Preferential expansion and survival of B lymphocytes based on VH framework 1 and framework 3 expression: "positive" selection in appendix of normal and VH-mutant rabbits.

B cells with a rearranged heavy-chain variable region VHa allotype-encoding VH1 gene segment predominate throughout the life of normal rabbits and appear to be the source of the majority of serum immunoglobulins, which thus bear VHa allotypes. The functional role(s) of these VH framework region (FR) allotypic structures has not been defined. We show here that B cells expressing surface immunoglobulin with VHa2 allotypic specificities are preferentially expanded and positively selected in the appendix of young rabbits. By flow cytometry, a higher proportion of a2+ B cells were progressing through the cell cycle (S/G2/M) compared to a2- B cells, most of which were in the G1/G0 phase of the cell cycle. The majority of appendix B cells in dark zones of germinal centers of normal 6-week-old rabbits were proliferating and very little apoptosis were observed. In contrast, in 6-week-old VH-mutant ali/ali rabbits, little cell proliferation and extensive apoptosis were observed. Nonetheless even in the absence of VH1, B cells with a2-like surface immunoglobulin had developed and expanded in the appendix of 11-week-old mutants. The numbers and tissue localization of B cells undergoing apoptosis then appeared similar to those found in 6-week-old normal appendix. Thus, B cells with immunoglobulin receptors lacking the VHa2 allotypic structures were less likely to undergo clonal expansion and maturation. These data suggest that "positive" selection of B lymphocytes through FR1 and FR3 VHa allotypic structures occurs during their development in the appendix.

VH gene expression and regulation in the mutant Alicia rabbit. Rescue of VHa2 allotype expression.

Rabbits of the Alicia strain, derived from rabbits expressing the VHa2 allotype, have a mutation in the H chain locus that has a cis effect upon the expression of VHa2 and VHa- genes. A small deletion at the most J-proximal (3') end of the VH locus leads to low expression of all the genes on the entire chromosome in heterozygous ali mutants and altered relative expression of VH genes in homozygotes. To study VH gene expression and regulation, we used the polymerase chain reaction to amplify the VH genes expressed in spleens of young and adult wild-type and mutant Alicia rabbits. The cDNA from reverse transcription of splenic mRNA was amplified and polymerase chain reaction libraries were constructed and screened with oligonucleotides from framework regions 1 and 3, as well as JH. Thirty-three VH-positive clones were sequenced and analyzed. We found that in mutant Alicia rabbits, products of the first functional VH gene (VH4a2), (or VH4a2-like genes) were expressed in 2- to 8-wk-olds. Expression of both the VHx and VHy types of VHa- genes was also elevated but the relative proportions of VHx and VHy, especially VHx, decreased whereas the relative levels of expression of VH4a2 or VH4a2-like genes increased with age. Our results suggest that the appearance of sequences resembling that of the VH1a2, which is deleted in the mutant ali rabbits, could be caused by alterations of the sequences of the rearranged VH4a2 genes by gene conversions and/or rearrangement of upstream VH1a2-like genes later in development.

Identification of enhancer sequences 3' of the rabbit Ig kappa L chain loci.

The rabbit is useful for studies of Ig L chain gene expression because of a great disparity in expression of two isotypic forms of the kappa L chain. Normally, K1 is expressed at high levels and K2 is almost silent; expression of K2 increases in mutant or experimentally allotype-suppressed animals. The reasons for the preferential utilization of the K1 isotype have not been fully elucidated. We were interested in looking for second enhancers 3' of the C kappa genes because the absence of a 3' enhancer in the K2 locus could explain the preferential utilization of the K1 isotype. However, we found a strong region of enhancer activity about 7 kb downstream of the C kappa 2 gene. Sequences in this region are highly conserved between rabbit, man, and mouse. There also appears to be a homologous 3' enhancer region in the rabbit K1 locus. We also confirmed earlier reports that the rabbit K1 intron enhancer is inactive in transient transfections into mouse B cells but find that the same construct has low but significant activity in a human B cell line. In a comparable construct the K2 intron enhancer is without activity suggesting possible differential activity of the intronic enhancers.

Mapping of the duplicated rabbit immunoglobulin kappa light chain locus.

