RESUMO
Development of effective human immunodeficiency virus 1 (HIV-1) vaccines requires synergy between innate and adaptive immune cells. Here we show that induction of the transcription factor CREB1 and its target genes by the recombinant canarypox vector ALVAC + Alum augments immunogenicity in non-human primates (NHPs) and predicts reduced HIV-1 acquisition in the RV144 trial. These target genes include those encoding cytokines/chemokines associated with heightened protection from simian immunodeficiency virus challenge in NHPs. Expression of CREB1 target genes probably results from direct cGAMP (STING agonist)-modulated p-CREB1 activity that drives the recruitment of CD4+ T cells and B cells to the site of antigen presentation. Importantly, unlike NHPs immunized with ALVAC + Alum, those immunized with ALVAC + MF59, the regimen in the HVTN702 trial that showed no protection from HIV infection, exhibited significantly reduced CREB1 target gene expression. Our integrated systems biology approach has validated CREB1 as a critical driver of vaccine efficacy and highlights that adjuvants that trigger CREB1 signaling may be critical for efficacious HIV-1 vaccines.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Imunogenicidade da Vacina/imunologia , Vacinas Virais/imunologia , Vacinas contra a AIDS/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Expressão Gênica/imunologia , Vetores Genéticos/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/virologia , Humanos , Imunização/métodos , Primatas/imunologia , Primatas/virologia , Vacinação/métodosRESUMO
Zika virus (ZIKV) is a mosquito-borne flavivirus that caused an epidemic in the Americas in 2016 and is linked to severe neonatal birth defects, including microcephaly and spontaneous abortion. To better understand the host response to ZIKV infection, we adapted the 10× Genomics Chromium single-cell RNA sequencing (scRNA-seq) assay to simultaneously capture viral RNA and host mRNA. Using this assay, we profiled the antiviral landscape in a population of human monocyte-derived dendritic cells infected with ZIKV at the single-cell level. The bystander cells, which lacked detectable viral RNA, expressed an antiviral state that was enriched for genes coinciding predominantly with a type I interferon (IFN) response. Within the infected cells, viral RNA negatively correlated with type I IFN-dependent and -independent genes (the antiviral module). We modeled the ZIKV-specific antiviral state at the protein level, leveraging experimentally derived protein interaction data. We identified a highly interconnected network between the antiviral module and other host proteins. In this work, we propose a new paradigm for evaluating the antiviral response to a specific virus, combining an unbiased list of genes that highly correlate with viral RNA on a per-cell basis with experimental protein interaction data. IMPORTANCE: Zika virus (ZIKV) remains a public health threat given its potential for re-emergence and the detrimental fetal outcomes associated with infection during pregnancy. Understanding the dynamics between ZIKV and its host is critical to understanding ZIKV pathogenesis. Through ZIKV-inclusive single-cell RNA sequencing (scRNA-seq), we demonstrate on the single-cell level the dynamic interplay between ZIKV and the host: the transcriptional program that restricts viral infection and ZIKV-mediated inhibition of that response. Our ZIKV-inclusive scRNA-seq assay will serve as a useful tool for gaining greater insight into the host response to ZIKV and can be applied more broadly to the flavivirus field.
