RESUMO
The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.
Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Sistemas CRISPR-Cas , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , RNA Viral/antagonistas & inibidores , Células A549 , Antibioticoprofilaxia/métodos , Sequência de Bases , Betacoronavirus/genética , Betacoronavirus/crescimento & desenvolvimento , COVID-19 , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Simulação por Computador , Sequência Conservada , Coronavirus/efeitos dos fármacos , Coronavirus/genética , Coronavirus/crescimento & desenvolvimento , Infecções por Coronavirus/tratamento farmacológico , Proteínas do Nucleocapsídeo de Coronavírus , RNA-Polimerase RNA-Dependente de Coronavírus , Células Epiteliais/virologia , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Pulmão/patologia , Pulmão/virologia , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , Filogenia , Pneumonia Viral/tratamento farmacológico , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2 , Proteínas não Estruturais Virais/genéticaRESUMO
γδ T cells are essential for immune defense and modulating physiological processes. While they have the potential to recognize large numbers of antigens through somatic gene rearrangement, the antigens which trigger most γδ T cell response remain unidentified, and the role of antigen recognition in γδ T cell function is contentious. Here, we show that some γδ T cell receptors (TCRs) exhibit polyspecificity, recognizing multiple ligands of diverse molecular nature. These ligands include haptens, metabolites, neurotransmitters, posttranslational modifications, as well as peptides and proteins of microbial and host origin. Polyspecific γδ T cells are enriched among activated cells in naive mice and the responding population in infection. They express diverse TCR sequences, have different functional potentials, and include the innate-like γδ T cells, such as the major IL-17 responders in various pathological/physiological conditions. We demonstrate that encountering their antigenic microbiome metabolite maintains their homeostasis and functional response, indicating that their ability to recognize multiple ligands is essential for their function. Human γδ T cells with similar polyspecificity also respond to various immune challenges. This study demonstrates that polyspecificity is a prevalent feature of γδ T cell antigen recognition, which enables rapid and robust T cell responses to a wide range of challenges, highlighting a unique function of γδ T cells.
Assuntos
Antígenos de Grupos Sanguíneos , Receptores de Antígenos de Linfócitos T gama-delta , Humanos , Camundongos , Animais , Antígenos , HaptenosRESUMO
Lifelong immunosuppression is required for allograft survival after kidney transplantation but may not ultimately prevent allograft loss resulting from chronic rejection. We developed an approach that attempts to abrogate immune rejection and the need for post-transplantation immunosuppression in three patients with Schimke immuno-osseous dysplasia who had both T-cell immunodeficiency and renal failure. Each patient received sequential transplants of αß T-cell-depleted and CD19 B-cell-depleted haploidentical hematopoietic stem cells and a kidney from the same donor. Full donor hematopoietic chimerism and functional ex vivo T-cell tolerance was achieved, and the patients continued to have normal renal function without immunosuppression at 22 to 34 months after kidney transplantation. (Funded by the Kruzn for a Kure Foundation.).
Assuntos
Transplante de Células-Tronco Hematopoéticas , Síndromes de Imunodeficiência , Transplante de Rim , Síndrome Nefrótica , Osteocondrodisplasias , Doenças da Imunodeficiência Primária , Arteriosclerose/genética , Arteriosclerose/terapia , Rejeição de Enxerto/prevenção & controle , Humanos , Síndromes de Imunodeficiência/terapia , Rim/fisiologia , Transplante de Rim/efeitos adversos , Síndrome Nefrótica/genética , Síndrome Nefrótica/terapia , Osteocondrodisplasias/genética , Osteocondrodisplasias/terapia , Doenças da Imunodeficiência Primária/genética , Doenças da Imunodeficiência Primária/terapia , Embolia Pulmonar/genética , Embolia Pulmonar/terapia , Condicionamento Pré-Transplante/métodosRESUMO
Sphingosine 1-phosphate (S1P) signaling regulates lymphocyte egress from lymphoid organs into systemic circulation. The sphingosine phosphate receptor 1 (S1P1) agonist FTY-720 (Gilenya) arrests immune trafficking and prevents multiple sclerosis (MS) relapses. However, alternative mechanisms of S1P-S1P1 signaling have been reported. Phosphoproteomic analysis of MS brain lesions revealed S1P1 phosphorylation on S351, a residue crucial for receptor internalization. Mutant mice harboring an S1pr1 gene encoding phosphorylation-deficient receptors (S1P1(S5A)) developed severe experimental autoimmune encephalomyelitis (EAE) due to autoimmunity mediated by interleukin 17 (IL-17)-producing helper T cells (TH17 cells) in the peripheral immune and nervous system. S1P1 directly activated the Jak-STAT3 signal-transduction pathway via IL-6. Impaired S1P1 phosphorylation enhances TH17 polarization and exacerbates autoimmune neuroinflammation. These mechanisms may be pathogenic in MS.
