VH gene usage in humans: biased usage of the VH6 gene in immature B lymphoid cells

Preferential usage of JH-proximal VH genes has been demonstrated in immature murine B cell repertoires. To determine whether this phenomenon is also evident in human repertoires, we studied utilization of VH6, the most JH-proximal human VH gene. Examination of VH gene usage in a panel of precursor B cell acute lymphoblastic leukemia samples indicated that 15% of the IgH rearrangements utilized VH6. VH6 is a single-member family in a total repertoire of 100-200 VH genes; thus, if usage were purely random, one would expect VH6 rearrangement frequency to be less than 1%. Analysis of VH gene usage in normal lymphoid tissues also revealed biased usage of VH6. VH6 was preferentially utilized in 16- to 24-week-old fetal liver as compared to adult peripheral blood mononuclear cells or spleen. Possible implications of the conservation of preferential usage of JH-proximal genes in both immature murine and human repertoires are discussed.     

Endogenous VH gene family expression in immunoglobulin-transgenic mice: evidence for selection of antibody repertoires

VH gene family expression in single cells of the emergent, available and actual B cell repertoires of C57BL/6 mice was compared to that of two immunoglobulin (Ig)-transgenic B6 lines (B6-Sp6 and M54). We found that less than 5% of bone marrow cells of transgenic mice express endogenous VH genes and that the vast majority (95%) of the peripheral, mature B cell repertoire in these animals is composed of cells expressing the VHJ558 transgenic family. Unimmunized transgenic mice, however, diversify VH gene family usage by 'background' Ig-secreting cells in the spleen, greater than 50% of which express endogenous VH genes. The pattern of endogenous VH gene family expression in the actual repertoire of B6-Sp6 mice is indistinguishable from that of normal B6 mice. In contrast, actual repertoires of M54 mice differ by a 4- to 5-fold higher representation of the VHQ52 family. These results demonstrate a powerful positive selection of B cells into the secretory compartments of unimmunized animals, show that actual and available repertoires differ very markedly, and suggest that V region interactions participate in the selection of 'natural antibody' repertoires.     

Contribution of the VH11 gene family to mitogen-responsive B cell repertoire in C57BL/6 mice

The contribution of VH11 gene family to the development of the primary B cell repertoire has been studied by analyzing 1.8 x 10(4) mitogen induced B lymphocyte colonies. The data demonstrate that VH11 family is predominantly expressed among neonatal splenic as well as adult peritoneal B cell colonies, both rich in Ly-1+ B cells. VH11 gene family expression among B splenocytes decreases during ontogeny and VH11 family pairs stochastically with different V kappa families among mitogen-activated neonatal B cell colonies, which are representative of an antigen unselected B cell repertoire. Thus, an increased VH11 expression among peritoneal and neonatal B cells points towards its biased expression among Ly-1+ B lymphocytes. The restricted V gene rearrangements and VH11-V kappa 9 pairing observed among anti-bromelain-treated mouse red blood cells autoantibodies are likely to be an outcome of both intrinsic gene recombination processes per se as well as selection by an autoantigen and/or local selective environmental factors.     

Skewed B cell VH family repertoire in Bcl-2-Ig transgenic mice.

The proto-oncogene Bcl-2 is normally expressed in B lineage cells in a stage specific manner and extends cell survival. Deregulated Bcl-2 expression has been shown to cause a major expansion in surface IgM and IgD positive B cells. In this report, the influence of deregulated expression of Bcl-2 on the VH repertoire of B cells was studied. This was accomplished by stimulating B cells from both adult and fetal Bcl-2-Ig transgenic mice and their normal littermates using the polyclonal activator lipopolysaccharide. Activated cells were then analyzed by in situ hybridization using radiolabeled C mu and VH gene probes. The D-proximal VH families 7183 and Q52 were preferentially expressed in the adult transgenic mice compared to their normal littermates. VH 7183 and Q52 were also over-represented in fetal transgenic mice but not to a greater extent than that observed with normal fetuses. These results demonstrate that the overproduction of Bcl-2, which prolongs cell survival independent of affecting proliferation, substantially alters the VH gene repertoire.     

