RESUMO
The COVID-19 pandemic has uncovered the high genetic variability of the SARS-CoV-2 virus and its ability to evade the immune responses that were induced by earlier viral variants. Only a few monoclonal antibodies that have been reported to date are capable of neutralizing a broad spectrum of SARS-CoV-2 variants. Here, we report the isolation of a new broadly neutralizing human monoclonal antibody, iC1. The antibody was identified through sorting the SARS-CoV-1 RBD-stained individual B cells that were isolated from the blood of a vaccinated donor following a breakthrough infection. In vitro, iC1 potently neutralizes pseudoviruses expressing a wide range of SARS-CoV-2 Spike variants, including those of the XBB sublineage. In an hACE2-transgenic mouse model, iC1 provided effective protection against the Wuhan strain of the virus as well as the BA.5 and XBB.1.5 variants. Therefore, iC1 can be considered as a potential component of the broadly neutralizing antibody cocktails resisting the SARS-CoV-2 mutation escape.
Assuntos
Enzima de Conversão de Angiotensina 2 , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , Camundongos Transgênicos , SARS-CoV-2 , Animais , SARS-CoV-2/imunologia , Humanos , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Enzima de Conversão de Angiotensina 2/imunologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Camundongos , Anticorpos Antivirais/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Pandemias/prevenção & controle , Betacoronavirus/imunologia , Betacoronavirus/genética , Anticorpos Amplamente Neutralizantes/imunologia , Modelos Animais de Doenças , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Pneumonia Viral/prevenção & controle , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Infecções por Coronavirus/prevenção & controleRESUMO
The emergence of SARS-CoV-2 mutant variants has posed a significant challenge to both the prevention and treatment of COVID-19 with anti-coronaviral neutralizing antibodies. The latest viral variants demonstrate pronounced resistance to the vast majority of human monoclonal antibodies raised against the ancestral Wuhan variant. Less is known about the susceptibility of the evolved virus to camelid nanobodies developed at the start of the pandemic. In this study, we compared nanobody repertoires raised in the same llama after immunization with Wuhan's RBD variant and after subsequent serial immunization with a variety of RBD variants, including that of SARS-CoV-1. We show that initial immunization induced highly potent nanobodies, which efficiently protected Syrian hamsters from infection with the ancestral Wuhan virus. These nanobodies, however, mostly lacked the activity against SARS-CoV-2 omicron-pseudotyped viruses. In contrast, serial immunization with different RBD variants resulted in the generation of nanobodies demonstrating a higher degree of somatic mutagenesis and a broad range of neutralization. Four nanobodies recognizing distinct epitopes were shown to potently neutralize a spectrum of omicron variants, including those of the XBB sublineage. Our data show that nanobodies broadly neutralizing SARS-CoV-2 variants may be readily induced by a serial variant RBD immunization.
RESUMO
SARS-CoV-2 has a relatively high mutation rate, with the frequent emergence of new variants of concern (VOCs). Each subsequent variant is more difficult to neutralize by the sera of vaccinated individuals and convalescents. Some decrease in neutralizing activity against new SARS-CoV-2 variants has also been observed in patients vaccinated with Gam-COVID-Vac. In the present study, we analyzed the interplay between the history of a patient's repeated exposure to SARS-CoV-2 antigens and the breadth of neutralization activity. Our study includes four cohorts of patients: Gam-COVID-Vac booster vaccinated individuals (revaccinated, RV), twice-infected unvaccinated individuals (reinfected, RI), breakthrough infected (BI), and vaccinated convalescents (VC). We assessed S-protein-specific antibody levels and the ability of sera to neutralize lentiviral particles pseudotyped with Spike protein from the original Wuhan variant, as well as the Omicron variants BA.1 and BA.4/5. Individuals with hybrid immunity (BI and VC cohorts) exhibited significantly higher levels of virus-binding IgG and enhanced breadth of virus-neutralizing activity compared to individuals from either the revaccination or reinfection (RV and RI) cohorts. These findings suggest that a combination of infection and vaccination, regardless of the sequence, results in significantly higher levels of S-protein-specific IgG antibodies and the enhanced neutralization of SARS-CoV-2 variants, thereby underscoring the importance of hybrid immunity in the context of emerging viral variants.
