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The Journal of Immunology, 2006, 177: 7007-7014.
Copyright © 2006 by The American Association of Immunologists, Inc.

VH Replacement Rescues Progenitor B Cells with Two Nonproductive VDJ Alleles1

Johannes Lutz*, Werner Müller{dagger} and Hans-Martin Jäck2,*

* Division of Molecular Immunology, Department of Internal Medicine III, Nikolaus-Fiebiger-Center of Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany; and {dagger} Department of Experimental Immunology, German Research Centre for Biotechnology, Braunschweig, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Inaccurate VDJ rearrangements generate a large number of progenitor (pro)-B cells with two nonproductive IgH alleles. Such cells lack essential survival signals mediated by surface IgM heavy chain (µH chain) expression and are normally eliminated. However, secondary rearrangements of upstream VH gene segments into assembled VDJ exons have been described in mice transgenic for productive µH chains, a process known as VH replacement. If VH replacement was independent of µH chain signals, it could also modify nonproductive VDJ exons and thus rescue pro-B cells with unsuccessful rearrangements on both alleles. To test this hypothesis, we homologously replaced the JH cluster of a mouse with a nonproductive VDJ exon. Surprisingly, B cell development in IgHVDJ–/VDJ– mice was only slightly impaired and significant numbers of IgM-positive B cells were produced. DNA sequencing confirmed that all VDJ sequences from µH chain-positive B lymphoid cells were generated by VH replacement in a RAG-dependent manner. Another unique feature of our transgenic mice was the presence of IgH chains with unusually long CDR3-H regions. Such IgH chains were functional and only modestly counter-selected, arguing against a strict length constraint for CDR3-H regions. In conclusion, VH replacement can occur in the absence of a µH chain signal and provides a potential rescue mechanism for pro-B cells with two nonproductive IgH alleles.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The generation of exons encoding the variable region of Ig receptors by recombination of V(D)J gene segments is the central mechanism to establish a divers Ab repertoire in mice and humans (reviewed in Ref. 1). V(D)J recombination is initiated by RAG1 and RAG2, which cleave highly conserved recombination signal sequences (RSS)3 adjacent to the V, D, and J segments. These RSS consist of a heptamer, a 12- or 23-bp spacer, and a nonamer sequence. After cleavage, the ends are processed by adding or removing nucleotides and religated by the nonhomologous end-joining repair system.

Rearrangement at the IgH locus starts in early progenitor (pro)-B cells with the generation of a DJ joint by recombining the 3' RSS of a D segment and the 5' RSS of a JH segment. Assembly of the VDJ exon is completed in late pro-B cells by recombining the 3' RSS of a VH segment with the 5' RSS of the DJ joint. If the VDJ exon is productive, an IgM heavy chain (µH chain) is synthesized, which associates with the surrogate L chain and the signaling components Ig{alpha}/Igβ to form a pre-BCR (reviewed in Ref. 2). However, due to nucleotide addition or deletion during recombination, the majority of VDJ joints are out-of-frame, and large numbers of pro-B cells contain nonproductive rearrangements on both IgH alleles (3). In addition, some productive VDJ rearrangements encode for so-called dysfunctional IgH chains unable to pair with either surrogate L chain or conventional IgL chain (4, 5). Both kinds of pro-B cells lack essential differentiation signals mediated by the pre-BCR and should be deleted by apoptosis. But most rearranged VDJ exons still represent a substrate for RAG1/2 as they contain a cryptic RSS (cRSS) consisting only of the conserved heptamer TACTGTG sequence present at the 3' end of most VH segments (6). Therefore, secondary RAG-mediated rearrangements between a classical RSS of an upstream VH segment and the cRSS in the VDJ exon can occur and replace the VH sequence of the VDJ exon by an upstream VH segment (7). This process of VH replacement was first described in murine and human pro-B cell lines (8, 9, 10) and later also found in transgenic mouse strains with a productively rearranged VDJ exon inserted into the JH cluster (6, 11, 12, 13). Although VH replacement has been detected in µH chain-negative pro-B tumor cell lines, in vivo VH replacement has only been observed in mice transgenic for a productive VDJ rearrangement and thus in the presence of a functional pre-BCR and BCR signaling pathway. Hence, it is not clear whether VH replacement in vivo requires a µH chain signal or whether it can also occur in the absence of a pre-BCR signal and might therefore be a mechanism to rescue pro-B cells with two nonproductively rearranged IgH alleles.

