RESUMO
Early etiological diagnosis is very important for the control of sudden viral infections, and requires antibodies with both high sensitivity and high specificity. Traditional antibody preparation methods have limitations, such as a long and arduous cycle, complicated operation, and high expenses. A chicken lymphoma cell line, DT40, is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture. Here, the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro. Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase (AID) gene, AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected. In this study, we generated a novel AID-inducible DT40 cell line (DT40-H7), in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system, and an inducible AID gene, based on the Tet-Off expression system, was stably transfected. AID expression was controlled in DT40-H7 cells in a simple and efficient manner; gene conversion and point mutations were observed only when AID was expressed. Using the antibody library generated from this cell line, we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus. The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.
Assuntos
Anticorpos Monoclonais/biossíntese , Linfócitos B/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linfócitos B/enzimologia , Linhagem Celular Tumoral , Galinhas , Chlorocebus aethiops , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Doxiciclina/farmacologia , Expressão Gênica/efeitos dos fármacos , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Hipermutação Somática de Imunoglobulina/genética , Células Vero , Proteínas não Estruturais Virais/imunologia , Zika virus/imunologia , Infecção por Zika virus/virologiaRESUMO
Activation-induced cytidine deaminase (AID) is a B-cell-specific enzyme that targets immunoglobulin genes to initiate class switch recombination and somatic hypermutation. In addition, through off-target activity, AID has a much broader effect on genomic instability by initiating oncogenic chromosomal translocations and mutations involved in the development and progression of lymphoma. AID expression is tightly regulated in B cells and its overexpression leads to enhanced genomic instability and lymphoma formation. The phosphatidylinositol 3-kinase δ (PI3Kδ) pathway regulates AID by suppressing its expression in B cells. Drugs for leukaemia or lymphoma therapy such as idelalisib, duvelisib and ibrutinib block PI3Kδ activity directly or indirectly, potentially affecting AID expression and, consequently, genomic stability in B cells. Here we show that treatment of primary mouse B cells with idelalisib or duvelisib, and to a lesser extent ibrutinib, enhanced the expression of AID and increased somatic hypermutation and chromosomal translocation frequency to the Igh locus and to several AID off-target sites. Both of these effects were completely abrogated in AID-deficient B cells. PI3Kδ inhibitors or ibrutinib increased the formation of AID-dependent tumours in pristane-treated mice. Consistently, PI3Kδ inhibitors enhanced AID expression and translocation frequency to IGH and AID off-target sites in human chronic lymphocytic leukaemia and mantle cell lymphoma cell lines, and patients treated with idelalisib, but not ibrutinib, showed increased somatic hypermutation in AID off-targets. In summary, we show that PI3Kδ or Bruton's tyrosine kinase inhibitors increase genomic instability in normal and neoplastic B cells by an AID-dependent mechanism. This effect should be carefully considered, as such inhibitors can be administered to patients for years.
Assuntos
Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Instabilidade Genômica/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Linfócitos B/enzimologia , Linfócitos B/patologia , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Citidina Desaminase/metabolismo , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Switching de Imunoglobulina/efeitos dos fármacos , Cadeias Pesadas de Imunoglobulinas/genética , Isoquinolinas/efeitos adversos , Isoquinolinas/farmacologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/patologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Piperidinas , Proteínas Tirosina Quinases/antagonistas & inibidores , Purinas/efeitos adversos , Purinas/farmacologia , Pirazóis/efeitos adversos , Pirazóis/farmacologia , Pirimidinas/efeitos adversos , Pirimidinas/farmacologia , Quinazolinonas/efeitos adversos , Quinazolinonas/farmacologia , Recombinação Genética/efeitos dos fármacos , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Translocação Genética/efeitos dos fármacosRESUMO
Clonal anergy is an enigmatic self-tolerance mechanism because no apparent purpose is served by retaining functionally silenced B cells bearing autoantibodies. Human autoantibodies with IGHV4-34*01 heavy chains bind to poly-N-acetyllactosamine carbohydrates (I/i antigen) on erythrocytes and B lymphocytes, cause cold agglutinin disease, and are carried by 5% of naive B cells that are anergic. We analyzed the specificity of three IGHV4-34*01 IgG antibodies isolated from healthy donors immunized against foreign rhesus D alloantigen or vaccinia virus. Each IgG was expressed and analyzed either in a hypermutated immune state or after reverting each antibody to its unmutated preimmune ancestor. In each case, the preimmune ancestor IgG bound intensely to normal human B cells bearing I/i antigen. Self-reactivity was removed by a single somatic mutation that paradoxically decreased binding to the foreign immunogen, whereas other mutations conferred increased foreign binding. These data demonstrate the existence of a mechanism for mutation away from self-reactivity in humans. Because 2.5% of switched memory B cells use IGHV4-34*01 and >43% of these have mutations that remove I/i binding, clonal redemption of anergic cells appears efficient during physiological human antibody responses.
