VH gene family repertoire of resting B cells. Preferential use of D-proximal families early in development may be due to distinct B cell subsets

The fetal VH gene repertoire was shown previously to be characterized by overrepresentation of D-proximal families, VH 7183 and VH Q52, compared with adult bone marrow B cells in which VH genes were expressed in a more stochastic fashion. To determine the underlying mechanisms of these findings, adult vs fetal progenitors were placed in the same supportive microenvironment and the resulting B lineage cells analyzed for VH gene family expression. The supportive microenvironment was provided by established adult bone marrow stromal cell layers. In this way the relative importance of environmental vs genetic influences could be determined. The fetal B cells and pre-B cells that developed on adult stromal cells maintained a fetal-like VH gene family repertoire with preference for D-proximal families VH 7183 and Q52. In contrast, adult cultured B cells maintained the adult-like repertoire with predominance of the largest family VH J558. Only after long-term incubation was there a change in the expression of particular VH gene families. These findings suggest that the D-proximal VH gene family preference observed early in ontogeny is associated more with the inherent genetic potential of B cell progenitors that predominate during fetal life and less with environmental influences.     

Neonatal and adult primary B cells use the same germ-line VH and V kappa genes in their (T,G)-A-L-specific repertoire

Although there is a nonrandom usage of VH gene families by primary B cells early in ontogeny, at issue is whether the preferential rearrangement of 3' germ-line VH genes, e.g., VH7183 and VHQ52 family genes, influences the neonatal B cell repertoire that can be expressed in response to Ag. In order to address this issue, and to determine whether neonatal B cells can use the same germ-line VH and V kappa genes as adult B cells in their primary response, we have analyzed at the molecular level the neonatal antibody response to (T,G)-A-L and compared it with the adult primary response. Among the TGB5 Id+, GT+ antibodies, which dominate the neonatal response to (T,G)-A-L, two VH gene families were used: J558 (high frequency) and 36-60 (low frequency). The majority of Id+ neonatal hybridomas used the same germ-line VH gene (H10, from the VHJ558 family), but with enormous diversity in the D region, and one of two germ-line V kappa 1 genes (V kappa 1A, V kappa 1C). These are the same germ-line V-genes used by most primary adult Id+ hybridomas, and the frequency of expression of this germ-line V-gene combination appears equivalent in the neonatal and adult primary repertoires. Therefore, it is clear from this study that as early as day 5, neonatal B cells can use the same germ-line V-genes as adult primary B cells in their Ag-specific repertoire.     

Direct quantitative in situ hybridization studies of Ig VH utilization. A comparison between unstimulated B cells from autoimmune and normal mice

This study examines Ig VH utilization in murine lupus with emphasis on the relative contribution of 3' and 5' gene families. We used in situ hybridization with 35S-labeled ssRNA probes to detect VH expression in individual spleen cells. Cells were taken from unmanipulated animals, and were not stimulated in vitro. This approach allows analysis of VH usage among only those B cells which have undergone activation in vivo, while minimizing the potential for skewing in vitro. We compared usage of the 3' 7183 and Q52 families with the more 5' J558 family in adult NZB, MRL-lpr/lpr, and nonautoimmune NIH Swiss mice. VH utilization in the autoimmune strains was proportionate to VH family size, and was not biased toward the 3' families when compared with the Swiss repertoire. Moreover, 3' skewing did not develop in NZB mice with increasing age. Thus, systemic autoimmunity is not associated with impaired normalization of the adult repertoire away from the 3' bias of early ontogeny. Instead, our data support a stochastic model for VH gene usage in the activated B cells and plasma cells of adult lupus mice.     

