RESUMO
The B cell antigen receptor, composed of membrane immunoglobulin (Ig) sheathed by the Igalpha/Igbeta heterodimer plays a critical role in mediating B cell development and responses to antigen. The cytoplasmic tails of Igalpha and Igbeta differ substantially but have been well conserved in evolution. Transfection experiments have revealed that, while these tails share an esssential tyrosine-based activation motif (ITAM), they perform differently in some but not all assays and have been proposed to recruit distinct downstream effectors. We have created transgenic mouse lines expressing chimeric receptors comprising an IgM fused to the cytoplasmic domain of each of the sheath polypeptides. IgM/alpha and IgM/beta chimeras (but not an IgM/beta with mutant ITAM) are each independently sufficient to mediate allelic exclusion, rescue B cell development in gene-targeted Igmu- mice that lack endogenous antigen receptors, as well as signal for B7 upregulation. While the (IgM/alpha) x (IgM/beta) double-transgenic mouse revealed somewhat more efficient allelic exclusion, our data indicate that each of the sheath polypeptides is sufficient to mediate many of the essential functions of the B cell antigen receptor, even if the combination gives optimal activity.
Assuntos
Linfócitos B/imunologia , Imunoconjugados , Imunoglobulina M/biossíntese , Receptores de Antígenos de Linfócitos B/biossíntese , Receptores de Antígenos de Linfócitos B/fisiologia , Abatacepte , Animais , Antígenos CD , Antígenos de Diferenciação/metabolismo , Antígeno CTLA-4 , Dimerização , Citometria de Fluxo , Interleucina-2/biossíntese , Ativação Linfocitária , Linfoma de Células B , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/química , Proteínas Recombinantes de Fusão/biossíntese , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas , TirosinaRESUMO
Mice were immunized with model xenogeneic (both the VH frameworks and the CH domains of human origin), chimeric (just VH frameworks human), or self antibodies, and the antiantibody responses were dissected. Only the self antibody did not elicit a response. A strong response was elicited by the most xenogeneic antibody with approximately 90% against the C and approximately 10% against the V. The anti-V response was not attenuated in the chimeric antibody, demonstrating that foreign VH frameworks can be sufficient to lead to a strong antiantibody response. The magnitude of this xenogeneic anti-VH response was similar to that of the allotypic response elicited by immunizing mice of the Igha allotype with an Ighb antibody. Thus, although chimerization can diminish antiantibody responses, attention should be paid both to V region immunogenicity and to polymorphism.
Assuntos
Anticorpos Anti-Idiotípicos/biossíntese , Animais , Feminino , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polimorfismo GenéticoRESUMO
Serum immunoglobulin (Ig)M provides the initial response to foreign antigen and plays a regulatory role in subsequent immune response development, accelerating the production of high-affinity IgG. Here we show that mice deficient in serum IgM have an increased propensity to spontaneous autoimmunity as judged by the development with age of serum IgG anti-DNA antibodies and the renal deposition of IgG and complement. They also exhibit augmented anti-DNA IgG production on exposure to lipopolysaccharide. Thus, deficiency in serum IgM leads to diminished responsiveness to foreign antigens but increased responsiveness to self-a paradoxical association reminiscent of that described in humans deficient in complement or IgA. We wondered whether serum IgM might play an analogous role with regard to the response to self-antigens. However, here-in contrast to the sluggish response to foreign antigens-we find that deficiency in serum IgM actually predisposes to the development of IgG antibodies to autoantigens.
Assuntos
Autoanticorpos/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Cadeias mu de Imunoglobulina/imunologia , Animais , Linhagem Celular , Imunoglobulina M/sangue , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Membrane IgM is expressed on the surface of B lymphocytes. It is not transported to the surface of transfected plasmacytoma or COS cells. Here, we show that mutation of four hydrophilic amino acids in the microm transmembrane is sufficient to overcome the intracellular retention of membrane IgM in non-B cells. This suggests that the B cell-specific IgM-associated proteins that have been postulated to assist the transport of membrane IgM to the cell surface (3) act either by forming a hydrophobic sheath that surrounds the microm transmembrane segment or by displacing an interaction with this segment that would otherwise cause retention. Experiments with a CD8/mu hybrid H chain indicate that the proteins that assist the transport of membrane IgM to the B cell surface at most need the mu CH4 and transmembrane/cytoplasmic portion for interaction.
