Ig gene rearrangement and expression in the progeny of B-cell progenitors in the course of clonal expansion in bone marrow cultures.

In cultures of murine bone marrow cells colonies of 10(3)-10(4) cells were identified which consisted to a large part of pre-B and B cells. Cell mixing experiments with genetically marked cells indicated that each colony is derived from a single progenitor cell not yet committed to the expression of either IgH locus. A concanavalin-A-mediated electrofusion method allowed us to rescue and amplify individual cells from a given colony by hybridization with X63.Ag8.653 cells. The molecular analysis of 12 such hybridomas revealed that all IgH loci were rearranged into DJH or productive or nonproductive VHDJH complexes. Most kappa and all lambda light chain loci were in germline configuration. Kappa chain expression was only seen in heavy (mu) chain expressing hybridomas. Hybridomas from a given colony were heterogeneous in terms of DJH and VHDJH rearrangements and in no cell was more than one productive VHDJH complex detected. None of the productive VHDJH complexes contained a VH gene of group 1 (J558), the largest VH gene family with about half of the VH genes. This is in marked contrast to VH gene usage in splenic B cells.     

Frequency and characterization of phenotypic Ig heavy chain allelically included IgM-expressing B cells in mice

Ig H chain (IgH) allelic exclusion remains a puzzling topic. Here, we address the following question: Do phenotypic IgH allelically included cells exist in normal mice and, if so, at what frequency? Sorted cells from heterozygous mice were evaluated for the expression of both IgM allotypes by double intracytoplasmic stainings. Dual expressors were found at a frequency of 1 in 104 splenic B cells. These data were confirmed by direct sequencing of IgH-rearranged alleles obtained after single cell (or clone) PCR on dual expressors. Typically, these cells have one rearranged J558 VH whereas, in the other allele, a D-proximal VH gene is used. Interestingly, dual expressors have rearranged IgH alleles with similar CDR3 lengths. These results show that, in contrast to the kappa L chain and the TCR beta-chain, IgH allelic exclusion is the result of an extremely stringent mechanism. We discuss two non-mutually exclusive scenarios for the origin of IgH dual expressors: 1) IgH allelically included cells arise when the first allele to rearrange productively is unable to form a pre-BCR; dual expressors could be a subset of this population in which, upon conventional L chain rearrangement, both IgH are expressed at the surface; and 2) synchronous rearrangement of the IgH alleles.     

Predominance of nonproductive rearrangements of VH81X gene segments evidences a dependence of B cell clonal maturation on the structure of nascent H chains

Ig H chain V regions using the VH81X gene segment were PCR amplified from genomic DNA obtained from either splenic B cells or surface (s)Ig- bone marrow cells of BALB/c mice. Sequence analysis demonstrated that 93% of VH81X containing H chain V region genes in splenic B cells were rearranged nonproductively. Furthermore, 74% of rearrangements of VH81X among sIg- bone marrow cells were nonproductive. This contrasts with previous results obtained for rearrangements of members of the VH36-60 gene segment family among sIg- cells wherein, as a consequence of extensive clonal expansion after productive H chain V gene rearrangement, 80% of rearrangements were productive. The low proportion of productive rearrangements of VH81X is interpreted as indicating that most productive rearrangements of VH81X cannot facilitate clonal expansion, which would support the hypothesis that selection for clonal expansion and maturation is dependent on the amino acid sequence of nascent H chains. Additionally, because most productive rearrangements of VH81X cannot facilitate clonal maturation but do appear to mediate allelic exclusion, these processes are likely to be regulated independently.     

Simultaneously arising Ly-1(CD5) B cell lymphomas have identical expressed IgH and kappa-genes but different nonproductive heavy chain rearrangements.

