A Human Immunoglobulin lambda  Locus Is Similarly Well Expressed in Mice and Humans

By Andrei V. Popov, Xiangang Zou, Jian Xian, Ian C. Nicholson, and Marianne Brüggemann

From the Laboratory of Developmental Immunology, The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom

    Abstract
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

Transgenic mice carrying a 380-kb region of the human immunoglobulin (Ig) lambda  light (L) chain locus in germline configuration were created. The introduced translocus on a yeast artificial chromosome (YAC) accommodates the most proximal Iglambda variable region (V) gene cluster, including 15 Vlambda genes that contribute to >60% of lambda  L chains in humans, all Jlambda -Clambda segments, and the 3' enhancer. HuIglambda YAC mice were bred with animals in which mouse Igkappa production was silenced by gene targeting. In the kappa -/- background, human Iglambda was expressed by ~84% of splenic B cells. A striking result was that human Iglambda was also produced at high levels in mice with normal kappa  locus. Analysis of bone marrow cells showed that human Iglambda and mouse Igkappa were expressed at similar levels throughout B cell development, suggesting that the Iglambda translocus and the endogenous kappa  locus rearrange independently and with equal efficiency at the same developmental stage. This is further supported by the finding that in hybridomas expressing human Iglambda the endogenous L chain loci were in germline configuration. The presence of somatic hypermutation in the human Vlambda genes indicated that the Iglambda -expressing cells function normally. The finding that human lambda  genes can be utilized with similar efficiency in mice and humans implies that L chain expression is critically dependent on the configuration of the locus.

Key words: human Iglambda translocus;  light chain expression levels;  pre-B cell activation;  V gene usage;  hypermutation
    Introduction
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

The light chain component of the Ig protein is encoded by two separate loci, Igkappa and Iglambda . The proportion of antibodies containing kappa  or lambda  L chains varies considerably between different species (1); in mice the kappa /lambda ratio is 95:5, compared with 60:40 in humans. Two models exist to account for the dominance of Igkappa expression in the mouse. From the observations that murine Iglambda -producing myelomas have rearranged kappa  L chain genes, whereas Igkappa -producing cells have the lambda  L chain locus in germline configuration, it was proposed initially that kappa  rearrangement must occur before lambda  rearrangement can begin (4, 5). Although the same observation applies for human B cells, the proportions of kappa - and lambda -producing cells are similar (4), suggesting that other factors are involved. The second proposal is that kappa  and lambda  loci are equally available for rearrangement at the same time, but the mouse kappa  locus is more efficient at engaging the rearrangement process (for review see reference 6). The occasional finding of cells with rearranged lambda  and the kappa  locus in germline configuration may support this (5, 7, 8). Any influence of antigen selection on the biased kappa /lambda ratio is discounted by the finding that the ratio is similar in fetal liver and in cells that have not encountered antigen (9).

L chain V-J rearrangement occurs at the transition from pre-B-II to immature B cells, where the surrogate L chain associated with membrane Igµ is replaced by kappa  or lambda  (14). Although the timing of L chain rearrangement is essentially defined, the processes that activate L chain locus rearrangement are not fully understood. From locus-silencing experiments, it is apparent that kappa  rearrangement is not a prerequisite for lambda  recombination (15), but instead that kappa  and lambda  rearrangements are independent events (16), the activation of which may be affected by differences in the strength of the respective enhancers. Targeted deletion of the kappa  3' enhancer in transgenic mice showed that this region is not essential for kappa  locus rearrangement or expression but is required for establishing the kappa /lambda ratio (17). A region that may regulate the accessibility of the human lambda  locus has been identified ~10 kb downstream of Clambda 7 (18, 19). Functional analysis using reporter gene assays identified a core enhancer region flanked by elements that can drastically reduce enhancer activity in pre-B cells (18). Although transfection studies showed that the kappa  and lambda  3' enhancer regions appear to be functionally equivalent, the core enhancer motifs are flanked by functional sequences that are remarkably dissimilar. The human Iglambda locus on chromosome 22q11.2 is 1.1 Mb in size and typically contains 70 Vlambda genes and 7 Jlambda -Clambda gene segments (20, 21). Approximately half of the Vlambda genes and Jlambda -Clambda 1, 2, 3, and 7 are regarded as functional. The Vlambda genes are organized in three clusters, with the members of a particular V gene family contained within the same cluster. There are 10 Vlambda gene families, with the largest, Vlambda III, having 23 members. In human peripheral blood lymphocytes, V gene segments of families I, II, and III from the J-C proximal cluster A are preferentially rearranged, with the contribution of the 2a2 Vlambda segment (2-14 using a position-based nomenclature; reference 22) being unusually high (23). All lambda  gene segments have the same polarity that allows deletional rearrangement (24). The diversity of the Iglambda repertoire is provided mainly by Vlambda -Jlambda combination. Additional CDR3 diversity due to N (nonencoded)1 or P (palindromic) nucleotide additions at the V to J junction is present in human sequences, although not as extensively as in IgH rearrangement, but is absent in sequences from mice (25), where the TdT (terminal deoxyribonucleotide transferase) activity is downregulated at the time of L chain rearrangement.