Assuntos
Linfócitos B/imunologia , Imunoglobulina M/análise , Cadeias mu de Imunoglobulina/análise , Receptores de Antígenos de Linfócitos B/análise , Sequência de Aminoácidos , Transporte Biológico , Humanos , Plasmocitoma/imunologiaRESUMO
CD22 is a B cell-specific transmembrane glycoprotein that acts to dampen signals generated through the B cell antigen receptor (BCR): B cells from CD22-deficient mice give increased Ca2+ fluxes on BCR ligation. Here we show that this B cell hyperresponsiveness correlates with the development of autoantibodies. After the age of eight months, CD22-deficient mice developed high titers of serum IgG directed against double-stranded DNA; these antibodies were of multiclonal origin, somatically mutated, and high affinity. Increased titers of antibodies to cardiolipin and myeloperoxidase were also noted. The results demonstrate that a single gene defect exclusive to B lymphocytes is, without additional contrivance, sufficient to trigger autoantibody development in a large proportion of aging animals. Thus, CD22 might have evolved specifically to regulate B cell triggering thresholds for the avoidance of autoimmunity.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Autoanticorpos/imunologia , Linfócitos B/metabolismo , Moléculas de Adesão Celular , Lectinas , Receptores de Superfície Celular/imunologia , Fatores Etários , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos B/genética , Autoanticorpos/química , Autoimunidade/imunologia , Técnicas Biossensoriais , DNA/imunologia , Marcação de Genes , Imunoglobulina G/sangue , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Cinética , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Receptores de Superfície Celular/genética , Lectina 2 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
Cell lines have been established that secrete a matched set of human chimeric IgM, IgG1, IgG2, IgG3, IgG4, IgE, and IgA2 antibodies that are directed against the hapten 4-hydroxy-3-nitrophenacetyl. These chimeric antibodies secreted from mouse plasmacytoma cells behave exactly like their authentic human counterparts in SDS-PAGE analysis, binding to protein A and in a wide range of serological assays. The antibodies have been compared in their ability to bind human C1q as well as in their efficacy in mediating lysis of human erythrocytes in the presence of human complement. A major conclusion to emerge is that whereas IgG3 bound C1q better than did IgG1, the chimeric IgG1 was much more effective than all the other IgG subclasses in complement-dependent hemolysis. The IgG1 antibody was also the most effective in mediating antibody-dependent cell-mediated cytotoxicity using both human effector and human target cells. These results suggest that IgG1 might be the favoured IgG subclass for therapeutic applications.
Assuntos
Anticorpos Monoclonais/imunologia , Imunoglobulinas/imunologia , Animais , Anticorpos Monoclonais/genética , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular , Enzimas Ativadoras do Complemento/imunologia , Complemento C1/imunologia , Complemento C1q , Proteínas do Sistema Complemento/imunologia , DNA Recombinante , Eletroforese em Gel de Poliacrilamida , Genes de Imunoglobulinas , Glicosilação , Haptenos , Hemólise , Humanos , Imunoglobulinas/genética , Camundongos , Nitrofenóis/imunologia , Fenilacetatos , Plasmocitoma/imunologia , Plasmídeos , Transfecção , Células Tumorais CultivadasRESUMO
Somatic hypermutation and isotype switch recombination occur in germinal center B cells, are linked to transcription, and are similarly affected by deficiency in MutS homologue (MSH)2. Class-switch recombination is abrogated by disruption of genes encoding components of the catalytic subunit of DNA-dependent protein kinase (DNA-PK(cs))/Ku complex and likely involves nonhomologous end joining (NHEJ). That somatic hypermutation might also be associated with end joining is suggested by its association with the creation of deletions, duplications, and sites accessible to terminal transferase. However, a requirement for NHEJ in the mutation process has not been demonstrated. Here we show that somatic mutation in mice deficient in NHEJ can be tested by introduction of rearranged immunoglobulin and T cell receptor transgenes: the transgene combination not only permits reconstitution of peripheral lymphoid compartments but also allows formation of germinal centers, despite the wholly monoclonal nature of the lymphocyte antigen receptors in these animals. Using this strategy, we confirm that somatic hypermutation like class-switching can occur in the absence of recombination-activating gene (RAG)1 but show that the two processes differ in that hypermutation can proceed essentially unaffected by deficiency in DNA-PK(cs) activity.
