Preferential rearrangement of V kappa 4 gene segments in pre-B cell lines.

Examination of the in vitro V kappa gene rearrangements of murine adult bone marrow-derived pre-B cell lines reveals that 21 of 25 (84%) cell lines have rearranged a member of the V kappa 4 family. In contrast, analysis of two V kappa cDNA libraries prepared from LPS-stimulated adult spleen cells indicates that only 17% of the Ig kappa cDNAs contain sequences belonging to the V kappa 4 gene family. Half of the pre-B cell lines examined also share an 8-kbp BamHI reciprocal product (rp). However, these rp do not involve the same V kappa gene, indicating that conserved BamHI sites exist 3' of some V kappa genes. This rp is also readily detected in DNA from normal adult spleen cells, suggesting that the in vitro rearrangements examined in this study are representative of kappa rearrangements that occur in vivo. We suggest that, unlike the diverse V kappa repertoire expressed by mature B cells, the germline V kappa segments involved in initial rearrangements of the Ig kappa locus are highly restricted, and that an initial V kappa 4 rearrangement is probably followed by other, more random recombination events.

Strain variation in the frequency of Abelson murine leukemia virus-transformed fetal liver pre-B cells bearing complete immunoglobulin heavy chain rearrangements.

Fetal liver Abelson pre-B cell lines obtained from CBA/Tufts.xid and (CBA/Tufts.xid x CBA/Tufts)F1 mice have complete VDJH rearrangements on at least one allele. Such high frequencies of VDJH rearrangements have previously been observed in adult derived but not fetal liver derived Abelson pre-B cell lines. Genetic analyses suggest that CBA/Tufts.xid carries an autosomal dominant gene(s) that determines the predominance of VDJH rearrangements among transformants. This autosomal gene(s) might affect the intrinsic development of the early B cell lineage in the fetus or the fetal microenvironment, expanding pre-B cells of the "more mature" VDJH phenotype.

The organization of the mouse Igh-V locus. Dispersion, interspersion, and the evolution of VH gene family clusters.

We have constructed a panel of Abelson murine leukemia virus-transformed pre-B cells to study the organization of the mouse VH gene families. Based on the analyses of VH gene deletions on 51 chromosomes with VH gene rearrangements, we have inferred a map order of the Igh locus that holds for both the Igha and Ighb haplotypes. We show that members of each VH gene family are generally clustered, although three family clusters (VHS107, VH36-60, VGAM3.8) are dispersed in two or three subregions of the locus. Members of two VH gene families, VHQ52 and VH7183, are extensively interspersed and map within the same subregion. An examination of the distribution of VH group members (VH II, I, and III) within the locus suggests that two major duplications may, in part, explain the dispersed pattern of VH family clusters. The relationship of VH organization and functional expression is discussed in terms of position-dependent and complexity-driven models.

Junctional diversification in the generation of the precursor of a discrete immune response.

Phosphocholine (PC)-specific antibodies that arise in the mouse in response to Proteus morganii (PM) and use V1-DFL16.1-JH1 are characterized by a number of recurring mutations. Most striking is an invariant A for G substitution in codon 95 of VH which results in an asparagine instead of aspartate at that position. Because of the apparent importance of this substitution in an anti-PC(PM) response, we wanted to determine the molecular basis for this base change. A cDNA library derived from pre-immune splenic B cells was examined for the frequency of VDJ containing the A substitution at 95 and the presence of additional point mutations in these sequences. Six different cDNA were isolated which contained an A substitution at the VD junction (frequency 0.00009); a seventh positive cDNA could not be examined. The V segments of four of these cDNA matched known germline genes and were, therefore, unmutated. Two others closely matched V in families whose members have not all been characterized, hence, it is not known whether the mutations observed are somatic or germline in origin. Sequences of 35 cDNA clones, containing the same V segment but differing in D, J and junctional nucleotides, revealed no mutations. These results indicate that the A substitution generated at codon 95 is most likely a product of V-DJ joining.

Germline diversity of the expressed BALB/c VhJ558 gene family.

Although the mouse immunoglobulin heavy chain (Igh) locus contains 15 heavy chain V (Vh) gene families, at least half of the Vh gene segments are members of the VhJ558 family. This large Vh gene family represents the least characterized germline coding regions of any of the mouse antigen receptor loci and the contribution of individual VhJ558 genes to the preimmune repertoire is poorly understood. In fact, relatively few germline VhJ558 sequences have been reported for BALB/c, the foundation strain for mouse immunoglobulin genetics and the prototypic strain of the Igh(a) haplotype. Here we present a database consisting of 66 sequences estimated to represent one-half of the total number of functional BALB/c VhJ558 genes. Our results indicate that a subset of the VhJ558 genes is highly expressed in the preimmune repertoire, with just nine Vh sequences accounting for nearly 50% of the VhJ558 heavy chains expressed by splenic B cells. We show that this disparity in the expressed Vh gene repertoire is not due to the position of the Vh genes relative to the Dh cluster or to multiple germline copies of the highly expressed VhJ558 genes. Together, these data constitute the first detailed analysis of functional BALB/c VhJ558 genes, demonstrate a striking bias in the use of particular VhJ558 genes in the preimmune repertoire, and provide sufficient information to study the regulation of the Dh-distal region of the Igh-V locus at the level of individual genes.