To address this question, we established a homozygous IgHVDJ–/VDJ– mouse by homologous replacement of the JH cluster with a nonproductive VDJ exon (VDJ– allele). Analysis of this mouse revealed that pro-B cells are capable of replacing the VH sequence of the VDJ– allele with upstream VH or VHD segments. The resulting pre-BCRs promote B cell development, yielding significant numbers of mature IgM-positive B cells. These data show that VH replacement can occur in vivo in the absence of a µH chain signal and can rescue pro-B cells with nonproductive IgH gene rearrangements.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of transgenic mice

To construct a nonproductive VDJ– allele, the VH17.2.25 exon in pµgpt (14) was mutated using the QuikChange Site-Directed Mutagenesis kit (Stratagene) and the primers ATG AAA TGA AGC TGG GTT ATC TTC TTC CTG GCG GCA GTG G (which mutates codon 3 of the leader peptide to an in-frame stop codon and codon 11 from Met to Ala) and CTT CAA CAT TAA AGA CAC CTA TGC GCA CTG GGT GAA GC (which mutates codon 53 from Met to Ala) and their complementary sequences. Presence of mutations was verified by DNA sequencing. The targeting vector was constructed as follows: first, a 1.6-kb NheI-BamHI fragment containing the DQ52 segment with flanking genomic sequences was isolated from the vector tv1 (11) and cloned into the XbaI and BamHI sites of pBSIIKS+ (Stratagene) followed by the insertion of a ClaI-ApaI lox-neo-lox fragment from pNeoflox8 and a SalI-SpeI linker into the ApaI site. The construction of the targeting vector was completed by inserting the 4.4-kb SalI-SpeI fragment containing the VDJ– allele. The targeting vector was linearized with SacII and SpeI and electroporated into IDG3.2 embryonic stem (ES) cells from (C57BL/6J x 129S6/SvEvTac)F1, which were established and provided by R. Kühn (Forschungszentrum für Umwelt und Gesundheit, München, Germany). Correct targeting was assessed in G418-resistant clones by Southern blotting and chimeric mice were derived by blastocyst injection. Offspring were screened by PCR for the presence of the VDJ– allele with primers VHQM forward TGC TAG ATA CTA TAG GTA CCC TTA CTA TGC and JH4 reverse ATC GGA TAC TGT ATA AAT GCT GTC AC. IgH wild-type configuration was detected with primers 5' of JH1 forward (GAA CAG AGG CAG AAC AGA GAC TGT G) and JH2 reverse (ACC TGA GGA GAC TGT GAG AGT GGT G) (15). Deletion of the neomycin phosphotransferase (neor) cassette was accomplished by crossing VDJ– transgenic mice with EIIa-cre mice (16), provided by M. Wegner (University Erlangen-Nürnberg, Erlangen, Germany) and confirmed by Southern blot analysis. VDJ– mice were bred with C57BL/6 mice obtained from Charles River Breeding Laboratories and were kept in individually ventilated cages under specific pathogen-free conditions at the animal housing of the Nikolaus-Fiebiger-Center following institutional guidelines.

Southern blotting, RT-PCR, and DNA sequencing

For Southern blot analysis, DNA was isolated, digested with appropriate restriction enzymes, gel-purified, and transferred to a nylon membrane as described (14). Filters were hybridized with DNA probes labeled with 32P using the Random Prime kit (Invitrogen Life Technologies). For RT-PCR, total RNA was purified with the RNeasy kit (Qiagen) from sorted cell populations of 10-wk-old mice and reverse transcribed with the SuperScript III RT-PCR kit (Invitrogen Life Technologies) using oligo(dT) primer. VH sequences were amplified with Taq polymerase (Genaxxon) using the VH family specific primers: VH7183 forward TGC GAG GTC GAC CTG GTG GAG TCT GGG (5), J558 leader1 ATG GGA TGG AGC TGG ATC TT (17), and J558 leader3 ATG GAA TGG AGC TGG GTC TT (17), the JH4 exon reverse primer ACG GTG ACT GAG GTT CCT TGA CC and the Cµ1 reverse primer GAA GGA AAT GGT GCT GGG CAG G. PCR products were gel-purified, cloned into the pCR2.1 vector, and sequenced. All VH family specific primers do not bind to the transgene.

