RESUMO
RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RCs from upstream VHs in a chromatin loop anchored by CTCF-binding elements (CBEs). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and thereby promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Proteínas de Homeodomínio/metabolismo , Recombinação V(D)J , Animais , Linhagem Celular , DNA Intergênico/genética , DNA Intergênico/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mutagênese , Sinais Direcionadores de Proteínas , RNA Guia de Cinetoplastídeos/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Immunoglobulin heavy chain (IgH) locus-associated G-rich long noncoding RNA (SµGLT) is important for physiological and pathological B cell DNA recombination. We demonstrate that the METTL3 enzyme-catalyzed N6-methyladenosine (m6A) RNA modification drives recognition and 3' end processing of SµGLT by the RNA exosome, promoting class switch recombination (CSR) and suppressing chromosomal translocations. The recognition is driven by interaction of the MPP6 adaptor protein with nuclear m6A reader YTHDC1. MPP6 and YTHDC1 promote CSR by recruiting AID and the RNA exosome to actively transcribe SµGLT. Direct suppression of m6A modification of SµGLT or of m6A reader YTHDC1 reduces CSR. Moreover, METTL3, an essential gene for B cell development in the bone marrow and germinal center, suppresses IgH-associated aberrant DNA breaks and prevents genomic instability. Taken together, we propose coordinated and central roles for MPP6, m6A modification, and m6A reader proteins in controlling long noncoding RNA processing, DNA recombination, and development in B cells.
Assuntos
Adenosina/análogos & derivados , Linfócitos B/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Processamento de Terminações 3' de RNA , RNA Longo não Codificante/metabolismo , Recombinação Genética , Adenosina/metabolismo , Animais , Linfócitos B/imunologia , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Feminino , Instabilidade Genômica , Células HEK293 , Humanos , Switching de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos Knockout , RNA Longo não Codificante/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismoRESUMO
Early B cell development is orchestrated by the combined activities of the transcriptional regulators E2A, EBF1, Foxo1 and Ikaros. However, how the genome-wide binding patterns of these regulators are modulated during B lineage development remains to be determined. Here we found that in lymphoid progenitor cells, the chromatin remodeler Brg1 specified the B cell fate. In committed pro-B cells, Brg1 regulated contraction of the locus encoding the immunoglobulin heavy chain (Igh) and controlled expression of the gene encoding the transcription factor c-Myc (Myc) to modulate the expression of genes encoding products that regulate ribosome biogenesis. In committed pro-B cells, Brg1 suppressed a pre-B lineage-specific pattern of gene expression. Finally, we found that Brg1 acted mechanistically to establish B cell fate and modulate cell growth by facilitating access of lineage-specific transcription factors to enhancer repertoires.
Assuntos
Linfócitos B/imunologia , Proliferação de Células , DNA Helicases/imunologia , Elementos Facilitadores Genéticos/imunologia , Proteínas Nucleares/imunologia , Fatores de Transcrição/imunologia , Animais , Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/imunologia , DNA Helicases/genética , DNA Helicases/metabolismo , Elementos Facilitadores Genéticos/genética , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Hibridização in Situ Fluorescente , Camundongos Knockout , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/metabolismo , Ligação Proteica/imunologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Acute systemic lupus erythematosus (SLE) courses with surges of antibody-secreting cells (ASCs) whose origin, diversity and contribution to serum autoantibodies remain unknown. Here, deep sequencing, proteomic profiling of autoantibodies and single-cell analysis demonstrated highly diversified ASCs punctuated by clones expressing the variable heavy-chain region VH4-34 that produced dominant serum autoantibodies. A fraction of ASC clones contained autoantibodies without mutation, a finding consistent with differentiation outside the germinal centers. A substantial ASC segment was derived from a distinct subset of newly activated naive cells of considerable clonality that persisted in the circulation for several months. Thus, selection of SLE autoreactivities occurred during polyclonal activation, with prolonged recruitment of recently activated naive B cells. Our findings shed light on the pathogenesis of SLE, help explain the benefit of agents that target B cells and should facilitate the design of future therapies.
