RESUMEN
The intestinal immune system interacts with commensal microbiota to maintain gut homeostasis. Furthermore, stress alters the microbiome composition, leading to impaired brain function; yet how the intestinal immune system mediates these effects remains elusive. Here we report that colonic γδ T cells modulate behavioral vulnerability to chronic social stress via dectin-1 signaling. We show that reduction in specific Lactobacillus species, which are involved in T cell differentiation to protect the host immune system, contributes to stress-induced social-avoidance behavior, consistent with our observations in patients with depression. Stress-susceptible behaviors derive from increased differentiation in colonic interleukin (IL)-17-producing γδ T cells (γδ17 T cells) and their meningeal accumulation. These stress-susceptible cellular and behavioral phenotypes are causally mediated by dectin-1, an innate immune receptor expressed in γδ T cells. Our results highlight the previously unrecognized role of intestinal γδ17 T cells in the modulation of psychological stress responses and the importance of dectin-1 as a potential therapeutic target for the treatment of stress-induced behaviors.
Asunto(s)
Intestinos , Lectinas Tipo C , Colon , Transducción de Señal , Receptores de Antígenos de Linfocitos T gamma-deltaRESUMEN
The IL-7R regulates the homeostasis, activation, and distribution of T cells in peripheral tissues. Although several transcriptional enhancers that regulate IL-7Rα expression in αß T cells have been identified, enhancers active in γδ T cells remain unknown. In this article, we discovered an evolutionarily conserved noncoding sequence (CNS) in intron 2 of the IL-7Rα-chain (IL-7Rα) locus and named this region CNS9. CNS9 contained a conserved retinoic acid receptor-related orphan receptor (ROR)-responsive element (RORE) and exerted RORγt-dependent enhancer activity in vitro. Mice harboring point mutations in the RORE in CNS9 (CNS9-RORmut) showed reduced IL-7Rα expression in IL-17-producing Vγ4+ γδ T cells. In addition, the cell number and IL-17A production of Vγ4+ γδ T cells were reduced in the adipose tissue of CNS9-RORmut mice. Consistent with the reduction in IL-17A, CNS9-RORmut mice exhibited decreased IL-33 expression in the adipose tissue, resulting in fewer regulatory T cells and glucose intolerance. The CNS9-ROR motif was partially responsible for IL-7Rα expression in RORγt+ regulatory T cells, whereas IL-7Rα expression was unaffected in RORγt-expressing Vγ2+ γδ T cells, Th17 cells, type 3 innate lymphoid cells, and invariant NKT cells. Our results indicate that CNS9 is a RORΕ-dependent, Vγ4+ γδ T cell-specific IL-7Rα enhancer that plays a critical role in adipose tissue homeostasis via regulatory T cells, suggesting that the evolutionarily conserved RORΕ in IL-7Rα intron 2 may influence the incidence of type 2 diabetes.
Asunto(s)
Elementos de Facilitación Genéticos , Intrones , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Receptores de Antígenos de Linfocitos T gamma-delta , Animales , Ratones , Intrones/genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Elementos de Facilitación Genéticos/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Glucosa/metabolismo , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/metabolismo , Ratones Endogámicos C57BL , Células Th17/inmunología , Interleucina-17/metabolismo , Interleucina-17/genética , Humanos , Tejido Adiposo/metabolismo , Tejido Adiposo/inmunologíaRESUMEN
Vγ6+ γδ T cells develop in the thymus at the perinatal stage and are exclusive IL-17A producers among γδ T cells. The loss of MHC class II led to the expansion of IL-17A+ Vγ6+ γδ T cells in the thymus. Thus, MHC class II in the thymus inhibits the generation of IL-17A+ Vγ6+ γδ T cells.
Asunto(s)
Genes MHC Clase II , Interleucina-17 , Receptores de Antígenos de Linfocitos T gamma-delta , Subgrupos de Linfocitos T , Timo , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Timo/citologíaRESUMEN
γδ T cells producing IL-17A (γδTh17 cells) are known to be involved in peritonitis induced by Escherichia coli infection in mice. In vivo treatment with Vγ6-specific mAb (1C10-1F7) significantly hampered resolution of E. coli infection. Thus, Vγ6+ γδTh17 cells mainly contributed to protection against E. coli infection.