RESUMO
Over the past decade, CAR T cell therapy for patients with B cell malignancies has evolved from an experimental technique to a clinically feasible option. To date, four CAR T cell products specific for a B cell surface marker, CD19, have been approved by the FDA. Despite the spectacular rates of complete remission in r/r ALL and NHL patients, a significant proportion of patients still relapse, frequently with the CD19 low/negative tumor phenotype. To address this issue, additional B cell surface molecules such as CD20 were proposed as targets for CAR T cells. Here, we performed a side-by-side comparison of the activity of CD20-specific CAR T cells based on the antigen-recognition modules derived from the murine antibodies, 1F5 and Leu16, and from the human antibody, 2F2. Whereas CD20-specific CAR T cells differed from CD19-specific CAR T cells in terms of subpopulation composition and cytokine secretion, they displayed similar in vitro and in vivo potency.
Assuntos
Antígenos CD20 , Imunoterapia Adotiva , Animais , Humanos , Camundongos , Citotoxicidade Imunológica , Imunoterapia Adotiva/métodos , Linfócitos T/imunologiaRESUMO
Immune evasion of SARS-CoV-2 undermines current strategies tocounteract the pandemic, with the efficacy of therapeutic virus-neutralizing monoclonal antibodies (nAbs) being affected the most. In this work, we asked whether two previously identified human cross-neutralizing nAbs, iB14 (class VH1-58) and iB20 (class VH3-53/66), are capable of neutralizing the recently emerged Omicron (BA.1) variant. Both nAbs were found to bind the Omicron RBD with a nanomolar affinity, yet they displayed contrasting functional features. When tested against Omicron, the neutralizing activity of iB14 was reduced 50-fold, whereas iB20 displayed a surprising increase in activity. Thus, iB20 is a unique representative of the VH3-53/66-class of nAbs in terms of breadth of neutralization, which establishes it as a candidate for COVID-19 therapy and prophylactics.
RESUMO
In the absence of virus-targeting small-molecule drugs approved for the treatment and prevention of COVID-19, broadening the repertoire of potent SARS-CoV-2-neutralizing antibodies represents an important area of research in response to the ongoing pandemic. Systematic analysis of such antibodies and their combinations can be particularly instrumental for identification of candidates that may prove resistant to the emerging viral escape variants. Here, we isolated a panel of 23 RBD-specific human monoclonal antibodies from the B cells of convalescent patients. A surprisingly large proportion of such antibodies displayed potent virus-neutralizing activity both in vitro and in vivo. Four of the isolated nAbs can be categorized as ultrapotent with an apparent IC100 below 16 ng/mL. We show that individual nAbs as well as dual combinations thereof retain activity against currently circulating SARS-CoV-2 variants of concern (such as B.1.1.7, B.1.351, B.1.617, and C.37), as well as against other viral variants. When used as a prophylactics or therapeutics, these nAbs could potently suppress viral replication and prevent lung pathology in SARS-CoV-2-infected hamsters. Our data contribute to the rational development of oligoclonal therapeutic nAb cocktails mitigating the risk of SARS-CoV-2 escape.
