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1.
Cochrane Database Syst Rev ; 7: CD012022, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32735048

RESUMO

BACKGROUND: Chronic lymphocytic leukaemia (CLL) is the most common cancer of the lymphatic system in Western countries. Several clinical and biological factors for CLL have been identified. However, it remains unclear which of the available prognostic models combining those factors can be used in clinical practice to predict long-term outcome in people newly-diagnosed with CLL. OBJECTIVES: To identify, describe and appraise all prognostic models developed to predict overall survival (OS), progression-free survival (PFS) or treatment-free survival (TFS) in newly-diagnosed (previously untreated) adults with CLL, and meta-analyse their predictive performances. SEARCH METHODS: We searched MEDLINE (from January 1950 to June 2019 via Ovid), Embase (from 1974 to June 2019) and registries of ongoing trials (to 5 March 2020) for development and validation studies of prognostic models for untreated adults with CLL. In addition, we screened the reference lists and citation indices of included studies. SELECTION CRITERIA: We included all prognostic models developed for CLL which predict OS, PFS, or TFS, provided they combined prognostic factors known before treatment initiation, and any studies that tested the performance of these models in individuals other than the ones included in model development (i.e. 'external model validation studies'). We included studies of adults with confirmed B-cell CLL who had not received treatment prior to the start of the study. We did not restrict the search based on study design. DATA COLLECTION AND ANALYSIS: We developed a data extraction form to collect information based on the Checklist for Critical Appraisal and Data Extraction for Systematic Reviews of Prediction Modelling Studies (CHARMS). Independent pairs of review authors screened references, extracted data and assessed risk of bias according to the Prediction model Risk Of Bias ASsessment Tool (PROBAST). For models that were externally validated at least three times, we aimed to perform a quantitative meta-analysis of their predictive performance, notably their calibration (proportion of people predicted to experience the outcome who do so) and discrimination (ability to differentiate between people with and without the event) using a random-effects model. When a model categorised individuals into risk categories, we pooled outcome frequencies per risk group (low, intermediate, high and very high). We did not apply GRADE as guidance is not yet available for reviews of prognostic models. MAIN RESULTS: From 52 eligible studies, we identified 12 externally validated models: six were developed for OS, one for PFS and five for TFS. In general, reporting of the studies was poor, especially predictive performance measures for calibration and discrimination; but also basic information, such as eligibility criteria and the recruitment period of participants was often missing. We rated almost all studies at high or unclear risk of bias according to PROBAST. Overall, the applicability of the models and their validation studies was low or unclear; the most common reasons were inappropriate handling of missing data and serious reporting deficiencies concerning eligibility criteria, recruitment period, observation time and prediction performance measures. We report the results for three models predicting OS, which had available data from more than three external validation studies: CLL International Prognostic Index (CLL-IPI) This score includes five prognostic factors: age, clinical stage, IgHV mutational status, B2-microglobulin and TP53 status. Calibration: for the low-, intermediate- and high-risk groups, the pooled five-year survival per risk group from validation studies corresponded to the frequencies observed in the model development study. In the very high-risk group, predicted survival from CLL-IPI was lower than observed from external validation studies. Discrimination: the pooled c-statistic of seven external validation studies (3307 participants, 917 events) was 0.72 (95% confidence interval (CI) 0.67 to 0.77). The 95% prediction interval (PI) of this model for the c-statistic, which describes the expected interval for the model's discriminative ability in a new external validation study, ranged from 0.59 to 0.83. Barcelona-Brno score Aimed at simplifying the CLL-IPI, this score includes three prognostic factors: IgHV mutational status, del(17p) and del(11q). Calibration: for the low- and intermediate-risk group, the pooled survival per risk group corresponded to the frequencies observed in the model development study, although the score seems to overestimate survival for the high-risk group. Discrimination: the pooled c-statistic of four external validation studies (1755 participants, 416 events) was 0.64 (95% CI 0.60 to 0.67); 95% PI 0.59 to 0.68. MDACC 2007 index score The authors presented two versions of this model including six prognostic factors to predict OS: age, B2-microglobulin, absolute lymphocyte count, gender, clinical stage and number of nodal groups. Only one validation study was available for the more comprehensive version of the model, a formula with a nomogram, while seven studies (5127 participants, 994 events) validated the simplified version of the model, the index score. Calibration: for the low- and intermediate-risk groups, the pooled survival per risk group corresponded to the frequencies observed in the model development study, although the score seems to overestimate survival for the high-risk group. Discrimination: the pooled c-statistic of the seven external validation studies for the index score was 0.65 (95% CI 0.60 to 0.70); 95% PI 0.51 to 0.77. AUTHORS' CONCLUSIONS: Despite the large number of published studies of prognostic models for OS, PFS or TFS for newly-diagnosed, untreated adults with CLL, only a minority of these (N = 12) have been externally validated for their respective primary outcome. Three models have undergone sufficient external validation to enable meta-analysis of the model's ability to predict survival outcomes. Lack of reporting prevented us from summarising calibration as recommended. Of the three models, the CLL-IPI shows the best discrimination, despite overestimation. However, performance of the models may change for individuals with CLL who receive improved treatment options, as the models included in this review were tested mostly on retrospective cohorts receiving a traditional treatment regimen. In conclusion, this review shows a clear need to improve the conducting and reporting of both prognostic model development and external validation studies. For prognostic models to be used as tools in clinical practice, the development of the models (and their subsequent validation studies) should adapt to include the latest therapy options to accurately predict performance. Adaptations should be timely.