Asunto(s)
Infecciones por Escherichia coli/inmunología , Inmunidad Innata/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Células Th17/inmunología , Animales , RatonesRESUMEN
Viral infection is a putative causal factor for the development of type 1 diabetes, but the exact pathogenic mechanism of virus-induced diabetes (VID) remains unclear. Here, to identify the critical factors that regulate VID, we analyzed encephalomyocarditis D (EMC-D) VID-sensitive DBA/2 mice in comparison with resistant B6 mice. EMC-D virus-induced cell death occurred more frequently in DBA/2 ß-cells than in B6 ß-cells with 100U/ml IFN-ß priming in vitro. We therefore purified ß-cells using flow cytometry from mice two days after EMC-D virus infection and subjected them to microarray analysis. As a results, innate immune response pathway was found to be enriched in B6 ß-cells. The signal transducer and activator of transcription 2 (Stat2) gene interacted with genes in the pathway. Stat2 gene expression levels were lower in DBA/2 mice than in B6 mice, restrictive to ß-cells. Moreover, administration of IFN-ß failed to upregulate Stat2 gene in DBA/2 ß-cells than in those of B6 in vivo. The viral titer significantly increased only in the DBA/2 pancreas. Thus, these provided data suggest that impaired upregulation of Stat2 gene restrictive to ß-cells at the early stage of infection is responsible for VID development in DBA/2 mice.
Asunto(s)
Infecciones por Cardiovirus/complicaciones , Diabetes Mellitus Tipo 1/virología , Células Secretoras de Insulina/virología , Factor de Transcripción STAT2/genética , Animales , Infecciones por Cardiovirus/tratamiento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/virología , Diabetes Mellitus Tipo 1/genética , Virus de la Encefalomiocarditis , Regulación de la Expresión Génica , Inmunidad Innata/genética , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/inmunología , Interferón Tipo I/farmacología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Factor de Transcripción STAT2/metabolismo , Regulación hacia ArribaRESUMEN
Interleukin 21 (IL-21) is a pleiotropic common cytokine receptor γ chain cytokine that promotes the effector functions of NK cells and CD8+ T cells and inhibits CD8+ T cell exhaustion during chronic infection. We found that the absolute number of short-lived effector CD8+ T cells (SLECs) (KLRG1high CD127low) decreased significantly in IL-21 receptor-deficient (IL-21R-/-) mice during Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. Early effector CD8+ T cells (EECs) (KLRG1low CD127low) were normally generated in IL-21R-/- mice after infection. Exhausted CD8+ T cells (PD-1high KLRG1low) were also normally generated in IL-21R-/- mice after infection. Mixed bone marrow (BM) chimera and transfer experiments showed that IL-21R on CD8+ T cells was essential for the proliferation of EECs, allowing them to differentiate into SLECs after BCG infection. On the other hand, the number of SLECs increased significantly after infection with recombinant BCG (rBCG) that secreted an antigen 85B (Ag85B)-IL-21 fusion protein (rBCG-Ag85B-IL-21), but the number of exhausted CD8+ T cells did not change after rBCG-Ag85B-IL-21 infection. These results suggest that IL-21 signaling drives the differentiation of SLECs from EECs but does not inhibit the exhaustion of CD8+ T cells following BCG infection in mice.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Interleucinas/metabolismo , Mycobacterium bovis/inmunología , Subgrupos de Linfocitos T/inmunología , Tuberculosis/inmunología , Animales , Linfocitos T CD8-positivos/química , Diferenciación Celular , Modelos Animales de Enfermedad , Inmunofenotipificación , Subunidad alfa del Receptor de Interleucina-7/análisis , Lectinas Tipo C , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Inmunológicos/análisis , Receptores de Interleucina-21/análisis , Receptores de Interleucina-21/deficiencia , Subgrupos de Linfocitos T/químicaRESUMEN
Notch signaling is an important regulator for the development and function of both αß and γδ T cells, whereas roles of Notch signaling in T cell maintenance remain unclear. We reported previously that the Notch-Hes1 pathway was involved in the intrathymic development of naturally occurring IL-17-producing (IL-17(+)) γδ T cells. To gain insight into additional roles for the Notch axis in the homeostasis of γδ T cells, we performed a genome-wide analysis of Notch target genes and identified the novel promoter site of IL-7Rα driven by the Notch-RBP-Jκ pathway. Constitutive Notch signaling had the potential to induce IL-7Rα expression on γδ T cells in vivo, as well as in vitro, whereas conditional deletion of RBP-Jκ abrogated IL-7Rα expression, but not Hes1 expression, by γδ T cells and selectively reduced the pool size of IL-7Rα(high) IL-17(+) γδ T cells in the periphery. In the absence of IL-7Rα-mediated signaling, IL-17(+) γδ T cells were barely maintained in adult mice. Addition of exogenous IL-7 in vitro selectively expanded IL-17(+) γδ T cells. Thus, our results revealed a novel role for the Notch-RBP-Jκ-IL-7Rα axis that is independent of Hes1 for homeostasis of IL-17(+) γδ T cells.