Antibodies

Fluorochrome-conjugated mAbs against CD19 (clone 1D3, PerCP), c-kit (clone ack45, PE), CD25 (clone PC61, PE), IgMa (clone DS-1, FITC), and IgMb (clone AF6-78, PE) were purchased from BD Pharmingen, and Cy5-conjugated goat Abs against mouse µH chain are from Southern Biotechnology Associates.

Flow cytometry

Single-cell suspensions were incubated with 0.15 M NH4Cl, 20 mM HEPES to remove erythrocytes. Cells were then membrane-stained with respective Abs for 30 min on ice. For intracellular staining, cells were first fixed and permeabilized using the Fix and Perm kit (An der Grub Biotechnologies). Stained cells were examined in a FACSCalibur (BD Biosciences) and data were analyzed with the CellQuest software (BD Biosciences). Cell sorting was performed on a MoFlo cell sorter (DakoCytomation).

Definition of CDR3-H region

The CDR3-H region was defined as the sequence between, but not including, the conserved cysteine codon TGT (IMGT position 104) at the 3' end of the germline VH segment and the conserved tryptophan codon TGG (IMGT position 118) at the 5' end of the JH4 segment (18).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation of a mouse with two nonproductive VDJ– alleles

To address the question whether VH replacement can occur in vivo in the absence of Ig receptor signals, we generated a mouse in which all pro-B cells carry two nonproductive VDJ– alleles. We first replaced the complete germline JH cluster in the ES cell line IDG3.2 by homologous recombination with a rearranged VDJ exon carrying a translational stop codon at aa position +3 of the leader peptide (VDJ–) (Fig. 1A). The resulting IgH chain gene is nonproductive because the premature translational stop codon allows only the translation of a dipeptide. DNA of 147 G418-resistant ES cell clones was digested with AseI and screened by Southern blot analysis with a DQ52 probe for homologous recombination of the targeting construct at the JH locus. Only one ES clone had the expected 5.8-kb fragment (data not shown). Mice derived from this clone were crossed with mice expressing cre recombinase under the ubiquitously active EIIa promoter (16), which resulted in the removal of the 1.2-kb loxP-flanked neomycin resistance cassette. The deletion was confirmed by the presence of a 4.6-kb band on Southern blots using the DQ52 probe (Fig. 1B). Offspring was genotyped by PCR using primer pairs (Fig. 1A) that detect either a 456-bp fragment of the targeted IgH locus (VDJ–-PCR) (Fig. 1C, left) or a 393-bp fragment of the wild-type JH locus (JH-PCR) (Fig. 1C, right).


Figure 1
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  		FIGURE 1. Generation of mice with nonproductive VDJ– alleles inserted into the JH locus (VDJ– mice). A, Schematic representation of the IgH locus and the targeting strategy. B, Homologous targeting and subsequent deletion of the neomycin phosphotransferase (neor) cassette were detected by Southern blot analysis of AseI-restricted murine DNA using a DQ52 probe. C, Genomic configurations in offspring were verified by PCR analysis of tail DNA using primers indicated in (A) as horizontal arrows.

 
B cell development in bone marrow and spleen of IgHVDJ–/VDJ– animals

If pro-B cells with two nonproductive VDJ– alleles did undergo secondary rearrangements at the IgH locus, we would expect B cell maturation beyond the pro-B cell stage and thus IgM-positive B cells in the bone marrow and the spleen of mice homozygous for the nonproductive VDJ– allele. Indeed, flow cytometry using Abs against markers characteristic for B lymphoid differentiation stages detected B cell populations beyond the CD19/c-kit double-positive pro-B stage in bone marrow (Fig. 2A) and spleen (Fig. 2B). Although the number of pro-B cells was increased about 2-fold in homozygous IgHVDJ–/VDJ– animals when compared with wild-type littermates, the numbers of CD19/CD25 double-positive pre-B and immature B cells were unchanged and mature surface IgM-positive B cells were slightly decreased in the bone marrow (Fig. 2D). Cellularity and number of surface IgM-positive B cells in the spleen were however reduced to 41 and 33%, respectively, of that found in wild-type littermates (Fig. 2D). One explanation for the more profound reduction of B cells in the periphery compared with the bone marrow could be the enrichment of autoreactive specificities in the replaced B cell population. Therefore, the number of B cells in the spleen might be a reflection of the decreased output of mature B cells from the bone marrow and a potential selection against autoreactive specificities. No major changes were seen in the marginal zone and the follicular B cell compartments in the spleen, as well as in the peritoneal B1 population of homozygous IgHVDJ–/VDJ– mice (data not shown).


