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Immunoglobulin (Ig) genes carry the unique ability to be reshaped in peripheral B lymphocytes after these cells encounter a specific antigen. B cells can then further improve their affinity, acquire new functions as memory cells and eventually end up as antibody-secreting cells. Ig class switching is an important change that occurs in this context, thanks to local DNA lesions initiated by the enzyme activation-induced deaminase (AID). Several cis-acting elements of the Ig heavy (IgH) chain locus make it accessible to the AID-mediated lesions that promote class switch recombination (CSR). DNA repeats, with a non-template strand rich in G-quadruplexes (G4)-DNA, are prominent cis-targets of AID and define the so-called "switch" (S) regions specifically targeted for CSR. By analyzing the structure of the human IgH locus, we uncover that abundant DNA repeats, some with a putative G4-rich template strand, are additionally present in downstream portions of the IgH coding genes. These like-S (LS) regions stand as 3' mirror-images of S regions and also show analogies to some previously reported repeats associated with the IgH locus 3' super-enhancer. A regulatory role of LS repeats is strongly suggested by their specific localization close to exons encoding the membrane form of Ig molecules, and by their conservation during mammalian evolution.
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Cadeias Pesadas de Imunoglobulinas , Ácidos Nucleicos , Humanos , Linfócitos B/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA/genética , Switching de Imunoglobulina/genética , Sequências Reguladoras de Ácido Nucleico , Cadeias Pesadas de Imunoglobulinas/genéticaRESUMO
Assessment of minimal residual disease in acute lymphoblastic leukemia by immune repertoire NGS requires spiking CDR3 sequences at known quantities into the patient's sample. Recently, the EuroClonality-NGS group released one of the most comprehensive protocols for this purpose. ARResT/Interrogate is a closed-source software for processing these NGS libraries, developed by this same group. Vidjil, an open-source alternative, currently cannot handle libraries prepared using this protocol. Here, we present a Vidjil add-on to solve this issue. EuroClonality-NGS prepared samples analyzed with Vidjil and ARResT/Interrogate were highly concordant (r = 0.998) and presented low error (root-mean-square error, RMSE = 0.112).
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Introduction: Normal CD30+ B cells represent a distinct B-cell differentiation stage with features of strong activation. We lack an in depth understanding of these cells, because they are not present in peripheral blood and are typically very rare in reactive lymphoid organs. CD30+ B cells have been discussed as a potential precursor population for the malignant CD30+ Hodgkin and Reed-Sternberg cells in classical Hodgkin lymphoma. As CD30+ B cells can be more numerous in some cases of reactive lymphadenitis, we aimed to characterize these CD30+ B cells in terms of their differentiation stage and clonal composition, also as a means to clarify whether such CD30+ B-cell populations may represent potential precursor lesions of Hodgkin lymphoma. Methods: We microdissected single CD30+ B cells from tissue sections of eight reactive lymph nodes with substantial numbers of such cells and sequenced their rearranged immunoglobulin (Ig) heavy chain V region (IGHV) genes. Results: The CD30+ B cells were polyclonal B cells in all instances, and they not only encompass post-germinal center (GC) B cells with mutated IGHV genes, but also include a substantial fraction of pre-germinal center B cells with unmutated IGHV genes. In five of the lymph nodes, mostly small clonal expansions were detected among the CD30+ B cells. Most of the expanded clones carried somatically mutated IGHV genes and about half of the mutated clones showed intraclonal diversity. Discussion: We conclude that in human reactive lymph nodes with relatively many CD30+ B cells, these cells are a heterogenous population of polyclonal B cells encompassing activated pre-GC B cells as well as GC and post-GC B cells, with some clonal expansions. Because of their polyclonality and frequent pre-GC differentiation stage, there is no indication that such cell-rich CD30+ B-cell populations represent precursor lesions of Hodgkin lymphoma.
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Doença de Hodgkin , Humanos , Doença de Hodgkin/genética , Genes de Imunoglobulinas , Linfonodos/patologia , Cadeias Pesadas de Imunoglobulinas/genética , Diferenciação Celular , Células ClonaisRESUMO
The tick-borne pathogen Neoehrlichia (N.) mikurensis is implicated in persistent infection of the vascular endothelium. B cells are crucial for the host defence to this infection. Chronic stimulation of B cells may result in B-cell transformation and lymphoma. Five patients with malignant B-cell lymphoma and concomitant N. mikurensis infection were investigated regarding clinical picture, lymphoma subtype, B-cell lymphoma immunophenotype and IGHV (variable region of the immunoglobulin heavy) gene repertoire. Three of the five patients improved markedly and ceased lymphoma treatment after doxycycline treatment to eliminate N. mikurensis. Sequencing the B-cell lymphoma IGHV genes revealed preferred usage of the IGHV1 (IGHV1-2, and -69) and IGHV3 (IGHV3-15, -21, -23) families. In conclusion, N. mikurensis infection may drive the development of malignant B-cell lymphomas. Eradication of the pathogen appears to induce remission with apparent curing of the lymphoma in some cases.