Asunto(s)
Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/inmunología , Interleucina-17/biosíntesis , Receptor Notch1/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores de Interleucina-7/inmunología , Animales , Anticuerpos/inmunología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Proliferación Celular/efectos de los fármacos , Estudio de Asociación del Genoma Completo , Proteínas de Homeodominio/biosíntesis , Homeostasis , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Interferón gamma/biosíntesis , Interleucina-7/biosíntesis , Interleucina-7/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Interleucina-7/biosíntesis , Transducción de Señal , Linfocitos T/inmunología , Factor de Transcripción HES-1RESUMEN
BACKGROUND: Klebsiella pneumoniae frequently causes life-threatening infection in children. Interleukin 17A (IL-17A) is known to be involved in protection against K. pneumoniae infection through activation of neutrophils. METHODS AND RESULTS: We found that IL-17A-producing γδ T cells existed more frequently in younger mice on examination of IL-17A-producing lymphocytes in the lung of naive mice at various ages. We hence compared the protective role of IL-17A-producing γδ T cells against pulmonary K. pneumoniae infection in young (3 weeks old) and adult (8-12 weeks old) mice. IL-17A-deficient mice were susceptible to K. pneumonia regardless of age. Cγ-, Vγ4/6-, or Vδ1-deficient mice were susceptible to K. pneumonia at young age, while interleukin 23p19 (IL-23p19)-deficient mice were susceptible at adult age. IL-17A-producing Vγ1-Vγ4- γδ T cells expressing canonical Vγ6/Vδ1 genes were dominant over IL-17A-producing Vγ4+ γδ T cells in the lungs of young mice after infection. The IL-17A-producing Vγ1-Vγ4- γδ T cells expressed an activation marker, CD69, and proliferated in an IL-23-independent manner, while the IL-17A-producing Vγ4+ γδ T cells expressing IL-23 receptor but no CD69 proliferated in IL-23-dependent manner. CONCLUSIONS: These results suggest that 2 types of IL-17A-producing γδ T cells are activated for host defense against K. pneumoniae infection by IL-23-dependent or independent mechanism.
Asunto(s)
Interleucina-17/metabolismo , Infecciones por Klebsiella/inmunología , Klebsiella pneumoniae/inmunología , Neumonía Bacteriana/inmunología , Linfocitos T/inmunología , Animales , Femenino , Interleucina-17/deficiencia , Subunidad p19 de la Interleucina-23/metabolismo , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
γδ T cells develop at the double-negative (DN) 2 and DN3 stages and acquire functions to produce IL-17 and IFN-γ in fetal thymus. However, the relationship between differentiation stages and their functions was unclear. In this study, we found that, although IFN-γ-producing and IL-17-producing γδ T cells developed from DN2 cells, only IFN-γ-producing γδ T cells developed from DN3 cells, indicating the direct generation of IL-17-producing γδ T cells from the DN2 stage, not through the DN3 stage. Single-cell analysis revealed that DN2 cells contained heterogeneous γδ T cell precursors with or without an ability to develop IL-17 producers. Inactivation of B cell leukemia/lymphoma 11b, a zinc finger transcription factor responsible for transition from early to late stages of DN2 cells, completely abrogated the development of IL-17-producing γδ T cells, although a unique subset of IFN-γ-producing γδ T cells expressing a high level of promyelocytic leukemia zinc finger was able to develop. Thus, our results reveal that γδ T cells are functionally differentiated to IFN-γ and IL-17 producers at different developmental stages in fetal thymus.
