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1.
Mol Immunol ; 114: 545-552, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31521018

RESUMO

Antibodies possessing high affinity and specificity are desired as therapeutic reagents and biosensor materials. Such antibodies are often obtained from immunized animals through the process referred to as affinity maturation where antibody affinity increases with time after immunization. Somatic hypermutation (SHM) was shown to be involved in this process; however, structural basis of affinity maturation has not well been understood yet. We analyzed the crystal structure of a high affinity anti-(4-hydroxy-3-nitrophenyl)acetyl antibody, C6, possessing Gly at position 95 of heavy chain and 17 amino acid replacements by SHM. Here, we discuss how the amino acid residues at position 95, introduced at a junction of VH and DH gene segments during gene-recombination, as well as those replaced by SHM contribute to increasing the affinity by comparing the C6 structure with that of a germline low affinity antibody, N1G9, possessing Tyr at position 95.


Assuntos
Anticorpos Monoclonais/química , Afinidade de Anticorpos/imunologia , Glicina/química , Cadeias Pesadas de Imunoglobulinas/química , Nitrofenóis/química , Sequência de Aminoácidos , Hipermutação Somática de Imunoglobulina/imunologia
2.
Nat Genet ; 51(3): 560-567, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30742113

RESUMO

Tumor-infiltrating B cells are an important component in the microenvironment but have unclear anti-tumor effects. We enhanced our previous computational algorithm TRUST to extract the B cell immunoglobulin hypervariable regions from bulk tumor RNA-sequencing data. TRUST assembled more than 30 million complementarity-determining region 3 sequences of the B cell heavy chain (IgH) from The Cancer Genome Atlas. Widespread B cell clonal expansions and immunoglobulin subclass switch events were observed in diverse human cancers. Prevalent somatic copy number alterations in the MICA and MICB genes related to antibody-dependent cell-mediated cytotoxicity were identified in tumors with elevated B cell activity. The IgG3-1 subclass switch interacts with B cell-receptor affinity maturation and defects in the antibody-dependent cell-mediated cytotoxicity pathway. Comprehensive pancancer analyses of tumor-infiltrating B cell-receptor repertoires identified novel tumor immune evasion mechanisms through genetic alterations. The IgH sequences identified here are potentially useful resources for future development of immunotherapies.


Assuntos
Linfócitos B/imunologia , Evasão da Resposta Imune/imunologia , Neoplasias/imunologia , Sequência de Bases , Regiões Determinantes de Complementaridade/imunologia , Humanos , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Análise de Sequência de RNA/métodos , Hipermutação Somática de Imunoglobulina/imunologia
3.
BMC Infect Dis ; 18(1): 613, 2018 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-30509199

RESUMO

BACKGROUND: Community-acquired pneumonia is a leading infectious cause of hospitalization. A few vaccines exist to prevent pneumococcal disease in adults, including a pneumococcal polysaccharide unconjugated vaccine and a protein conjugated polysaccharide vaccine. Previous studies on the human immune response to the unconjugated vaccine showed that the vaccine boosted the existing memory B cells. In the present study, we investigated the human B cell immune response following pneumococcal polysaccharide conjugate vaccination. METHODS: Plasmablast B cells from a pneumococcal polysaccharide conjugate vaccinee were isolated and cloned for analysis. In response to primary vaccination, identical sequences from the plasmablast-derived antibodies were identified from multiple B cells, demonstrating evidence of clonal expansion. We evaluated the binding specificity of these human monoclonal antibodies in immunoassays, and tested there in vitro function in a multiplexed opsonophagocytic assay (MOPA). To characterize the plasmablast B cell response to the pneumococcal conjugated vaccine, the germline usage and the variable region somatic hypermutations on these antibodies were analyzed. Furthermore, a serotype 4 polysaccharide-specific antibody was tested in an animal challenge study to explore the in vivo functional activity. RESULTS: The data suggests that the pneumococcal polysaccharide conjugate vaccine boosted memory B cell responses, likely derived from previous pneumococcal exposure. The majority of the plasmablast-derived antibodies contained higher numbers of variable region somatic hypermutations and evidence for selection, as demonstrated by replacement to silent ratio's (R/S) greater than 2.9 in the complementarity-determining regions (CDRs). In addition, we found that VH3/JH4 was the predominant germline sequence used in these polysaccharide-specific B cells. All of the tested antibodies demonstrated narrow polysaccharide specificity in ELISA binding, and demonstrated functional opsonophagocytic killing (OPK) activity in the MOPA assay. The in-vivo animal challenge study showed that the tested serotype 4 polysaccharide-specific antibody demonstrated a potent protective effect when administered prior to bacterial challenge. CONCLUSIONS: The findings on the pneumococcal polysaccharide conjugate vaccine responses from a vaccinated subject reported in this study are similar to previously published data on the pneumococcal polysaccharide unconjugated vaccine responses. In both vaccine regimens, the pre-existing human memory B cells were expanded after vaccination with preferential use of the germline VH3/JH4 genes.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Linfócitos B/imunologia , Memória Imunológica , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/uso terapêutico , Hipermutação Somática de Imunoglobulina , Adulto , Animais , Anticorpos Antibacterianos/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Feminino , Rearranjo Gênico do Linfócito B/genética , Rearranjo Gênico do Linfócito B/imunologia , Humanos , Memória Imunológica/genética , Memória Imunológica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções Pneumocócicas/genética , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas/imunologia , Sorogrupo , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Streptococcus pneumoniae/imunologia , Vacinação , Vacinas Conjugadas/imunologia , Vacinas Conjugadas/uso terapêutico
6.
Proc Natl Acad Sci U S A ; 115(19): 4921-4926, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29669924

