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1.
Front Immunol ; 13: 837246, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36569888

RESUMO

The mechanisms of B-cell diversification differ greatly between aves and mammals, but both produce B cells and antibodies capable of supporting an effective immune response. To see how differences in the generation of diversity might affect overall repertoire diversity, we have compared the diversity characteristics of immunoglobulin genes from domestic chickens to those from humans. Both use V(D)J gene rearrangement and somatic hypermutation, but only chickens use somatic gene conversion. A range of diversity analysis tools were used to investigate multiple aspects of amino acid diversity at both the germline and repertoire levels. The effect of differing amino acid usages on antibody characteristics was assessed. At both the germline and repertoire levels, chickens exhibited lower amino acid diversity in comparison to the human immunoglobulin genes, especially outside of the complementarity-determining region (CDR). Chickens were also found to possess much larger and more hydrophilic CDR3s with a higher predicted protein binding potential, suggesting that the antigen-binding site in chicken antibodies is more flexible and more polyreactive than that seen in human antibodies.


Assuntos
Galinhas , Regiões Determinantes de Complementaridade , Humanos , Animais , Regiões Determinantes de Complementaridade/genética , Galinhas/genética , Genes de Imunoglobulinas , Aminoácidos/genética , Diversidade de Anticorpos/genética , Anticorpos/genética , Mamíferos
2.
Mol Immunol ; 151: 231-241, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36179605

RESUMO

The antibody repertoire (Rep-seq) sequencing revolutionized the diversity of antigen B cell receptor studies, allowing deep and quantitative analysis to decipher the role of adaptive immunity in health and disease. Particularly, horse (Equus caballus) polyclonal antibodies have been produced and used since the century XIX to treat and prophylaxis diphtheria, tuberculosis, tetanus, pneumonia, and, more recently, COVID-19. However, our knowledge about the horse B cell receptors repertories is minimal. We present a deep horse antibody heavy chain repertoire (IGH) characterization of non-infected horses using NGS (Next generation sequencing). This study obtained a mean of 248,169 unique IgM clones and 66,141 unique IgG clones from four domestic adult horses. Rarefaction analysis showed sequence coverage was between 52 % and 82 % in IgM and IgG isotypes. We observed that besides horses antibody can use all functional IGHV genes, around 80 % of their antibodies use only three IGHV gene segments, and around 55 % use only one IGHJ gene segment. This limited VJ diversity seems to be compensated by the junctional diversity of these antibodies. We observed that the junctional diversity in horse antibodies is widespread, present in more than 90 % of horse antibodies. Besides this, the length of this region seems to be higher in horse antibodies than in other species. N1 and N2 nucleotides addition range from 0 to 111 nucleotides. In addition, around 45 % of the antibody clones have more than ten nucleotides in both the N1 and N2 junction regions. This diversity mechanism may be one of the most important in providing variability to the equine antibody repertoire. This study provides new insights regarding horse antibody composition, diversity generation, and particularities compared to other species, such as the frequency and length of N nucleotide addition. This study also points out the urgent need to better characterize TdT in horses and other species to better understand antibody repertoire characteristics.


Assuntos
COVID-19 , Animais , Diversidade de Anticorpos , Cavalos , Imunoglobulina G/genética , Imunoglobulina M/genética , Nucleotídeos , Receptores de Antígenos de Linfócitos B/genética
3.
Proc Natl Acad Sci U S A ; 119(36): e2205470119, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-36037353

RESUMO

Recombination of antibody genes in B cells can involve distant genomic loci and contribute a foreign antigen-binding element to form hybrid antibodies with broad reactivity for Plasmodium falciparum. So far, antibodies containing the extracellular domain of the LAIR1 and LILRB1 receptors represent unique examples of cross-chromosomal antibody diversification. Here, we devise a technique to profile non-VDJ elements from distant genes in antibody transcripts. Independent of the preexposure of donors to malaria parasites, non-VDJ inserts were detected in 80% of individuals at frequencies of 1 in 104 to 105 B cells. We detected insertions in heavy, but not in light chain or T cell receptor transcripts. We classify the insertions into four types depending on the insert origin and destination: 1) mitochondrial and 2) nuclear DNA inserts integrated at VDJ junctions; 3) inserts originating from telomere proximal genes; and 4) fragile sites incorporated between J-to-constant junctions. The latter class of inserts was exclusively found in memory and in in vitro activated B cells, while all other classes were already detected in naïve B cells. More than 10% of inserts preserved the reading frame, including transcripts with signs of antigen-driven affinity maturation. Collectively, our study unravels a mechanism of antibody diversification that is layered on the classical V(D)J and switch recombination.


