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1.
Sci Immunol ; 5(45)2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-32139586

RESUMO

B cells in human food allergy have been studied predominantly in the blood. Little is known about IgE+ B cells or plasma cells in tissues exposed to dietary antigens. We characterized IgE+ clones in blood, stomach, duodenum, and esophagus of 19 peanut-allergic patients, using high-throughput DNA sequencing. IgE+ cells in allergic patients are enriched in stomach and duodenum, and have a plasma cell phenotype. Clonally related IgE+ and non-IgE-expressing cell frequencies in tissues suggest local isotype switching, including transitions between IgA and IgE isotypes. Highly similar antibody sequences specific for peanut allergen Ara h 2 are shared between patients, indicating that common immunoglobulin genetic rearrangements may contribute to pathogenesis. These data define the gastrointestinal tract as a reservoir of IgE+ B lineage cells in food allergy.

2.
Nat Immunol ; 21(2): 199-209, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959979

RESUMO

A goal of HIV vaccine development is to elicit antibodies with neutralizing breadth. Broadly neutralizing antibodies (bNAbs) to HIV often have unusual sequences with long heavy-chain complementarity-determining region loops, high somatic mutation rates and polyreactivity. A subset of HIV-infected individuals develops such antibodies, but it is unclear whether this reflects systematic differences in their antibody repertoires or is a consequence of rare stochastic events involving individual clones. We sequenced antibody heavy-chain repertoires in a large cohort of HIV-infected individuals with bNAb responses or no neutralization breadth and uninfected controls, identifying consistent features of bNAb repertoires, encompassing thousands of B cell clones per individual, with correlated T cell phenotypes. These repertoire features were not observed during chronic cytomegalovirus infection in an independent cohort. Our data indicate that the development of numerous B cell lineages with antibody features associated with autoreactivity may be a key aspect in the development of HIV neutralizing antibody breadth.


Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia
3.
Am J Transplant ; 2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31811777

RESUMO

Cardiac allograft vasculopathy (CAV) is associated with intragraft B cell infiltrates. Here, we studied the clonal composition of B cell infiltrates using 4 graft specimens with CAV. Using deep sequencing, we analyzed the immunoglobulin heavy chain variable region repertoire in both graft and blood. Results showed robust B cell clonal expansion in the graft but not in the blood for all cases. Several expanded B cell clones, characterized by their uniquely rearranged complementarity-determining region 3, were detected in different locations in the graft. Sequences from intragraft B cells also showed elevated levels of mutated rearrangements in the graft compared to blood B cells. The number of somatic mutations per rearrangement was also higher in the graft than in the blood, suggesting that B cells continued maturing in situ. Overall, our studies demonstrated B cell clonal expansion in human cardiac allografts with CAV. This local B cell response may contribute to the pathophysiology of CAV through a mechanism that needs to be identified.

4.
Sci Transl Med ; 11(481)2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814336

RESUMO

Antigenic exposures at epithelial sites in infancy and early childhood are thought to influence the maturation of humoral immunity and modulate the risk of developing immunoglobulin E (IgE)-mediated allergic disease. How different kinds of environmental exposures influence B cell isotype switching to IgE, IgG, or IgA, and the somatic mutation maturation of these antibody pools, is not fully understood. We sequenced antibody repertoires in longitudinal blood samples in a birth cohort from infancy through the first 3 years of life and found that, whereas IgG and IgA show linear increases in mutational maturation with age, IgM and IgD mutations are more closely tied to pathogen exposure. IgE mutation frequencies are primarily increased in children with impaired skin barrier conditions such as eczema, suggesting that IgE affinity maturation could provide a mechanistic link between epithelial barrier failure and allergy development.


