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1.
Mol Immunol ; 116: 63-72, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31622795

RESUMO

Somatic hypermutation (SHM) of Ig genes is initiated by activation-induced cytidine deaminase (AID) and requires target gene transcription. A splice isoform of SRSF1, SRSF1-3, is necessary for AID-dependent SHM of IgV genes. Nevertheless, its exact molecular mechanism of action in SHM remains unknown. Our in silico studies show that, unlike SRSF1, SRSF1-3 lacks a strong nuclear localization domain. We show that the absence of RS domain in SRSF1-3 affects its nuclear localization, as compared to SRSF1. Consequently, SRSF1-3 is predominantly present in the cytoplasm. Remarkably, co-immunoprecipitation studies showed that SRSF1-3 interacts with Topoisomerase 1 (TOP1), a crucial regulator of SHM that assists in generating ssDNA for AID activity. Moreover, the immunofluorescence studies confirmed that SRSF1-3 and TOP1 are co-localized in the nucleus. Furthermore, Proximity Ligation Assay corroborated the direct interaction between SRSF1-3 and TOP1. An interaction between SRSF1-3 and TOP1 suggests that SRSF1-3 likely influences the TOP1 activity and consequently can aid in SHM. Accordingly, SRSF1-3 probably acts as a link between TOP1 and SHM, by spatially regulating TOP1 activity at the Ig locus. We also confirmed the interaction between SRSF1-3 and AID in chicken B-cells. Thus, SRSF1-3 shows dual-regulation of SHM, via interacting with AID as well as TOP1.


Assuntos
Citidina Desaminase/genética , DNA Topoisomerases Tipo I/genética , Genes de Imunoglobulinas/genética , Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Hipermutação Somática de Imunoglobulina/genética , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Linhagem Celular , Núcleo Celular/genética , Galinhas/genética , Switching de Imunoglobulina , Imunoprecipitação/métodos , Camundongos , Isoformas de Proteínas/genética
2.
Nature ; 574(7776): 122-126, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31554970

RESUMO

B cells are important in the pathogenesis of many, and perhaps all, immune-mediated diseases. Each B cell expresses a single B cell receptor (BCR)1, and the diverse range of BCRs expressed by the total B cell population of an individual is termed the 'BCR repertoire'. Our understanding of the BCR repertoire in the context of immune-mediated diseases is incomplete, and defining this could provide new insights into pathogenesis and therapy. Here, we compared the BCR repertoire in systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Crohn's disease, Behçet's disease, eosinophilic granulomatosis with polyangiitis, and immunoglobulin A (IgA) vasculitis by analysing BCR clonality, use of immunoglobulin heavy-chain variable region (IGHV) genes and-in particular-isotype use. An increase in clonality in systemic lupus erythematosus and Crohn's disease that was dominated by the IgA isotype, together with skewed use of the IGHV genes in these and other diseases, suggested a microbial contribution to pathogenesis. Different immunosuppressive treatments had specific and distinct effects on the repertoire; B cells that persisted after treatment with rituximab were predominately isotype-switched and clonally expanded, whereas the inverse was true for B cells that persisted after treatment with mycophenolate mofetil. Our comparative analysis of the BCR repertoire in immune-mediated disease reveals a complex B cell architecture, providing a platform for understanding pathological mechanisms and designing treatment strategies.