Assuntos
Células Dendríticas , Análise de Célula Única , Infecção por Zika virus , Zika virus , Humanos , Zika virus/fisiologia , Infecção por Zika virus/virologia , Infecção por Zika virus/imunologia , Células Dendríticas/virologia , Células Dendríticas/imunologia , RNA Viral/metabolismo , RNA Viral/genética , Interferon Tipo I/metabolismo , Interações Hospedeiro-Patógeno , Análise de Sequência de RNARESUMO
It has been estimated that more than 390 million people are infected with Dengue virus every year; around 96 millions of these infections result in clinical pathologies. To date, there is only one licensed viral vector-based Dengue virus vaccine CYD-TDV approved for use in dengue endemic areas. While initially approved for administration independent of serostatus, the current guidance only recommends the use of this vaccine for seropositive individuals. Therefore, there is a critical need for investigating the influence of Dengue virus serostatus and immunological mechanisms that influence vaccine outcome. Here, we provide comprehensive evaluation of sero-status and host immune factors that correlate with robust immune responses to a Dengue virus vector based tetravalent vaccine (TV003) in a Phase II clinical cohort of human participants. We observed that sero-positive individuals demonstrate a much stronger immune response to the TV003 vaccine. Our multi-layered immune profiling revealed that sero-positive subjects have increased baseline/pre-vaccination frequencies of circulating T follicular helper (cTfh) cells and the Tfh related chemokine CXCL13/BLC. Importantly, this baseline/pre-vaccination cTfh profile correlated with the vaccinees' ability to launch neutralizing antibody response against all four sero-types of Dengue virus, an important endpoint for Dengue vaccine clinical trials. Overall, we provide novel insights into the favorable cTfh related immune status that persists in Dengue virus sero-positive individuals that correlate with their ability to mount robust vaccine specific immune responses. Such detailed interrogation of cTfh cell biology in the context of clinical vaccinology will help uncover mechanisms and targets for favorable immuno-modulatory agents.
Assuntos
Anticorpos Antivirais/imunologia , Vacinas contra Dengue/imunologia , Imunogenicidade da Vacina/imunologia , Células T Auxiliares Foliculares/imunologia , Anticorpos Neutralizantes/imunologia , Dengue/prevenção & controle , Feminino , Humanos , Masculino , Vacinas Combinadas/imunologiaRESUMO
In response to microbial stimulation, monocytes can differentiate into macrophages or monocyte-derived dendritic cells (MoDCs) but the molecular requirements guiding these possible fates are poorly understood. In addition, the physiological importance of MoDCs in the host cellular and immune responses to microbes remains elusive. Here, we demonstrate that the nuclear orphan receptor NR4A3 is required for the proper differentiation of MoDCs but not for other types of DCs. Indeed, the generation of DC-SIGN+ MoDCs in response to LPS was severely impaired in Nr4a3-/- mice, which resulted in the inability to mount optimal CD8+ T cell responses to gram-negative bacteria. Transcriptomic analyses revealed that NR4A3 is required to skew monocyte differentiation toward MoDCs, at the expense of macrophages, and allows the acquisition of migratory characteristics required for MoDC function. Altogether, our data identify that the NR4A3 transcription factor is required to guide the fate of monocytes toward MoDCs.
Assuntos
Linhagem da Célula/imunologia , Proteínas de Ligação a DNA/genética , Células Dendríticas/imunologia , Lipopolissacarídeos/farmacologia , Monócitos/imunologia , Proteínas do Tecido Nervoso/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Diferenciação Celular , Linhagem da Célula/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/imunologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interleucina-4/farmacologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Ativação Linfocitária , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Monócitos/citologia , Monócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/imunologia , Cultura Primária de Células , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Esteroides/deficiência , Receptores de Esteroides/imunologia , Receptores dos Hormônios Tireóideos/deficiência , Receptores dos Hormônios Tireóideos/imunologia , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Susceptibility to autoimmune diabetes is a complex genetic trait. Linkage analyses exploiting the NOD mouse, which spontaneously develops autoimmune diabetes, have proved to be a useful tool for the characterization of some of these traits. In a linkage analysis using 3A9 TCR transgenic mice on both B10.BR and NOD.H2k backgrounds, we previously determined that both the Idd2 and Idd13 loci were linked to the proportion of immunoregulatory CD4-CD8- double negative (DN) T cells. In addition to Idd2 and Idd13, five other loci showed weak linkage to the proportion of DN T cells. Of interest, in an interim analysis, a locus on chromosome 12 is linked to DN T cell proportion in both the spleen and the lymph nodes. To determine the impact of this locus on DN T cells, we generated two congenic sublines, which we named Chr12P and Chr12D for proximal and distal, respectively. While 3A9 TCR:insHEL NOD.H2k-Chr12D mice were protected from diabetes, 3A9 TCR:insHEL NOD.H2k-Chr12P showed an increase in diabetes incidence. Yet, the proportion of DN T cells was similar to the parental 3A9 TCR NOD.H2k strain for both of these congenic sublines. A genome-wide two dimensional LOD score analysis reveals genetic epistasis between chromosome 12 and the Idd13 locus. Altogether, this study identified further complex genetic interactions in defining the proportion of DN T cells, along with evidence of genetic epistasis within a locus on chromosome 12 influencing autoimmune susceptibility.