Assuntos
Diversidade de Anticorpos/imunologia , Células Produtoras de Anticorpos/imunologia , Autoanticorpos/imunologia , Proliferação de Células , Lúpus Eritematoso Sistêmico/imunologia , Doença Aguda , Sequência de Aminoácidos , Diversidade de Anticorpos/genética , Células Produtoras de Anticorpos/metabolismo , Autoanticorpos/genética , Autoanticorpos/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sequência de Bases , Células Clonais/imunologia , Células Clonais/metabolismo , Citometria de Fluxo , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Vacinas contra Influenza/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Dados de Sequência Molecular , Proteoma/análise , Proteoma/imunologia , Proteômica/métodos , Homologia de Sequência de Aminoácidos , Análise de Célula Única/métodos , Espectrometria de Massas em Tandem , Toxoide Tetânico/imunologiaRESUMO
Class switch recombination (CSR) at the immunoglobulin heavy-chain (IgH) locus is associated with the formation of R-loop structures over switch (S) regions. While these often occur co-transcriptionally between nascent RNA and template DNA, we now show that they also form as part of a post-transcriptional mechanism targeting AID to IgH S-regions. This depends on the RNA helicase DDX1 that is also required for CSR in vivo. DDX1 binds to G-quadruplex (G4) structures present in intronic switch transcripts and converts them into S-region R-loops. This in turn targets the cytidine deaminase enzyme AID to S-regions so promoting CSR. Notably R-loop levels over S-regions are diminished by chemical stabilization of G4 RNA or by the expression of a DDX1 ATPase-deficient mutant that acts as a dominant-negative protein to reduce CSR efficiency. In effect, we provide evidence for how S-region transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH locus.
Assuntos
Adenosina Trifosfatases/metabolismo , RNA Helicases DEAD-box/fisiologia , Quadruplex G , Cadeias Pesadas de Imunoglobulinas/genética , Região de Troca de Imunoglobulinas/genética , RNA/química , Adenosina Trifosfatases/genética , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Replicação do DNA , Switching de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA/genética , Recombinação GenéticaRESUMO
BACKGROUND: RNA-DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive. RESULTS: Here, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2'-O-methylation (2'-OMe). Moreover, we find that 2'-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2'-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2'-OMe, R-loop stability and CSR. Surprisingly, FBL, AID's interaction partner and aSNORD1C promoted AID targeting to the IgH locus. CONCLUSION: Taken together, our results suggest that 2'-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability.
Assuntos
Citidina Desaminase , Switching de Imunoglobulina , Estruturas R-Loop , Switching de Imunoglobulina/genética , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/química , Animais , Camundongos , Metilação , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Recombinação Genética , RNA/metabolismo , RNA/genéticaRESUMO
Since the initial report of V (D) J "allelic exclusion/inclusion" (allelic exclusion rearrangement or allelic inclusion rearrangement) and the concept of the "dual B cell receptor (BCR)" in 1961, despite ongoing discoveries, the precise proportion and source mechanism of dual BCR under physiological conditions have been puzzling immuologists. This study takes advantage of the single cell B cell receptor sequencing (scBCR-seq) technology, which can perfectly match the heavy and light chains of BCR at the level of a single B cell, and obtain the full length mRNA sequence of the complementary determining region 3 (CDR3). Through analyzing the pairing of functional IGH (immunoglobulin heavy chain) and IGL (immunoglobulin light chain) in single B cell from both human and mouse bone marrow and peripheral blood, it was observed that dual BCR B cells exhibit stable and high levels of expression. Among them, the human bone marrow and peripheral blood contain about 10% dual (or multiple) BCR B cells, while in mouse peripheral blood and bone marrow memory B cells, this proportion reaches around 20%. At the same time, we innovatively found that in each research sample of humans and mice, there are three (or more) functional rearrangements (mRNA level) of a single chain in a single B cell. By analyzing the position, direction and other compositional characteristics of the V(D)J gene family, we found that at least two (or more) of them are derived from over two (or more) specific allelic inclusion rearrangements of a single chromosome (mRNA molecular level evidence), our findings also highlighted the necessity of classified single cell sequencing data based on single, dual (or multiple) and cannot be assembled into BCR when analyzing the B cell repertoire. The results of this article provides new methods and modeling references for evaluating the proportion and source mechanisms of dual BCR B cells, as well as potential significance of allelic inclusion (exclusion escape) of V(D)J rearrangement.
