Mutation pattern of immunoglobulin transgenes is compatible with a model of somatic hypermutation in which targeting of the mutator is linked to the direction of DNA replication.

We have previously demonstrated that B lymphocyte specific somatic mutations are introduced into the variable regions of immunoglobulin kappa transgenes in two independent transgenic mouse lines. The frequency, distribution and nature of these mutations strongly suggest that they arose as a result of the process of somatic hypermutation, which is responsible, in part, for affinity maturation during an immune response. Unexpectedly, in these multiple copy transgenic lines, many of the transgene copies showed no evidence of somatic mutation. This paradox was addressed by determining the sequence of each transgene copy in several B cell hybridomas derived from a mouse line carrying three copies of the kappa transgene. It was found that the somatic hypermutation process in different B cells from the same mouse preferentially targets one, but not the same, transgene copy. We present a model, based on the pattern of this targeting, which links somatic hypermutation to the orientation of the Ig gene relative to the direction of DNA replication.     

Influence of a V kappa 8 L chain transgene on endogenous rearrangements and the immune response to the HA(Sb) determinant on influenza virus

A rearranged murine V kappa 8/J kappa 5 L chain gene that codes for the L chain of most antibodies generated in the primary response of BALB/c mice to the antigenic site, Sb, of the hemagglutinin (HA) molecule of influenza virus A/PR/8/34 (PR8) has been cloned. Three transgenic lines were generated by microinjecting the gene. Lines Ga and L each contain a single copy of the transgene whereas line Gb contains three complete copies. Mice of the Ga lineage showed increased V kappa 8-specific mRNA levels only in spleen, but not in nonlymphoid organs and therefore displayed apparently normal lymphoid-specific regulation of the Ig transgene. B cell hybridomas generated from these mice were analyzed for rearrangements of endogenous V kappa genes. Greater than 90% of the C kappa alleles were retained in germ-line configuration in the Ga line, compared with only 0 to 18% in the L line. Thus, a wide variation in the frequency of endogenous rearrangements is seen among mice of different lineages using the same transgene construct. None of more than 150 hybridomas derived from LPS-stimulated splenic B cells of Ga mice exhibited HA-binding activity although they expressed the transgene and, in most cases, excluded endogenous V kappa rearrangements. In contrast, a large fraction of hybridomas isolated after primary immunization with PR8 were HA(Sb)-specific. This indicated that the transgene was functional but formed HA-specific antibodies with a more restricted set of H chains than previously hypothesized. The primary anti-HA response to immunization with PR8 was diminished in all lines compared with normal mice except for a slightly accelerated but transient burst of anti-HA antibody formation in two out of three lines (Ga and Gb). This early response in G lineage mice was largely specific for HA(Sb) and thus appeared to be composed of transgene-expressing antibodies. No differences in serum titers were observed in the secondary anti-HA responses to booster inoculation with PR8 between transgenic and normal mice.     

Somatic DNA rearrangement generates functional rat immunoglobulin kappa chain genes: the J kappa gene cluster is longer in rat than in mouse

The kappa immunoglobulin (Ig) genes from rat kidney and from rat myeloma cells were cloned and analyzed. In kidney DNA one C kappa species is observed by Southern blotting and cloning in phage vectors; this gene most likely represents the embryonic configuration. In the IR52 myeloma DNA two C kappa species are observed: one in the same configuration seen in kidney and one which has undergone a rearrangement. This somatic rearrangement has brought the expressed V region to within 2.7 kb 5' of the C kappa coding region; the rearrangement site is within the J kappa cluster which we have mapped. The rat somatic Ig rearrangement, therefore, closely resembles that seen in mouse Ig genes. In the rat embryonic fragment two J kappa segments were mapped at 2 and 4.3 kb 5' from the C kappa coding region. Therefore, the rat J kappa cluster extends over about 2.3 kb, a region much longer than the 1.4 kb of the mouse and human J kappa clusters. In the region between C kappa and the expressed J kappa of IR52 myeloma DNA, and XbaI site present in the embryonic kappa gene has been lost. A somatic mutation has therefore occurred in the intervening sequence DNA approx. 0.7 kb 3' from the V/J recombination site. Southern blots of rat kidney DNA hybridized with different rat V kappa probes showed non-overlapping sets of bands which correspond to different subgroups, each composed of 8-10 closely related V kappa genes.     

