Restricted utilization of VH and DH genes in leukemic rabbit B cells

Polyclonal B cell leukemias have been generated in 17- to 20-day old Emu-myc transgenic rabbits. To analyze the repertoire of VH genes utilized in early B cells, eight VDJ genes were cloned from these leukemic cells. The nucleotide sequences of these genes indicated that seven of the eight VDJ genes encoded prototype VHa1, VHa2 or VHa3 allotypes. The two VDJ genes encoding VHa1 molecules had VH segments with identical nucleotide sequences; similarly, the VH segments of the four VDJ genes encoding VHa2 molecules were identical, with the exception of a single base pair. These data suggest that a limited repertoire of VH genes were utilized in the generation of these VDJ genes. The DH segments of these genes were limited to two DH families, D1 and D2, indicating that a restricted repertoire of DH genes also had been utilized. Since these leukemic cells probably developed early in ontogeny, we suggest that this restricted utilization of VH and DH genes is representative of B cells from developmentally immature rabbits.     

CDR3 regions in the preimmune VH B cell repertoire of lpr mice.

Previous studies have suggested that the CDR3 genetic element of the heavy chain variable region of autoantibodies is important in determining reactivity against self antigens, particularly against DNA. The lpr mutation was recently found to encode for a defective form of the fas protein, a molecule important for the transmission of the apoptotic signal into cells. Our aim was to determine whether CDR3 elements similar to those described for autoantibody-producing hybridomas derived from lupus-prone strains could be found in the preimmune repertoire of B cells in mice with the lpr mutation. The analysis of the junctions of the VH-C mu functional rearrangements derived by polymerase chain reaction (PCR) amplification of RNA obtained from splenic small, resting cells stimulated with lipopolysaccharide (LPS) from male lpr mice showed that a large proportion of them expressed D genes in the unusual reading frames 2 and 3. Two of the lpr joints were formed by D-D fusions. Similarly, nearly half of the lpr sequences had arginines, an amino acid which promotes binding to dsDNA and is seldom observed in normal junctions. Our results show that the preimmune repertoire of lpr animals has abnormal CDR3 elements which may result from a failure at different levels of selection. The antigen-dependent selection of such elements that leads to the expansion of specific, high-affinity anti-dsDNA antibody-producing clones might depend on other genetic factors not found in the C57B1/6-lpr strains but in the MRL-lpr.     

Comparable profiles of the immunoglobulin heavy chain complementarity determining region (CDR)-3 in CD5+ and CD5- human cord blood B lymphocytes.

It has been suggested that CD5+ B cells are closely related to the pathogenesis of autoimmune disease or chronic lymphocytic leukaemia, whereas the origin and physiological role of CD5+ B cells remain controversial. To study the molecular differences between CD5+ and CD5- B cells in terms of immunoglobulin gene structure, we sorted both subsets from new-born cord blood and analysed the complementarity determining region (CDR)-3 profiles of the immunoglobulin heavy chain (IgH) gene. The CDR-3 sequences from both CD5+ and CD5- B cells represented the same incidence and length of N-region, and the same usage of D and JH segments. When translated into amino acids, 24 of 32 clones (75%) and 25 of 37 clones (65.8%) from their respective subset were productive, and the composition of the deduced amino acids were similar between both subsets. These data suggest that CD5+ B cells are not a distinct lineage presenting a biased immunoglobulin repertoire in B cells.     

An immunoglobulin heavy chain variable region (VH) marker associated with cross-reactive idiotypes in man.

The VH specificities of two rabbit antisera raised against the H chains of two human IgM cold agglutinins were studied with the aid of proteins on which the VH sequence data are available. They were found to recognize a new VH antigen (VHMar), which may represent a subgroup of VHI. This antigen is found on anti-Ii antibodies which have cross-reactive idiotypes. It is moderately well-expressed in normal gamma globulin and strongly expressed on 8% of unselected myeloma and macroglobulinaemia proteins. There is evidence to suggest that a proportion of the cross-reactive idiotypes among the anti-Ii antibodies involve this VH antigen.     

Receptor revision of immunoglobulin heavy chain genes in human MALT lymphomas.