Assuntos
Doenças do Sistema Imunitário/imunologia , Isotipos de Imunoglobulinas/análise , Isotipos de Imunoglobulinas/imunologia , Receptores de Antígenos de Linfócitos B/análise , Receptores de Antígenos de Linfócitos B/imunologia , Adulto , Idoso , Células Clonais/citologia , Células Clonais/imunologia , Humanos , Imunoglobulina A/análise , Imunoglobulina A/imunologia , Switching de Imunoglobulina/imunologia , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Pessoa de Meia-Idade , Adulto Jovem
3.
Nat Commun ; 10(1): 3859, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455769

RESUMO

Induction of long-lived antibody responses during infection or vaccination is often essential for subsequent protection, but the relative contributions of T follicular helper (Tfh) cells and T helper 1 (Th1) cells for induction of antigen specific antibody responses to viruses are unclear. Here, we establish an acute Zika virus (ZIKV) infection model in immunocompetent mice, and show that ZIKV infection elicits robust Th1-like Tfh cell and protective antibody responses. While these Th1-like Tfh cells share phenotypic and transcriptomic profiles with both Tfh and Th1 cells, they also have unique surface markers and gene expression characteristics, and are dependent on T-bet for their development. Th1-like Tfh cells, but not Th1 cells, are essential for class switching of ZIKV-specific IgG2c antibodies and maintenance of long-term neutralizing antibody responses. Our study suggests that specific modulation of the Th1-like Tfh cell response during infection or vaccination may augment the induction of antiviral antibody response to ZIKV and other viruses.


Assuntos
Switching de Imunoglobulina/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Interações entre Hospedeiro e Microrganismos/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Camundongos , Camundongos Knockout , Transdução de Sinais/imunologia , Proteínas com Domínio T/metabolismo , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Células Vero , Infecção por Zika virus/virologia
4.
Immunity ; 51(2): 206-208, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31433966

RESUMO

Class-switch recombination (CSR) provides diverse effector functions to B cells. In this issue of Immunity, Roco et al. (2019) refute long-standing dogma that germinal centers are the principal location for CSR; instead, CSR predominantly occurs early post immunization and declines during germinal center formation.


Assuntos
Centro Germinativo , Switching de Imunoglobulina , Linfócitos B
5.
Infect Immun ; 87(11)2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31427447

RESUMO

Coxiella burnetii is an obligate intracellular Gram-negative bacterium which causes human Q fever. An acidified citrate cysteine medium (ACCM-2) has been developed which mimics the intracellular replicative niche of C. burnetii and allows axenic growth of the bacteria. To determine if C. burnetii cultured in ACCM-2 retains immunogenicity, we compared the protective efficacies of formalin-inactivated C. burnetii Nine Mile phase I (PIV) and phase II (PIIV) vaccines derived from axenic culture 7, 14, and 28 days postvaccination. PIV conferred significant protection against virulent C. burnetii as early as 7 days postvaccination, which suggests that ACCM-2-derived PIV retains immunogenicity and protectivity. We analyzed the cellular immune response in spleens from PIV- and PIIV-vaccinated mice by flow cytometry at 7 and 14 days postvaccination and found significantly more granulocytes in PIV-vaccinated mice than in PIIV-vaccinated mice. Interestingly, we found these infiltrating granulocytes to be SSChigh CD11b+ CD125+ Siglec-F+ (where SSChigh indicates a high side scatter phenotype) eosinophils. There was no change in the number of eosinophils in PIV-vaccinated CD4-deficient mice compared to the level in controls, which suggests that eosinophil accumulation is CD4+ T cell dependent. To evaluate the importance of eosinophils in PIV-mediated protection, we vaccinated and challenged eosinophil-deficient ΔdblGATA mice. ΔdblGATA mice had significantly worse disease than their wild-type counterparts when challenged 7 days postvaccination, while no significant difference was seen at 28 days postvaccination. Nevertheless, ΔdblGATA mice had elevated serum IgM with decreased IgG1 and IgG2a whether mice were challenged at 7 or 28 days postvaccination. These results suggest that eosinophils may play a role in early vaccine protection against C. burnetii and contribute to antibody isotype switching.