CD5 and immunoglobulin VH gene expression in B-cell lines from patients with autoimmune diseases.

We studied the CD5 mRNA expression and VH gene family usage in Epstein-Barr virus (EBV)-immortalized B-cell lines derived from the blood of patients with type 1 diabetes (IDDM) of recent onset and of patients with polyneuritis cranialis multiplex (cranial neuritis; CN). After immortalization with EBV, at least 10 cell lines from each subject were tested for surface CD5 and CD20. mRNA expression was studied using cDNA probes for the six VH families as well as for CD5. The EBV lines from the IDDM patients used the VHIV family more frequently and VHI and VHII families less frequently than lines from controls. EBV lines from CN patients expressed the VHI and VHII families more often than those of the controls. When the IDDM and CN lines were compared, the lines derived from IDDM patients were found to use VH families I and II less frequently and VH families IV and V more frequently than lines from CN patients. There were no significant differences in the mean numbers of CD5+ B cells in the cell lines tested. More than half of the lines from each patient expressed CD5 at the mRNA level. No correlation was seen between the expression of surface CD5 and the level of CD5 mRNA expression. There was, however, a positive correlation between the usage of VH families III, V and VI, and the CD5 mRNA expression. In conclusion, the usage of VH families I to VI seemed to differ in patients with IDDM and CN. No differences were seen in the surface CD5 expression, but the lines expressing CD5 mRNA preferentially used the VH families III, V and VI.     

Expression of members of the immunoglobulin VH3 gene families is not restricted at the level of individual genes in human chronic lymphocytic leukemia.

Previous studies on the repertoire of Ig VH genes utilized in the malignant cells of chronic lymphocytic leukemia (CLL) have suggested a non-random expression pattern. In particular, individual genes from the VH1, VH5, and VH6 gene families have been frequently found in CLL. With regard to other VH gene families, including the large VH3 family which is expressed in greater than 50% of CLL cases, it is unknown whether CLL cells utilize either a broad or a restricted repertoire of VH genes. In the present paper, we analyzed the VH genes expressed in a collection of 11 CLL cases. The results of these experiments demonstrate that there is no apparent restriction in the usage of individual members of the VH3 gene families in CLL.     

Increased utilization of the VH6 gene family in patients with autoimmune idiopathic thrombocytopenic purpura

The VH gene family utilization pattern among pokeweed mitogen-stimulated immunocompetent B cells (available repertoire) and naturally activated B cells (actual repertoire) from the spleen was analysed in a group of patients with autoimmune idiopathic thrombocytopenic purpura (AITP). For this purpose a non-radioactive RNA in situ hybridization technique was employed, allowing detection of VH gene family expression in single cells. The results show that the VH gene family expression pattern in patients with AITP does not correlate with genomic complexity of the VH genes. Furthermore, the pattern of VH gene family utilization in AITP patients is statistically different from that of healthy controls in the available, but not in the actual repertoire. The VH5 gene family is used at a frequency of 11.3% in patients' resting B lymphocytes, compared to 23.9% in controls. The VH6 gene family is used more frequently in patients (23.0% compared to 3.8% in controls). The increase in VH6 gene expression is not reflected in the actual repertoire, where the frequency of expression is 6.4%, and can therefore not be directly related to the presence of disease specific autoantibodies.     

VH gene repertoire

In this review, we have assembled some results on VH gene usage by mouse and human. We conclude that there is an early bias in usage of certain VH genes in both mouse and human. This biased usage has a strain dependent component as evidenced by its continued presence in the adult repertoire of some mouse strains, notably BALB/c, and not in others. The reason for the fetal bias is uncertain. However, the finding that the VH gene segments used in the human fetal repertoire are similar in sequence but not in chromosomal position to those expressed in the mouse fetal-repertoire leads us to suggest that the bias is not due to chromosomal location but rather may be reflecting the functioning of these gene products early in ontogeny.     