Figure 2
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  		FIGURE 2. Flow cytometric analysis of B cell maturation in wild-type and transgenic VDJ– mice. Bone marrow (A and C) and spleen cells (B) from 6-wk-old mice were surface (CD19, c-kit, surface IgM) or cytoplasmic (cIgM) stained with the indicated fluorochrome-conjugated abs. Fluorescence intensities (FI) of cells falling into the lymphocyte gate were determined by FACS. Values shown with each plot (right) indicate percentages of cells in marked gates. Results are representative of six experiments with independent litters. D, Numbers of B lymphoid cells in bone marrow (BM) and spleen of six littermates with two animals per genotype.

 
The absence of B cell populations beyond the pro-B cell stage and cytoplasmic µH chain-positive cells in VDJ– transgenic animals on a RAG2–/– background (Fig. 2C) clearly shows that the production of IgM-positive B cells in RAG-proficient IgHVDJ–/VDJ– mice depends on a RAG-mediated modification of the transgene, likely by VH replacement, and excludes RAG-independent mechanisms as a source of productive µH chain genes, as well as synthesis of µH chain protein from the nonproductive transgene.

Sequence analysis of productive µH chain genes

To determine whether productive VDJ rearrangements in µH chain-positive cells from homozygous IgHVDJ–/VDJ– mice were generated by VH replacement of the transgene, we sorted B lymphoid cells of various developmental stages, prepared cDNA and amplified the VDJ exon with a forward primer specific for either the D-proximal VH7183 family or the most distal VHJ558 family and a reverse primer annealing to the Cµ1 exon. All 114 sequences analyzed had replaced the VH segment of the transgenic VDJ– exon with an upstream Ig segment, using the cRSS TACTGTG in the transgenic VDJ– exon and the regular RSS of the upstream Ig gene segment (Fig. 3). The junctions were further diversified by addition of nontemplated and palindromic nucleotides, as well as by removal of bases from the 5' end of the residual VDJ– exon.


Figure 3
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  		FIGURE 3. VH replacement at the VDJ– allele. A, The inserted VDJ– transgene is cleaved 3' of the cryptic heptamer TACTGTG sequence (boldface in B) and joined via a RSS either directly (a) to an upstream VH segment (dark gray) or in two steps (b) to a D segment (light gray) and a VH segment. B, Alignment of the unmodified VDJ– allele with productive VH sequences from IgHVDJ–/VDJ– animals. VDJ rearrangements were amplified by RT-PCR with forward primers specific for VH7183 or VHJ558 family members. In 6 of 114 sequences (a), the transgenic VH region was replaced by an upstream VH segment prototypic for a VH replacement in wild-type mice. However, in the majority of sequences (108 of 114 sequences) (b), the transgenic VH region was replaced by both an upstream VH and D segment. Six representative sequences are shown. In most rearrangements, nontemplated (N) and palindromic (P) nucleotides were added at the junctions and bases were removed 3' of the recombination site. The CDR3-H region between the TGT in VH and the TGG in JH segments is indicated by dotted lines.

 
Productive VDJ sequences could be divided into two groups. The smaller one (6 of 114) replaced the VH segment of the transgenic VDJ exon with an upstream VH segment and represents the VH replacement prototypic for wild-type mice (Fig. 3B, a), indicating that V-to-VDJ replacements are infrequent in our mouse. In contrast, the larger group of sequences (108 of 114) replaced the VH segment with both an upstream VH and D segment (Fig. 3B, b). The latter is possible because in this transgenic mouse, upstream D segments, which are normally deleted during the primary VDJ rearrangement, are still present and available for recombination.