Assuntos
Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/imunologia , Coxiella burnetii/imunologia , Eosinófilos/fisiologia , Switching de Imunoglobulina/imunologia , Febre Q/prevenção & controle , Animais , Sistema Livre de Células , Imunidade Celular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Baço/citologia , Vacinação
6.
Immunity ; 51(2): 337-350.e7, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31375460

RESUMO

Class-switch recombination (CSR) is a DNA recombination process that replaces the immunoglobulin (Ig) constant region for the isotype that can best protect against the pathogen. Dysregulation of CSR can cause self-reactive BCRs and B cell lymphomas; understanding the timing and location of CSR is therefore important. Although CSR commences upon T cell priming, it is generally considered a hallmark of germinal centers (GCs). Here, we have used multiple approaches to show that CSR is triggered prior to differentiation into GC B cells or plasmablasts and is greatly diminished in GCs. Despite finding a small percentage of GC B cells expressing germline transcripts, phylogenetic trees of GC BCRs from secondary lymphoid organs revealed that the vast majority of CSR events occurred prior to the onset of somatic hypermutation. As such, we have demonstrated the existence of IgM-dominated GCs, which are unlikely to occur under the assumption of ongoing switching.


Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Switching de Imunoglobulina , Plasmócitos/imunologia , Linfoma Plasmablástico/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Filogenia , Receptores de Antígenos de Linfócitos B/metabolismo
7.
Scand J Immunol ; 90(5): e12809, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31322747

RESUMO

We evaluated 18 DiGeorge syndrome (DGS) patients and aimed to investigate the immunological changes in this population. DGS patients with low naive CD4+ T and CD8+ T cells were defined as high-risk (HR) patients, whereas patients with normal numbers of naive CD4+ and CD8+ T cells were defined as standard risk (SR) patients. Level of serum IgM, CD3+ T cell counts and percentages of class-switched memory B cells were significantly low in HR group compared to SR ones. Severe infections and persistent hypoparathyroidism were detected significantly higher in HR group. Patients with reduced percentages of class-switched B cells had earlier onset of infection, lower blood IgM, lower CD4+ and CD8+ T counts than patients with normal class-switched memory B cells. Decreased levels of IgM were associated with low numbers of naive CD4+ and recent thymic emigrants T cells. Monitoring the immune changes of patients with DGS would be useful to predict the severe phenotype of disease.


Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Síndrome de DiGeorge/imunologia , Imunoglobulina M/sangue , Adolescente , Adulto , Criança , Pré-Escolar , Síndrome de DiGeorge/genética , Feminino , Humanos , Hipoparatireoidismo/diagnóstico , Switching de Imunoglobulina/imunologia , Memória Imunológica/imunologia , Lactente , Contagem de Linfócitos , Masculino
8.
Immunohorizons ; 3(3): 88-93, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-31342012

RESUMO

The CD1d-binding glycolipid α-galactosylceramide (α-GC) is a potent adjuvant that activates NKT cells and in turn enhances T-dependent humoral immunity. Very little is known about how NKT cells and the NKT follicular helper (NKTfh) subset influence the immune response to T-independent polysaccharides. In this study, we used a Cre-Lox approach to generate mice devoid of the Bcl6 master transcription factor in CD4 lineage cells and thus devoid of NKTfh cells but not total NKT cells. It was observed that α-GC-driven IgG1 class switch against a polysaccharide Ag was dependent on the NKTfh subset. However, α-GC was unable to stimulate a polysaccharide-specific Ab recall response. It was observed that NKT-derived IL-21 was able to exert limited influence on the IgG1 response and was therefore likely to work in concert with other factors. This work shows that α-GC-driven NKTfh cells can direct polysaccharide-specific B cell responses by promoting IgG1 class switch but do not provide signals needed for generation of polysaccharide-specific B cell memory.