Assuntos
Encéfalo/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Interleucina-17/metabolismo , Lisofosfolipídeos/metabolismo , Esclerose Múltipla/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais/imunologia , Esfingosina/análogos & derivados , Animais , Autopsia , Encéfalo/imunologia , Encéfalo/patologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Inflamação , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-6/metabolismo , Janus Quinases/genética , Janus Quinases/imunologia , Janus Quinases/metabolismo , Lisofosfolipídeos/imunologia , Camundongos , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Fosforilação , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Esfingosina/imunologia , Esfingosina/metabolismo , Células Th17RESUMO
Water mixing is a critical mechanism in marine habitats that governs many important processes, including nutrient transport. Physical mechanisms, such as winds or tides, are primarily responsible for mixing effects in shallow coastal systems, but the sheltered habitats adjacent to mangroves experience very low turbulence and vertical mixing. The significance of biogenic mixing in pelagic habitats has been investigated but remains unclear. In this study, we show that the upside-down jellyfish Cassiopea sp. plays a significant role with respect to biogenic contributions to water column mixing within its shallow natural habitat ([Formula: see text] m deep). The mixing contribution was determined by high-resolution flow velocimetry methods in both the laboratory and the natural environment. We demonstrate that Cassiopea sp. continuously pump water from the benthos upward in a vertical jet with flow velocities on the scale of centimeters per second. The volumetric flow rate was calculated to be 212 Lâ h-1 for average-sized animals (8.6 cm bell diameter), which translates to turnover of the entire water column every 15 min for a median population density (29 animals per m2). In addition, we found Cassiopea sp. are capable of releasing porewater into the water column at an average rate of 2.64 mLâ h-1 per individual. The release of nutrient-rich benthic porewater combined with strong contributions to water column mixing suggests a role for Cassiopea sp. as an ecosystem engineer in mangrove habitats.
Assuntos
Ecossistema , Meio Ambiente , Cifozoários/fisiologia , Água/fisiologia , Animais , Densidade DemográficaRESUMO
BACKGROUND: Data on the safety and efficacy of coronavirus disease 2019 (COVID-19) vaccination in people with a range of primary immunodeficiencies (PIDs) are lacking because these patients were excluded from COVID-19 vaccine trials. This information may help in clinical management of this vulnerable patient group. OBJECTIVE: We assessed humoral and T-cell immune responses after 2 doses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA (mRNA) vaccines in patients with PID and functional B-cell defects. METHODS: A double-center retrospective review was performed of patients with PID who completed COVID-19 mRNA vaccination and who had humoral responses assessed through SARS-CoV-2 spike protein receptor binding domain (RBD) IgG antibody levels with reflex assessment of the antibody to block RBD binding to angiotensin-converting enzyme 2 (ACE2; hereafter referred to as ACE2 receptor blocking activity, as a surrogate test for neutralization) and T-cell response evaluated by an IFN-γ release assay. Immunization reactogenicity was also reviewed. RESULTS: A total of 33 patients with humoral defect were evaluated; 69.6% received BNT162b2 vaccine (Pfizer-BioNTech) and 30.3% received mRNA-1273 (Moderna). The mRNA vaccines were generally well tolerated without severe reactions. The IFN-γ release assay result was positive in 24 (77.4%) of 31 patients. Sixteen of 33 subjects had detectable RBD-specific IgG responses, but only 2 of these 16 subjects had an ACE2 receptor blocking activity level of ≥50%. CONCLUSION: Vaccination of this cohort of patients with PID with COVID-19 mRNA vaccines was safe, and cellular immunity was stimulated in most subjects. However, antibody responses to the spike protein RBD were less consistent, and, when detected, were not effective at ACE2 blocking.
