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1.
Immunity ; 51(6): 1088-1101.e5, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31732168

RESUMO

The B cell response to Ehrlichia muris is dominated by plasmablasts (PBs), with few-if any-germinal centers (GCs), yet it generates protective immunoglobulin M (IgM) memory B cells (MBCs) that express the transcription factor T-bet and harbor V-region mutations. Because Ehrlichia prominently infects the liver, we investigated the nature of liver B cell response and that of the spleen. B cells within infected livers proliferated and underwent somatic hypermutation (SHM). Vh-region sequencing revealed trafficking of clones between the spleen and liver and often subsequent local clonal expansion and intraparenchymal localization of T-bet+ MBCs. T-bet+ MBCs expressed MBC subset markers CD80 and PD-L2. Many T-bet+ MBCs lacked CD11b or CD11c expression but had marginal zone (MZ) B cell phenotypes and colonized the splenic MZ, revealing T-bet+ MBC plasticity. Hence, liver and spleen are generative sites of B cell responses, and they include V-region mutation and result in liver MBC localization.


Assuntos
Linfócitos B/imunologia , Ehrlichia/imunologia , Ehrlichiose/imunologia , Imunoglobulina M/imunologia , Fígado/imunologia , Baço/imunologia , Animais , Antígeno B7-1/biossíntese , Região Variável de Imunoglobulina/genética , Memória Imunológica/imunologia , Fígado/citologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína 2 Ligante de Morte Celular Programada 1/biossíntese , Hipermutação Somática de Imunoglobulina/genética , Baço/citologia , Proteínas com Domínio T/metabolismo
2.
BMC Cancer ; 19(1): 809, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31412798

RESUMO

BACKGROUND: Eradication of minimal residual disease (MRD), at the end of Fludarabine-Cyclophosphamide-Rituximab (FCR) treatment, is a validated surrogate marker for progression-free and overall survival in chronic lymphocytic leukaemia. But such deep responses are also associated with severe immuno-depletion, leading to infections and the development of secondary cancers. METHODS: We assessed, blood MRD and normal immune cell levels at the end of treatment, in 162 first-line FCR patients, and analysed survival and adverse event. RESULTS: Multivariate Landmark analysis 3 months after FCR completion identified unmutated IGHV status (HR, 2.03, p = 0.043), the level of MRD reached (intermediate versus low, HR, 2.43, p = 0.002; high versus low, HR, 4.56, p = 0.002) and CD4 > 200/mm3 (HR, 3.30, p <  0.001) as factors independently associated with progression-free survival (PFS); neither CD8 nor NK counts were associated with PFS. The CD4 count was associated with PFS irrespective of IGHV mutational status, but only in patients with detectable MRD (HR, 3.51, p = 0.0004, whereas it had no prognostic impact in MRD < 10- 4 patients: p = 0.6998). We next used a competitive risk model to investigate whether immune cell subsets could be associated with the risk of infection and found no association between CD4, CD8 and NK cells and infection. CONCLUSIONS: Consolidation/maintenance trials based on detectable MRD after FCR should investigate CD4 T-cell numbers both as a selection and a response criterion, and consolidation treatments should target B-cell/T-cell interactions.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos T CD4-Positivos/patologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Ciclofosfamida/efeitos adversos , Ciclofosfamida/uso terapêutico , Feminino , Seguimentos , Humanos , Região Variável de Imunoglobulina/genética , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasia Residual , Prognóstico , Rituximab/efeitos adversos , Rituximab/uso terapêutico , Análise de Sobrevida , Resultado do Tratamento , Vidarabina/efeitos adversos , Vidarabina/análogos & derivados , Vidarabina/uso terapêutico
3.
PLoS Comput Biol ; 15(8): e1007207, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31442220