Assuntos
Switching de Imunoglobulina/imunologia , Imunoglobulina G/imunologia , Memória Imunológica , Células T Matadoras Naturais/imunologia , Polissacarídeos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Comunicação Celular , Feminino , Galactosilceramidas/imunologia , Imunização , Switching de Imunoglobulina/genética , Imunoglobulina G/genética , Ativação Linfocitária , Masculino , Camundongos , Camundongos Transgênicos , Células T Matadoras Naturais/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo
9.
EMBO J ; 38(14): e101260, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31304630

RESUMO

Tissue-resident iNKT cells maintain tissue homeostasis and peripheral surveillance against pathogens; however, studying these cells is challenging due to their low abundance and poor recovery from tissues. We here show that iNKT transnuclear mice, generated by somatic cell nuclear transfer, have increased tissue resident iNKT cells. We examined expression of PLZF, T-bet, and RORγt, as well as cytokine/chemokine profiles, and found that both monoclonal and polyclonal iNKT cells differentiated into functional subsets that faithfully replicated those seen in wild-type mice. We detected iNKT cells from tissues in which they are rare, including adipose, lung, skin-draining lymph nodes, and a previously undescribed population in Peyer's patches (PP). PP-NKT cells produce the majority of the IL-4 in Peyer's patches and provide indirect help for B-cell class switching to IgG1 in both transnuclear and wild-type mice. Oral vaccination with α-galactosylceramide shows enhanced fecal IgG1 titers in iNKT cell-sufficient mice. Transcriptional profiling reveals a unique signature of PP-NKT cells, characterized by tissue residency. We thus define PP-NKT as potentially important for surveillance for mucosal pathogens.


Assuntos
Perfilação da Expressão Gênica/métodos , Switching de Imunoglobulina , Imunoglobulina G/genética , Células T Matadoras Naturais/metabolismo , Nódulos Linfáticos Agregados/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Galactosilceramidas/administração & dosagem , Galactosilceramidas/imunologia , Interleucina-4/genética , Camundongos , Células T Matadoras Naturais/citologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Técnicas de Transferência Nuclear , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteínas com Domínio T/genética , Vacinação
10.
Mol Immunol ; 112: 198-205, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31176199

RESUMO

AID initiates both somatic hypermutation (SHM) and class switch recombination (CSR) in Ig genes. AID-induced mutations are linked with transcription initiation and elongation. Transcription occurs in the context of chromatin and thus RNA PolII and AID need to deal with nucleosomes. Both nucleosome stability and positioning significantly influence the accessibility of AID to Ig genes and the SHM pattern. Interestingly, in the nucleosome, SHM process seems to have a preference for the top strand. To know whether the preferential targeting of SHM to the top strand is due to a post-AID event, we expressed an inhibitor of Uracil DNA glycosylase (UNG), Ugi, into DT40 cells containing the nucleosome positioning sequence (MP2) and compared the SHM pattern. We observed a similar preference to the top strand for the high-affinity nucleosome positioning sequence in UNG inhibited cells. Furthermore, to understand whether the primary sequence of nucleosome sequence is influencing preferential targeting, we introduced two copies of MP2 sequence in the reverse orientation (MP2R) into a variable Ig gene. We observed that the MP2R cells also demonstrated preferential targeting of the non-transcribed strand in nucleosome as compared to the transcribed strand, confirming that in nucleosome sequences AID has better access to Cs on the top strand. The preferential targeting of AID on the top strand suggests that RNA Pol-II stalls while it transcribes the stable nucleosomes, thus giving ample opportunity for the transcribed strand to form R-loops with the nascent RNA, thereby gives limited access to AID on the bottom strand.


Assuntos
Citidina Desaminase/genética , Nucleossomos/genética , Animais , Linfócitos B/fisiologia , Linhagem Celular , Galinhas , Genes de Imunoglobulinas/genética , Switching de Imunoglobulina/genética , RNA Polimerase II/genética , Hipermutação Somática de Imunoglobulina/genética , Uracila-DNA Glicosidase/genética
11.
PLoS Genet ; 15(4): e1008101, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30946744