Assuntos
Linfócitos B , Receptores de Antígenos de Linfócitos B , Camundongos , Humanos , Animais , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , RNA Mensageiro/genéticaRESUMO
Microorganisms have coevolved diverse mechanisms to impair host defenses. A major one, superantigens, can result in devastating effects on the immune system. While all known superantigens induce vast immune cell proliferation and come from opportunistic pathogens, recently, proteins with similar broad specificity to antibody variable (V) domain families were identified in a commensal microbiota. These proteins, identified in the human commensal Ruminococcus gnavus, are called immunoglobulin-binding protein (Ibp) A and B and have been shown to activate B cells in vitro expressing either human VH3 or murine VH5/6/7. Here, we provide molecular and functional studies revealing the basis of this Ibp/immunoglobulin (Ig) interaction. The crystal structure and biochemical assays of a truncated IbpA construct in complex with mouse VH5 antigen-binding fragment (Fab) shows a binding of Ig heavy chain framework residues to the Ibp Domain D and the C-terminal heavy chain binding domain (HCBD). We used targeted mutagenesis of contact residues and affinity measurements and performed studies of the Fab-IbpA complex to determine the stoichiometry between Ibp and VH domains, suggesting Ibp may serve to cluster full-length IgA antibodies in vivo. Furthermore, in vitro stimulation experiments indicate that binding of the Ibp HCBD alone is sufficient to activate responsive murine B cell receptors. The presence of these proteins in a commensal microbe suggest that binding a broad repertoire of immunoglobulins, particularly in the gut/microbiome environment, may provide an important function in the maintenance of host/microbiome homeostasis contrasting with the pathogenic role of structurally homologous superantigens expressed by pathogens.
Assuntos
Anticorpos Monoclonais/metabolismo , Linfócitos B/metabolismo , Clostridiales/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Superantígenos/metabolismo , Animais , Anticorpos Monoclonais/química , Linfócitos B/imunologia , Sítios de Ligação , Clostridiales/crescimento & desenvolvimento , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos B/química , Superantígenos/químicaRESUMO
IgZ or its equivalent IgT is a newly discovered teleost specific Ig class that is highly specialized in mucosal immunity. However, whether this IgZ/IgT class participates in other biological processes remains unclear. In this study, we unexpectedly discovered that IgZ is highly expressed in zebrafish ovary, accumulates in unfertilized eggs, and is transmitted to offspring from eggs to zygotes. Maternally transferred IgZ in zygotes is found at the outer and inner layers of chorion, perivitelline space, periphery of embryo body, and yolk, providing different lines of defense against pathogen infection. A considerable number of IgZ+ B cells are found in ovarian connective tissues distributed between eggs. Moreover, pIgR, the transporter of IgZ, is also expressed in the ovary and colocalizes with IgZ in the zona radiata of eggs. Thus, IgZ is possibly secreted by ovarian IgZ+ B cells and transported to eggs through association with pIgR in a paracrine manner. Maternal IgZ in zygotes showed a broad bacteriostatic activity to different microbes examined, and this reactivity can be manipulated by orchestrating desired bacteria in water where parent fish live or immunizing the parent fish through vaccination. These observations suggest that maternal IgZ may represent a group of polyclonal Abs, providing protection against various environmental microbes encountered by a parent fish that were potentially high risk to offspring. To our knowledge, our findings provide novel insights into a previously unrecognized functional role of IgZ/IgT Ig in the maternal transfer of immunity in fish, greatly enriching current knowledge about this ancient Ig class.