Assuntos
Leucemia Linfocítica Crônica de Células B/mortalidade , Modelos Teóricos , Adulto , Fatores Etários , Viés , Biomarcadores Tumorais , Calibragem , Intervalos de Confiança , Análise Discriminante , Intervalo Livre de Doença , Feminino , Genes p53/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Estadiamento de Neoplasias , Prognóstico , Intervalo Livre de Progressão , Receptores de Antígenos de Linfócitos B/genética , Reprodutibilidade dos Testes , Proteína Supressora de Tumor p53/genética
2.
PLoS One ; 15(8): e0236477, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756607

RESUMO

Antibodies function by binding to antigens. Antibodies must be cloned and expressed to determine their binding characteristics, but current methods for high-throughput antibody sequencing yield antibody DNA pooled from many cells and do not readily permit cloning of antibodies from single B cells. We present a strategy for retrieving and cloning antibody DNA from single cells within a pooled library of cells. Our strategy, called selective PCR for antibody retrieval (SPAR), takes advantage of the unique sequence barcodes attached to individual cDNA molecules during sample preparation to enable specific amplification by PCR of antibody heavy- and light-chain cDNA originating from a single cell. We show through computational analysis that most human antibodies sequenced using typical high-throughput methods can be retrieved using SPAR, and experimentally demonstrate retrieval of full-length antibody variable region cDNA from three cells within pools of ~5,000 cells. SPAR enables rapid low-cost cloning and expression of native human antibodies from pooled single-cell sequence libraries for functional characterization.


Assuntos
Anticorpos/genética , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Aminoácidos/genética , Técnicas de Visualização da Superfície Celular , Clonagem Molecular , DNA Complementar/genética , Biblioteca Gênica , Vetores Genéticos/genética , Humanos , Análise de Célula Única
3.
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
4.
Science ; 369(6507): 1119-1123, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661058