Community values in Vermont health planning.

KIE: This is one of a set of six short articles, grouped under the umbrella title "Grassroots bioethics revisited: health care priorities and community values," with a very brief introduction by Bruce Jennings. The articles focus on the involvement of community health decisions projects with public policy issues of access to health care, allocation of resources, setting health care priorities, cost containment, and rationing.^ieng

Utilization of V kappa families and V kappa exons. Implications for the available B cell repertoire.

A molecular cloning approach was used to determine the relative utilization of 2 individual V kappa 21 genes, 13 V kappa gene families, and the 4 functional J kappa gene segments among splenic B cells of nonimmunized BALB/c mice. Based on the observed frequency of individual V kappa gene expression, we estimate that the mouse genome encodes 150 to 180 functional V kappa genes, and we suggest that most functional V kappa exons are expressed at comparable frequencies in the preimmune antibody repertoire. In contrast, clear differences in J kappa segment utilization were observed, J kappa 4 being consistently underrepresented with respect to the other J kappa segments.

Regulation of thymus-independent responses: unresponsiveness to a second challenge of TNP-Ficoll is mediated by hapten-specific antibodies.

CBA mice immunized with the TI-2 antigen TNP-Ficoll are unresponsive to a second challenge of the same antigen. In addition, spleen cells from unresponsive mice fail to respond to any dose of TNP-Ficoll in vitro. This hapten-specific unresponsive state is T cell independent and is due to hapten-specific IgM-mediated inhibition of B cell triggering. The mechanism of inhibition, although unknown, is unlikely to be antigen masking. In vitro responses to the TI-1 antigen TNP-LPS are not inhibited by concentrations of anti-TNP antibody, which completely suppress responses to TNP-Ficoll.

Accessibility changes across the mouse Igh-V locus during B cell development.

During their development, B and T lymphocytes are thought to undergo several cycles of chromatin remodeling at their antigen receptor loci that serve to regulate access of a common V(D)J recombinase to particular gene segments. We used germ-line transcription and susceptibility to DNasel as markers to examine tissue and stage-specific changes in chromatin structure surrounding genes of the VHJ558, VH10, and VHS107 families, whose members are located at discreet subregions of the locus. Germ-line VH transcripts from all three families were detectable at pro- and pre-B cell stages. Transcripts from the VH10 and VHS107 families, but not VHJ558, remained detectable at the immature and mature B cell stages. Unexpectedly, none of the germ-line VH loci examined were markedly nuclease sensitive, regardless of cell type or transcriptional activity. A modest degree of nuclease sensitivity was noted at the VHJ558 loci of pro-B and pre-B cells, however. Our data suggest that the entire Igh-V locus becomes accessible at early B cell stages, and returns thereafter to an inaccessible state. However, the timing of these accessibility changes does not occur uniformly across the VH array. These results imply that multiple long-range elements are involved in targeting VH genes for rearrangement.

Rearrangement and selection in the developing Vkappa repertoire of the mouse: an analysis of the usage of two Vkappa gene segments.

Detailed analysis of the rearrangement and expression of two mouse Vkappa genes has been used to examine B cell repertoire development. The Vkappa1-A gene is used by a large proportion (9.6%) of splenic B cells in the adult primary repertoire, whereas the Vkappa22 gene is used at a much lower frequency (0.16%). Consistent with these results, quantitative PCR (Q-PCR) assays revealed that the number of splenic B cells with rearranged Vkappa1-A genes is much greater than the number with rearranged Vkappa22 genes. Q-PCR was also performed on both normal bone marrow pre-B cells and transformed pre-B cells induced to rearrange their kappa loci at high frequency. In contrast to splenic B cell rearrangements, the numbers of Vkappa1-A and Vkappa22 rearrangements in pre-B cells differ by only two- or threefold, suggesting that the intrinsic rearrangement frequencies of these two Vkappa genes are not significantly different. Further evidence of disproportionate selection was obtained by comparing the percentages of productive rearrangements amplified from genomic splenic DNA. Sequence analysis showed 84% (37 of 44) of the Vkappa1-A rearrangements but only 57% (29 of 51) of the Vkappa22 rearrangements to be in-frame. Together these results suggest that B cells expressing Vkappa1-A-encoded light chains are preferentially selected either in the periphery or in the transition from pre-B to B cell. Sequence data also reveal a surprisingly restricted diversity of VJ junctions, apparently due to biases introduced by the rearrangement mechanism.