Assuntos
Anticorpos Antivirais/imunologia , Autoanticorpos/imunologia , Linfócitos B/imunologia , Imunização , Imunoglobulina G/imunologia , Sistema do Grupo Sanguíneo Rh-Hr/administração & dosagem , Hipermutação Somática de Imunoglobulina/imunologia , Vaccinia virus/imunologia , Anergia Clonal/imunologia , Feminino , Humanos , Memória Imunológica/efeitos dos fármacos , Memória Imunológica/imunologia , Masculino , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Hipermutação Somática de Imunoglobulina/efeitos dos fármacosRESUMO
PrimPol is a DNA damage tolerance enzyme possessing both translesion synthesis (TLS) and primase activities. To uncover its potential role in TLS-mediated IgVλ hypermutation and define its interplay with other TLS polymerases, PrimPol(-/-) and PrimPol(-/-)/Polη(-/-)/Polζ (-/-) gene knockouts were generated in avian cells. Loss of PrimPol had no significant impact on the rate of hypermutation or the mutation spectrum of IgVλ. However, PrimPol(-/-) cells were sensitive to methylmethane sulfonate, suggesting that it may bypass abasic sites at the IgVλ segment by repriming DNA synthesis downstream of these sites. PrimPol(-/-) cells were also sensitive to cisplatin and hydroxyurea, indicating that it assists in maintaining / restarting replication at a variety of lesions. To accurately measure the relative contribution of the TLS and primase activities, we examined DNA damage sensitivity in PrimPol(-/-) cells complemented with polymerase or primase-deficient PrimPol. Polymerase-defective, but not primase-deficient, PrimPol suppresses the hypersensitivity of PrimPol(-/-) cells. This indicates that its primase, rather than TLS activity, is pivotal for DNA damage tolerance. Loss of TLS polymerases, Polη and Polζ has an additive effect on the sensitivity of PrimPol(-/-) cells. Moreover, we found that PrimPol and Polη-Polζ redundantly prevented cell death and facilitated unperturbed cell cycle progression. PrimPol(-/-) cells also exhibited increased sensitivity to a wide variety of chain-terminating nucleoside analogs (CTNAs). PrimPol could perform close-coupled repriming downstream of CTNAs and oxidative damage in vitro. Together, these results indicate that PrimPol's repriming activity plays a central role in reinitiating replication downstream from CTNAs and other specific DNA lesions.