Assuntos
Doenças Transmissíveis/imunologia , Meio Ambiente , Receptores de Antígenos de Linfócitos B/metabolismo , Adulto , Envelhecimento , Anticorpos/genética , Antígenos/imunologia , Linfócitos B/imunologia , Carbanilidas , Pré-Escolar , Células Clonais , Eczema/imunologia , Características da Família , Feminino , Humanos , Hipersensibilidade/imunologia , Switching de Imunoglobulina , Imunoglobulina E/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Lactente , Masculino , Hipermutação Somática de Imunoglobulina , Vacinas/imunologia
5.
Blood Adv ; 2(21): 2814-2828, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30373888

RESUMO

Systemic mastocytosis (SM) is a highly heterogeneous disease with indolent and aggressive forms, with the mechanisms leading to malignant transformation still remaining to be elucidated. Here, we investigated the presence and frequency of genetic variants in 34 SM patients with multilineal KIT D816V mutations. Initial screening was performed by targeted sequencing of 410 genes in DNA extracted from purified bone marrow cells and hair from 12 patients with nonadvanced SM and 8 patients with advanced SM, followed by whole-genome sequencing (WGS) in 4 cases. Somatic mutations were further investigated in another 14 patients with advanced SM. Despite the fact that no common mutation other than KIT D816V was found in WGS analyses, targeted next-generation sequencing identified 67 nonsynonymous genetic variants involving 39 genes. Half of the mutations were somatic (mostly multilineal), whereas the other half were germline variants. The presence of ≥1 multilineal somatic mutation involving genes other than KIT D816V, ≥3 germline variants, and ≥1 multilineal mutation in the SRSF2, ASXL1, RUNX1, and/or EZH2 genes (S/A/R/E genes), in addition to skin lesions, splenomegaly, thrombocytopenia, low hemoglobin levels, and increased alkaline phosphatase and ß2-microglobulin serum levels, were associated with a poorer patient outcome. However, the presence of ≥1 multilineal mutation, particularly involving S/A/R/E genes, was the only independent predictor for progression-free survival and overall survival in our cohort.


Assuntos
Mastocitose Sistêmica/diagnóstico , Proteínas Proto-Oncogênicas c-kit/genética , Adolescente , Adulto , Idoso , Fosfatase Alcalina/sangue , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Criança , Intervalo Livre de Doença , Feminino , Variação Genética , Mutação em Linhagem Germinativa , Hemoglobinas/análise , Humanos , Recém-Nascido , Masculino , Mastocitose Sistêmica/genética , Mastocitose Sistêmica/mortalidade , Mastocitose Sistêmica/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Polimorfismo de Nucleotídeo Único , Microglobulina beta-2/sangue
6.
Cancer Genet ; 216-217: 10-15, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29025582

RESUMO

FLT3 fusions are associated with myeloid and lymphoid neoplasms with eosinophilia. We describe a patient presenting with clinicopathologic features of both chronic eosinophilic leukemia, not otherwise specified (CEL, NOS) and systemic mastocytosis (SM). The bone marrow demonstrated a myeloproliferative neoplasm with eosinophilia and aggregates of atypical mast cells. Cytogenetic analysis revealed a t(13;14)(q12;q32), which was subsequently molecularly characterized as a novel TRIP11-FLT3 rearrangement. A KIT D816V mutation was also identified. The patient rapidly transformed to T-lymphoblastic leukemia/lymphoma and expired shortly after diagnosis. This is the fifth FLT3 fusion gene described in the literature; the presence of both myeloid and lymphoid neoplasms implicates involvement of an early hematopoietic progenitor by rearranged FLT3. We suggest that leukemias and lymphomas with FLT3 fusion genes exhibit similar clinicopathologic features to, and should be included in, the WHO category of "Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1, or with PCM1-JAK2."