Assuntos
Diversidade de Anticorpos , Linfócitos B , Genes de Imunoglobulinas , Anticorpos Antiprotozoários/genética , Antígenos CD/imunologia , Linfócitos B/imunologia , Genômica , Humanos , Cadeias Leves de Imunoglobulina/genética , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/imunologia , Mutagênese Insercional , Plasmodium falciparum , Receptores de Antígenos de Linfócitos T/genética , Receptores Imunológicos/imunologia
4.
mBio ; 13(4): e0183922, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-35856618

RESUMO

Inactivated influenza vaccines induce greater antibody responses in females than males among both humans and mice. To test the breadth of protection, we used recombinant mouse-adapted A/California/2009 (maA/Cal/09) H1N1 viruses containing mutations at one (1M), two (2M), or three (3M) antigenic sites, in addition to a virus containing the 1M mutation and a substitution of the Ca2 antigenic site (Sub) with one derived from an H5 hemagglutinin (HA) to challenge mice of both sexes. Following maA/Cal/09 vaccination, females produced greater virus-specific, class-switched total IgG and IgG2c antibodies against the vaccine and all mutant viruses, and antibodies from females recognized a greater number of unique, linear HA epitopes than did antibodies from males. While females had greater neutralizing antibody titers against the vaccine virus, both sexes showed a lower neutralization capacity against mutant viruses. After virus challenge, vaccinated females had lower pulmonary virus titers and reduced morbidity than males for the 1M and 2M viruses, but not the Sub virus. Females generated greater numbers of germinal center (GC) B cells containing superior somatic hypermutation (SHM) frequencies than vaccinated males. Deletion of activation-induced cytidine deaminase (Aicda) eliminated female-biased immunity and protection against the 2M virus. Harnessing methods to improve GC B cell responses and frequencies of SHM, especially in males, should be considered in the development of universal influenza vaccines. IMPORTANCE Adult females develop greater antibody responses to influenza vaccines than males. We hypothesized that female-biased immunity and protection would be dependent on the extent of virus diversity as well as molecular mechanisms in B cells which constrain the breadth of epitope recognition. We developed a panel of mouse-adapted (ma) A/Cal/09 viruses that had mutations in the immunodominant hemagglutinin. Following vaccination against maA/Cal/09, females were better able to neutralize maA/Cal/09 than males, but neutralization of mutant maA/Cal/09 viruses was equally poor in both sexes, despite vaccinated females being better protected against these viruses. Vaccinated females benefited from the greater production of class-switched, somatically hypermutated antibodies generated in germinal center B cells, which increased recognition of more diverse maA/Cal/09 hemagglutinin antigen epitopes. Female-biased protection against influenza infection and disease after vaccination is driven by differential mechanisms in males versus females and should be considered in the design of novel vaccine platforms.


Assuntos
Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza , Influenza Humana , Infecções por Orthomyxoviridae , Animais , Anticorpos Antivirais , Diversidade de Anticorpos , Epitopos , Feminino , Centro Germinativo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hemaglutininas , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Masculino , Camundongos , Vacinas de Produtos Inativados
5.
Genome Res ; 32(6): 1152-1169, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35545447

RESUMO

The V(D)J recombination process rearranges the variable (V), diversity (D), and joining (J) genes in the immunoglobulin (IG) loci to generate antibody repertoires. Annotation of these loci across various species and predicting the V, D, and J genes (IG genes) are critical for studies of the adaptive immune system. However, because the standard gene finding algorithms are not suitable for predicting IG genes, they have been semimanually annotated in very few species. We developed the IGDetective algorithm for predicting IG genes and applied it to species with the assembled IG loci. IGDetective generated the first large collection of IG genes across many species and enabled their evolutionary analysis, including the analysis of the "bat IG diversity" hypothesis. This analysis revealed extremely conserved V genes in evolutionary distant species, indicating that these genes may be subjected to the same selective pressure, for example, pressure driven by common pathogens. IGDetective also revealed extremely diverged V genes and a new family of evolutionary conserved V genes in bats with unusual noncanonical cysteines. Moreover, unlike all other previously reported antibodies, these cysteines are located within complementarity-determining regions. Because cysteines form disulfide bonds, we hypothesize that these cysteine-rich V genes might generate antibodies with noncanonical conformations and could potentially form a unique part of the immune repertoire in bats. We also analyzed the diversity landscape of the recombination signal sequences and revealed their features that trigger the high/low usage of the IG genes.