RESUMO

Class switch recombination (CSR) requires activation-induced cytidine deaminase (AID) to trigger DNA double strand breaks (DSBs) at the immunoglobulin heavy chain (IGH) in B cells. Joining of AID-dependent DSBs within IGH facilitate CSR and effective humoral immunity, but ligation to DSBs in non-IGH chromosomes leads to chromosomal translocations. Thus, the mechanism by which AID-dependent DSBs are repaired requires careful examination. The random activity of AID in IGH leads to a spectrum of DSB structures. In this report, we investigated how DSB structure impacts end-joining leading to CSR and chromosomal translocations in human B cells, for which models of CSR are inefficient and not readily available. Using CRISPR/Cas9 to model AID-dependent DSBs in IGH and non-IGH genes, we found that DSBs with 5' and 3' overhangs led to increased processing during end-joining compared to blunt DSBs. We observed that 5' overhangs were removed and 3' overhangs were filled in at recombination junctions, suggesting that different subsets of enzymes are required for repair based on DSB polarity. Surprisingly, while Cas9-mediated switching preferentially utilized NHEJ regardless of DSB structure, A-EJ strongly preferred repairing blunt DSBs leading to translocations in the absence of NHEJ. We found that DSB polarity influenced frequency of Cas9-mediated switching and translocations more than overhang length. Lastly, recombination junctions from staggered DSBs exhibited templated insertions, suggesting iterative resection and filling in during repair. Our results demonstrate that DSB structure biases repair towards NHEJ or A-EJ to complete recombination leading to CSR and translocations, thus helping to elucidate the mechanism of genome rearrangements in human B cells.


Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Quebras de DNA de Cadeia Dupla , Switching de Imunoglobulina , Translocação Genética , Sequência de Bases , Sistemas CRISPR-Cas , Linhagem Celular , Citidina Desaminase/metabolismo , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA por Junção de Extremidades/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Modelos Genéticos , Proteínas Proto-Oncogênicas c-bcl-6/genética , Recombinação Genética
12.
J Immunol Res ; 2019: 9561350, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30906792

RESUMO

Neutrophils have recently been proposed as cells with high functional plasticity and are involved in the pathogenesis of infections, malignancy, and autoimmune diseases. However, less is known about the role of neutrophil in humoral response. In this study, we examined the importance of neutrophils and the neutrophil-derived DAMP protein, MRP14, in antibody production. Splenic neutrophils and MRP14 that are present in the splenic peri-MZ region have a close contact with MZ B cells and promote their differentiation into plasma cells. Using neutrophil-depleting mice and an MRP14-blocking compound, we showed that the presence of neutrophil and MRP14 is required for class switch, plasma cell maintenance, and antibody production in the spleen. We found that MRP14 could also be produced by neutrophils in the bone marrow and support the maintenance of bone marrow plasma cells. MRP14 binding could enhance the effect of the BAFF signal and protect primary multiple myeloma cells from doxorubicin-induced apoptosis. Our data demonstrate the effects of neutrophils on neighboring B cells and plasma cells, which provides new insights into the connection between neutrophil and humoral responses.


Assuntos
Alarminas/metabolismo , Linfócitos B/fisiologia , Células da Medula Óssea/fisiologia , Calgranulina B/metabolismo , Imunidade Humoral , Neutrófilos/imunologia , Plasmócitos/fisiologia , Animais , Anticorpos/metabolismo , Comunicação Celular , Diferenciação Celular , Plasticidade Celular , Células Cultivadas , Humanos , Switching de Imunoglobulina , Camundongos , Camundongos Endogâmicos C57BL
13.
Nucleic Acids Res ; 47(9): 4569-4585, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30838409