Assuntos
Eosinofilia/complicações , Linfoma/complicações , Linfoma/genética , Transtornos Mieloproliferativos/complicações , Transtornos Mieloproliferativos/genética , Proteínas de Fusão Oncogênica/genética , Tirosina Quinase 3 Semelhante a fms/genética , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
7.
Nat Immunol ; 17(10): 1226-34, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27525369

RESUMO

Antigen-specific B cells bifurcate into antibody-secreting cells (ASCs) and memory B cells (MBCs) after infection or vaccination. ASCs (plasmablasts) have been extensively studied in humans, but less is known about B cells that become activated but do not differentiate into plasmablasts. Here we have defined the phenotype and transcriptional program of a subset of antigen-specific B cells, which we have called 'activated B cells' (ABCs), that were distinct from ASCs and were committed to the MBC lineage. We detected ABCs in humans after infection with Ebola virus or influenza virus and also after vaccination. By simultaneously analyzing antigen-specific ASCs and ABCs in human blood after vaccination against influenza virus, we investigated the clonal overlap and extent of somatic hypermutation (SHM) in the ASC (effector) and ABC (memory) lineages. Longitudinal tracking of vaccination-induced hemagglutinin (HA)-specific clones revealed no overall increase in SHM over time, which suggested that repeated annual immunization might have limitations in enhancing the quality of influenza-virus-specific antibody.


Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Ebolavirus/fisiologia , Doença pelo Vírus Ebola/imunologia , Vírus da Influenza A/fisiologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Fator de Transcrição PAX5/metabolismo , Plasmócitos/imunologia , Adulto , Anticorpos Antivirais/sangue , Diferenciação Celular , Células Clonais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Memória Imunológica , Ativação Linfocitária , Hipermutação Somática de Imunoglobulina/genética , Vacinação , Adulto Jovem
8.
J Allergy Clin Immunol ; 137(2): 579-586.e7, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26309181

RESUMO

BACKGROUND: B cells expressing IgE contribute to immunity against parasites and venoms and are the source of antigen specificity in allergic patients, yet the developmental pathways producing these B cells in human subjects remain a subject of debate. Much of our knowledge of IgE lineage development derives from model studies in mice rather than from human subjects. OBJECTIVE: We evaluate models for isotype switching to IgE in human subjects using immunoglobulin heavy chain (IGH) mutational lineage data. METHODS: We analyzed IGH repertoires in 9 allergic and 24 healthy adults using high-throughput DNA sequencing of 15,843,270 IGH rearrangements to identify clonal lineages of B cells containing members expressing IgE. Somatic mutations in IGH inherited from common ancestors within the clonal lineage are used to infer the relationships between B cells. RESULTS: Data from 613,641 multi-isotype B-cell clonal lineages, of which 592 include an IgE member, are consistent with indirect switching to IgE from IgG- or IgA-expressing lineage members in human subjects. We also find that these inferred isotype switching frequencies are similar in healthy and allergic subjects. CONCLUSIONS: We found evidence that secondary isotype switching of mutated IgG1-expressing B cells is the primary source of IgE in human subjects, with lesser contributions from precursors expressing other switched isotypes and rarely IgM or IgD, suggesting that IgE is derived from previously antigen-experienced B cells rather than naive B cells that typically express low-affinity unmutated antibodies. These data provide a basis from which to evaluate allergen-specific human antibody repertoires in healthy and diseased subjects.


Assuntos
Linfócitos B/metabolismo , Switching de Imunoglobulina/genética , Imunoglobulina E/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/imunologia , Sequência de Bases , Estudos de Casos e Controles , Análise por Conglomerados , Biologia Computacional/métodos , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Switching de Imunoglobulina/imunologia , Imunoglobulina E/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Isotipos de Imunoglobulinas/genética , Isotipos de Imunoglobulinas/imunologia , Masculino , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Dados de Sequência Molecular , Alinhamento de Sequência , Hipermutação Somática de Imunoglobulina , Adulto Jovem
9.
J Allergy Clin Immunol ; 137(1): 157-167, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26152318