Assuntos
Diversidade de Anticorpos , Recombinação V(D)J , Anticorpos , Regiões Determinantes de Complementaridade/genética , Genes de Imunoglobulinas
6.
BMC Immunol ; 23(1): 12, 2022 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-35287587

RESUMO

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) causes impairment of T and B cell responses, which begins during the acute phase of infection and is not completely restored by antiretroviral treatment. Regulatory T cell (Tregs) can improve overall disease outcome by controlling chronic inflammation but may also suppress beneficial HIV-1 specific immune responses. We aimed to analyze the profile of Tregs and their correlation with the status of T cells activation, the expression of IL-2 and IFNγ and the profile of HIV-1 specific antibodies response in Mozambican people living chronically with HIV-1 (PLWH-C). RESULTS: In PLWH-C, the proportion of total Tregs was positively correlated with the proportion of IL-2+CD4 T cells (r = 0.647; p = 0.032) and IL-2+IFNγ+CD8 T cells (r = 0.551; p = 0.014), while the proportions of Helios+Tregs correlated inversely with levels of IL-2+CD8 T cells (r = - 0.541; p = 0.017). Overall, PLWH-C, with (82%) or without virologic suppression (64%), were seronegative for at least HIV-1 p31, gp160 or p24, and the breadth of antibody responses was positively correlated with proportions of CD38+HLA-DR+CD8 T cells (r = 0.620; p = 0.012), viral load (r = 0.452; p = 0.040) and inversely with absolute CD4 T cells count (r = - 0.481; p = 0.027). Analysis of all individuals living HIV-1 showed that the breadth of HIV-1 antibody responses was inversely correlated with the proportion of Helios+Tregs (r = - 0.45; p = 0.02). CONCLUSION: Among Mozambican people living with HIV-1, seronegativity to some HIV-1 proteins is common, particularly in virologically suppressed individuals. Furthermore, lower diversity of HIV-specific antibodies is correlated to lower immune activation, lower viral replication and higher CD4 counts, in PLWH-C. Elevation in the proportion of Helios+Tregs is related to a reduction of CD8 T expressing intracellular IL-2, in PLWH-C, but may contribute to impairment of B cell function.


Assuntos
Infecções por HIV , HIV-1 , Diversidade de Anticorpos , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Humanos , Interleucina-2/metabolismo , Ativação Linfocitária , Moçambique , Linfócitos T Reguladores
7.
Elife ; 112022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-35129436

RESUMO

Aging individuals exhibit a pervasive decline in adaptive immune function, with important implications for health and lifespan. Previous studies have found a pervasive loss of immune-repertoire diversity in human peripheral blood during aging; however, little is known about repertoire aging in other immune compartments, or in species other than humans. Here, we perform the first study of immune-repertoire aging in an emerging model of vertebrate aging, the African turquoise killifish (Nothobranchius furzeri). Despite their extremely short lifespans, these killifish exhibit complex and individualized heavy-chain repertoires, with a generative process capable of producing millions of distinct productive sequences. Whole-body killifish repertoires decline rapidly in within-individual diversity with age, while between-individual variability increases. Large, expanded B-cell clones exhibit far greater diversity loss with age than small clones, suggesting important differences in how age affects different B-cell populations. The immune repertoires of isolated intestinal samples exhibit especially dramatic age-related diversity loss, related to an elevated prevalence of expanded clones. Lower intestinal repertoire diversity was also associated with transcriptomic signatures of reduced B-cell activity, supporting a functional role for diversity changes in killifish immunosenescence. Our results highlight important differences in systemic vs. organ-specific aging dynamics in the adaptive immune system.


Assuntos
Diversidade de Anticorpos/imunologia , Fundulidae/imunologia , Imunossenescência/imunologia , Imunidade Adaptativa/imunologia , Envelhecimento/imunologia , Animais , Linfócitos B/imunologia , Humanos , Longevidade/imunologia , Microbiota/imunologia , Modelos Animais
8.
J Immunol ; 208(1): 143-154, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34862258

RESUMO

Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.