Here we have introduced a 410-kb yeast artificial chromosome (YAC), which contains most of the Vlambda genes of cluster A and all the Jlambda -Clambda segments in germline configuration, into mice that have one or both endogenous Igkappa alleles disrupted. The translocus shows high expression in both backgrounds, and is able to compete equally with the endogenous mouse kappa  locus.

    Materials and Methods
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

The HuIglambda YAC, Introduction into Embryonic Stem Cells, and Derivation of Transgenic Mice.

The 410-kb HuIglambda YAC, accommodating a 380-kb region (Vlambda -JClambda ) of the human lambda  L chain locus with V, J, and C genes in germline configuration, was constructed as previously described (29). To allow selection, two copies of the neomycin resistance gene (NEOr) were site-specifically integrated into the ampicillin gene on the left (centromeric) YAC arm. YAC-containing yeast cells were fused with HM-1 embryonic stem (ES) cells (a gift from D. Melton, Department of Pathology, University Medical School, Edinburgh, UK), as previously described (30), and G418-resistant colonies were picked and analyzed 2-3 wk after protoplast fusion. ES cells containing a complete HuIglambda YAC copy, confirmed by Southern hybridization, were used for blastocyst injection to produce chimeric animals (31). Breeding of chimeric animals with BALB/c mice resulted in germline transmission. Further breeding with kappa -/- mice (32) established the lines for analysis.

Southern Blot Analysis.

Conventional DNA was obtained (33) or high molecular weight DNA was prepared in agarose blocks (34). For the preparation of testis DNA, tissues were homogenized and passed through 70 µM nylon mesh. Pulsed-field gel electrophoresis (PFGE) conditions to separate in the 50-900 kb range were 1% agarose, 180 V, 70 s switch time, and 30 h running time at 3.5°C. Hybridization probes were Clambda 2+3 and the left YAC arm probe (LA) comprising LYS2 (29).

Hybridoma Production and ELISA Assay.

Hybridomas were obtained from 3-mo-old HuIglambda YAC/kappa +/- animals by fusion of splenocytes with NS0 myeloma cells (35). After fusion, cells were plated on 96-well plates such as to obtain single clones. Human and mouse antibody production was determined in sandwich ELISA assays (36) on MaxiSorp plates (Nalge Nunc, Denmark). For the detection of human or mouse Iglambda , coating reagents were a 1:500 dilution of anti-human lambda  L chain mAb HP-6054 (L 6522; Sigma Chemical Co.) or a 1:500 dilution of the 2.3 mg/ml rat anti-mouse lambda  mAB (L 2280; Sigma Chemical Co.), respectively. Respective binding was detected with biotinylated antibodies: polyclonal anti-human lambda  (B 0900; Sigma Chemical Co.), a 1:1,000 dilution of polyclonal anti-mouse lambda  (RPN 1178; Amersham International) or rat anti-mouse Iglambda (No. 021172D; PharMingen) followed by streptavidin-conjugated horseradish peroxidase (Amersham International). Mouse IgG2alambda myeloma protein from HOPC1 (M 6034; Sigma Chemical Co.) and human serum IgGlambda (I 4014; Sigma Chemical Co.) were used to standardize the assays. To determine mouse kappa  L chain levels, plates were coated with a 1:1,000 dilution of rat anti-mouse kappa , clone EM34.1 (K 2132; Sigma Chemical Co.), and bound Ig was detected using biotinylated rat mAb anti-mouse Igkappa (Cat. No. 04-6640; Zymed). Mouse myeloma proteins IgG2akappa and IgG1kappa (UPC10, M 9144, and MOPC21, M 9269; Sigma Chemical Co.) were used as standards. For detection of mouse IgM, plates were coated with polyclonal anti-mouse µ (The Binding Site, UK) and bound Ig was detected with biotinylated goat anti-mouse µ (RPN1176; Amersham International) followed by streptavidin-conjugated horseradish peroxidase. Mouse plasmacytoma TEPC183, IgMkappa (M 3795; Sigma Chemical Co.) was used as a standard.