RESUMO

UNG is the major uracil-DNA glycosylase in mammalian cells and is involved in both error-free base excision repair of genomic uracil and mutagenic uracil-processing at the antibody genes. However, the regulation of UNG in these different processes is currently not well understood. The UNG gene encodes two isoforms, UNG1 and UNG2, each possessing unique N-termini that mediate translocation to the mitochondria and the nucleus, respectively. A strict subcellular localization of each isoform has been widely accepted despite a lack of models to study them individually. To determine the roles of each isoform, we generated and characterized several UNG isoform-specific mouse and human cell lines. We identified a distinct UNG1 isoform variant that is targeted to the cell nucleus where it supports antibody class switching and repairs genomic uracil. We propose that the nuclear UNG1 variant, which in contrast to UNG2 lacks a PCNA-binding motif, may be specialized to act on ssDNA through its ability to bind RPA. RPA-coated ssDNA regions include both transcribed antibody genes that are targets for deamination by AID and regions in front of the moving replication forks. Our findings provide new insights into the function of UNG isoforms in adaptive immunity and DNA repair.


Assuntos
DNA Glicosilases/genética , Reparo do DNA/genética , Switching de Imunoglobulina/genética , Recombinação Genética/genética , Uracila-DNA Glicosidase/genética , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Núcleo Celular/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Técnicas de Inativação de Genes , Genoma/genética , Humanos , Camundongos , Antígeno Nuclear de Célula em Proliferação/genética , Isoformas de Proteínas/genética , Uracila/metabolismo
14.
Immun Inflamm Dis ; 7(2): 74-85, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30859748

RESUMO

INTRODUCTION: Allergen-specific immunoglobulin isotype formation associated with immunoglobulin class-switching during the lactation period is the immunological background for food allergy in infants. We analyzed the serial changes in the production of feeding type-related egg- and milk-specific immunoglobulin isotypes from birth to 6 months of age with or without eczema in 84 infants. METHODS: Allergen-specific immunoglobulin G1 (IgG1), IgG2, IgG3, IgG4, IgA, and IgE levels of hen's egg and bovine milk were measured in cord blood and blood samples from infants at 2, 4, and 6 months of age by the densely carboxylated protein microarray. RESULTS: Formula and mixed feeding were associated with a rapid increase in cow's milk allergen-specific immunoglobulins and feeding type-related significant differences in casein-specific immunoglobulin levels were detected. Breast and mixed feeding were associated with slow but significant increase in ovalbumin-specific IgG1 and IgE levels, but not other immunoglobulins. We found two different immunoglobulin isotype formation at 6 months of age with low- or high-affinity IgE against ovalbumin. One isotype formation pattern had relatively high ovalbumin-specific IgG1 levels, detectable IgG2, and low-affinity IgE, while the other had low ovalbumin-specific IgG1 levels, undetectable IgG2, and high levels of high-affinity IgE. The incidence of eczema was significantly higher in the latter pattern (84.6%), compared with the remaining infants (42.2%). CONCLUSIONS: Feeding practice-related allergen sensitization and immunoglobulin isotype formation were identified during the lactation period. The development of eczema during the lactation period could potentially modify the immunoglobulin isotype formation with high levels of high-affinity IgE.


Assuntos
Alérgenos/imunologia , Eczema/imunologia , Hipersensibilidade a Ovo/imunologia , Ovos/efeitos adversos , Switching de Imunoglobulina/imunologia , Imunoglobulina E/imunologia , Hipersensibilidade a Leite/imunologia , Leite/efeitos adversos , Fatores Etários , Animais , Afinidade de Anticorpos/imunologia , Formação de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Bovinos , Galinhas , Eczema/complicações , Hipersensibilidade a Ovo/complicações , Hipersensibilidade a Ovo/genética , Feminino , Humanos , Isotipos de Imunoglobulinas/genética , Isotipos de Imunoglobulinas/imunologia , Lactente , Recém-Nascido , Masculino , Hipersensibilidade a Leite/complicações , Hipersensibilidade a Leite/genética , Gravidez
15.
J Biol Chem ; 294(13): 5198-5207, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30705092