A single VH gene is utilized predominantly in anti-BrMRBC hybridomas derived from purified Ly-1 B cells. Definition of the VH11 family

By establishing hybridomas from two distinct surface IgM+ splenic B cell populations, Ly-1 B cells and "conventional" (Ly-1-) B cells, we found that the Ly-1 B population includes a 30 to 70 times higher frequency (1 to 2%) of cells with specificity for bromelain treated autologous red blood cells (anti-BrMRBC) when compared with conventional B cells (0.03%). We cloned and sequenced the V genes encoding anti-BrMRBC antibody from two hybridomas made with Ly-1 B cells sorted from the spleen of SM/J mice. The VH sequence (for both) is identical with the previously reported sequence associated with this specificity and belongs to a new VH gene family. This gene family, defined here as VH11, has only two members and is the predominant VH rearranged in a collection of Ly-1 B derived anti-BrMRBC hybridomas, always in association with a single VL gene (a member of the V kappa 9 family). Furthermore, analysis of hybridomas made with Ly-1 B cells sorted from the peritoneum reveals a yet higher increased frequency of VH11-encoded anti-BrMRBC specificity (30%). This variation in frequency of anti-BrMRBC in the Ly-1 population depending on location, together with the repeated association of VH11 with a particular V kappa gene suggest that antigen driven selection is (at least in part) responsible for the biased V gene expression seen in this population. Furthermore, a mechanism that might contribute to biased expression, preferential rearrangement due to close proximity to J (as seen in pre-B lines), is excluded by localization of VH11 5' to several of the more J-proximal families (Q52, 7183).     

Interspersion of the VHQ52 and VH7183 gene families in the NFS/N mouse

Deletion mapping analysis has shown that members of the VH7183 and VHQ52 gene families are interspersed in the NFS/N mouse. To obtain direct evidence that members of these gene families are physically linked, an NFS/N liver library was constructed and genomic clones were analyzed for hybridization to both VHQ52 and VH7183 gene probes. Four clones were identified which contained both VHQ52 and VH7183 hybridizable restriction fragments. Two clones containing rearranged VHQ52 genes were also found to hybridize with the VH7183 gene probe. Sequence analysis of three of the VH7183-containing restriction fragments indicate that all are pseudogenes which contain interruptions at either the 5' and/or 3' ends of the VH coding region. Given the D-proximal location of at least a portion of the VHQ52 gene family relative to VH7183 in NFS/N mice, and the known correlation between D proximity and the frequency of VH gene utilization, 22 NFS/N-derived pre-B cell lines were analyzed for VHQ52 gene utilization. More than 40% of the identified H chain (VHDJH) rearrangements in this survey used members of this gene family. Furthermore, analysis of poly(A)+ RNA from NFS/N fetal liver and adult spleen also indicates preferential utilization of VHQ52 family in fetal liver. Kinetic studies show, however, that there are no changes in relative utilization throughout fetal ontogeny. The implications of these findings for the expression and randomization of the VH repertoire are discussed.     

V-region directed selection in differentiating B lymphocytes.

We here analyse the repertoire of VH7183 rearrangements isolated from different stages of B cell differentiation in adult mice. The nucleotide sequence analyses of VH7183-D-JH rearrangements derived from large pre-B cells (B220+, mu-), small pre-B cells (B220+, mu-) and mature B cells (B220+, mu+) isolated from adult bone marrow revealed a sequential accumulation, among functional rearrangements, of D segments of the FL16 family and a depletion of D segments using the second and the third reading frame (RF). One member (VH7183.1) of the VH7183 gene family was utilized in 60-80% of the rearrangements of all populations analysed. In neonates the majority of the rearrangements utilizing this gene was found to be functional. In contrast, > 96% of the VH7183 rearrangements isolated from adult spleen were non-functional. These data provide evidence for cellular selection of VH regions acting at different points of the B cell differentiation pathway and at the transition of B cells from the bone marrow to the periphery.     

