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1.
Sci Rep ; 10(1): 17718, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077873

RESUMO

COVID-19 has been widely spreading. We aimed to examine adaptive immune cells in non-severe patients with persistent SARS-CoV-2 shedding. 37 non-severe patients with persistent SARS-CoV-2 presence that were transferred to Zhongnan hospital of Wuhan University were retrospectively recruited to the PP (persistently positive) group, which was further allocated to PPP group (n = 19) and PPN group (n = 18), according to their testing results after 7 days (N = negative). Epidemiological, demographic, clinical and laboratory data were collected and analyzed. Data from age- and sex-matched non-severe patients at disease onset (PA [positive on admission] patients, n = 37), and lymphocyte subpopulation measurements from matched 54 healthy subjects were extracted for comparison (HC). Compared with PA patients, PP patients had much improved laboratory findings. The absolute numbers of CD3+ T cells, CD4+ T cells, and NK cells were significantly higher in PP group than that in PA group, and were comparable to that in healthy controls. PPP subgroup had markedly reduced B cells and T cells compared to PPN group and healthy subjects. Finally, paired results of these lymphocyte subpopulations from 10 PPN patients demonstrated that the number of T cells and B cells significantly increased when the SARS-CoV-2 tests turned negative. Persistent SARS-CoV-2 presence in non-severe COVID-19 patients is associated with reduced numbers of adaptive immune cells. Monitoring lymphocyte subpopulations could be clinically meaningful in identifying fully recovered COVID-19 patients.


Assuntos
Linfócitos B/citologia , Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Linfócitos T/citologia , Adulto , Linfócitos B/imunologia , Linfócitos B/metabolismo , Betacoronavirus/isolamento & purificação , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Estudos de Casos e Controles , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Masculino , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Estudos Retrospectivos , Índice de Gravidade de Doença , Linfócitos T/imunologia , Linfócitos T/metabolismo
2.
PLoS One ; 15(9): e0235183, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32956421

RESUMO

Members of the broad complex, tram track, bric-a-brac and zinc finger (BTB-ZF) family of transcription factors, such as BCL-6, ZBTB20, and ZBTB32, regulate antigen-specific B cell differentiation, plasma cell longevity, and the duration of antibody production. We found that ZBTB38, a different member of the BTB-ZF family that binds methylated DNA at CpG motifs, is highly expressed by germinal center B cells and plasma cells. To define the functional role of ZBTB38 in B cell responses, we generated mice conditionally deficient in this transcription factor. Germinal center B cells lacking ZBTB38 dysregulated very few genes relative to wild-type and heterozygous littermate controls. Accordingly, mice with hematopoietic-specific deletion of Zbtb38 showed normal germinal center B cell numbers and antibody responses following immunization with hapten-protein conjugates. Memory B cells from these animals functioned normally in secondary recall responses. Despite expression of ZBTB38 in hematopoietic stem cells, progenitors and mature myeloid and lymphoid lineages were also present in normal numbers in mutant mice. These data demonstrate that ZBTB38 is dispensable for hematopoiesis and antibody responses. These conditional knockout mice may instead be useful in defining the functional importance of ZBTB38 in other cell types and contexts.


Assuntos
Formação de Anticorpos , Linfócitos B/imunologia , Proteínas Repressoras/imunologia , Animais , Linfócitos B/citologia , Deleção de Genes , Expressão Gênica , Hematopoese , Imunização , Camundongos , Camundongos Knockout , Proteínas Repressoras/genética
3.
Nature ; 584(7820): 274-278, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760003

RESUMO

Colonization by the microbiota causes a marked stimulation of B cells and induction of immunoglobulin, but mammals colonized with many taxa have highly complex and individualized immunoglobulin repertoires1,2. Here we use a simplified model of defined transient exposures to different microbial taxa in germ-free mice3 to deconstruct how the microbiota shapes the B cell pool and its functional responsiveness. We followed the development of the immunoglobulin repertoire in B cell populations, as well as single cells by deep sequencing. Microbial exposures at the intestinal mucosa generated oligoclonal responses that differed from those of germ-free mice, and from the diverse repertoire that was generated after intravenous systemic exposure to microbiota. The IgA repertoire-predominantly to cell-surface antigens-did not expand after dose escalation, whereas increased systemic exposure broadened the IgG repertoire to both microbial cytoplasmic and cell-surface antigens. These microbial exposures induced characteristic immunoglobulin heavy-chain repertoires in B cells, mainly at memory and plasma cell stages. Whereas sequential systemic exposure to different microbial taxa diversified the IgG repertoire and facilitated alternative specific responses, sequential mucosal exposure produced limited overlapping repertoires and the attrition of initial IgA binding specificities. This shows a contrast between a flexible response to systemic exposure with the need to avoid fatal sepsis, and a restricted response to mucosal exposure that reflects the generic nature of host-microbial mutualism in the mucosa.


Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Imunidade nas Mucosas/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Simbiose/imunologia , Administração Intravenosa , Administração Oral , Animais , Clostridiales/imunologia , Clostridiales/isolamento & purificação , Escherichia coli/imunologia , Escherichia coli/isolamento & purificação , Feminino , Vida Livre de Germes , Imunoglobulina A/química , Imunoglobulina A/imunologia , Imunoglobulina G/química , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Memória Imunológica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasmócitos/citologia , Plasmócitos/imunologia , Priming de Repetição
4.
Nat Protoc ; 15(9): 2920-2955, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32788719

RESUMO

Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8-10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.


Assuntos
Sistema Imunitário/citologia , Dispositivos Lab-On-A-Chip , Fenótipo , Análise de Célula Única/instrumentação , Animais , Linfócitos B/citologia , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia de Fluorescência
5.
Nature ; 584(7819): 142-147, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32612238

RESUMO

Nuclear processes, such as V(D)J recombination, are orchestrated by the three-dimensional organization of chromosomes at multiple levels, including compartments1 and topologically associated domains (TADs)2,3 consisting of chromatin loops4. TADs are formed by chromatin-loop extrusion5-7, which depends on the loop-extrusion function of the ring-shaped cohesin complex8-12. Conversely, the cohesin-release factor Wapl13,14 restricts loop extension10,15. The generation of a diverse antibody repertoire, providing humoral immunity to pathogens, requires the participation of all V genes in V(D)J recombination16, which depends on contraction of the 2.8-Mb-long immunoglobulin heavy chain (Igh) locus by Pax517,18. However, how Pax5 controls Igh contraction in pro-B cells remains unknown. Here we demonstrate that locus contraction is caused by loop extrusion across the entire Igh locus. Notably, the expression of Wapl is repressed by Pax5 specifically in pro-B and pre-B cells, facilitating extended loop extrusion by increasing the residence time of cohesin on chromatin. Pax5 mediates the transcriptional repression of Wapl through a single Pax5-binding site by recruiting the polycomb repressive complex 2 to induce bivalent chromatin at the Wapl promoter. Reduced Wapl expression causes global alterations in the chromosome architecture, indicating that the potential to recombine all V genes entails structural changes of the entire genome in pro-B cells.


Assuntos
Genes de Cadeia Pesada de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Fator de Transcrição PAX5/metabolismo , Proteínas/genética , Proteínas Repressoras/metabolismo , Recombinação V(D)J/genética , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Cadeias Pesadas de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Camundongos , Complexo Repressor Polycomb 2/metabolismo , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Regiões Promotoras Genéticas/genética
6.
Nat Commun ; 11(1): 3677, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699279

RESUMO

Through the formation of concentration gradients, morphogens drive graded responses to extracellular signals, thereby fine-tuning cell behaviors in complex tissues. Here we show that the chemokine CXCL13 forms both soluble and immobilized gradients. Specifically, CXCL13+ follicular reticular cells form a small-world network of guidance structures, with computer simulations and optimization analysis predicting that immobilized gradients created by this network promote B cell trafficking. Consistent with this prediction, imaging analysis show that CXCL13 binds to extracellular matrix components in situ, constraining its diffusion. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable product. Mice lacking cathepsin B display aberrant follicular architecture, a phenotype associated with effective B cell homing to but not within lymph nodes. Our data thus suggest that reticular cells of the B cell zone generate microenvironments that shape both immobilized and soluble CXCL13 gradients.