Assuntos
Proteínas Aviárias/metabolismo , Subpopulações de Linfócitos B/imunologia , Linfoma de Burkitt/imunologia , Elementos Facilitadores Genéticos/genética , Imunoglobulinas/metabolismo , RNA Polimerase II/metabolismo , Animais , Diversidade de Anticorpos , Proteínas Aviárias/genética , Linfoma de Burkitt/genética , Galinhas , Citidina Desaminase/genética , Humanos , Imunoglobulinas/genética , Ativação Linfocitária , Mutagênese Sítio-Dirigida , Mutação/genética , RNA Polimerase II/genética , Hipermutação Somática de Imunoglobulina , Transcrição Genética
9.
Immunohorizons ; 5(8): 675-686, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34433623

RESUMO

Ab repertoire diversity plays a critical role in the host's ability to fight pathogens. CDR3 is partially responsible for Ab-Ag binding and is a significant source of diversity in the repertoire. CDR3 diversity is generated during VDJ rearrangement because of gene segment selection, gene segment trimming and splicing, and the addition of nucleotides. We analyzed the Ab repertoire diversity across multiple experiments examining the effects of spaceflight on the Ab repertoire after vaccination. Five datasets from four experiments were analyzed using rank-abundance curves and Shannon indices as measures of diversity. We discovered a trend toward lower diversity as a result of spaceflight but did not find the same decrease in our physiological model of microgravity in either the spleen or bone marrow. However, the bone marrow repertoire showed a reduction in diversity after vaccination. We also detected differences in Shannon indices between experiments and tissues. We did not detect a pattern of CDR3 usage across the experiments. Overall, we were able to find differences in the Ab repertoire diversity across experimental groups and tissues.


Assuntos
Medula Óssea/imunologia , Regiões Determinantes de Complementaridade/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Voo Espacial/métodos , Baço/imunologia , Vacinação/métodos , Sequência de Aminoácidos , Animais , Diversidade de Anticorpos/genética , Diversidade de Anticorpos/imunologia , Medula Óssea/metabolismo , Regiões Determinantes de Complementaridade/genética , Feminino , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos Endogâmicos C57BL , RNA-Seq/métodos , Baço/metabolismo
10.
Front Immunol ; 12: 705381, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34349765

RESUMO

The role of B cells in the tumor microenvironment (TME) has largely been under investigated, and data regarding the antibody repertoire encoded by B cells in the TME and the adjacent lymphoid organs are scarce. Here, we utilized B cell receptor high-throughput sequencing (BCR-Seq) to profile the antibody repertoire signature of tumor-infiltrating lymphocyte B cells (TIL-Bs) in comparison to B cells from three anatomic compartments in a mouse model of triple-negative breast cancer. We found that TIL-Bs exhibit distinct antibody repertoire measures, including high clonal polarization and elevated somatic hypermutation rates, suggesting a local antigen-driven B-cell response. Importantly, TIL-Bs were highly mutated but non-class switched, suggesting that class-switch recombination may be inhibited in the TME. Tracing the distribution of TIL-B clones across various compartments indicated that they migrate to and from the TME. The data thus suggests that antibody repertoire signatures can serve as indicators for identifying tumor-reactive B cells.


Assuntos
Diversidade de Anticorpos , Subpopulações de Linfócitos B/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Mamárias Experimentais/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Microambiente Tumoral/imunologia , Animais , Células Sanguíneas/imunologia , Medula Óssea/patologia , Linhagem Celular Tumoral/transplante , Movimento Celular , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Região Variável de Imunoglobulina/genética , Linfonodos/patologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Receptores de Antígenos de Linfócitos B/genética , Hipermutação Somática de Imunoglobulina , Neoplasias de Mama Triplo Negativas/patologia
11.
Int Immunopharmacol ; 99: 107964, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34271417