Eleven MRL-lpr/lpr anti-DNA autoantibodies are encoded by genes from four VH gene families: a potentially biased usage of VH genes

The genes encoding 11 independently derived anti-DNA autoantibodies from the lupus-prone mouse strain, MRL-lpr/lpr, were examined with VH, D, and JH gene probes. These autoantibodies do not define new VH gene families, since all of the autoantibodies were encoded by VH genes from four of the nine known gene families. A minimum of nine different VH genes encoded this panel of 11 anti-DNA autoantibodies. These results are consistent with the stochastic use of the VH gene repertoire and the expression of multiple VH genes. However, the data is also consistent with a biased usage of the VH gene repertoire. First, two pairs of autoantibodies, one from the J558 family and one from the 7183 family, appear to express identical or closely related VH genes as determined by the position of two restriction enzyme sites 5' of the expressed VH genes. In addition, three autoantibodies that appear to be sister clones might define a third VH gene that is used repeatedly. Secondly, about 45% of the panel is encoded by the Q52 and 7183 families, which are the 3' most families. These families have been shown to be preferentially rearranged early in B cell ontogeny. This suggests that some anti-DNA autoantibodies might originate from a population of B cells that predominate early in ontogeny. An alternative hypothesis is that the potential bias in VH gene and gene family usage could be due to antigen selection. All four JH genes are expressed, although the JH1 gene appears to be underutilized in both expressed and unexpressed rearrangements. Two members of the panel that bind double-stranded DNA were encoded by two different VH gene families, the S107 family and the J558 family.     

Intraclonal diversity in the VH genes expressed by CD5- chronic lymphocytic leukemia-producing pathologic IgM rheumatoid factor.

The leukemic cells from 95% of patients with B cell chronic lymphocytic leukemia (CLL) coexpress B cell differentiation antigens and the pan-T lymphocyte surface antigen CD5 (Leu 1). As such, CLL generally may be considered a malignancy of the CD5 B cell, a minor B cell subpopulation implicated in the production of autoantibodies. Recent data indicate that CD5+ leukemic cells may express autoantibody-associated V region genes with little or no somatic mutation. We examined the Heavy chain V genes expressed by an unusual CLL that secretes rheumatoid factor (RF) autoantibodies but does not express the CD5 surface Ag. Nucleic acid sequence analyses of the rearranged VH genes of three independent rDNA clones demonstrated intraclonal diversity not apparent in previously studied cases of CD5+ CLL. Comparison of the rearranged VH genes reveals that they belong to the VH4 gene subgroup and share highest homology with a rearranged VH gene (Ab44) that encodes a polyreactive autoantibody. That these productively rearranged VH genes encode the RF generated by this unusual CLL population is demonstrated by immunoblotting of the RF paraprotein using primary sequence dependent antipeptide antisera. These results indicate that CD5- CLL, like their CD5+ counterparts, may produce RF. However, unlike CD5+ CLL examined to date, CD5- CLL may have intraclonal diversity in their expressed Ig genes.     

Ig VH gene family repertoire of plasma cells derived from lupus-prone MRL/lpr and MRL/++ mice

VH gene family usage was determined in both spontaneous, in vivo activated plasma cells and LPS-induced plasma cells from individual MRL/lpr mice by using in situ hybridization. It was found that VH gene family expression in spontaneous plasma cells varied from mouse to mouse. Some mice expressed VH families in an apparently random manner similar to that obtained with polyclonal activation. Other mice showed an exaggerated expression of particular VH gene families. VH J558 was overrepresented most frequently, but overrepresentation of VH 7183, Q52, and 36-60 was also observed. Importantly, LPS-induced VH gene family expression in these same mice displaying biased VH family usage in spontaneous plasma cells, appeared normal with no evidence for similar biases in the LPS-induced repertoire. Anti-DNA antibody concentrations and the degree of glomerulonephritis were determined for each mouse to measure the severity of disease. The level of expression of the J558 family was positively correlated with disease severity. The results suggest that the initial autoantibody response is highly diverse but becomes more restricted as the disease progresses.     

Circulating human B cells that express surrogate light chains display a unique antibody repertoire

Circulating human B cells that coexpress V-preB and conventional L chains (V-preB+L+ B cells) are a recently described subset of B cells that express Abs with features of self-reactivity. Initial analysis of V-preB+L+ B cells was limited to Ig-kappa and to the small, underused VH5 family. To determine whether Abs commonly expressed by V-preB+L+ B cells show similar features, we analyzed Ig H chains from three highly expressed VH families, VH1, VH3, and VH4, and Ig-lambda. We find that VH1 and VH3 Abs expressed by V-preB+L+ B cells resemble VH5 in that they display increased JH6 use, long CDR3s, and an increased frequency of D-D fusions. Abs in all three of these VH families also show skewed D reading frame use resulting in predominance of hydrophobic amino acids, which are counterselected in conventional B cells. Like Ig-kappa genes, the Ig-lambda genes in V-preB+L+ B cells show long CDR3s, but they differ from Ig-kappa genes in that they display no evidence of receptor editing. We conclude that a large number of H and L chain Abs expressed by V-preB+L+ B cells display features associated with self-reactive Abs.     