The rabbit has two isotypic forms of the immunoglobulin kappa light chain, K1 and K2, which probably arose by duplication. In the normal rabbit, only traces of K2 light chains are produced. However, K2 levels are elevated in allotype-suppressed rabbits and in the Basilea strain which does not produce K1 because of a K1 mRNA splice site mutation. Previous cloning and sequencing showed that each isotype has its own set of J kappa genes but it was not known whether the two isotypes utilize shared or separate sets of V kappa genes. In addition, although genetic linkage of allotypes associated with the K1 and K2 genes has been demonstrated, physical linkage had not been previously demonstrated by overlapping cosmid or phage clones. We used pulsed field and transverse alternating field electrophoresis to obtain megabase maps and to estimate the size of the duplication of the rabbit kappa light chain locus. We found that the two C kappa genes are about 1 megabase apart. One explanation for the poor expression of K2, could be great physical distance from V kappa genes. However, we found that there are V kappa, J kappa and C kappa 2 genes within a approximately 105-kb fragment. Thus, physical distance of V kappa from C kappa 2 may not be the basis for poor K2 expression.

Preferrential rearrangement in normal rabbits of the 3' VHa allotype gene that is deleted in Alicia mutants; somatic hypermutation/conversion may play a major role in generating the heterogeneity of rabbit heavy chain variable region sequences.

The rabbit is unique in having well-defined allotypes in the variable region of the heavy chain. Products of the VHa locus, (with alleles a1, a2, and a3), account for the majority of the serum immunoglobulins. A small percentage of the serum immunoglobulins are a-negative. In 1986, Kelus and Weiss described a mutation that depressed the expression of the Ig VH a2 genes in an a1/a2 rabbit. From this animal the Alicia rabbit strain was developed and the mutation was termed ali. We previously showed, using Southern analysis and the transverse alternating field electrophoresis technique, that the difference between the ali rabbit and normal is a relatively small deletion including some of the most 3' VH genes. The most JH proximal 3' VH1 genes in DNA from normal rabbits of a1, a2 and a3 haplotypes encode a1, a2 and a3 molecules respectively, and it has been suggested that these genes are responsible for allelic inheritance of VHa allotypes. The present study suggests that the 3' end of the VH locus probably plays a key role in regulation of VH gene expression in rabbits because VH gene(s) in this region are the target(s) of preferential VDJ rearrangements. This raises the possibility that mechanisms such as somatic gene conversion and hypermutation are at work to generate the antibody repertoire in this species. Our data support the view that the 3' VH1 gene may be the preferred target for rearrangement in normal rabbits, and for the normal chromosome in heterozygous ali animals. However, homozygous ali rabbits with a deletion that removed the a2-encoding VH1 on both chromosomes do survive, rearrange other VH genes and produce normal levels of immunoglobulins as well as a significant percentage of B cells which bear the a2 allotype. This challenges the view that one VH gene, VH1, is solely responsible for the inheritance pattern of VHa allotypes.

Molecular analysis of recombination sites within the immunoglobulin heavy chain locus of the rabbit.

Previously, recombinations involving genes of the rabbit immunoglobulin heavy chain locus have been documented serologically. These data indicated that the sites at which the causative recombination events occurred could have been anywhere from within the VH gene cluster up to, or 3' of, C mu. Since these sites could not be localized further by serological methods, we attempted to do this using techniques of molecular biology. DNAs from homozygous recombinant rabbits and from the appropriate non-recombinant parental haplotypes were characterized using Southern blots hybridized with a panel of probes derived from cloned regions of the rabbit immunoglobulin heavy chain gene complex. In all three recombinants, the site was downstream of the entire VH cluster and upstream of the JH cluster within an approximately 50 kilobase (kb) region containing expanses of repetitive-sequence DNA as well as DH genes. DH-specific probes further showed that in two of the recombinants, the recombination appears to have occurred within or 5' of DH1 and 5' of DH2 genes; in the third it occurred 3' of the DH2 genes but at least approximately 5 kb 5' of the JH region.

cDNA clones encoding immunoglobulin lambda chains from rabbit expressing the phenotype c7.