Assuntos
Vacina de mRNA-1273 contra 2019-nCoV/imunologia , Vacina BNT162/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Doenças da Imunodeficiência Primária/imunologia , Vacina de mRNA-1273 contra 2019-nCoV/administração & dosagem , Vacina de mRNA-1273 contra 2019-nCoV/efeitos adversos , Adulto , Idoso , Anticorpos Antivirais/biossíntese , Linfócitos B/imunologia , Vacina BNT162/administração & dosagem , Vacina BNT162/efeitos adversos , Feminino , Humanos , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/biossíntese , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T/imunologia , Adulto JovemRESUMO
Wetlands provide essential ecosystem services, including nutrient cycling, flood protection, and biodiversity support, that are sensitive to changes in wetland hydrology. Wetland hydrological inputs come from precipitation, groundwater discharge, and surface run-off. Changes to these inputs via climate variation, groundwater extraction, and land development may alter the timing and magnitude of wetland inundation. Here, we use a long-term (14-year) comparative study of 152 depressional wetlands in west-central Florida to identify sources of variation in wetland inundation during two key time periods, 2005-2009 and 2010-2018. These time periods are separated by the enactment of water conservation policies in 2009, which included regional reductions in groundwater extraction. We investigated the response of wetland inundation to the interactive effects of precipitation, groundwater extraction, surrounding land development, basin geomorphology, and wetland vegetation class. Results show that water levels were lower and hydroperiods were shorter in wetlands of all vegetation classes during the first (2005-2009) time period, which corresponded with low rainfall conditions and high rates of groundwater extraction. Under water conservation policies enacted in the second (2010-2018) time period, median wetland water depths increased 1.35 m and median hydroperiods increased from 46 % to 83 %. Water-level variation was additionally less sensitive to groundwater extraction. The increase in inundation differed among vegetation classes with some wetlands not displaying signs of hydrological recovery. After accounting for effects of several explanatory factors, inundation still varied considerably among wetlands, suggesting a diversity of hydrological regimes, and thus ecological function, among individual wetlands across the landscape. Policies seeking to balance human water demand with the preservation of depressional wetlands would benefit by recognizing the heightened sensitivity of wetland inundation to groundwater extraction during periods of low precipitation.
Assuntos
Água Subterrânea , Áreas Alagadas , Humanos , Ecossistema , Água Doce , ÁguaRESUMO
The capacity to respond to environmental challenges ultimately relies on phenotypic variation which manifests from complex interactions of genetic and nongenetic mechanisms through development. While we know something about genetic variation and structure of many species of conservation importance, we know very little about the nongenetic contributions to variation. Rhizophora mangle is a foundation species that occurs in coastal estuarine habitats throughout the neotropics where it provides critical ecosystem functions and is potentially threatened by anthropogenic environmental changes. Several studies have documented landscape-level patterns of genetic variation in this species, but we know virtually nothing about the inheritance of nongenetic variation. To assess one type of nongenetic variation, we examined the patterns of DNA sequence and DNA methylation in maternal plants and offspring from natural populations of R. mangle from the Gulf Coast of Florida. We used a reduced representation bisulfite sequencing approach (epi-genotyping by sequencing; epiGBS) to address the following questions: (a) What are the levels of genetic and epigenetic diversity in natural populations of R. mangle? (b) How are genetic and epigenetic variation structured within and among populations? (c) How faithfully is epigenetic variation inherited? We found low genetic diversity but high epigenetic diversity from natural populations of maternal plants in the field. In addition, a large portion (up to ~25%) of epigenetic differences among offspring grown in common garden was explained by maternal family. Therefore, epigenetic variation could be an important source of response to challenging environments in the genetically depauperate populations of this foundation species.
Assuntos
Rhizophoraceae , Animais , Metilação de DNA , Ecossistema , Epigênese Genética , Rhizophoraceae/genéticaRESUMO
Motivation: Multiple biological clocks govern a healthy pregnancy. These biological mechanisms produce immunologic, metabolomic, proteomic, genomic and microbiomic adaptations during the course of pregnancy. Modeling the chronology of these adaptations during full-term pregnancy provides the frameworks for future studies examining deviations implicated in pregnancy-related pathologies including preterm birth and preeclampsia. Results: We performed a multiomics analysis of 51 samples from 17 pregnant women, delivering at term. The datasets included measurements from the immunome, transcriptome, microbiome, proteome and metabolome of samples obtained simultaneously from the same patients. Multivariate predictive modeling using the Elastic Net (EN) algorithm was used to measure the ability of each dataset to predict gestational age. Using stacked generalization, these datasets were combined into a single model. This model not only significantly increased predictive power by combining all datasets, but also revealed novel interactions between different biological modalities. Future work includes expansion of the cohort to preterm-enriched populations and in vivo analysis of immune-modulating interventions based on the mechanisms identified. Availability and implementation: Datasets and scripts for reproduction of results are available through: https://nalab.stanford.edu/multiomics-pregnancy/. Supplementary information: Supplementary data are available at Bioinformatics online.