Development of VH81X transgene-bearing B cells in fetus and adult: sites for expansion and deletion in conventional and CD5/B1 cells

The most D-proximal functional VH gene, VH81X, is preferentially expressed in the mouse fetal B cell repertoire; however, it is expressed in few B cells in the adult. To determine when VH81X gene expression affects size and phenotype of particular stages in B cell differentiation, transgenic mice have been developed expressing a germline fetal liver-derived VH81X-mu rearrangement. Comparative analysis of B lymphopoiesis reveals similarities and differences between fetal liver and adult bone marrow which pinpoint developmental stages in mice during which VH81X-expressing B cell progenitors expand or deplete compartment sizes. These include a similar reduction in c- kitR+ and establishment of a predominant CD43low/HSAhigh phenotype within the B220+ CD43+ compartment which is dependent on the association of the transgene with lambda 5. In contrast, the CD43- pre- B and immature B cell compartments are expanded in the fetus but not in the adult. In addition, there are other factors that later disfavor the survival of VH81X-expressing B1 and B2 cells. Thus the failure to detect VH81X-bearing B cells in the adult is the result of a multistep selection process occurring at all stages during B repertoire expansion.      

Immunoglobulin VH gene expression in human aging.

Immune responses change in aging humans, but it is not known whether there is an age-associated change in the expressed B cell repertoire. We compared Ig VH cDNA libraries from circulating B cells of five elderly and three young human adults. As in young persons, nearly two-thirds of the cDNA clones from older subjects had zero to three V(H) mutations, although there was more individual variation among the elderly. V(H)4 family expression increased in older subjects, both in unmutated and in mutated cDNA clones, whereas V(H)3 family expression predominated in young adults. To test for bias toward activated cells in the cDNA libraries, we studied two older persons by both cDNA library analysis and single-cell RT-PCR. In one subject, more than 85% of VH segments were unmutated by either analysis. In the second, mutated Ig segments were much more frequent in cDNA clones than in consecutive single cells; however, V(H) family usage and high representation of particular genes were similar in both analyses. While aging humans continue to produce naive B cells with unmutated Ig genes, a shift to greater use of the V(H)4 family members and expression of particular genes may reflect changes in selection of developing B cells before affinity maturation toward reactivity with foreign antigen.     

B cell repertoire in adult antigen-free and conventional neonatal BALB/c mice. II. Analysis of antigen-binding capacities in relation to VH gene usage

Hybridomas were derived from lipopolysaccharide-reactive splenic B cells of adult germ-free BALB/c mice fed a chemically defined ultrafiltered "antigen-free" diet (GF-CD) and from splenic B cells of 5-day-old conventional (CV-NEO) BALB/c mice. The monoclonal antibodies (mAb) from both collections of hybridomas were tested for reactivity against a large panel of antigens of exogenous and endogenous origin. As a source of natural exogenous antigens 36 different bacteria and 9 different viruses were used, while as endogenous antigens frozen tissue sections of stomach, liver and kidney, the Hep-2 cell line and the anti-idiotopic mAb Ac38 and Ac146 were used. In both collections of mAb approximately 70% reacted with one or more bacterial antigens, while no reactivity could be detected against the viral antigens. Of the GF-CD and CV-NEO hybridomas, 16% and 19%, respectively, reacted with one or more frozen tissue sections. Overall 56% and 68% of the GF-CD and CV-NEO hybridomas, respectively, were producing multireactive antibodies reactive to several exogenous and/or endogenous antigens. Among the GF-CD hybridomas a correlation was found between multireactivity and the usage of the VH gene family PC7183. In CV-NEO hybridomas, however, the preferential utilization of the VH gene family PC7183 was found among both mono- and multireactive hybridomas. The results suggest (a) that the actual B cell repertoire of neonatal mice consists of a large proportion of multireactive B cells which are reactive with autoantigens and bacterial antigens, but not viral antigens and (b) that in antigen-deprived mice the neonatal repertoire is largely preserved during maturation of the mice.     