Assuntos
Linhagem da Célula , Diabetes Mellitus Tipo 1/genética , Epistasia Genética , Predisposição Genética para Doença , Linfócitos T/imunologia , Animais , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Feminino , Ligação Genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos TransgênicosRESUMO
Natural killer cells constitute potent innate lymphoid cells that play a major role in both tumor immunosurveillance and viral clearance via their effector functions. A four-stage model of NK cell functional maturation has been established according to the expression of CD11b and CD27, separating mature NK (mNK) cells into distinct populations that exhibit specific phenotypic and functional properties. To identify genetic factors involved in the regulation of NK cell functional maturation, we performed a linkage analysis on F2 (B6.Rag1-/- × NOD.Rag1-/- intercross) mice. We identified six loci on chromosomes 2, 4, 7, 10, 11, and 18 that were linked to one or more mNK cell subsets. Subsequently, we performed an in silico analysis exploiting mNK cell subset microarray data, highlighting various genes and microRNAs as potential regulators of the functional maturation of NK cells. Together, the combination of our unbiased genetic linkage study and the in silico analysis positions genes known to affect NK cell biology along the specific stages of NK cell functional maturation. Moreover, this approach allowed us to uncover a novel candidate gene in the regulation of NK cell maturation, namely Trp53 Using mice deficient for Trp53, we confirm that this tumor suppressor regulates NK cell functional maturation. Additional candidate genes revealed in this study may eventually serve as targets for the modulation of NK cell functional maturation to potentiate both tumor immunosurveillance and viral clearance.
Assuntos
Regulação da Expressão Gênica , Ligação Genética , Células Matadoras Naturais/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Antígeno CD11b/imunologia , Diferenciação Celular , Processos de Crescimento Celular , Células Cultivadas , Simulação por Computador , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos NOD , MicroRNAs/genética , MicroRNAs/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
NK cells play a crucial role in innate immunity due to their direct cytotoxicity toward tumors, virally infected cells, and stressed cells, and they also contribute to the orchestration of the adaptive response by their ability to produce immunoregulatory cytokines. In secondary lymphoid organs, NK cells compose the third most abundant lymphocyte subset after T cells and B cells. In this study, we perform an unbiased linkage analysis to determine the genetic loci that may limit the size of the NK cell compartment. Specifically, we exploit differences in NK cell proportion and absolute number between the C57BL/6 and the NOD mice. In addition to the previously identified linkage to chromosome 8, we find that a locus on chromosome 17, which encompasses the MHC locus, impacts NK cell number. Moreover, we identify a locus on mouse chromosome 9 that is strongly linked to the proportion and absolute number of NK cells. Using NOD congenic mice, we validate that both the MHC and the chromosome 9 loci influence the proportion and absolute number of NK cells. We have thus identified additional loci specifically linked to the proportion of NK cells and present some of the potential candidate genes comprised within these loci.