Assuntos
Metaboloma , Microbiota , Gravidez , Proteoma , Transcriptoma , Biologia Computacional , Feminino , HumanosRESUMO
NF-κB signaling through its NFKB1-dependent canonical and NFKB2-dependent noncanonical pathways plays distinctive roles in a diverse range of immune processes. Recently, mutations in these 2 genes have been associated with common variable immunodeficiency (CVID). While studying patients with genetically uncharacterized primary immunodeficiencies, we detected 2 novel nonsense gain-of-function (GOF) NFKB2 mutations (E418X and R635X) in 3 patients from 2 families, and a novel missense change (S866R) in another patient. Their immunophenotype was assessed by flow cytometry and protein expression; activation of canonical and noncanonical pathways was examined in peripheral blood mononuclear cells and transfected HEK293T cells through immunoblotting, immunohistochemistry, luciferase activity, real-time polymerase chain reaction, and multiplex assays. The S866R change disrupted a C-terminal NF-κΒ2 critical site affecting protein phosphorylation and nuclear translocation, resulting in CVID with adrenocorticotropic hormone deficiency, growth hormone deficiency, and mild ectodermal dysplasia as previously described. In contrast, the nonsense mutations E418X and R635X observed in 3 patients led to constitutive nuclear localization and activation of both canonical and noncanonical NF-κΒ pathways, resulting in a combined immunodeficiency (CID) without endocrine or ectodermal manifestations. These changes were also found in 2 asymptomatic relatives. Thus, these novel NFKB2 GOF mutations produce a nonfully penetrant CID phenotype through a different pathophysiologic mechanism than previously described for mutations in NFKB2.
Assuntos
Códon sem Sentido , Subunidade p52 de NF-kappa B/genética , Imunodeficiência Combinada Severa/genética , Insuficiência Adrenal/genética , Imunodeficiência de Variável Comum/genética , Displasia Ectodérmica/genética , Hormônio do Crescimento/deficiência , Células HEK293 , Humanos , Mutação de Sentido Incorreto , FenótipoRESUMO
OPINION STATEMENT: The thymus is a key organ involved in establishing central immune tolerance. Thymic epithelial tumors (TETs) include thymomas and thymic carcinomas. Thymomas, which are histologically distinct from thymic carcinomas, lead to dysregulated thymopoiesis via decreased thymic epithelial expression of AIRE and MHC Class II, as well as via alterations in thymic architecture, thereby resulting in autoimmune complications that manifest as paraneoplastic disorders (PNDs). Although progress has been made in elucidating the mechanisms underlying thymoma-associated PNDs, there remains a great need to further define the underlying mechanisms and to identify additional immune biomarkers, such as novel antibodies (in "seronegative" cases) to facilitate diagnosis and monitoring of patients. In addition, a better understanding of the pathogenesis of PNDs could lead to improved treatment strategies for both thymomas and their immune complications. In advanced, refractory cases of TETs (both thymoma and thymic carcinoma), additional therapeutic approaches are needed. Immune checkpoint inhibitors have revolutionized the treatment of several malignancies and hold promise in the treatment of TETs; however, the risks for immune-related adverse events (especially for inducing PNDs as well as in the setting of pre-existing PNDs) underscore the need to optimize patient selection and improve clinical management before there can be widespread acceptance of checkpoint inhibitor therapy in patients with TETs.