Immunoglobulin VH gene expression following hemorrhage

Hemorrhage has multiple effects on immunologic response, including alteration of B cell repertoires. In limiting dilution studies, decreased absolute frequencies of splenic clonal precursors specific for bacterial antigens were found after blood loss. In order to better define the effects of hemorrhage on B cell function, we examined immunoglobulin VH gene family expression following blood loss using both in situ hybridization and the RNA colony blot technique. No changes in VH gene family utilization were found after hemorrhage. These results demonstrate that the hemorrhage induced alteration in B cell function involves all VH gene families, without modifying distributive frequencies in VH gene family expression.     

Biased VH gene expression in murine CD5 B cells results from age-dependent cellular selection.

Flow cytometry-purified, peritoneal and splenic CD5+ and CD5- B cells from neonatal and adult C57BL/6 mice were studied for expression of VH and Vx gene families in RNA colony blot assays, and for frequencies of clones secreting antibodies to bromelain-treated mouse red blood cells (BrMRBC), single-stranded DNA, trimethyl ammonium and bovine gamma-globulin, by limiting dilution. The results show few overall differences between the two B cell subsets, which both manifest ontogenic D-proximal VH preferences that are lost with age. Biased VH11 expression in CD5 B cells is high in adult peritoneum and spleen but absent in newborns. It only partly correlates with the selection of anti-BrMRBC reactivity, which is considerably higher in peritoneum than in spleen. No particular Vx bias was observed in any of the populations studied with the possible exception of Vx22 in peritoneal CD5+ B cells. We conclude that the antibody repertoire expressed by peritoneal CD5+ B cells of adult mice is not the result of a genetic program, but rather the consequence of local, age-dependent cellular selection mechanisms.     

VH gene family repertoires of "viable motheaten" (mev) mice

Mutant viable motheaten (mev) mice provide an useful experimental model to study the origin and molecular properties of autoantibodies. In the present investigation we have compared by in situ hybridization VH gene family usage in lipopolysaccharide-activated B cells (available repertoire) and spontaneously immunoglobulin-secreting (actual repertoire) B cells in the spleen of 6-8-week-old BALB/c and mutant BALB/c-mev mice. We have found that while sharing identical available splenic repertoires and expressing a diversified set of VH families, mev mice differ from control BALB/c animals in VH family representation in the actual plasma cell repertoires where they showed a decreased utilization of VH7183 genes and an increased representation of the VHJ606 family when compared to control BALB/c animals. These results indicate that selection of actual repertoires may indeed differ between autoimmune and control mice, but do not establish whether such changes are the primary cause of the disease or whether they are secondary to the initiating of the autoimmune process.     

Selective Usage of VH Genes in Adult Human B Lymphocyte Repertoires

The authors have compared the VH gene utilization patterns among small resting immunocompetent B cells and large naturally activated B lymphocytes of healthy human adults. They employed a non-radioactive RNA in situ hybridization technique that allows detection of VH gene family expression at the single cell level. Pokeweed mitogen stimulated and unmanipulated mononuclear cells from peripheral blood and spleen of unrelated individuals were hybridized to digoxigenin-labelled antisense RNA probes specific for human VH families 1–6 and for the constant region genes Cμ and Cγ. The observed VH gene family utilization patterns did not correlate with the genomic complexity of human VH genes. The VH3 gene family was most frequently used among resting B cells in both peripheral blood and spleen. Among naturally activated lymphocytes the VH6 gene was markedly over-represented, while expression of the VH1 and VH3 gene families was decreased. The data show that V-region mediated selection participates in shaping the peripheral antibody repertoire in healthy adults.     