RESUMO

OBJECTIVE: The pro-inflammatory cytokine IL-32 has high susceptibility to develop cancer. But no previous meta-analysis was done to provide firm evidence. This systematic review and meta-analysis was designed to evaluate the association of IL-32 gene polymorphisms (rs28372698 and rs12934561) with cancer. METHOD: Eligible studies were selected using authentic databases searching from January 2013 to January 2021. Demographic data and genotypic information were extracted and organized from the selected studies. Review Manager (RevMan) version 5.4 was used to perform data analysis and data arrangement for meta-analysis. RESULTS: A total of seven studies with 3395 patients and 3781 controls were included in this study. IL-32 rs28372698 polymorphism implied that mutant allele (TT) carriers had a significantly higher risk of cancer (OR = 1.43, p = 0.032). Codominant 3, recessive and allele models also showed 1.36-, 1.38- and 1.11-fold increased risk, respectively (p < 0.05). Besides, the Asian population showed a significantly increased risk in codominant 2 (OR = 1.74), codominant 3 (OR = 1.78), recessive (OR = 1.76) and allele model (OR = 1.16). IL-32 rs12934561 showed significantly reduced cancer risk in codominant 1 (OR = 0.66. p = 0.035), codominant 2 (OR = 0.76, p = 0.007), and dominant model (OR = 0.72, p = 0.012). After subgroup analysis, an association of rs12934561 was found in Asians (codominant 1: OR = 0.54, p = 7.28 × 10-8; codominant 2: OR = 1.40, p = 0.019; codominant 3: OR = 0.76, p = 0.0006; dominant model: OR = 0.64, p = 1.12 × 10-5; overdominant model: OR = 0.64, p = 3.92 × 10-7) but not in Caucasians. After stratifying with the control source, a significant (p < 0.05) association of rs28372698 and rs12934561 was found with cancer in population-based controls. No publication bias was found, and the outcome of this meta-analysis was not influenced by any individual study confirmed from sensitivity analysis. Moreover, trial sequential analysis (TSA) established a link between rs28372698 and rs12934561 polymorphisms and cancer. CONCLUSION: The outcome of this meta-analysis revealed that IL-32 rs28372698 and rs12934561 polymorphisms are associated with cancer. Moreover, the Asian dynasty had a significant association compared to Caucasians.


Assuntos
Carcinogênese/genética , Interleucinas/genética , Neoplasias/genética , Animais , Diversidade de Anticorpos , Humanos , Polimorfismo Genético
12.
Cell Rep ; 36(2): 109349, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34260907

RESUMO

Generation of the primary antibody repertoire requires V(D)J recombination of hundreds of gene segments in the immunoglobulin heavy chain (Igh) locus. The role of interleukin-7 receptor (IL-7R) signaling in Igh recombination has been difficult to partition from its role in B cell survival and proliferation. With a detailed description of the Igh repertoire in murine IL-7Rα-/- bone marrow B cells, we demonstrate that IL-7R signaling profoundly influences VH gene selection during VH-to-DJH recombination. We find skewing toward 3' VH genes during de novo VH-to-DJH recombination more severe than the fetal liver (FL) repertoire and uncover a role for IL-7R signaling in DH-to-JH recombination. Transcriptome and accessibility analyses suggest reduced expression of B lineage transcription factors (TFs) and targets and loss of DH and VH antisense transcription in IL-7Rα-/- B cells. Thus, in addition to its roles in survival and proliferation, IL-7R signaling shapes the Igh repertoire by activating underpinning mechanisms.


Assuntos
Diversidade de Anticorpos/genética , Linfócitos B/metabolismo , Medula Óssea/metabolismo , Genes de Cadeia Pesada de Imunoglobulina , Região Variável de Imunoglobulina/genética , Receptores de Interleucina-7/metabolismo , Transdução de Sinais , Animais , Sequência de Bases , Linhagem da Célula/genética , Cromatina/metabolismo , DNA Intergênico/genética , Feto/metabolismo , Fígado/embriologia , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Motivos de Nucleotídeos/genética , Fator de Transcrição PAX5/metabolismo , Transativadores/metabolismo , Transcrição Genética
13.
J Immunol Methods ; 496: 113089, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34181966

RESUMO

Immune hosts are valuable sources for antibody discovery. To construct in vitro display antibody libraries from immune repertoires, singleplex or multiplex PCR amplification were employed using primers targeting multiple immunoglobulin genes. However, during this process, the B cell receptor repertoire is distorted due to interactions between multiple target genes and primers. To minimize this alternation, we devised a new method for harvesting immunoglobulin genes and tested its performance in rabbit variable heavy chain (VH) and variable kappa light chain (VK) genes. Double-stranded cDNA was synthesized using primers containing V/J gene-specific regions and universal sequence parts for in vitro display. VH and VK gene libraries were obtained through subsequent PCR amplification using primers with universal sequences. Next-generation sequencing analysis confirmed that universal PCR libraries had more diverse VH and VK clonotypes, and a less biased clonal distribution, than conventional singleplex or multiplex gene-specific PCR libraries.