RESUMO

Activation-induced deaminase (AID) initiates hypermutation of Ig genes in activated B cells by converting C:G into U:G base pairs. G1-phase variants of uracil base excision repair (BER) and mismatch repair (MMR) then deploy translesion polymerases including REV1 and Pol η, which exacerbates mutation. dNTP paucity may contribute to hypermutation, because dNTP levels are reduced in G1 phase to inhibit viral replication. To derestrict G1-phase dNTP supply, we CRISPR-inactivated SAMHD1 (which degrades dNTPs) in germinal center B cells. Samhd1 inactivation increased B cell virus susceptibility, increased transition mutations at C:G base pairs, and substantially decreased transversion mutations at A:T and C:G base pairs in both strands. We conclude that SAMHD1's restriction of dNTP supply enhances AID's mutagenicity and that the evolution of Ig hypermutation included the repurposing of antiviral mechanisms based on dNTP starvation.


Assuntos
Linfócitos B/imunologia , Fase G1/imunologia , Ativação Linfocitária , Mutação , Proteína 1 com Domínio SAM e Domínio HD , Hipermutação Somática de Imunoglobulina/imunologia , Animais , Linfócitos B/citologia , Citidina Desaminase/imunologia , Fase G1/genética , Masculino , Camundongos , Camundongos Transgênicos , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/imunologia
7.
Immunity ; 48(3): 500-513.e6, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29548671

RESUMO

Virtually the entire surface of the HIV-1-envelope trimer is recognized by neutralizing antibodies, except for a highly glycosylated region at the center of the "silent face" on the gp120 subunit. From an HIV-1-infected donor, #74, we identified antibody VRC-PG05, which neutralized 27% of HIV-1 strains. The crystal structure of the antigen-binding fragment of VRC-PG05 in complex with gp120 revealed an epitope comprised primarily of N-linked glycans from N262, N295, and N448 at the silent face center. Somatic hypermutation occurred preferentially at antibody residues that interacted with these glycans, suggesting somatic development of glycan recognition. Resistance to VRC-PG05 in donor #74 involved shifting of glycan-N448 to N446 or mutation of glycan-proximal residue E293. HIV-1 neutralization can thus be achieved at the silent face center by glycan-recognizing antibody; along with other known epitopes, the VRC-PG05 epitope completes coverage by neutralizing antibody of all major exposed regions of the prefusion closed trimer.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Polissacarídeos/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Antígenos Virais/química , Antígenos Virais/imunologia , Sítios de Ligação , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Glicopeptídeos/química , Glicopeptídeos/imunologia , Glicosilação , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Polissacarídeos/química , Ligação Proteica/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Relação Estrutura-Atividade
8.
Arthritis Rheumatol ; 70(7): 1102-1113, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29457375