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Infecções por Anaplasmataceae , Linfoma de Células B , Doenças Transmitidas por Carrapatos , Infecções por Anaplasmataceae/complicações , Infecções por Anaplasmataceae/tratamento farmacológico , Infecções por Anaplasmataceae/microbiologia , Linfoma de Células B/etiologia , Linfoma de Células B/genética , Doenças Transmitidas por Carrapatos/microbiologia , Receptores de Antígenos de Linfócitos B , Doxiciclina/uso terapêutico , Antibacterianos/uso terapêutico , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Análise de Sequência de DNA , ImunofenotipagemRESUMO
B cell receptor (BcR) immunoglobulins (IG) display a tremendous diversity due to complex DNA rearrangements, the V(D)J recombination, further enhanced by the somatic hypermutation process. In chronic lymphocytic leukemia (CLL), the mutational load of the clonal BcR IG expressed by the leukemic cells constitutes an important prognostic and predictive biomarker. Here, we provide a reliable methodology capable of determining the mutational status of IG genes in CLL using high-throughput sequencing, starting from leukemic cell DNA or RNA.
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Leucemia Linfocítica Crônica de Células B , Genes de Imunoglobulinas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulinas/genética , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Antígenos de Linfócitos B/genéticaRESUMO
Nanobubble technology has shown appealing technical benefits and potential applications in aquaculture. We recently found that treatment with ozone nanobubbles (NB-O3) activated expression of several immune-related genes leading to effective response to subsequent exposure to fish pathogens. In this study, we investigated whether pre-treatment of Nile tilapia (Oreochromis niloticus) with NB-O3 can enhance specific immune responses and improve efficacy of immersion vaccination against Streptococcus agalactiae. Spleen and head kidney of fish in the vaccinated groups showed a substantial upregulation in expression levels of pro-inflammatory cytokine genes (IL-1ß, TNF-α, IL-6) and immunoglobulin classes (IgM, IgD, IgT) compared with the unvaccinated control groups. The mRNA transcript of pro-inflammatory cytokine genes was greatest (approx. 2.8-3.3 folds) on day 7 post-vaccination, whereas the relative expression of immunoglobulin genes was greatest (approx. 3.2-4.1 folds) on day 21 post-immunization. Both systemic and mucosal IgM antibodies were elicited in vaccinated groups. As the result, the cumulative survival rate of the vaccinated groups was found to be higher than that of the unvaccinated groups, with a relative percent survival (RPS) ranging from 52.9 to 70.5%. However, fish in the vaccinated groups that received pre-treatment with NB-O3, bacterial antigen uptakes, expression levels of IL-1ß, TNF-α, IL-6,IgM, IgD, and IgT, as well as the specific-IgM antibody levels and percent survival, were all slightly or significantly higher than that of the vaccinated group without pre-treatment with NB-O3. Taken together, our findings suggest that utilizing pre-treatment with NB-O3 may improve the immune response and efficacy of immersion vaccination in Nile tilapia.