Asunto(s)
Interferón gamma/inmunología , Interleucina-17/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Timo/embriología , Animales , Interferón gamma/genética , Interleucina-17/genética , Ratones , Ratones Mutantes , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Proteínas Represoras/genética , Proteínas Represoras/inmunología , Linfocitos T/citología , Timo/inmunología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/inmunologíaRESUMEN
Sepsis is a systemic inflammatory response to a severe, life-threatening infection with organ dysfunction. Although there is no effective treatment for this fatal illness, a deeper understanding of the pathophysiological basis of sepsis and its underlying mechanisms could lead to the development of new treatment approaches. Here, we demonstrate that the selective Bruton's tyrosine kinase (Btk) inhibitor acalabrutinib augments survival rates in a lipopolysaccharide (LPS)-induced septic model. Our in vitro and in vivo findings both indicate that acalabrutinib reduces IL-6 production specifically in marginal zone B (MZ B) cells rather than in macrophages. Furthermore, Btk-deficient MZ B cells exhibited suppressed LPS-induced IL-6 production in vitro. Nuclear factor-kappa B (NF-κB) signaling, which is the downstream signaling cascade of Toll-like receptor 4 (TLR4), was also severely attenuated in Btk-deficient MZ B cells. These findings suggest that Btk blockade may prevent sepsis by inhibiting IL-6 production in MZ B cells. In addition, although Btk inhibition may adversely affect B cell maturation and humoral immunity, antibody responses were not impaired when acalabrutinib was administered for a short period after immunization with T-cell-independent (TI) and T-cell-dependent (TD) antigens. In contrast, long-term administration of acalabrutinib slightly impaired humoral immunity. Therefore, these findings suggest that Btk inhibitors may be a potential option for alleviating endotoxic shock without compromising humoral immunity and emphasize the importance of maintaining a delicate balance between immunomodulation and inflammation suppression.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa , Linfocitos B , Interleucina-6 , Choque Séptico , Animales , Ratones , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Benzamidas , Lipopolisacáridos/toxicidad , FN-kappa B , Pirazinas , Choque Séptico/inducido químicamente , Choque Séptico/tratamiento farmacológico , Linfocitos B/inmunologíaRESUMEN
IL-17A-producing γδ T cells in mice consist primarily of Vγ6+ tissue-resident cells and Vγ4+ circulating cells. How these γδ T cell subsets are regulated during homeostasis and cancer remains poorly understood. Using single-cell RNA sequencing and flow cytommetry, we show that lung Vγ4+ and Vγ6+ cells from tumor-free and tumor-bearing mice express contrasting cell surface molecules as well as distinct co-inhibitory molecules, which function to suppress their expansion. Vγ6+ cells express constitutively high levels of PD-1, whereas Vγ4+ cells upregulate TIM-3 in response to tumor-derived IL-1ß and IL-23. Inhibition of either PD-1 or TIM-3 in mammary tumor-bearing mice increased Vγ6+ and Vγ4+ cell numbers, respectively. We found that genetic deletion of γδ T cells elicits responsiveness to anti-PD-1 and anti-TIM-3 immunotherapy in a mammary tumor model that is refractory to T cell checkpoint inhibitors, indicating that IL-17A-producing γδ T cells instigate resistance to immunotherapy. Together, these data demonstrate how lung IL-17A-producing γδ T cell subsets are differentially controlled by PD-1 and TIM-3 in steady-state and cancer.
