RESUMEN
The intracellular pathogen Mycobacterium tuberculosis (Mtb) and its human host have long co-evolved. Although the host cellular immune response is critical to the control of the bacterium information on the specific contribution of different immune cell subsets in humans is incomplete. Mucosal associated invariant T (MAIT) cells are a prevalent and unique T-cell population in humans with the capacity to detect intracellular infection with bacteria including Mtb. MAIT cells detect bacterially derived metabolites presented by the evolutionarily conserved major histocompatibility complex-like molecule MR1. Here, we review recent advances in our understanding of this T-cell subset and address the potential roles for MR1-restricted T cells in the control, diagnosis, and therapy of tuberculosis.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/inmunología , Interacciones Huésped-Patógeno/inmunología , Membrana Mucosa/citología , Membrana Mucosa/inmunología , Mycobacterium tuberculosis/inmunología , Subgrupos de Linfocitos T/inmunología , Tuberculosis/inmunología , Animales , Evolución Biológica , Susceptibilidad a Enfermedades , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Ligandos , Membrana Mucosa/metabolismo , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/microbiología , Tuberculosis/metabolismo , Tuberculosis/microbiologíaRESUMEN
The nonclassical HLA molecule MHC-related protein 1 (MR1) presents metabolites of the vitamin B synthesis pathways to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells. This new class of Ags represents a variation on the classical paradigm of self/non-self discrimination because these T cells are activated through their TCR by small organic compounds generated during microbial vitamin B2 synthesis. Beyond the fundamental significance, the invariant nature of MR1 across the human population is a tantalizing feature for the potential development of universal immune therapeutic and diagnostic tools. However, many aspects of MR1 Ag presentation and MR1-restricted T cell biology remain unknown, and the ubiquitous expression of MR1 across tissues and cell lines can be a confounding factor for experimental purposes. In this study, we report the development of a novel CRISPR/Cas9 genome editing lentiviral system and its use to efficiently disrupt MR1 expression in A459, THP-1, and K562 cell lines. We generated isogenic MR1(-/-) clonal derivatives of the A549 lung carcinoma and THP-1 monocytic cell lines and used these to study T cell responses to intracellular pathogens. We confirmed that MAIT cell clones were unable to respond to MR1(-/-) clones infected with bacteria whereas Ag presentation by classical and other nonclassical HLAs was unaffected. This system represents a robust and efficient method to disrupt the expression of MR1 and should facilitate investigations into the processing and presentation of MR1 Ags as well as into the biology of MAIT cells.
Asunto(s)
Presentación de Antígeno/inmunología , Edición Génica/métodos , Antígenos de Histocompatibilidad Clase I/inmunología , Activación de Linfocitos/inmunología , Antígenos de Histocompatibilidad Menor/inmunología , Linfocitos T/inmunología , Línea Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Citometría de Flujo , Vectores Genéticos , Humanos , Lentivirus , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination.
Asunto(s)
Evasión Inmune , Infecciones por Poxviridae/inmunología , Poxviridae/patogenicidad , Linfocitos T/inmunología , Linfocitos T/virología , Proteínas Virales/fisiología , Animales , Células CHO , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Cricetulus , Femenino , Células HEK293 , Humanos , Evasión Inmune/genética , Células Jurkat , Macaca mulatta , Ratones , Ratones Endogámicos BALB C , Mpox/inmunología , Poxviridae/genética , Poxviridae/inmunologíaRESUMEN
Mucosa-associated invariant T (MAIT) cells express the semi-invariant T-cell receptor TRAV1-2 and detect a range of bacteria and fungi through the MHC-like molecule MR1. However, knowledge of the function and phenotype of bacteria-reactive MR1-restricted TRAV1-2(+) MAIT cells from human blood is limited. We broadly characterized the function of MR1-restricted MAIT cells in response to bacteria-infected targets and defined a phenotypic panel to identify these cells in the circulation. We demonstrated that bacteria-reactive MR1-restricted T cells shared effector functions of cytolytic effector CD8(+) T cells. By analysing an extensive panel of phenotypic markers, we determined that CD26 and CD161 were most strongly associated with these T cells. Using FACS to sort phenotypically defined CD8(+) subsets we demonstrated that high expression of CD26 on CD8(+) TRAV1-2(+) cells identified with high specificity and sensitivity, bacteria-reactive MR1-restricted T cells from human blood. CD161(hi) was also specific for but lacked sensitivity in identifying all bacteria-reactive MR1-restricted T cells, some of which were CD161(dim) . Using cell surface expression of CD8, TRAV1-2, and CD26(hi) in the absence of stimulation we confirm that bacteria-reactive T cells are lacking in the blood of individuals with active tuberculosis and are restored in the blood of individuals undergoing treatment for tuberculosis.
