Repertoire of human antibodies against the polysaccharide capsule of Streptococcus pneumoniae serotype 6B.
(1/2048)
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)
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.
(2/2048)
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)
Biased JH usage in plasma cell immunoglobulin gene sequences from colonic mucosa in ulcerative colitis but not in Crohn's disease.
(3/2048)
BACKGROUND: Ulcerative colitis is an inflammatory disease of the colonic and rectal mucosa. Autoantibodies have been observed in ulcerative colitis which may have a role in the pathogenesis of the disease. Evidence also suggests that there is an hereditary predisposition towards the disease, although no individual genes have been identified. AIMS: This is a pilot study of immunoglobulin heavy chain genes (IgH) in ulcerative colitis to determine whether they have any particular genetic characteristics which may lead to a better understanding of the disease aetiology. SUBJECTS: Colonic or rectal tissue was obtained from five children with ulcerative colitis. Tissue was also obtained from five children with Crohn's disease and five children who did not have inflammatory bowel disease as controls. METHODS: B cells and IgD+ B cells were identified by immunohistochemistry on frozen sections. Areas of lamina propria containing plasma cells, and areas of IgD+ B cells were microdissected. The immunoglobulin genes were PCR amplified, cloned, and sequenced. Sequences were analysed for content of somatic mutations and composition of heavy chain. RESULTS: An increase in the use of JH6 and DXP'1, and a decrease in the use of JH4, gene segments in immunoglobulin genes from lamina propria plasma cells, and from virgin IgD+ B cells, was found in patients with ulcerative colitis. These biases were not present in the control groups. CONCLUSIONS: There is a fundamental difference in the immunoglobulin genes from patients with ulcerative colitis. Whether this is caused by a difference in content of immunoglobulin gene segments in the germline or a difference in the recombination mechanism is not known. (+info)
A novel stromal cell-dependent B lymphoid stem-like cell line that induces immunoglobulin gene rearrangement.
(4/2048)
A stroma-dependent B lymphoid cell line (B31-1) has been established by coculturing sorted stem cells on a novel bone marrow stromal cell line (TBR31-1). B31-1 cells express B220, but do not express other B lymphoid differentiation markers including CD43, heat stable antigen (HSA), or surface immunoglobulin (Ig) M (sIgM), and their Ig heavy chain (IgH) gene loci are germ-line in configuration. The addition of interleukin (IL)-7 or coculture with another stromal cell line, ST2, induces D-J rearrangement of the IgH gene and B lymphocyte differentiation markers. B31-1 cells restore an in vivo repopulation activity to lethally irradiated mice, and the repopulated cells differentiate to HSA+ pre-B cells.Continuous coculture results in two distinct populations, B220(-) c-Kit+ cells and B220(+) c-Kit+ cells; B220(-) c-Kit+ cells are self-renewed and differentiate to B220(+) c-Kit+ cells, while B220(+) c-Kit+ cells produce only B220(+) c-Kit+ cells. Both B220(-) and B220(+) cells similarly express the IgH germ-line transcript (Imu), mRNAs for recombinase (TdT, Rag-1, and Rag-2), and lymphoid-specific transcription factors (Pax-5, EBF, E12/E47, Oct-2, and Ikaros), but the DNA binding activity of Pax-5, EBF, Oct-2, and E2A are low in B220(-) cells and while high in B220(+) cells. These results suggest the existence of at least two active states in the IgH locus before the induction of IgH gene rearrangement during B lymphopoietic development. (+info)
Induction of Ig light chain gene rearrangement in heavy chain-deficient B cells by activated Ras.
(5/2048)
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.
(6/2048)
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)
Immunoglobulin VH gene expression among extranodal marginal zone B-cell lymphomas of the ocular adnexa.
(7/2048)
PURPOSE: Most lymphomas of the ocular adnexa are primary extranodal non-Hodgkin's lymphomas of the B-cell type, with the most common lymphoma subtype being the extranodal marginal-zone B-cell lymphoma (EMZL). Analysis of somatic mutations in the variable (V) region of the Ig heavy (H)-chain gene segment suggests that EMZL development in other locations is dependent on antigen stimulation. The purpose of this study was to analyze the presence of somatic hypermutations in clonally rearranged Ig H-chain V genes of this lymphoma entity in the ocular adnexa and to estimate whether the mutation pattern is compatible with antigen selection. METHODS: Twenty-six cases of EMZL of the ocular adnexa were diagnosed on the basis of morphology, histology, and immunohistology. A nested polymerase chain reaction (PCR) was performed on DNA extracted from paraffin sections. The isolated PCR products were sequenced and compared with published VH germline segments to determine the number of somatic mutations in the complementarity-determining region (CDR) 2 and framework (FW) region 3. RESULTS: The number of somatic mutations in the cases of EMZL varied between 0 and 24: Five cases involved 0 to 3 somatic mutations, and the remaining 21 cases involved 4 to 24 mutations. Based on the ratio of replacement (R) to silent (S) mutations in the CDR2 or FW3 regions, antigen selection seems to have occurred in 60% of ocular adnexal EMZL. The VH3 family was the most commonly expressed germline VH family (54%), followed by VH4 (23%), with biased usage of the latter. Some germline VH1 genes used included DP-8, DP-10, DP-53, DP-63 (VH4.21), and DP-49, which are frequently used by autoantibodies (e.g., rheumatoid factors) and natural autoantibodies. CONCLUSIONS: EMZLs of the ocular adnexa have an Ig H-chain mutation pattern that supports the concept that they represent a clonal expansion of post-germinal-center memory B-cells in most instances. In two thirds of cases, antigen selection may have occurred, and autoantibodies may have a role in their development. (+info)
Type I IFN sets the stringency of B cell repertoire selection in the bone marrow.
(8/2048)
Locally produced type I interferon (IFN-I) enhances the sensitivity of bone marrow B cell to IgM receptor ligation. The establishment of B cell repertoires, on the other hand, seems to involve selective processes that are critically dependent on B cell receptor (BCR) ligation. In order to assess the importance of BCR triggering thresholds on the selection of polyclonal unmanipulated B cell populations, we compared VH gene expression and reactivity repertoires in various B cell compartments of wild-type and IFN-I receptor-deficient mice (IFN-I-R-/-). These analyses demonstrate that increased B cell sensitivity to BCR ligation mediated by IFN-I in the bone marrow (BM) has consequences on the stringency of B cell repertoire selection. Thus, the normal counter-selection of both VH7183 gene family expression and multireactivity was impaired among immature BM B cells from mutant mice. Furthermore, as a result of reduced efficiency of BCR ligation-dependent inhibition of terminal differentiation, IFN-I-R-/- animals produce, in BM and thymus, higher numbers of plasma cells secreting antibodies that are more multireactive than wild-type animals. Finally, mutant serum IgM natural antibodies display a more reactive repertoire than controls, a likely reflection of the BM resident plasma cell repertoire. The present observations demonstrate, therefore, that local modulation of BCR triggering thresholds leads to important modifications in the generation and/or selection of normal B cell populations. (+info)