RESUMO
The aim of this study was to fill important gaps in the evolutionary history of immunoglobulins by examining the structure and diversity of IgL genes in non-teleost ray-finned fish. First, based on the bioinformatic analysis of recent transcriptomic and genomic resources, we experimentally characterized the IgL genes in the chondrostean fish, Acipenser ruthenus (sterlet). We show that this species has three loci encoding IgL kappa-like chains with a translocon-type gene organization and a single VJC cluster, encoding homogeneous lambda-like light chain. In addition, sterlet possesses sigma-like VL and J-CL genes, which are transcribed separately and both encode protein products with cleavable leader peptides. The Acipenseriformes IgL dataset was extended by the sequences mined in the databases of species belonging to other non-teleost lineages of ray-finned fish: Holostei and Polypteriformes. Inclusion of these new data into phylogenetic analysis showed a clear subdivision of IgL chains into five groups. The isotype described previously as the teleostean IgL lambda turned out to be a kappa and lambda chain paralog that emerged before the radiation of ray-finned fish. We designate this isotype as lambda-2. The phylogeny also showed that sigma-2 IgL chains initially regarded as specific for cartilaginous fish are present in holosteans, polypterids, and even in turtles. We conclude that there were five ancient IgL isotypes, which evolved differentially in various lineages of jawed vertebrates.
Assuntos
Peixes/genética , Genes de Cadeia Leve de Imunoglobulina , Variação Genética , Isotipos de Imunoglobulinas/genética , Sequência de Aminoácidos , Animais , Peixes/classificação , Perfilação da Expressão Gênica , Loci Gênicos , Genoma , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Hibridização in Situ Fluorescente , Filogenia , Transcriptoma , Recombinação V(D)JRESUMO
CD150 (IPO3/SLAM) belongs to the SLAM family of receptors and serves as a major entry receptor for measles virus. CD150 is expressed on normal and malignant cells of the immune system. However, little is known about its expression outside the hematopoietic system, especially tumors of the central nervous system (CNS). Although CD150 was not found in different regions of normal brain tissues, our immunohistochemical study revealed its expression in 77.6% of human CNS tumors, including glioblastoma, anaplastic astrocytoma, diffuse astrocytoma, ependymoma, and others. CD150 was detected in the cytoplasm, but not on the cell surface of glioma cell lines, and it was colocalized with the endoplasmic reticulum and Golgi complex markers. In addition to the full length mRNA of the mCD150 splice isoform, in glioma cells we found a highly expressed novel CD150 transcript (nCD150), containing an 83 bp insert. The insert is derived from a previously unrecognized exon designated Cyt-new, which is located 510 bp downstream of the transmembrane region exon, and is a specific feature of primate SLAMF1. Both mCD150 and nCD150 cDNA variants did not contain any mutations and had the leader sequence. The nCD150 transcript was also detected in normal and malignant B lymphocytes, primary T cells, dendritic cells and macrophages; however, in glioma cells nCD150 was found to be the predominant CD150 isoform. Similarly to mCD150, cell surface expression of nCD150 allows wild type measles virus entry to the cell. Our data indicate that CD150 expression in CNS tumors can be considered a new diagnostic marker and potential target for novel therapeutic approaches.
Assuntos
Antígenos CD/genética , Regulação da Expressão Gênica , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Antígenos CD/química , Antígenos CD/metabolismo , Linfócitos B/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , Neoplasias do Sistema Nervoso Central/metabolismo , Neoplasias do Sistema Nervoso Central/patologia , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Vírus do Sarampo/fisiologia , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
SLAMF9 is a member of the signaling lymphocyte-activating molecule (SLAM) immunoreceptor family. The SLAM family receptors are expressed in a broad range of immune cells and play an important role in immunity. To date, SLAMF9 is the least studied member of this family. Its ligand, signaling properties, and cells on whose surface it is expressed are unknown. We generated hybridoma clones 6E11 and 7G5 secreting monoclonal antibodies specific to human SLAMF9. BALB/c mice were immunized with Escherichia coli-expressed purified SLAMF9 protein; splenocytes from these mice were fused with mouse myeloma cell line NS-1. Based on isotyping of the MAbs, clone 6E11 was referred to the IgG1 subclass, while 7G5 to IgG2b. The specificity of these MAbs was assessed by ELISA, immunoblotting, immunohistochemistry, and flow cytometry. According to the results of epitope analysis, clone 6E11 reacts with the C2-like domain, whereas 7G5 is specific to the V-like domain of the SLAMF9 molecule. The generated MAbs were demonstrated to be applicable in various immunochemical analyses. They may be useful tools in studies clarifying the expression and function of human SLAMF9.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Animais , Especificidade de Anticorpos , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Citometria de Fluxo , Humanos , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Terciária de Proteína/genética , Família de Moléculas de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