A cDNA library derived from spleen cells of an unimmunized rabbit expressing the c7 phenotype of Ig lambda chains (c7+, c21-) was screened with V lambda or C lambda probes of a lambda light chain bearing c21 epitopes. The nucleotide sequences of three hybridizing clones were found to be identical within the V lambda, J lambda and C lambda regions. The V lambda region was 97% similar to that of the functional germ-line gene V lambda 2, and the C lambda region was identical to that of gene C lambda 6, recently identified. Gene C lambda 6 exhibited four codon differences when compared with gene C lambda 5, the latter encoding c21 epitopes. The data presented here and in the accompanying report (Jaton, J.-C. et al., Eur. J. Immunol. 1990, 20:2713) support the view that gene C lambda 6 encodes the C region of c7 lambda chains and that c7 and c21 markers designate two distinct isotypic forms of lambda chains. On the basis of comparative Southern blotting analyses and restriction maps of cloned genomic regions containing V lambda and C lambda genes, a scheme is proposed to account for the c7- and c21- phenotypes.

Chemical and immunochemical identification of the second major rabbit immunoglobulin lambda chain polypeptide bearing c7 epitopes.

The purification of lambda chain-containing IgG fraction from pooled sera of Basilea rabbits, which were bred and selected for the expression of a high level of lambda chains positive for c7 but negative for c21, was carried out. On the basis of specific binding to anti-c7 antiserum, the c7 lambda chain fraction in serum IgG was shown to account for up to 70% of the total immunoglobulin light chains, the remaining 30%, bearing the expected k2 bas isotype. Peptide mapping of the mixed light chains (lambda + k2 bas) followed by microsequencing of the constant region fragments indicated that the C lambda region originated from the cDNA sequence derived from a c7+,c21- Basilea rabbit (i.e. identical to gene C lambda 6), as described in the preceding report (Hayzer, D. J. et al., Eur. J. Immunol. 1990. 20: 2707). In addition, a synthetic peptide encompassing residues 139-159 of the constant region derived from the predicted sequence of gene C lambda 6 was shown to partially inhibit the c7-anti-c7 binding reaction in a sensitive enzyme-linked immunosorbent assay. Taken together, the chemical and immunochemical data clearly demonstrate that gene C lambda 6 indeed encodes c7 epitopes.

Altered phenotypic expression of immunoglobulin heavy-chain variable-region (VH) genes in Alicia rabbits probably reflects a small deletion in the VH genes closest to the joining region.

Rabbits of the Alicia strain have a mutation (ali) that segregates with the immunoglobulin heavy-chain (lgh) locus and has a cis effect upon the expression of heavy-chain variable-region (VH) genes encoding the a2 allotype. In heterozygous a1/ali or a3/ali rabbits, serum immunoglobulins are almost entirely the products of the normal a1 or a3 allele and only traces of a2 immunoglobulin are detectable. Adult homozygous ali/ali rabbits likewise have normal immunoglobulin levels resulting from increased production of a-negative immunoglobulins and some residual ability to produce the a2 allotype. By contrast, the majority of the immunoglobulins of wild-type a2 rabbits are a2-positive and only a small percentage are a-negative. Genomic DNAs from homozygous mutant and wild-type animals were indistinguishable by Southern analyses using a variety of restriction enzyme digests and lgh probes. However, when digests with infrequently cutting enzymes were analyzed by transverse alternating-field electrophoresis, the ali DNA fragments were 10-15 kilobases smaller than the wild type. These fragments hybridized to probes both for VH and for a region of DNA a few kilobases downstream of the VH genes nearest the joining region. We suggest that this relatively small deletion affects a segment containing 3' VH genes with important regulatory functions, the loss of which leads to the ali phenotype. These results, and the fact that the 3' VH genes rearrange early in B-cell development, indicate that the 3' end of the VH locus probably plays a key role in regulation of VH gene expression.

Genetic analyses of restriction fragment length polymorphisms using high molecular weight DNA from sperm or lymphocytes embedded in agarose.