Flow Cytometry Analysis.

Cell suspensions were obtained from bone marrow, spleen, and Peyer's patches (PPs). Multicolor staining was then carried out with the following reagents in combinations (illustrated in Fig. 4): FITC-conjugated anti-human lambda  (F 5266; Sigma Chemical Co.), PE-conjugated anti-mouse c-kit (CD117) receptor (clone ACK45, cat. No. 09995B; PharMingen), PE-conjugated anti-mouse CD25 (IL-2 receptor) (clone 3C7, P 3317; Sigma Chemical Co.), biotin-conjugated anti- human kappa  (clone G20-193, cat. No. 08172D, PharMingen), biotin-conjugated anti-mouse CD19 (clone 1D3, cat. No. 09654D; PharMingen), followed by Streptavidin-Quantum red (S 2899; Sigma Chemical Co.) or Streptavidin-PerCP (cat. No. 340130; Becton Dickinson) and rat monoclonal anti-mouse kappa  L chain (clone MRC-OX-20, cat. MCA152; Serotec, UK) coupled according to the manufacturer's recommendations with allophycocyanin (APC) (PJ25C; ProZyme). Data were collected from 106 stained cells on a FACScalibur® flow cytometer (Becton Dickinson) as previously described (32). Cells were first gated on forward and side scatter to exclude dead cells. To obtain accurate percentage distribution for comparison, cells from normal mice were stained in parallel. In addition, human peripheral blood lymphocytes were purified on Ficoll gradients (1.077 g/ml) and stained with PE-conjugated anti-human CD19 antibody (P 7437, clone SJ25-C1; Sigma Chemical Co.), biotinylated anti-human kappa  followed by Streptavidin-Quantum red, and FITC-conjugated anti-human lambda  antibodies as above.


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Fig. 4.   Flow cytometric analysis of L chain expression during B cell development. (A) Distribution of kappa  and lambda  L chain expression of CD19+ human peripheral lymphocytes and B220+ mouse spleen cells from HuIglambda YAC/Mokappa +/-, HuIglambda YAC/Mokappa +/-, and HuIglambda YAC/Mokappa -/- mice. (B) Mouse Igkappa and human Iglambda L chain expression in CD19+/c-kit+ (top) and CD19+/CD25+ (bottom) HuIglambda YAC/Mokappa +/- bone marrow cells.

For reverse transcriptase (RT)-PCR cloning of Vlambda genes, PP cells were stained with FITC-conjugated peanut agglutinin (PNA) (L 7381; Sigma Chemical Co.) and PE-conjugated anti-mouse B220 antibodies (P 3567; Sigma Chemical Co.). Double-positive cells were sorted on the FACStarPlus flow cytometer (Becton Dickinson) as previously described (32) and 5 × 103 cells were lysed in denaturing solution (37). 5'RACE was carried out as described below with one modification: 2 µg of carrier RNA was added to the cell lysates before RNA extraction and precipitation.

Cloning and Sequencing of 5'RACE Products.