RESUMO

Molecular understanding of neutralizing antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could accelerate vaccine design and drug discovery. We analyzed 294 anti-SARS-CoV-2 antibodies and found that immunoglobulin G heavy-chain variable region 3-53 (IGHV3-53) is the most frequently used IGHV gene for targeting the receptor-binding domain (RBD) of the spike protein. Co-crystal structures of two IGHV3-53-neutralizing antibodies with RBD, with or without Fab CR3022, at 2.33- to 3.20-angstrom resolution revealed that the germline-encoded residues dominate recognition of the angiotensin I converting enzyme 2 (ACE2)-binding site. This binding mode limits the IGHV3-53 antibodies to short complementarity-determining region H3 loops but accommodates light-chain diversity. These IGHV3-53 antibodies show minimal affinity maturation and high potency, which is promising for vaccine design. Knowledge of these structural motifs and binding mode should facilitate the design of antigens that elicit this type of neutralizing response.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Formação de Anticorpos , Betacoronavirus/imunologia , Regiões Determinantes de Complementaridade/química , Infecções por Coronavirus/prevenção & controle , Cadeias Pesadas de Imunoglobulinas/química , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Sítios de Ligação , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Cristalografia por Raios X , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Pneumonia Viral/imunologia , Domínios Proteicos , Vacinas Virais/química , Vacinas Virais/genética , Vacinas Virais/imunologia
5.
PLoS Pathog ; 16(4): e1008438, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32353066

RESUMO

One of the defining characteristics of the B cell receptor (BCR) is the extensive diversity in the repertoire of immunoglobulin genes that make up the BCR, resulting in broad range of specificity. Gammaherpesviruses are B lymphotropic viruses that establish life-long infection in B cells, and although the B cell receptor plays a central role in B cell biology, very little is known about the immunoglobulin repertoire of gammaherpesvirus infected cells. To begin to characterize the Ig genes expressed by murine gammaherpesvirus 68 (MHV68) infected cells, we utilized single cell sorting to sequence and clone the Ig variable regions of infected germinal center (GC) B cells and plasma cells. We show that MHV68 infection is biased towards cells that express the Igλ light chain along with a single heavy chain variable gene, IGHV10-1*01. This population arises through clonal expansion but is not viral antigen specific. Furthermore, we show that class-switching in MHV68 infected cells differs from that of uninfected cells. Fewer infected GC B cells are class-switched compared to uninfected GC B cells, while more infected plasma cells are class-switched compared to uninfected plasma cells. Additionally, although they are germinal center derived, the majority of class switched plasma cells display no somatic hypermutation regardless of infection status. Taken together, these data indicate that selection of infected B cells with a specific BCR, as well as virus mediated manipulation of class switching and somatic hypermutation, are critical aspects in establishing life-long gammaherpesvirus infection.


Assuntos
Linfócitos B/imunologia , Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/veterinária , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Cadeias lambda de Imunoglobulina/imunologia , Doenças dos Roedores/imunologia , Animais , Linfócitos B/virologia , Feminino , Gammaherpesvirinae/genética , Centro Germinativo/imunologia , Centro Germinativo/virologia , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Plasmócitos/imunologia , Plasmócitos/virologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Doenças dos Roedores/genética , Doenças dos Roedores/virologia
6.
Proc Natl Acad Sci U S A ; 117(21): 11624-11635, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32385154

RESUMO

Activation-induced cytidine deaminase (AID) is the key enzyme for class switch recombination (CSR) and somatic hypermutation (SHM) to generate antibody memory. Previously, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was shown to be required for AID-dependent DNA breaks. Here, we defined the function of major RNA-binding motifs of hnRNP K, GXXGs and RGGs in the K-homology (KH) and the K-protein-interaction (KI) domains, respectively. Mutation of GXXG, RGG, or both impaired CSR, SHM, and cMyc/IgH translocation equally, showing that these motifs were necessary for AID-dependent DNA breaks. AID-hnRNP K interaction is dependent on RNA; hence, mutation of these RNA-binding motifs abolished the interaction with AID, as expected. Some of the polypyrimidine sequence-carrying prototypical hnRNP K-binding RNAs, which participate in DNA breaks or repair bound to hnRNP K in a GXXG and RGG motif-dependent manner. Mutation of the GXXG and RGG motifs decreased nuclear retention of hnRNP K. Together with the previous finding that nuclear localization of AID is necessary for its function, lower nuclear retention of these mutants may worsen their functional deficiency, which is also caused by their decreased RNA-binding capacity. In summary, hnRNP K contributed to AID-dependent DNA breaks with all of its major RNA-binding motifs.