RESUMO

BACKGROUND: The frequencies, cellular phenotypes, epitope specificity, and clonal diversity of allergen-specific B cells in patients with food allergy are not fully understood but are of major pathogenic and therapeutic significance. OBJECTIVE: We sought to characterize peanut allergen-specific B-cell populations and the sequences and binding activities of their antibodies before and during immunotherapy. METHODS: B cells binding fluorescently labeled Ara h 1 or Ara h 2 were phenotyped and isolated by means of flow cytometric sorting from 18 patients at baseline and 13 patients during therapy. Fifty-seven mAbs derived from allergen-binding single B cells were evaluated by using ELISA, Western blotting, and peptide epitope mapping. Deep sequencing of the B-cell repertoires identified additional members of the allergen-specific B-cell clones. RESULTS: Median allergen-binding B-cell frequencies were 0.0097% (Ara h 1) or 0.029% (Ara h 2) of B cells in baseline blood from allergic patients and approximately 3-fold higher during immunotherapy. Five of 57 allergen-specific cells belonged to clones containing IgE-expressing members. Almost all allergen-specific antibodies were mutated, and binding to both conformational and linear allergen epitopes was detected. Increasing somatic mutation of IgG4 members of a clone was seen in immunotherapy, whereas IgE mutation levels in the clone did not increase. CONCLUSION: Most peanut allergen-binding B cells isolated by means of antigen-specific flow sorting express mutated and isotype-switched antibodies. Immunotherapy increases their frequency in the blood, and even narrowly defined allergen epitopes are recognized by numerous distinct B-cell clones in a patient. The results also suggest that oral immunotherapy can stimulate somatic mutation of allergen-specific IgG4.


Assuntos
Albuminas 2S de Plantas/imunologia , Alérgenos/imunologia , Antígenos de Plantas/imunologia , Linfócitos B/imunologia , Glicoproteínas/imunologia , Hipersensibilidade a Amendoim/imunologia , Proteínas de Plantas/imunologia , Adolescente , Adulto , Criança , Pré-Escolar , Dessensibilização Imunológica , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulina E/genética , Imunoglobulina G/genética , Masculino , Proteínas de Membrana , Mutação , Hipersensibilidade a Amendoim/terapia , Adulto Jovem
10.
Methods Mol Biol ; 1343: 219-33, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26420720

RESUMO

High-throughput DNA sequencing techniques have greatly accelerated the pace of research into the repertoires of antibody and T cell receptor gene rearrangements that confer antigen specificity to adaptive immune responses. Studies of aging-related changes in human B cell repertoires have benefited from the ability to detect and quantify thousands to millions of B cell clones in human samples, and study the mutational lineages and isotype switching relationships within each clonal lineage. Correlation of repertoire analysis with antibody gene data from antigen-specific B cells is poised to give much greater insight into clinically relevant B cell responses and memory storage. Here, we describe strategies for preparing and analyzing human antibody gene libraries for studying B cell repertoires.


Assuntos
Linfócitos B/metabolismo , Genes de Imunoglobulinas , Sequenciamento de Nucleotídeos em Larga Escala , Anticorpos/genética , Biologia Computacional/métodos , Biblioteca Gênica , Rearranjo Gênico do Linfócito B , Humanos , Análise de Sequência de DNA
11.
Sci Transl Med ; 7(302): 302ra135, 2015 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-26311730

RESUMO

Common variable immune deficiency (CVID) is the most common symptomatic primary immune deficiency, affecting ~1 in 25,000 persons. These patients suffer from impaired antibody responses, autoimmunity, and susceptibility to lymphoid cancers. To explore the cellular basis for these clinical phenotypes, we conducted high-throughput DNA sequencing of immunoglobulin heavy chain gene rearrangements from 93 CVID patients and 105 control subjects and sorted naïve and memory B cells from 13 of the CVID patients and 10 of the control subjects. The CVID patients showed abnormal VDJ rearrangement and abnormal formation of complementarity-determining region 3 (CDR3). We observed a decreased selection against antibodies with long CDR3s in memory repertoires and decreased variable gene replacement, offering possible mechanisms for increased patient autoreactivity. Our data indicate that patient immunodeficiency might derive from both decreased diversity of the naïve B cell pool and decreased somatic hypermutation in memory repertoires. The CVID patients also exhibited an abnormal clonal expansion of unmutated B cells relative to the controls. Although impaired B cell germinal center activation is commonly viewed as causative in CVID, these data indicate that CVID B cells diverge from controls as early as the pro-B stage, cell and suggest possible explanations for the increased incidence of autoimmunity, immunodeficiency, and lymphoma CVID patients.