Assuntos
Antineoplásicos Imunológicos/efeitos adversos , Biomarcadores Tumorais/imunologia , Síndromes Paraneoplásicas/imunologia , Timoma/imunologia , Timoma/terapia , Neoplasias do Timo/imunologia , Neoplasias do Timo/terapia , Antineoplásicos Imunológicos/uso terapêutico , Humanos , Síndromes Paraneoplásicas/patologiaRESUMO
Preterm labor and infections are the leading causes of neonatal deaths worldwide. During pregnancy, immunological cross talk between the mother and her fetus is critical for the maintenance of pregnancy and the delivery of an immunocompetent neonate. A precise understanding of healthy fetomaternal immunity is the important first step to identifying dysregulated immune mechanisms driving adverse maternal or neonatal outcomes. This study combined single-cell mass cytometry of paired peripheral and umbilical cord blood samples from mothers and their neonates with a graphical approach developed for the visualization of high-dimensional data to provide a high-resolution reference map of the cellular composition and functional organization of the healthy fetal and maternal immune systems at birth. The approach enabled mapping of known phenotypical and functional characteristics of fetal immunity (including the functional hyperresponsiveness of CD4+ and CD8+ T cells and the global blunting of innate immune responses). It also allowed discovery of new properties that distinguish the fetal and maternal immune systems. For example, examination of paired samples revealed differences in endogenous signaling tone that are unique to a mother and her offspring, including increased ERK1/2, MAPK-activated protein kinase 2, rpS6, and CREB phosphorylation in fetal Tbet+CD4+ T cells, CD8+ T cells, B cells, and CD56loCD16+ NK cells and decreased ERK1/2, MAPK-activated protein kinase 2, and STAT1 phosphorylation in fetal intermediate and nonclassical monocytes. This highly interactive functional map of healthy fetomaternal immunity builds the core reference for a growing data repository that will allow inferring deviations from normal associated with adverse maternal and neonatal outcomes.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunidade Inata/fisiologia , Células Matadoras Naturais/imunologia , Placenta/imunologia , Gravidez/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Feminino , Humanos , Proteínas da Gravidez/imunologia , Fator de Transcrição STAT1/imunologiaAssuntos
Doenças Autoimunes , Síndrome Linfoproliferativa Autoimune , Doença Relacionada a Imunoglobulina G4 , Transtornos Linfoproliferativos , Adulto , Síndrome Linfoproliferativa Autoimune/complicações , Síndrome Linfoproliferativa Autoimune/diagnóstico , Síndrome Linfoproliferativa Autoimune/genética , Humanos , Imunoglobulina G , Contagem de Linfócitos , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/etiologia , Linfócitos TRESUMO
BACKGROUND: Activation of Toll-like receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in patients with systemic lupus erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has not been described. OBJECTIVE: We sought to characterize TLR activation across multiple immune cell subsets and subjects, with the goal of establishing a reference framework against which to compare pathologic processes. METHODS: Peripheral whole-blood samples were stimulated with TLR ligands and analyzed by means of mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied 17 healthy volunteer donors and 8 patients with newly diagnosed and untreated SLE. RESULTS: Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of natural killer cells and T cells selectively induced nuclear factor κ light chain enhancer of activated B cells in response to TLR2 ligands. CD14(hi) monocytes exhibited the most polyfunctional cytokine expression patterns, with more than 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared among a group of healthy donors, with minimal intraindividual and interindividual variability. Furthermore, autoimmune disease altered baseline cytokine production; newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity. CONCLUSION: Mass cytometry defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in patients with inflammatory diseases, such as SLE.
Assuntos
Células Matadoras Naturais/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Monócitos/imunologia , Linfócitos T/imunologia , Receptores Toll-Like/metabolismo , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Lúpus Eritematoso Sistêmico/genética , Ativação Linfocitária , NF-kappa B/metabolismo , Especificidade de Órgãos , Transdução de Sinais , Análise de Célula Única/métodos , TranscriptomaRESUMO
Schimke immuno-osseous dysplasia (SIOD) is an autosomal recessive, fatal childhood disorder associated with skeletal dysplasia, renal dysfunction, and T-cell immunodeficiency. This disease is linked to biallelic loss-of-function mutations of the SMARCAL1 gene. Although recurrent infection, due to T-cell deficiency, is a leading cause of morbidity and mortality, the etiology of the T-cell immunodeficiency is unclear. Here, we demonstrate that the T cells of SIOD patients have undetectable levels of protein and mRNA for the IL-7 receptor alpha chain (IL7Rα) and are unresponsive to stimulation with IL-7, indicating a loss of functional receptor. No pathogenic mutations were detected in the exons of IL7R in these patients; however, CpG sites in the IL7R promoter were hypermethylated in SIOD T cells. We propose therefore that the lack of IL7Rα expression, associated with hypermethylation of the IL7R promoter, in T cells and possibly their earlier progenitors, restricts T-cell development in SIOD patients.