Assuntos
Imunidade Adaptativa/genética , Cromossomos Humanos Par 17/imunologia , Cromossomos Humanos Par 8/imunologia , Cromossomos Humanos Par 9/imunologia , Células Matadoras Naturais/imunologia , Animais , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 8/genética , Cromossomos Humanos Par 9/genética , Citometria de Fluxo , Ligação Genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL/genética , Camundongos Endogâmicos C57BL/imunologia , Camundongos Endogâmicos NOD/genética , Camundongos Endogâmicos NOD/imunologia , Camundongos TransgênicosRESUMO
Autoimmune diseases result from a break in immune tolerance. Various mechanisms of peripheral tolerance can protect against autoimmunity, including immunoregulatory CD4(-)CD8(-) double-negative (DN) T cells. Indeed, we have previously shown that diabetes-prone mouse strains exhibit a low proportion of DN T cells relative to that of diabetes-resistant mice, and that a single autologous transfer of DN T cells can impede autoimmune diabetes development, at least in the 3A9 TCR transgenic setting. In this study, we aim to understand the genetic basis for the difference in DN T cell proportion between diabetes-resistant and diabetes-prone mice. We thus perform an unbiased linkage analysis in 3A9 TCR F2 (NOD.H2(k) × B10.BR) mice and reveal that a locus on chromosome 9, which coincides with Idd2, is linked to the proportion of DN T cells in the lymph nodes. We generate two NOD.H2(k).B10-Chr9 congenic mouse strains and validate the role of this genetic interval in defining the proportion of DN T cells. Moreover, we find that the increased proportion of DN T cells in lymphoid organs is associated with a decrease in both diabetes incidence and serum IgG Ab levels. Together, the data suggest that Idd2 is linked to DN T cell proportion and that a physiological increase in DN T cell number may be sufficient to confer resistance to autoimmune diabetes. Altogether, these findings could help identify new candidate genes for the development of therapeutic avenues aimed at modulating DN T cell number for the prevention of autoimmune diseases.
Assuntos
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Linfonodos/imunologia , Locos de Características Quantitativas/imunologia , Animais , Diabetes Mellitus Tipo 1/prevenção & controle , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos TRESUMO
Plasmacytoid dendritic cells (pDC) compose one of the many distinct dendritic cell subsets. The primary function of pDC is to potently produce type 1 IFNs upon stimulation, which is highly relevant in antiviral responses. Consequently, the ability to manipulate the size of the pDC compartment in vivo may increase the capacity to clear viral infections. In an attempt to identify genetic loci affecting the size of the pDC compartment, defined by both the proportion and absolute number of pDC, we undertook an unbiased genetic approach. Linkage analysis using inbred mouse strains identified a locus on chromosome 7 (Pdcc1) significantly linked to both the proportion and the absolute number of pDC in the spleen. Moreover, loci on either chromosome 11 (Pdcc2) or 9 (Pdcc3) modified the effect of Pdcc1 on chromosome 7 for the proportion and absolute number of pDC, respectively. Further analysis using mice congenic for chromosome 7 confirmed Pdcc1, demonstrating that variation within this genetic interval can regulate the size of the pDC compartment. Finally, mixed bone marrow chimera experiments showed that both the proportion and the absolute number of pDC are regulated by cell-intrinsic hematopoietic factors. Our findings highlight the multigenic regulation of the size of the pDC compartment and will facilitate the identification of genes linked to this trait.
Assuntos
Compartimento Celular/imunologia , Cromossomos de Mamíferos/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Genes Dominantes/imunologia , Animais , Compartimento Celular/genética , Cromossomos de Mamíferos/genética , Feminino , Genes Dominantes/genética , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Endogâmicos NZB , Camundongos Knockout , Quimera por Radiação , Baço/citologia , Baço/imunologiaRESUMO
Zika virus (ZIKV) is a mosquito-borne flavivirus that caused an epidemic in the Americas in 2016 and is linked to severe neonatal birth defects, including microcephaly and spontaneous abortion. To better understand the host response to ZIKV infection, we adapted the 10x Genomics Chromium single cell RNA sequencing (scRNA-seq) assay to simultaneously capture viral RNA and host mRNA. Using this assay, we profiled the antiviral landscape in a population of human moDCs infected with ZIKV at the single cell level. The bystander cells, which lacked detectable viral RNA, expressed an antiviral state that was enriched for genes coinciding predominantly with a type I interferon (IFN) response. Within the infected cells, viral RNA negatively correlated with type I IFN dependent and independent genes (antiviral module). We modeled the ZIKV specific antiviral state at the protein level leveraging experimentally derived protein-interaction data. We identified a highly interconnected network between the antiviral module and other host proteins. In this work, we propose a new paradigm for evaluating the antiviral response to a specific virus, combining an unbiased list of genes that highly correlate with viral RNA on a per cell basis with experimental protein interaction data. Our ZIKV-inclusive scRNA-seq assay will serve as a useful tool to gaining greater insight into the host response to ZIKV and can be applied more broadly to the flavivirus field.