A group of CD5(Ly-1) B cell lymphomas are described. They were derived from mice which received a common pool of syngeneic mouse spleen cells. Southern blot analysis revealed that the lymphomas exhibited an unusual set of Ig gene rearrangements. Six lymphomas analyzed had either of two rearrangement patterns. EcoRI restriction digests of tumor DNA probed for rearrangements in the JH region, resulted in restriction fragments of 4.7 and 5.6 kb or of 4.7 and 8.5 kb. Each had an identical HindIII restriction fragment identified when probed for kappa gene rearrangements. Inasmuch as several B cell lymphomas from mice receiving a common pool of spleen cells had identical kappa-rearrangements and one identical IgH rearrangement, it was important to determine the DNA sequence of expressed IgH and kappa-genes. Each tumor was found to have identical nucleotide sequences of VH-DH-JH and VK-JK. The nonproductive IgH rearrangements each consisted of incomplete DH-JH rearrangements. The 8.5-kb EcoRI fragment was generated from a DFL16 gene segment rearranged into JH3, and the 5.6-kb fragment was generated from DQ52 rearranged into JH)1. We conclude that these Ly-1 B tumors are most likely derived from a single clone of cells which underwent a secondary rearrangement on the nonproductive allele after kappa-rearrangement had occurred. The alternate possibility of independently arising lymphomas with identical expressed VH and VK sequences is discussed.     

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.     

The transition of pre-BI to pre-BII cells is dependent on the VH structure of the mu/surrogate L chain receptor.

We have demonstrated previously that the majority ( > 90%) of VH12 B cells are absent from the adult peripheral repertoire, and that most that remain have the fourth position at the D-J function (designated 10/G4). We report here that most VH 12-expressing pre-B cells are lost during the transition from the pre-BI to the pre-BII cell stage in normal mice, and that pre-BII cell productive (P) rearrangements ar enriched in 10/G4 CDR3. This coincides with the initial expression of H chain and the generation of the mu/surrogate L chain (SL) receptor. In contrast, there is not enrichment for 10/G4 CDR3 in mu MT mice, and the frequency of P rearrangements is as expected from a random rearrangement mechanism, ruling out a biased rearrangement mechanism unique to VH12. We have also demonstrated that non-10/G4 mu chains can associate with SL and be expressed on the cell surface, suggesting that they are available on the cell surface for selection. Thus, transition of pre-BI to pre-BII cells is dependent on the structure of the VH domain.     

Developmental changes in the human heavy chain CDR3

The CDR3 of the Ig H chain (CDR3(H)) is significantly different in fetal and adult repertoires. To understand the mechanisms involved in the developmental changes in the CDR3(H) of Ig H chains, sets of nonproductive V(H)DJ(H) rearrangements obtained from fetal, full-term neonates and adult single B cells were analyzed and compared with the corresponding productive repertoires. Analysis of the nonproductive repertoires was particularly informative in assessing developmental changes in the molecular mechanisms of V(H)DJ(H) recombination because these rearrangements did not encode a protein and therefore their distribution was not affected by selection. Although a number of differences were noted, the major reasons that fetal B cells expressed Ig H chains with shorter CDR3(H) were both diminished TdT activity in the DJ(H) junction and the preferential use of the short J(H) proximal D segment D7-27. The enhanced usage of D7-27 by fetal B cells appeared to relate to its position in the locus rather than its short length. The CDR3(H) progressively acquired a more adult phenotype during ontogeny. In fetal B cells, there was decreased recurrent DJ(H) rearrangements before V(H)-DJ(H) rearrangement and increased usage of junctional microhomologies both of which also converted to the adult pattern during ontogeny. Overall, these results indicate that the decreased length and complexity of the CDR3(H) of fetal B cells primarily reflect limited enzymatic modifications of the joins as well as a tendency to use proximal D and J(H) segments during DJ(H) rearrangements.     

Selection of individual VH genes occurs at the pro-B to pre-B cell transition

B cells are subjected to selection at multiple checkpoints during their development. The selection of Ab H chains is difficult to study because of the large diversity of the CDR3. To study the selection of individual Ab H chain V region genes (V(H)), we performed CDR3 spectratyping of ～ 75-300 rearrangements per individual V(H) in C57BL6/J mice. We measured the fraction of rearrangements that were in-frame in B cell DNA. We demonstrate that individual V(H)s have different fractions of in-frame rearrangements (IF fractions) ranging from 10 to 90% and that these IF fractions are reproducible in different mice. For most V(H)s, the IF fraction in pro-B cells approximated 33% and then shifted to the nearly final (mature) B cell value by the cycling pre-B cell stage. The frequency of high in-frame (IF) V(H) usage increased in cycling pre-B cells compared with that in pro-B cells, whereas this did not occur for low IF V(H)s. The IF fraction did not shift as much in BCR-expressing B cells and was minimally affected by L chain usage for most V(H). High IF clan II/III V(H)s share more positively charged CDR2 sequences, whereas high IF clan I J558 CDR2 sequences are diverse. These data indicate that individual V(H)s are subjected to differential selection, that V(H) IF fraction is mainly established through pre-BCR-mediated selection, that it may operate differently in clan I versus II/III V(H)s, and that it has a lasting influence on the Ab repertoire.     