Assuntos
Linfócitos B/imunologia , Imunodeficiência de Variável Comum/genética , Cadeias Pesadas de Imunoglobulinas/genética , Imunodeficiência de Variável Comum/imunologia , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Mutação
12.
Philos Trans R Soc Lond B Biol Sci ; 370(1676)2015 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-26194750

RESUMO

The human and mouse antibody repertoires are formed by identical processes, but like all small animals, mice only have sufficient lymphocytes to express a small part of the potential antibody repertoire. In this study, we determined how the heavy chain repertoires of two mouse strains are generated. Analysis of IgM- and IgG-associated VDJ rearrangements generated by high-throughput sequencing confirmed the presence of 99 functional immunoglobulin heavy chain variable (IGHV) genes in the C57BL/6 genome, and inferred the presence of 164 IGHV genes in the BALB/c genome. Remarkably, only five IGHV sequences were common to both strains. Compared with humans, little N nucleotide addition was seen in the junctions of mouse VDJ genes. Germline human IgG-associated IGHV genes are rare, but many murine IgG-associated IGHV genes were unmutated. Together these results suggest that the expressed mouse repertoire is more germline-focused than the human repertoire. The apparently divergent germline repertoires of the mouse strains are discussed with reference to reports that inbred mouse strains carry blocks of genes derived from each of the three subspecies of the house mouse. We hypothesize that the germline genes of BALB/c and C57BL/6 mice may originally have evolved to generate distinct germline-focused antibody repertoires in the different mouse subspecies.


Assuntos
Diversidade de Anticorpos/genética , Cadeias Pesadas de Imunoglobulinas/genética , Animais , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Região Variável de Imunoglobulina/genética , Imunofenotipagem , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Especificidade da Espécie
13.
Proc Natl Acad Sci U S A ; 112(2): 500-5, 2015 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-25535378

RESUMO

Adaptive immune responses in humans rely on somatic genetic rearrangements of Ig and T-cell receptor loci to generate diverse antigen receptors. It is unclear to what extent an individual's genetic background affects the characteristics of the antibody repertoire used in responding to vaccination or infection. We studied the B-cell repertoires and clonal expansions in response to attenuated varicella-zoster vaccination in four pairs of adult identical twins and found that the global antibody repertoires of twin pair members showed high similarity in antibody heavy chain V, D, and J gene segment use, and in the length and features of the complementarity-determining region 3, a major determinant of antigen binding. These twin similarities were most pronounced in the IgM-expressing B-cell pools, but were seen to a lesser extent in IgG-expressing B cells. In addition, the degree of antibody somatic mutation accumulated in the B-cell repertoire was highly correlated within twin pair members. Twin pair members had greater numbers of shared convergent antibody sequences, including mutated sequences, suggesting similarity among memory B-cell clonal lineages. Despite these similarities in the memory repertoire, the B-cell clones used in acute responses to ZOSTAVAX vaccination were largely unique to each individual. Taken together, these results suggest that the overall B-cell repertoire is significantly shaped by the underlying germ-line genome, but that stochastic or individual-specific effects dominate the selection of clones in response to an acute antigenic stimulus.