Assuntos
Anticorpos , Citidina Desaminase , Quebras de DNA , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Motivos de Ligação ao RNA/genética , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Switching de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Camundongos , Hipermutação Somática de Imunoglobulina/genética
7.
Immunogenetics ; 72(5): 279-294, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32367185

RESUMO

Protection and neutralization of a vast array of pathogens is accomplished by the tremendous diversity of the B cell receptor (BCR) repertoire. For jawed vertebrates, this diversity is initiated via the somatic recombination of immunoglobulin (Ig) germline elements. While it is clear that the number of these germline segments differs from species to species, the extent of cross-species sequence diversity remains largely uncharacterized. Here we use extensive computational and statistical methods to investigate the sequence diversity and evolutionary relationship between Ig variable (V), diversity (D), and joining (J) germline segments across nine commonly studied species ranging from zebrafish to human. Metrics such as guanine-cytosine (GC) content showed low redundancy across Ig germline genes within a given species. Other comparisons, including amino acid motifs, evolutionary selection, and sequence diversity, revealed species-specific properties. Additionally, we showed that the germline-encoded diversity differs across antibody (recombined V-D-J) repertoires of various B cell subsets. To facilitate future comparative immunogenomics analysis, we created VDJgermlines, an R package that contains the germline sequences from multiple species. Our study informs strategies for the humanization and engineering of therapeutic antibodies.


Assuntos
Variação Genética , Região Variável de Imunoglobulina/genética , Filogenia , Motivos de Aminoácidos , Animais , Diversidade de Anticorpos/genética , Subpopulações de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Composição de Bases , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/química , Seleção Genética , Especificidade da Espécie , Recombinação V(D)J/genética , Vertebrados
8.
Proc Biol Sci ; 287(1927): 20200489, 2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32396805

RESUMO

The evolution of the adaptive immune system has provided vertebrates with a uniquely sophisticated immune toolkit, enabling them to mount precise immune responses against a staggeringly diverse range of antigens. Like other vertebrates, teleost fishes possess a complex and functional adaptive immune system; however, our knowledge of the complex antigen-receptor genes underlying its functionality has been restricted to a small number of experimental and agricultural species, preventing systematic investigation into how these crucial gene loci evolve. Here, we analyse the genomic structure of the immunoglobulin heavy chain (IGH) gene loci in the cyprinodontiforms, a diverse and important group of teleosts present in many different habitats across the world. We reconstruct the complete IGH loci of the turquoise killifish (Nothobranchius furzeri) and the southern platyfish (Xiphophorus maculatus) and analyse their in vivo gene expression, revealing the presence of species-specific splice isoforms of transmembrane IGHM. We further characterize the IGH constant regions of 10 additional cyprinodontiform species, including guppy, Amazon molly, mummichog and mangrove killifish. Phylogenetic analysis of these constant regions suggests multiple independent rounds of duplication and deletion of the teleost-specific antibody class IGHZ in the cyprinodontiform lineage, demonstrating the extreme volatility of IGH evolution. Focusing on the cyprinodontiforms as a model taxon for comparative evolutionary immunology, this work provides novel genomic resources for studying adaptive immunity and sheds light on the evolutionary history of the adaptive immune system.


Assuntos
Evolução Biológica , Ciprinodontiformes/fisiologia , Cadeias Pesadas de Imunoglobulinas/genética , Animais , Ciprinodontiformes/genética , Genoma , Cadeias Pesadas de Imunoglobulinas/imunologia , Filogenia , Volatilização
9.
Scand J Immunol ; 92(2): e12912, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32458431