Assuntos
Resistência à Doença/imunologia , Doenças dos Peixes/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Isotipos de Imunoglobulinas/imunologia , Proteínas de Peixe-Zebra/imunologia , Peixe-Zebra/imunologia , Aeromonas hydrophila/imunologia , Aeromonas hydrophila/fisiologia , Animais , Resistência à Doença/genética , Embrião não Mamífero/embriologia , Embrião não Mamífero/imunologia , Embrião não Mamífero/microbiologia , Feminino , Doenças dos Peixes/microbiologia , Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Isotipos de Imunoglobulinas/genética , Isotipos de Imunoglobulinas/metabolismo , Masculino , Herança Materna/genética , Herança Materna/imunologia , Vibrio/classificação , Vibrio/imunologia , Vibrio/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/microbiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Zigoto/imunologia , Zigoto/metabolismo , Zigoto/microbiologiaRESUMO
Central nervous system B cells have several potential roles in multiple sclerosis (MS): secretors of proinflammatory cytokines and chemokines, presenters of autoantigens to T cells, producers of pathogenic antibodies, and reservoirs for viruses that trigger demyelination. To interrogate these roles, single-cell RNA sequencing (scRNA-Seq) was performed on paired cerebrospinal fluid (CSF) and blood from subjects with relapsing-remitting MS (RRMS; n = 12), other neurologic diseases (ONDs; n = 1), and healthy controls (HCs; n = 3). Single-cell immunoglobulin sequencing (scIg-Seq) was performed on a subset of these subjects and additional RRMS (n = 4), clinically isolated syndrome (n = 2), and OND (n = 2) subjects. Further, paired CSF and blood B cell subsets (RRMS; n = 7) were isolated using fluorescence activated cell sorting for bulk RNA sequencing (RNA-Seq). Independent analyses across technologies demonstrated that nuclear factor kappa B (NF-κB) and cholesterol biosynthesis pathways were activated, and specific cytokine and chemokine receptors were up-regulated in CSF memory B cells. Further, SMAD/TGF-ß1 signaling was down-regulated in CSF plasmablasts/plasma cells. Clonally expanded, somatically hypermutated IgM+ and IgG1+ CSF B cells were associated with inflammation, blood-brain barrier breakdown, and intrathecal Ig synthesis. While we identified memory B cells and plasmablast/plasma cells with highly similar Ig heavy-chain sequences across MS subjects, similarities were also identified with ONDs and HCs. No viral transcripts, including from Epstein-Barr virus, were detected. Our findings support the hypothesis that in MS, CSF B cells are driven to an inflammatory and clonally expanded memory and plasmablast/plasma cell phenotype.
Assuntos
Linfócitos B/imunologia , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Adulto , Linfócitos B/metabolismo , Sistema Nervoso Central/imunologia , Quimiocinas/metabolismo , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Humanos , Imunoglobulina G/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , TranscriptomaRESUMO
Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged Igh allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive Igh allele is expressed, a phenomenon known as Igh allelic exclusion. In contrast to a productively rearranged Igh allele, the Igh messenger RNA (mRNA) (IgHR) from a nonproductively rearranged Igh allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable IgHR to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the IghTer5H∆TM mouse model from IghTer5H mice having a premature termination codon at position +5 in leader exon of IghTer5H allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of IghTer5H message, indicating that previous conclusions regarding a role of IgHR in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the IghTer5H∆TM knock-in allele, which generated stable IgHR but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not IgHR expression.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Alelos , Animais , Biomarcadores/metabolismo , Loci Gênicos , Camundongos Endogâmicos C57BL , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos TestesRESUMO
To generate antibodies with different effector functions, B cells undergo Immunoglobulin Heavy Chain (IgH) class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical nonhomologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable â¼25% of CSR can be achieved by the alternative end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ-mediated repair of endonuclease-generated breaks requires DNA end resection, we show that CtIP-mediated DNA end resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppresses resection without altering the MH pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ-mediated CSR by suppressing interchromosomal translocations independent of end resection. Finally, temporal analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favored substrates for MH-mediated end-joining contributing to the robustness and resection independence of A-EJ-mediated CSR.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA por Junção de Extremidades , Switching de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas/genética , Motivos de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linfócitos B/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Camundongos , Fosforilação , Recombinação GenéticaRESUMO
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/genéticaRESUMO
An 85-year-old man with a history of aortic dissection suddenly fainted, underwent cardiac heart arrest, and died. An autopsy was performed, but the cause of death was not grossly identified. Congo red staining detected amyloid deposits in systemic organs, including the heart, lungs, liver, and kidneys. Immunohistochemical (IHC) analysis revealed immunoglobulin (Ig) λ light chain (-λ) in systemic blood vessels and transthyretin (TTR) in the heart and lungs. Ig-λ was predominantly positive in the blood vessels of the lungs, while TTR was detected in the alveolar septum. In the heart, Ig-λ was positive in the endocardium and blood vessels, and TTR was positive in nodular deposits between cardiomyocytes. The concurrent deposition of Ig-λ and TTR in the heart was further substantiated by laser microdissection (LMD)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) at each deposition site. Despite systemic deposition of Ig-λ, bone marrow biopsy findings were not diagnostic for multiple myeloma. In summary, we present an autopsy case of concurrent Ig-λ and TTR deposition as revealed by IHC and LC-MS/MS. When Congo red staining and IHC results are indeterminate due to the deposition of multiple amyloid proteins, LMD-LC-MS/MS is useful for determining the precursor protein.