Spleen RNA was prepared as previously described (37) and for cDNA preparation 2-3 µg of RNA was ethanol precipitated and air dried. For rapid amplification of 5' cDNA ends (5'RACE) (38) first strand cDNA was primed with oligo(dT)22, and 100 U of Super Script II reverse transcriptase (GIBCO BRL) was used at 46°C according to the manufacturer's instructions with 20 U of placental RNAse inhibitor (Promega). The DNA/RNA duplex was passed through 1 ml G-50 equilibrated with TE (10 mM Tris-HCl, pH 7.8, and 1 mM EDTA) in a hypodermic syringe to remove excess oligo(dT). For G-tailing, 20 U of TdT (Cambio, UK) was used according to standard protocols (39). Double-stranded cDNA was obtained from G-tailed single-stranded cDNA by addition of oligonucleotide Pr1 (see below), 100 µM dNTP, and 2.5 U of Klenow fragment (Cambio), followed by incubation for 10 min at 40°C. After heating the reaction for 1 min at 94°C and extraction with phenol-chloroform, the double-stranded cDNA was passed through G-50 to remove primer Pr1. PCR amplifications, 35 cycles, were carried out in the RoboCycler Gradient 96 Thermal Cycler (Stratagene) using oligonucleotides Pr2 and Pr3. For PCR of PP cDNA 50 cycles were used: 40 cycles in the first amplification and 10 cycles in additional amplifications. Pfu Thermostable Polymerase (Stratagene) was used instead of Taq polymerase to reduce PCR error rates. The amplification products were purified using a GENECLEAN II kit (BIO 101) and reamplified for five cycles with primers Pr2 and Pr4 to allow cloning into EcoRI sites. Oligonucleotide for 5'RACE of Vlambda genes were: Pr1, 5'-AATTCTAAAACTACAAACTGCCCCCCCCA/T/G-3'; Pr2, 5'-AATTCTAAAACTACAAACTGC-3' (sense); Pr3, 5'-CTCCCGGGTAGAAGTCAC-3' (reverse); and Pr4, 5'-AATTCGTGTGGCCTTGTTGGCT-3' (reverse nested).

Vlambda PCR products of ~500 bp were cut out from agarose gels and purified on GENECLEAN II. The DNA was incubated in 50 mM Tris-HCl, pH 7.4, and 10 mM MgCl2, with 100 µM dGTP/dCTP and 1 U of Klenow fragment for 10 min at room temperature. Under these conditions the Klenow fragment removed the 3' ends of the PCR products (AATT) leaving ligatable EcoRI overhangs. DNA was ligated with EcoRI-restricted pUC19, transformed into competent E. coli XL1Blue, and colonies were selected on X-Gal/IPTG/amp plates. Plasmid DNA prepared from white colonies was used for sequencing. Sequencing of both strands was done on the ABI 373 automated sequencer (Applied Biosystems, Inc.) in the Babraham Institute Microchemical Facility.

    Results
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
The Transgenic Human Iglambda Locus.

The human Iglambda translocus (Fig. 1) was assembled as a YAC by recombining one YAC containing about half of the human Vlambda gene segments with three overlapping cosmids containing Vlambda and Jlambda -Clambda gene segments and the 3' enhancer (29). This produced a 410-kb YAC accommodating a 380-kb region of the human lambda  L chain locus containing 15 Vlambda genes regarded as functional, 3 Vlambda s with open reading frames not found to be expressed, and 13 Vlambda pseudogenes (40). This HuIglambda YAC was introduced into ES cells by protoplast fusion (30) and chimeric mice were produced by blastocyst injection (31). The ES cell clone used for blastocyst injection showed a 450-kb NotI fragment corresponding to HuIglambda YAC, as identified by PFGE and Southern hybridization with probes to the 3' end of the construct, identifying the Clambda 2+3 regions, and to the left centromeric YAC arm at the 5' end, identifying the LYS2 gene (data not shown). Germline transmission was obtained, and PFGE analysis of testis DNA from one animal is illustrated in Fig. 2. A NotI fragment larger than 380 kb is necessary to accommodate this region of the HuIglambda YAC, and the 450-kb band obtained indicates random integration involving the single NotI site 3' of Jlambda -Clambda and a NotI site in the mouse chromosome. Digests with EcoRI/HindIII and hybridization with the Clambda 2+3 probe further confirmed the integrity of the transferred HuIglambda YAC (Fig. 2). The results indicated that one complete copy of the HuIglambda YAC was integrated in the mouse genome.