B cell development in the presence of long CDR3-H regions

As most VH replacements of the VDJ– allele contain a second D segment, they should encode for µH chains with CDR3-H regions considerably longer than those found in µH chains encoded by rearrangements of a wild-type IgH allele. To address this question, we compared CDR3-H regions of productive VDJ sequences in splenic B cells from heterozygous IgHVDJ–/wt mice, in which the two IgH alleles can be distinguished by their IgM allotype. In these animals, IgM-positive B cells have either performed VH replacement on the transgenic IgMa allele (replaced VH sequences) or rearranged the wild-type IgMb allele. RNA was isolated from FACS-sorted IgMa- and IgMb-positive splenocytes, amplified by RT-PCR, cloned and sequenced. As developmental changes in CDR3-H length have been analyzed in great detail for VH7183 family members (18), we used a forward primer specific for this D-proximal family. VDJ sequences using the VH7183 family member VH81X were omitted because IgH chains carrying VH81X sequences very often fail to pair with surrogate L chain and conventional L chain (4, 19). Because all replaced sequences contain the JH4 segment of the residual transgene, we used a JH4 reverse primer to amplify wild-type VDJ sequences, so that all compared CDR3-H regions use the same JH segment. The analyzed sequences from IgMa-positive B cells with a replaced V exon contained CDR3-H regions with an average length of 18.0 aas. In contrast IgMb-positive B cells with wild-type rearrangements used CDR3-H regions with an average of 12.5 aas (Fig. 4), a value identical with previous reports for VH7183 sequences in splenocytes from wild-type mice (18).


Figure 4
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  		FIGURE 4. Changes in CDR3-H region length of replaced µH chains during B cell development in VDJ– transgenic mice. RNA was prepared from spleen and bone marrow (BM) B cells sorted for the respective surface markers from animals heterozygous or homozygous for the transgenic VDJ– allele. VDJ sequences were amplified by RT-PCR with primers specific for the VH7183 or VHJ558 family, cloned and sequenced. The length of the CDR3-H regions was determined according to Fig. 3 and plotted for each sequence of the respective cell population. The mean values are indicated as lines. B cells from heterozygous mice were divided into IgMa+ and IgMb+ populations expressing the replaced IgMa or the wild-type IgMb allele, respectively. In the homozygous mice, all cells expressed replaced sequences. The observed decrease in CDR3-H length from the surface µH chain-negative stages to the surface µH chain-positive stages is not significant (p = 0.05).

 
Next, we ask whether long CDR3-H regions are disadvantageous and counter-selected during B cell development. We, therefore, compared expressed VDJ sequences of FACS-sorted pro-B (CD19+ and c-kit+), pre-B (CD19+, CD25+, and surface IgM-negative), and immature B cells (CD19+, CD25+, and surface IgM-positive) from the bone marrow, as well as surface IgM-positive mature B cells from the spleen of homozygous IgHVDJ–/VDJ– animals. This analysis confirmed a considerable increase in the average length of CDR3-H regions in all B cell developmental stages (Fig. 4). Pro-B and pre-B cells contained µH chains with similar CDR3-H lengths of 17.8 and 18.1 aas, respectively. In contrast, the transition from the pre-BCR-positive pre-B to the BCR-positive immature B cell stage was accompanied by a decrease in CDR3-H length to 16.5 aas. No further change in the average CDR3-H length was observed during the transition from the immature B cell stage in the bone marrow to the mature B cell stage in the spleen. The appearance of longer CDR3-H regions was not restricted to rearrangements using VH segments of the VH7183 family, as VDJ rearrangements with VHJ558 family members also showed increased CDR3-H regions with an average CDR3-H length of 15.5 aas in splenic B cells (Fig. 4). Thus, CDR3-H regions longer than 16 aas are only modestly counter-selected, likely through pairing with IgL chains.