Assuntos
Dano ao DNA , DNA Primase/metabolismo , Replicação do DNA , Nucleosídeos/metabolismo , Animais , Biocatálise/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Galinhas , Cisplatino/farmacologia , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/efeitos da radiação , DNA Polimerase Dirigida por DNA/metabolismo , Região Variável de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/genética , Metanossulfonato de Metila/farmacologia , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Raios UltravioletaRESUMO
Class-switch DNA recombination (CSR) and somatic hypermutation (SHM), which require activation-induced cytidine deaminase (AID), and plasma cell differentiation, which requires B lymphocyte-induced maturation protein-1 (Blimp-1), are critical for the generation of class-switched and hypermutated (mature) Ab and autoantibody responses. We show that histone deacetylase inhibitors valproic acid and butyrate dampened AICDA/Aicda (AID) and PRDM1/Prdm1 (Blimp-1) mRNAs by upregulating miR-155, miR-181b, and miR-361 to silence AICDA/Aicda, and miR-23b, miR-30a, and miR-125b to silence PRDM1/Prdm1, in human and mouse B cells. This led to downregulation of AID, Blimp-1, and X-box binding protein 1, thereby inhibiting CSR, SHM, and plasma cell differentiation without altering B cell viability or proliferation. The selectivity of histone deacetylase inhibitor-mediated silencing of AICDA/Aicda and PRDM1/Prdm1 was emphasized by unchanged expression of HoxC4 and Irf4 (important inducers/modulators of AICDA/Aicda), Rev1 and Ung (central elements for CSR/SHM), and Bcl6, Bach2, or Pax5 (repressors of PRDM1/Prdm1 expression), as well as unchanged expression of miR-19a/b, miR-20a, and miR-25, which are not known to regulate AICDA/Aicda or PRDM1/Prdm1. Through these B cell-intrinsic epigenetic mechanisms, valproic acid blunted class-switched and hypermutated T-dependent and T-independent Ab responses in C57BL/6 mice. In addition, it decreased class-switched and hypermutated autoantibodies, ameliorated disease, and extended survival in lupus MRL/Fas(lpr/lpr) mice. Our findings outline epigenetic mechanisms that modulate expression of an enzyme (AID) and transcription factors (Blimp-1 and X-box binding protein 1) that are critical to the B cell differentiation processes that underpin Ab and autoantibody responses. They also provide therapeutic proof-of-principle in autoantibody-mediated autoimmunity.
Assuntos
Formação de Anticorpos , Linfócitos B/efeitos dos fármacos , Linfócitos B/fisiologia , Citidina Desaminase/genética , Epigênese Genética , Inativação Gênica , Inibidores de Histona Desacetilases/farmacologia , MicroRNAs/genética , Proteínas Repressoras/genética , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Autoanticorpos/biossíntese , Autoanticorpos/imunologia , Linfócitos B/citologia , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Switching de Imunoglobulina/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos MRL lpr , Plasmócitos/citologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Plasmócitos/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Fatores de Transcrição de Fator Regulador X , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
AIMS: In the present study we have investigated the comparative switching propensity of murine peritoneal and splenic B cell subpopulations to IgA in presence of retinoic acid (RA) and TGF-ß. METHODS AND RESULTS: To study the influence of RA and TGF-ß on switching of B cell subpopulations to IgA, peritoneal (B1a, B1b and B2 cells) and splenic (B1a, marginal zone, and B2) B cells from normal BALB/c mice were FACS purified, cultured for 4 days in presence of RA and TGF-ß and the number of IgA producing cells was determined by ELISPOT assay or FACS analysis. In presence of TGF-ß, peritoneal B1b cells switched to IgA more potently than other peritoneal B cell subpopulations. When TGF-ß was combined with retinoic acid (RA), switching to IgA was even more pronounced. Under these conditions, "innate" B cells like peritoneal and splenic B1 cells and MZ B cells produced IgA more readily than B2 cells. Additionally, high frequency of nucleotide exchanges indicating somatic hypermutation in VH regions was observed. Besides IgA induction, RA treatment of sorted PEC and splenic B cells led to expression of gut homing molecules - α4ß7 and CCR9. Intraperitoneal transfer of RA-treated B1 cells into Rag1(-/-) recipients resulted in IgA in serum and gut lavage, most efficiently amongst B1b cell recipients. CONCLUSION: Present study demonstrates the differential and synergistic effect of RA and TGF-ß on switching of different B cell subpopulations to IgA and establishes the prominence of peritoneal B1b cells in switching to IgA under the influence of these two factors. Our study extends our knowledge about the existing differences among B cell subpopulations with regards to IgA production and indicates towards their differential contribution to gut associated humoral immunity.