Assuntos
Arteriosclerose/genética , Síndromes de Imunodeficiência/genética , Síndrome Nefrótica/genética , Osteocondrodisplasias/genética , Embolia Pulmonar/genética , Receptores de Interleucina-7/genética , Linfócitos T/metabolismo , Adolescente , Adulto , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Células Cultivadas , Criança , Pré-Escolar , DNA Helicases/genética , Metilação de DNA , Citometria de Fluxo , Expressão Gênica , Humanos , Imuno-Histoquímica , Síndromes de Imunodeficiência/metabolismo , Síndromes de Imunodeficiência/patologia , Interleucina-17/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Mutação , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patologia , Doenças da Imunodeficiência Primária , Regiões Promotoras Genéticas/genética , Embolia Pulmonar/metabolismo , Embolia Pulmonar/patologia , Receptores de Interleucina-7/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Adulto JovemAssuntos
Antígeno CTLA-4/genética , Síndromes de Imunodeficiência/diagnóstico , Pneumocystis carinii/fisiologia , Pneumonia por Pneumocystis/genética , Adolescente , Haploinsuficiência , Humanos , Hospedeiro Imunocomprometido , Síndromes de Imunodeficiência/genética , Masculino , Osteomielite , Pneumonia por Pneumocystis/diagnósticoRESUMO
Single-cell technologies have immense potential to shed light on molecular and biological processes that drive human diseases. Mass cytometry (or Cytometry by Time Of Flight mass spectrometry, CyTOF) has already been employed in clinical studies to comprehensively survey patients' circulating immune system. As interest in the "bedside" application of mass cytometry is growing, the delineation of relevant methodological issues is called for. This report uses a newly generated dataset to discuss important methodological considerations when mass cytometry is implemented in a clinical study. Specifically, the use of whole blood samples versus peripheral blood mononuclear cells (PBMCs), design of mass-tagged antibody panels, technical and analytical implications of sample barcoding, and application of traditional and unsupervised approaches to analyze high-dimensional mass cytometry datasets are discussed. A mass cytometry assay was implemented in a cross-sectional study of 19 women with a history of term or preterm birth to determine whether immune traits in peripheral blood differentiate the two groups in the absence of pregnancy. Twenty-seven phenotypic and 11 intracellular markers were simultaneously analyzed in whole blood samples stimulated with lipopolysaccharide (LPS at 0, 0.1, 1, 10, and 100 ng mL(-1)) to examine dose-dependent signaling responses within the toll-like receptor 4 (TLR4) pathway. Complementary analyses, grounded in traditional or unsupervised gating strategies of immune cell subsets, indicated that the prpS6 and pMAPKAPK2 responses in classical monocytes are accentuated in women with a history of preterm birth (FDR<1%). The results suggest that women predisposed to preterm birth may be prone to mount an exacerbated TLR4 response during the course of pregnancy. This important hypothesis-generating finding points to the power of single-cell mass cytometry to detect biologically important differences in a relatively small patient cohort.