Assuntos
Proteínas de Ligação a DNA , Genes RAG-1 , Mutagênese , Proteínas Serina-Treonina Quinases/genética , Recombinação Genética , Animais , Sequência de Bases , Domínio Catalítico , Proteína Quinase Ativada por DNA , Rearranjo Gênico , Centro Germinativo , Proteínas de Homeodomínio , Imunoglobulinas/genética , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Muramidase/imunologia , Subunidades Proteicas , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos T/genética , Transposases/genéticaRESUMO
How do B cells generate the enormous diversity of antibodies that are able to recognize and bind to whichever antigen a B cell might happen to encounter in the body? Several genetic mechanisms that manipulate different combinations of immunoglobulin genes are known. In their Perspective, Neuberger and Scott, highlight another genetic mechanism called RNA editing now shown to be involved in the production of antibody diversity.
Assuntos
Diversidade de Anticorpos , Linfócitos B/imunologia , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Edição de RNA , Desaminase APOBEC-1 , Animais , Linfócitos B/enzimologia , Catálise , Citidina Desaminase/química , Evolução Molecular , Genes de Imunoglobulinas , Humanos , Switching de Imunoglobulina , Ativação Linfocitária , Camundongos , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
CD22 is a surface glycoprotein of B lymphocytes that is rapidly phosphorylated on cytoplasmic tyrosines after antigen receptor cross-linking. Splenic B cells from mice with a disrupted CD22 gene were found to be hyperresponsive to receptor signaling: Heightened calcium fluxes and cell proliferation were obtained at lower ligand concentrations. The mice gave an augmented immune response, had an expanded peritoneal B-1 cell population, and contained increased serum titers of autoantibody. Thus, CD22 is a negative regulator of antigen receptor signaling whose onset of expression at the mature B cell stage may serve to raise the antigen concentration threshold required for B cell triggering.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos B/imunologia , Moléculas de Adesão Celular , Lectinas , Animais , Anticorpos Antinucleares/sangue , Formação de Anticorpos , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Cálcio/metabolismo , Feminino , Marcação de Genes , Imunização , Imunoglobulina M/sangue , Imunofenotipagem , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fosforilação , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/fisiologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Transdução de Sinais , TransfecçãoRESUMO
Multiple DNA polymerases exist in eukaryotes. Polymerases alpha, delta and epsilon are mainly responsible for chromosomal DNA replication in the nucleus and are required for proliferation. In contrast, the repair polymerases beta and eta are not essential for cellular proliferation in yeast or mice, but a lack of either polymerase can lead, respectively, to defects in base excision repair or the ability to replicate past lesions induced by ultraviolet (UV) radiation [1-3]. Here, we have focused on polymerase zeta. This was first described as a non-essential product of the yeast REV3/REV7 genes involved in UV-induced mutagenesis, and was later implicated in trans-lesion synthesis [4,5]. Unlike in yeast, the mouse homologue (mRev3) was found to be essential for life. Homozygous mutant mice died in utero. Mutant embryos were considerably reduced in size at day 10.5 of development and usually aborted around day 12.5. It is likely that this block reflects a need for mRev3 in proliferative clonal expansion (rather than in the production of a particular cell type) as mutant blastocysts showed greatly diminished expansion of the inner cell mass in culture. Thus, mRev3 could be required to repair a form of externally induced DNA damage that otherwise accumulates during clonal expansion or, consistent with the high homology shared between its Rev7 partner and the mitotic checkpoint gene product Mad2 [6], mRev3 might play a role in cell proliferation and genomic stability even in the absence of environmentally induced damage.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Desenvolvimento Embrionário e Fetal/genética , Proteínas Fúngicas/genética , Genes Letais , Proteínas de Saccharomyces cerevisiae , Animais , Sequência de Bases , Primers do DNA , Camundongos , Camundongos MutantesRESUMO
For the generation of secondary response antibodies, immunoglobulin genes are subjected to hypermutation. Cells expressing antibodies with higher affinity are then selected by antigen. Recent clues to the mechanism of hypermutation come from experiments using transgenic mice enabling analysis of the controlling cis-acting elements and the intrinsic features of the hypermutation, dissociated from the effects of antigenic selection.