Competition between B cells with replaced and wild-type µH chains in heterozygous IgHVDJ–/wt mice

To directly compare the developmental capacity of B cells with longer CDR3-H regions to that of B cells with CDR3-H regions of wild-type length, we used again heterozygous IgHVDJ–/wt mice. As described, cells expressing replaced transgenic IgMa alleles with long CDR3-H regions can be distinguished from cells expressing wild-type IgMb alleles with normal CDR3-H length by their allotype. If BCRs with long CDR3-H regions are disadvantageous, the frequency of B cells expressing the transgenic IgMa allele should strongly decrease in the IgM+ population during transition from the immature to the mature B cell stage. However, we detected only a reduction in the frequency of IgMa-positive B cells by one-third, i.e., from 14.0% of the IgM+ population in the bone marrow to 9.2% in the spleen (Fig. 5). Hence, B cells with CDR3-H regions considerably longer than the average of 11.5 aas found in the VH repertoire (20) developed in competition with B cells using shorter CDR3-H regions, suggesting that this increase in CDR3-H length does not severely affect the functional properties of a µH chain and thereby the signaling capacity of a pre-BCR and a BCR. At the same time, the predominance of IgMb-expressing cells in the bone marrow of heterozygous IgHVDJ–/wt mice shows clearly that VDJ recombination of the endogenous wild-type IgH allele is more efficient than VH replacements of the transgene.


Figure 5
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  		FIGURE 5. Changes in the frequencies of B cells expressing replaced or wild-type µH chains during B cell development in heterozygous VDJ– mice. A, Bone marrow (BM) and spleen cells were surface-stained with Abs distinguishing replaced µH chains with the allotype of the targeted IgH allele (IgMa) and wild-type (wt) µH chains with the allotype of the wild-type allele (IgMb). B, Frequencies of IgMa-positive cells are shown for six heterozygous VDJ– mice from two independent litters. The mean values are represented by a horizontal line.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In vivo VH replacement in pro-B cells in the absence of µH chain expression

In light of the error-prone VDJ rearrangement mechanism, VH replacement might be an important mechanism to rescue not only B cells carrying an autoreactive BCR but also pro-B cells with either nonproductive VDJ rearrangements or productive rearrangements encoding a transport incompetent, i.e., a dysfunctional µH chain (reviewed in Ref. 13). This mechanism, however, would require that VH replacement occurs in pro-B cells in the absence of pre-BCR signals.

Ongoing VH replacement has been discovered in several murine and human pro-B and pre-B cell lines with nonproductive IgH alleles (8, 9, 10), indicating that VH replacements can occur in transformed cell lines in the absence of a µH chain signal. Additional evidence for a µH chain-independent VH replacement comes from mice transgenic for functional µH chain genes. In these mice, VH replacements were detected by PCR as early as in large pro-B cells (Hardy fraction C), most of which normally lack µH chain expression (12). Furthermore, VH replacements often include nontemplated nucleotides added by the TdT, which is normally only expressed at the pro-B cell stage. However, as B cells in these transgenic mouse strains start their development already with a productively rearranged VDJ exon, expression of the transgenic µH chain could precede normal µH chain expression and coincide with pro-B cell markers.

To unequivocally assess the occurrence of VH replacements of nonproductive VDJ– exons in the absence of a µH chain signal, we generated a mouse with a nonproductive VDJ– knockin at the IgH locus similar to the QM mouse (11), which has been used extensively to study VH replacement (11, 21, 22, 23, 24, 25). Analyses of homozygous IgHVDJ–/VDJ– mice clearly demonstrated that pro-B cells with two nonproductive VDJ– rearrangements can use VH replacement in vivo to generate productive VDJ joints and develop into mature B cells. In addition, VH replacements in homozygous IgHVDJ–/VDJ– mice could only be observed in RAG-proficient animals, which is in line with previous reports (22) and verified the requirement of RAG in the VH replacement process. This observation also excluded other DNA recombination events such as activation-induced cytidine deaminase-mediated gene conversion, a mechanism diversifying the Ig repertoire in birds and some mammals (26, 27).