Assuntos
Linfócitos B/imunologia , Microambiente Celular/imunologia , Quimiocina CXCL13/metabolismo , Células Dendríticas Foliculares/imunologia , Imunidade Adaptativa , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Linhagem Celular , Quimiocina CXCL13/imunologia , Simulação por Computador , Células Dendríticas Foliculares/citologia , Células Dendríticas Foliculares/metabolismo , Matriz Extracelular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Modelos Biológicos , Tonsila Palatina/citologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Células Estromais/imunologia , Células Estromais/metabolismo
8.
PLoS One ; 15(7): e0236664, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32722684

RESUMO

BACKGROUND: Systemic lupus erythematosus (SLE) is a severe autoimmune disease in which immune tolerance defects drive production of pathogenic anti-nuclear autoantibodies. Anergic B cells are considered a potential source of these autoantibodies due to their autoreactivity and overrepresentation in SLE patients. Studies of lupus-prone mice have shown that genetic defects mediating autoimmunity can breach B cell anergy, but how this breach occurs with regards to endogenous nuclear antigen remains unclear. We investigated whether B and T cell defects in congenic mice (c1) derived from the lupus-prone New Zealand Black strain can breach tolerance to nuclear self-antigen in the presence of knock-in genes (Vκ8/3H9; dKI) that generate a ssDNA-reactive, anergic B cell population. METHODS: Flow cytometry was used to assess splenic B and T cells from 8-month-old c1 dKI mice and serum autoantibodies were measured by ELISA. dKI B cells stimulated in vitro with anti-IgM were assessed for proliferation and activation by examining CFSE decay and CD86. Cytokine-producing T cells were identified by flow cytometry following culture of dKI splenocytes with PMA and ionomycin. dKI B cells from 6-8-week-old mice were adoptively transferred into 4-month-old wild type recipients and assessed after 7 days via flow cytometry and immunofluorescence microscopy. RESULTS: c1 dKI mice exhibited B cell proliferation indicative of impaired anergy, but had attenuated autoantibodies and germinal centres compared to wild type littermates. This attenuation appeared to stem from a decrease in PD-1hi T helper cells in the dKI strains, as c1 dKI B cells were recruited to germinal centres when adoptively transferred into c1 wild type mice. CONCLUSION: Anergic, DNA-specific autoreactive B cells only seem to drive profound autoimmunity in the presence of concomitant defects in the T cell subsets that support high-affinity plasma cell production.


Assuntos
Anticorpos Antinucleares/genética , Anticorpos Antifosfolipídeos/genética , Antígenos/imunologia , Linfócitos B/imunologia , Anergia Clonal , Lúpus Eritematoso Sistêmico/imunologia , Animais , Linfócitos B/citologia , Proliferação de Células , DNA de Cadeia Simples/imunologia , Suscetibilidade a Doenças , Técnicas de Introdução de Genes , Lúpus Eritematoso Sistêmico/genética , Camundongos
9.
J Leukoc Biol ; 107(6): 1137-1154, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32533638

RESUMO

The chemokine CCL20 is broadly produced by endothelial cells in the liver, the lung, in lymph nodes and mucosal lymphoid tissues, and recruits CCR6 expressing leukocytes, particularly dendritic cells, mature B cells, and subpopulations of T cells. How CCL20 is systemically scavenged is currently unknown. Here, we identify that fluorescently labeled human and mouse CCL20 are efficiently taken-up by the atypical chemokine receptor ACKR4. CCL20 shares ACKR4 with the homeostatic chemokines CCL19, CCL21, and CCL25, although with a lower affinity. We demonstrate that all 4 human chemokines recruit ß-arrestin1 and ß-arrestin2 to human ACKR4. Similarly, mouse CCL19, CCL21, and CCL25 equally activate the human receptor. Interestingly, at the same chemokine concentration, mouse CCL20 did not recruit ß-arrestins to human ACKR4. Further cross-species analysis suggests that human ACKR4 preferentially takes-up human CCL20, whereas mouse ACKR4 similarly internalizes mouse and human CCL20. Furthermore, we engineered a fluorescently labeled chimeric chemokine consisting of the N-terminus of mouse CCL25 and the body of mouse CCL19, termed CCL25_19, which interacts with and is taken-up by human and mouse ACKR4.