Asunto(s)
Dipeptidil Peptidasa 4/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Membrana Mucosa/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Células Cultivadas , Dipeptidil Peptidasa 4/metabolismo , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Antígenos de Histocompatibilidad Menor , Mycobacterium smegmatis/inmunología , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Subgrupos de Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
For many intracellular bacteria, both adaptively acquired and innately encoded effector T cells play a central role in the control, and in some cases, clearance of these pathogens. Through the rapid identification of those cells harboring intracellular bacteria, effector T cells have the capacity to both directly control the infection and shape the immune response to the pathogen. Here, we review the mechanisms by which effector T cells control intracellular infection and emphasize the means by which they recognize their targets. As will become evident, the diversity of both redundant and non-redundant effector mechanisms in conjunction with broad recognition of both protein and non-protein antigens allows for the identification of a broad array of bacterial pathogens and lessens the likelihood of immune evasion.
Asunto(s)
Presentación de Antígeno , Enfermedades Transmisibles/inmunología , Linfocitos T/inmunología , HumanosRESUMEN
Control of infection with Mycobacterium tuberculosis (Mtb) requires Th1-type immunity, of which CD8+ T cells play a unique role. High frequency Mtb-reactive CD8+ T cells are present in both Mtb-infected and uninfected humans. We show by limiting dilution analysis that nonclassically restricted CD8+ T cells are universally present, but predominate in Mtb-uninfected individuals. Interestingly, these Mtb-reactive cells expressed the Valpha7.2 T-cell receptor (TCR), were restricted by the nonclassical MHC (HLA-Ib) molecule MR1, and were activated in a transporter associated with antigen processing and presentation (TAP) independent manner. These properties are all characteristics of mucosal associated invariant T cells (MAIT), an "innate" T-cell population of previously unknown function. These MAIT cells also detect cells infected with other bacteria. Direct ex vivo analysis demonstrates that Mtb-reactive MAIT cells are decreased in peripheral blood mononuclear cells (PBMCs) from individuals with active tuberculosis, are enriched in human lung, and respond to Mtb-infected MR1-expressing lung epithelial cells. Overall, these findings suggest a generalized role for MAIT cells in the detection of bacterially infected cells, and potentially in the control of bacterial infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Membrana Mucosa/inmunología , Mycobacterium tuberculosis/inmunología , Secuencia de Aminoácidos , Células Clonales , Regiones Determinantes de Complementariedad , Reacciones Cruzadas , Antígenos HLA/inmunología , Humanos , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Mucosal-associated Invariant T (MAIT) cells are an innate-like T cell subset that recognize a broad array of microbial pathogens, including respiratory pathogens. Here we investigate the transcriptional profile of MAIT cells localized to the human lung, and postulate that MAIT cells may play a role in maintaining homeostasis at this mucosal barrier. Using the MR1/5-OP-RU tetramer, we identified MAIT cells and non-MAIT CD8+ T cells in lung tissue not suitable for transplant from human donors. We used RNA-sequencing of MAIT cells compared to non-MAIT CD8+ T cells to define the transcriptome of MAIT cells in the human lung. We show that, as a population, lung MAIT cells are polycytotoxic, secrete the directly antimicrobial molecule IL-26, express genes associated with persistence, and selectively express cytokine and chemokine- related molecules distinct from other lung-resident CD8+ T cells, such as interferon-γ- and IL-12- receptors. These data highlight MAIT cells' predisposition to rapid pro-inflammatory cytokine responsiveness and antimicrobial mechanisms in human lung tissue, concordant with findings of blood-derived counterparts, and support a function for MAIT cells as early sensors in the defense of respiratory barrier function.