Assuntos
Linfócitos B/imunologia , Genes de Imunoglobulinas , Região Variável de Imunoglobulina/genética , Mutação , Animais , Humanos , CamundongosRESUMO
Burkitt's lymphomas harbour chromosomal translocations bringing c-MYC into the vicinity of one of the immunoglobulin gene loci. Point mutations have been described within c-MYC in several Burkitt's lymphomas and it has been proposed that translocation into the Ig loci might have transformed c-MYC into a substrate for the antibody hypermutation mechanism. Here we test this hypothesis by exploiting a Burkitt's lymphoma line (Ramos) that we have previously shown to hypermutate its immunoglobulin genes constitutively. We find that, during in vitro culture, Ramos mutates the c-MYC allele that is translocated into the IgH locus whilst leaving the untranslocated c-MYC and other control genes essentially unaffected. The mutations are introduced downstream of the c-MYC transcription start with the pattern of substitutions being characteristic of the antibody hypermutation mechanism; the mutation frequency is 2-3-fold lower than for the endogenous functional IgH allele. Thus chromosomal translocations involving the Ig loci may not only contribute to transformation by deregulating oncogene expression but could also act by potentiating subsequent oncogene hypermutation.
Assuntos
Alelos , Linfoma de Burkitt/genética , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Mutação , Proteínas Proto-Oncogênicas c-myc/genética , Translocação Genética , Sequência de Bases , Humanos , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
The canonical form of the B cell antigen receptor is composed of membrane immunoglobulin sheathed by the alpha/beta heterodimer. Whereas membrane IgM cannot be transported to the cell surface in the absence of alpha/beta, both IgD and IgG2b can be expressed naked (i.e. without alpha/beta) on the surface of myeloma transfectants. In the case of one cell-line, such naked IgD has been shown to be inserted into the membrane by a glycosyl-phosphatidylinositol anchor. Here, however, we show that both IgD and IgG2b (but not IgM) can be expressed on the surface of myeloma transfectants without either sheath or anchor. This distinction between the isotypes is attributable to differences in the region of the transmembrane segment.
Assuntos
Imunoglobulina D/química , Imunoglobulina G/química , Proteínas de Membrana/química , Plasmocitoma/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Imunoglobulina D/genética , Imunoglobulina D/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Cadeias Pesadas de Imunoglobulinas/química , Isotipos de Imunoglobulinas , Camundongos , Dados de Sequência Molecular , Fosfatidilinositol Diacilglicerol-Liase , Diester Fosfórico Hidrolases/farmacologia , Plasmocitoma/genética , Plasmídeos , Transfecção , Células Tumorais CultivadasRESUMO
Myeloma DNA expression systems can be used for the synthesis and secretion of antibody/enzyme recombinant molecules. Here we describe the construction of a myeloma cell-line that secretes a hapten-specific antibody/enzyme hybrid molecule, in which the antibody Fc portion has been replaced by the Klenow fragment of Escherichia coli DNA polymerase I (PolIk). This Fab-PolIk hybrid molecule is secreted in good yield from the myeloma transfectants, can be purified to homogeneity in a single step on hapten-Sepharose columns, and exhibits PolIk activity as judged by its use in dideoxy nucleotide sequencing. Thus Fab-PolIk can be used for the same purposes as conventional PolIk but has the advantage that it is easily purified to homogeneity in a one-step purification from culture medium.
Assuntos
DNA Polimerase I/genética , Fragmentos de Peptídeos/genética , Proteínas Recombinantes/biossíntese , Animais , Cromatografia de Afinidade , Clonagem Molecular , DNA Polimerase I/biossíntese , DNA Polimerase I/isolamento & purificação , Imunoglobulina G/genética , Camundongos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/isolamento & purificação , Relação Estrutura-Atividade , TransfecçãoRESUMO
Here we compare human monoclonal antibody (MAb) production from mouse strains that carry disruptions of their endogenous mouse IgH/IgK loci and harbor human IgM + Igkappa(BABkappa) or human IgM + Igkappa + IgA transloci (BABkappa,lambda). We found that whereas both strains proved effective for the isolation of antigen-specific IgM antibodies, many of the IgM MAbs elicited from BABkappa comprise human mu chains that are associated with mouse lambda chains. In contrast, BABkappa,lambda mice gave rise to fully functional, polymeric human IgM antibodies comprising both human IgH and human IgL chains. Therefore, the inclusion of a human Iglambda translocus (in addition to the human IgH + Igkappa transloci) not only diminishes problems of endogenous mouse Iglambda expression but also provides a strain of mice that yields fully human MAbs to a wide range of antigens, as witnessed by the isolation of MAbs to human blood cells, tumor cell lines, and an immunoglobulin idiotype.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Imunoglobulina M/biossíntese , Imunoglobulina M/genética , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/genética , Animais , Anticorpos Monoclonais/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Hibridomas/imunologia , Hibridomas/fisiologia , Imunoglobulina M/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Monócitos/imunologia , Monócitos/fisiologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da Espécie , Baço/imunologiaRESUMO
The physiological mechanism for producing antigen-specific antibodies is based on a two-phase neo-Darwinian process: the first phase consists of diversity generation (formation of the repertoire), and the second phase is antigen-mediated selection. In this article, we consider how the natural immunoglobulin gene-diversification processes can be exploited both in vivo and in vitro in order to allow the generation of novel antibody (and heterologous protein) repertoires.
