Repertoire of human antibodies against the polysaccharide capsule of Streptococcus pneumoniae serotype 6B.
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We examined the repertoire of antibodies to Streptococcus pneumoniae 6B capsular polysaccharide induced with the conventional polysaccharide vaccine in adults at the molecular level two ways. In the first, we purified from the sera of seven vaccinees antipneumococcal antibodies and determined their amino acid sequences. Their VH regions are mainly the products of VH3 family genes (candidate genes, 3-23, 3-07, 3-66, and 3-74), but the product of a VH1 family gene (candidate gene, 1-03) is occasionally used. All seven individuals have small amounts of polyclonal kappa+ antibodies (Vkappa1 to Vkappa4 families), although kappa+ antibodies are occasionally dominated by antibodies formed with the product of the A27 Vkappa gene. In contrast, lambda+ anti-6B antibodies are dominated by the antibodies derived from one of 3 very similar Vlambda2 family genes (candidate genes, 2c, 2e, and 2a2) and Clambda1 gene product. The Vlambda2(+) antibodies express the 8.12 idiotype, which is expressed on anti-double-stranded-DNA antibodies. In one case, Vlambda is derived from a rarely expressed Vlambda gene, 10a. In the second approach, we studied a human hybridoma (Dob1) producing anti-6B antibody. Its VH region sequence is closely related to those of the 3-15 VH gene (88% nucleotide homology) and JH4 (92% homology). Its VL region is homologous to the 2a2 Vlambda2 gene (91%) and Jlambda1/Clambda1. Taken together, the V region of human anti-6B antibodies is commonly formed by a VH3 and a Vlambda2 family gene product. (+info)
Insertion of excised IgH switch sequences causes overexpression of cyclin D1 in a myeloma tumor cell.
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Oncogenes are often dysregulated in B cell tumors as a result of a reciprocal translocation involving an immunoglobulin locus. The translocations are caused by errors in two developmentally regulated DNA recombination processes: V(D)J and IgH switch recombination. Both processes share the property of joining discontinuous sequences from one chromosome and releasing intervening sequences as circles that are lost from progeny cells. Here we show that these intervening sequences may instead insert in the genome and that during productive IgH mu-epsilon switch recombination in U266 myeloma tumor cells, a portion of the excised IgH switch intervening sequences containing the 3' alpha-1 enhancer has inserted on chromosome 11q13, resulting in overexpression of the adjacent cyclin D1 oncogene. (+info)
Analysis of V(H)-D-J(H) gene transcripts in B cells infiltrating the salivary glands and lymph node tissues of patients with Sjogren's syndrome.
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OBJECTIVE: In patients with Sjogren's syndrome (SS), B lymphocytes have been found to infiltrate salivary glands, resulting in sialadenitis and keratoconjunctivitis. The disease is frequently associated with benign and neoplastic lymphoproliferation. The present study was undertaken to investigate whether clonal B cell expansion takes place in lymphocytic infiltrations of salivary glands under (auto- [?]) antigen stimulation, by analyzing in more detail the variable part (V(H)-D-J(H)) of the immunoglobulin heavy chain genes expressed in these B cells. METHODS: Biopsies of the labial salivary glands and lymph nodes were performed on 2 female patients with SS. The Ig gene rearrangements in these tissues were amplified by reverse transcriptase-polymerase chain reaction using specific primers. RESULTS: A total of 94 V(H)-D-J(H) transcripts were cloned and sequenced. Our data suggest a polyclonal origin of the B cell infiltrates. In 92 of the transcripts, V(H) genes were modified by somatic mutation. Further analysis showed counterselection for replacement mutations within the framework regions, suggesting that those B cells were stimulated and selected for functional expression of a surface Ig. In labial salivary glands from both patients, clonally related B cells became evident. Members of 1 particular clone were found in both the lip and lymph node material. CONCLUSION: These data provide evidence, on the nucleotide sequence level, that an antigen-triggered clonal B cell expansion takes place in the salivary glands of patients with SS who do not have histologic evidence of developing lymphoma. It may be speculated that those B cell clones expand during disease progression, resulting in lymphomagenesis. (+info)
The role of homophilic binding in anti-tumor antibody R24 recognition of molecular surfaces. Demonstration of an intermolecular beta-sheet interaction between vh domains.
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The murine antibody R24 and mouse-human Fv-IgG1(kappa) chimeric antibody chR24 are specific for the cell-surface tumor antigen disialoganglioside GD3. X-ray diffraction and surface plasmon resonance experiments have been employed to study the mechanism of "homophilic binding," in which molecules of R24 recognize and bind to other molecules of R24 though their heavy chain variable domains. R24 exhibits strong binding to liposomes containing disialoganglioside GD3; however, the kinetics are unusual in that saturation of binding is not observed. The binding of chR24 to GD3-bearing liposomes is significantly weaker, suggesting that cooperative interactions involving antibody constant regions contribute to R24 binding of membrane-bound GD3. The crystal structures of the Fabs from R24 and chR24 reveal the mechanism for homophilic binding and confirm that the homophilic and antigen-binding idiotopes are distinct. The homophilic binding idiotope is formed largely by an anti-parallel beta-sheet dimerization between the H2 complementarity determining region (CDR) loops of two Fabs, while the antigen-binding idiotope is a pocket formed by the three CDR loops on the heavy chain. The formation of homophilic dimers requires the presence of a canonical conformation for the H2 CDR in conjunction with participation of side chains. The relative positions of the homophilic and antigen-binding sites allows for a lattice of GD3-specific antibodies to be constructed, which is stabilized by the presence of the cell membrane. This model provides for the selective recognition by R24 of cells that overexpress GD3 on the cell surface. (+info)
Human triclonal anti-IgG gammopathy. I. Iso-electric focusing characteristics of the IgG, IgA and IgM anti-IgG and their heavy and light chains.