Asunto(s)
Antiinfecciosos , Células T Invariantes Asociadas a Mucosa , Antibacterianos , Linfocitos T CD8-positivos , Citocinas , Humanos , PulmónRESUMEN
The control of Mycobacterium tuberculosis (Mtb) infection is heavily dependent on the adaptive Th1 cellular immune response. Paradoxically, optimal priming of the Th1 response requires activation of priming dendritic cells with Th1 cytokine IFN-gamma. At present, the innate cellular mechanisms required for the generation of an optimal Th1 T cell response remain poorly characterized. We hypothesized that innate Mtb-reactive T cells provide an early source of IFN-gamma to fully activate Mtb-exposed dendritic cells. Here, we report the identification of a novel population of Mtb-reactive CD4(-) alphabetaTCR(+) innate thymocytes. These cells are present at high frequencies, respond to Mtb-infected cells by producing IFN-gamma directly ex vivo, and display characteristics of effector memory T cells. This novel innate population of Mtb-reactive T cells will drive further investigation into the role of these cells in the containment of Mtb following infectious exposure. Furthermore, this is the first demonstration of a human innate pathogen-specific alphabetaTCR(+) T cell and is likely to inspire further investigation into innate T cells recognizing other important human pathogens.
Asunto(s)
Células Dendríticas/inmunología , Mycobacterium tuberculosis/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología , Timo/inmunología , Tuberculosis/inmunología , Recuento de Células , Células Dendríticas/metabolismo , Células Dendríticas/microbiología , Humanos , Inmunidad Innata , Lactante , Recién Nacido , Interferón Tipo I/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/microbiología , Activación de Linfocitos , Subgrupos de Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/microbiología , Timo/citología , Timo/metabolismoRESUMEN
Mucosal-associated invariant T (MAIT) cells typically express a TRAV1-2+ semi-invariant TCRα that enables recognition of bacterial, mycobacterial, and fungal riboflavin metabolites presented by MR1. MAIT cells are associated with immune control of bacterial and mycobacterial infections in murine models. Here, we report that a population of pro-inflammatory TRAV1-2+ CD8+ T cells are present in the airways and lungs of healthy individuals and are enriched in bronchoalveolar fluid of patients with active pulmonary tuberculosis (TB). High-throughput T cell receptor analysis reveals oligoclonal expansions of canonical and donor-unique TRAV1-2+ MAIT-consistent TCRα sequences within this population. Some of these cells demonstrate MR1-restricted mycobacterial reactivity and phenotypes suggestive of MAIT cell identity. These findings demonstrate enrichment of TRAV1-2+ CD8+ T cells with MAIT or MAIT-like features in the airways during active TB and suggest a role for these cells in the human pulmonary immune response to Mycobacterium tuberculosis.
Asunto(s)
Linfocitos T CD8-positivos/citología , Células T Invariantes Asociadas a Mucosa/inmunología , Tuberculosis Pulmonar/inmunología , Animales , Bronquios/microbiología , Líquido del Lavado Bronquioalveolar , Broncoscopía , Linfocitos T CD8-positivos/microbiología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Sistema Inmunológico , Inflamación , Intestinos/inmunología , Pulmón/inmunología , Pulmón/microbiología , Ratones , Antígenos de Histocompatibilidad Menor/inmunología , Células T Invariantes Asociadas a Mucosa/microbiología , Mycobacterium tuberculosis/inmunología , Oregon , Fenotipo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Sudáfrica , Tuberculosis Pulmonar/microbiologíaRESUMEN
Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRα. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRα, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2(+) MAIT cells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAIT antigens are derived from riboflavin metabolites, this suggests that TRAV12-2(+) clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites.