Sequence analysis of 114 productive VDJ exons revealed that all VH replacements had used the cRSS in the VH segment of the VDJ– exon. Similar to VH replacements in other transgenic mouse strains (6, 11, 12), we observed two kinds of productive VH replacements. In six sequences the transgenic VH region was replaced by an upstream VH region, an event prototypic for VH replacements in wild-type mice. However, in the remaining 108 sequences the replacement introduced both an upstream VH and D region, a process that is only possible because IgHVDJ–/VDJ– mice still carry the D cluster upstream of the VDJ– transgene. One explanation for the preferential recombination of the cRSS with upstream D segments, which are deleted in wild-type mice during the primary VDJ recombination, could be the different length of the spacer between the nonamer and heptamer in RSS 3' of D and VH segments, which measure 12- and 23-bp, respectively. Generally, V(D)J recombination occurs preferentially between a RSS with a 12-bp spacer and a RSS with a 23-bp spacer (28). For the cRSS, however, it is unclear whether a spacer is associated with the heptamer. Chen et al. (6) described a potential nonamer motif 12-bp upstream of the cryptic heptamer. But in this case, the 12/23 rule would predict a better compatibility of the putative 12-bp spacer of the cRSS with the 23-bp spacer of a VH-RSS than with the 12-bp spacer of the preferentially used D-RSS. Therefore, it seems likely that VH replacements using additional D segments mainly reflect the shorter distance between the transgene and the D cluster, as well as the ordered chromatin opening at the IgH locus (29, 30). Due to the latter, D segments are already available for recombination at the early pro-B cell stage where D-to-JH rearrangements normally take place, whereas the VH cluster only becomes accessible at the subsequent late pro-B cell stage where VH-to-DJH rearrangements occur. VH replacements of the prearranged, transgenic VDJ using D segments could therefore have already occurred at the early pro-B cell stage. However, sequences with direct V-to-VDJ replacements in our transgenic mouse document that VH replacement can occur after the opening of the VH cluster at the late pro-B cell stage and could potentially rescue pro-B cells with two nonproductive IgH alleles in wild-type animals, even though V-to-VDJ replacement is an infrequent event with a lower efficiency than conventional VDJ recombination. During revision of our report, a study using a similar transgenic knockin mouse also reported V-to-VDJ replacements of nonproductive alleles and verified that these replacements are rare (31).

The notion that VH replacement might be frequently used during B cell development comes from a computational analysis of human Ig sequences. This analysis detected in 5–13% of the DNA sequences potential footprints of VH replacements, which could have occurred at various developmental stages (10). But these data are contrasted by a study that measured only a low recombination frequency on recombination substrates with cRSS (32). Moreover, VH replacement is a rare event in TdT-deficient mice (33). Therefore, the extent of VH replacement at the pro-B cell stage in nontransgenic mice and its role in the fate of pro-B cells with two nonproductive VDJ– exons remain to be elucidated.

Development of B cells with long CDR3-H regions

Interestingly, µH chains formed by VH replacement in IgHVDJ–/VDJ– transgenic mice have on average considerably longer CDR3-H regions than µH chains of wild-type B cells, offering the opportunity to study the implications of long CDR3-H regions on B cell development and Ig receptor functionality.

The CDR3-H region is the major determinant for the Ag specificity of an Ab and comprises the D segment and the adjacent ends of the VH and JH segments. In wild-type mice, VDJ recombination can generate CDR3-H regions of various lengths that increase with the onset of TdT activity in adult B cells (34). Still, the lengths of CDR3-H regions in the peripheral repertoire show only a narrow Gaussian distribution (20). This narrow length distribution with a mean value of 11.5 aas in wild-type mice (20) could have two different reasons. It could merely reflect intrinsic limitations of the VDJ recombination mechanism in mice, like D and JH segment length or TdT activity. However, it could also be the result of µH chain selection for preferred structural or functional properties, as is indicated by an increase in CDR3-H length during adult B cell development (18, 35). Previous studies have shown that the structure of CDR3-H regions does indeed affect the binding of µH chains to surrogate L chain and conventional L chain, and thereby the assembly of signal-competent pre-BCRs and BCRs, respectively (4, 19, 36). Moreover, the structure of a CDR3-H region could also control the abundance of pre-BCRs and BCRs on the cell surface, and thus their signal strength, via its affinity to the endoplasmic reticulum-resident chaperone BiP (K. Herrmann, C. Vettermann, and H.-M. Jäck, unpublished data). Finally, CDR3-H regions could also contribute to autoreactive specificities.