Assuntos
Imunoglobulina A/metabolismo , Peritônio/citologia , Fator de Crescimento Transformador beta/farmacologia , Tretinoína/farmacologia , Animais , Contagem de Células , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Humanos , Imunoglobulina A/sangue , Switching de Imunoglobulina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Baço/citologia , Baço/efeitos dos fármacosRESUMO
Activation-induced cytidine deaminase (AID) is essential for class switch recombination and somatic hypermutation. Its deregulated expression acts as a genomic mutator that can contribute to the development of various malignancies. During treatment with imatinib mesylate (IM), patients with chronic myeloid leukemia often develop hypogammaglobulinemia, the mechanism of which has not yet been clarified. Here, we provide evidence that class switch recombination on B-cell activation is apparently inhibited by IM through down-regulation of AID. Furthermore, expression of E2A, a key transcription factor for AID induction, was markedly suppressed by IM. These results elucidate not only the underlying mechanism of IM-induced hypogammaglobulinemia but also its potential efficacy as an AID suppressor.
Assuntos
Citidina Desaminase/antagonistas & inibidores , Switching de Imunoglobulina/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia , Animais , Benzamidas , Citidina Desaminase/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Avaliação Pré-Clínica de Medicamentos , Mesilato de Imatinib , Imunossupressores/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Recombinação Genética/efeitos dos fármacos , Recombinação Genética/imunologia , Ovinos , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Resultado do TratamentoRESUMO
BACKGROUND: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. METHODOLOGY/PRINCIPAL FINDINGS: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer and Activator of Transcription 5 (STAT5). Active STAT5 inhibits the differentiation of B cells while increasing their replicative life span. We obtained cloned B cell lines, which produced antibodies in the presence of interleukin 21 after turning off STAT5. We used this method to obtain monoclonal antibodies against the model antigen tetanus toxin. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel and relatively simple method of immortalizing antigen-specific human B cells for isolation of human monoclonal antibodies. These results show that STAT5 overexpression can be employed to isolate antigen specific antibodies from human memory B cells.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Linfócitos B/imunologia , Epitopos/imunologia , Fator de Transcrição STAT5/imunologia , Adulto , Animais , Linfócitos B/efeitos dos fármacos , Linhagem Celular , Separação Celular , Células Clonais , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Memória Imunológica/imunologia , Interleucinas/farmacologia , Camundongos , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Hipermutação Somática de Imunoglobulina/genética , Toxina Tetânica/imunologiaRESUMO
Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates class switch recombination and somatic hypermutation of immunoglobulin genes (Ig) in B lymphocytes. However, AID also produces off-target DNA damage, including mutations in oncogenes and double-stranded breaks that can serve as substrates for oncogenic chromosomal translocations. AID is strictly regulated by a number of mechanisms, including phosphorylation at serine 38 and threonine 140, which increase activity. Here we show that phosphorylation can also suppress AID activity in vivo. Serine 3 is a novel phospho-acceptor which, when mutated to alanine, leads to increased class switching and c-myc/IgH translocations without affecting AID levels or catalytic activity. Conversely, increasing AID phosphorylation specifically on serine 3 by interfering with serine/threonine protein phosphatase 2A (PP2A) leads to decreased class switching. We conclude that AID activity and its oncogenic potential can be downregulated by phosphorylation of serine 3 and that this process is controlled by PP2A.