Assuntos
Anticorpos/genética , Biblioteca Gênica , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Reação em Cadeia da Polimerase Multiplex , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Anticorpos/imunologia , Diversidade de Anticorpos , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Coelhos
14.
Epilepsia ; 62(6): 1416-1428, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33949696

RESUMO

OBJECTIVE: Focal cortical dysplasia (FCD) is a major cause of difficult-to-treat epilepsy in children and young adults, and the diagnosis is currently based on microscopic review of surgical brain tissue using the International League Against Epilepsy classification scheme of 2011. We developed an iterative histopathological agreement trial with genetic testing to identify areas of diagnostic challenges in this widely used classification scheme. METHODS: Four web-based digital pathology trials were completed by 20 neuropathologists from 15 countries using a consecutive series of 196 surgical tissue blocks obtained from 22 epilepsy patients at a single center. Five independent genetic laboratories performed screening or validation sequencing of FCD-relevant genes in paired brain and blood samples from the same 22 epilepsy patients. RESULTS: Histopathology agreement based solely on hematoxylin and eosin stainings was low in Round 1, and gradually increased by adding a panel of immunostainings in Round 2 and the Delphi consensus method in Round 3. Interobserver agreement was good in Round 4 (kappa = .65), when the results of genetic tests were disclosed, namely, MTOR, AKT3, and SLC35A2 brain somatic mutations in five cases and germline mutations in DEPDC5 and NPRL3 in two cases. SIGNIFICANCE: The diagnoses of FCD 1 and 3 subtypes remained most challenging and were often difficult to differentiate from a normal homotypic or heterotypic cortical architecture. Immunohistochemistry was helpful, however, to confirm the diagnosis of FCD or no lesion. We observed a genotype-phenotype association for brain somatic mutations in SLC35A2 in two cases with mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy. Our results suggest that the current FCD classification should recognize a panel of immunohistochemical stainings for a better histopathological workup and definition of FCD subtypes. We also propose adding the level of genetic findings to obtain a comprehensive, reliable, and integrative genotype-phenotype diagnosis in the near future.


Assuntos
Malformações do Desenvolvimento Cortical/diagnóstico por imagem , Malformações do Desenvolvimento Cortical/patologia , Adolescente , Adulto , Idade de Início , Diversidade de Anticorpos , Encéfalo/patologia , Criança , Pré-Escolar , Técnica Delfos , Feminino , Genótipo , Humanos , Imuno-Histoquímica , Lactente , Imageamento por Ressonância Magnética , Masculino , Malformações do Desenvolvimento Cortical/cirurgia , Pessoa de Meia-Idade , Mutação/genética , Procedimentos Neurocirúrgicos , Variações Dependentes do Observador , Fenótipo , Convulsões/etiologia , Adulto Jovem
15.
Acta Neuropathol ; 142(1): 191-210, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33929593

RESUMO

The genetic basis of brain tumor development is poorly understood. Here, leukocyte DNA of 21 patients from 15 families with ≥ 2 glioma cases each was analyzed by whole-genome or targeted sequencing. As a result, we identified two families with rare germline variants, p.(A592T) or p.(A817V), in the E-cadherin gene CDH1 that co-segregate with the tumor phenotype, consisting primarily of oligodendrogliomas, WHO grade II/III, IDH-mutant, 1p/19q-codeleted (ODs). Rare CDH1 variants, previously shown to predispose to gastric and breast cancer, were significantly overrepresented in these glioma families (13.3%) versus controls (1.7%). In 68 individuals from 28 gastric cancer families with pathogenic CDH1 germline variants, brain tumors, including a pituitary adenoma, were observed in three cases (4.4%), a significantly higher prevalence than in the general population (0.2%). Furthermore, rare CDH1 variants were identified in tumor DNA of 6/99 (6%) ODs. CDH1 expression was detected in undifferentiated and differentiating oligodendroglial cells isolated from rat brain. Functional studies using CRISPR/Cas9-mediated knock-in or stably transfected cell models demonstrated that the identified CDH1 germline variants affect cell membrane expression, cell migration and aggregation. E-cadherin ectodomain containing variant p.(A592T) had an increased intramolecular flexibility in a molecular dynamics simulation model. E-cadherin harboring intracellular variant p.(A817V) showed reduced ß-catenin binding resulting in increased cytosolic and nuclear ß-catenin levels reverted by treatment with the MAPK interacting serine/threonine kinase 1 inhibitor CGP 57380. Our data provide evidence for a role of deactivating CDH1 variants in the risk and tumorigenesis of neuroepithelial and epithelial brain tumors, particularly ODs, possibly via WNT/ß-catenin signaling.