Assuntos
Citometria de Fluxo/métodos , Leucócitos Mononucleares/imunologia , Testes Imediatos , Nascimento Prematuro/diagnóstico , Nascimento Prematuro/imunologia , Nascimento a Termo/imunologia , Adulto , Estudos Transversais , Feminino , Humanos , Recém-Nascido , GravidezRESUMO
Compared with older children and adults, human neonates have reduced and delayed CD4(+) T cell immunity to certain pathogens, but the mechanisms for these developmental differences in immune function remain poorly understood. We investigated the hypothesis that impaired human neonatal CD4(+) T cell immunity was due to reduced signaling by naive CD4(+) T cells following engagement of the αß-TCR/CD3 complex and CD28. Surprisingly, calcium flux following engagement of CD3 was significantly higher in neonatal naive CD4(+) T cells from umbilical cord blood (CB) compared with naive CD4(+) T cells from adult peripheral blood. Enhanced calcium flux was also observed in adult CD4(+) recent thymic emigrants. Neonatal naive CD4(+) T cells also had higher activation-induced Erk phosphorylation. The microRNA miR-181a, which enhances activation-induced calcium flux in murine thymocytes, was expressed at significantly higher levels in CB naive CD4(+) T cells compared with adult cells. Overexpression of miR-181a in adult naive CD4(+) T cells increased activation-induced calcium flux, implying that the increased miR-181a levels of CB naive CD4(+) T cells contributed to their enhanced signaling. In contrast, AP-1-dependent transcription, which is downstream of Erk and required for full T cell activation, was decreased in CB naive CD4(+) T cells compared with adult cells. Thus, CB naive CD4(+) T cells have enhanced activation-dependent calcium flux, indicative of the retention of a thymocyte-like phenotype. Enhanced calcium signaling and Erk phosphorylation are decoupled from downstream AP-1-dependent transcription, which is reduced and likely contributes to limitations of human fetal and neonatal CD4(+) T cell immunity.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Ativação Linfocitária/imunologia , MicroRNAs/sangue , Transdução de Sinais/imunologia , Regulação para Cima/imunologia , Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Humanos , Recém-Nascido , Ativação Linfocitária/genética , MicroRNAs/biossíntese , MicroRNAs/genética , Transdução de Sinais/genética , Regulação para Cima/genéticaRESUMO
Whereas thymic education eliminates most self-reactive T cells, additional mechanisms to promote tolerance in the periphery are critical to prevent excessive immune responses against benign environmental Ags and some self-Ags. In this study we show that murine CD4(+) recent thymic emigrants (RTEs) are programmed to facilitate tolerance in the periphery. Both in vitro and in vivo, naive RTEs more readily upregulate Foxp3 than do mature naive cells after stimulation under tolerogenic conditions. In RTEs, a relatively high sensitivity to retinoic acid contributes to decreased IFN-γ production, permitting the expression of Foxp3. Conversely, mature naive CD4 cells have a lower sensitivity to retinoic acid, resulting in increased IFN-γ production and subsequent IFN-γ-mediated silencing of Foxp3 expression. Enhanced retinoic acid signaling and Foxp3 induction in RTEs upon Ag encounter in the periphery may serve as form of secondary education that complements thymic education and helps avoid inappropriate immune responses. This mechanism for tolerance may be particularly important in settings where RTEs comprise a large fraction of the peripheral T cell pool, such as in newborns or after umbilical cord blood transplant.
Assuntos
Movimento Celular/imunologia , Hipersensibilidade/imunologia , Tolerância Imunológica , Tretinoína/fisiologia , Animais , Animais Recém-Nascidos , Autoantígenos/imunologia , Autoantígenos/metabolismo , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/biossíntese , Hipersensibilidade/metabolismo , Hipersensibilidade/patologia , Interferon gama/antagonistas & inibidores , Interferon gama/biossíntese , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Baço/citologia , Baço/imunologia , Baço/metabolismo , Timo/citologia , Timo/imunologia , Timo/metabolismo , Fatores de Tempo , Tretinoína/metabolismoRESUMO
BACKGROUND: Autosomal recessive loss-of-function mutations in dedicator of cytokinesis 8 (DOCK8) cause a combined immunodeficiency characterized by atopy, recurrent infections, and cancer susceptibility. A genotype-phenotype explanation for the variable disease expression is lacking. OBJECTIVE: We investigated whether reversions contributed to the variable disease expression. METHODS: Patients followed at the National Institutes of Health's Clinical Center were studied. We performed detailed genetic analyses and intracellular flow cytometry to detect DOCK8 protein expression within lymphocyte subsets. RESULTS: We identified 17 of 34 DOCK8-deficient patients who had germline mutations with variable degrees of reversion caused by somatic repair. Somatic repair of the DOCK8 mutations resulted from second-site mutation, original-site mutation, gene conversion, and intragenic crossover. Higher degrees of reversion were associated with recombination-mediated repair. DOCK8 expression was restored primarily within antigen-experienced T cells or natural killer cells but less so in naive T or B cells. Several patients exhibited multiple different repair events. Patients who had reversions were older and had less severe allergic disease, although infection susceptibility persisted. No patients were cured without hematopoietic cell transplantation. CONCLUSIONS: In patients with DOCK8 deficiency, only certain combinations of germline mutations supported secondary somatic repair. Those patients had an ameliorated disease course with longer survival but still had fatal complications or required hematopoietic cell transplantation. These observations support the concept that some DOCK8-immunodeficient patients have mutable mosaic genomes that can modulate disease phenotype over time.