Asunto(s)
Receptor 2 Celular del Virus de la Hepatitis A , Interleucina-17 , Neoplasias , Receptor de Muerte Celular Programada 1 , Subgrupos de Linfocitos T , Animales , Ratones , Receptor de Muerte Celular Programada 1/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/metabolismoRESUMEN
Trans-chromosomic (Tc) mice carrying mini-chromosomes with megabase-sized human immunoglobulin (Ig) loci have contributed to the development of fully human therapeutic monoclonal antibodies, but mitotic instability of human mini-chromosomes in mice may limit the efficiency of hybridoma production. Here, we establish human antibody-producing Tc mice (TC-mAb mice) that stably maintain a mouse-derived, engineered chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig-knockout background. Comprehensive, high-throughput DNA sequencing shows that the human Ig repertoire, including variable gene usage, is well recapitulated in TC-mAb mice. Despite slightly altered B cell development and a delayed immune response, TC-mAb mice have more subsets of antigen-specific plasmablast and plasma cells than wild-type mice, leading to efficient hybridoma production. Our results thus suggest that TC-mAb mice offer a valuable platform for obtaining fully human therapeutic antibodies, and a useful model for elucidating the regulation of human Ig repertoire formation.
Asunto(s)
Anticuerpos Monoclonales , Cadenas Pesadas de Inmunoglobulina , Animales , Cromosomas Artificiales de Levadura , Humanos , Hibridomas , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Ratones , Ratones TransgénicosRESUMEN
Although murine γδ T cells are largely considered innate immune cells, they have recently been reported to form long-lived memory populations. Much remains unknown about the biology and specificity of memory γδ T cells. Here, we interrogated intestinal memory Vγ4 Vδ1 T cells generated after foodborne Listeria monocytogenes (Lm) infection to uncover an unanticipated complexity in the specificity of these cells. Deep TCR sequencing revealed that a subset of non-canonical Vδ1 clones are selected by Lm infection, consistent with antigen-specific clonal expansion. Ex vivo stimulations and in vivo heterologous challenge infections with diverse pathogenic bacteria revealed that Lm-elicited memory Vγ4 Vδ1 T cells are broadly reactive. The Vγ4 Vδ1 T cell recall response to Lm, Salmonella enterica serovar Typhimurium (STm) and Citrobacter rodentium was largely mediated by the γδTCR as internalizing the γδTCR prevented T cell expansion. Both broadly-reactive canonical and pathogen-selected non-canonical Vδ1 clones contributed to memory responses to Lm and STm. Interestingly, some non-canonical γδ T cell clones selected by Lm infection also responded after STm infection, suggesting some level of cross-reactivity. These findings underscore the promiscuous nature of memory γδ T cells and suggest that pathogen-elicited memory γδ T cells are potential targets for broad-spectrum anti-infective vaccines.
Asunto(s)
Infecciones Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Citrobacter rodentium/fisiología , Listeria monocytogenes/fisiología , Células T de Memoria/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Salmonella typhi/fisiología , Animales , Antígenos Bacterianos/inmunología , Células Cultivadas , Reacciones Cruzadas , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunidad Heteróloga , Células T de Memoria/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Especificidad del Receptor de Antígeno de Linfocitos TRESUMEN
There are seven Vγ gene segments in the TCR γ chain loci of mice. We developed monoclonal antibodies (mAbs) specific to the Vγ6 chain (Heilig & Tonegawa nomenclature). By immunizing Vγ4/6 KO mice with complementarity-determining region peptides in Vγ6 chains, we generated three hybridomas. These hybridomas produced mAbs capable of cell surface staining of Vγ6/Vδ1 gene-transfected T-cell line lacking TCR as well as of Vγ1- Vγ4- Vγ5- Vγ7- γδ T cells and the CD3high TCRδint γδ T cells in various organs. The location of Vγ6+ γδ T cells, which peaked in the newborn thymus, was associated with mTEC. In vivo administration of clone 1C10-1F7 mAb impaired protection against Klebsiella pneumoniae infection but ameliorated psoriasis-like dermatitis induced by imiquimod treatment. These new mAbs are useful to elucidate the development, location, and functions of Vγ6 γδ T cells in mice.