RESUMO

OBJECTIVE: To better understand the role of B cells, the potential mechanisms responsible for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjögren's syndrome (SS), this study explored patterns of selection pressure and sites of N-glycosylation acquired by somatic mutation (acN-glyc) in the IgG variable (V) regions of antibody-secreting cells (ASCs) isolated from the minor salivary glands of patients with SS and non-SS control patients with sicca symptoms. METHODS: A novel method to produce and characterize recombinant monoclonal antibodies (mAb) from single cell-sorted ASC infiltrates was applied to concurrently probe expressed genes (all heavy- and light-chain isotypes as well as any other gene of interest not related to immunoglobulin) in the labial salivary glands of patients with SS and non-SS controls. V regions were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed for the incidence of N-glycosylation and selection pressure. For specificity testing, the amplified regions were expressed as either the native mAb or mutant mAb lacking the acN-glyc motif. Protein modeling was used to demonstrate how even an acN-glyc site outside of the complementarity-determining region could participate in, or inhibit, antigen binding. RESULTS: V-region sequence analyses revealed clonal expansions and evidence of secondary light-chain editing and allelic inclusion, of which neither of the latter two have previously been reported in patients with SS. Increased frequencies of acN-glyc were found in the sequences from patients with SS, and these acN-glyc regions were associated with an increased number of replacement mutations and lowered selection pressure. A clonal set of polyreactive mAb with differential framework region 1 acN-glyc motifs was also identified, and removal of the acN-glyc could nearly abolish binding to autoantigens. CONCLUSION: These findings support the notion of an alternative mechanism for the selection and proliferation of some autoreactive B cells, involving V-region N-glycosylation, in patients with SS.


Assuntos
Células Produtoras de Anticorpos/metabolismo , Linfócitos B/imunologia , Glândulas Salivares/imunologia , Síndrome de Sjogren/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Adulto , Idoso , Proliferação de Células/genética , Feminino , Glicosilação , Humanos , Ativação Linfocitária/fisiologia , Masculino , Pessoa de Meia-Idade , Glândulas Salivares/citologia , Síndrome de Sjogren/genética
9.
Int J Mol Sci ; 18(9)2017 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-28867784

RESUMO

Lymphocytes are endowed with unique and specialized enzymatic mutagenic properties that allow them to diversify their antigen receptors, which are crucial sensors for pathogens and mediators of adaptive immunity. During lymphocyte development, the antigen receptors expressed by B and T lymphocytes are assembled in an antigen-independent fashion by ordered variable gene segment recombinations (V(D)J recombination), which is a highly ordered and regulated process that requires the recombination activating gene products 1 & 2 (RAG1, RAG2). Upon activation by antigen, B lymphocytes undergo additional diversifications of their immunoglobulin B-cell receptors. Enzymatically induced somatic hypermutation (SHM) and immunoglobulin class switch recombination (CSR) improves the affinity for antigen and shape the effector function of the humoral immune response, respectively. The activation-induced cytidine deaminase (AID) enzyme is crucial for both SHM and CSR. These processes have evolved to both utilize as well as evade different DNA repair and DNA damage response pathways. The delicate balance between enzymatic mutagenesis and DNA repair is crucial for effective immune responses and the maintenance of genomic integrity. Not surprisingly, disturbances in this balance are at the basis of lymphoid malignancies by provoking the formation of oncogenic mutations and chromosomal aberrations. In this review, we discuss recent mechanistic insight into the regulation of RAG1/2 and AID expression and activity in lymphocytes and the complex interplay between these mutagenic enzymes and DNA repair and DNA damage response pathways, focusing on the base excision repair and mismatch repair pathways. We discuss how disturbances of this interplay induce genomic instability and contribute to oncogenesis.