Asunto(s)
Antígenos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Menor/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Riboflavina/inmunología , Streptococcus pyogenes/inmunología , Subgrupos de Linfocitos T/inmunología , Células A549 , Presentación de Antígeno/inmunología , Línea Celular , Células Cultivadas , Antígenos de Histocompatibilidad Clase I/metabolismo , Interacciones Huésped-Patógeno/inmunología , Humanos , Antígenos de Histocompatibilidad Menor/metabolismo , Células T Invariantes Asociadas a Mucosa/inmunología , Células T Invariantes Asociadas a Mucosa/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Riboflavina/metabolismo , Infecciones Estreptocócicas/diagnóstico , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/fisiología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Mucosal associated invariant T (MAIT) cells are an innate-like T cell subset prevalent in humans and distributed throughout the blood and mucosal sites. Human MAIT cells are defined by the expression of the semi-invariant TCRα chain TRAV1-2/TRAJ12/20/33 and are restricted by the non-polymorphic major histocompatibility complex (MHC) class I-like molecule, MHC-related protein 1, MR1. MAIT cells are activated by small organic molecules, derived from the riboflavin biosynthesis pathway of bacteria and fungi, presented by MR1. Traditionally, MAIT cells were thought to recognize a limited number of antigens due to usage of an invariant TCRα chain and restriction by a non-polymorphic MHC molecule. However, recent studies demonstrate that the TCR repertoire of MAIT cells is more heterogeneous, suggesting there is a more diverse array of MR1 antigens that MAIT cells can recognize. In response to infected cells, MAIT cells produce the pro-inflammatory cytokines, IFN-γ and TNF, and are cytolytic. Studies performed in MR1-deficient mice suggest that MAIT cells can provide anti-bacterial control within the first few days post-infection, as well as contribute to enhanced adaptive immunity in murine models of respiratory infections. In humans, the role of MAIT cells is unclear; however, evidence points to interplay between MAIT cells and microbial infections, including Mycobacterium tuberculosis. Given that MAIT cells are pro-inflammatory, serve in early control of bacterial infections, and appear enriched at tissue sites where microbes interface and gain access to the body, we postulate that they play an important role in antimicrobial immune responses. In this review, we discuss the most recent studies on the function and phenotype of MAIT cells, including their TCR diversity and antigenic repertoire, with a focus on the contribution of human MAIT cells in the immune response to microbial infection.