Assuntos
Citidina Desaminase/química , Citidina Desaminase/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Translocação Genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Switching de Imunoglobulina/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/metabolismo , Recombinação Genética/efeitos dos fármacos , Recombinação Genética/genética , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Hipermutação Somática de Imunoglobulina/genética , Translocação Genética/efeitos dos fármacosRESUMO
TLR9 activation by unmethylated CpG provides a homeostatic mechanism to maintain B cell memory in the absence of Ag. In this study, we demonstrate that CpG also triggers the generation of somatically mutated memory B cells from immature transitional B cells. In response to CpG, a fraction of transitional B cells proliferates and introduces somatic hypermutations in the H chain V regions. The nonproliferating pool of transitional B cells mostly maintains germline configurations. Mutations are VH specific: VH5 is the least mutated family, whereas VH1 and VH4/6 are the most mutated families. CpG stimulation also results in upregulation of VH5 transcripts in proliferating cells. Therefore, early recognition of bacterial DNA preferentially expands VH5-expressing B cells while inducing somatic hypermutations in other families. The mutation frequency, range, and type of substitutions observed in vitro are comparable to those found in memory B cells from the peripheral blood of Hyper IgM type 1 patients and the spleen of normal infants. The process triggered by TLRs may represent a first step leading to additional diversification of the germline repertoire and to the generation of memory B cells that will further refine their repertoire and specificity in the germinal centers.
Assuntos
Adjuvantes Imunológicos/farmacologia , Linfócitos B/imunologia , Imunoglobulina M/imunologia , Memória Imunológica/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Receptor Toll-Like 9/imunologia , Linfócitos B/citologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Centro Germinativo/citologia , Centro Germinativo/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Memória Imunológica/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Receptor Toll-Like 9/agonistas , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
Genetic variation at immunoglobulin (Ig) gene variable regions in B-cells is created through a multi-step process involving deamination of cytosine bases by activation-induced cytidine deaminase (AID) and their subsequent mutagenic repair. To protect the genome from dangerous, potentially oncogenic effects of off-target mutations, both AID activity and mutagenic repair are targeted specifically to the Ig genes. However, the mechanisms of targeting are unknown and recent data have highlighted the role of regulating mutagenic repair to limit the accumulation of somatic mutations resulting from the more widely distributed AID-induced lesions to the Ig genes. Here we investigated the role of the DNA damage sensor poly-(ADPribose)-polymerase-1 (PARP-1) in the repair of AID-induced DNA lesions. We show through sequencing of the diversifying Ig genes in PARP-1(-/-) DT40 B-cells that PARP-1 deficiency results in a marked reduction in gene conversion events and enhanced high-fidelity repair of AID-induced lesions at both Ig heavy and light chains. To further characterize the role of PARP-1 in the mutagenic repair of AID-induced lesions, we performed functional analyses comparing the role of engineered PARP-1 variants in high-fidelity repair of DNA damage induced by methyl methane sulfonate (MMS) and the mutagenic repair of lesions at the Ig genes induced by AID. This revealed a requirement for the previously uncharacterized BRCT domain of PARP-1 to reconstitute both gene conversion and a normal rate of somatic mutation at Ig genes, while being dispensable for the high-fidelity base excision repair. From these data we conclude that the BRCT domain of PARP-1 is required to initiate a significant proportion of the mutagenic repair specific to diversifying antibody genes. This role is distinct from the known roles of PARP-1 in high-fidelity DNA repair, suggesting that the PARP-1 BRCT domain has a specialized role in assembling mutagenic DNA repair complexes involved in antibody diversification.
Assuntos
Conversão Gênica/genética , Genes de Imunoglobulinas/genética , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Citidina Desaminase/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Conversão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutação/genética , Poli(ADP-Ribose) Polimerases/deficiência , Estrutura Terciária de Proteína , Recombinação Genética/efeitos dos fármacos , Recombinação Genética/genética , Hipermutação Somática de Imunoglobulina/efeitos dos fármacosRESUMO
OBJECTIVE: Transient B cell depletion by rituximab has been used with clinical efficacy in the treatment of patients with rheumatoid arthritis (RA). Previous studies of B cell repopulation have shown long-term numerical reduction in memory B cells. Non-class-switched IgD+CD27+ memory B cells, in particular, repopulate slowly. This study was undertaken to determine whether mutational acquisition in individual B cell receptors in repopulating class-switched and non-class-switched memory B cells is affected by rituximab. METHODS: Cells obtained from 16 RA patients, 4 healthy donors, and 3 patients who underwent allogeneic stem cell transplantation (ASCT) were analyzed using single B cell sorting followed by nested polymerase chain reaction and Ig V(H)3 sequencing. RESULTS: There was a delayed acquisition of mutations in Ig receptors of IgD+ memory B cells over a period of 6 years after a single course of rituximab. One year after rituximab treatment, 84% of single repopulating IgD+CD27+ B cells were unmutated, and no highly mutated Ig receptors were found (compared with 52% before therapy). Over time, increasing numbers of mutations were detected. Even 6 years after rituximab treatment, however, mutations in IgD+ memory B cells were still significantly reduced. In contrast, class-switched memory B cells repopulated with quantitatively normal mutations. In comparison, in patients undergoing ASCT, IgD+ memory cells repopulated earlier with higher mutations in Ig receptors. CONCLUSION: Our data suggest that IgD+ memory B cells are particularly susceptible to the effects of rituximab, with delayed acquisition of mutations in their Ig receptors still evident 6 years after a single course of rituximab. Our findings indicate that these cells have different requirements for mutational acquisition compared with class-switched memory B cells.
