbl RING finger domain. These mice exhibit complete thymic deletion by young adulthood, which is not caused by a developmental block, lack of progenitors or peripheral T-cell activation. Rather, this phenotype correlates with greatly increased expression of the CD5 and CD69 activation markers and increased sensitivity to anti-CD3-induced cell death. Thymic loss contrasts the normal fate of the c-Cbl-/- thymus, even though thymocytes from both mutant mice show equivalent enhancement in proximal TCR signaling, Erk activation and calcium mobilization. Remarkably, only the RING finger mutant thymocytes show prominent TCR-directed activation of Akt. We show that the mutant c-Cbl protein itself is essential for activating this pathway by recruiting the p85 regulatory subunit of PI 3-kinase. This study provides a unique model for analyzing high-intensity TCR signals that cause thymocyte deletion and highlights multiple roles of c-Cbl in regulating this process.
Asunto(s)
Proteínas Proto-Oncogénicas c-cbl/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal , Timo/inmunología , Sustitución de Aminoácidos , Animales , Antígenos CD/análisis , Antígenos de Diferenciación de Linfocitos T/análisis , Apoptosis , Complejo CD3/análisis , Antígenos CD5/análisis , Quinasas MAP Reguladas por Señal Extracelular/análisis , Lectinas Tipo C , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/análisis , Ratones , Ratones Transgénicos , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-cbl/química , Proteínas Proto-Oncogénicas c-cbl/genética , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Dendritic cells (DCs) regulate various aspects of innate immunity, including natural killer (NK) cell function. Here we define the mechanisms involved in DC-NK cell interactions during viral infection. NK cells were efficiently activated by murine cytomegalovirus (MCMV)-infected CD11b(+) DCs. NK cell cytotoxicity required interferon-alpha and interactions between the NKG2D activating receptor and NKG2D ligand, whereas the production of interferon-gamma by NK cells relied mainly on DC-derived interleukin 18. Although Toll-like receptor 9 contributes to antiviral immunity, we found that signaling pathways independent of Toll-like receptor 9 were important in generating immune responses to MCMV, including the production of interferon-alpha and the induction of NK cell cytotoxicity. Notably, adoptive transfer of MCMV-activated CD11b(+) DCs resulted in improved control of MCMV infection, indicating that these cells participate in controlling viral replication in vivo.
Asunto(s)
Células Dendríticas/inmunología , Infecciones por Herpesviridae/inmunología , Células Asesinas Naturales/inmunología , Muromegalovirus/inmunología , Animales , Antígenos CD11/biosíntesis , Citotoxicidad Inmunológica , Interferón-alfa/biosíntesis , Interferón gamma/biosíntesis , Interleucina-18 , Ligandos , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Subfamilia K de Receptores Similares a Lectina de Células NK , Receptores Inmunológicos/metabolismo , Receptores de Células Asesinas NaturalesRESUMEN
Effective natural killer (NK) cell recognition of murine cytomegalovirus (MCMV)-infected cells depends on binding of the Ly49H NK cell activation receptor to the m157 viral glycoprotein. Here we addressed the immunological consequences of variation in m157 sequence and function. We found that most strains of MCMV possess forms of m157 that evade Ly49H-dependent NK cell activation. Importantly, repeated passage of MCMV through resistant Ly49H+ mice resulted in the rapid emergence of m157 mutants that elude Ly49H-dependent NK cell responses. These data provide the first molecular evidence that NK cells can exert sufficient immunological pressure on a DNA virus, such that it undergoes rapid and specific mutation in an NK cell ligand enabling it to evade efficient NK cell surveillance.
Asunto(s)
Células Asesinas Naturales/inmunología , Muromegalovirus/genética , Mutación , Animales , Antígenos Ly/genética , Células Cultivadas , Citotoxicidad Inmunológica , ADN/metabolismo , Femenino , Fibroblastos/metabolismo , Variación Genética , Glicoproteínas/química , Inmunidad Innata , Lectinas Tipo C , Ligandos , Subgrupos Linfocitarios , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Subfamilia A de Receptores Similares a Lectina de Células NK , Reacción en Cadena de la Polimerasa , Receptores Inmunológicos , Receptores Similares a Lectina de Células NK , Bazo/virología , Transfección , beta-Galactosidasa/metabolismoRESUMEN
The haematopoietic-specific RhoGTPase, Rac2, has been indirectly implicated in T-lymphocyte development and function, and as a pivotal regulator of T Helper 1 (T(H)1) responses. In other haematopoietic cells it regulates cytoskeletal rearrangement downstream of extracellular signals. Here we demonstrate that Rac2 deficiency results in an abnormal distribution of T lymphocytes in vivo and defects in T-lymphocyte migration and filamentous actin generation in response to chemoattractants in vitro. To investigate the requirement for Rac2 in IFN-gamma production and TH1 responses in vivo, Rac2-deficient mice were challenged with Leishmania major and immunized with ovalbumin-expressing cytomegalovirus. Despite a minor skewing towards a T(H)2 phenotype, Rac2-deficient mice displayed no increased susceptibility to L. major infection. Cytotoxic T-lymphocyte responses to cytomegalovirus and ovalbumin were also normal. Although Rac2 is required for normal T-lymphocyte migration, its role in the generation of T(H)1 responses to infection in vivo is largely redundant.