RESUMEN
Mycobacterium tuberculosis (Mtb) is transmitted via inhalation of aerosolized particles. While alveolar macrophages are thought to play a central role in the acquisition and control of this infection, Mtb also has ample opportunity to interact with the airway epithelium. In this regard, we have recently shown that the upper airways are enriched with a population of non-classical, MR1-restricted, Mtb-reactive CD8⺠T cells (MAIT cells). Additionally, we have demonstrated that Mtb-infected epithelial cells lining the upper airways are capable of stimulating IFNγ production by MAIT cells. In this study, we demonstrate that airway epithelial cells efficiently stimulate IFNγ release by MAIT cells as well as HLA-B45 and HLA-E restricted T cell clones. Characterization of the intracellular localization of Mtb in epithelial cells indicates that the vacuole occupied by Mtb in epithelial cells is distinct from DC in that it acquires Rab7 molecules and does not retain markers of early endosomes such as Rab5. The Mtb vacuole is also heterogeneous as there is a varying degree of association with Lamp1 and HLA-I. Although the Mtb vacuole shares markers associated with the late endosome, it does not acidify, and the bacteria are able to replicate within the cell. This work demonstrates that Mtb infected lung epithelial cells are surprisingly efficient at stimulating IFNγ release by CD8⺠T cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Endosomas/inmunología , Células Epiteliales/inmunología , Mycobacterium tuberculosis/inmunología , Mucosa Respiratoria/inmunología , Vacuolas/inmunología , Linfocitos T CD8-positivos/microbiología , Linfocitos T CD8-positivos/patología , Línea Celular , Técnicas de Cocultivo , Endosomas/microbiología , Endosomas/patología , Células Epiteliales/microbiología , Células Epiteliales/patología , Expresión Génica , Antígenos HLA-B/genética , Antígenos HLA-B/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/inmunología , Cultivo Primario de Células , Mucosa Respiratoria/microbiología , Mucosa Respiratoria/patología , Vacuolas/microbiología , Vacuolas/patología , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/inmunología , Proteínas de Unión a GTP rab7 , Antígenos HLA-ERESUMEN
Mucosal-associated invariant T (MAIT) cells express a semi-invariant T cell receptor (TCR) that detects microbial metabolites presented by the nonpolymorphic major histocompatibility complex (MHC)-like molecule MR1. The highly conserved nature of MR1 in conjunction with biased MAIT TCRα chain usage is widely thought to indicate limited ligand presentation and discrimination within a pattern-like recognition system. Here, we evaluated the TCR repertoire of MAIT cells responsive to three classes of microbes. Substantial diversity and heterogeneity were apparent across the functional MAIT cell repertoire as a whole, especially for TCRß chain sequences. Moreover, different pathogen-specific responses were characterized by distinct TCR usage, both between and within individuals, suggesting that MAIT cell adaptation was a direct consequence of exposure to various exogenous MR1-restricted epitopes. In line with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of specific repertoires shaped by microbial exposure.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/metabolismo , Bacterias/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Membrana Mucosa/citología , Membrana Mucosa/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Bacterias/efectos de los fármacos , Línea Celular , Células Clonales , Regiones Determinantes de Complementariedad/química , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T/genética , Humanos , Ligandos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Membrana Mucosa/efectos de los fármacos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Homología de Secuencia de Aminoácido , Linfocitos T/efectos de los fármacos , Complejo Vitamínico B/farmacologíaRESUMEN
Mucosal-associated invariant T (MAIT) cells are a unique T cell subset in mammals. They are present at high frequencies at mucosal tissue sites and have an intrinsic capacity to respond to microbial infections. The semi-invariant antigen recognition receptor of MAIT cells detects the non-polymorphic antigen-presenting molecule major histocompatibility complex class I-related protein 1 (MR1), which can bind microorganism-derived riboflavin metabolites. The striking evolutionary conservation in both the MR1 molecule and the MAIT T cell receptor suggests that strong selective pressures maintain this T cell pattern recognition system which detects microbial infection.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/inmunología , Inmunidad Mucosa , Receptores de Antígenos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Evolución Molecular , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Mamíferos , Modelos Biológicos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Riboflavina/metabolismo , Selección GenéticaRESUMEN
Mucosal associated invariant T cells are unique T cells localized at high frequencies at the portals of entry for many pathogens. Mucosal associated invariant T cells display a variety of characteristics that suggest their function is to act as effectors in the initial control of microbial infection at mucosal sites.
Asunto(s)
Infecciones Bacterianas/inmunología , Linfocitos T/inmunología , Animales , Humanos , Inmunidad Mucosa/inmunología , RatonesRESUMEN
Neonates are clearly more susceptible to severe disease following infection with a variety of pathogens than are adults. However, the causes for this are unclear and are often attributed to immunological immaturity. While several aspects of immunity differ between adults and neonates, the capacity of dendritic cells in neonates to process and present antigen to CD8+ T cells remains to be addressed. We used human CD8+ T cell clones to compare the ability of neonatal and adult monocyte-derived dendritic cells to present or process and present antigen using the MHC class I pathway. Specifically, we assessed the ability of dendritic cells to present antigenic peptide, present an HLA-E-restricted antigen, process and present an MHC class I-restricted antigen through the classical MHC class I pathway, and cross present cell-associated antigen via MHC class I. We found no defect in neonatal dendritic cells to perform any of these processing and presentation functions and conclude that the MHC class I antigen processing and presentation pathway is functional in neonatal dendritic cells and hence may not account for the diminished control of pathogens.