BACKGROUND/AIMS: Rearrangement of immunoglobulin gene segments, leading to B cells with functional receptors, is thought to be largely restricted to developing immature B cells in bone marrow. However, accumulating evidence suggests that mature B cells occasionally modify their antigen specificity by VH segment replacements during the germinal centre reaction to enhance antigen affinity, or to overcome self reactive antigen receptors. Although malignant B cells maintain the features of their normal counterparts in most instances, to date, such replacements have not been described for human B cell lymphomas. METHODS: Rearranged immunoglobulin heavy chain genes from two extranodal marginal zone B cell lymphomas were amplified, cloned, and sequenced. Sequences with identical CDR3 regions were selected and aligned to each other and public databases. RESULTS: VH replacements were seen in two extranodal marginal zone B cell lymphomas. In line with the hypothesis that in mature B cells these replacements are associated with active somatic hypermutation, in addition to VH replacement, different mutation patterns were seen in the revised VH portions. In the remaining common 3'-VH regions, these mutations could be used to establish a phylogenetic relation between the sequences, rendering the possibility of artefactual chimaeric polymerase chain reaction products very unlikely. CONCLUSIONS: These results support the view that VH replacements are a further mechanism for reshaping antigen affinity and specificity, and indicate that these receptor modifications are not restricted to normal and reactive germinal centre B cells, but may also occur in close association with the development of malignant B cell lymphomas.     

A single VH family and long CDR3s are the targets for hypermutation in bovine immunoglobulin heavy chains

Summary: Bovine immunoglobulins are made from genes belonging to a small family of closely related Vh, genes. In this respect cattle resemble all species of domesticated mammals, which also use one VH family The family, named BoVH1, is homologous to the mouse Q52 family, and there are no more than 20 genes of this family in the bovine genome. Another feature of bovine heavy chains is the use of long CDR3s, which have an average of 21 codons. It seems that there are several families of long, closely related D genes rich in glycine and tyrosine responsible for this length. Sequences described as targets for mutations in other species can be found in CDR1, CDR2, and the putative D genes. The mutation mechanism starts at some point between late fetal stage and birth and seems to be antigen Independent. Diversity seems to be generated by hypermutation, although other mechanistns cannot be discomited at this time. Contrary to humans and mice, which have several Vh gene families comprising more than 100 genes, cattle use only a few genes and long CDR3s followed by somatic mutation to generate the necessary diversity to recognize the universe of antigens they will encounter during their life.     

Evaluation of clonal immunoglobulin heavy chain rearrangements in Hodgkin's disease using the polymerase chain reaction (PCR)

We have correlated histologic type of Hodgkin's disease, degree of Hodgkin and Reed-Sternberg cell infiltration, percentage of Hodgkin and Reed-Sternberg cell positivity for latent membrane protein, immunophenotype of Hodgkin and Reed-Sternberg cells, and immunoglobulin heavy chain (IgH) gene rearrangements detected by polymerase chain reaction (PCR) in 56 unselected Hodgkin's disease cases. Two protocols were used for amplification of IgH gene using Fr2 or Fr3 V-region primers, in conjunction with nested primers directed to the JH region. PCR products were run on polyacrylamide gels. Immunohistochemical studies were performed on paraffin sections using monoclonal antibodies for CD20 and latent membrane protein, and polyclonal antibody to CD3. Using both primer combinations we detected a definitive clonal band in 23.2% of the Hodgkin's disease cases. Clonal IgH rearrangements were detected in 23.6% of nodular sclerosis type and in 28.5% of mixed cellularity type. Using a highly sensitive method such as PCR, more than 20% of unselected cases of Hodgkin's disease were found to contain B-cell clonal proliferations, but there was no correlation between histological and immunological parameters and molecular analysis results.     

Regulation of immunoglobulin light chain gene rearrangements during early B cell development in the human.

Southern blot analyses of immunoglobulin light chain gene rearrangements in human leukemias and myelomas indicated that lambda loci in kappa-producing cells are largely unrearranged while kappa loci in lambda producers are often rearranged and inactivated by rearrangements of the kappa-deleting element (KDE). For a systematic analysis of the regulation of light chain rearrangements during early B cell development in normal human B cells also considering functionality of the rearrangements, we used FACS-sorted single naive kappa- and lambda-expressing B cells from peripheral blood of healthy humans. V(kappa)J(kappa) and V(lambda)J(lambda) joints and rearrangements involving the KDE were amplified simultaneously from single cells and sequenced. Whereas only 2 - 3 % of kappa-expressing cells carry V(lambda)J(lambda) joints, nearly all lambda-expressing cells have rearranged kappa loci and indeed carry V(kappa)J(kappa) joints. The V(kappa)J(kappa) joints in lambda-expressing cells exhibit preferential J(kappa)4 and J(kappa)5 over J(kappa)1 and J(kappa)2 usage compared to kappa-expressing cells. Thirty percent of the V(kappa)J(kappa) joints in lambda producers are rearranged in-frame. These data indicate extensive sequential V(kappa)-J(kappa) rearrangements and inactivation of functional V(kappa)J(kappa) joints in lambda-expressing cells, presumably before V(lambda)J(lambda) joining.     