RESUMO

Antibodies developed for research and clinical applications may exhibit suboptimal stability, expressibility, or affinity. Existing optimization strategies focus on surface mutations, whereas natural affinity maturation also introduces mutations in the antibody core, simultaneously improving stability and affinity. To systematically map the mutational tolerance of an antibody variable fragment (Fv), we performed yeast display and applied deep mutational scanning to an anti-lysozyme antibody and found that many of the affinity-enhancing mutations clustered at the variable light-heavy chain interface, within the antibody core. Rosetta design combined enhancing mutations, yielding a variant with tenfold higher affinity and substantially improved stability. To make this approach broadly accessible, we developed AbLIFT, an automated web server that designs multipoint core mutations to improve contacts between specific Fv light and heavy chains (http://AbLIFT.weizmann.ac.il). We applied AbLIFT to two unrelated antibodies targeting the human antigens VEGF and QSOX1. Strikingly, the designs improved stability, affinity, and expression yields. The results provide proof-of-principle for bypassing laborious cycles of antibody engineering through automated computational affinity and stability design.


Assuntos
Afinidade de Anticorpos , Desenho de Drogas , Região Variável de Imunoglobulina/genética , Engenharia de Proteínas/métodos , Animais , Afinidade de Anticorpos/genética , Biologia Computacional , Células HEK293 , Humanos , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/imunologia , Biblioteca de Peptídeos , Engenharia de Proteínas/estatística & dados numéricos , Estabilidade Proteica , Software , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologia
4.
Vet Immunol Immunopathol ; 215: 109903, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31420067

RESUMO

Sensitivity of clonality analysis based on immunoglobulin heavy chain (IGH) in canine cutaneous plasmacytoma is lower than that in diffuse large B cell lymphoma (DLBCL) because of somatic hypermutation occurring at the IGH locus. Therefore, this study aimed to improve the sensitivity of clonality analysis for canine cutaneous plasmacytoma. To achieve this, clonality analysis based on the immunoglobulin kappa chain (IGK) locus was established. Sensitivity and specificity were examined in genomic DNA extracted from formalin-fixed paraffin-embedded sections of cutaneous plasmacytomas, DLBCLs, and lymph nodes without lymphoma. Forward primers were designed based on the IGKV genes, and reverse primers were designed based on the IGKJ genes and kappa deleting element (Kde). Analysis using IGKV and IGKJ primers demonstrated clonality in 24 of 29 cutaneous plasmacytomas (82.8%), while analysis with primers for IGKV and Kde showed clonality in 16 of 29 cases (55.2%). In DLBCL, the IGKV and IGKJ primer set yielded clonality in 18 of 23 cases (78.3%), and the IGKV and Kde primer set yielded 9 of 23 cases (39.1%). No clonal results were obtained from 23 lymph nodes without lymphoma. Sensitivity of the IGKV and IGKJ primer set was significantly higher than that of the IGH primers reported previously. Thus, clonality analysis based on the IGK locus can be utilized for canine B cell tumors. In conclusion, clonality testing based on IGH and IGK may be beneficial as an adjunct tool for diagnosis of canine B cell tumors including cutaneous plasmacytoma.


Assuntos
Doenças do Cão/imunologia , Cadeias kappa de Imunoglobulina/genética , Linfoma de Células B/veterinária , Plasmocitoma/veterinária , Neoplasias Cutâneas/veterinária , Animais , Células Clonais , DNA de Neoplasias , Doenças do Cão/genética , Cães , Genes de Imunoglobulinas , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Linfonodos/imunologia , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Plasmocitoma/genética , Plasmocitoma/imunologia , Plasmocitoma/patologia , Sensibilidade e Especificidade , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia
5.
Vet Immunol Immunopathol ; 215: 109913, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31420069