Asunto(s)
Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Línea Celular , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/virología , Citometría de Flujo , Antígeno HLA-A2/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Recién Nacido , Interferón gamma/inmunología , Fosfoproteínas/inmunología , Virus Vaccinia/crecimiento & desarrollo , Virus Vaccinia/inmunología , Proteínas de la Matriz Viral/inmunologíaRESUMEN
Neonates are more susceptible than adults to viral and bacterial diseases. We hypothesized that plasmacytoid dendritic cells, the cells that provide large amounts of IFN-alpha in response to Toll-like receptor 9 (TLR9) agonists, are defective in neonates. To assess the intrinsic functionality of plasmacytoid dendritic cells from neonates we compared IFN-alpha production by plasmacytoid dendritic cells derived from neonates versus adults in both whole blood and in purified plasmacytoid dendritic cells. TLR9-stimulation of whole blood from adults and neonates resulted in comparable amounts of IFN-alpha production. However, we observed small but significant differences in IFN-alpha production from purified CD123+ plasmacytoid dendritic cells from neonates after stimulation with the TLR9 ligand CpG-DNA. Furthermore, we assessed surface expression of co-stimulatory molecules on plasmacytoid dendritic cells after stimulation. While purified CD123+ plasmacytoid dendritic cells from adults up-regulated co-stimulatory molecules CD80 and CD86 with IL-3 alone those from neonates required the addition of CpG-DNA to reach adult levels. Therefore, the intrinsic deficiencies of neonatal plasmacytoid dendritic cells can be mitigated by TLR9 agonists. These results are consistent with the observation that vaccines that effect strong adjuvant activity on dendritic cells can induce protective responses in neonates.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Interferón-alfa/metabolismo , Oligodesoxirribonucleótidos/farmacología , Receptor Toll-Like 9/agonistas , Adulto , Envejecimiento/inmunología , Envejecimiento/fisiología , Antígeno B7-1/análisis , Antígeno B7-1/genética , Antígeno B7-2/análisis , Antígeno B7-2/genética , Secuencia de Bases , Diferenciación Celular/inmunología , Diferenciación Celular/fisiología , Separación Celular , Células Cultivadas , ADN/genética , Células Dendríticas/inmunología , Humanos , Recién Nacido , Interleucina-3/fisiología , Subunidad alfa del Receptor de Interleucina-3 , Oligodesoxirribonucleótidos/genética , Receptores de Interleucina-3/análisis , Receptor Toll-Like 9/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Human CMV establishes a lifelong latent infection in the majority of people worldwide. Although most infections are asymptomatic, immunocompetent hosts devote an extraordinary amount of immune resources to virus control. To increase our understanding of CMV immunobiology in an animal model, we used a genomic approach to comprehensively map the C57BL/6 CD8 T cell response to murine CMV (MCMV). Responses to 27 viral proteins were detectable directly ex vivo, the most diverse CD8 T cell response yet described within an individual animal. Twenty-four peptide epitopes were mapped from 18 Ags, which together account for most of the MCMV-specific response. Most Ags were from genes expressed at early times, after viral genes that interfere with Ag presentation are expressed, consistent with the hypothesis that the CD8 T cell response to MCMV is largely driven by cross-presented Ag. Titration of peptide epitopes in a direct ex vivo intracellular cytokine staining assay revealed a wide range of functional avidities, with no obvious correlation between functional avidity and the strength of the response. The immunodominance hierarchy varied only slightly between mice and between experiments. However, H-2(b)-expressing mice with different genetic backgrounds responded preferentially to different epitopes, indicating that non-MHC-encoded factors contribute to immunodominance in the CD8 T cell response to MCMV.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Genoma Viral/genética , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Muromegalovirus/genética , Muromegalovirus/inmunología , Enfermedad Aguda , Algoritmos , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Computadores , Epítopos/química , Epítopos/inmunología , Biblioteca de Genes , Antígenos de Histocompatibilidad/inmunología , Ratones , Datos de Secuencia Molecular , Muromegalovirus/fisiología , Sistemas de Lectura Abierta/genética , Péptidos/química , Péptidos/inmunologíaRESUMEN
