RESUMO
Immunoglobulin G (IgG) is one of the five antibody classes produced in mammals as part of the humoral responses accountable for protecting the organisms from infection. Its antibody heavy chain constant region is encoded by the Ig heavy-chain gamma gene (IGHG). In humans, there are four IGHG genes which encode the four subclasses, each with a specialized effector function. Although four subclasses of IgG proteins have also been reported in macaques, this does not appear to be the rule for all primates. In Platyrrhini, IgG has been stated to be encoded by a single-copy gene. To date, it remains unknown how the IGHG has expanded or contracted in the primate order; consequently, we have analyzed data from 38 primate genome sequences to identify IGHG genes and describe the evolution of IGHG genes in primate order. IGHG belongs to a multigene family that evolves by the birth-death evolutionary model in primates. Whereas Strepsirrhini and Platyrrhini have a single-copy gene, in Catarrhini, it has expanded to several paralogs in their genomes; some deleted and others pseudogenized. Furthermore, episodic positive selection may have promoted a species-specific IgG effector function. We propose that IgG evolved to reach an optimal number of copies per genome to adapt their humoral immune responses to different environmental conditions. This study has implications for biomedical trials using non-human primates.
Assuntos
Regiões Constantes de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas , Animais , Regiões Constantes de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina G/genética , Evolução Molecular , Platirrinos , Filogenia , MamíferosRESUMO
Over the last two decades, the variable domains from heavy chain-only antibodies in camelids (nanobodies) have emerged as valuable immunoreagents for analytical and diagnostic applications. One prominent use of nanobodies is for the detection of small molecules due to their ease of production, resistance to solvents used in sample extraction, facile genetic manipulation, and small size. These last two properties make it possible to produce biotinylated nanobodies in vivo, which can be loaded in an orientated manner on magnetic beads covered with avidin, creating high-density immunoadsorbenpi twbch ""ts. The method described here details the use of nanobody-based adsorbents to concentrate small molecular weight analytes for subsequent quantitative analysis by MALDI-TOF mass spectrometry. Quantitation requires the inclusion of an internal standard (IS), a compound with properties similar to those of the analyte, enabling compensation for uneven distribution during crystallization of the MALDI-TOF matrix. Since nanobody generation against small compounds requires conjugation to carrier proteins, the same conjugation chemistry can be used to synthesize the IS. By design the IS cross reacts with the capture nanobody and can be preloaded in the immunoadsorbent, facilitating quantitative detection of the target compound.
Assuntos
Anticorpos de Domínio Único , Cadeias Pesadas de Imunoglobulinas , Separação Imunomagnética , Fenômenos Magnéticos , Anticorpos de Domínio Único/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Chronic lymphocytic leukemia (CLL) is the most common mature B-cell neoplasm in the West. IGHV4-34 is one of the most frequently used genes in CLL patients, which usually display an indolent outcome. In this study, we explored the mutational profile of CLL patients expressing IGHV4-34 within different stereotypes and their association with prognostic factors and clinical outcome. A multi-institutional cohort of unselected 1444 CLL patients was analyzed by RT-PCR and bidirectional sequencing. Cytogenetics and molecular cytogenetics analyses were also performed. We identified 144 (10%) IGHV4-34 expressing cases, 119 mutated (M), 44 of them with stereotyped B-cell receptors. Subset #4 was the most frequent (56.8% of cases) followed by subsets #16 (13.6%), #29 (6.8%), and #201 (2.3%), with different distribution among countries. Analysis of somatic hypermutation profile showed significant differences among stereotyped subsets for G28>D/E, P45>S, E55>Q, and S64>I changes (p < 0.01) and high frequency of disruption of the glycosylation motif in the VH CDR2 region. All stereotyped IGHV4-34 cases showed normal karyotypes. Deletion 13q14 as a sole alteration was present in 42.8% of stereotyped cases with a different distribution among subsets. A shorter time to first treatment was found in non-stereotyped vs. stereotyped M-IGHV4-34 patients (p = 0.034). Our results add new information supporting the importance of recurrent amino acid changes at particular positions, contributing to refine the molecular characterization of South American CLL patients.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Leucemia Linfocítica Crônica de Células B/genética , Idoso , Estudos de Coortes , Feminino , Rearranjo Gênico , Humanos , Leucemia Linfocítica Crônica de Células B/epidemiologia , Masculino , Pessoa de Meia-Idade , Mutação , Receptores de Antígenos de Linfócitos B/genética , Hipermutação Somática de Imunoglobulina , América do Sul/epidemiologiaRESUMO