RESUMO

The development of a rapid and efficient system to generate porcine monoclonal antibodies (mAbs) is an important step toward the discovery of critical neutralizing targets for designing rational vaccines against porcine viruses. In this study, we established a platform for producing porcine mAbs based on single cell technologies. First, we singled out an optimal donor from 507 pigs based on serum antibody neutralizing activity against porcine reproductive and respiratory syndrome virus (PRRSV). After identifying the contribution of IgG to the neutralizing activity, single CD45R+IgG+Ag+ B cells were sorted from peripheral blood mononuclear cells (PBMCs). Single B cell RT-PCR was performed using primers designed to cover the germline repertoire of the porcine VH/VL gene segments. Paired VH/VLs were cloned into a eukaryotic expression vector and transfected into 293T cells. We demonstrate that full-length porcine mAbs were produced, and antigen-specific mAbs were obtained after further validation. The approach reported in this study can be applied to generate porcine mAbs against any given antigen and may help with the screening of neutralizing antibodies against porcine pathogens.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Suínos/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Especificidade de Anticorpos , Linfócitos B/imunologia , Células HEK293 , Humanos , Região Variável de Imunoglobulina/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Transfecção , Recombinação V(D)J
7.
Hematol Oncol ; 37(4): 368-374, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31325190

RESUMO

In 2009, the four laboratories of the Fondazione Italiana Linfomi (FIL) minimal residual disease (MRD) Network started a collaborative effort to harmonize and standardize their methodologies at the national level, performing quality control (QC) rounds for follicular lymphoma (FL) and mantle cell lymphoma (MCL) MRD assessment. In 16 QC rounds between 2010 and 2017, the four laboratories received 208 bone marrow (BM) samples (126 FL; 82 MCL); 187 were analyzed, according to the EuroMRD Consortium guidelines, by both nested (NEST) polymerase chain reaction (PCR) and real-time quantitative (RQ) PCR for BCL2/IGH MBR or IGHV rearrangements. Here, we aimed at analyzing the samples that challenged the interlaboratory reproducibility and data interpretation. Overall, 156/187 BM samples (83%) were concordantly classified as NEST+/RQ+ or NEST-/RQ- by all the four laboratories. The remaining 31 samples (17%) resulted alternatively positive and negative in the interlaboratory evaluations, independently of the method and the type of rearrangement, and were defined "borderline" (brd) samples: 12 proved NEST brd/RQ brd, 7 NEST-/RQ brd, 10 NEST brd/RQ positive not quantifiable (PNQ), and 2 NEST brd/RQ-. Results did not change even increasing the number of replicates/sample. In 6/31 brd samples, droplet digital PCR (ddPCR) was tested and showed no interlaboratory discordance. Despite the high interlaboratory reproducibility in the MRD analysis obtained and maintained by the QC round strategy, samples with the lowest MRD levels can still represent a challenge: 17% (31/187) of our samples showed discordant results in interlaboratory assessments, with 6.4% (12/187) remained brd even applying the two methods. Thus, although representing a minority, brd samples are still problematic, especially when a clinically oriented interpretation of MRD results is required. Alternative, novel methods such as ddPCR and next-generation sequencing have the potential to overcome the current limitations.


Assuntos
Exame de Medula Óssea , Medula Óssea/patologia , Ensaio de Proficiência Laboratorial , Linfoma não Hodgkin/patologia , Reação em Cadeia da Polimerase , Exame de Medula Óssea/normas , Células Clonais , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Genes de Imunoglobulinas , Genes bcl-2 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Itália/epidemiologia , Linfoma não Hodgkin/genética , Neoplasia Residual , Proteínas de Fusão Oncogênica/análise , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Garantia da Qualidade dos Cuidados de Saúde , Reprodutibilidade dos Testes , Translocação Genética
8.
PLoS Negl Trop Dis ; 13(7): e0007595, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31356611