Ig lambda-producing B cells do not show feedback inhibition of gene rearrangement

In order to study the regulation of expression of Ig lambda genes we have analyzed lambda-producing hybridomas derived from transgenic mice which harbor a functionally rearranged kappa transgene. We also analyzed lambda-producing hybridomas from nontransgenic mice. Surprisingly, all but one of the transgenic lambda-hybridomas co-produce kappa L chains. Also, in contrast to transgenic kappa-hybridomas, most lambda-hybridomas have rearranged endogenous kappa genes despite the presence of transgenic kappa-chains and endogenous H chains. Analysis of spleen cells and hybridomas from nontransgenic mice shows that about 20% of lambda-producing B cells in the spleen co-produce kappa, and a similar proportion of lambda-hybridomas from normal spleens produce both kappa- and lambda-chains. The data argue strongly against the strictly sequential expression of kappa and lambda genes. We present a new model for the regulation of kappa and lambda gene expression, whose key feature is the distinction between a kappa cell lineage in which Ig gene rearrangement is susceptible to feedback by a complete antibody molecule at the pre-B cell stage, and a kappa lambda B cell lineage which does not show feedback inhibition during B cell development.     

Gene conversion-like sequence transfers between transgenic antibody V genes are independent of RAD54

Homology-based Ig gene conversion is a major mechanism for Ab diversification in chickens and the Rad54 DNA repair protein plays an important role in this process. In mice, although gene conversion appears to be rare among endogenous Ig genes, Ab H chain transgenes undergo isotype switching and gene conversion-like sequence transfer processes that also appear to involve homologous recombination or gene conversion. Furthermore, homology-based DNA repair has been suggested to be important for somatic mutation of endogenous mouse Ig genes. To assess the role of Rad54 in these mouse B cell processes, we have analyzed H chain transgene isotype switching, sequence transfer, and somatic hypermutation in mice that lack RAD54. We find that Rad54 is not required for either transgene switching or transgene hypermutation. Furthermore, even transgene sequence transfers that are known to require homology-based recombinations are Rad54 independent. These results indicate that mouse B cells must use factors for promoting homologous recombination that are distinct from the Rad54 proteins important in homology-based chicken Ab gene recombinations. Our findings also suggest that mouse H chain transgene sequence transfers might be more closely related to an error-prone homology-based somatic hypermutational mechanism than to the hyperconversion mechanism that operates in chicken B cells.     

Immunoglobulin kappa light chain gene promoter and enhancer are not responsible for B-cell restricted gene rearrangement.

We have produced transgenic mice which synthesize chimeric mouse-rabbit immunoglobulin (Ig) kappa light chains following in vivo recombination of an injected unrearranged kappa gene. The exogenous gene construct contained a mouse germ-line kappa variable (V kappa) gene segment, the mouse germ-line joining (J kappa) locus including the enhancer, and the rabbit b9 constant (C kappa) region. A high level of V-J recombination of the kappa transgene was observed in spleen of the transgenic mice. Surprisingly, a particularly high degree of variability in the exact site of recombination and the presence of non germ-line encoded nucleotides (N-regions) were found at the V-J junction of the rearranged kappa transgene. Furthermore, unlike endogenous kappa genes, rearrangement of the exogenous gene occurred in T-cells of the transgenic mice. These results show that additional sequences, other than the heptamer-nonamer signal sequences and the promoter and enhancer elements, are required to obtain stage- and lineage- specific regulation of Ig kappa light chain gene rearrangement in vivo.     

A pivotal role for DNase I-sensitive regions 3b and/or 4 in the induction of somatic hypermutation of IgH genes.

Chimeric mice were prepared from embryonic stem cells transfected with IgH genes as transgenes and RAG-2-deficient blastocysts for the purpose of identifying the cis-acting elements responsible for the induction of somatic hypermutation. Among the three transgene constructs used, the V(H) promoter, the rearranged V(H)-D-J(H), an intron enhancer/matrix attachment region, and human Cmu were common to all, but the 3'-untranslated region in each construct was different. After immunization of mice with a T cell-dependent Ag, the distribution and frequency of hypermutation in transgenes were analyzed. The transgene lacking the 3' untranslated region showed a marginal degree of hypermutation. Addition of the 3' enhancer resulted in a slight increase in the number of mutations. However, the transgene containing DNase I-sensitive regions 3b and 4 in addition to the 3' enhancer showed more than a 10-fold increase in hypermutation, reaching levels comparable to those observed in endogenous V(H)186.2 genes of C57BL/6 mice.     

Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.

BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents. MATERIALS AND METHODS: Rearranged V kappa region genes from 10 kappa-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three kappa-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined. RESULTS: All BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged V kappa genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines). CONCLUSIONS: The detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for mu chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher.     