Autoimmunity may have its origins of early repertoire selection in developmental B cells. Such a primary repertoire is probably shaped by selecting B cells that can efficiently perform productive signaling, stimulated by self-antigens in the bone marrow, such as DNA. In support of that idea, we previously found a V segment from VH10 family that can form antibodies that bind to DNA independent of CDR3 usage. In this paper we designed four antibody fragments in a novel single-chain pre-BCR (scpre-BCR) format containing germinal V gene segments from families known to bind DNA (VH10) or not (VH4) connected to a murine surrogate light chain (SLC), lacking the highly charged unique region (UR), by a hydrophilic peptide linker. We also tested the influence of CDR2 on DNA reactivity by shuffling the CDR2 loop. The scpre-BCRs were expressed in bacteria. VH10 bearing scpre-BCR could bind DNA, while scpre-BCR carrying the VH4 segment did not. The CDR2 loop shuffling hampered VH10 reactivity while displaying a gain-of-function in the nonbinding VH4 germline. We modeled the binding sites demonstrating the conservation of a positivity charged pocket in the VH10 CDR2 as the possible cross-reactive structural element. We presented evidence of DNA reactivity hardwired in a V gene, suggesting a structural mechanism for innate autoreactivity. Therefore, while autoreactivity to DNA can lead to autoimmunity, efficiently signaling for B cell development is likely a trade-off mechanism leading to the selection of potentially autoreactive repertoires.
Assuntos
Região Variável de Imunoglobulina/genética , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Sequência de Aminoácidos/genética , Animais , Anticorpos Antinucleares/genética , Arginina/genética , Arginina/metabolismo , Autoantígenos/genética , Autoimunidade/imunologia , Sequência de Bases/genética , DNA/imunologia , Células Germinativas/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/ultraestrutura , Camundongos , Anticorpos de Domínio Único/ultraestrutura , Relação Estrutura-AtividadeRESUMO
INTRODUCTION: Minimal residual disease (MRD) is a cornerstone for stratification of upfront B-lymphoblastic leukemia (B-ALL) treatment protocols to decrease relapse risk. Although its detection by flow cytometry (FC) and real-time quantitative polymerase has clinical usefulness, evidence suggests that methods with increased sensitivity could lead to improved outcomes. The aim of this study was to develop an amplicon-based assay followed by high-throughput sequencing of the immunoglobulin heavy chain variable region for MRD detection in B-ALL. METHODS: We analyzed 84 samples, 27 from diagnosis, 5 from relapse, 40 from post-treatment samples, and 12 from healthy controls. RESULTS: Our assay was able to identify more neoplastic clones at diagnosis than Sanger sequencing including incomplete DJ rearrangements. From the 40 MRD samples evaluated 21 were positive by our new approach on high-throughput sequencing assay, but only 15 of these were positive by FC. The remaining 19 were negative by the two techniques. CONCLUSION: We have developed a novel approach on high-sensitive assay for MRD detection in B-ALL, which could add clinical value in the management of patients, especially in cases negative for MRD by FC.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Neoplasia Residual/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasia Residual/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnósticoRESUMO
Nile tilapia (Oreochromis niloticus) is a freshwater fish, which is extensively cultivated worldwide and constitutes one of the model species for the study of fish immunology. Monoclonal antibodies are very advantageous molecular tools for studying teleost immune system. Specifically, monoclonal antibodies that react with immunoglobulins are used successfully in the study of the humoral immune response of several fish species. In the present study, we produced and characterized a monoclonal antibody against tilapia IgM heavy chain using a peptide-based strategy. The peptide sequence was selected from the surface-exposed region between CH3-CH4 domains. The specificity of the polyclonal serum and the hybridoma culture supernatant obtained by immunization with the peptide conjugated to keyhole limpet hemocyanin were evaluated by western blotting, both showing reactivity against tilapia serum IgM. The purified mAb was able to recognize secreted IgM by western blotting and ELISA and membrane IgM by flow cytometry. We also demonstrated that the antibody doesn't cross-react with a recombinant IgT fragment. This tool allowed us to study for the first time the stimulation of mucosal immunity after Pituitary Adenylate Cyclase Activating Polypeptide administration. Overall, the results demonstrated the utility of this mAb to characterize humoral immune response in O. niloticus.