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Fig. 1.   The HuIglambda YAC accommodates a 380-kb region of the human lambda  L chain locus in authentic configuration with all Vlambda genes of cluster A (21, 22, 40), the Jlambda -Clambda segments, and the 3' enhancer (17). Black boxes represent functional Vlambda genes (3-27, 3-25, 2-23, 3-22, 3-21, 3-19, 2-18, 3-16, 2-14, 2-11, 3-10, 3-9, 2-8, 4-3, and 3-1) and white boxes show Vlambda genes with open reading frames (2-33, 3-32, and 3-12) that have not been identified in productive rearrangements of human lymphocytes (40). Pseudogenes are not shown. Black triangles (black-triangle) indicate V gene use in functionally Iglambda rearrangements (mutated [see Fig. 5] and unmutated) found by RT-PCR in spleen and sorted PP cells from HuIglambda mice. Rearrangement to Jlambda 1 was found in 5 sequences, Jlambda 2 in 18, and Jlambda 3 in 8. The unique NotI restriction site is indicated. Probes to assess the integrity of the HuIglambda YAC, LA (left arm) and Clambda 2+3 are indicated.


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Fig. 2.   Southern blot analysis of HuIglambda YAC integration. (Left) NotI-digested testis DNA resolved on PFGE and hybridized with the Clambda 2+3 probe. The same size band was obtained with the left arm probe (data not shown). The majority of the hybridization signal remains in the compression band (CB) presumably due to protection of the NotI site by methylation. (Right) EcoRI/HindIII digests hybridized with the Clambda 2+3 probe. Lane 1, HuIglambda YAC ES cell DNA from a protoplast fusion clone; lane 2, normal ES cell DNA; lane 3, human genomic DNA (XZ); lane 4, human KB carcinoma (53) DNA; lanes 5 and 6, tail DNA from two HuIglambda YAC germline transmission mice. Note that the human DNA shows an additional 5.2-kb band that represents an allelic variation (54).


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Fig. 5.   Hypermutated human Vlambda sequences from sorted B220+ and PNA+ PP B cells from HuIglambda +YAC/kappa +/- mice. The sequences are a representative selection of the functional Vlambda -Jlambda rearrangements (indicated by the triangles in Fig. 1) isolated from RT-PCR.

Human Iglambda Expression Is Dominant in Mouse kappa -/- Animals.

To assess the human lambda  L chain repertoire for the production of authentic human antibodies, the HuIglambda YAC mice were bred with mice in which endogenous Igkappa production was silenced by gene targeting (32). In these kappa -/- mice, the mouse Iglambda titers are elevated compared with kappa +/+ strains (32, 41). Serum titrations (Fig. 3) showed that human Iglambda antibody titers in HuIglambda YAC/kappa -/- mice are between 1 and 2 mg/ml, which is up to 10-fold higher than average mouse Iglambda levels. Interestingly, in the HuIglambda YAC/kappa -/- mice, the mouse Iglambda production returned to levels similar to that found in normal mice. High numbers of human Iglambda + cells were also identified in flow cytometric analysis of splenic B cells from HuIglambda YAC/kappa -/- mice (Fig. 4 A), with human lambda  expressed on the surface of ~84% of the B cells and mouse Iglambda + expressed on <5% (data not shown).


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Fig. 3.   Human Iglambda , mouse Igkappa , and mouse Iglambda serum titers for HuIglambda YAC/Mokappa +/- and HuIglambda YAC/Mokappa -/- mice (five to six mice per group kept in germfree conditions and five human sera). Antibody levels presented were obtained from 2-3-mo-old animals but the serum titers from older mice were similar. From the five HuIglambda YAC/Mokappa +/- mice tested, three animals had somewhat higher mouse Igkappa titers than human Iglambda , whereas two animals showed higher human Iglambda levels. The controls show L chain distribution in human and normal mouse serum. Total Ig levels are in good agreement with the sum of individual titers (data not shown).

Human Iglambda Expression Equals Mouse Igkappa Production.