RESUMO

Immune processes in liver transplantation remain poorly understood. Acute allograft rejection in liver transplantation is a kind of T cell-mediated inflammatory disease accompanied by inflammatory cell infiltration. However, the effect of acute allograft rejection on the immunological characteristics of TCRs in peripheral blood mononuclear cell is unknown. In this study, we characterized the pattern of the human T cell receptor beta chain (TRB) and immunoglobulin heavy chain (IGH) complementarity-determining region 3 (CDR3) repertoires via high-throughput sequencing in 11 acute allograft rejection (AG) cases, 23 patients with stable allograft liver function (ST) who had liver transplantation performed and 20 healthy controls (HC). The diversity of TRB-CDR3 was significantly reduced in the AG group compared with the ST group and healthy controls (HC). The CDR3 and N-addition length distribution were not significantly different between the AG and ST groups. However, N-addition length distribution was significantly changed compared to HC. It seemed that AG used more short N-additions and healthy people used more long N-additions in TRB-CDR3 repertoire. Our findings suggested that the TRB-CDR3 region of AG had distinctive V gene use compared with that of HC. The characteristics of ST seemed to be in between those of AG and HC although the difference is not significant. Cluster analysis showed that the TRB repertoire could not effectively distinguish AG from ST. This research might give to a better understanding of the immune process of liver transplantation.


Assuntos
Regiões Determinantes de Complementaridade/imunologia , Rejeição de Enxerto/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Transplante de Fígado , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Adulto , Regiões Determinantes de Complementaridade/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T alfa-beta/genética
10.
Pediatr Blood Cancer ; 67(6): e28280, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32277801

RESUMO

Acute lymphoblastic leukemia (ALL) is often composed of numerous subclones. Here we test whether the clonal composition of the blood is representative of the bone marrow at leukemia onset. Using ultra-deep IGH sequencing, we detected 28 clones across 16 patients; 5/28 were only in the marrow. In four patients, the most abundant clones differed between sites, including three in which the dominant medullary clones were minimally detectable in the blood. These findings demonstrate that the peripheral blood often underrepresents the genetic heterogeneity in a B-ALL and highlight the potential impact of tissue site selection on the detection of minor subclones.


Assuntos
Biomarcadores Tumorais/genética , Medula Óssea/patologia , Evolução Clonal , Células Clonais/patologia , Cadeias Pesadas de Imunoglobulinas/genética , Leucócitos Mononucleares/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Adolescente , Adulto , Medula Óssea/metabolismo , Criança , Pré-Escolar , Células Clonais/metabolismo , Feminino , Seguimentos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Prognóstico
11.
Proc Natl Acad Sci U S A ; 117(8): 4320-4327, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32047037

RESUMO

The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21-derived heavy chains (HCs) with IGLV3-21-derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110-expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR-BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01-expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110-expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.


Assuntos
Cadeias lambda de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Linfócitos B/imunologia , Estudos de Coortes , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Predisposição Genética para Doença , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias lambda de Imunoglobulina/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Mutação Puntual , Receptores de Antígenos de Linfócitos B/genética
12.
Proc Natl Acad Sci U S A ; 117(10): 5453-5462, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32098847

RESUMO

Developing lymphocytes diversify their antigen receptor (AgR) loci by variable (diversity) joining (V[D]J) recombination. Here, using the micrococcal nuclease (MNase)-based chromatin accessibility (MACC) assay with low-cell count input, we profile both small-scale (kilobase) and large-scale (megabase) changes in chromatin accessibility and nucleosome occupancy in primary cells during lymphoid development, tracking the changes as different AgR loci become primed for recombination. The three distinct chromatin structures identified in this work define unique features of immunoglobulin H (IgH), Igκ, and T cell receptor-α (TCRα) loci during B lymphopoiesis. In particular, we find locus-specific temporal changes in accessibility both across megabase-long AgR loci and locally at the recombination signal sequences (RSSs). These changes seem to be regulated independently and can occur prior to lineage commitment. Large-scale changes in chromatin accessibility occur without significant change in nucleosome density and represent key features of AgR loci not previously described. We further identify local dynamic repositioning of individual RSS-associated nucleosomes at IgH and Igκ loci while they become primed for recombination during B cell commitment. These changes in chromatin at AgR loci are regulated in a locus-, lineage-, and stage-specific manner during B lymphopoiesis, serving either to facilitate or to impose a barrier to V(D)J recombination. We suggest that local and global changes in chromatin openness in concert with nucleosome occupancy and placement of histone modifications facilitate the temporal order of AgR recombination. Our data have implications for the organizing principles that govern assembly of these large loci as well as for mechanisms that might contribute to aberrant V(D)J recombination and the development of lymphoid tumors.