Although in vitro studies have shown that herpesviruses, including murine CMV (MCMV), encode genes that interfere with the MHC class I pathway, their effects on the CTL response in vivo is unclear. We identified a D(b)-restricted CTL epitope from MCMV M45 by screening an MCMV genomic library using CTL clones isolated from mice infected with MCMV lacking m152. Because m152 severely inhibits CTL recognition of M45 in vitro, we questioned whether an M45-specific response would be generated in mice infected with wild-type MCMV expressing m152. Mice infected with wild-type MCMV or MCMVDelta(m)152 made similar responses to the M45 Ag. Moreover, we saw no skewing of the proportion of M45-specific CD8 T cells within the total MCMV-specific response after infection with MCMV with m152. Despite the profound effect m152 has on presentation of M45 in vitro, it does not affect the immunodominance of M45 in the CTL response in vivo.
Asunto(s)
Adyuvantes Inmunológicos/genética , Presentación de Antígeno/genética , Epítopos Inmunodominantes/inmunología , Glicoproteínas de Membrana/genética , Muromegalovirus/genética , Muromegalovirus/inmunología , Ribonucleótido Reductasas/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas Virales , Adyuvantes Inmunológicos/fisiología , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Línea Celular , Línea Celular Transformada , Separación Celular , Células Clonales , Epítopos de Linfocito T/inmunología , Femenino , Antígenos H-2/inmunología , Infecciones por Herpesviridae/inmunología , Antígeno de Histocompatibilidad H-2D , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/inmunología , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Muromegalovirus/fisiología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Ribonucleótido Reductasas/metabolismo , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/virologíaRESUMEN
Macrophages play an important role in murine cytomegalovirus (MCMV) infection in vivo, both in disseminating infection and in harboring latent virus. MCMV encodes three immune evasion genes (m4, m6, and m152) that interfere with the ability of cytotoxic T cells (CTL) to detect virus-infected fibroblasts, but the efficacy of immune evasion in macrophages has been controversial. Here we show that MCMV immune evasion genes function in H-2(b) primary bone marrow macrophages (BMMphi) in the same way that they do in fibroblasts. Metabolic labeling experiments showed that class I is retained in the endoplasmic reticulum by MCMV infection and associates with m4/gp34 to a similar extent in fibroblasts and BMMphi. We tested a series of K(b)- and D(b)-restricted CTL clones specific for MCMV early genes against a panel of MCMV wild-type virus and mutants lacking m152, m4, or m6. MCMV immune evasion genes effectively inhibited antigen presentation. m152 appeared sufficient to abolish D(b)-restricted presentation in infected macrophages, as has been previously observed in infected fibroblasts. However, for inhibition of recognition of infected macrophages by K(b)-restricted CTL, m4, m6, and m152 were all required. The contribution of m4 to inhibition of recognition appeared much more important in macrophages than in fibroblasts. Thus, MCMV immune evasion genes function effectively in primary macrophages to prevent CTL recognition of early antigens and show the same pattern of major histocompatibility complex class I allele discrimination as is seen in fibroblasts. Furthermore, for inhibition of K(b)-restricted presentation, a strong synergistic effect was noted among m152, m4, and m6.