Assuntos
Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/genética , Linfócitos B/imunologia , Vacina contra Herpes Zoster/imunologia , Herpesvirus Humano 3/imunologia , Gêmeos Monozigóticos/genética , Estudos de Coortes , Regiões Determinantes de Complementaridade/genética , Feminino , Humanos , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina M/genética , Memória Imunológica/genética , Masculino , Pessoa de Meia-Idade , Mutação
14.
Cell Host Microbe ; 16(2): 215-226, 2014 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-25121750

RESUMO

Monoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env.


Assuntos
Anticorpos Anti-HIV/metabolismo , Proteína gp41 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Íleo/imunologia , Microbiota/imunologia , Especificidade de Anticorpos , Antígenos de Bactérias/imunologia , Reações Cruzadas , Infecções por HIV/virologia , Humanos , Íleo/patologia , Íleo/virologia , Dados de Sequência Molecular , Plasmócitos/imunologia , Plasmócitos/virologia , Ligação Proteica
15.
Cell Host Microbe ; 16(1): 105-14, 2014 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-24981332

RESUMO

B cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individual's secreted antibody response to the vaccine, and the expanded clones are enriched with those expressing influenza-specific monoclonal antibodies. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.


Assuntos
Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Rearranjo Gênico do Linfócito B , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Formação de Anticorpos , Humanos , Vacinas contra Influenza/administração & dosagem
16.
J Immunol ; 192(2): 603-11, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24337376

RESUMO

Elderly humans show decreased humoral immunity to pathogens and vaccines, yet the effects of aging on B cells are not fully known. Chronic viral infection by CMV is implicated as a driver of clonal T cell proliferations in some aging humans, but whether CMV or EBV infection contributes to alterations in the B cell repertoire with age is unclear. We have used high-throughput DNA sequencing of IGH gene rearrangements to study the BCR repertoires over two successive years in 27 individuals ranging in age from 20 to 89 y. Some features of the B cell repertoire remain stable with age, but elderly subjects show increased numbers of B cells with long CDR3 regions, a trend toward accumulation of more highly mutated IgM and IgG Ig genes, and persistent clonal B cell populations in the blood. Seropositivity for CMV or EBV infection alters B cell repertoires, regardless of the individual's age: EBV infection correlates with the presence of persistent clonal B cell expansions, whereas CMV infection correlates with the proportion of highly mutated Ab genes. These findings isolate effects of aging from those of chronic viral infection on B cell repertoires and provide a baseline for understanding human B cell responses to vaccination or infectious stimuli.


Assuntos
Envelhecimento/imunologia , Linfócitos B/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Linfócitos B/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Infecções por Vírus Epstein-Barr/virologia , Genes de Imunoglobulinas/genética , Genes de Imunoglobulinas/imunologia , Herpesvirus Humano 4/genética , Humanos , Pessoa de Meia-Idade , Mutação/genética , Mutação/imunologia , Adulto Jovem
17.
Haematologica ; 98(11): 1689-96, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23872309

RESUMO

In order to identify novel somatic mutations associated with classic BCR/ABL1-negative myeloproliferative neoplasms, we performed high-coverage genome sequencing of DNA from peripheral blood granulocytes and cultured skin fibroblasts from a patient with MPL W515K-positive primary myelofibrosis. The primary myelofibrosis genome had a low somatic mutation rate, consistent with that observed in similar hematopoietic tumor genomes. Interfacing of whole-genome DNA sequence data with RNA expression data identified three somatic mutations of potential functional significance: i) a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; ii) a 19-base pair deletion involving a potential regulatory region in the 5'-untranslated region of BRD2, implicated in transcriptional regulation and cell cycle control; and iii) a non-synonymous point mutation in KIAA0355, an uncharacterized protein. Additional mutations in three genes (CAP2, SOX30, and MFRP) were also evident, albeit with no support for expression at the RNA level. Re-sequencing of these six genes in 178 patients with polycythemia vera, essential thrombocythemia, and myelofibrosis did not identify recurrent somatic mutations in these genes. Finally, we describe methods for reducing false-positive variant calls in the analysis of hematologic malignancies with a low somatic mutation rate. This trial is registered with ClinicalTrials.gov (NCT01108159).