RESUMO

The base excision repair (BER) pathway is an important DNA repair pathway and is essential for immune responses. In fact, it regulates both the antigen-stimulated somatic hypermutation (SHM) process and plays a central function in the process of class switch recombination (CSR). For both processes, a central role for apurinic/apyrimidinic endonuclease 1 (APE1) has been demonstrated. APE1 acts also as a master regulator of gene expression through its redox activity. APE1's redox activity stimulates the DNA-binding activity of several transcription factors, including NF-κB and a few others involved in inflammation and in immune responses. Therefore, it is possible that APE1 has a role in regulating the CSR through its function as a redox coactivator. The present study was undertaken to address this question. Using the CSR-competent mouse B-cell line CH12F3 and a combination of specific inhibitors of APE1's redox (APX3330) and repair (compound 3) activities, APE1-deficient or -reconstituted cell lines expressing redox-deficient or endonuclease-deficient proteins, and APX3330-treated mice, we determined the contributions of both endonuclease and redox functions of APE1 in CSR. We found that APE1's endonuclease activity is essential for IgA-class switch recombination. We provide evidence that the redox function of APE1 appears to play a role in regulating CSR through the interleukin-6 signaling pathway and in proper IgA expression. Our results shed light on APE1's redox function in the control of cancer growth through modulation of the IgA CSR process.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Imunoglobulina A/genética , Switching de Imunoglobulina , Animais , Linfócitos B/metabolismo , Linhagem Celular , Reparo do DNA , Humanos , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Transdução de Sinais
16.
Science ; 363(6428): 748-753, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30765568

RESUMO

B2 cells engage in classical antibody responses, whereas B1 cells are considered carriers of innate immunity, biased toward recognizing epitopes present on the surfaces of common pathogens and self antigens. To explore the role of B cell antigen receptor (BCR) specificity in driving B1 cell differentiation, we developed a transgenic system allowing us to change BCR specificity in B cells in an inducible and programmed manner. Mature B2 cells differentiated into bona fide B1 cells upon acquisition of a B1 cell-typical self-reactive BCR through a phase of proliferative expansion. Thus, B2 cells have B1 cell differentiation potential in addition to their classical capacity to differentiate into memory and plasma cells, and B1 differentiation can be instructed by BCR-mediated self-reactivity, in the absence of B1-lineage precommitment.


Assuntos
Subpopulações de Linfócitos B/citologia , Diferenciação Celular/imunologia , Plasticidade Celular/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Diferenciação Celular/genética , Linhagem da Célula , Plasticidade Celular/genética , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/genética , Transcriptoma
17.
J Cutan Pathol ; 46(5): 380-382, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30681733

RESUMO

We report a case of IgG/IgA pemphigus presenting as pemphigus foliaceus following diagnosis and treatment of classic IgG-mediated pemphigus vulgaris. The dual presentation of IgG and IgA positivity on direct immunofluorescence (DIF) constitutes a rare form of pemphigus with a wide variety of clinicopathologic manifestations. The progression of pemphigus vulgaris is commonly because of epitope spreading. However, the patient's disease was partially refractory to rituximab and showed a change in the DIF with dual staining for IgG and IgA. This indicates that class-switching may have occurred with epitope spreading or that there was autoreactive IgA at the onset of disease below the threshold of detection by DIF. Our case indicates that in progressive autoimmune disease refractory to treatment, re-evaluation of the patient for antibody isotypes absent on initial diagnosis may offer key information in better identifying the cause of progression as well as in directing the necessary treatment.