Assuntos
Antígenos CD/genética , Neoplasias Encefálicas/genética , Caderinas/genética , Carcinoma/genética , Neoplasias Neuroepiteliomatosas/genética , Adenoma/genética , Adenoma/patologia , Compostos de Anilina/uso terapêutico , Animais , Diversidade de Anticorpos , Neoplasias Encefálicas/tratamento farmacológico , Carcinoma/tratamento farmacológico , DNA de Neoplasias/genética , Técnicas de Introdução de Genes , Variação Genética , Células HEK293 , Humanos , Neoplasias Neuroepiteliomatosas/tratamento farmacológico , Oligodendroglioma/genética , Oligodendroglioma/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Purinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Sequenciamento Completo do Genoma
16.
PLoS Comput Biol ; 17(3): e1008781, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33647011

RESUMO

The naïve antibody/B-cell receptor (BCR) repertoires of different individuals ought to exhibit significant functional commonality, given that most pathogens trigger an effective antibody response to immunodominant epitopes. Sequence-based repertoire analysis has so far offered little evidence for this phenomenon. For example, a recent study estimated the number of shared ('public') antibody clonotypes in circulating baseline repertoires to be around 0.02% across ten unrelated individuals. However, to engage the same epitope, antibodies only require a similar binding site structure and the presence of key paratope interactions, which can occur even when their sequences are dissimilar. Here, we search for evidence of geometric similarity/convergence across human antibody repertoires. We first structurally profile naïve ('baseline') antibody diversity using snapshots from 41 unrelated individuals, predicting all modellable distinct structures within each repertoire. This analysis uncovers a high (much greater than random) degree of structural commonality. For instance, around 3% of distinct structures are common to the ten most diverse individual samples ('Public Baseline' structures). Our approach is the first computational method to find levels of BCR commonality commensurate with epitope immunodominance and could therefore be harnessed to find more genetically distant antibodies with same-epitope complementarity. We then apply the same structural profiling approach to repertoire snapshots from three individuals before and after flu vaccination, detecting a convergent structural drift indicative of recognising similar epitopes ('Public Response' structures). We show that Antibody Model Libraries derived from Public Baseline and Public Response structures represent a powerful geometric basis set of low-immunogenicity candidates exploitable for general or target-focused therapeutic antibody screening.


Assuntos
Anticorpos , Diversidade de Anticorpos , Linfócitos B , Bases de Dados Genéticas , Epitopos Imunodominantes , Linfócitos B/química , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biologia Computacional , Humanos
17.
MAbs ; 13(1): 1882028, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33602056

RESUMO

The human IgG3 subclass is conspicuously absent among the formats for approved monoclonal antibody therapies and Fc fusion protein biologics. Concern about the potential for rapid degradation, reduced plasma half-life, and increased immunogenicity due to marked variation in allotypes has apparently outweighed the potential advantages of IgG3, which include high affinity for activating Fcγ receptors, effective complement fixation, and a long hinge that appears better suited for low abundance targets. This review aims to highlight distinguishing features of IgG3 and to explore its functional role in the immune response. We present studies of natural immunity and recombinant antibody therapies that elucidate key contributions of IgG3 and discuss historical roadblocks that no longer remain clearly relevant. Collectively, this body of evidence motivates thoughtful reconsideration of the clinical advancement of this distinctive antibody subclass for treatment of human diseases. Abbreviations: ADCC - Antibody-Dependent Cell-mediated CytotoxicityADE - Antibody-dependent enhancementAID - Activation-Induced Cytidine DeaminaseCH - Constant HeavyCHF - Complement factor HCSR - Class Switch RecombinationEM - Electron MicroscopyFab - Fragment, antigen bindingFc - Fragment, crystallizableFcRn - Neonatal Fc ReceptorFcγR - Fc gamma ReceptorHIV - Human Immunodeficiency VirusIg - ImmunoglobulinIgH - Immunoglobulin Heavy chain geneNHP - Non-Human Primate.


Assuntos
Imunidade Humoral , Imunoglobulina G/uso terapêutico , Vacinas/uso terapêutico , Vacinas contra a AIDS/uso terapêutico , Animais , Diversidade de Anticorpos , Especificidade de Anticorpos , Vacinas Anticâncer/uso terapêutico , Glicosilação , Humanos , Switching de Imunoglobulina , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Vacinas Pneumocócicas/uso terapêutico , Engenharia de Proteínas , Processamento de Proteína Pós-Traducional , Relação Estrutura-Atividade , Vacinas/genética , Vacinas/imunologia , Vacinas/metabolismo
18.
Nat Immunol ; 22(3): 279-286, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33495652

RESUMO

The constituents of the gut microbiome are determined by the local habitat, which itself is shaped by immunological pressures, such as mucosal IgA. Using a mouse model of restricted antibody repertoire, we identified a role for antibody-microbe interactions in shaping a community of bacteria with an enhanced capacity to metabolize L-tyrosine. This model led to increased concentrations of p-cresol sulfate (PCS), which protected the host against allergic airway inflammation. PCS selectively reduced CCL20 production by airway epithelial cells due to an uncoupling of epidermal growth factor receptor (EGFR) and Toll-like receptor 4 (TLR4) signaling. Together, these data reveal a gut microbe-derived metabolite pathway that acts distally on the airway epithelium to reduce allergic airway responses, such as those underpinning asthma.