Assessment of human Iglambda production in heterozygous HuIglambda YAC+/kappa +/- mice allowed a detailed comparison of expression and activation of endogenous versus transgenic L chain loci present at equal functional numbers. Serum analysis (Fig. 3) of mice capable of expressing both human lambda  and mouse kappa  showed similar titers for human and mouse L chains. Human Iglambda levels in HuIglambda YAC/kappa +/+ transgenic mice were similar to those in HuIglambda YAC/kappa +/- mice. Total Ig levels in HuIglambda YAC+/kappa +/- mice were 1-2 mg/ml, with an average contribution of  ~51% mouse Igkappa , 43% human Iglambda , and 6% mouse Iglambda . As is also seen in human serum, the analysis of individual HuIglambda YAC/kappa +/- animals showed there were variations in the lambda /kappa ratios. Three of the HuIglambda YAC/kappa +/- mice produced somewhat higher kappa  levels, whereas in two mice the human lambda  levels were higher than the Igkappa titers. In HuIglambda YAC/kappa +/- mice, high translocus expression was also found in B220+ B cells from different tissues, with 38% of spleen cells expressing human lambda  and 45% expressing mouse kappa  (Fig. 4 A). As illustrated, these values closely resemble the levels in human volunteers with 34% Iglambda + versus 51% Igkappa + in CD19+ peripheral blood lymphocytes. In HuIglambda YAC/ kappa +/+ mice, which carry a wild-type kappa  locus, the levels of Iglambda are ~25% and endogenous kappa  levels are ~60% (Fig. 4 A). It is likely that these differences in expression levels are dependent on the number of active gene loci.

To assess the developmental stage at which the high contribution of the human lambda  translocus becomes established, we examined surface L chain expression by bone marrow cells of HuIglambda YAC/kappa +/- mice. For this, B cell lineage marker CD19 and specific antibodies to human lambda  and mouse kappa  were used in four-color staining with the early B cell markers c-kit (CD117) and CD25. Fig. 4 B shows that surface L chain expression (human lambda  or mouse kappa ) was detectable on a similar small proportion of B cells at each of these stages of development, which suggests that human and mouse L chain rearrangements are simultaneous. The specificity of the staining detecting mouse kappa  and human lambda  on small numbers of early B cells, which has been reported independently (42), was verified by the absence of similar positive cells in the analysis of bone marrow from control mice (data not shown).

DNA Rearrangement and Diversification of a Highly Active Human lambda  Translocus.

To further clarify the potential of the L chain translocus to contribute to the antibody repertoire, we analyzed human lambda  and mouse kappa  L chain production using individual hybridoma clones from HuIglambda YAC/ kappa +/- mice. Results from two fusions suggest that human lambda  and mouse kappa  L chain-producing cells were present in the spleen of HuIglambda YAC/kappa -/+ mice at similar frequencies. Furthermore, in the hybridomas the amounts of human Iglambda (2-20 µg/ml) or mouse Igkappa (4-25 µg/ml) were very similar. To determine whether Igkappa rearrangement precedes Iglambda , as found in mice and humans (4, 5), the configuration of the endogenous Igkappa and the human lambda  translocus were analyzed in these hybridomas. Southern blot hybridization of randomly picked hybridoma clones showed that in 11 human Iglambda expressers, 7 had the mouse kappa  locus in germline configuration, 1 clone had mouse Igkappa rearranged, and 3 clones had the mouse kappa  locus deleted, whereas in 19 mouse Igkappa expressers, all but 2 had the human Iglambda locus in germline configuration. This result suggests that there is no locus activation bias and further emphasizes that the human lambda  translocus performs with similar efficiency as the endogenous kappa  locus.

The capacity of the human lambda  locus to produce a diverse antibody repertoire is further documented by the V-J rearrangement. Sequences were isolated from spleen and PP cells by 5'RACE PCR amplification to avoid bias from specific V gene primers. The use of individual Vlambda genes is indicated by the triangles in Fig. 1, and shows that a substantial proportion of the Vlambda genes on the translocus are being used in productive rearrangements, with Vlambda 3-1 and Vlambda 3-10 being most frequently expressed. In Vlambda -Jlambda rearrangements, Jlambda 2 was used preferentially and Jlambda 3 and 1 were used less frequently, whereas, as expected, Jlambda 4, 5, and 6 were not used as they are adjacent to psi Cs. Extensive variability due to N or P sequence additions, which is found in human but not mouse L chain sequences (25, 27, 28), was not observed. Sequences obtained by RT-PCR from FACS®-sorted PP germinal center B cells (B220+/PNA+) revealed that somatic hypermutation is operative in HuIglambda YAC mice (Fig. 5). We identified 11 unique Vlambda -Jlambda rearrangements with two or more changes in the V region, excluding the CDR3, which may be affected by Vlambda -Jlambda recombination. The majority of mutations lead to amino acid replacements, but there was no preferential distribution in CDR1 and CDR2.