Repetitive usage of immunoglobulin VH and D gene segments in CD5+ Ly-1 B clones of (NZB x NZW)F1 mice.

The usually small Ly-1 B cell population is markedly increased in older mice by expansion of certain clones. This results in a cellular picture very similar to human B chronic lymphocytic leukemia. Here we report a molecular analysis of the immunoglobulin gene rearrangements of the Ly-1 B cell populations in (NZB x NZW)F1 females. We find that (i) the number of clones found in the peritoneum (a major tissue source of Ly-1 B cells) decreases with age till mono- or biclonality is common by approximately 6 months, (ii) many clones from different mice show the same size rearrangements at both the Ig heavy and light chain loci and (iii) the IgH rearrangements found in a clone isolated from the spleen of one mouse are a subset of those found in the peritoneum of the same mouse, implying migration occurs from the peritoneum to the spleen. Molecular cloning and sequencing of the IgH rearrangements from the peritoneal clones of one B/W mouse revealed that all productive rearrangements used the identical unmutated VH and D elements joined to different JHS. Indeed, two VDJH4 rearrangements were recovered which were identical but for six junctional (N region) nucleotides. The conservation of VH and D segment usage in the rearrangements of these Ly-1 B cell clones could indicate some strong selective pressure for clonal expansion (for example antigen selection) operates via the immunoglobulin molecules of these cells. Southern analyses of other (NZB x NZW)F1 mice with this cloned VH and the usage of the same or similar VH genes among a number of Ly-1 B origin tumors in other mouse strains indicate the generality of this repetitive VH gene usage in individual mice.     

Mice triallelic for the Ig heavy chain locus: implications for VHDJH recombination

V(H)DJ(H) recombination has been extensively studied in mice carrying an Ig heavy chain rearranged transgene. In most models, inhibition of endogenous Ig rearrangement occurs, consistently with the feedback model of IgH recombination. Nonetheless, an incomplete IgH allelic exclusion is a recurrent observation in these animals. Furthermore, transgene expression in ontogeny is likely to start before somatic recombination, thus limiting the use of Ig-transgenic mice to access the dynamics of V(H)DJ(H) recombination. As an alternative approach, we challenged the regulation of somatic recombination with the introduction of an extra IgH locus in germline configuration. This was achieved by reconstitution of RAG2(-/-) mice with fetal liver cells trisomic for chromosome 12 (Ts12). We found that all three alleles can recombine and that the ratio of Ig allotype-expressing B cells follows the allotypic ratio in trisomic cells. Although these cells are able to rearrange the three alleles, the levels of Ig phenotypic allelic exclusion are not altered when compared with euploid cells. Likewise, we find that most VDJ rearrangements of the silenced allele are unable to encode a functional mu-chain, indicating that the majority of these cells are also genetically excluded. These results provide additional support for the feedback model of allelic exclusion.     

A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins.

We have generated transgenic mice that express a diverse repertoire of human sequence immunoglobulins. The expression of this repertoire is directed by light and heavy chain minilocus transgenes comprised of human protein coding sequences in an unrearranged, germ-line configuration. In this paper we describe the construction of these miniloci and the composition of the CDR3 repertoire generated by the transgenic mice. The largest transgene discussed is a heavy chain minilocus that includes human mu and gamma 1 coding sequences together with their respective switch regions. It consists of a single 61 kb DNA fragment propagated in a bacterial plasmid vector. Both human heavy chain classes are expressed in animals that carry the transgene. In light chain transgenic animals the unrearranged minilocus sequences recombine to form VJ joints that use all five human J kappa segments, resulting in a diversity of human-like CDR3 regions. Similarly, in heavy chain transgenics the inserted sequences undergo VDJ joining complete with N region addition to generate a human-like VH CDR3 repertoire. All six human JH segments and at least eight of the ten transgene encoded human D segments are expressed. The transgenic animals described in this paper represent a potential source of human sequence antibodies for in vivo therapeutic applications.     