RESUMO

Ebolaviruses cause an often rapidly fatal syndrome known as Ebola virus disease (EVD), with average case fatality rates of ~50%. There is no licensed vaccine or treatment for EVD, underscoring the urgent need to develop new anti-ebolavirus agents, especially in the face of an ongoing outbreak in the Democratic Republic of the Congo and the largest ever outbreak in Western Africa in 2013-2016. Lectins have been investigated as potential antiviral agents as they bind glycans present on viral surface glycoproteins, but clinical use of them has been slowed by concerns regarding their mitogenicity, i.e. ability to cause immune cell proliferation. We previously engineered a banana lectin (BanLec), a carbohydrate-binding protein, such that it retained antiviral activity but lost mitogenicity by mutating a single amino acid, yielding H84T BanLec (H84T). H84T shows activity against viruses containing high-mannose N-glycans, including influenza A and B, HIV-1 and -2, and hepatitis C virus. Since ebolavirus surface glycoproteins also contain many high-mannose N-glycans, we assessed whether H84T could inhibit ebolavirus replication. H84T inhibited Ebola virus (EBOV) replication in cell cultures. In cells, H84T inhibited both virus-like particle (VLP) entry and transcription/replication of the EBOV mini-genome at high micromolar concentrations, while inhibiting infection by transcription- and replication-competent VLPs, which measures the full viral life cycle, in the low micromolar range. H84T did not inhibit assembly, budding, or release of VLPs. These findings suggest that H84T may exert its anti-ebolavirus effect(s) by blocking both entry and transcription/replication. In a mouse model, H84T partially (maximally, ~50-80%) protected mice from an otherwise lethal mouse-adapted EBOV infection. Interestingly, a single dose of H84T pre-exposure to EBOV protected ~80% of mice. Thus, H84T shows promise as a new anti-ebolavirus agent with potential to be used in combination with vaccination or other agents in a prophylactic or therapeutic regimen.


Assuntos
Antivirais/farmacologia , Ebolavirus/efeitos dos fármacos , Musa/química , Lectinas de Plantas/farmacologia , Animais , Antivirais/síntese química , Linhagem Celular Tumoral , Ebolavirus/genética , Ebolavirus/imunologia , Escherichia coli , Feminino , Engenharia Genética , Células HEK293 , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Lectinas de Plantas/síntese química , Replicação Viral/efeitos dos fármacos
10.
PLoS One ; 14(6): e0218717, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31233538

RESUMO

The diversity of antibody variable regions makes cDNA sequencing challenging, and conventional monoclonal antibody cDNA amplification requires the use of degenerate primers. Here, we describe a simplified workflow for amplification of IgG antibody variable regions from hybridoma RNA by a specialized RT-PCR followed by Sanger sequencing. We perform three separate reactions for each hybridoma: one each for kappa, lambda, and heavy chain transcripts. We prime reverse transcription with a primer specific to the respective constant region and use a template-switch oligonucleotide, which creates a custom sequence at the 5' end of the antibody cDNA. This template-switching circumvents the issue of low sequence homology and the need for degenerate primers. Instead, subsequent PCR amplification of the antibody cDNA molecules requires only two primers: one primer specific for the template-switch oligonucleotide sequence and a nested primer to the respective constant region. We successfully sequenced the variable regions of five mouse monoclonal IgG antibodies using this method, which enabled us to design chimeric mouse/human antibody expression plasmids for recombinant antibody production in mammalian cell culture expression systems. All five recombinant antibodies bind their respective antigens with high affinity, confirming that the amino acid sequences determined by our method are correct and demonstrating the high success rate of our method. Furthermore, we also designed RT-PCR primers and amplified the variable regions from RNA of cells transfected with chimeric mouse/human antibody expression plasmids, showing that our approach is also applicable to IgG antibodies of human origin. Our monoclonal antibody sequencing method is highly accurate, user-friendly, and very cost-effective.


Assuntos
Anticorpos Monoclonais/genética , Região Variável de Imunoglobulina/genética , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Reações Antígeno-Anticorpo , Primers do DNA/genética , DNA Complementar/genética , Células HEK293 , Humanos , Hibridomas/imunologia , Imunoglobulina G/genética , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fluxo de Trabalho
11.
Nat Commun ; 10(1): 2771, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235807

RESUMO

Diverse antibody repertoires are generated through remote genomic interactions involving immunoglobulin variable (VH), diversity (DH) and joining (JH) gene segments. How such interactions are orchestrated remains unknown. Here we develop a strategy to track VH-DHJH motion in B-lymphocytes. We find that VH and DHJH segments are trapped in configurations that allow only local motion, such that spatially proximal segments remain in proximity, while spatially remote segments remain remote. Within a subset of cells, however, abrupt changes in VH-DHJH motion are observed, plausibly caused by temporal alterations in chromatin configurations. Comparison of experimental and simulated data suggests that constrained motion is imposed by a network of cross-linked chromatin chains characteristic of a gel phase, yet poised near the sol phase, a solution of independent chromatin chains. These results suggest that chromosome organization near the sol-gel phase transition dictates the timing of genomic interactions to orchestrate gene expression and somatic recombination.


Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Regulação da Expressão Gênica/fisiologia , Genes de Imunoglobulinas/genética , Recombinação V(D)J/fisiologia , Animais , Linfócitos B/metabolismo , Linhagem Celular , Cromossomos/genética , Proteínas de Ligação a DNA/deficiência , Genômica , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Camundongos , Transição de Fase
12.
Nucleic Acids Res ; 47(14): 7418-7429, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31127309

RESUMO

Affinity maturation of the humoral immune response depends on somatic hypermutation (SHM) of immunoglobulin (Ig) genes, which is initiated by targeted lesion introduction by activation-induced deaminase (AID), followed by error-prone DNA repair. Stringent regulation of this process is essential to prevent genetic instability, but no negative feedback control has been identified to date. Here we show that poly(ADP-ribose) polymerase-1 (PARP-1) is a key factor restricting AID activity during somatic hypermutation. Poly(ADP-ribose) (PAR) chains formed at DNA breaks trigger AID-PAR association, thus preventing excessive DNA damage induction at sites of AID action. Accordingly, AID activity and somatic hypermutation at the Ig variable region is decreased by PARP-1 activity. In addition, PARP-1 regulates DNA lesion processing by affecting strand biased A:T mutagenesis. Our study establishes a novel function of the ancestral genome maintenance factor PARP-1 as a critical local feedback regulator of both AID activity and DNA repair during Ig gene diversification.


Assuntos
Citidina Desaminase/genética , Genes de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Poli(ADP-Ribose) Polimerase-1/genética , Hipermutação Somática de Imunoglobulina/genética , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Linhagem Celular Tumoral , Células Cultivadas , Citidina Desaminase/metabolismo , Dano ao DNA , Reparo do DNA , Humanos , Camundongos , Mutação , Poli(ADP-Ribose) Polimerase-1/metabolismo
13.
Medicine (Baltimore) ; 98(22): e15811, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31145313

RESUMO

Immunoglobulin heavy chain variable region (IGHV) gene mutation status is a biomarker for the prognosis of chronic lymphocytic leukemia, whether it is associated with the diagnosis, staging, and prognosis of patients with mantle cell lymphoma (MCL) remains to be determined.The IGHV gene mutations of 52 MCL patients were determined by DNA sequencing and compared with published IGHV germline sequences.DNA sequence alignment of IGHV variable regions with published IGHV germline sequences showed that the coincidence rate was 94% to 100%. Ten cases (21%) were significantly mutated with the rate of 96.9% to 94.0%. The overall survival time of patients was negatively correlated with the degree of IGHV gene mutation. Further survival analysis with log-rank test demonstrated that the patients with significant IGHV gene mutations showed a trend towards poor survival.The mutation rate of the IGHV variant region may be determined to assess the prognosis and overall survival time of MCL patients.


Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/mortalidade , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Feminino , Humanos , Linfoma de Célula do Manto/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Análise de Sobrevida
14.
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
15.
MAbs ; 11(4): 735-746, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900945

RESUMO

Traditional hybridoma and B cell cloning antibody discovery platforms have inherent limits in immune repertoire sampling depth. One consequence is that monoclonal antibody (mAb) leads often lack the necessary affinity for therapeutic applications, thus requiring labor-intensive and time-consuming affinity in vitro engineering optimization steps. Here, we show that high-affinity variants of mouse-derived mAbs can be rapidly obtained by testing of somatic sequence variants obtained by deep sequencing of antibody variable regions in immune repertories from immunized mice, even with a relatively sparse sampling of sequence variants from large sequence datasets. Affinity improvements can be achieved for mAbs with a wide range of affinities. The optimized antibody variants derived from immune repertoire mining have no detectable in vitro off-target binding and have in vivo clearance comparable to the parental mAbs, essential properties in therapeutic antibody leads. As generation of antibody variants in vitro is replaced by mining of variants generated in vivo, the procedure can be applied to rapidly identify affinity-optimized mAb variants.