FCRLA is an ER-resident B-cell specific protein. The exact function of this protein remains unclear although human FCRLA has been recently shown to interact with IgM, IgG and IgA. The retention of FCRLA in ER is mediated by the N-terminal domain. The major human FCRLA isoform is encoded by five exons, of which one encodes a short signal peptide (SSP) and the others code four protein domains. Here we show that human tissues also produce transcripts which contain an additional exon and encode proteins with signal peptide that is six residues longer (LSP). Transfection experiments demonstrated that the extension of the signal peptide had no visible effect on the topology and molecular mass of the processed four-domain FCRLA isoform. However, the length of the signal peptide was found to affect processing of two-domain FCRLA isoforms composed of the third and fourth domains (FCRLAd2). The signal peptide was not cleaved in the SSP-FCRLAd2 and this isoform was found to accumulate in the ER. In contrast, the LSP-containing FCRLAd2 isoform was processed, O-glycosylated and secreted. The secreted FCRLAd2 isoform did not interact with IgG- or IgM-immunosorbents.
Assuntos
Isoformas de Proteínas/genética , Sinais Direcionadores de Proteínas/genética , Receptores Imunológicos/genética , Processamento Alternativo/genética , Sequência de Aminoácidos , Linhagem Celular Tumoral , Células HEK293 , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Dados de Sequência Molecular , Isoformas de Proteínas/metabolismo , Receptores Fc , Receptores Imunológicos/metabolismoRESUMO
FCRLA is an intracellular B cell protein that belongs to the FcR-like family. Using newly generated FCRLA-specific antibodies, we studied the constitutive expression pattern of mouse FCRLA and monitored changes during an immune response and following in vitro B cell activation. All B cell subpopulations examined expressed FCRLA. However, the level of FCRLA expression is determined by the stage of B cell differentiation. Low expression of FCRLA is characteristic of naïve follicular and marginal zone B cells. High expression was detected in a small fraction of activated B cells scattered along migratory pathways in the lymphoid tissues. FCRLA-bright cells could be subdivided into two subpopulations, with high and low/undetectable level of intracellular immunoglobulins, which phenotypically resemble either plasma or memory B cells. High expression of FCRLA in subset(s) of terminally differentiated B-cells suggests that, being an ER protein, FCRLA may participate in the regulation of immunoglobulin assembly and secretion.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Receptores Imunológicos/biossíntese , Receptores Imunológicos/imunologia , Animais , Anticorpos/imunologia , Medula Óssea/imunologia , Medula Óssea/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Feminino , Imunoglobulinas/imunologia , Imunoglobulinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Ativação Linfocitária , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Receptores Imunológicos/genética , Transdução de SinaisRESUMO
We studied the evolution of the CD2 family in tetrapods by extracting and analyzing CD2-like genes from the genome of the amphibian species Silurana (Xenopus) tropicalis. An exhaustive analysis of the genomic and cDNA databases resulted in the identification of at least 70 CD2-like genes. The predicted receptors mostly maintain the typical VC2 ectodomains, but are highly diverse in their C-termini, which suggests a broad range of signaling capacities. Apart from the presumed monomeric receptors with ITSM and/or ITIM motifs, the Silurana family includes secreted proteins. Furthermore, a fraction of the receptors contain a conserved TM subtype with the NxxR motif that is known to promote an association with the FcRγ subunit and that was previously found in the members of the FcR- and KIR-related receptors. The expression analysis of a sample of the genes showed broad tissue distribution and gene-specific expression patterns. Phylogenetic analysis predicted that the CD58, CD150/SLAM, and SLAMF8 genes were maintained as single-copy genes in both mammals and amphibians, while others expanded/contracted in a lineage-specific manner.