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Human IgG, IgA and IgM anti-IgG autoantibodies have been isolated from the serum of an individual with Felty's syndrome. These were initially noted as soluble circulating serum complexes by analytical ultracentrifugation. Isolation was accomplished by solid phase immunoadsorption and each of the three antibody populations obtained was shown to be of restricted heterogeneity by liquid and polyacrylamide gel electrofocussing methods. Type kappa light chains were obtained from each protein. Co-isoelectric focusing experiments of all possible pairs of these light chains showed them to have identical net charge characteristics. Heavy chains obtained from each protein were also monoclonal and of differing isoelectric point. The availability of this serum provides a human model with which to study the changes which may occur in autoantibodies during the autoimmune response. (+info)
Ialpha exon-replacement mice synthesize a spliced HPRT-C(alpha) transcript which may explain their ability to switch to IgA. Inhibition of switching to IgG in these mice.
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Antibody class switching is regulated by transcription of unrearranged C(H) genes to produce germline (GL) transcripts which direct the choice of isotype and are required for switching. However, their role is unknown. GL transcripts are initiated at the I exons located upstream of each switch region. Although deletion of the I exon by gene targeting prevents switch recombination to that CH gene, the Ialpha exon can be replaced by an entirely different DNA segment, a minigene driven by the phosphoglycerate kinase (PGK) promoter and encoding hypoxanthine phosphoribosyl transferase (HPRT), oriented in the sense direction, without reducing antibody class switching to IgA. To understand why HPRT substitution of the Ialpha exon does not disrupt switch recombination, we have analyzed the structure of the transcript from the targeted allele in these mice. We identify a spliced transcript in which the HPRT exons are spliced to the C(alpha) gene segments, resulting in a structure similar to normal GL transcripts. The abundance of this transcript is similar to that of the normal alpha GL RNA. We also demonstrate that switching to the four IgG subclasses in B cells from these mice is reduced in comparison to wild-type mice. We discuss the possibility that the strong PGK promoter inserted at the Ig alpha locus may interfere with interaction of the promoters for gamma GL transcripts with the 3' IgH enhancer. (+info)
Induction of Ig light chain gene rearrangement in heavy chain-deficient B cells by activated Ras.
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During B cell development, rearrangement and expression of Ig heavy chain (HC) genes promote development and expansion of pre-B cells accompanied by the onset of Ig light chain (LC) variable region gene assembly. To elucidate the signaling pathways that control these events, we have tested the ability of activated Ras expression to promote B cell differentiation to the stage of LC gene rearrangement in the absence of Ig HC gene expression. For this purpose, we introduced an activated Ras expression construct into JH-deleted embryonic stem cells that lack the ability to assemble HC variable region genes and assayed differentiation potential by recombination activating gene (RAG) 2-deficient blastocyst complementation. We found that activated Ras expression induces the progression of B lineage cells beyond the developmental checkpoint ordinarily controlled by mu HC. Such Ras/JH-deleted B cells accumulate in the periphery but continue to express markers associated with precursor B cells including RAG gene products. These peripheral Ras/JH-deleted B cell populations show extensive Ig LC gene rearrangement but maintain an extent of kappa LC gene rearrangement and a preference for kappa over lambda LC gene rearrangement similar to that of wild-type B cells. We discuss these findings in the context of potential mechanisms that may regulate Ig LC gene rearrangement. (+info)
Predominant VH genes expressed in innate antibodies are associated with distinctive antigen-binding sites.
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Antibodies to phosphatidylcholine (PtC), a common constituent of mammalian and bacterial cell membranes, represent a large proportion of the natural antibody repertoire in mice. Previous studies of several mouse strains (e.g., C57BL/6) have shown that anti-PtC antibodies are mainly encoded by the VH11 and VH12 immunoglobulin heavy chain variable region gene families. We show here, however, that VH11 and VH12 encode only a small proportion of the anti-PtC antibodies in BALB/c mice. Instead, VHQ52-encoded antibodies predominate in this strain. In addition, two-thirds of the cells expressing VHQ52 family genes use a single gene (which, interestingly, has been previously shown to predominate in the anti-oxazolone response). We also show here that in anti-PtC antibodies from all strains, the distinctive antigen-binding sites associated with VHQ52 differ substantially from those associated with VH11 and VH12. That is, VHQ52-containing transcripts preferentially use the joining region JH4 rather than JH1 and exhibit more diverse complementarity-determining region 3 (CDR3) junctions with more N-region nucleotide additions at the gene segment junctions. Thus, the VH gene family that predominates in the anti-PtC repertoire differs among mouse strains, whereas the distinctive VHDJH rearrangements (CDR3, JH) associated with each VH gene family are similar in all strains. We discuss these findings in the context of a recent hypothesis suggesting that CDR3 structure, independent of VH framework, is sufficient to define the specificity of an antibody. (+info)