Assuntos
Amiloidose , Autopsia , Imunoglobulinas/metabolismo , Pré-Albumina/metabolismo , Idoso de 80 Anos ou mais , Amiloide/metabolismo , Amiloidose/diagnóstico , Amiloidose/patologia , Cromatografia Líquida , Diagnóstico Diferencial , Humanos , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Rim/metabolismo , Rim/patologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Espectrometria de Massas em TandemRESUMO
A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine-murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human-murine chimeric anti-CD20 antibody Rituximab.
Assuntos
Anticorpos Monoclonais/química , Antígenos CD20/imunologia , Espectrometria de Massa com Troca Hidrogênio-Deutério , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Sítios de Ligação de Anticorpos , Linhagem Celular Tumoral , Cromatografia Líquida , Cães , Humanos , Imunoglobulina G/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Cinética , Biblioteca de Peptídeos , Proteínas Recombinantes de Fusão , Espectrometria de Massas em TandemRESUMO
B cell isotype switching plays an important role in modulating adaptive immune responses. It occurs in response to specific signals that often induce different isotype (I) promoters driving transcription of switch regions, located upstream of the Ig heavy chain (IgH) constant genes. The transcribed switch regions can recombine, leading to a change of the constant gene and, consequently, of antibody isotype. Switch transcription is controlled by the superenhancer 3' regulatory region (3'RR) that establishes long-range chromatin cis-interactions with I promoters. Most stimuli induce more than one I promoter, and switch transcription can occur on both chromosomes. Therefore, it is presently unknown whether induced I promoters compete for the 3'RR on the same chromosome. Here we performed single-chromosome RT-qPCR assays to examine switch transcription monoallelically in the endogenous context. We show that there are two modes of 3'RR-mediated activation of I promoters: coactivation and competition. The nature of the inducing signal plays a pivotal role in determining the mode of activation. Furthermore, we provide evidence that, in its endogenous setting, the 3'RR has a bidirectional activity. We propose that the coactivation and competition modes mediated by the 3'RR may have evolved to cope with the different kinetics of primary immune responses.
Assuntos
Imunidade Adaptativa , Linfócitos B/imunologia , Elementos Facilitadores Genéticos/genética , Switching de Imunoglobulina/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Regiões 3' não Traduzidas/genética , Alelos , Animais , Linfócitos B/metabolismo , Células Cultivadas , Elementos Facilitadores Genéticos/imunologia , Feminino , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Masculino , Camundongos , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica/imunologiaRESUMO
KEY MESSAGE: Present review summarizes the current applications of nanobodies in plant science and biotechnology, including plant expression of nanobodies, plant biotechnological applications, nanobody-based immunodetection, and nanobody-mediated resistance against plant pathogens. Nanobodies (Nbs) are variable domains of heavy chain-only antibodies (HCAbs) isolated from camelids. In spite of their single domain structure, nanobodies display many unique features, such as small size, high stability, and cryptic epitopes accessibility, which make them ideal for sophisticated applications in plants and animals. In this review, we summarize the current applications of nanobodies in plant science and biotechnology, focusing on nanobody expression in plants, plant biotechnological applications, determination of plant toxins and pathogens, and nanobody-mediated resistance against plant pathogens. Prospects and challenges of nanobody applications in plants are also discussed.