Assuntos
Reparo do DNA/genética , Imunidade Humoral/genética , Hipermutação Somática de Imunoglobulina/genética , Recombinação V(D)J/genética , Linfócitos B/imunologia , Citidina Desaminase/genética , Dano ao DNA/genética , Dano ao DNA/imunologia , Reparo do DNA/imunologia , Rearranjo Gênico/genética , Rearranjo Gênico/imunologia , Humanos , Mutagênese/genética , Mutagênese/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Linfócitos T/imunologia , Recombinação V(D)J/imunologia
10.
Proc Natl Acad Sci U S A ; 114(32): 8614-8619, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28747530

RESUMO

Variable regions of Ig chains provide the antigen recognition portion of B-cell receptors and derivative antibodies. Ig heavy-chain variable region exons are assembled developmentally from V, D, J gene segments. Each variable region contains three antigen-contacting complementarity-determining regions (CDRs), with CDR1 and CDR2 encoded by the V segment and CDR3 encoded by the V(D)J junction region. Antigen-stimulated germinal center (GC) B cells undergo somatic hypermutation (SHM) of V(D)J exons followed by selection for SHMs that increase antigen-binding affinity. Some HIV-1-infected human subjects develop broadly neutralizing antibodies (bnAbs), such as the potent VRC01-class bnAbs, that neutralize diverse HIV-1 strains. Mature VRC01-class bnAbs, including VRC-PG04, accumulate very high SHM levels, a property that hinders development of vaccine strategies to elicit them. Because many VRC01-class bnAb SHMs are not required for broad neutralization, high overall SHM may be required to achieve certain functional SHMs. To elucidate such requirements, we used a V(D)J passenger allele system to assay, in mouse GC B cells, sequence-intrinsic SHM-targeting rates of nucleotides across substrates representing maturation stages of human VRC-PG04. We identify rate-limiting SHM positions for VRC-PG04 maturation, as well as SHM hotspots and intrinsically frequent deletions associated with SHM. We find that mature VRC-PG04 has low SHM capability due to hotspot saturation but also demonstrate that generation of new SHM hotspots and saturation of existing hotspot regions (e.g., CDR3) does not majorly influence intrinsic SHM in unmutated portions of VRC-PG04 progenitor sequences. We discuss implications of our findings for bnAb affinity maturation mechanisms.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Anticorpos Neutralizantes/imunologia , Linfócitos B/metabolismo , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Mutação , Hipermutação Somática de Imunoglobulina/imunologia , Animais , Anticorpos Monoclonais Murinos/genética , Anticorpos Neutralizantes/genética , Anticorpos Anti-HIV/genética , Camundongos
11.
Immunotherapy ; 9(8): 659-667, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28653569

RESUMO

Mutable viruses, such as HIV, pose difficult obstacles to prevention and/or control by vaccination. Mutable viruses rapidly diversify in populations and in individuals, impeding development of effective vaccines. We devised the 'mutable vaccine' to appropriate the properties of mutable viruses that undermine conventional strategies. The vaccine consists of a DNA construct encoding viral antigen and regulatory sequences that upon delivery to B cells target the enzymatic apparatus of 'somatic hypermutation' causing the construct to mutate one million-times baseline rates and allowing production and presentation of antigen variants. We postulate the mutable vaccine might thus anticipate diversification of mutable viruses, allowing direct control or slowing of evolution. Initial work presented here should encourage consideration of this novel approach.


Assuntos
Vacinas contra a AIDS , Antígenos Virais , Infecções por HIV , HIV-1 , Mutação , Vacinas de DNA , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Vacinas contra a AIDS/uso terapêutico , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Linfócitos B/imunologia , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/terapia , HIV-1/genética , HIV-1/imunologia , Humanos , Hipermutação Somática de Imunoglobulina/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêutico
12.
Immunol Lett ; 188: 46-52, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28610800

RESUMO

Humanized mouse models present an important tool for preclinical evaluation of new vaccines and therapeutics. Here we show the human variable repertoire of antibody sequences cloned from a previously described human immune system (HIS) mouse model that possesses functional human CD4+ T cells and B cells, namely HIS-CD4/B mice. We sequenced variable IgG genes from single memory B-cell and plasma-cell sorted from splenocytes or whole blood lymphocytes of HIS-CD4/B mice that were vaccinated with a human plasmodial antigen, a recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP). We demonstrate that rPfCSP immunization triggers a diverse B-cell IgG repertoire composed of various human VH family genes and distinct V(D)J recombinations that constitute diverse CDR3 sequences similar to humans, although low hypermutated sequences were generated. These results demonstrate the substantial genetic diversity of responding human B cells of HIS-CD4/B mice and their capacity to mount human IgG class-switched antibody response upon vaccination.