Molecular cloning of the primary IgH repertoire: a quantitative analysis of VH gene usage in adult mice.

The generation of the primary antibody repertoire requires the somatic rearrangement of germline gene segments. It is not known, however, whether all functional V and J gene segments have an equal probability of contributing to this initial set of antibody specificities. To address this issue, we have examined the relative utilization of VH and JH gene segments of the mouse. We have constructed VH cDNA phage libraries from C mu transcripts obtained from polyclonally activated spleen cells of the BALB/c and C57BL/6 strains. We show that probes specific for either one, two or three functional VH gene segments hybridize to cDNAs at frequencies directly proportional to the number of functional germline VH genes detected by each probe. In contrast, the representation of 10 VH gene families within each library indicates that certain families are under-represented relative to their estimated germline gene number. These families must either have extraordinary proportions of nonfunctional genes or are influenced by as yet unidentified regulatory mechanisms or constraints on rearrangement.

The immunoglobulin heavy chain variable region (Igh-V) locus in the mouse. I. One hundred Igh-V genes comprise seven families of homologous genes.

Seven families of Igh-V genes have been defined by Southern blot analysis of genomic DNA from eighteen inbred strains of mice. Each of twenty-four cloned Vh genes hybridized to one of seven nonoverlapping sets of Eco RI restriction fragments. These families contain from 2 to approximately 40 hybridizing fragments. From these data we estimate that the mouse Igh-V locus consists of one hundred Vh genes. Genes within a Vh family share greater than 80% sequence homology while the sequence homology between families is generally less than 70%. There is extensive restriction fragment length polymorphism among the strains analyzed allowing the assignment of complete (Igh-V + Igh-C) Igh haplotypes for eighteen inbred mouse strains.

The utilization of individual VH exons in the primary repertoire of adult BALB/c mice.

The utilization of VH gene families is severely biased during fetal development, such that D-region proximal VH genes are overrepresented. After birth the VH repertoire becomes more equally representative of the total inherited set of functional VH genes. To investigate the extent of this normalization process, we have determined the relative utilization of individual VH exons in the pre-immune repertoire of adult BALB/c spleen cells. Large samples of IgM heavy chain transcripts from polyclonally activated B cells were captured in a cDNA phage library and screened by hybridization using highly specific oligonucleotide probes. These studies revealed that the utilization of particular VH exons can differ by an order of magnitude. Significantly, the VH genes most under-represented in the pre-immune repertoire are located in the region of the Igh locus most distal to the DjhCh region. We suggest that the chromosomal position of a particular VH gene may influence its utilization and that the normalization of the adult repertoire is incomplete.

Deletion mapping of the mouse ornithine decarboxylase-related locus Odc-rs8 within Igh-V.

The Odc-rs8 locus belongs to a family of mouse DNA sequences related to the gene encoding ornithine decarboxylase (ODC). Odc-rs8 was mapped by recombinant inbred (RI) strain analysis to the region of Chromosome (Chr) 12 occupied by the variable region genes of the immunoglobulin heavy chain (Igh) complex. In the present study, alleles at Odc-rs8 were shown to cosegregate with those for Igh variable region (Igh-V or VH) genes among 37 inbred mouse strains that had been characterized previously for their haplotypes at Igh. For a more precise definition of the location of Odc-rs8 relative to Igh-V, DNAs from 17 Abelson murine leukemia virus (A-MuLV)-transformed pre-B cell lines cultured from mice heterozygous at Igh and Odc-rs8 were analyzed for the presence of DNA restriction fragments (RFs) derived from each parental Odc-rs8 allele. These cell lines, each of which has rearranged one or both Igh genes, previously were employed in mapping members of nine VH gene families by deletion analysis (Brodeur et al. 1988). Comparing the deletion profiles of the cell lines for Odc-rs8 with those for the VH gene families has located Odc-rs8b within the VHJ558/VH3609 gene cluster and Odc-rs8c either within or upstream of the 5'-most 9% of VHJ558, identifying Odc-rs8 as a potentially useful marker for the 5' end of the Igh complex.

Organization and expression of VH gene families preferentially expressed by Ly-1+ (CD5) B cells.

Previously we have shown that a high proportion (5%-15%) of peritoneal Ly-1+ B cells that bind the common membrane phospholipid, phosphatidylcholine, express either of two VH genes. One of these genes, CH34VH, belongs to the recently described VH11 family, whereas the other VH gene, CH27VH, is reported here to belong to a new, single-member family which we designate VH12. We have characterized these new VH families in terms of strain polymorphism, map position relative to nine other VH gene families, and frequency of expression in polyclonally stimulated splenic B cell populations. We find that VH11 and VH12 genes are expressed at frequencies similar to the VH genes of other families in the spleen, and that these genes are not located among the J-proximal VH segments which are preferentially expressed during early development. These results suggest that the VH11 and VH12 genes are not preferentially rearranged, and support our earlier studies which suggested that the high frequency of VH11 and VH12 expression among peritoneal Ly-1 B cells is due to antigen selection.