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Receptores de Antígenos de Linfocitos T gamma-delta/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/inmunología , Formación de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Línea Celular Tumoral , Femenino , Inmunización , Inmunofenotipificación , Ratones , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Proteins coating Plasmodium merozoite surface and secreted from its apical organelles are considered as promising vaccine candidates for blood-stage malaria. The rhoptry neck protein 12 of Plasmodium falciparum (PfRON12) was recently reported as a protein specifically expressed in schizonts and localized to the rhoptry neck of merozoites. Here, we assessed its potential as a vaccine candidate. We expressed a recombinant PfRON12 protein by a wheat germ cell-free system to obtain anti-PfRON12 antibody. Immunoblot analysis of schizont lysates detected a single band at approximately 40â¯kDa under reducing conditions, consistent with the predicted molecular weight. Additionally, anti-PfRON12 antibody recognized a single band around 80â¯kDa under non-reducing conditions, suggesting native PfRON12 forms a disulfide-bond-mediated multimer. Immunofluorescence assay and immunoelectron microscopy revealed that PfRON12 localized to the rhoptry neck of merozoites in schizonts and to the surface of free merozoites. The biological activity of anti-PfRON12 antibody was tested by in vitro growth inhibition assay (GIA), and the rabbit antibodies significantly inhibited merozoite invasion of erythrocytes. We then investigated whether PfRON12 is immunogenic in P. falciparum-infected individuals. The sera from P. falciparum infected individuals in Thailand and Mali reacted with the recombinant PfRON12. Furthermore, human anti-PfRON12 antibodies affinity-purified from Malian serum samples inhibited merozoite invasion of erythrocytes in vitro. Moreover, pfron12 is highly conserved with only 4 non-synonymous mutations in the coding sequence from approximately 200 isolates deposited in PlasmoDB. These results suggest that PfRON12 might be a potential blood-stage vaccine candidate antigen against P. falciparum.
Asunto(s)
Antígenos de Protozoos/inmunología , Eritrocitos/parasitología , Merozoítos/fisiología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/genética , Infecciones Asintomáticas/epidemiología , Ensayo de Inmunoadsorción Enzimática , Eritrocitos/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Vacunas contra la Malaria/genética , Vacunas contra la Malaria/inmunología , Malaria Falciparum/epidemiología , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Malaria Falciparum/prevención & control , Malí/epidemiología , Merozoítos/inmunología , Conejos , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Esquizontes/química , Tailandia/epidemiologíaRESUMEN
The pluripotential cell-specific gene Nanog encodes a homeodomain-bearing transcription factor required for maintaining the undifferentiated state of stem cells. However, the molecular mechanisms that regulate Nanog gene expression are largely unknown. To address this important issue, we used luciferase assays to monitor the relative activities of deletion fragments from the 5'-flanking region of the gene. An adjacent pair of highly conserved Octamer- and Sox-binding sites was found to be essential for activating pluripotential state-specific gene expression. Furthermore, the 5'-end fragment encompassing the Octamer/Sox element was sufficient for inducing the proper expression of a green fluorescent protein reporter gene even in human embryonic stem (ES) cells. The potential of OCT4 and SOX2 to bind to this element was verified by electrophoretic mobility shift assays with extracts from F9 embryonal carcinoma cells and embryonic germ cells derived from embryonic day 12.5 embryos. However, in ES cell extracts, a complex of OCT4 with an undefined factor preferentially bound to the Octamer/Sox element. Thus, Nanog transcription may be regulated through an interaction between Oct4 and Sox2 or a novel pluripotential cell-specific Sox element-binding factor which is prominent in ES cells.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas Nucleares/fisiología , Elementos de Respuesta/genética , Células Madre/metabolismo , Factores de Transcripción/fisiología , Región de Flanqueo 5'/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Embrión de Mamíferos/citología , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas HMGB , Humanos , Luciferasas/análisis , Luciferasas/genética , Ratones , Datos de Secuencia Molecular , Mutación/genética , Proteína Homeótica Nanog , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factor 3 de Transcripción de Unión a Octámeros , Factores de Transcripción SOXB1 , Eliminación de Secuencia/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
We recently found that a unique subset of innate-like γδ T cells develops from the DN2a stage of the fetal thymus independently of the zinc-finger transcription factor B cell leukemia/lymphoma 11b (Bcl11b). Herein, we characterize these Bcl11b-independent γδ T cells in the periphery as CD5-NK1.1+ and Granzyme B+, and we show that they are capable of producing interferon (IFN)-γ upon T cell receptor stimulation without Ca2+ influx. In wild-type mice, these cells were sparse in lymphoid tissues but abundant in non-lymphoid tissues, such as the liver. Bcl11b-independent CD5-NK1.1+ γδ T cells appeared and contributed to early protection before Bcl11b-dependent CD5+NK1.1- γδ T cells following Listeria monocytogenes infection, resembling their sequential appearance during development in the thymus.