Assuntos
Antígenos de Protozoários/imunologia , Sistema Imunitário/imunologia , Imunoglobulina G/imunologia , Malária/imunologia , Plasmodium/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Modelos Animais de Doenças , Amplificação de Genes , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Xenoenxertos , Humanos , Imunoglobulina G/genética , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Malária/parasitologia , Camundongos , Camundongos Transgênicos , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
13.
Oncotarget ; 8(25): 40079-40089, 2017 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-28445143

RESUMO

The immunoglobulin heavy chain (IGH) gene loci are subject to specific recombination events during B-cell differentiation including somatic hypermutation and class switch recombination which mark the end of immunoglobulin gene maturation in germinal centers of secondary lymph nodes. These two events rely on the activity of activation-induced cytidine deaminase (AID) which requires DNA double strand breaks be created, a potential danger to the cell. Applying 3D-fluorescence in situ hybridization coupled with immunofluorescence staining to a previously described experimental system recapitulating normal B-cell differentiation ex vivo, we have kinetically analyzed the radial positioning of the two IGH gene loci as well as their proximity with the nucleolus, heterochromatin and γH2AX foci. Our observations are consistent with the proposal that these IGH gene rearrangements take place in a specific perinucleolar "recombination compartment" where AID could be sequestered thus limiting the extent of its potentially deleterious off-target effects.


Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Nucléolo Celular/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Nucléolo Celular/metabolismo , Células Cultivadas , Citidina Desaminase/imunologia , Citidina Desaminase/metabolismo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Humanos , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Hibridização in Situ Fluorescente/métodos , Ativação Linfocitária/imunologia , Microscopia Confocal , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia
14.
Mol Immunol ; 87: 47-59, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28407558

RESUMO

The integrin CD11b, which is encoded by the integrin subunit alpha M (ITGAM), is primarily expressed on the surface of innate immune cells. Genetic variations in ITGAM are among the strongest risk factors for systemic lupus erythematosus, an autoimmune disease characterized by the presence of autoantibodies. However, the regulatory function of CD11b in the antibody responses remains unclear. Here, we report the induction of CD11b in activated B2 B cells and define its unexpected role in immunoglobulin heavy chain class switch recombination (CSR). LPS-activated B cells lacking CD11b yielded fewer IgG subtypes such as IgG1 and IgG2a in vitro, and immunization-dependent CSR and affinity maturation of antibodies were severely impaired in CD11b-deficient mice. Notably, we observed the reduced expression of activation-induced cytidine deaminase (AID), an enzyme that initiates CSR and somatic hypermutation, and ectopic expression of AID was sufficient to rescue the defective CSR of CD11b-deficient B cells. LPS-induced phosphorylation of NF-κB p65 and IκBα was attenuated in CD11b-deficient B cells, and hyperactivation of IκB kinase 2 restored the defective AID expression and CSR, which implied that CD11b regulates the NF-κB-dependent induction of AID. Overall, our experimental evidence emphasized the function of CD11b in antibody responses and the role of CD11b as a vital regulator of CSR.


Assuntos
Anticorpos/imunologia , Formação de Anticorpos/imunologia , Antígeno CD11b/imunologia , Citidina Desaminase/imunologia , Switching de Imunoglobulina/imunologia , Animais , Linfócitos B , Regulação da Expressão Gênica/imunologia , Quinase I-kappa B/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/imunologia , Recombinação Genética/imunologia , Hipermutação Somática de Imunoglobulina/imunologia
15.
Clin Immunol ; 176: 77-86, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28104464

RESUMO

BACKGROUND: Mutations in PIK3CD and PIK3R1 cause activated PI3K-δ syndrome (APDS) by dysregulation of the PI3K-AKT pathway. METHODS: We studied precursor and peripheral B-cell differentiation and apoptosis via flowcytometry. Furthermore, we performed AKT-phosphorylation assays and somatic hypermutations (SHM) and class switch recombination (CSR) analysis. RESULTS: We identified 13 patients of whom 3 had new mutations in PIK3CD or PIK3R1. Patients had low total B-cell numbers with increased frequencies of transitional B cells and plasmablasts, while the precursor B-cell compartment in bone marrow was relatively normal. Basal AKT phosphorylation was increased in lymphocytes from APDS patients and natural effector B cells where most affected. PI3K mutations resulted in altered SHM and CSR and increased apoptosis. CONCLUSIONS: The B-cell compartment in APDS patients is affected by the mutations in PI3K. There is reduced differentiation beyond the transitional stage, increased AKT phosphorylation and increased apoptosis. This B-cell phenotype contributes to the clinical phenotype.