A Postimplantation Lethal Mutation Induced by Transgene Insertion on Mouse Chromosome 8

We have produced three lines of transgenic mice that contain additional copies of the mouse phosphoglycerate kinase 1 (Pgk1) gene. Two of these lines, 94-A and 94-K, which are descendants of a common founder, did not produce liveborn progeny carrying two copies of these transgenes (i.e., A/A, K/K, or A/K). Genotyping of midgestation embryos showed that A/K embryos are dead by Embryonic Day 10. Comparison of the level of transgene expression in the three transgenic lines ruled out PGK1 toxicity as the cause of death of A/A, A/K, and K/K embryos. The death of A/A, K/K, and A/K transgenic mice was therefore attributed to an insertional mutation disrupting a gene or genes essential for normal embryogenesis. Analysis of the structure of the 94-A and 94-K transgenes indicated that they differ in the number of tandem repeats and in the positions of the transgene-cellular DNA junctions. To determine if the two transgenes represent a single integration followed by a rearrangement or two independent integration events, we cloned the endogenous sequences surrounding the 94-A and 94-K transgene insertion sites. Restriction analysis of the isolated genomic clones indicated that the endogenous sequences abutting the 3′ ends of the 94-A and 94-K transgenes are separated by less than 20 kb, providing strong support for the single integration model. Further analysis indicated that the 94-A transgene is associated with a deletion of at least 18 kb and is located in the vicinity of a widely transcribed endogenous gene. Chromosomal mapping of the endogenous sequences flanking the 94-A and 94-K transgene insertions using mouse-hamster somatic cell hybrids and a (C57BL/6J × SPRET/Ei)F1 × SPRET/Ei backcross panel allowed us to assign the 94-A(K) transgene insertion to the subcentral region of mouse chromosome 8.     

Precursors of both conventional and Ly-1 B cells can escape feedback inhibition of Ig gene rearrangement

Experiments with transgenic mice carrying rearranged Ig transgenes have shown that membrane bound Ig molecules cause feedback inhibition of endogenous Ig gene rearrangement. However, this inhibition is never complete. It has been postulated that escape from feedback may be a property of the Ly-1 B cell subset, whereas rearrangement of endogenous Ig genes may be completely inhibited in conventional B cells. This possibility was investigated in transgenic mice carrying a lambda transgene under the control of the H chain enhancer. It was found that kappa producing B cells in these lambda transgenic mice were for the most part, although not exclusively, of the conventional B cell phenotype. Examination of peritoneal exudate cells revealed that a large proportion of Ly-1 B cells also express kappa. Adoptive transfer of bone marrow from adult lambda transgenic mice, a source of conventional B cell precursors, resulted in the production of relatively high levels of serum kappa 2 to 3 mo after transfer into recipient SCID mice. A high proportion of donor B cells in the spleen produced endogenous kappa protein with or without co-production of lambda. It is concluded that precursors of both conventional and Ly-1 B cells can escape feedback inhibition of L chain gene rearrangement.     

In vitro and in vivo expression of a nephritogenic Ig heavy chain determinant: pathogenic autoreactivity requires permissive light chains

Lymphocyte antigen receptors are promising targets for immune intervention strategies in disorders marked by repertoire skewing or expansion of lymphocyte subsets. Appropriate application of immune receptor modulation is predicated on understanding the role of a particular receptor in pathogenesis and disease regulation. The VHB/W16 gene, restricted to mice carrying the j haplotype for the J558 family, is overexpressed by murine lupus anti-DNA Ig. This gene is also expressed recurrently among nephritogenic anti-DNA Ig recovered from several autoimmune strains, suggesting that cells expressing this pathogenic receptor are positively selected during disease progression. To explore the extent and mechanisms by which Ig H chains expressing this gene contribute to autoimmunity, an Ig H chain gene was engineered for in vitro and in vivo recombination studies. Site-directed mutagenesis generated unique restriction sites to link PCR-amplified V region (VDJ) cDNA to previously isolated genomic fragments containing Ig regulatory and signal sequences. The new 3 kb VDJ gene was then ligated to a 9 kb fragment encoding the IgM constant region. Transfection of H chain loss variant myeloma with the complete 12 kb construct, termed 238H-Cmicro, resulted in secretion of intact Ig pairing 238H-Cmicro, with a lambda L chain; however, transfectant Ig lacked autoreactivity and pathogenicity. Introduction of the 238H-Cmicro H chain as a transgene onto the non-autoimmune C57BL/6 background resulted in abundant B cell surface expression of 238H-Cmicro, however, four transgenic Ig recovered by fusion of LPS-stimulated splenocytes and formed by combination of 238H-Cmicro, with endogenous kappa chains do not bind DNA or laminin. These results indicate that the antigen binding sites encoded by this disease-associated gene and/or H chain must associate with permissive L chains to specify autoimmunity. The 238H-Cmicro, transgenic model should prove useful in dissecting the in vivo fate of 238H-Cmicro, L combinations that produce pathogenic autoreactive receptors and in evaluating receptor-targeted interventions.     