Assuntos
Antígenos CD/genética , Antígenos CD2/genética , Receptores de Superfície Celular/genética , Xenopus/imunologia , Sequência de Aminoácidos , Animais , Antígenos CD/classificação , Antígenos CD2/classificação , Evolução Molecular , Dados de Sequência Molecular , Filogenia , Receptores de Superfície Celular/classificação , Alinhamento de Sequência , Transdução de Sinais , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Xenopus/genéticaRESUMO
Fc receptor-like A (FCRLA) is an unusual member of the extended Fc receptor family. FCRLA has homology to receptors for the Fc portion of Ig (FCR) and to other FCRL proteins. However, unlike these other family representatives, which are typically transmembrane receptors with extracellular ligand-binding domains, FCRLA has no predicted transmembrane domain or N-linked glycosylation sites and is an intracellular protein. We show by confocal microscopy and biochemical assays that FCRLA is a soluble resident endoplasmic reticulum (ER) protein, but it does not possess the amino acid sequence KDEL as an ER retention motif in its C-terminus. Using a series of deletion mutants, we found that its ER retention is most likely mediated by the amino terminal partial Ig-like domain. We have identified ER-localized Ig as the FCRLA ligand. FCRLA is unique among the large family of Fc receptors, in that it is capable of associating with multiple Ig isotypes, IgM, IgG and IgA. Among hemopoietic cells, FCRLA expression is restricted to the B lineage and is most abundant in germinal center B lymphocytes. The studies reported here demonstrate that FCRLA is more broadly expressed among human B lineage cells than originally reported; it is found at significant levels in resting blood B cells and at varying levels in all B-cell subsets in tonsil.
Assuntos
Linfócitos B/imunologia , Retículo Endoplasmático/imunologia , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Receptores Imunológicos/imunologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Receptores Fc , Linfócitos T/imunologiaRESUMO
FCRL6 receptor is a more recently identified representative of the FCRL family. We generated a panel of mouse mAbs to baculovirus-derived recombinant FCRL6 protein. The clone 7B2 was found to specifically recognize a 63kDa protein expressed preferentially on the surface of CD8 T and CD56 NK cells in human peripheral blood and spleen. The clone 7B2 reacts with FCRL6 in Western blotting, FACS, and immunohistochemistry. In the T cell lineage, FCRL6 functions in antigen-experienced cells. Mitogenic stimulation of PB leukocytes in vitro resulted in an abrogation of the FCRL6 gene expression. We found a significant decrease in the FCRL6 gene expression in peripheral T cells of patients with certain autoimmune and blood diseases, and its upregulation at the late stages of HIV infection. Study of the FCRL6 association with signaling molecules showed its ability to recruit SHP-1, SHP-2, SHIP-1, and SHIP-2 phosphatases, and also adaptor protein Grb2 through phosphorylated cytoplasmic tyrosines. The current results demonstrate inhibitory potential of FCRL6 and suggest its possible involvement in modulation of CTL effector functions in various immune disorders.
Assuntos
Proteínas de Transporte/imunologia , Regulação da Expressão Gênica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Processamento Alternativo , Sequência de Aminoácidos , Doenças Autoimunes/imunologia , Células Sanguíneas/citologia , Linfócitos T CD8-Positivos/imunologia , Doenças Hematológicas/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Células Matadoras Naturais/imunologia , Dados de Sequência Molecular , RNA Mensageiro/imunologia , Alinhamento de Sequência , Baço/citologiaRESUMO
In this study, we searched the amphibian species Xenopus laevis and Silurana (Xenopus) tropicalis for the presence of genes homologous to mammalian KIRs and avian CHIRs (KRIR family). By experimental and computational procedures, we identified four related ILR (Ig-like Receptors) genes in S. tropicalis and three in X. laevis. ILRs encode type I transmembrane receptors with 3-4 Ig-like extracellular domains. All predicted ILR proteins appear to be activating receptors. ILRs have a broad expression pattern, the gene transcripts were found in both lymphoid and non-lymphoid tissues. Phylogenetic analysis shows that the amphibian KRIR family receptors evolved independently from their mammalian and avian counterparts. The only conserved structural element of tetrapod KRIRs is the NxxR motif-containing transmembrane domain that facilitates association with FcRgamma subunit. Our findings suggest that if KRIRs of various vertebrates have any common function at all, such a function is activating rather than inhibitory.