Assuntos
Linfócitos B/fisiologia , Cromatina/metabolismo , Rearranjo Gênico do Linfócito B , Linfopoese/genética , Receptores de Antígenos/genética , Recombinação V(D)J , Animais , Cromatina/química , Loci Gênicos , Testes Genéticos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/genética , Linfoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Nuclease do Micrococo , Nucleossomos , Receptores de Antígenos de Linfócitos T alfa-beta/genética
13.
Nucleic Acids Res ; 48(7): 3553-3566, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32086526

RESUMO

Developing B cells undergo V(D)J recombination to generate a vast repertoire of Ig molecules. V(D)J recombination is initiated by the RAG1/RAG2 complex in recombination centres (RCs), where gene segments become accessible to the complex. Whether transcription is the causal factor of accessibility or whether it is a side product of other processes that generate accessibility remains a controversial issue. At the IgH locus, V(D)J recombination is controlled by Eµ enhancer, which directs the transcriptional, epigenetic and recombinational events in the IgH RC. Deletion of Eµ enhancer affects both transcription and recombination, making it difficult to conclude if Eµ controls the two processes through the same or different mechanisms. By using a mouse line carrying a CpG-rich sequence upstream of Eµ enhancer and analyzing transcription and recombination at the single-cell level, we found that recombination could occur in the RC in the absence of detectable transcription, suggesting that Eµ controls transcription and recombination through distinct mechanisms. Moreover, while the normally Eµ-dependent transcription and demethylating activities were impaired, recruitment of chromatin remodeling complexes was unaffected. RAG1 was efficiently recruited, thus compensating for the defective transcription-associated recruitment of RAG2, and providing a mechanistic basis for RAG1/RAG2 assembly to initiate V(D)J recombination.


Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Transcrição Genética , Recombinação V(D)J , Alelos , Animais , DNA Helicases/metabolismo , Metilação de DNA , Elementos Facilitadores Genéticos , Proteínas de Homeodomínio/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo
14.
Emerg Microbes Infect ; 9(1): 111-123, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31906823

RESUMO

The Zika virus (ZIKV) is a mosquito-borne flavivirus that causes neonatal abnormalities and other disorders. Antibodies to the ZIKV envelope (E) protein can block infection. In this study, next-generation sequencing (NGS) of immunoglobulin heavy chain (IgH) mRNA transcripts was combined with single-cell PCR cloning of E-binding monoclonal antibodies for analysing antibody response in a patient from the early stages of infection to more than one year after the clearance of the virus. The patient's IgH repertoire 14 and 64 days after symptom onset showed dramatic dominant clonal expansion but low clonal diversity. IgH repertoire 6 months after disease-free status had few dominant clones but increased diversity. E-binding antibodies appeared abundantly in the repertoire during the early stages of infection but quickly declined after clearance of the virus. Certain VH genes such as VH5-10-1 and VH4-39 appeared to be preferentially enlisted for a rapid antibody response to ZIKV infection. Most of these antibodies require relatively few somatic hypermutations to acquire the ability to bind to the E protein, pointing to a possible mechanism for rapid defence against ZIKV infection. This study provides a unique and holistic view of the dynamic changes and characteristics of the antibody response to ZIKV infection.