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Ciclídeos , Doenças dos Peixes , Ozônio , Infecções Estreptocócicas , Animais , Temperatura Alta , Imersão , Imunoglobulina D , Imunoglobulina M , Interleucina-6 , Infecções Estreptocócicas/prevenção & controle , Infecções Estreptocócicas/veterinária , Streptococcus agalactiae , Fator de Necrose Tumoral alfa , Vacinas de Produtos InativadosRESUMO
Classification of patients with chronic lymphocytic leukemia (CLL) based on the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene has established predictive and prognostic relevance. The SHM status is assessed based on the number of mutations within the IG heavy variable domain sequence, albeit only over the rearranged IGHV gene excluding the variable heavy complementarity determining region 3 (VH CDR3). This may lead to an underestimation of the actual impact of SHM, in fact overlooking the most critical region for antigen-antibody interactions, i.e. the VH CDR3. Here we investigated whether SHM may be present within the VH CDR3 of cases bearing 'truly unmutated' IGHV genes (i.e. 100% germline identity across VH FR1-VH FR3) employing Next Generation Sequencing. We studied 16 patients bearing a 'truly unmutated' CLL clone assigned to stereotyped subsets #1 (n=12) and #6 (n=4). We report the existence of SHM within the germline-encoded 3'IGHV, IGHD, 5'IGHJ regions of the VH CDR3 in both the main IGHV-IGHD-IGHJ gene clonotype and its variants. Recurrent somatic mutations were identified between different patients of the same subset, supporting the notion that they represent true mutational events rather than technical artefacts; moreover, they were located adjacent to/within AID hotspots, pointing to SHM as the underlying mechanism. In conclusion, we provide immunogenetic evidence for intra-VH CDR3 variations, attributed to SHM, in CLL patients carrying 'truly unmutated' IGHV genes. Although the clinical implications of this observation remain to be defined, our findings offer a new perspective into the immunobiology of CLL, alluding to the operation of VH CDR3-restricted SHM in U-CLL.
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The repertoire of antibodies (Abs) produced upon vaccination against a particular antigenic site is rarely studied due to the complexity of the immunogens. We received such an opportunity when one rhesus macaque was immunized six times at 0, 4, 10, 16, 32, and 143 weeks with C4-447 peptide containing the 8-mer epitope for human monoclonal Ab (mAb) 447-52D specific to the V3 region of gp120 HIV-1. Strong anti-V3 antibody responses reached 50% binding titer in serum of 10-5 at week 10 that declined to 10-3 by week 70. After an additional boost of C4-447 peptide at week 143, titers rebounded to 10-5 at week 146, or 2.7 years after the first immunization. Using the blood sample at week 146, we produced 41 V3-specific recombinant mAbs by single B cell isolation and cloning. Sequence analysis revealed 21B cell lineages, single and clonally related, based on immunoglobulin gene usage and CDR3s. The broad repertoire of Abs directed to a small antigenic site shows the targeting potency of a vaccine-elicited immune response in rhesus macaques.
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Vacinas contra a AIDS , Infecções por HIV , HIV-1 , Animais , Anticorpos Neutralizantes , Linhagem da Célula , Epitopos , Anticorpos Anti-HIV , Proteína gp120 do Envelope de HIV , Humanos , Macaca mulattaRESUMO
Cancer genomes harbor numerous genomic alterations and many cancers accumulate thousands of nucleotide sequence variations. A prominent fraction of these mutations arises as a consequence of the off-target activity of DNA/RNA editing cytosine deaminases followed by the replication/repair of edited sites by DNA polymerases (pol), as deduced from the analysis of the DNA sequence context of mutations in different tumor tissues. We have used the weight matrix (sequence profile) approach to analyze mutagenesis due to Activation Induced Deaminase (AID) and two error-prone DNA polymerases. Control experiments using shuffled weight matrices and somatic mutations in immunoglobulin genes confirmed the power of this method. Analysis of somatic mutations in various cancers suggested that AID and DNA polymerases η and θ contribute to mutagenesis in contexts that almost universally correlate with the context of mutations in A:T and G:C sites during the affinity maturation of immunoglobulin genes. Previously, we demonstrated that AID contributes to mutagenesis in (de)methylated genomic DNA in various cancers. Our current analysis of methylation data from malignant lymphomas suggests that driver genes are subject to different (de)methylation processes than non-driver genes and, in addition to AID, the activity of pols η and θ contributes to the establishment of methylation-dependent mutation profiles. This may reflect the functional importance of interplay between mutagenesis in cancer and (de)methylation processes in different groups of genes. The resulting changes in CpG methylation levels and chromatin modifications are likely to cause changes in the expression levels of driver genes that may affect cancer initiation and/or progression.