In VDJ– transgenic animals most VH replacements on the VDJ– allele include a second D segment and create VDDJ joints with a CDR3-H length of up to 22 aas. At the same time, CDR3-H regions of wild-type length are generated by direct V-to-VDJ replacements or removal of bases downstream of the opened cRSS. If the observed mean CDR3-H length of 11.5 aas in peripheral B cells of wild-type mice (20) was the result of a selection process, a shift from longer to shorter CDR3-H regions should be evident during B cell development in homozygous IgHVDJ–/VDJ– animals. In contrast, if the observed CDR3-H length distribution was merely a reflection of mechanistic processes and limitations and if CDR3-H regions with increased length were equally functional, the CDR3-H length distribution should be similar in pro-B and mature B cells.

To test these predictions, we focused on VDJ exons using the VH7183 gene segments because changes in CDR3-H lengths of this VH family have been studied in great detail (18). In wild-type mice their CDR3-H length profile peaks at 11.4 and 12.5 aas in pro-B cells and mature B cells, respectively (18), a finding that was confirmed in our study for splenic wild-type VDJ sequences from heterozygous IgHVDJ–/wt mice. This finding is in contrast to homozygous VDJ– animals, where the average CDR3-H length is increased to 17.8 aas in unselected pro-B cells and to 16.5 aas in splenic B cells. Strikingly, the average CDR3-H length of VH-replaced transgenic µH chains was comparable in splenic B cells that had developed in the presence (heterozygous mouse) or absence (homozygous mouse) of competing B cells with CDR3-H regions of wild-type length. Concomitantly, in heterozygous IgHVDJ–/wt mice the frequency of B cells expressing a replaced BCR decreased only by one-third during the transition from the bone marrow to the spleen. B cells with longer CDR3-H regions were also functional in respect to their ability to develop into Ab-secreting plasma cells, as serum IgM, IgA, and IgG1 levels in IgHVDJ–/VDJ– mice were comparable to those detected in wild-type littermates (data not shown). Furthermore, even though long CDR3-H regions are frequently associated with antinuclear specificities and autoreactivity (34, 37, 38), tolerance was not affected, as we could not detect Abs against dsDNA in homozygous 6-mo-old female IgHVDJ–/VDJ– mice by ELISA (data not shown). These findings indicate that the low frequency of B cells expressing µH chains with CDR3-H regions longer than 16 aas in wild-type mice is rather due to the VDJ recombination process than the result of rigorous counter-selection against those B cells or functional inadequacies of longer CDR3-H regions.

In summary, we show that VH replacement can occur in pro-B cells in the absence of an Ig receptor signal, offering a potential rescue mechanism for pro-B cells with two nonproductively rearranged IgH loci. In addition, we provide evidence that the narrow CDR3-H length distribution in wild-type mice is not exclusively due to negative selection against longer CDR3-H regions but rather represents mechanistic constraints during VDJ recombination.


   Acknowledgments
 
We thank Christian Vettermann for many helpful discussions, Dirk Mielenz for critical reading of the manuscript, Edith Roth for excellent technical assistance, and Anke Samuels, Maria Ebel, and Martin Hafner for help in the ES cell culture and for the generation of germline competent chimeric mice. We also thank Ralf Kühn (München, Germany) for the unpublished ES cell line and Michael Wegner (Erlangen, Germany) for EIIa-cre transgenic mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The work was supported in part by the Project Grant SFB466 and the Research Grant JA 968/2 from the Deutsche Forschungsgemeinschaft (to H.-M.J.) and by the German National Genome Network Grant 01GR0439 (to W.M.). Back

2 Address correspondence and reprint requests to Dr. Hans-Martin Jäck, Division of Molecular Immunology, Nikolaus-Fiebiger-Center of Molecular Medicine, Friedrich-Alexander-University of Erlangen-Nürnberg, Glückstrasse 6, D-91054 Erlangen, Germany. E-mail address: hjaeck{at}molmed.uni-erlangen.de Back

3 Abbreviations used in this paper: RSS, recombination signal sequence; cRSS, cryptic RSS; pro, progenitor; µH chain, IgM heavy chain; ES, embryonic stem. Back

Received for publication July 12, 2006. Accepted for publication August 24, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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