Assuntos
Agamaglobulinemia/genética , Agamaglobulinemia/imunologia , Linfócitos B/imunologia , Diferenciação Celular/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/genética , Adolescente , Adulto , Diferenciação Celular/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Mutação/genética , Mutação/imunologia , Fosforilação/genética , Plasmócitos/imunologia , Células Precursoras de Linfócitos B/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Recidiva , Transdução de Sinais/genética , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Adulto Jovem
16.
PLoS One ; 11(11): e0164567, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27828971

RESUMO

We have developed a new bioinformatics framework for the analysis of rearranged bovine heavy chain immunoglobulin (Ig) variable regions by combining and refining widely used alignment algorithms. This bioinformatics framework allowed us to investigate alignments of heavy chain framework regions (FRHs) and the separate alignments of FRHs and heavy chain complementarity determining regions (CDRHs) to determine their germline origin in the four cattle breeds Aubrac, German Black Pied, German Simmental, and Holstein Friesian. Now it is also possible to specifically analyze Ig heavy chains possessing exceptionally long CDR3Hs. In order to gain more insight into breed specific differences in Ig combinatorial diversity, somatic hypermutations and putative gene conversions of IgG, we compared the dominantly transcribed variable (IGHV), diversity (IGHD), and joining (IGHJ) segments and their recombination in the four cattle breeds. The analysis revealed the use of 15 different IGHV segments, 21 IGHD segments, and two IGHJ segments with significant different transcription levels within the breeds. Furthermore, there are preferred rearrangements within the three groups of CDR3H lengths. In the sequences of group 2 (CDR3H lengths (L) of 11-47 amino acid residues (aa)) a higher number of recombination was observed than in sequences of group 1 (L≤10 aa) and 3 (L≥48 aa). The combinatorial diversity of germline IGHV, IGHD, and IGHJ-segments revealed 162 rearrangements that were significantly different. The few preferably rearranged gene segments within group 3 CDR3H regions may indicate specialized antibodies because this length is unique in cattle. The most important finding of this study, which was enabled by using the bioinformatics framework, is the discovery of strong evidence for gene conversion as a rare event using pseudogenes fulfilling all definitions for this particular diversification mechanism.


Assuntos
Diversidade de Anticorpos/genética , Bovinos/genética , Biologia Computacional/métodos , Conversão Gênica , Cadeias Pesadas de Imunoglobulinas/genética , Algoritmos , Animais , Diversidade de Anticorpos/imunologia , Cruzamento , Bovinos/classificação , Bovinos/imunologia , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Rearranjo Gênico do Linfócito B/genética , Rearranjo Gênico do Linfócito B/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Análise de Sequência de DNA , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Especificidade da Espécie
17.
Mol Immunol ; 80: 78-90, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27835756

RESUMO

Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates somatic hypermutation (SH) and class switch recombination (CSR) by deaminating cytosine to uracil. The targeting of AID and therefore SH and CSR to Ig genes is a central process of the immune system, but the trans-acting factors mediating the specific targeting have remained elusive. Here we show that defective calmodulin inhibition of the transcription factor E2A after activation of the B cell receptor (BCR) leads to reduced BCR, IL4 plus CD40 ligand stimulated CSR to IgE and instead CSR to other Ig classes. AID that initiates CSR is shown to be in a complex with the transcription factors E2A, PAX5 and IRF4 on key sequences of the Igh locus. Calmodulin shows proximity with each of them after BCR stimulation. BCR signaling reduces binding of the proteins to some of the target sites on the Igh locus, and calmodulin resistance of E2A blocks these reductions. AID binds directly to the bHLH domain of E2A and to the PD domain of PAX5. E2A, AID, PAX5 and IRF4 are components of a CSR complex that is redistributed on the Igh locus by BCR signaling through calmodulin binding.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Citidina Desaminase/genética , Genes de Cadeia Pesada de Imunoglobulina/genética , Switching de Imunoglobulina/genética , Fatores Reguladores de Interferon/genética , Fator de Transcrição PAX5/genética , Animais , Linfócitos B/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Calmodulina/genética , Calmodulina/imunologia , Imunoprecipitação da Cromatina , Citidina Desaminase/imunologia , Citometria de Fluxo , Genes de Cadeia Pesada de Imunoglobulina/imunologia , Switching de Imunoglobulina/imunologia , Fatores Reguladores de Interferon/imunologia , Camundongos , Fator de Transcrição PAX5/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Ativação Transcricional
18.
J Clin Invest ; 126(11): 4289-4302, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27701145