Assuntos
Receptores KIR/genética , Receptores KIR/imunologia , Proteínas de Xenopus/genética , Proteínas de Xenopus/imunologia , Xenopus/genética , Xenopus/imunologia , Sequência de Aminoácidos , Animais , Evolução Biológica , Southern Blotting , Galinhas , Mineração de Dados , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Transfecção , Xenopus laevisRESUMO
BACKGROUND: Recent studies have revealed an unexpected diversity of domain architecture among FcR-like receptors that presumably fulfill regulatory functions in the immune system. Different species of mammals, as well as chicken and catfish have been found to possess strikingly different sets of these receptors. To better understand the evolutionary history of paired receptors, we extended the study of FcR-like genes in amphibian representatives Xenopus tropicalis and Xenopus laevis. RESULTS: The diploid genome of X. tropicalis contains at least 75 genes encoding paired FcR-related receptors designated XFLs. The allotetraploid X. laevis displays many similar genes primarily expressed in lymphoid tissues. Up to 35 domain architectures generated by combinatorial joining of six Ig-domain subtypes and two subtypes of the transmembrane regions were found in XFLs. None of these variants are shared by FcR-related proteins from other studied species. Putative activating XFLs associate with the FcRgamma subunit, and their transmembrane domains are highly similar to those of activating mammalian KIR-related receptors. This argues in favor of a common origin for the FcR and the KIR families. Phylogenetic analysis shows that the entire repertoires of the Xenopus and mammalian FcR-related proteins have emerged after the amphibian-amniotes split. CONCLUSION: FcR- and KIR-related receptors evolved through continual species-specific diversification, most likely by extensive domain shuffling and birth-and-death processes. This mode of evolution raises the possibility that the ancestral function of these paired receptors was a direct interaction with pathogens and that many physiological functions found in the mammalian receptors were secondary acquisitions or specializations.
Assuntos
Evolução Molecular , Variação Genética , Receptores Fc/genética , Xenopus/genética , Sequência de Aminoácidos , Animais , Expressão Gênica , Humanos , Camundongos , Filogenia , Receptores Fc/classificação , Receptores KIR/genética , Alinhamento de Sequência , Especificidade da Espécie , Xenopus/classificação , Xenopus/imunologia , Xenopus laevis/genética , Xenopus laevis/imunologiaRESUMO
We describe a simple and efficient method to detect antibodies against native epitopes following immunization with denatured proteins and peptides. With this method, soluble antigens genetically fused with placental alkaline phosphatase (AP) are used as probes to detect antibodies immobilized on nitrocellulose membranes. The AP-tagged proteins can be produced in sufficient amounts using transient transfection of eukaryotic cells with an appropriate cDNA fragment in a commercial AP-tag vector. The intrinsic thermo-stable phosphatase activity of a tagged protein obviates the need for its purification. To evaluate the method, three recently identified proteins of the FcR family, FCRLA, FCRL1, and FCRL4, were fused with AP and tested in a reaction with various polyclonal and monoclonal antibodies raised by immunization with bacterially produced antigens and peptide conjugates. All the three probes demonstrated high specificity in analysis of immune sera and hybridoma supernatants. Sensitivity of the assay varied depending on antibody tested and, in some cases, was in the subnanogram range. The results obtained show that AP-tagged proteins are useful tools for discrimination of antibodies against native epitopes when production of antigen in its native conformation is laborious and expensive.