Assuntos
Biotecnologia/métodos , Nanotecnologia/métodos , Plantas , Anticorpos de Domínio Único , Epitopos , Cadeias Pesadas de Imunoglobulinas/classificação , Cadeias Pesadas de Imunoglobulinas/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismoRESUMO
A translocation involving the cyclin-dependent kinase 6 (CDK6) gene [t(CDK6)] is a rare but recurrent abnormality in B-cell neoplasms. To further characterise this aberration, we studied 57 cases; the largest series reported to date. Fluorescence in situ hybridisation analysis confirmed the involvement of CDK6 in all cases, including t(2;7)(p11;q21) immunoglobulin kappa locus (IGK)/CDK6 (n = 51), t(7;14)(q21;q32) CDK6/immunoglobulin heavy locus (IGH) (n = 2) and the previously undescribed t(7;14)(q21;q11) CDK6/T-cell receptor alpha locus (TRA)/T-cell receptor delta locus (TRD) (n = 4). In total, 10 patients were diagnosed with chronic lymphocytic leukaemia, monoclonal B-cell lymphocytosis or small lymphocytic lymphoma, and 47 had small B-cell lymphoma (SmBL) including 36 cases of marginal zone lymphoma (MZL; 34 splenic MZLs, one nodal MZL and one bronchus-associated lymphoid tissue lymphoma). In all, 18 of the 26 cytologically reviewed cases of MZL (69%) had an atypical aspect with prolymphocytic cells. Among the 47 patients with MZL/SmBL, CD5 expression was found in 26 (55%) and the tumour protein p53 (TP53) deletion in 22 (47%). The TP53 gene was mutated in 10/30 (33%); the 7q deletion was detected in only one case, and no Notch receptor 2 (NOTCH2) mutations were found. Immunoglobulin heavy-chain variable-region (IGHV) locus sequencing revealed that none harboured an IGHV1-02*04 gene. Overall survival was 82% at 10 years and not influenced by TP53 aberration. Our present findings suggest that most t(CDK6)+ neoplasms correspond to a particular subgroup of indolent marginal zone B-cell lymphomas with distinctive features.
Assuntos
Antígenos CD5/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfoma de Zona Marginal Tipo Células B/metabolismo , Neoplasias Esplênicas/patologia , Proteína Supressora de Tumor p53/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Brônquicas/diagnóstico , Neoplasias Brônquicas/metabolismo , Diferenciação Celular , Aberrações Cromossômicas , Feminino , Genes p53/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/metabolismo , Hibridização in Situ Fluorescente/métodos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Linfoma de Zona Marginal Tipo Células B/diagnóstico , Linfoma de Zona Marginal Tipo Células B/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Análise de Sobrevida , Estruturas Linfoides Terciárias/patologia , Translocação Genética/genética , Trissomia/genéticaRESUMO
Immunoglobulin secretion is modulated by competition between the use of a weak promoter-proximal poly(A) site and a nonconsensus splice site in the final secretory-specific exon of the heavy chain pre-mRNA. The RNA polymerase II transcription elongation factor ELL2, which is induced in plasma cells, enhanced both polyadenylation and exon skipping with the gene encoding the immunoglobulin heavy-chain complex (Igh) and reporter constructs. Lowering ELL2 expression by transfection of heterogenous ribonucleoprotein F (hnRNP F) or small interfering RNA resulted in lower abundance of secretory-specific forms of immunoglobulin heavy-chain mRNA. ELL2 and the polyadenylation factor CstF-64 tracked together with RNA polymerase II across the Igh mu- and gamma-gene segments; the association of both factors was blocked by ELL2-specific small interfering RNA. Thus, loading of ELL2 and CstF-64 on RNA polymerase II was linked, caused enhanced use of the proximal poly(A) site and was necessary for processing of immunoglobulin heavy-chain mRNA.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Plasmócitos/metabolismo , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA/imunologia , Fatores de Elongação da Transcrição/imunologia , Animais , Fator Estimulador de Clivagem/genética , Fator Estimulador de Clivagem/imunologia , Fator Estimulador de Clivagem/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Plasmócitos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , TransfecçãoRESUMO
The coordination of nutrient and energy availability with cell growth and division is essential for proper immune cell development and function. By using a chemical mutagenesis strategy in mice, we identified a pedigree that has a complete block in B cell development at the pre-B cell stage resulting from a deletion in the Fnip1 gene. Enforced expression of an immunoglobulin transgene failed to rescue B cell development. Whereas essential pre-B cell signaling molecules were activated normally in Fnip1-null pre-B cells, the metabolic regulators AMPK and mTOR were dysregulated, resulting in excessive cell growth and enhanced sensitivity to apoptosis in response to metabolic stress (pre-B cell receptor crosslinking, oncogene activation). These results indicate that Folliculin-interacting protein 1 (Fnip1) is vital for B cell development and metabolic homeostasis and reveal a metabolic checkpoint that may ensure that pre-B cells have sufficient metabolic capacity to support division, while limiting lymphomagenesis caused by deregulated growth.