RESUMO

Patients with mutations in AICDA, which encodes activation-induced cytidine deaminase (AID), display an impaired peripheral B cell tolerance. AID mediates class-switch recombination (CSR) and somatic hypermutation (SHM) in B cells, but the mechanism by which AID prevents the accumulation of autoreactive B cells in blood is unclear. Here, we analyzed B cell tolerance in AID-deficient patients, patients with autosomal dominant AID mutations (AD-AID), asymptomatic AICDA heterozygotes (AID+/-), and patients with uracil N-glycosylase (UNG) deficiency, which impairs CSR but not SHM. The low frequency of autoreactive mature naive B cells in UNG-deficient patients resembled that of healthy subjects, revealing that impaired CSR does not interfere with the peripheral B cell tolerance checkpoint. In contrast, we observed decreased frequencies of SHM in memory B cells from AD-AID patients and AID+/- subjects, who were unable to prevent the accumulation of autoreactive mature naive B cells. In addition, the individuals with AICDA mutations, but not UNG-deficient patients, displayed Tregs with defective suppressive capacity that correlated with increases in circulating T follicular helper cells and enhanced cytokine production. We conclude that SHM, but not CSR, regulates peripheral B cell tolerance through the production of mutated antibodies that clear antigens and prevent sustained interleukin secretions that interfere with Treg function.


Assuntos
Linfócitos B/imunologia , Pontos de Checagem do Ciclo Celular/imunologia , Citidina Desaminase/deficiência , Tolerância Imunológica , Memória Imunológica , Mutação , Hipermutação Somática de Imunoglobulina/imunologia , Linfócitos B/patologia , Pontos de Checagem do Ciclo Celular/genética , Citidina Desaminase/imunologia , Feminino , Humanos , Masculino , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia
19.
J Exp Med ; 213(7): 1255-65, 2016 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-27298445

RESUMO

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 , Vírus Vaccinia/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 fármacos
20.
J Exp Med ; 213(6): 921-8, 2016 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-27217538

RESUMO

Activation-induced cytidine deaminase (AID), the enzyme responsible for induction of sequence variation in immunoglobulins (Igs) during the process of somatic hypermutation (SHM) and also Ig class switching, can have a potent mutator phenotype in the development of lymphoma. Using various Epstein-Barr virus (EBV) recombinants, we provide definitive evidence that the viral nuclear protein EBNA3C is essential in EBV-infected primary B cells for the induction of AID mRNA and protein. Using lymphoblastoid cell lines (LCLs) established with EBV recombinants conditional for EBNA3C function, this was confirmed, and it was shown that transactivation of the AID gene (AICDA) is associated with EBNA3C binding to highly conserved regulatory elements located proximal to and upstream of the AICDA transcription start site. EBNA3C binding initiated epigenetic changes to chromatin at specific sites across the AICDA locus. Deep sequencing of cDNA corresponding to the IgH V-D-J region from the conditional LCL was used to formally show that SHM is activated by functional EBNA3C and induction of AID. These data, showing the direct targeting and induction of functional AID by EBNA3C, suggest a novel role for EBV in the etiology of B cell cancers, including endemic Burkitt lymphoma.


Assuntos
Linfoma de Burkitt/imunologia , Citidina Desaminase/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Rearranjo Gênico do Linfócito B/imunologia , Herpesvirus Humano 4/imunologia , Proteínas de Neoplasias/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Linfoma de Burkitt/genética , Linhagem Celular , Citidina Desaminase/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Rearranjo Gênico do Linfócito B/genética , Herpesvirus Humano 4/genética , Humanos , Masculino , Proteínas de Neoplasias/genética , Elementos de Resposta/imunologia , Hipermutação Somática de Imunoglobulina/genética
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