ImmunoGlobulin galaxy (IGGalaxy) for simple determination and quantitation of immunoglobulin heavy chain rearrangements from NGS

Background

Toxic shock syndrome (TSS) is caused by an overwhelming host-mediated response to bacterial superantigens produced mainly by Staphylococcus aureus and Streptococcus pyogenes. TSS is characterized by aberrant activation of T cells and excessive release of pro-inflammatory cytokines ultimately resulting in capillary leak, septic shock, multiple organ dysfunction and high mortality rates. No therapeutic or vaccine has been approved by the U.S. Food and Drug Administration for TSS, and novel therapeutic strategies to improve clinical outcome are needed. Mesenchymal stromal (stem) cells (MSCs) are stromal cells capable of self-renewal and differentiation. Moreover, MSCs have immunomodulatory properties, including profound effects on activities of T cells and macrophages in specific contexts. Based on the critical role of host-derived immune mediators in TSS, we hypothesized that MSCs could modulate the host-derived proinflammatory response triggered by Staphylococcal enterotoxin B (SEB) and improve survival in experimental TSS.

Methods

Effects of MSCs on proinflammatory cytokines in peripheral blood were measured in wild-type C57BL/6 mice injected with 50 μg of SEB. Effects of MSCs on survival were monitored in fatal experimental TSS induced by consecutive doses of D-galactosamine (10 mg) and SEB (10 μg) in HLA-DR4 transgenic mice.

Results

Despite significantly decreasing serum levels of IL-2, IL-6 and TNF induced by SEB in wild-type mice, human MSCs failed to improve survival in experimental TSS in HLA-DR4 transgenic mice. Similarly, a previously described downstream mediator of human MSCs, TNF-stimulated gene 6 (TSG-6), did not significantly improve survival in experimental TSS. Furthermore, murine MSCs, whether unstimulated or pre-treated with IFNγ, failed to improve survival in experimental TSS.

Conclusions

Our results suggest that the immunomodulatory effects of MSCs are insufficient to rescue mice from experimental TSS, and that mediators other than IL-2, IL-6 and TNF are likely to play critical mechanistic roles in the pathogenesis of experimental TSS.     

Monomorphic organization of human diversity (DH) heavy chain variable genes.

While recent evidence indicates that human immunoglobulin heavy chain variable (VH) genes exhibit a high degree of heterogeneity, little is known concerning the polymorphism of the diversity (DH) gene complex. This locus comprises two clusters, major and minor, which are physically linked with the VH locus. In assessing the variability of the human major and minor DH clusters, we found no evidence for a substantial restriction site polymorphism. We also noted that, in contrast to what was found in the Japanese population, the DH1 gene is not deleted in an appreciable proportion of the European population. We propose that, because DH genes impart the critical functions associated with the third complementarity-determining region, the genomic organization of the human DH locus has been evolutionarily conserved through selection pressure mechanisms.     

DNA polymorphism 5' to the JH region of the human immunoglobulin heavy chain gene and immunoglobulin gene rearrangements in leukemia

DNA samples of two patients with acute myeloblastic leukemia were noted to have a variant band on digestion with HindIII and hybridization to a probe for the joining region of the immunoglobulin (Ig) heavy chain gene (JH). Further analysis showed that the variant band represented a constitutional DNA polymorphism rather than an Ig gene rearrangement. The HindIII fragment detected by the JH probe includes a highly polymorphic region 5' to the Ig heavy chain locus, and hence variant germline bands may be seen on autoradiography that can be mistaken for Ig gene rearrangements. The use of several different restriction enzymes and the analysis of the constitutional DNA allows a clear distinction to be made between somatic gene rearrangements and DNA polymorphisms.     

Selection against N-region diversity in immunoglobulin heavy chain variable regions during the development of pre-immune B cell repertoires.