Assuntos
Quimiocina CCL19/metabolismo , Quimiocina CCL20/metabolismo , Quimiocina CCL21/metabolismo , Quimiocinas CC/metabolismo , Receptores CCR/metabolismo , beta-Arrestinas/genética , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Sítios de Ligação , Linhagem Celular , Quimiocina CCL19/química , Quimiocina CCL19/genética , Quimiocina CCL20/química , Quimiocina CCL20/genética , Quimiocina CCL21/química , Quimiocina CCL21/genética , Quimiocinas CC/química , Quimiocinas CC/genética , Células HEK293 , Células HeLa , Humanos , Ligantes , Camundongos , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Receptores CCR/química , Receptores CCR/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Transfecção , beta-Arrestinas/metabolismo
10.
Mol Cell Biol ; 40(16)2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32513818

RESUMO

Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) class switch recombination (CSR), somatic hypermutation (SHM), and gene conversion by converting DNA cytosines to uracils at specific genomic regions. In this study, we examined AID footprints across the entire length of an engineered switch region in cells ablated for uracil repair. We found that AID deamination occurs predominantly at WRC hot spots (where W is A or T and R is A or G) and that the deamination frequency remains constant across the entire switch region. Importantly, we analyzed monoallelic AID deamination footprints on both DNA strands occurring within a single cell cycle. We found that AID generates few and mostly isolated uracils in the switch region, although processive AID deaminations are evident in some molecules. The frequency of molecules containing deamination on both DNA strands at the acceptor switch region correlates with the class switch efficiency, raising the possibility that the minimal requirement for DNA double-strand break (DSB) formation is as low as even one AID deamination event on both DNA strands.


Assuntos
Linfócitos B/citologia , Citosina/metabolismo , Switching de Imunoglobulina/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Animais , Citidina Desaminase/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Desaminação/imunologia , Recombinação Genética/genética
11.
Nat Commun ; 11(1): 3013, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541654

RESUMO

B lymphoid development is initiated by the differentiation of hematopoietic stem cells into lineage committed progenitors, ultimately generating mature B cells. This highly regulated process generates clonal immunological diversity via recombination of immunoglobulin V, D and J gene segments. While several transcription factors that control B cell development and V(D)J recombination have been defined, how these processes are initiated and coordinated into a precise regulatory network remains poorly understood. Here, we show that the transcription factor ETS Related Gene (Erg) is essential for early B lymphoid differentiation. Erg initiates a transcriptional network involving the B cell lineage defining genes, Ebf1 and Pax5, which directly promotes expression of key genes involved in V(D)J recombination and formation of the B cell receptor. Complementation of Erg deficiency with a productively rearranged immunoglobulin gene rescued B lineage development, demonstrating that Erg is an essential and stage-specific regulator of the gene regulatory network controlling B lymphopoiesis.


Assuntos
Linfócitos B/metabolismo , Diferenciação Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Linfopoese/genética , Proteínas Oncogênicas/genética , Transcrição Genética , Regulador Transcricional ERG/genética , Animais , Linfócitos B/citologia , Linhagem da Célula/genética , Células Cultivadas , Redes Reguladoras de Genes/genética , Células-Tronco Hematopoéticas/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Oncogênicas/metabolismo , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulador Transcricional ERG/metabolismo , Recombinação V(D)J/genética
12.
Cytometry A ; 97(8): 772-776, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32542842

RESUMO

A reduced peripheral blood absolute lymphocyte count with an elevated neutrophil count has been a consistent observation in hospitalized coronavirus disease 2019 (COVID-19) patients. In this brief meta-analysis, the reduction of lymphocyte subset counts in COVID-19 patients was investigated across 20 peer-reviewed studies meeting criteria for reporting lymphocyte subset counts and COVID-19 disease severity. CD4+ T cell, CD8+ T cell, B cell, NK cell, and total lymphocyte cell counts all showed statistically significant reduction in patients with severe/critical COVID-19 disease compared to mild/moderate disease. T-cell subsets showed the largest standardized magnitude of change. In some studies, multivariate analysis has shown that CD4 and/or CD8 T-cells counts are independently predictive of patient outcomes. © 2020 International Society for Advancement of Cytometry.