Assuntos
Anticorpos Monoclonais/imunologia , Ciclídeos/imunologia , Proteínas de Peixes/imunologia , Imunidade Humoral , Cadeias Pesadas de Imunoglobulinas/imunologia , Imunoglobulina M/imunologia , Sequência de Aminoácidos , Animais , Alinhamento de SequênciaRESUMO
Antibodies, T cell receptors and major histocompatibility complex molecules are members of the immunoglobulin superfamily and have pivotal roles in the immune system. The fine interrelation between them regulates several immune functions. Here, we describe lesser-known functions ascribed to these molecules in generating and maintaining immune response. Particularly, we outline the contribution of antibody- and T cell receptor-derived complementarity-determining region neoantigens, antigenized antibodies, as well as major histocompatibility complex class I molecules-derived epitopes to the induction of protective/therapeutic immune responses against pathogens and cancer. We discuss findings of our own and other studies describing protective mechanisms, based on immunogenic properties of immunoglobulin superfamily members, and evaluate the perspectives of application of this class of immunogens in molecular vaccines design.
Assuntos
Anticorpos/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Sequência de Aminoácidos/genética , Animais , Formação de Anticorpos , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunidade/imunologia , Cadeias Pesadas de Imunoglobulinas , Região Variável de Imunoglobulina , Imunoglobulinas/imunologia , Imunoglobulinas/metabolismoRESUMO
The origin and function of blood IgM+IgD+CD27+ B cells is controversial, and they are considered a heterogeneous population. Previous staining of circulating B cells of healthy donors with rotavirus fluorescent virus-like particles allowed us to differentiate two subsets of IgM+IgD+CD27+: IgMhi and IgMlo B cells. Here, we confirmed this finding and compared the phenotype, transcriptome, in vitro function, and Ig gene repertoire of these two subsets. Eleven markers phenotypically discriminated both subsets (CD1c, CD69, IL21R, CD27, MTG, CD45RB, CD5, CD184, CD23, BAFFR, and CD38) with the IgMhi phenotypically resembling previously reported marginal zone B cells and the IgMlo resembling both naïve and memory B cells. Transcriptomic analysis showed that both subpopulations clustered close to germinal center-experienced IgM only B cells with a Principal Component Analysis, but differed in expression of 78 genes. Moreover, IgMhi B cells expressed genes characteristic of previously reported marginal zone B cells. After stimulation with CpG and cytokines, significantly (p < 0.05) higher frequencies (62.5%) of IgMhi B cells proliferated, compared with IgMlo B cells (35.37%), and differentiated to antibody secreting cells (14.22% for IgMhi and 7.19% for IgMlo). IgMhi B cells had significantly (p < 0.0007) higher frequencies of mutations in IGHV and IGKV regions, IgMlo B cells had higher usage of IGHJ6 genes (p < 0.0001), and both subsets differed in their HCDR3 properties. IgMhi B cells shared most of their shared IGH clonotypes with IgM only memory B cells, and IgMlo B cells with IgMhi B cells. These results support the notion that differential expression of IgM and IgD discriminates two subpopulations of human circulating IgM+IgD+CD27+ B cells, with the IgMhi B cells having similarities with previously described marginal zone B cells that passed through germinal centers, and the IgMlo B cells being the least differentiated amongst the IgM+CD27+ subsets.
Assuntos
Subpopulações de Linfócitos B/fisiologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunoglobulina D/metabolismo , Imunoglobulina M/metabolismo , Adulto , Perfilação da Expressão Gênica , Humanos , Imunoglobulina D/genética , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina M/genética , Região Variável de Imunoglobulina/genética , Imunofenotipagem , Fenótipo , Análise de Componente Principal , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Antibodies that block interaction of immune checkpoint receptors with its ligands have revolutionized the treatment of several cancers. Despite the success of this approach, the high cost has been restricted the use of this class of drugs. In this context, the development of biosimilar can be an important strategy for reducing prices and expanding access after patent has been dropped. Here, we evaluated the use of HEK293 cells for transient expression of an immune checkpoint-blocking antibody as a first step for biosimilar development. Antibody light and heavy chain genes were cloned into pCI-neo vector and transiently expressed in HEK293 cells. The culture supernatant was then subjected to protein A affinity chromatography, which allowed to obtain the antibody with high homogeneity. For physicochemical comparability, biosimilar antibody and reference drug were analyzed by SDS-PAGE, isoelectric focusing, circular dichroism and fluorescence spectroscopy. The results indicated that the both antibodies have a high degree of structural similarity. Lastly, the biosimilar antibody binding capacity to target receptor was shown to be similar to reference product in ELISA and flow cytometry assays. These data demonstrate that the HEK293 system can be used as an important tool for candidate selection and early development of biosimilar antibodies.