The generation of Ig heavy chain chain diversity is dependent on the ordered rearrangement of three different, i.e. variable (VH), diversity (DH), and joining (JH), germline gene segments, exonuclease nibbling of the terminals of these gene segments, and the addition of template-independent nucleotide (N-sequences) in the junctions of these segments. The latter process has recently been reported to be limited within B cells formed during early ontogeny. In this study, we have analysed a large number of VHDJH rearrangements isolated from genomic DNA of adult and neonatal C57BI/6 mice using the polymerase chain reaction (PCR) technique. A comparison of functional versus non-functional VHDJH rearrangements derived from these PCR libraries, or from a set of previously published clones of BALB/c origin, revealed a selection against N-region diversity both in neonatal and adult B cell repertoires. This selection process is most pronounced in the early development of the immune system but can still be observed in the adult. Furthermore, selection against N-sequence additions was evident amongst neonatal VHDJH rearrangements utilizing both VH 7183 and VH J558 genes, but only in VH 7183 utilizing clones of adult origin. These results imply that in addition to a developmentally controlled onset of N-sequence additions, cellular selection against N-region diversity exist both in the neonatal and adult immune system.     

Properties of B cell stage specific and ubiquitous nuclear factors binding to immunoglobulin heavy chain gene switch regions.

The Ig heavy chain (IgH) constant region (CH) class switch is manifested by DNA deletions which exchange the C mu gene of a functional VDJ-CH rearrangement for a C gamma, C epsilon or C alpha gene. Repetitive sequences (S regions) 5' of each CH gene mediate CH gene switch recombination by an illegitimate mechanism. S mu can be subdivided into S mu 5' (non-repetitive) and S mu 3' (repetitive) components with recombination occurring in either part. Here, we describe the properties of ubiquitous and B cell stage specific S mu binding factors NFS mu-U1 and NFS mu-B1 respectively. U1 only bound to S mu 5' sequences, and B1 to S mu 5', S mu 3' sequences and to other S regions with varying affinities. DMS and OP-Cu footprinting revealed the sequence AAAAAGCATGGCTGA in the U1 site while the B1 S mu 5' site overlapped the 3' end of the U1 binding site and also contained additional 3' flanking S mu repeat motifs (GAGCTGAGATGGGTGGGCT). Binding site competition assays reveal that NFS mu-B1 is either very related or identical to S alpha BP (described by Waters et al., Mol. Cell Biol. 9:5594, 1989) and BSAP (identified by Barberis et al., Genes Devl. 4:849, 1990) which were shown to bind to two sequences upstream of the S alpha repeats and within the promoters of sea urchin histone genes respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hodgkin's disease: immunoglobulin heavy and light chain gene rearrangements revealed in single Hodgkin/Reed-Sternberg cells.

AIM: To corroborate and investigate the nature of Hodgkin/Reed-Sternberg cells (H/R-S) of various subtypes of Hodgkin's disease. METHOD: Single H/R-S cells were micro-picked from frozen sections of tissues affected by Hodgkin's disease. The DNA from these cells was amplified by the polymerase chain reaction (PCR) with immunoglobulin heavy chain (IgH) gene FRIIIa/JH primers and light chain gene family specific primers. RESULTS: Fifty two of 135 isolated cells gave specific reaction products (36%). IgH and V kappa 4 gene rearrangements were found repeatedly in many H/R-S cells from one case of lymphocyte predominant Hodgkin's disease. Repeated V kappa 4 and individual IgH/V kappa 4,2 rearrangements were seen in one case, and individual IgH and V lambda 3/V kappa 4 rearrangements were seen in another case of nodular sclerosis-type Hodgkin's disease. Repeated IgH/V lambda 3 and individual V lambda 2,4 rearrangements, repeated V kappa 4 and individual IgH/V kappa 3 rearrangements, and repeated IgH and individual V kappa 3/V kappa 4 rearrangement were detected, respectively, in three cases of mixed cellularity-type Hodgkin's disease. Repeated and individual IgH rearrangements were found in another two cases of mixed cellularity-type Hodgkin's disease. CONCLUSION: The H/R-S cells isolated from lymphocyte predominant Hodgkin's disease had IgH and V kappa 4 gene rearrangements, which supports the conclusion that this disease results from a proliferation of neoplastic B cells. The IgH and kappa and/or lambda gene rearrangements seen in H/R-S cells isolated from classic Hodgkin's disease (mixed cellularity-type and nodular sclerosis-type) support the theory that these cells derive from B lineage cells at various stages of differentiation. To our knowledge, this is first time that lambda gene rearrangements have been detected in H/R-S cells.