Assuntos
Diversidade de Anticorpos/genética , Rearranjo Gênico do Linfócito B , Seleção Genética , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Afinidade de Anticorpos/genética , Especificidade de Anticorpos/genética , Linhagem Celular , Genes de Imunoglobulinas , Humanos , Camundongos , Camundongos Transgênicos , MutaçãoRESUMO
We have previously described a strategy for integrating selectable marker genes into yeast artificial chromosomes (YACs) to facilitate their transfer into embryonic stem (ES) cells. Here we apply this technology to create mice carrying the core region of the human immunoglobulin (Ig) kappa light chain locus. A YAC was isolated which contains a 300 kb insert spanning three V kappa segments, the J kappa cluster, the C kappa region and extending downstream of the Kde element. After modification of this YAC to integrate the selectable neo marker gene, the YAC was introduced into ES cells by protoplast fusion. Several ES cell clones were obtained which appeared to harbor one complete copy of the YAC while retaining little or no other yeast DNA. The ES cells were injected into blastocysts and the chimaeric mice were shown to rearrange the introduced human light chain genes with the resultant production of antibodies containing human kappa light chains in the serum.
Assuntos
Cromossomos Fúngicos , Genoma Humano , Cadeias kappa de Imunoglobulina/genética , Animais , Sequência de Bases , Blastocisto , Quimera , Biblioteca Gênica , Rearranjo Gênico , Marcadores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Protoplastos , TransfecçãoAssuntos
Anticorpos/genética , Imunidade/imunologia , Linfócitos B/metabolismo , Sítios de Ligação , Diferenciação Celular/genética , Evolução Molecular , Humanos , Região Variável de Imunoglobulina/imunologia , Modelos Genéticos , Modelos Moleculares , Conformação Proteica , Células-Tronco/metabolismoAssuntos
Bacteriófago lambda/genética , Klebsiella pneumoniae/genética , Óperon , Transdução Genética , Arabinose/análogos & derivados , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Bacterianos , Klebsiella pneumoniae/enzimologia , Hibridização de Ácido Nucleico , Fosfotransferases/metabolismo , Ribitol , Desidrogenase do Álcool de Açúcar/metabolismo , Álcoois Açúcares , Transcrição GênicaRESUMO
Novel proteins have been elaborated over evolutionary time by an iterative alternation of mutation and selection. In a similar way, the humoral immune system also uses an iterative alternation of mutation and selection to generate novel antibodies that display a high affinity for their cognate antigen -- but this is achieved in a matter of a days. Gene rearrangement is used to produce a primary repertoire of antibodies and, on entering the body, antigen triggers the clonal expansion of those B lymphocytes that express a cognate antibody, albeit one of low affinity. Rapid and specific affinity maturation is then achieved by subjecting the immunoglobulin genes in the rapidly expanding B cells to a period of intense mutation. The intensity of this mutational assault is tolerated because it is targeted specifically to the immunoglobulin genes, causing relatively little damage to other loci. Antigen-mediated selection then allows the preferential expansion of those mutants expressing antibodies displaying improved binding characteristics. Here, studies are described that have been performed to glean insight into the mechanisms of the hypermutation and selection processes. Experiments are also described in which an attempt has been made to recapitulate aspects of physiological antibody generation in vitro, allowing the development of novel approaches to the generation of proteins with high-affinity binding sites.