    Discussion
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Materials and Methods
Results
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References

The ratio of lambda  to kappa  L chain expression varies considerably between different species (1, 43, 44), and in mice the low lambda  L chain levels are believed to be a result of inefficient activation of the mouse lambda  locus during B cell differentiation (for review see reference 6). The Iglambda (~40%) to Igkappa (~60%) ratio in humans is more balanced and suggests that both lambda  and kappa  play an equally important role in immune responses. This is supported by the finding that the mouse Vlambda genes are most similar to the less frequently used distal human Vlambda gene families, whereas no genes comparable to the major contributors to the human Vlambda repertoire are present in the mouse locus (40). With the HuIglambda YAC, these Vlambda genes are available, and are able to make a significant contribution to the antibody repertoire. The 410-kb HuIglambda YAC translocus accommodates V gene region cluster A containing at least 15 functional Vlambda genes (see Fig. 1). In humans, cluster A is the main contributor to the lambda  antibody repertoire, with Vlambda 2-14 (2a2) expressed most frequently at 27% in blood lymphocytes (23). We also find expression of Vlambda 2-14 in the translocus mice, but the main contributors to the lambda  L chain repertoire were 3-1 (the Vlambda gene most proximal to the J-C region) and 3-10, both of which are expressed at ~3% in humans. Although the validity of conclusions about the contribution of different genes is dependent on the numbers examined, the overexpression of Vlambda 3-1 (11 sequences) and Vlambda 3-10 (8 sequences) in the 31 sequences obtained may imply that rearrangement or selection preferences are different in mice and humans. Analysis of recombination signal sequences (RSS) in mouse L chains showed that kappa  and lambda  RSS differ significantly, and that those genes with the highest similarity to consensus RSS rearrange most frequently (45). The RSS of Vlambda 3-1 and Vlambda 3-10 show a 100% match with the mouse consensus sequence, which may explain their frequent expression in the translocus mice. In addition, most human Vlambda RSS match the established consensus sequence significantly better than mouse Vlambda s (21, 45).

We found extensive somatic hypermutation of many rearranged human Iglambda sequences, indicating that they are able to participate in normal immune responses. The levels of mutation in B220+/PNA+ PP cells from HuIglambda YAC translocus mice were similar to what has been reported for mouse L chains (46). Rather unexpected was the pattern of somatic hypermutation with similar numbers of silent and replacement point mutations found in the complementarity-determining and framework regions. Somatic hypermutation is usually associated with a higher level of replacement mutations in CDRs and more silent mutations in the framework regions, and the distribution observed here may argue against efficient antigen selection having taken place. Interestingly, however, lambda  L chain sequences obtained from human peripheral blood lymphocytes also showed high numbers of mutations in framework 2 (23). Part of framework 2 lies at the interface of the VL and VH domains and it has been suggested that this region may be important for optimal H and lambda  L chain interaction and, in particular, interaction of the human lambda  L chain and the endogenous mouse H chain (26).

In the mouse, unlike in humans, L chain diversification due to untemplated nucleotide addition is essentially absent, because TdT expression has been downregulated by the stage at which L chain rearrangement takes place (28, 47). This concept is challenged by the observation that mouse L chain rearrangement can occur at the same time as VH to DJH rearrangements (48) or even earlier (42). Our results also show L chain rearrangement at the pre-B cell stage with a similar number of human lambda - or mouse kappa -expressing B cells also expressing c-kit+ or CD25+ (see Fig. 4). Although the cell numbers are small, the results suggest that there is no preferential activation of either the human lambda  translocus or the endogenous kappa  locus. However, despite this early activation, there is no accumulation of N or P nucleotide diversity in the rearranged human lambda  L chains, unlike rearranged lambda  L chains from human peripheral B cells (27). The small number (<1%) of human Iglambda +/mouse Igkappa + double positive spleen and bone marrow cells may indicate that haplotype exclusion at the L chain level is less strictly controlled than is IgH exclusion (49).