Asunto(s)
Antígenos Ly/metabolismo , Antígenos CD5/genética , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Proteínas Represoras/metabolismo , Linfocitos T/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Antígenos CD5/deficiencia , Células Cultivadas , Femenino , Expresión Génica , Granzimas/metabolismo , Interferón gamma/análisis , Interferón gamma/metabolismo , Interleucina-17/análisis , Interleucina-17/metabolismo , Listeria monocytogenes/fisiología , Listeriosis/inmunología , Listeriosis/prevención & control , Hígado/inmunología , Hígado/metabolismo , Hígado/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Linfocitos T/citología , Linfocitos T/inmunología , Timo/citología , Timo/inmunología , Timo/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
Nanog is a novel pluripotential cell-specific gene that plays a crucial role in maintaining the undifferentiated state of early postimplantation embryos and embryonic stem (ES) cells. We have explored the expression pattern and function of Nanog and a Nanog-homologue, Nanog-ps1.Nanog-ps1 was mapped on Chromosome 7 and shown to be a pseudogene. Immunocytochemical analysis in vivo showed that the NANOG protein was absent in unfertilized oocytes, and was detected in cells of morula-stage embryos, the inner cell mass of blastocysts and the epiblast of E6.5 and E7.5 embryos, but not in primordial germ cells of early postimplantation embryos. In monkey and human ES cells, NANOG expression was restricted to undifferentiated cells. Furthermore, reactivation of the somatic cell-derived Nanog was tightly linked with nuclear reprogramming induced by cell hybridization with ES cells and by nuclear transplantation into enucleated oocytes. Notably, mouse Nanog (+/-) ES cells, which produced approximately half the amount of NANOG produced by wild-type ES cells, readily differentiated to multi-lineage cells in culture medium including LIF. The labile undifferentiated state was fully rescued by constitutive expression of exogenous Nanog. Thus, the activity of Nanog is tightly correlated with an undifferentiated state of cells even in nuclear reprogrammed somatic cells. Nanog may function as a key regulator for sustaining pluripotency in a dose-dependent manner.
Asunto(s)
Blastocisto/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Homeodominio/genética , Células Madre Pluripotentes/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Quimera , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/metabolismo , Femenino , Haplorrinos , Proteínas de Homeodominio/metabolismo , Humanos , Células Híbridas , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mórula/metabolismo , Proteína Homeótica Nanog , Células Madre Pluripotentes/citología , Seudogenes , Homología de Secuencia de AminoácidoRESUMEN
Interleukin 7 (IL-7) has an important function in the development and maintenance of IL-17A+ γδ T cells. We here constructed a recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing antigen 85B (Ag85B)-IL-7 fusion protein (rBCG-Ag85B-IL-7). The Ag85B-IL-7 fusion protein and IL-7 were detected in the bacterial lysate of rBCG-Ag85B-IL-7. rBCG-Ag85B-IL-7 was the same in number as control rBCG expressing Ag85B (rBCG-Ag85B) in the lung at the early stage after intravenous inoculation, whereas the numbers of IL-17A+ γδ T cells and Ag-specific Th1 cells were significantly higher in the lungs of mice inoculated with rBCG-Ag85B-IL-7 than those inoculated with rBCG-Ag85B. The Ag-specific Th1 cell response was impaired in mice lacking IL-17A+ γδ T cells after inoculation with rBCG-Ag85B-IL-7. Thus, rBCG-Ag85B-IL-7 increases the pool size of IL-17A+ γδ T cells, which subsequently augment the Th1 response to mycobacterial infection.