Assuntos
Estudos de Associação Genética/métodos , Variação Genética/genética , Estudo de Associação Genômica Ampla/métodos , Mutação/genética , Mielofibrose Primária/diagnóstico , Mielofibrose Primária/genética , Células Cultivadas , Humanos , Masculino , Pessoa de Meia-Idade
18.
Cell Host Microbe ; 13(6): 691-700, 2013 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-23768493

RESUMO

Dengue is the most prevalent mosquito-borne viral disease in humans, and the lack of early prognostics, vaccines, and therapeutics contributes to immense disease burden. To identify patterns that could be used for sequence-based monitoring of the antibody response to dengue, we examined antibody heavy-chain gene rearrangements in longitudinal peripheral blood samples from 60 dengue patients. Comparing signatures between acute dengue, postrecovery, and healthy samples, we found increased expansion of B cell clones in acute dengue patients, with higher overall clonality in secondary infection. Additionally, we observed consistent antibody sequence features in acute dengue in the highly variable major antigen-binding determinant, complementarity-determining region 3 (CDR3), with specific CDR3 sequences highly enriched in acute samples compared to postrecovery, healthy, or non-dengue samples. Dengue thus provides a striking example of a human viral infection where convergent immune signatures can be identified in multiple individuals. Such signatures could facilitate surveillance of immunological memory in communities.


Assuntos
Anticorpos Antivirais/sangue , Vírus da Dengue/imunologia , Dengue/imunologia , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Regiões Determinantes de Complementaridade/imunologia , Humanos , Memória Imunológica
19.
Nature ; 496(7446): 469-76, 2013 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-23552890

RESUMO

Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Evolução Molecular , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , HIV-1/química , HIV-1/imunologia , Vacinas contra a AIDS/imunologia , África , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/genética , Antígenos CD4/química , Antígenos CD4/imunologia , Linhagem da Célula , Células Cultivadas , Células Clonais/citologia , Reações Cruzadas/imunologia , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Anticorpos Anti-HIV/genética , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/classificação , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Testes de Neutralização , Filogenia , Estrutura Terciária de Proteína
20.
BMC Bioinformatics ; 12: 144, 2011 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-21569267

RESUMO

BACKGROUND: Continuing research into the global multiple sequence alignment problem has resulted in more sophisticated and principled alignment methods. Unfortunately these new algorithms often require large amounts of time and memory to run, making it nearly impossible to run these algorithms on large datasets. As a solution, we present two general methods, Crumble and Prune, for breaking a phylogenetic alignment problem into smaller, more tractable sub-problems. We call Crumble and Prune meta-alignment methods because they use existing alignment algorithms and can be used with many current alignment programs. Crumble breaks long alignment problems into shorter sub-problems. Prune divides the phylogenetic tree into a collection of smaller trees to reduce the number of sequences in each alignment problem. These methods are orthogonal: they can be applied together to provide better scaling in terms of sequence length and in sequence depth. Both methods partition the problem such that many of the sub-problems can be solved independently. The results are then combined to form a solution to the full alignment problem. RESULTS: Crumble and Prune each provide a significant performance improvement with little loss of accuracy. In some cases, a gain in accuracy was observed. Crumble and Prune were tested on real and simulated data. Furthermore, we have implemented a system called Job-tree that allows hierarchical sub-problems to be solved in parallel on a compute cluster, significantly shortening the run-time. CONCLUSIONS: These methods enabled us to solve gigabase alignment problems. These methods could enable a new generation of biologically realistic alignment algorithms to be applied to real world, large scale alignment problems.


Assuntos
Algoritmos , Alinhamento de Sequência/métodos , Animais , Sequência de Bases , Análise por Conglomerados , Genoma , Humanos , Fatores de Tempo
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