A simple and efficient method for determining restriction fragment length polymorphism types on large numbers of individuals using small samples of peripheral blood or sperm cells is described. Whole cells embedded in low gelling/melting temperature agarose were treated with a series of enzyme, detergent, and washing steps to release high molecular weight DNA that was then digested with standard restriction enzymes such as EcoRI and PstI, electrophoresed, blotted, and probed as in normal Southern analyses. The technique should be readily adaptable to any application requiring DNA from small numbers of cells for Southern analyses or pulsed field gel electrophoresis.

Linked genetic markers of the rabbit kappa light chain are not linked to the Tcr beta chain genes.

In order to investigate linkage, we used serum allotypes of the two rabbit C kappa isotypes and restriction fragment length polymorphisms (RFLPs) of the genes for V kappa, C kappa, and T-cell receptor C beta. The inheritance of these genetic markers was studied through backcross and F2 matings. Southern analysis and hybridization of genomic DNA with a C kappa probe detected a 5 kb Pst I fragment linked to expression of the K2bas1 allotype and the presence of the kappa 1bbas gene and a 6.6 kb Pst I fragment linked to the expression of the K1b9 allotype, the presence of the kappa 2bas2 gene and lack of expression of the K2bas1 allotype. A V kappa probe detected a 1.3 kb Eco RI fragment linked to the presence of the kappa 1bbas gene and expression of the K2bas1 allotype. In contrast, the 9 or 14 kb Eco RI RFLP (C beta a or C beta b) detected with a Tcr beta chain probe segregated independently from C kappa allotypes and RFLPs. It has previously been found that C kappa and C beta are also unlinked in man, whereas in the mouse they are linked at a distance of approximately 8 centimorgans.

Anti-hapten IgG antibodies bound to cell surface hapten: anti-IgG antibody prevents dissociation as measured with fluid phase hapten.

We investigated the dissociation by fluid phase hapten of IgG antibodies bound to cell surface hapten in the presence and absence of anti-IgG antibodies. Dissociation was quantitated with fluid phase hapten, preventing reassociation of the anti-hapten antibodies. More than 90% of the anti-hapten IgG alone was prevented from reassociation by low concentrations of fluid phase hapten (nanogram to microgram range). In contrast, no dissociation of some IgG-anti-IgG complexes could be measured even at 24 hr incubation in the presence of very large excess of fluid phase hapten (100 mg/ml). We excluded aggregate formation between anti-hapten antibodies due to cross-linking by anti-antibodies as a cause for decreased dissociability by 1) performing the experiments in large excess of anti-antibody, 2) showing that the phenomenon was independent of anti-hapten antibody density, 3) showing that decreased dissociation also occurred at 4 degrees C, and 4) showing that aggregation by protein A did induce decreased dissociability, albeit three orders of magnitude lower than the anti-antibody. It was concluded that anti-antibody directly affected the "avidity" of cell hapten bound anti-hapten IgG in an unknown manner.

The structural and genetic basis for expression of normal and latent VHa allotypes of the rabbit.

The immunoglobulin heavy chain variable regions of the rabbit are unusual in having genetically controlled, serologically detectable alternative forms, the VHa allotypes, as well as minor VH allotypes of the x, y and w groups. New insights into the probable structural basis for the VHa allotypes have come from re-examination of earlier protein sequence data in the light of newly deduced protein sequences derived from sequencing cloned cDNAs and genomic DNAs encoding VH regions. Here we review this sequence information, and define the allotype-correlated differences at seven positions in framework region 1 and 10 positions in framework region 3 that may lead to the serologically detectable allotypic determinants (allotopes). Most alternative amino acids at allotype-correlated positions can be derived from each other by single-base changes. Thus somatic mutations and/or gene conversion-like events must be considered along with other serological and genetic explanations for various reported observations of the production of latent VHa allotypes. The proximity of rabbit VH genes (approximately 3 kb apart) might enhance the likelihood of conversion-like events in both germline and somatic cells.

Genetics and expression of kappa-type light chains in Basilea rabbits.