Ig V region gene expression in small lymphocytic lymphoma with little or no somatic hypermutation

Using the polymerase chain reaction we examined for specific Ig kappa-L chain V region gene (V kappa gene) rearrangement in small lymphocytic non-Hodgkin's lymphomas that express Ig bearing a major kappa-L chain associated cross-reactive Id, designated 17.109. Previously, we identified the 17.109-cross-reactive Id in chronic lymphocytic leukemia as a serologic marker for expression of a highly conserved V kappa gene, designated Humkv325. Using sense-strand oligonucleotides specific for the 5'-end of this V kappa gene and antisense oligonucleotide specific for a J kappa region consensus sequence, we could amplify specifically Humkv325 when juxtaposed with J kappa through Ig gene rearrangement. This allowed us to amplify rearranged V kappa genes from DNA isolated from minute amounts of lymphoma biopsy material for molecular analyses. Our studies demonstrate that 17.109-reactive SL NHL, with or without associated CLL, rearrange, and presumably express, Humkv325 without substantial somatic diversification. Our data suggest that malignant B cells in SL NHL, in contrast to NHL of follicular center cell origin, may express immunoglobulin variable region genes with little or no somatic hypermutation.     

Lack of detectable somatic hypermutation in the V region of the Ig H chain gene of a human chronic B lymphocytic leukemia

Monoclonal human B cell tumors are a model system for the study of somatic hypermutation of the Ig genes of humans. It was previously shown that a number of B cell lymphomas exhibited striking V region point mutation, hypothesized to result from the somatic hypermutation mechanism. In this study we have extended the analysis to chronic lymphocytic leukemia. We have cloned and sequenced the productive Vh representing five different cells from a monoclonal chronic lymphocytic leukemia. All five Vh sequences were identical. Therefore, the Vh region in this leukemia was not the subject of detectable somatic mutation. These data suggest that chronic lymphocytic leukemia might lack the mechanism for somatic hypermutation and represent a stage of normal B lymphocyte differentiation in which the somatic hypermutation mechanism is not active.     

Hypermutation in Ig V genes from mice deficient in the MLH1 mismatch repair protein

During somatic hypermutation of Ig V genes, mismatched nucleotide substitutions become candidates for removal by the DNA mismatch repair pathway. Previous studies have shown that V genes from mice deficient for the MSH2 and PMS2 mismatch repair proteins have frequencies of mutation that are comparable with those from wild-type (wt) mice; however, the pattern of mutation is altered. Because the absence of MSH2 and PMS2 produced different mutational spectra, we examined the role of another protein involved in mismatch repair, MLH1, on the frequency and pattern of hypermutation. MLH1-deficient mice were immunized with oxazolone Ag, and splenic B cells were analyzed for mutations in their V kappa Ox1 light chain genes. Although the frequency of mutation in MLH1-deficient mice was twofold lower than in wt mice, the pattern of mutation in Mlh1-/- clones was similar to wt clones. These findings suggest that the MLH1 protein has no direct effect on the mutational spectrum.     

A 5' differentially methylated sequence and the 3'-flanking region are necessary for H19 transgene imprinting.

The mouse H19 gene is expressed exclusively from the maternal allele. The imprinted expression of the endogenous gene can be recapitulated in mice by using a 14-kb transgene encompassing 4 kb of 5'-flanking sequence, 8 kb of 3'-flanking sequence, which includes the two endoderm-specific enhancers, and an internally deleted structural gene. We have generated multiple transgenic lines with this 14-kb transgene and found that high-copy-number transgenes most closely follow the imprinted expression of the endogenous gene. To determine which sequences are important for imprinted expression, deletions were introduced into the transgene. Deletion of the 5' region, where a differentially methylated sequence proposed to be important in determining parental-specific expression is located, resulted in transgenes that were expressed and hypomethylated, regardless of parental origin. A 6-kb transgene, which contains most of the differentially methylated sequence but lacks the 8-kb 3' region, was not expressed and also not methylated. These results indicate that expression of either the H19 transgene or a 3' DNA sequence is key to establishing the differential methylation pattern observed at the endogenous locus. Finally, methylation analysis of transgenic sperm DNA from the lines that are not imprinted reveals that the transgenes are not capable of establishing and maintaining the paternal methylation pattern observed for imprinted transgenes and the endogenous paternal allele. Thus, the imprinting of the H19 gene requires a complex set of elements including the region of differential methylation and the 3'-flanking sequence.