Assuntos
Anticorpos Monoclonais/efeitos adversos , Antirreumáticos/uso terapêutico , Linfócitos B/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Receptores Fc/efeitos dos fármacos , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Adulto , Idoso , Anticorpos Monoclonais Murinos , Linfócitos B/imunologia , Rearranjo Gênico do Linfócito B/efeitos dos fármacos , Rearranjo Gênico do Linfócito B/imunologia , Humanos , Memória Imunológica/imunologia , Pessoa de Meia-Idade , Receptores Fc/imunologia , Rituximab , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Mismatch repair plays an essential role in reducing the cellular mutation load. Paradoxically, proteins in this pathway produce A . T mutations during the somatic hypermutation of immunoglobulin genes. Although recent evidence implicates the translesional DNA polymerase eta in producing these mutations, it is unknown how this or other translesional polymerases are recruited to immunoglobulin genes, since these enzymes are not normally utilized in conventional mismatch repair. In this report, we demonstrate that A . T mutations were closely associated with transversion mutations at a deoxycytidine. Furthermore, deficiency in uracil-N-glycolase (UNG) or mismatch repair reduced this association. These data reveal a previously unknown interaction between the base excision and mismatch repair pathways and indicate that an abasic site generated by UNG within the mismatch repair tract recruits an error-prone polymerase, which then introduces A . T mutations. Our analysis further indicates that repair tracts typically are approximately 200 nucleotides long and that polymerase eta makes approximately 1 error per 300 T nucleotides. The concerted action of Msh2 and UNG in stimulating A . T mutations also may have implications for mutagenesis at sites of spontaneous cytidine deamination.
Assuntos
Adenina/metabolismo , Pareamento de Bases/genética , Proteína 2 Homóloga a MutS/metabolismo , Hipermutação Somática de Imunoglobulina/genética , Timina/metabolismo , Uracila-DNA Glicosidase/metabolismo , Animais , Citidina Desaminase/metabolismo , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Imunoglobulinas/genética , Camundongos , Modelos Biológicos , Mutagênese/efeitos dos fármacos , Mutação/genética , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Uracila-DNA Glicosidase/antagonistas & inibidoresRESUMO
The control of systemic infection by encapsulated microorganisms requires T-independent type II (TI-2) Ab responses to bacterial polysaccharides. To understand how such responses evolve, we explored the function of transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI), a member of the TNFR family, required for TI-2 Ab production. Quasimonoclonal (QM) mice produce robust TI-2 responses to 4-hydroxy-3-nitrophenylacetate (NP)-Ficoll, owing to the high precursor frequency of NP-specific B cells in the marginal zone of the spleen. QM mice that lack TACI produce decreased numbers of IgM (2-fold) and IgG (1.6-fold) NP-specific ASCs, compared with TACI-positive QM mice in response to immunization with NP-Ficoll. Our studies indicate that TACI acts at a remote time from activation because TACI is not necessary for activation and proliferation of B cells both in vitro and in vivo. Instead, TACI-deficient QM B cells remained in the cell cycle longer than TACI-proficient QM cells and had impaired plasma cell differentiation in response to NP-Ficoll. We conclude that TACI has dual B cell-autonomous functions, inhibiting prolonged B cell proliferation and stimulating plasma cell differentiation, thus resolving the longstanding paradox that TACI may have both B cell-inhibitory and -stimulatory functions. By promoting plasma cell differentiation earlier during clonal expansion, TACI may decrease the chances of autoantibody production by somatic hypermutation of Ig genes in response to T-independent Ags.