Assuntos
Linfócitos B/citologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Infecções por Coronavirus/sangue , Células Matadoras Naturais/citologia , Pneumonia Viral/sangue , Subpopulações de Linfócitos T/citologia , Betacoronavirus , Humanos , Contagem de Linfócitos , Neutrófilos/citologia , Pandemias
13.
J Biol Regul Homeost Agents ; 34(2): 319-326, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32431140

RESUMO

The relationship between allergic diseases and cancer is a very controversial topic, widely discussed in the last decades. Many studies have demonstrated inverse association between allergy and cancer, but others have reached neutral conclusions or have indicated a positive role of allergy in the development of cancer. However, either inhibiting or favoring, many cells and molecules relevant in the allergic process play a role in tumorigenesis. On the one hand, activated immune cells, like classically activated macrophages "M1", activated dendritic cells, IL-33 and amphiregulin stimulated Innate Lymphoid Cells (ILC2), Th1, IFN-γ producing T CD8+ and B lymphocytes have inhibitory effects on tumorigenesis and tumor progression. On the other hand, tolerogenic immune cells, like alternatively activated macrophages "M2" (M2a, M2b and M2c), tolerogenic dendritic cells, ILC3, T regulatory and B regulatory lymphocytes, while inhibiting allergic sensitization and response, appear to favour carcinogenesis. Furthermore, M2 subtypes macrophages (M2a, M2b), IL-25 stimulated ILC2 and Th2 lymphocytes have a role both in inducing allergic reactions and in favouring cancer progression. In addition, mast cells, pivotal cells in allergy, have a different effect of tumorigenesis based on their location - they can promote cancer progression or inhibit it. Finally, eosinophils have shown a prevalent tumoricidal function mediated by α-defensins, TNF-α, granzymes A and IL-18. Better understanding the role of various cells on carcinogenesis can help in developing new strategies (diagnostic, therapeutic and of follow up) against tumor.


Assuntos
Hipersensibilidade/complicações , Imunidade Inata , Neoplasias/complicações , Linfócitos B/citologia , Carcinogênese , Transformação Celular Neoplásica , Eosinófilos/citologia , Humanos , Macrófagos/citologia , Linfócitos T/citologia
14.
Proc Natl Acad Sci U S A ; 117(23): 12693-12699, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32457160

RESUMO

Natural environments can present diverse challenges, but some genotypes remain fit across many environments. Such "generalists" can be hard to evolve, outcompeted by specialists fitter in any particular environment. Here, inspired by the search for broadly neutralizing antibodies during B cell affinity maturation, we demonstrate that environmental changes on an intermediate timescale can reliably evolve generalists, even when faster or slower environmental changes are unable to do so. We find that changing environments on timescales comparable with evolutionary transients in a population enhance the rate of evolving generalists from specialists, without enhancing the reverse process. The yield of generalists is further increased in more complex dynamic environments, such as a "chirp" of increasing frequency. Our work offers design principles for how nonequilibrium fitness "seascapes" can dynamically funnel populations to genotypes unobtainable in static environments.


Assuntos
Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos/genética , Meio Ambiente , Evolução Molecular , Modelos Genéticos , Animais , Anticorpos Neutralizantes/genética , Especificidade de Anticorpos/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Diferenciação Celular , Genótipo , Humanos
15.
Nature ; 584(7819): 115-119, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32454513

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global health emergency that is in urgent need of intervention1-3. The entry of SARS-CoV-2 into its target cells depends on binding between the receptor-binding domain (RBD) of the viral spike protein and its cellular receptor, angiotensin-converting enzyme 2 (ACE2)2,4-6. Here we report the isolation and characterization of 206 RBD-specific monoclonal antibodies derived from single B cells from 8 individuals infected with SARS-CoV-2. We identified antibodies that potently neutralize SARS-CoV-2; this activity correlates with competition with ACE2 for binding to RBD. Unexpectedly, the anti-SARS-CoV-2 antibodies and the infected plasma did not cross-react with the RBDs of SARS-CoV or Middle East respiratory syndrome-related coronavirus (MERS-CoV), although there was substantial plasma cross-reactivity to their trimeric spike proteins. Analysis of the crystal structure of RBD-bound antibody revealed that steric hindrance inhibits viral engagement with ACE2, thereby blocking viral entry. These findings suggest that anti-RBD antibodies are largely viral-species-specific inhibitors. The antibodies identified here may be candidates for development of clinical interventions against SARS-CoV-2.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Linfócitos B/citologia , Linfócitos B/imunologia , Betacoronavirus/química , Criança , Células Clonais/citologia , Células Clonais/imunologia , Reações Cruzadas , Cristalização , Cristalografia por Raios X , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Testes de Neutralização , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Plasma/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
16.
PLoS One ; 15(5): e0233794, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470013