The Ig VH repertoire of fetal liver-derived pre-B cells is influenced by the expression of a gene linked to X-linked immune deficiency.

Ig VH repertoire differences between normal and x-linked immune deficiency- (xid) expressing mice are well established. To test the hypothesis that such differences might exist as early as the pre-B stage of ontogeny we generated panels of xid fetal liver derived Abelson murine leukemia virus transformants with H chain Ig VDJ rearrangements. Cells from CBA/Tufts.xid mice used VH genes from many families, with no demonstrable preference for 3' genes. Analysis of cells derived from (CBA/Tufts.xid X CBA/Tufts)F1 mice showed preferential usage of 3' family genes in the phenotypically normal females, even though V to DJ joins were made in vivo. The defective male mice did not show this marked preferential usage. A similar, but less marked, effect on VH gene usage was seen in mice with X-linked immune deficiency and a BALB/c background. Taken together, these results show that either X-linked immune deficiency, or a closely linked gene, affects fetal pre-B cells such that the usual pattern of predominant usage of 3' family genes is altered.

Deletional mapping of fifteen mouse VH gene families reveals a common organization for three Igh haplotypes.

In addition to the content of germ-line variable gene segments, the organization of V genes has been implicated in the development of the Ab repertoire. We have searched the expressed VH genes of BALB/c mice for additional VH gene families and utilized deletion mapping to explore the extent of VH gene family interspersion. We have identified and characterized one new VH gene family (VH15) and extended our previous studies of the Igha and Ighb haplotypes to include a third haplotype (Ighj) using a newly developed panel of pre-B cell lines (CXCB cell lines). We conclude that the Igha, Ighb, and Ighj haplotypes have a similar Igh-V locus structure. A refined deletional map for 15 VH gene families and an individual member of the VHSM7 family (H10) has been constructed based on the deletion profiles of 72 rearranged heavy chain loci. These results demonstrate previously unrecognized examples of interspersion among members of the VHS107, VH10, and VHSM7 families.

Germline structure and differential utilization of Igha and Ighb VH10 genes.

Ab heavy chains encoded by mouse VH10 genes have been of particular interest due to their frequent association with DNA binding. We reported previously that VH10 sequences are over-represented in the preimmune repertoire considering the apparent number of germline-encoded VH10 gene segments. In this report, we show that the VH10 family consists of three and two germline genes in the Igha and Ighb haplotypes, respectively. The complete nucleotide sequences of these five genes, including promoters and recombination signal sequences, were determined and allow unambiguous assignment of allelic relationships. The usage of individual VH10 genes varied significantly and ranged from 0.2% to an extraordinary 7.2% of the VH genes expressed by splenic B cells. Since the promoter and recombination signal sequence elements of all five VH10 genes are identical, we suggest that the few amino acid differences encoded by these five germline VH10 genes determine their representation in the preimmune repertoire. Rearrangements of the most frequently used VH10 gene have an apparent bias for histidine at position 95 of complementarity-determining region-3 (CDR3). These CDR3s are also biased for asparagine, an amino acid associated with the CDRs of DNA binding Abs. Together, these results suggest that high VH10 gene use is the result of B cell receptor-mediated selection and may involve DNA and/or ligands that share antigenic features with DNA.

The cell-mediated response to schistosomal antigens at the clonal level: development and characterization of a panel of egg antigen-specific murine T cell clones.

The cellular basis of the immune response underlying the granulomatous hypersensitivity in experimental murine schistosomiasis caused by Schistosoma mansoni was investigated by examining a panel of 16 egg antigen-specific T cell clones. The clones were derived from a sensitized T cell line by limiting dilution, and were selected on the basis of their strong responses against schistosomal egg antigens. By cytofluorographic analysis, it was determined that all clones were T helper cells and expressed the CD3+CD4+CD8- phenotype. Lymphokine analysis revealed that some clones secreted either interleukin (IL)-2 or IL-4, but a surprisingly large number were double producers. Southern blot analysis verified the clonality of these T cells and indicated that the clones examined included at least five independent clones by the criterion of T cell receptor beta gene rearrangements. Despite their diversity, the clones responded strongly, and virtually exclusively, to egg antigen components with isoelectric points in the limited range of 4.7 to 5.2. The relevant antigenic egg molecules were shown to require processing by accessory cells for presentation to, and stimulation of, the T cell clones.