Assuntos
Anticorpos Monoclonais/metabolismo , Linfócitos B/imunologia , Região Variável de Imunoglobulina/genética , Doença de Parkinson/terapia , alfa-Sinucleína/imunologia , Animais , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Células Clonais , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridomas , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Doença de Parkinson/imunologia , Hipermutação Somática de Imunoglobulina
16.
J Immunol ; 202(8): 2220-2228, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30850477

RESUMO

Abs can acquire N-linked glycans in their V regions during Ag-specific B cell responses. Among others, these N-linked glycans can affect Ag binding and Ab stability. Elevated N-linked glycosylation has furthermore been associated with several B cell-associated pathologies. Basic knowledge about patterns of V region glycosylation at different stages of B cell development is scarce. The aim of the current study is to establish patterns of N-glycosylation sites in Ab V regions of naive and memory B cell subsets. We analyzed the distribution and acquisition of N-glycosylation sites within Ab V regions of peripheral blood and bone marrow B cells of 12 healthy individuals, eight myasthenia gravis patients, and six systemic lupus erythematosus patients, obtained by next-generation sequencing. N-glycosylation sites are clustered around CDRs and the DE loop for both H and L chains, with similar frequencies for healthy donors and patients. No evidence was found for an overall selection bias against acquiring an N-glycosylation site, except for the CDR3 of the H chain. Interestingly, both IgE and IgG4 subsets have a 2-fold higher propensity to acquire Fab glycans compared with IgG1 or IgA. When expressed as rmAb, 35 out of 38 (92%) nongermline N-glycosylation sites became occupied. These results point toward a differential selection pressure of N-glycosylation site acquisition during affinity maturation of B cells, which depends on the location within the V region and is isotype and subclass dependent. Elevated Fab glycosylation represents an additional hallmark of TH2-like IgG4/IgE responses.


Assuntos
Linfócitos B/imunologia , Imunoglobulina G , Região Variável de Imunoglobulina , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária/genética , Linfócitos B/patologia , Glicosilação , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Lúpus Eritematoso Sistêmico/patologia
17.
Nat Commun ; 10(1): 628, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30733445

RESUMO

Analysis of antibody repertoires by high-throughput sequencing is of major importance in understanding adaptive immune responses. Our knowledge of variations in the genomic loci encoding immunoglobulin genes is incomplete, resulting in conflicting VDJ gene assignments and biased genotype and haplotype inference. Haplotypes can be inferred using IGHJ6 heterozygosity, observed in one third of the people. Here, we propose a robust novel method for determining VDJ haplotypes by adapting a Bayesian framework. Our method extends haplotype inference to IGHD- and IGHV-based analysis, enabling inference of deletions and copy number variations in the entire population. To test this method, we generated a multi-individual data set of naive B-cell repertoires, and found allele usage bias, as well as a mosaic, tiled pattern of deleted IGHD and IGHV genes. The inferred haplotypes may have clinical implications for genetic disease predispositions. Our findings expand the knowledge that can be extracted from antibody repertoire sequencing data.


Assuntos
Teorema de Bayes , Variações do Número de Cópias de DNA/genética , Haplótipos/genética , Alelos , Genótipo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética
18.
Methods Mol Biol ; 1956: 61-75, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30779030

RESUMO

Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the isolated cells, rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of V gene subgroup-specific primers recognizing nearly all V genes together with primers for the J genes. By sequence analysis of V region genes from distinct cells, the clonal relationship of the B lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D, and J gene usage. Moreover, the presence and pattern of somatic Ig V gene mutations give valuable insight into the stage of differentiation of the B cells.