Assuntos
Anticorpos Antivirais/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Adulto , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Formação de Anticorpos , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Estudos Longitudinais , Masculino , Testes de Neutralização , Zika virus/genética , Infecção por Zika virus/virologia
15.
Cancer Genet ; 242: 15-24, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31980417

RESUMO

The diagnosis and risk stratification of multiple myeloma (MM) is based on clinical and cytogenetic tests. Magnetic CD138 enrichment followed by interphase FISH (fluorescence in situ hybridisation) is the gold standard to identify prognostic translocations and copy number alterations (CNA). Although clinical implications of gene expression profiling (GEP) or panel based sequencing results are evident, those tests have not yet reached routine clinical application. We set up a single workflow to analyse MM of 211 patients at first diagnosis by whole genome sequencing (WGS) and RNA-Seq and validate the results by FISH analysis. We observed a 96% concordance of FISH and WGS results when assessing translocations involving the IGH locus and an overall concordance of FISH and WGS of 92% when assessing CNA. WGS analysis resulted in the identification of 17 additional MYC-translocations that were missed by FISH analysis. RNA-Seq followed by supervised clustering grouped patients in their expected genetically defined subgroup and prompted the assessment of WGS data in cases that were not congruent with FISH. This allowed the identification of additional IGH-translocations and hyperdiploid cases. We show the reliability of WGS an RNA-Seq in a clinical setting, which is a prerequisite for a novel routine diagnostic test.


Assuntos
Mieloma Múltiplo/diagnóstico , RNA-Seq , Sequenciamento Completo do Genoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Medula Óssea/patologia , Variações do Número de Cópias de DNA , Testes Diagnósticos de Rotina , Feminino , Perfilação da Expressão Gênica , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Medição de Risco , Deleção de Sequência , Sindecana-1/genética , Translocação Genética
17.
Hematol Oncol ; 38(2): 171-180, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31955451

RESUMO

We described four patients with diffuse large B-cell lymphoma (DLBCL) carrying t(9;14)(p13;q32) that places the PAX5 adjacent to the immunoglobulin heavy chain (IGH) gene. Ages ranged between 63 and 80, and three were female. One developed a nodal disease, and the other three involved extranodal organs. The lymphoma cells were CD10- /BCL6- /MUM1+ in three and CD10+ /BCL6+ /MUM1+ in one. BCL2 was weak or negative. All had t(9;14)(p13;q32), and three had additional 14q32/IGH translocations or +der(14)t(9;14)(p13;q32). Fluorescence in situ hybridization using the PAX5 break-apart probe showed that the locus was disrupted between the 5' and 3' probes or within the 5' probe. Immunohistochemistry (IHC) using a monoclonal antibody against PAX5 showed strong nuclear positivity in all four patients. Cell block IHC of a CD30+ DLBCL cell line, KIS-1, which carried the t(9;14)(p13;q32) and PAX5-IGH fusion gene, reproduced the CD10- /BCL6- /MUM1+ immunophenotype, low-level BCL2, and strong nuclear PAX5. Uniform nuclear positivity of MUM1 in all four cases and KIS-1 cells suggest that these lymphomas arose at a late stage of B-cell differentiation, where expression of PAX5 physiologically becomes downregulated. It is therefore possible that high-level PAX5 resulting from t(9;14)(p13;q32) at this stage of differentiation perturbs the plasma cell differentiation program initiated by PAX5 repression, thereby contributing to the development of a fraction of DLBCL.


Assuntos
Núcleo Celular/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Imuno-Histoquímica/métodos , Fatores Reguladores de Interferon/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Fator de Transcrição PAX5/metabolismo , Translocação Genética , Idoso , Idoso de 80 Anos ou mais , Núcleo Celular/genética , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 9/genética , Feminino , Humanos , Fatores Reguladores de Interferon/genética , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Fator de Transcrição PAX5/genética , Prognóstico
18.
PLoS One ; 15(1): e0228164, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31995598

RESUMO

Most of the approved monoclonal antibodies used in the clinic were initially discovered in mice. However, many targets of therapeutic interest are highly conserved proteins that do not elicit a robust immune response in mice. There is a need for non-mammalian antibody discovery platforms which would allow researchers to access epitopes that are not recognized in mammalian hosts. Recently, we introduced the OmniChicken®, a transgenic animal carrying human VH3-23 and VK3-15 at its immunoglobulin loci. Here, we describe a new version of the OmniChicken which carries VH3-23 and either VL1-44 or VL3-19 at its heavy and light chain loci, respectively. The Vλ-expressing birds showed normal B and T populations in the periphery. A panel of monoclonal antibodies demonstrated comparable epitope coverage of a model antigen compared to both wild-type and Vκ-expressing OmniChickens. Kinetic analysis identified binders in the picomolar range. The Vλ-expressing bird increases the antibody diversity available in the OmniChicken platform, further enabling discovery of therapeutic leads.