Immunoglobulin VH gene expression in Ly-1+ and conventional B lymphocytes

Lymphocyte populations in which Ly-1 B cells are differentially represented were studied for the expression of ten VH gene families, either by an RNA colony blot assay or by in situ hybridization of single cells, in BALB/c and C57BL/6 mice. The comparisons of cells from lymph nodes, Peyer's patches and adult spleen (poor in Ly-1 B cells) with cells from peritoneal cavity and neonatal spleen (rich in Ly-1 B cells) were confirmed by the analysis of adult peritoneal Ly-1- and Ly-1+ B cells sorted on the fluorescence-activated cell sorter. The results indicate that the peritoneal Ly-1+ B subset uses the whole spectrum of known VH gene families, and shows a preferential utilization of CP12 VH genes, most likely as a result of a selective process during life.     

A new germline VH36-60 gene is used in the neonatal primary and adult memory response to (T,G)-A--L.

Our previous studies of the neonatal primary response to (T,G)-A--L showed that the majority of anti-(T,G)-A--L antibodies bind the copolymer L-Glu:L-Tyr (GT), share idiotypy (Id), and use the H10 germline VH gene from the VHJ558 family and a V kappa 1 gene. We also identified two hybridomas from different neonatal donors that produced GT+, Id+ antibodies using a V kappa 1 gene with a VH gene from the VH36-60 family. In the study reported here, we show that both neonatal hybridomas use the same germline VH gene from the VH36-60 gene family. However, the VH gene sequence is different from previously identified germline genes of the VH36-60 gene family. To determine whether the expressed heavy chain gene had undergone somatic mutation, we isolated the corresponding germline gene from kidney DNA. Sequence analysis of this gene shows that it is a new member of the VH36-60 family which is not mutated in the neonatal antibodies. Furthermore, the deduced amino acid sequences of the two neonatal antibodies are identical not only in the VH region but also in the VH-D-JH joins, suggesting that there is a strong selection for CDRIII among neonatal anti-(T,G)-A--L antibodies using this germline gene (designated here as VH3A1) with a V kappa 1 gene. Also, the VH gene from the VH36-60 family that we showed previously was used by an adult memory B cell clone specific for (T,G)-A--L, can now be identified as a rearrangement of the VH3A1 germline gene. Elucidation of the germline variable region genes that are used in the antigen-specific neonatal response will help us understand the mechanisms that shape the preimmune B cell repertoire during B cell development.     

VH gene usage and CDR3 analysis of B cell receptor in the peripheral blood of patients with PBC

We have analyzed the IgM, IgG and IgA BCR repertoire in PBC patients by means of quantitative RT-PCR and CDR3-spectratyping with immunoscope technology. PBMC from 35 PBC patients and 18 normal controls were analyzed. Quantitative B cell repertoire analysis of IgM from healthy donors showed the preferential usage of VH3a, VH3b and VH4 families. Very similar VH family usage was observed in IgM B cells from PBC patients. CDR3- spectratyping of IgM BCR rearrangements showed a Gaussian distribution for dominant VH families in control donors, and similar diversity was found for the VH3b family in PBC patients. In contrast, VH3a and VH4 families showed oligoclonal expansions in some patients. Quantitative B cell repertoire analysis of IgG and IgA did not reveal any difference in VH chain distribution in PBC patients as compared to the control donors. Immunoscope profiles of CDR3 length distribution showed several peak expansions in B cells from control donors, particularly for the VH3a and VH4 families. CDR3 length distribution profiles of IgG and IgA from PBC patients were oligoclonal too, with expansions throughout the various VH chains. However, no common expansions within the CDR3 region were found intraindividually between IgG, IgA and IgM, and between patients. In conclusion, immunoscope technology does provide, for the first time, a sensitive and rapid method for detailed immunoglobulin gene usage analysis in peripheral B cells from PBC patients. This study failed to demonstrate preferential B cell rearrangements in the blood of patients with PBC, but this technology may be more successful if applied to the analysis of compartmental B cells (i.e. liver infiltrating B cells).