RESUMO

The domestic ferret (Mustela putorius furo) provides a critical animal model to study human respiratory diseases. However immunological insights are restricted due to a lack of ferret-specific reagents and limited genetic information about ferret B and T cell receptors. Here, variable, diversity and joining genes within the ferret kappa, lambda and heavy chain immunoglobulin loci were annotated using available genomic information. A multiplex PCR approach was derived that facilitated the recovery of paired heavy and light chain immunoglobulin sequences from single sorted ferret B cells, allowing validation of predicted germline gene sequences and the identification of putative novel germlines. Eukaryotic expression vectors were developed that enabled the generation of recombinant ferret monoclonal antibodies. This work advances the ferret as an informative immunological model for viral diseases by allowing the in-depth interrogation of antibody-based immunity.


Assuntos
Linfócitos B/imunologia , Modelos Animais de Doenças , Furões , Cadeias Leves de Imunoglobulina/genética , Receptores de Antígenos de Linfócitos B/genética , Animais , Anticorpos Monoclonais/biossíntese , Linfócitos B/citologia , Sequência de Bases , Furões/genética , Furões/imunologia , Genoma , Proteínas Recombinantes de Fusão/biossíntese
17.
Nat Commun ; 11(1): 2570, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444631

RESUMO

At present, it is not clear how memory B lymphocytes are maintained over time, and whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility.


Assuntos
Linfócitos B/imunologia , Células da Medula Óssea/imunologia , Switching de Imunoglobulina , Memória Imunológica , Baço/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Animais Selvagens/imunologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Células da Medula Óssea/citologia , Ciclo Celular , Proliferação de Células/genética , Regulação da Expressão Gênica/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Baço/citologia , Células Estromais/citologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
18.
Nat Commun ; 11(1): 2564, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444798

RESUMO

Chromosome structure is a crucial regulatory factor for a wide range of nuclear processes. Chromosome conformation capture (3C)-based experiments combined with computational modelling are pivotal for unveiling 3D chromosome structure. Here, we introduce TADdyn, a tool that integrates time-course 3C data, restraint-based modelling, and molecular dynamics to simulate the structural rearrangements of genomic loci in a completely data-driven way. We apply TADdyn on in situ Hi-C time-course experiments studying the reprogramming of murine B cells to pluripotent cells, and characterize the structural rearrangements that take place upon changes in the transcriptional state of 21 genomic loci of diverse expression dynamics. By measuring various structural and dynamical properties, we find that during gene activation, the transcription starting site contacts with open and active regions in 3D chromatin domains. We propose that these 3D hubs of open and active chromatin may constitute a general feature to trigger and maintain gene transcription.


Assuntos
Linfócitos B/metabolismo , Reprogramação Celular , Cromatina/química , Ativação Transcricional , Animais , Linfócitos B/química , Linfócitos B/citologia , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Camundongos , Células-Tronco Pluripotentes/química , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
19.
PLoS One ; 15(5): e0233630, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32459819

RESUMO

Characterization of distinct histone methylation and acetylation binding patterns in promoters and prediction of novel regulatory regions remains an important area of genomic research, as it is hypothesized that distinct chromatin signatures may specify unique genomic functions. However, methods that have been proposed in the literature are either descriptive in nature or are fully parametric and hence more restrictive in pattern discovery. In this article, we propose a two-step non-parametric statistical inference procedure to characterize unique histone modification patterns and apply it to analyzing the binding patterns of four histone marks, H3K4me2, H3K4me3, H3K9ac, and H4K20me1, in human B-lymphoblastoid cells. In the first step, we used a functional principal component analysis method to represent the concatenated binding patterns of these four histone marks around the transcription start sites as smooth curves. In the second step, we clustered these curves to reveal several unique classes of binding patterns. These uncovered patterns were used in turn to scan the whole-genome to predict novel and alternative promoters. Our analyses show that there are three distinct promoter binding patterns of active genes. Further, 19654 regions not within known gene promoters were found to overlap with human ESTs, CpG islands, or common SNPs, indicative of their potential role in gene regulation, including being potential novel promoter regions.