Patterns of Ig V region expression among neonatal hemagglutinin-responsive B cells. Evidence for non-random VH gene representation during later stages of development

Hybridoma libraries were established whose specificities reflect those within the BALB/c hemagglutinin-responsive B cell repertoire at 1 or 2 wk of age. These libraries were generated through chronic immunization regimes that induce responses dominated by clonotypes available at the age of initial immunization. Dot blot analyses of cytoplasmic RNA from these hybridomas were performed to determine the Ig H chain V region (VH) families associated with the repertoire at each age. Although genes from most known VH families can generate hemagglutinin-specific antibodies, clonotypes prevalent during the first week of life disproportionately use VH7183 gene segments. In contrast, hybridomas representative of the repertoire in 2-wk-old individuals preferentially use VHS107, VH36-60, and VHX24 gene segments. These results demonstrate changes in VH gene family predominance that correlate with the age-related patterns of clonal emergence and turnover previously shown in the hemagglutinin-reactive B cell pool. Taken together, these findings suggest that the very early neonatal Ag-responsive B cell pool closely reflects preferential VH gene rearrangements within the pre-B cell compartment. Further, they suggest that either non-random strategies of VH gene expression, or selective clonal expansion strategies based on VH, operate even at later stages of development.     

Analysis of VH gene replacement events in a B cell lymphoma

We have analyzed a series of recombinational events at the IgH chain locus of the B cell lymphoma, NFS-5. Each of these recombinational events results in the replacement of the VH gene segment of the rearranged H chain gene (VhDJh) with that of an upstream germline gene segment. Replacements on the productive and nonproductive alleles have been observed. In each case, the recombination occurs in close proximity to a highly conserved heptameric sequence (5'TACTGTG3') which is located at the 3' end of the VH coding region. In the two examples of recombination on the productive allele that have been analyzed, the initial VHQ52 gene is replaced by different VH7183 genes. On the non-productive allele, sequential replacement events have been analyzed: the initial VHQ52 rearrangement is first replaced by a closely related VHQ52 gene, followed by a second replacement using a VHQ52 pseudogene. Southern blot analysis using VH probes indicates that these recombinations may be accompanied by the deletion of germline VH genes belonging to both the VHQ52 and VH7183 families, suggesting that these gene families are interspersed in the NFS/N mouse.     

Immunodominance of the VH1-46 antibody gene segment in the primary repertoire of human rotavirus-specific B cells is reduced in the memory compartment through somatic mutation of nondominant clones

Detailed characterization of Ag-specific naive and memory B cell Ab repertoires elucidates the molecular basis for the generation of Ab diversity and the optimization of Ab structures that bind microbial Ags. In this study, we analyzed the immunophenotype and VH gene repertoire of rotavirus (RV) VP6-specific B cells in three circulating naive or memory B cell subsets (CD19+IgD+CD27-, CD19+IgD+CD27+, or CD19+IgD-CD27+) at the single-cell level. We aimed to investigate the influence of antigenic exposure on the molecular features of the two RV-specific memory B cell subsets. We found an increased frequency of CD19+IgD+CD27+ unclass-switched memory B cells and a low frequency of somatic mutations in CD19+IgD-CD27+ class-switched memory B cells in RV-specific memory B cells, suggesting a reduced frequency of isotype switching and somatic mutation in RV VP6-specific memory B cells compared with other memory B cells. Furthermore, we found that dominance of the VH1-46 gene segment was a prominent feature in the VH gene repertoire of RV VP6-specific naive B cells, but this dominance was reduced in memory B cells. Increased diversity in the VH gene repertoire of the two memory B cell groups derived from broader usage of VH gene segments, increased junctional diversity that was introduced by differential TdT activities, and somatic hypermutation.     