Assuntos
Anticorpos/metabolismo , Bactérias/metabolismo , Cresóis/metabolismo , Microbioma Gastrointestinal , Intestinos/microbiologia , Pulmão/metabolismo , Pneumonia/prevenção & controle , Hipersensibilidade Respiratória/prevenção & controle , Ésteres do Ácido Sulfúrico/metabolismo , Tirosina/metabolismo , Administração Oral , Alérgenos , Animais , Anticorpos/imunologia , Diversidade de Anticorpos , Bactérias/imunologia , Células Cultivadas , Quimiocina CCL20/metabolismo , Técnicas de Cocultura , Cresóis/administração & dosagem , Modelos Animais de Doenças , Receptores ErbB/metabolismo , Feminino , Interações Hospedeiro-Patógeno , Injeções Intravenosas , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pneumonia/imunologia , Pneumonia/metabolismo , Pneumonia/microbiologia , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/microbiologia , Transdução de Sinais , Ésteres do Ácido Sulfúrico/administração & dosagem , Receptor 4 Toll-Like/metabolismo , Tirosina/administração & dosagem
19.
Pharmacogenomics ; 22(2): 73-80, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33448876

RESUMO

The North Carolina Precision Health Collaborative is an interdisciplinary, public-private consortium of precision health experts who strategically align statewide resources and strengths to elevate precision health in the state and beyond. Pharmacogenomics (PGx) is a key area of focus for the North Carolina Precision Health Collaborative. Experts from Atrium Health's Levine Cancer Institute, Duke University/Duke Health System, Mission Health and the University of North Carolina (UNC) at Chapel Hill/UNC Health System have collaborated since 2017 to implement strategic PGx initiatives, including basic sciences research, translational research and clinical implementation of germline testing into practice and policy. This institutional profile highlights major PGx programs and initiatives across these organizations and how the collaborative is working together to advance PGx science and implementation.


Assuntos
Farmacogenética , Medicina de Precisão , Diversidade de Anticorpos , Hospitais Universitários , Humanos , North Carolina , Parcerias Público-Privadas , Pesquisa , Pesquisa Translacional Biomédica
20.
Front Immunol ; 12: 827256, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35126381

RESUMO

Virus-like particles (VLPs) are used in different marketed vaccines and are able to induce potent antibody responses. The innate pattern recognition receptors TLR7/8 recognize single stranded (ss) RNA naturally packaged into some VLPs and have been shown to enhance the production of IgG antibodies upon immunization. Here we demonstrate that, upon immunization with RNA-loaded bacteriophage-derived VLP Qß, TLR7 signaling accelerates germinal center formation, promotes affinity/avidity maturation of VLP-specific IgG and isotype switching to IgG2b/2c. These findings extrapolated to antigens displayed on Qß; as Fel d 1, the major cat allergen, chemically attached to Qß also induced higher affinity/avidity IgG2b/2c antibodies in a TLR7-dependent fashion. Chimeric mice lacking TLR7-expression exclusively in B cells demonstrated that the enhanced IgG responses were driven by a B cell intrinsic mechanism. Importantly, deep sequencing of the BCR repertoire of antigen-specific B cells demonstrated higher diversity in mice with TLR7 signaling in B cells, suggesting that TLR7-signaling drives BCR repertoire development and diversity. Furthermore, the current data demonstrate that high levels of clonal diversity are reached early in the response and maintained by TLR7 signaling. In conclusion, TLR7 signaling enhances levels and quality of IgG antibodies, and this finding has major implications for vaccine design.


Assuntos
Anticorpos Antivirais/imunologia , Diversidade de Anticorpos/imunologia , Transdução de Sinais , Receptor 7 Toll-Like/metabolismo , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Glicoproteínas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunização , Imunoglobulina G/imunologia , Imunofenotipagem , Camundongos , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Proteínas Recombinantes/imunologia
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