Assuntos
Animais Geneticamente Modificados/genética , Galinhas/genética , Cadeias lambda de Imunoglobulina/genética , Animais , Animais Geneticamente Modificados/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Galinhas/imunologia , Humanos , Imunidade Humoral , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/imunologia , Progranulinas/imunologia , Linfócitos T/imunologia , Transgenes/genética
19.
Immunogenetics ; 72(3): 165-179, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31838542

RESUMO

Non-human primates have been used as animal models because of their phylogenetic closeness to humans. However, the genetic differences between humans and non-human primates must be considered to select the appropriate animal models. Recently, New World monkeys (Platyrrhines) have generated a higher interest in biomedical research, especially in assessing vaccine safety and immunogenicity. Given the continued and renewed interest in Platyrrhines as biomedical models, it is a necessary to have a better and more complete understanding of their immune system and its implications for research. Immunoglobulins (Ig) are the main proteins that mediate humoral immunity. These proteins have evolved as part of an adaptive immune response system derived from ancient vertebrates. There are at least four Ig classes in Prosimians, whereas five have been reported in Catarrhines. Information on the structure and evolution of the loci containing immunoglobulin heavy chain constant genes (Igh) in Platyrrhines, however, is limited. Here, Igh loci were characterized in 10 Platyrrhines using the available whole genome sequences. Human and Macaca Igh loci were also assessed to compare them with their Platyrrhines counterparts. Differences in Igh locus structure were observed between Platyrrhines and Catarrhines. Noteworthy changes occur in the γ gene, which encodes a key Ig involved in organism defense that would favor protection after vaccination. The remarkable differences between the immunoglobulin proteins of Platyrrhines and Catarrhines warrant a cautionary message to biomedical researchers.


Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Platirrinos/genética , Platirrinos/imunologia , Animais , Evolução Biológica , Evolução Molecular , Genoma/genética , Genômica/métodos , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Filogenia , Primatas/genética , Vertebrados/genética
20.
J Exp Med ; 217(2)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31704807

RESUMO

Well-ordered HIV-1 envelope glycoprotein (Env) trimers are prioritized for clinical evaluation, and there is a need for an improved understanding about how elicited B cell responses evolve following immunization. To accomplish this, we prime-boosted rhesus macaques with clade C NFL trimers and identified 180 unique Ab lineages from ∼1,000 single-sorted Env-specific memory B cells. We traced all lineages in high-throughput heavy chain (HC) repertoire (Rep-seq) data generated from multiple immune compartments and time points and expressed several as monoclonal Abs (mAbs). Our results revealed broad dissemination and high levels of somatic hypermutation (SHM) of most lineages, including tier 2 virus neutralizing lineages, following boosting. SHM was highest in the Ab complementarity determining regions (CDRs) but also surprisingly high in the framework regions (FRs), especially FR3. Our results demonstrate the capacity of the immune system to affinity-mature large numbers of Env-specific B cell lineages simultaneously, supporting the use of regimens consisting of repeated boosts to improve each Ab, even those belonging to less expanded lineages.


Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos B/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Vacinação , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Regiões Determinantes de Complementaridade/genética , Feminino , Anticorpos Anti-HIV/imunologia , Infecções por HIV/virologia , HIV-1/química , Sequenciamento de Nucleotídeos em Larga Escala , Cadeias Pesadas de Imunoglobulinas/genética , Macaca mulatta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Célula Única , Hipermutação Somática de Imunoglobulina
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