Assuntos
Fosfatase Alcalina/imunologia , Epitopos/imunologia , Receptores Fc/imunologia , Receptores Imunológicos/imunologia , Fosfatase Alcalina/genética , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Western Blotting , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Receptores Fc/genética , Receptores Imunológicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
FCRLA is a recently identified intracellular protein structurally related to the classic Fc receptors and expressed primarily in the germinal centers of B cells. We generated six monoclonal antibodies (MAbs) specific to the human protein. The MAbs recognize three different epitopes, which were shown to be localized on the D3 domain of the FCRLA molecule. The clones M101 and M616 were demonstrated to be applicable in various immunochemical analyses, such as immunoblotting, immunohistochemistry, and immunoprecipitation. In addition, this pair of antibodies was used for development of a sandwich version of ELISA to quantitatively detect FCRLA in cell lysates. Using these MAbs, we studied FCRLA expression in a panel of human B cell lines, such as Raji, Daudi, Bjab, BL-2, RPMI 1788, RPMI 8226, IM-9, and SKW6.4. It was found that all these lines, except RPMI 8226, produce FCRLA but may vary in the proportion of FCRLA-positive cells. The MAbs we established can be a useful tool to investigate the functional role of FCRLA and its applicability as a B cell development and malignant transformation marker.
Assuntos
Anticorpos Monoclonais/biossíntese , Receptores Fc/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Linfócitos B/metabolismo , Linhagem Celular , Chlorocebus aethiops , Epitopos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Receptores Fc/biossíntese , Proteínas Recombinantes/imunologiaRESUMO
In primates and rodents, the extended FcR family is comprised of three subsets: classical FcRs, structurally diverse cell surface receptors currently designated FCRL1-FCRL6, and intracellular proteins FCRLA and FCRLB. Using bioinformatic analysis, we revealed the FcR-like genes of the same three subsets in the genome of dog, another representative of placental mammals, and in the genome of short-tailed opossum, a representative of marsupials. In contrast, a single FcR-like gene was found in the current version of the chicken genome. This in silico finding was confirmed by the gene cloning and subsequent Southern blot hybridization. The chicken FCRL gene encodes a cell surface receptor with the extracellular region composed of four Ig-like domains of the D1-, D2-, D3-, and D4-subtypes. The gene is expressed in lymphoid and non-lymphoid tissues. Phylogenetic analysis of the mammalian and chicken genes suggested that classical FcRs, FCRLA, and FCRLB emerged after the mammalian-avian split but before the eutherian-marsupial radiation. The data obtained show that the repertoire of the classical FcRs and surface FcR-like proteins in mammalian species was shaped by an extensive recombination process, which resulted in domain shuffling and species-specific gain and loss of distinct exons or entire genes.
Assuntos
Evolução Biológica , Galinhas/genética , Cães/genética , Gambás/genética , Filogenia , Receptores Fc/genética , Animais , Clonagem Molecular , Biologia Computacional , Variação Genética , Humanos , Alinhamento de Sequência , Especificidade da EspécieRESUMO
The transmembrane signaling subunits (TSSs) bearing the immunoreceptor tyrosine-based activation motif (ITAM) play a crucial role in triggering the effector functions of mammalian leukocytes. The involvement in key immune reactions and obvious extension through duplication events make TSSs valuable markers of the evolution of the immune system. We surveyed the genomic sequences of the teleostean fish Fugu rubripes for the presence of genes encoding these accessory molecules. Automatic gene prediction was not efficient because of the poor ability of the programs used to recognize the short exons encoding the intracellular regions of TSSs. However, the unique compactness of the Fugu genome and the conservation of the exon/intron arrangements of the TSS genes facilitated their recognition by visual inspection of the candidate genomic sequences. Evidence for the presence of the CD3epsilon, CD3gamma/delta, CD79a, CD79b, TCRzeta, FcRgamma, DAP12 and DAP10 genes in the Fugu genome was obtained. Furthermore, conserved synteny for the short regions including the TSS genes was revealed by comparison of the Fugu and human genomes. The data demonstrate that the set of TSSs arose before the teleost-tetrapod split and provide a starting point for experimental investigation of the molecular evolution of the leukocyte-activating receptor complexes from fish species to mammals.