In transgenic mice carrying Ig regions in germline configuration on minigene constructs, efficient DNA rearrangement and high antibody expression levels are rarely achieved. Competition with the endogenous locus can be eliminated using Ig knockout strains, in which transgene expression is usually improved (50). Poor transloci expression levels could be a result of the failure of human sequences to work efficiently in the mouse background or, alternatively, of the absence of locus-specific control regions that are more likely to be included on larger transgenic regions (51). Recently we addressed this question in transgenic mice by the introduction of different sized minigene- and YAC-based human kappa  L chain loci (53). The result showed that neither the size of the V gene cluster nor the V gene numbers present were relevant to achieving high translocus expression levels. The YAC-based loci contained downstream regions of the human kappa  locus, and it is possible that the presence of an undefined region with cis-controlled regulatory sequences may have been crucial in determining expressibility and subsequently L chain choice. The HuIglambda YAC contains equivalent regions from the human Iglambda locus, which may promote the use of the translocus in the L chain repertoire. Hybridomas from HuIglambda YAC+/ kappa +/- mice show no evidence for a bias in L chain locus selection during development, as demonstrated by the absence of rearrangement of the nonexpressed locus. This is in contrast with what is seen in Ig expressing mouse and human B cell clones (4, 5), and supports the model that lambda  and kappa  rearrangements are indeed independent (15, 54) and that poor Iglambda expression levels in mice may be the result of inefficient signals acting during recombination (16). A possible signal that initiates L chain recombination has been identified through gene targeting experiments where the 3' kappa  enhancer was deleted (17). In these mice, the kappa /lambda ratio was reduced from 20:1 in normal mice down to 1:1, and the kappa  locus was largely in germline configuration in lambda -expressing cells, as we also see in the HuIglambda YAC+/kappa +/- hybridoma clones. The high level of human Iglambda expression in the HuIglambda YAC+/kappa +/- mice could be due to the strength of the downstream enhancer of the human lambda  locus. An analysis of human L chain enhancer activities identified three synergistic modules at the 3' end of the lambda  locus which constitute a powerful pre-B cell specific enhancer that appears to be stronger than the corresponding kappa  enhancer (55). Analysis of the mouse lambda  3' enhancer suggests the biased kappa /lambda ratio in mice may be a direct result of the differences in locus specific regulation provided by the respective enhancers (19, 56). The results suggest that strength and ability of the human 3' lambda  enhancer to function in the mouse background may be the reason that lambda  and kappa  loci can compete equally at the pre-B cell stage to initiate L chain rearrangement, resulting in the similar levels of human Iglambda and mouse Igkappa seen in the HuIglambda YAC+/kappa +/- mice.

    Footnotes

Address correspondence to Marianne Brüggemann, Laboratory of Developmental Immunology, Department of Development and Genetics, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK. Phone: 44-1223-496-304; Fax: 44-1223-496-030; E-mail: marianne.bruggemann{at}bbsrc.ac.uk

Received for publication 14 December 1998 and in revised form 8 March 1999.

   A.V. Popov and X. Zou contributed equally to this work.

We thank Drs. I. Tomlinson, G. Winter, and O. Ignatovich for access to their database of human Vlambda sequences and helpful discussions. We are grateful to Drs. D. Melton for provision of the HM-1 ES cells, E. Corps for hybridoma production, B. Goyenechea for help with the Southern hybridization, and N. Miller for helping with the flow cytometry.

This work was supported by the Biotechnology and Biological Sciences Research Council and the Babraham Institute.

Abbreviations used in this paper ES, embryonic stem; Hu, human; N, nonencoded; P, palindromic; PFGE, pulsed-field gel electrophoresis; PNA, peanut agglutinin; PP, Peyer's patch; RACE, rapid amplification of cDNA ends; RSS, recombination signal sequence(s); RT, reverse transcriptase; TdT, terminal deoxyribonucleotide transferase; YAC, yeast artificial chromosome.

    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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