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Analyses of IGHV gene mutations in chronic lymphocytic leukemia (CLL) have had a major impact on the prognostication and treatment of this disease. A hallmark of IGHV-mutation status is that it very rarely changes clonally over time. Nevertheless, targeted and deep DNA sequencing of IGHV-IGHD-IGHJ regions has revealed intraclonal heterogeneity. We used a DNA sequencing approach that achieves considerable depth and minimizes artefacts and amplification bias to identify IGHV-IGHD-IGHJ subclones in patients with prolonged temporal follow-up. Our findings extend previous studies, revealing intraclonal IGHV-IGHD-IGHJ diversification in almost all CLL clones. Also, they indicate that some subclones with additional IGHV-IGHD-IGHJ mutations can become a large fraction of the leukemic burden, reaching numerical criteria for monoclonal B-cell lymphocytosis. Notably, the occurrence and complexity of post-transformation IGHV-IGHD-IGHJ heterogeneity and the expansion of diversified subclones are similar among U-CLL and M-CLL patients. The molecular characteristics of the mutations present in the parental, clinically dominant CLL clone (CDC) differed from those developing post-transformation (post-CDC). Post-CDC mutations exhibit significantly lower fractions of mutations bearing signatures of activation induced deaminase (AID) and of error-prone repair by Polη, and most of the mutations were not ascribable to those enzymes. Additionally, post-CDC mutations displayed a lower percentage of nucleotide transitions compared with transversions that was also not like the action of AID. Finally, the post-CDC mutations led to significantly lower ratios of replacement to silent mutations in VH CDRs and higher ratios in VH FRs, distributions different from mutations found in normal B-cell subsets undergoing an AID-mediated process. Based on these findings, we propose that post-transformation mutations in CLL cells either reflect a dysfunctional standard somatic mutational process or point to the action of another mutational process not previously associated with IG V gene loci. If the former option is the case, post-CDC mutations could lead to a lesser dependence on antigen dependent BCR signaling and potentially a greater influence of off-target, non-IG genomic mutations. Alternatively, the latter activity could add a new stimulatory survival/growth advantage mediated by the BCR through structurally altered FRs, such as that occurring by superantigen binding and stimulation.
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Minimal residual disease (MRD) determined by classic polymerase chain reaction (PCR) methods is a powerful outcome predictor in mantle cell lymphoma (MCL). Nevertheless, some technical pitfalls can reduce the rate of of molecular markers. Therefore, we applied the EuroClonality-NGS IGH (next-generation sequencing immunoglobulin heavy chain) method (previously published in acute lymphoblastic leukaemia) to 20 MCL patients enrolled in an Italian phase III trial sponsored by Fondazione Italiana Linfomi. Results from this preliminary investigation show that EuroClonality-NGS IGH method is feasible in the MCL context, detecting a molecular IGH target in 19/20 investigated cases, allowing MRD monitoring also in those patients lacking a molecular marker for classical screening approaches.
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Rearranjo Gênico , Sequenciamento de Nucleotídeos em Larga Escala , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma de Célula do Manto/genética , Biomarcadores Tumorais/genética , Genes de Imunoglobulinas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Itália/epidemiologia , Linfoma de Célula do Manto/diagnóstico , Linfoma de Célula do Manto/epidemiologia , Neoplasia Residual/diagnóstico , Neoplasia Residual/epidemiologia , Neoplasia Residual/genéticaRESUMO
BIOMED-2 Concerted Action BMH4-CT98-3936 (BIOMED-2) PCR protocols are an important diagnostic tool in the evaluation of cutaneous lymphomas. The aim of this study was to assess the diagnostic value of the genotyping results obtained by these techniques in daily clinical practice. A total of 360 paraffin-embedded skin samples were retrospectively reviewed from 114 cutaneous T-cell lymphomas and 35 cutaneous B-cell lym-phomas. A total of 249 biopsies from 180 patients with benign lymphoid infiltrates served as controls. T-cell receptor and immunoglobulin gene rearrangements were assessed using the BIOMED-2 method. A combined T-cell receptor gamma and beta assay approach reliably distinguished cutaneous T-cell lymphomas from benign skin T-cell infiltrates (sensitivity 89.4%; specificity 81.5%). Analysis of complete immunoglobulin heavy chain rearrangements also differentiated cutaneous B-cell lymphomas from benign B-cell infiltrates (sensitivity 85.7%; specificity 82.4%). In conclusion, the full BIOMED-2 protocol is a useful aid combined with clinical, histological and immunophenotypical findings for assessment of lymphoid clonality in skin lymphoid proliferations.