Qa-2 expression in the adult murine thymus. A unique marker for a mature thymic subset

The MHC Ag, Qa-2, is expressed on all peripheral T cells, a subset of bone marrow cells, and to a lesser extent on B cells. The Qa-2 Ag is also expressed on 5 to 6% of normal adult murine thymocytes. Through the use of flow cytometry, counterflow centrifugal elutriation and acridine orange staining, we have analyzed the cell surface phenotype, cell size, and cell cycle status of this thymic population. Our studies indicate that Qa-2+ thymocytes are large, non-mitotic, G1 cells which have the cell surface phenotype of CD5+, CD3+, J11dLO and lack receptors for peanut agglutinin. This population can be further subdivided into three categories; CD4+/CD8-, CD4-/CD8+, and CD4-/CD8-. These data indicate that Qa-2 surface expression can only be detected on thymocytes in the final stages of differentiation. The Qa-2 Ag can be used as a cell surface marker to identify a unique subset of mature thymocytes.     

Studies of CD4- CD8- alpha beta bone marrow T cells with suppressor activity.

The predominant T cell subset in the bone marrow of specific pathogen-free C57BL/Ka and BALB/c mice expressed the alpha beta+ TCR CD4- CD8- surface phenotype. Purified C57BL/Ka alpha beta+ TCR CD4- CD8- marrow cells obtained by cell sorting suppressed the MLR of C57BL/Ka responder and BALB/c stimulator spleen cells. Although the percentage of typical T cells in the spleen was markedly reduced in adult nude mice or normal neonatal mice as compared to the normal adult, the percentage of alpha beta+ TCR CD4- CD8- cells in the spleen and marrow was not. The percentage of "self-reactive" V beta 5+ T cells in the BALB/c spleen was markedly reduced as compared to that in the C57BL/Ka spleen. However, the percentages in the bone marrow were similar. The results indicate that the predominant subset of marrow T cells in these pathogen-free mice differ with regard to surface marker phenotype, function, dependence on the adult thymus, and deletion of certain self-reactive V beta receptors as compared to typical spleen T cells. The marrow T cells appear to develop directly from marrow precursors without rearranged beta chain genes during a 48 hour in vitro culture.     

VH gene expression by nontransformed pre-B cells upon differentiation in vitro

Mice have more than 1000 VH gene segments, and each pre-B cell must choose a single one for rearrangement to encode the V portion of the antibody H chain. Presumably, all or most of the functional VH gene segments must be chosen by the population of B lymphocytes if the organism is to express the diversity that is observed in the immune system. Control of the selection of a VH gene segment for expression is not understood. We have found that the members of the VH gene family closest to the constant genes, the 7183 family, are transcribed in a manner that is specific for the stage of B cell development after pre-B cells derived from spleens of 6- to 8-wk-old nude mice are induced to differentiate in vitro by a mixture of dendritic cells and mitogen-activated T lymphocytes (DC-T). DC-T from spleens and lymph nodes induce transient high levels of synthesis of RNA from the 7183 VH family, whereas DC-T from Peyer's patches of mice of the same age as those from which spleen and lymph node DC-T were prepared did not induce the expression of RNA from that gene family. Spleen and Peyer's patch DC-T induce secretion of similar total amounts of antibody. Therefore, the RNA synthesis from members of at least one VH gene family is specific both for the lymphoid tissue in which B cell differentiation occurs and for the developmental stage of the B lymphoid cells.     

A new murine Ig VH gene family

A novel murine VH gene family, termed VH10, has been found and characterized. Based on RFLP analysis, this family exhibits extensive polymorphism among inbred strains of mice and encompasses two to five members, depending on the Igh haplotype. Analyses of recombinant inbred strains suggest a map position of this family 5' to the 7183 and Q52 VH gene families. A VH10 gene has been found to encode anti-DNA autoantibodies from lupus mice; another one may be a pseudogene.     