Induced kappa receptor editing shows no allelic preference in a mouse pre-B cell line

B cell Ag receptor editing is a process that can change kappa antigen recognition specificity of a B cell receptor through secondary gene rearrangements on the same allele. In this study we used a model mouse pre-B cell line (38B9) to examine factors that might affect allelic targeting of secondary rearrangements of the kappa locus. We isolated clones that showed both productive and nonproductive rearrangements of one kappa allele, while retaining the other kappa allele in the germline configuration (kappa(+)/kappa degrees or kappa(-)/kappa degrees ). In the absence of any selective pressures, subsequent rearrangement of the germline alleles occurred at the same frequency as secondary rearrangement of the productive or nonproductive rearranged alleles. Because 38B9 cells lack Ig heavy chains, we stably expressed mu heavy chain protein in 38B9 cells to determine whether heavy-light pairing might affect allelic targeting of secondary kappa rearrangements. Although the expression of heavy chain was found to both pair with and stabilize kappa protein in these cells, it had no effect on preferential targeting Vkappa-Jkappa receptor editing compared with rearrangement of a germline allele. These studies suggest that in the absence of selection to eliminate autoreactive Vkappa-Jkappa genes, there is no allelic preference for secondary rearrangement events in 38B9 cells.     

The ontogeny of diversification at the immunoglobulin heavy chain locus in Xenopus.

Since the larval and adult antibody responses are distinct and restricted in the clawed toad Xenopus, it offers a near ideal model for studying the ontogeny of antibody repertoires and the mechanisms involved. Immunoglobulin heavy chain (IgH) cDNA clones and B cell IgH DNA clones from various larval and adult libraries have been analysed in isogenic Xenopus. Some features are similar in adults and tadpoles, while others differ and explain the particularities observed previously at the protein level. Among the similarities we found are: (i) the mode of rearrangements (there are approximately 50% abortive events in B cells from both stages), (ii) VH family usage (10 of 11 known VH families are expressed proportionally to the number of VH elements per family), and (iii) JH usage (of the eight to nine Xenopus JH elements, two are used in approximately 70% of the VH regions in both stages of development). We found that there is relatively higher membrane exon expression in tadpoles compared with adults; and that most of the differences come from the diversification of CDR3 through DH usage and N diversification. Unlike in mammals, Xenopus DH elements are used with a remarkable flexibility with inversion, fusions and usage in different reading frames, but tadpoles show a strong bias for the usage of only a few DH elements and of a preferred reading frame. There is N diversification, which further increases CDR3 heterogeneity, in adult Xenopus but virtually none in tadpoles. These observations can account for the fact that larval antibody responses are less heterogeneous than those of adults.     

Combination of 3' and 5' IgH regulatory elements mimics the B-specific endogenous expression pattern of IgH genes from pro-B cells to mature B cells in a transgenic mouse model

To ensure the B cell differentiation stage specificity of the intronic Emu element and of the locus control region (LCR) that lies downstream of the IgH chain locus, we generated transgenic mice harboring a V(H) promoter-GFP reporter gene linked to the 3'LCR region and the Emu element. By flow cytometry, GFP(+) lymphocytes were observed amongst pro-B cells (B220(+)CD43(+)CD117(+)) and at all stages of differentiation up to mature B cells (B220(+)IgM(+)IgD(+)). Expression was strictly confined to cells committed to the B lymphocyte lineage as judged by the lack of GFP(+)Thy1,2(+) cells (T lymphocytes) and GFP(+)B220(-)CD117(+)CD43(+) cells (uncommitted lymphohematopoietic progenitors). Therefore, the Emu-GFP-3'LCR transgene is not expressed by hematopoietic stem cells, begins its expression in pro-B cells and is specifically active at all stages of B cell maturation. The combination of 3' and 5' IgH regulatory elements thus appears as a potentially useful cassette in transgenes that require a stringent and early B lineage-specific expression.