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)
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)
Immunoglobulin VH gene expression among extranodal marginal zone B-cell lymphomas of the ocular adnexa.
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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)
Molecular detection of acute lymphoblastic leukaemia in boys with testicular relapse.
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AIMS: To determine the role of polymerase chain reaction (PCR) based minimal residual disease (MRD) detection of leukaemia specific DNA in testicular relapse in childhood acute lymphoblastic leukaemia. METHODS: DNA was obtained from archival testicular and bone marrow samples from boys with acute lymphoblastic leukaemia who relapsed in the testes. Overlapping DJH clone specific primers derived from clonal immunoglobulin heavy chain (IgH) gene rearrangement in each case were used to analyse testicular or bone marrow DNA. RESULTS: Histologically normal end of treatment testicular biopsies in the five patients in longterm remission were all MRD negative, but MRD positive in three of six boys with subsequent testicular relapse. Histologically normal bone marrow samples taken at the end of treatment were MRD negative in five of seven cases, but MRD positive in all cases at the time of isolated testicular relapse. Three boys with unilateral testicular relapse underwent unilateral orchidectomy, rather than bilateral testicular irradiation, as part of their treatment. Two of these boys were MRD positive in the histologically uninvolved testes, and both had subsequent relapses either in the testes or the bone marrow, while the MRD negative patient has not had a testicular relapse. CONCLUSIONS: The presence of MRD in testicular tissue can be assayed with a PCR based method to detect clone specific antigen receptor gene rearrangements. In this setting, PCR is more sensitive than conventional testicular histology for predicting clinical outcomes. MRD assays might be useful in the management of boys at the time of isolated testicular relapse, to confirm the presence of unilateral testicular disease. (+info)
Extended duration of DH-JH rearrangement in immunoglobulin heavy chain transgenic mice: implications for regulation of allelic exclusion.
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Here we show that suppression of VH-DJH rearrangement in mice bearing a mu heavy (H) chain transgene (mu-tg mice) is associated with an extended period of DH-JH rearrangement, the first step of Immunoglobulin H chain gene rearrangement. Whereas DH-JH rearrangement is normally initiated and completed at the pro-B cell stage, in mu-tg mice it continues beyond this stage and occurs most frequently at the small (late) pre-B stage. Despite ongoing DH-JH rearrangement in late pre-B cells of mu-tg mice, VH-DJH rearrangement is not detectable in these cells. We infer that the lack of VH-DJH rearrangement primarily reflects tg-induced acceleration of B cell differentiation past the stage at which rearrangement of VH elements is permissible. In support of this inference, we find that the normal representation of early B lineage subsets is markedly altered in mu-tg mice. We suggest that the effect of a productive VH-DJH rearrangement at an endogenous H chain allele may be similar to that of a mu-tg; i.e., cells that make a productive VH-DJH rearrangement on the first attempt rapidly progress to a developmental stage that precludes VH-DJH rearrangement at the other allele (allelic exclusion). (+info)
Characteristics of sequences around individual nucleotide substitutions in IgVH genes suggest different GC and AT mutators.
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Somatic hypermutation affects Ig genes during T-dependent B cell responses and is characterized by a high frequency of single base substitutions. Hypermutation is not a completely random process; a study of mutations in different systems has revealed the presence of sequence motifs that target mutation. In a recent analysis of the sequences surrounding individual mutated bases in out-of-frame human IgVH genes, we found that the target motifs around mutated G's and C's are reverse complements of each other. This finding suggests that hypermutation acts on both strands of DNA, which contradicts evidence of a strand-dependent mechanism as suggested by an observed bias in A and T mutations and the involvement of transcriptional machinery. We have now extended our database of out-of-frame genes and determined the sequence motifs flanking mutated A and T nucleotides. In addition, we have analyzed the flanking sequences for different types of nucleotide substitutions separately. Our results confirm the relationship between the motifs for G and C mutations and show that the motifs surrounding mutated A's and T's are weaker and do not have the same relationship. Taken together with our observation of A/T strand bias in out-of-frame genes, this observation suggests that there is a semitargeted G/C mutator that is strand-independent and a separate A/T mutator that is strand-dependent and is less reliant on the local target sequence. (+info)
IgG1 production by sIgD+ splenic B cells and peritoneal B-1 cells in response to IL-5 and CD38 ligation.
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CD38 ligation on mouse B cells by CS/2, an anti-mouse CD38 mAb, induces proliferation, IL-5 receptor alpha chain expression and tyrosine phosphorylation of Bruton's tyrosine kinase. Furthermore, stimulation of splenic B cells with IL-5 together with CS/2 induces Blimp-1 expression and differentiation into Ig-producing cells. Here we examined the role of IL-5 in IgG1 and IgA production by B cells isolated from the spleen and peritoneal cavity. CD38 recognized by CS/2 was expressed in the follicular mantle B cells surrounding the germinal center, sIgD+ splenic B cells and peritoneal B cells. IL-5 induced IgG1 production in splenic sIgD+ B cells stimulated with CS/2, while it was ineffective to induce IgA production. Among the various cytokines tested, only IL-5 had a synergistic effect on IgG1 production with CS/2. IL-5 could induce the generation of S micro-Sgamma1 reciprocal recombination DNA products in CS/2-stimulated B cells. IL-4 was ineffective to induce either micro-gamma1 switch recombination or IgG1 secretion with CS/2, demonstrating that IL-5 promotes both micro-gamma1 switch recombination and IgG1 secretion in an IL-4-independent manner. The peritoneal B-2 cells exhibited both IgG1 and IgA production in response to IL-5 plus CS/2, while B-1 cells produced IgG1. These results imply that the pattern of differentiation to Ig-producing cells seen with peritoneal B cells is not identical to the pattern seen with splenic B cells and that peritoneal B-2 cells contain precursors of IgA-producing cells responding to IL-5 plus CS/2. (+info)
Genetic pathway to recurrent chromosome translocations in murine lymphoma involves V(D)J recombinase.
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Chromosome translocations involving antigen receptor loci are a genetic hallmark of non-Hodgkin's lymphomas in humans. Most commonly, these translocations result in juxtaposition of the immunoglobulin heavy-chain (IgH) locus with one of several cellular proto-oncogenes, leading to deregulated oncogene expression. The V(D)J recombinase, which mediates physiologic rearrangements of antigen receptor genes, may play a mechanistic role in some lymphoma translocations, although evidence is indirect. A high incidence of B-lineage lymphomas has been observed in mice with severe combined immunodeficiency (SCID) and p53-null mutations. We show that these tumors are characteristic of the pro-B-cell stage of development and that they harbor recurrent translocations involving chromosomes 12 and 15. Fluorescence in situ hybridization (FISH) shows retention of IgH sequences on the derivative chromosome 12, implying that breakpoints involve the IgH locus. Pro-B-cell lymphomas were suppressed in SCID p53(-/-) mice by a Rag-2-null mutation, demonstrating that DNA breaks generated during V(D)J recombination are required for oncogenic transformation, and suggesting that t(12;15) arise during attempted IgH rearrangement in pro-B cells. These studies indicate that the oncogenic potential inherent in antigen receptor diversification is controlled in vivo by efficient rejoining of DNA ends generated during V(D)J recombination and an intact cellular response to DNA damage. (+info)