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Linfoma de Células B , Linfoma Cutâneo de Células T , Neoplasias Cutâneas , Humanos , Linfoma de Células B/diagnóstico , Linfoma de Células B/genética , Linfoma Cutâneo de Células T/diagnóstico , Linfoma Cutâneo de Células T/genética , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/genéticaRESUMO
The vertebrate immune system provides a powerful defense because of the ability to potentially recognize an unlimited number of pathogens. The antibody molecule, also termed immunoglobulin (Ig) is one of the major mediators of the immune response. It is built up from two types of Ig domains: the variable domain, which provides the capability to recognize and bind a potentially infinite range of foreign substances, and the constant domains, which exert the effector functions. In the last 20 years, advances in our understanding of the molecular mechanisms and structural features of antibody in mammals and in a variety of other organisms have uncovered the underlying principles and complexity of this fundamental molecule. One notable evolutionary topic is the origin and evolution of antibody. Many aspects have been clearly stated, but some others remain limited or obscure. By considering a wide range of prokaryotic and eukaryotic organisms through a literature survey about the topic, we have provided an integrated view of the emergence of antibodies in evolution and underlined the very ancient origins.
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Advanced genome-wide association studies (GWAS) identified several transforming mutations in susceptible loci which are recognized as valuable prognostic markers in chronic lymphocytic leukemia (CLL) and B cell lymphoma (BCL). Alongside, robust genetic manipulations facilitated the generation of preclinical mouse models to validate mutations associated with poor prognosis and refractory B cell malignancies. Taken together, these studies identified new prognostic markers that could achieve characteristics of precision biomarkers for molecular diagnosis. On the contrary, the idea of augmented B cell antigen receptor (BCR) signaling as a transforming cue has somewhat receded despite the efficacy of Btk and Syk inhibitors. Recent studies from several research groups pointed out that acquired mutations in BCR components serve as faithful biomarkers, which become important for precision diagnostics and therapy, due to their relevant role in augmented BCR signaling and CLL pathogenesis. For example, we showed that expression of a single point mutated immunoglobulin light chain (LC) recombined through the variable gene segment IGLV3-21, named IGLV3-21R110, marks severe CLL cases. In this perspective, we summarize the molecular mechanisms fine-tuning B cell transformation, focusing on immunoglobulin point mutations and recurrent mutations in tumor suppressors. We present a stochastic model for gain-of-autonomous BCR signaling and subsequent neoplastic transformation. Of note, additional mutational analyses on immunoglobulin heavy chain (HC) derived from non-subset #2 CLL IGLV3-21R110 cases endorses our perspective. Altogether, we propose a model of malignant transformation in which the augmented BCR signaling creates a conducive platform for the appearance of transforming mutations.
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Sequencing antibody repertoires has steadily become cheaper and easier. Sequencing methods usually rely on some form of amplification, often a massively multiplexed PCR prior to sequencing. To eliminate potential biases and create a data set that could be used for other studies, our lab compared unamplified sequencing results from the splenic heavy-chain repertoire in the mouse to those processed through two commercial applications. We also compared the use of mRNA vs total RNA, reverse transcriptase, and primer usage for cDNA synthesis and submission. The use of mRNA for cDNA synthesis resulted in higher read counts but reverse transcriptase and primer usage had no statistical effects on read count. Although most of the amplified data sets contained more antibody reads than the unamplified data set, we detected more unique V-gene segments in the unamplified data set. Although unique CDR3 detection was much lower in the unamplified data set, RNASeq detected 98% of the high frequency CDR3s. We have shown that unamplified profiling of the antibody repertoire is possible, detects more V-gene segments, and detects high frequency clones in the repertoire.
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Immunoglobulin (IG) gene remodeling by V(D)J recombination plays a central role in the generation of normal B cells, and somatic hypermutation and class switching of IG genes are key processes during antigen-driven B cell differentiation. However, errors of these processes are involved in the development of B cell lymphomas. IG locus-associated translocations of proto-oncogenes are a hallmark of many B cell malignancies. Additional transforming events include inactivating mutations in various tumor suppressor genes and also latent infection of B cells with viruses, such as Epstein-Barr virus. Many B cell lymphomas require B cell antigen receptor expression, and in several instances, chronic antigenic stimulation plays a role in lymphoma development and/or sustaining tumor growth. Often, survival and proliferation signals provided by other cells in the microenvironment are a further critical factor in lymphoma development and pathophysiology. Many B cell malignancies derive from germinal center B cells, most likely because of the high proliferation rate of these cells and the high activity of mutagenic processes.