In contrast to rabbits of b4, b5, b6, and b9 allotypes whose serum immunoglobulins (Igs) are predominantly composed of kappa-type light chains, rabbits of the mutant Basilea strain have serum Igs that are largely of lambda type. We prepared several antisera that recognized a minor K2 (bas) light chain that is produced by Basilea rabbits. With these antisera we identified the K2 (bas) isotype in the serum of the original b9/b9 male rabbit whose offspring displayed the Basilea mutant phenotype. It was present in one half of his nonmutant offspring which inherited b9 from him and another b allotype from their mothers. Breeding was conducted both in Basel and at the NIH to develop and maintain colonies of mutant Basilea strain rabbits. The data obtained during colony development confirm that the trait of expression of the bas allotype maps to the same genetic region (b locus) that is known to control the allelic b allotypes b4, b5, b6 and b9. Homozygotes or heterozygotes of b4, b5 or b6 allotype (bb/bb) were mated with homozygous bbas / bbas rabbits to produce F1s , and then F2s as well as progeny of backcrosses to both homozygous parental types (bb/bb and bbas / bbas ) were produced. The bas allotype segregates as an allele (or pseudoallele ) at the b locus although there was a deficiency in recovery of homozygous bas offspring in both the F2 and backcross matings to bbas / bbas parental type in the NIH colony. This selective deficiency may reflect a deleterious effect on survival of homozygous bas progeny.

Expression of kappa chain allotypes at the cell surface and in serum immunoglobulins of normal and allotype-suppressed heterozygous rabbits.

The kappa light chain allotypes b4 and b5 were measured in the serum and on the surfaces of peripheral blood lymphocytes of normal and allotype-suppressed heterozygous rabbits. Surface immunoglobulins (sIg) were detected by fluorescence microscopy and additional quantitative data were obtained by flow microfluorometry. Although a few b5-suppressed animals had no b5 in serum or on cell surfaces for years, most b5-suppressed and all b4-suppressed animals studied had some cells with sIg of the suppressed type by 1 year of age. In suppressed animals the level of serum Ig remained depressed throughout life but cells with sIg appeared in disproportionately large numbers. The effect was particularly striking in those animals suppressed for the b4 type.

A subpopulation of small pre-B cells in rabbit bone marrow expresses kappa light chains and exhibits allelic exclusion of b locus allotypes.

The expression of immunoglobulin heavy and light chains by pre-B cells and B lymphocytes was examined by two-color immunoflourescence in heterozygous b5b9 rabbits. Allelic exclusion of b5 and b9 kappa light chain allotypes was observed for both surface immunoglobulin-negative pre-B cells and surface immunoglobulin-positive B lymphocytes. In newborn bone marrow, pre-B cells and immature B lymphocytes expressing b9 were as numerous as those expressing b5. In contrast, circulating B cells and bone marrow plasma cells expressing the b5 marker outnumber b9+ cells by 2 to 1 in adult b5b9 animals. Whereas most B lymphocytes expressed kappa light chain b allotypes, approximately 80% of the micro heavy chain-positive pre-B cells did not. The pre-B cells that expressed detectable light chains were relatively small lymphocytes. A model is presented which includes a "transitional" pre-B cell that expresses both micro chains and kappa chains.

Immunofluorescence studies on the expression of VH a allotypes by pre-B and B cells of homozygous and heterozygous rabbits.

Fluorochrome-conjugated antibodies specific for C mu determinants and VH a allotypes were used to examine pre-B cells and B lymphocytes for expression of these markers. The majority of mu+ bone marrow pre-B cells were shown to express a allotypic determinants in conjunction with C mu. Both pre-B and B cells from a2 a3 heterozygous rabbits showed allelic exclusion of these allotypes. Pre-B cells expressing the a2 or a3 specificities appeared to be generated in approximately equal numbers in heterozygotes, while B lymphocytes expressing a3 appeared to undergo preferential clonal expansion very early in development. It was also observed that rabbit bone marrow and blood contained a population of myeloid cells which, in a2 a3 heterozygotes, stained for both a2 and a3 determinants. The frequencies of this cell type, which exhibited bright immunofluorescence staining for a allotypes, could not be reduced by prolonged incubation at 37 degrees C but were readily reduced after cell suspensions were treated with low pH buffer. It is concluded that these myeloid cells bear high avidity Fc receptors for serum immunoglobulin and may be the culprits in studies which have found production of two a or b allotypes by individual B lymphocytes.