Assuntos
Rearranjo Gênico de Cadeia Pesada de Linfócito B , Doença de Hodgkin/genética , Região Variável de Imunoglobulina/genética , Microdissecção e Captura a Laser/métodos , Linfoma de Células B/genética , Reação em Cadeia da Polimerase/métodos , Análise de Célula Única/métodos , Linfócitos B/metabolismo , Linfócitos B/patologia , Genes de Imunoglobulinas , Doença de Hodgkin/patologia , Humanos , Linfoma de Células B/patologia
19.
Methods Mol Biol ; 1956: 105-125, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30779032

RESUMO

The majority of lymphomas originate from B cells at the germinal center stage. Preferential selection of B-cell clones by a limited set of antigens has been suggested to drive lymphoma development. While recent studies in chronic lymphocytic leukemia have shown that self-reactive B-cell receptors (BCR) can generate cell-autonomous signaling and proliferation, our knowledge about the role of BCRs for the development or survival of other lymphomas remains limited. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows the unbiased characterization of the human antibody repertoire at single-cell level through the generation of recombinant monoclonal antibodies from single primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow-cytometric isolation of single human B cells to the reverse transcription-polymerase chain reaction (RT-PCR)-based amplification of the expressed immunoglobulin (Ig) transcripts (IGH, IGK, and IGL) and their subsequent cloning into expression vectors for the in vitro production of recombinant monoclonal antibodies. The strategy may be used to obtain information on the clonal evolution of B-cell lymphomas by single-cell sequencing of Ig transcripts and on the antibody reactivity of human lymphoma B cells.


Assuntos
Anticorpos Monoclonais/genética , Linfócitos B/metabolismo , Clonagem Molecular/métodos , Citometria de Fluxo/métodos , Imunoglobulinas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Separação Celular/métodos , Vetores Genéticos/genética , Células HEK293 , Humanos , Cadeias J de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Proteínas Recombinantes/genética , Análise de Célula Única/métodos
20.
Methods Mol Biol ; 1956: 139-155, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30779034

RESUMO

Comprehensive analysis of the clonotypic B cell receptor immunoglobulin (BcR IG) gene rearrangement sequences in patients with mature B cell neoplasms has led to the identification of significant repertoire restrictions, culminating in the discovery of subsets of patients expressing highly similar, stereotyped BcR IG. This finding strongly supports selection by common epitopes or classes of structurally similar epitopes in the ontogeny of these tumors. BcR IG stereotypy was initially described in chronic lymphocytic leukemia (CLL), where the stereotyped fraction of the disease accounts for a remarkable one-third of patients. However, subsequent studies showed that stereotyped BcR IG are also present in other neoplasms of mature B cells, including mantle cell lymphoma (MCL) and splenic marginal zone lymphoma (SMZL). Subsequent cross-entity comparisons led to the conclusion that stereotyped IG are mostly "disease-specific," implicating distinct immunopathogenetic processes. Interestingly, mounting evidence suggests that a molecular subclassification of lymphomas based on BcR IG stereotypy is biologically and clinically relevant. Indeed, particularly in CLL, patients assigned to the same subset due to expressing a particular stereotyped BcR IG display remarkably consistent biological background and clinical course, at least for major and well-studied subsets. Thus, the robust assignment to stereotyped subsets may assist in the identification of mechanisms underlying disease onset and progression, while also refining risk stratification. In this book chapter, we provide an overview of the recent BcR IG stereotypy studies in mature B cell malignancies and outline previous and current methodological approaches used for the identification of stereotyped IG.


Assuntos
Genes de Imunoglobulinas , Linfoma de Células B/genética , Receptores de Antígenos de Linfócitos B/genética , Sequência de Aminoácidos , Animais , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Humanos , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Linfoma de Células B/patologia , Receptores de Antígenos de Linfócitos B/química
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