RESUMO
Protective immunity to dengue virus (DENV) requires antibody response to all four serotypes. Systems vaccinology identifies a multi-OMICs pre-vaccination signature and mechanisms predictive of broad antibody responses after immunization with a tetravalent live attenuated DENV vaccine candidate (Butantan-DV/TV003). Anti-inflammatory pathways, including TGF-ß signaling expressed by CD68low monocytes, and the metabolites phosphatidylcholine (PC) and phosphatidylethanolamine (PE) positively correlate with broadly neutralizing antibody responses against DENV. In contrast, expression of pro-inflammatory pathways and cytokines (IFN and IL-1) in CD68hi monocytes and primary and secondary bile acids negatively correlates with broad DENV-specific antibody responses. Induction of TGF-ß and IFNs is done respectively by PC/PE and bile acids in CD68low and CD68hi monocytes. The inhibition of viral sensing by PC/PE-induced TGF-ß is confirmed in vitro. Our studies show that the balance between metabolites and the pro- or anti-inflammatory state of innate immune cells drives broad and protective B cell response to a live attenuated dengue vaccine.
Assuntos
Anticorpos Antivirais , Vacinas contra Dengue , Vírus da Dengue , Vacinas contra Dengue/imunologia , Humanos , Vírus da Dengue/imunologia , Anticorpos Antivirais/imunologia , Dengue/imunologia , Dengue/prevenção & controle , Dengue/virologia , Monócitos/imunologia , Monócitos/metabolismo , Formação de Anticorpos/imunologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/imunologia , Vacinação , Anticorpos Neutralizantes/imunologiaRESUMO
When antigens derived from apoptotic cells are presented by conventional dendritic cells (cDC), T cell tolerance is induced. Surprisingly, the presentation of apoptotic cell antigens by an unconventional DC subset, termed merocytic dendritic cells (mcDC), can reverse T cell anergy. The potency of mcDC at breaking T cell tolerance has been demonstrated in the context of tumors and autoimmunity, suggesting that modulating the number of mcDC in vivo may be of clinical interest. To identify the genetic determinants that define the number of mcDC, we performed a linkage analysis between NOD and C57BL/6 mouse strains, where autoimmune-prone NOD mice show an increased proportion of mcDC relative to the non-autoimmune-prone C57BL/6 mice. We identified a locus on chromosome 2 significantly linked to both the proportion and the absolute number of mcDC in the spleen. Interestingly, the dominant interval on chromosome 2 overlaps with a locus previously associated with diabetes protection, namely Idd13. Using NOD.Idd13 congenic mice, we validate the impact of the Idd13 congenic interval in defining the proportion and number of mcDC in the spleen. These results show that the decreased number of mcDC is conferred by C57BL/6 alleles at the Idd13 locus, which is linked to diabetes resistance.
Assuntos
Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Animais , Contagem de Células , Mapeamento Cromossômico , Células Dendríticas/classificação , Células Dendríticas/patologia , Diabetes Mellitus Tipo 1/patologia , Feminino , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Imunofenotipagem , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos KnockoutRESUMO
IL-12 and IL-23 cytokines respectively drive Th1 and Th17 type responses. Yet, little is known regarding the biology of these receptors. As the IL-12 and IL-23 receptors share a common subunit, it has been assumed that these receptors are co-expressed. Surprisingly, we find that the expression of each of these receptors is restricted to specific cell types, in both mouse and human. Indeed, although IL-12Rß2 is expressed by NK cells and a subset of γδ T cells, the expression of IL-23R is restricted to specific T cell subsets, a small number of B cells and innate lymphoid cells. By exploiting an IL-12- and IL-23-dependent mouse model of innate inflammation, we demonstrate an intricate interplay between IL-12Rß2 NK cells and IL-23R innate lymphoid cells with respectively dominant roles in the regulation of systemic versus local inflammatory responses. Together, these findings support an unforeseen lineage-specific dichotomy in the in vivo role of both the IL-12 and IL-23 pathways in pathological inflammatory states, which may allow more accurate dissection of the roles of these receptors in chronic inflammatory diseases in humans.