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Linfócitos B/patologia , Linfoma de Células B/patologia , Animais , Linfócitos B/imunologia , Linfócitos B/virologia , Epigênese Genética , Infecções por Herpesviridae/complicações , Herpesvirus Humano 4/isolamento & purificação , Herpesvirus Humano 8/isolamento & purificação , Humanos , Imunidade , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Linfoma de Células B/virologia , Mutação , Translocação Genética , Microambiente TumoralRESUMO
High-throughput mRNA sequencing (RNA-Seq) provides both qualitative and quantitative evaluation of the transcriptome. This method uses complementary DNA (cDNA) to generate several millions of short sequence reads that are aligned to a reference genome allowing the comprehensive characterization of the transcripts in a cell. RNA-Seq has a wide variety of applications which lead to a pervasive adoption of this method well beyond the genomics community and a deployment of this technique as a standard part of the toolkit applied in life sciences. This chapter describes a protocol to perform mRNA sequencing using the Illumina NextSeq or MiSeq platforms, presents sequencing data quality metrics, and outlines a bioinformatic pipeline for sequence alignment, digital gene expression, identification of gene fusions, detection of transcript isoforms, description and annotation of genetic variants, and de novo immunoglobulin gene assembly.
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Genômica/métodos , Linfoma de Células B/genética , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Processamento Alternativo , Perfilação da Expressão Gênica/métodos , Fusão Gênica , Genes de Imunoglobulinas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação , Polimorfismo de Nucleotídeo Único , Software , TranscriptomaRESUMO
Influenza is a leading cause of death in the elderly, and the vaccine protects only a fraction of this population. A key aspect of antibody-mediated anti-influenza virus immunity is adaptation to antigenically distinct epitopes on emerging strains. We examined factors contributing to reduced influenza vaccine efficacy in the elderly and uncovered a dramatic reduction in the accumulation of de novo immunoglobulin gene somatic mutations upon vaccination. This reduction is associated with a significant decrease in the capacity of antibodies to target the viral glycoprotein, hemagglutinin (HA), and critical protective epitopes surrounding the HA receptor-binding domain. Immune escape by antigenic drift, in which viruses generate mutations in key antigenic epitopes, becomes highly exaggerated. Because of this reduced adaptability, most B cells activated in the elderly cohort target highly conserved but less potent epitopes. Given these findings, vaccines driving immunoglobulin gene somatic hypermutation should be a priority to protect elderly individuals.
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Linfócitos B/imunologia , Epitopos/imunologia , Imunidade Humoral , Vacinas contra Influenza/imunologia , Orthomyxoviridae/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Epitopos/genética , Voluntários Saudáveis , Humanos , Vacinas contra Influenza/administração & dosagem , Pessoa de Meia-Idade , Mutação , Orthomyxoviridae/genética , Adulto JovemRESUMO
Sequencing antibody repertoires has steadily become cheaper and easier. Sequencing methods usually rely on some form of amplification, often a massively multiplexed PCR prior to sequencing. To eliminate potential biases and create a data set that could be used for other studies, our laboratory compared unamplified sequencing results from the splenic heavy-chain repertoire in the mouse to those processed through two commercial applications. We also compared the use of mRNA vs total RNA, reverse transcriptase, and primer usage for cDNA synthesis and submission. The use of mRNA for cDNA synthesis resulted in higher read counts but reverse transcriptase and primer usage had no statistical effects on read count. Although most of the amplified data sets contained more antibody reads than the unamplified data set, we detected more unique variable (V)-gene segments in the unamplified data set. Although unique CDR3 detection was much lower in the unamplified data set, RNASeq detected 98% of the high-frequency CDR3s. We have shown that unamplified profiling of the antibody repertoire is possible, detects more V-gene segments, and detects high-frequency clones in the repertoire.
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Five classes of immunoglobulins are known to exist in mammals. The number of isotypes of classes G, E and A varies among species for unknown reasons. Here, a study of the presence of immunoglobulin genes in Primates was carried out from the genomes and transcriptomes deposited in the NCBI repository. For this, a machine learning application based upon neural networks was implemented that scans the genomes and identifies the exon sequences that encode the immunoglobulin CH domains. From these exons, the immunoglobulins that each species possess can be inferred. Also, the presence of sequences outside the IGHC locus was found which were produced by retrotranscription of RNA that are probably not viable. From this study, the distribution of immunoglobulin genes across primate orders is described in detail. In Prosimians, IgD genes are not found; in Platyrrhines, a gene is identified for each of the immunoglobulin classes but the IgD gene does not have the CH2 exon; in the Cercopithecidae family, a gene is detected for each class in the Colobinae family, while in Cercopithecidae the genes for IgG have been duplicated several times. In hominids, a greater number of duplications that include the genes that code for IgA and IgE are observed. These results indicate that from the appearance of the Cercopithecidae, there is an evolutionary instability in the Ig locus.