Assuntos
Linfócitos B/metabolismo , Ilhas de CpG , Histonas/metabolismo , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Transcrição Genética , Linfócitos B/citologia , Histonas/genética , Humanos
20.
Zhonghua Bing Li Xue Za Zhi ; 49(6): 576-582, 2020 Jun 08.
Artigo em Chinês | MEDLINE | ID: covidwho-548023

RESUMO

Objective: To study the pathological changes of the spleen in patients with COVID-19 and to analyze the relationship between the weakened immune system and splenic lesions. Methods: Postmortem needle autopsies from the spleen were carried out on 10 patients who died from COVID-19 in Wuhan. Routine hematoxylin and eosin (HE) staining was used to observe the pathological changes. The changes of lymphocytes were studied further with immunohistochemistry.RT-PCR was used to detect 2019-nCoV RNA in the spleen. In addition,the Epstein-Barr virus (EBV) was detected by in situ hybridization, and coronavirus particles were detected by transmission electron microscopy in 2 cases. Results: There were 7 males and 3 females, with an average age of 68.3 years.Of the 10 cases, 4 had cancer history and another 4 had other underlying diseases respectively.Cough, fever, malaise and dyspnea were the main clinical symptoms.The time from onset to death was 15-45 days.Ten cases patients had normal or slight increase in peripheral blood leukocyte count in the early stage of the disease, 6 cases had significant increase before death. Five patients' peripheral blood lymphocyte count decreased in the early stage of the disease, and 10 patients' peripheral blood lymphocyte count decreased significantly before the disease progressed or died. Seven cases were treated with corticosteroid (methylprednisolone ≤40 mg/d, not more than 5 days). Histopathological examination showed that the cell composition of the spleen decreased, white pulp atrophied at different levels, meanwhile lymphoid follicles decreased or absent;in addition, the ratio of red pulp to white pulp increased with varying degrees. In 7 cases, more neutrophil infiltration was found, and in 5 cases, scattered plasma cell infiltration was found. Macrophage proliferation and hemophagocytic phenomena in a few cells were found in a case. Meanwhile, necrosis and lymphocyte apoptosis were detected in 2 cases, small artery thrombosis and spleen infarction in 1 case, and fungal infection in 1 case. The results of immunohistochemistry showed that the T and B lymphocyte components of the spleen in all cases decreased in varying degrees. CD20(+) B cells were found to accumulate in the lymphoid sheath around the splenic artery in 8 cases. However, CD20 and CD21 immunostaining in 2 cases showed that the number of white pulp was almost normal, and splenic nodules were atrophic. CD3(+), CD4(+) and CD8(+)T cells were decreased. In 9 cases,CD68(+) macrophages were no significant changes in the distribution and quantity. While more CD68(+) cells were found in the medullary sinuses of 1 case (related to fungal infection). Few CD56(+) cells were found. EBV was negative by in situ hybridization. RT-PCR was used to detect the nucleic acid of 2019-nCoV. One of 10 cases was positive, 39 years old,who was the youngest patient in this group, and the other 9 cases were negative. Coronavirus particles were found in the cytoplasm of macrophage under electron microscope in 2 cases. Conclusions: The death of COVID-19 occurs mainly in the elderly, and some cases have no underlying diseases. Spleen may be one of the organs directly attacked by the virus in some patients who died from COVID-19. T and B lymphocyte in the spleen decrease in varying degrees, lymphoid follicles are atrophied, decreased or absent, and the number of NK cells do not change significantly. And the pathological changes of the spleen are not related to the use of low dose corticosteroid, which may be related to the direct attack of virus and the attack of immune system on its own tissues.


Assuntos
Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Baço/patologia , Adulto , Idoso , Autopsia , Linfócitos B/citologia , Betacoronavirus , Feminino , Humanos , Masculino , Pandemias , Baço/virologia , Linfócitos T/citologia
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