Assuntos
Autoanticorpos/metabolismo , Epitopos/metabolismo , Imunoglobulina A/metabolismo , Switching de Imunoglobulina , Imunoglobulina G/metabolismo , Pênfigo , Técnica Direta de Fluorescência para Anticorpo , Humanos , Masculino , Pessoa de Meia-Idade , Pênfigo/metabolismo , Pênfigo/patologia
18.
PLoS One ; 14(1): e0210526, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30629682

RESUMO

C9orf82 protein, or conserved anti-apoptotic protein 1 or caspase activity and apoptosis inhibitor 1 (CAAP1) has been implicated as a negative regulator of the intrinsic apoptosis pathway by modulating caspase expression and activity. In contrast, an independent genome wide screen for factors capable of driving drug resistance to the topoisomerase II (Topo II) poisons doxorubicin and etoposide, implicated a role for the nuclear protein C9orf82 in delaying DSBs repair downstream of Topo II, hereby sensitizing cells to DSB induced apoptosis. To determine its function in a genetically defined setting in vivo and ex vivo, we here employed CRISPR/Cas9 technology in zygotes to generate a C9orf82 knockout mouse model. C9orf82ko/ko mice were born at a Mendelian ratio and did not display any overt macroscopic or histological abnormalities. DSBs repair dependent processes like lymphocyte development and class switch recombination (CSR) appeared normal, arguing against a link between the C9orf82 encoded protein and V(D)J recombination or CSR. Most relevant, primary pre-B cell cultures and Tp53 transformed mouse embryo fibroblasts (MEFs) derived from C9orf82ko/ko E14.5 and wild type embryos displayed comparable sensitivity to a number of DNA lesions, including DSBs breaks induced by the topoisomerase II inhibitors, etoposide and doxorubicin. Likewise, the kinetics of γH2AX formation and resolution in response to etoposide of C9orf82 protein proficient, deficient and overexpressing MEFs were indistinguishable. These data argue against a direct role of C9orf82 protein in delaying repair of Topo II generated DSBs and regulating apoptosis. The genetically defined systems generated in this study will be of value to determine the actual function of C9orf82 protein.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo II/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Linfócitos B/citologia , Linfócitos B/metabolismo , Sistemas CRISPR-Cas , Caspase 3/metabolismo , Proliferação de Células , Células Cultivadas , Dano ao DNA , Reparo do DNA , Switching de Imunoglobulina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/citologia , Linfócitos T/metabolismo
20.
J Immunol ; 202(2): 579-590, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30530594

RESUMO

During an adaptive immune response, activated mature B cells give rise to Ab-secreting plasma cells to fight infection. B cells undergo Ab class switching to produce different classes of Abs with varying effector functions. The mammalian/mechanistic target of rapamycin (mTOR) signaling pathway is activated during this process, and disrupting mTOR complex 1 (mTORC1) in B cells impairs class switching by a poorly understood mechanism. In particular, it is unclear which mTORC1 downstream substrates control this process. In this study, we used an in vitro murine model in which the mTORC1 inhibitor rapamycin, when added after a B cell has committed to divide, suppresses class switching while preserving proliferation. Investigation of mTORC1 substrates revealed a role for eukaryotic translation initiation factor 4E (eIF4E) and eIF4E-binding proteins in class switching. Mechanistically, we show that genetic or pharmacological disruption of eIF4E binding to eIF4G reduced cap-dependent translation, which specifically affected the expression of activation-induced cytidine deaminase protein but not Aicda mRNA. This translational impairment decreased Ab class switching independently of proliferation. These results uncover a previously undescribed role for mTORC1 and the eIF4E-binding proteins/eIF4E axis in activation-induced cytidine deaminase protein expression and Ab class switching in mouse B cells, suggesting that cap-dependent translation regulates key steps in B cell differentiation.


Assuntos
Linfócitos B/imunologia , Proteínas de Transporte/imunologia , Fator de Iniciação 4E em Eucariotos/imunologia , Switching de Imunoglobulina , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Fosfoproteínas/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linfócitos B/efeitos dos fármacos , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Células Cultivadas , Fator de Iniciação 4E em Eucariotos/genética , Fatores de Iniciação em Eucariotos , Regulação da Expressão Gênica , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoproteínas/genética , Ligação Proteica , Biossíntese de Proteínas , Transdução de Sinais , Sirolimo/farmacologia
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