Assuntos
Anticorpos Antibacterianos/imunologia , Cápsulas Bacterianas/imunologia , Diferenciação Celular/imunologia , Ativação Linfocitária/imunologia , Plasmócitos/imunologia , Proteína Transmembrana Ativadora e Interagente do CAML/imunologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Autoanticorpos/imunologia , Cápsulas Bacterianas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Ficoll/imunologia , Ficoll/farmacologia , Imunização , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Camundongos , Camundongos Knockout , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Proteína Transmembrana Ativadora e Interagente do CAML/deficiênciaRESUMO
Somatic hypermutation, which occurs in antigen-activated germinal centre B lymphocytes, diversifies the genes that encode immunoglobulin variable regions and leads to the 'affinity maturation' of the humoral immune response. Hypermutation affects dC/dG and dA/dT pairs with approximately equal frequency in vivo. DNA polymerase-theta contributes to hypermutagenesis at dC/dG pairs and DNA polymerase-eta is substantially involved in the generation of hypermutations at dA/dT pairs. The biochemical properties of polymerases-theta and -eta indicate that their DNA synthetic activities are potentially susceptible to inhibition by nucleoside analogues, so it is feasible that nucleoside analogues reduce the accumulation of dC/dG- and dA/dT-targeted hypermutations in vivo. Nucleoside analogues could hence impair the humoral adaptive immune response of HIV-infected patients who are prescribed these chemotherapeutic agents.
Assuntos
Nucleosídeos/genética , Hipermutação Somática de Imunoglobulina , Linfócitos B/imunologia , Replicação do DNA/imunologia , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/imunologia , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1/genética , HIV-1/imunologia , Humanos , Nucleosídeos/imunologia , Nucleosídeos/farmacologia , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , DNA Polimerase tetaRESUMO
OBJECTIVES: To determine whether the immunoglobulin V(H) gene mutational status has an effect on the activation, proliferation and surface antigen expression of chronic lymphocytic leukemia (CLL) cells when stimulated in vitro. METHODS: The proliferation and activation responses of CLL cells were studied in 22-immunoglobulin gene V(H) unmutated (UM-CLL) and 12 hypermutated (M-CLL) CLL cases in 4-day cultures. As the mitogen responses have been previously shown to be diverse in CLL, a case-specific strategy based on optimized mitogen combinations (OMCs) of interleukin-2 (IL-2), 12-O-tetradecanoylphorbol 13-acetate (TPA), Staphylococcus aureus Cowan 1 (SAC), and human recombinant tumor necrosis factor alpha (TNF) was applied in cell stimulation. The expression of 23 surface membrane antigens (CD5, CD11c, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD38, CD40, CD45, CD45RA, CD45RO, CD79b, CD80, CD95, CD124, CD126, CD130, FMC7, IgD, and IgM) was studied by flow cytometry at days 0 and 4. RESULTS: The proliferation and activation responses were similar in UM-CLL and M-CLL when OMCs contained IL-2, TPA or TNF. SAC induced faster proliferation in UM-CLL than in M-CLL. OMC stimulation induced preferential down-regulation of growth- promoting cell surface receptors CD5, CD21, and CD124 and preferential up-regulation of growth-inhibiting antigen CD80 in M-CLL. CONCLUSIONS: Difference in immunophenotypic evolution of UM-CLL and M-CLL can be demonstrated if appropriate matrix signals are provided. The pathways for CD5, CD21, CD124 (IL4R), and CD80 (B7-1) regulation should be further explored in relation with somatic hypermutation and outcome of CLL.