RESUMEN
Increased IgE is a typical feature of allergic rhinitis. Local class-switch recombination has been intimated but B cell precursors and mechanisms remain elusive. Here we describe the dynamics underlying the generation of IgE-antibody secreting cells (ASC) in human nasal polyps (NP), mucosal tissues rich in ASC without germinal centers (GC). Using VH next generation sequencing, we identified an extrafollicular (EF) mucosal IgD+ naïve-like intermediate B cell population with high connectivity to the mucosal IgE ASC. Mucosal IgD+ B cells, express germline epsilon transcripts and predominantly co-express IgM. However, a small but significant fraction co-express IgG or IgA instead which also show connectivity to ASC IgE. Phenotypically, NP IgD+ B cells display an activated profile and molecular evidence of BCR engagement. Transcriptionally, mucosal IgD+ B cells reveal an intermediate profile between naïve B cells and ASC. Single cell IgE ASC analysis demonstrates lower mutational frequencies relative to IgG, IgA, and IgD ASC consistent with IgE ASC derivation from mucosal IgD+ B cell with low mutational load. In conclusion, we describe a novel mechanism of GC-independent, extrafollicular IgE ASC formation at the nasal mucosa whereby activated IgD+ naïve B cells locally undergo direct and indirect (through IgG and IgA), IgE class switch.
Asunto(s)
Formación de Anticuerpos/inmunología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Inmunoglobulina D/inmunología , Inmunoglobulina E/inmunología , Mucosa Nasal/inmunología , Mucosa Nasal/metabolismo , Adulto , Formación de Anticuerpos/genética , Células Productoras de Anticuerpos/inmunología , Células Productoras de Anticuerpos/metabolismo , Biología Computacional , Perfilación de la Expresión Génica , Centro Germinal/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hipersensibilidad/etiología , Hipersensibilidad/metabolismo , Cambio de Clase de Inmunoglobulina/genética , Cambio de Clase de Inmunoglobulina/inmunología , Isotipos de Inmunoglobulinas/genética , Isotipos de Inmunoglobulinas/inmunología , Inmunofenotipificación , Pólipos Nasales/etiología , Pólipos Nasales/metabolismo , Pólipos Nasales/patología , Polen/inmunología , Estaciones del Año , Hipermutación Somática de InmunoglobulinaRESUMEN
The epitheliochorial placenta of swine is considered a barrier to Ag and selective transport of IgG, so this species should be an excellent model with which to determine whether switch recombination is Ag dependent. Analysis of Ig levels and Ig isotype profiles in >150 normal and virus-infected fetuses from 38-110 days of gestation (DG) suggested that IgG, IgA, and IgM were most likely the result of de novo fetal synthesis. Although transcripts for IgM could be recovered at DG 50 (114 DG is full gestation) in all major fetal lymphoid tissues, those for IgG and IgA first became prominent at 60 DG in thymus, and transcription and spontaneous secretion became especially pronounced in this organ in older fetuses. Data on transcription, secretion, and serum isotype profiles suggest that although all fetal IgA and IgM may result from de novo synthesis, some IgG may result from low-level selective transport. The complementarity-determining region 3 spectratypes of thymic IgA and IgG transcripts at 70 and 90 days, respectively, were as polyclonal as that of IgM, indicating a broad repertoire of switched B cells although the VDJs transcribed with these switched isotypes in normal fetuses were not diversified in comparison to those from animals exposed to environmental Ags such as age-matched, virus-infected fetuses, colonized isolator piglets, and conventional adults. However, VDJs expressed with switched isotypes were more diversified than those expressed with IgM. Thus, switch recombination in fetal life does not appear to be driven by environmental Ag and is only weakly coupled to VDJ diversification. These findings, and the fact that the oligoclonal IgA and IgM repertoires in a noninductive site of the mucosal immune system (parotid gland) become polyclonal in piglets reared germfree, suggest that initial expansion of the switched cells in the B cell compartment of fetal and neonatal piglets is not driven by environmental Ag.
Asunto(s)
Diversidad de Anticuerpos , Sangre Fetal/inmunología , Cambio de Clase de Inmunoglobulina , Isotipos de Inmunoglobulinas/genética , Porcinos/inmunología , Animales , Antígenos/inmunología , Antígenos Virales/inmunología , Calostro/inmunología , ADN Nucleotidiltransferasas/metabolismo , Ambiente , Femenino , Enfermedades Fetales/embriología , Enfermedades Fetales/inmunología , Enfermedades Fetales/veterinaria , Vida Libre de Gérmenes , Edad Gestacional , Inmunoglobulina A/biosíntesis , Inmunoglobulina A/sangre , Inmunoglobulina A/genética , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Inmunoglobulina G/genética , Isotipos de Inmunoglobulinas/sangre , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/sangre , Inmunoglobulina M/genética , Región de Cambio de la Inmunoglobulina , Masculino , Intercambio Materno-Fetal , Glándula Parótida/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/embriología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Embarazo , Recombinación Genética , Porcinos/embriología , Porcinos/crecimiento & desarrollo , Enfermedades de los Porcinos/embriología , Enfermedades de los Porcinos/inmunología , Timo/embriología , Timo/inmunología , Transcripción Genética , VDJ RecombinasasRESUMEN
OBJECTIVE: To examine anti-double-stranded DNA (anti-dsDNA) IgG autoantibodies from the bone marrow of individuals with systemic lupus erythematosus (SLE). METHODS: A library of single-chain variable fragments (scFv) was constructed from SLE bone marrow complementary DNA of gamma, kappa, and lambda isotype by cloning into the pHENIX phagemid vector. The library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, and their heavy (H)- and light (L)-chain recombinants, were prepared, purified, and analyzed for binding to DNA by enzyme-linked immunosorbent assay. RESULTS: DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respectively) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of replacement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both single-stranded DNA and dsDNA. These 2 Ig subunits contained third complementarity-determining region arginines and had acquired the majority of replacement mutations. CONCLUSION: Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patients exploit diverse V genes and cationic V-D-J and V-J junctions for DNA binding, and accumulate replacement mutations that enhance binding.
Asunto(s)
ADN/inmunología , Adulto , Anticuerpos Antinucleares/genética , Arginina/química , Secuencia de Bases , Células de la Médula Ósea/inmunología , ADN/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Isotipos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Lupus Eritematoso Sistémico/inmunología , Biblioteca de Péptidos , ARN Mensajero/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
Previously, the specificity of human immune responses to Dactylis pollen was analyzed in 26 plasma samples with high levels of grass-pollen-specific IgG4 ('IgG4+ plasma', largely from grass-pollen-allergic patients), as compared to 25 plasma samples with low grass-pollen-specific IgG4 ('normal plasma', from nonatopic individuals). In the present study, a quantification of the Dactylis-pollen-specific IgE, IgM, IgA class and IgG subclass antibodies in these plasma samples is proposed. Isotypic distribution in IgG4+ plasma was 68% IgG [IgG2 (38%) > IgG4 (30%) > IgG1 (19%) > IgG3 (13%)], 27% IgM, 4% IgA and 0.05% IgE. In normal plasma it was 73% IgM, 20% IgG [IgG3 (38%) > IgG2 (33%) > IgG1 (29%) > IgG4 (0%)], 6% IgA and 0.006% IgE. In IgG4+ plasma, specific IgE, IgG1, IgG2 and IgG4 concentrations were positively correlated between each other. Finally, the present study clearly confirmed the possible role of the CH gene regulation in allergic diseases.