Isotype, VH genes, and antigen-binding analysis of hybridomas from newborn normal BALB/c mice

In this study we report on the characterization of a panel of 62 hybridomas generated by fusing unstimulated spleen cells from neonatal (less than 24 hr old) normal BALB/c mice with the non-secreting Sp 2/0 cell line. The vast majority (98%) of these hybridomas secreted Ig but only 20% produced IgM. The isotype of the remaining hybridomas was determined as being IgG2b. Interestingly, when splenocytes from 1-day-old mice were stimulated with LPS for 48 h prior to the fusion event, 84% of the hybridomas were secreting IgM. The hybridoma supernatants were screened either by ELISA or RIA for binding reactivity using a panel of 17 Ag, proportionally divided between self and non-self. A binding reactivity could be assigned in 44% of cases. Of these, 29% were monoreactive, i.e., reactivity occurred with one Ag only, while the remaining 15% were multireactive. The majority (21 of 27) of hybridomas with a defined reactivity were directed against self-Ag. These included autologous red blood cells, DNA, histone H1, thyroglobulin, and Ag of the cell surface of T cells. The frequency of utilization of VH genes was determined using DNA probes for eight VH gene families. While all VH gene families appeared to have been used, one, VH 7183, had a slight but significant (p less than 0.02) higher utilization than expected by random expression. The frequency of all the other VH gene families was not significantly different from random utilization. No correlation was found between Ag reactivity in the supernatants and the utilization of a particular VH gene family. These findings indicate that early in the ontogeny the predominant reactivity of B cells is for self-Ag and, unlike what it is commonly believed, the IgM isotype is not dominant within these endogenously activated B cells at this time of ontogeny when genes from all VH families are utilized.     

Distribution of ecto-5'-nucleotidase on subsets of human T and B lymphocytes as detected by indirect immunofluorescence using goat antibodies

Human T and B lymphocyte subsets were characterized for ecto-5'-nucleotidase (ecto-5'-NT) expression by two-color immunofluorescence by using polyclonal goat antibodies to 5'-NT and murine monoclonal antibodies to T and B cell subsets. Anti-5'-NT antibodies were prepared by immunizing a goat with purified human placental 5'-NT. Lymphocyte surface 5'-NT was detected with F(ab')2 fragments of immune goat IgG followed by biotinylated F(ab')2 rabbit anti-goat IgG and fluorescein isothiocyanate-avidin. Lymphocyte cell surface antigens were detected with phycoerythrin (PE)-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD16, and anti-CD19. HB-4, an antigen present on a major subset of human peripheral blood B cells, was detected with murine monoclonal anti-HB-4 and PE-anti-mouse-kappa. Analysis showed that ecto-5'-NT was expressed on 32 +/- 7% of CD3+, 19 +/- 6% of CD4+, and 50 +/- 21% of CD8+ T cells, but not on CD16+ lymphocytes. Ecto-5'-NT was also expressed on 81 +/- 8% of adult peripheral blood B cells as defined by PE-anti-CD19; HB-4 was expressed on 84 +/- 7% of CD19+ cells. The two populations of B cells were not identical, however, because HB-4 was co-expressed on only 79 +/- 18% of ecto-5'-NT+ B cells. Two-color immunofluorescent staining of T cells from a patient with congenital agammaglobulinemia and low T cell ecto-5'-NT activity revealed reduced percentages of ecto-5'-NT+ cells in his CD3+, CD4+, and CD8+ populations. Thus, reduced ecto-5'-NT activity by enzyme assay was paralleled by reduced numbers of 5'-NT molecules on the cell surface. Two-color immunofluorescent staining of B cells from a patient with hypogammaglobulinemia and low B cell ecto-5'-NT activity also revealed markedly reduced expression of 5'-NT. HB-4 expression was normal, however, suggesting that the patient's B cells were blocked in maturation subsequent to the acquisition of HB-4 but prior to that of ecto-5'-NT. These results demonstrate that anti-5'-NT antibodies will be valuable tools for analyzing ecto-5'-NT expression and lymphocyte maturation in patients with immuno-deficiency diseases.