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Related Collections Immunobiology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 February 2001, Vol. 97, No. 4, pp. 1001-1008 IMMUNOBIOLOGY Ordered recombination of immunoglobulin light chain genes occurs at the IGK locus but seems less strict at the IGL locus Mirjam van der Burg, Talip Tümkaya, Marjan Boerma, Sandra de Bruin-Versteeg, Anton W. Langerak, and Jacques J. M. van Dongen From the Department of Immunology, Erasmus University Rotterdam/ University Hospital Rotterdam, Rotterdam, The Netherlands.     Abstract Top Abstract Introduction Materials and methods Results Discussion References Regulation of allelic and isotypic exclusion of human immunoglobulin (Ig) light-chain genes was studied in 113 chronic B-cell leukemias as a "single-cell" model that allowed complete analysis of each light chain allele. Our data show that monospecific Ig light chain expression is in about 90% of cases determined by ordered recombination: Ig gene (IGK) rearrangements, followed by IGK deletions and Ig gene (IGL) rearrangements, resulting in the presence of only one functional Ig light chain rearrangement. In about 10% (10 cases), 2 functional Ig light chain rearrangements (IGK/IGL or IGL/IGL, but not IGK/IGK) were identified. This might be explained by the fact that regulation of the ordered recombination process is not fully strict, particularly when the IGL locus is involved. Unfavorable somatic mutations followed by receptor editing might have contributed to this finding. Eight of these 10 cases indeed contained somatic mutations. In cases with 2 functional Ig light chain rearrangements, both alleles were transcribed, but monospecific Ig expression was still maintained. This suggests that in these cases allelelic exclusion is not regulated at the messenger RNA level but either at the level of translation or protein stability or via preferential pairing of Ig light and Ig heavy chains. Nevertheless, ordered rearrangement processes are the main determinant for monospecific Ig light chain expression. (Blood. 2001;97:1001-1008) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References In normal and malignant human B cells, functional expression of immunoglobulin  (Ig) genes (IGK) occurs more frequently than functional expression of Ig genes (IGL), resulting in an Ig/ distribution of approximately 1.4.1 The observed ratio between Ig- and Ig-expressing B cells in mice is significantly higher (by about 10).2 The difference in / ratio between mice and man might be related to differences in the organization of the IGK and IGL loci. Although the human IGK locus is organized like the murine IGK locus, the organization of the IGL loci differs.3,4 Mice only have 3 V and 4 J segments, each followed by a C segment, which are arranged in 2 clusters.5,6 In human beings, about 30 functional V gene segments are located upstream of 4 functional J-C clusters.7,8 Thus, the theoretical combinatorial repertoire of V-J rearrangements in humans is 10-fold higher than in mice, which might explain the difference in / ratio. Two models have been proposed for explaining the relative "overrepresentation" of IGK genes in both species: the ordered model and the stochastic model.9-12 The ordered model proposes that IGK genes rearrange prior to IGL; the stochastic model postulates that, in principle, the 2 types of Ig light chain genes rearrange totally independently but that other factors render IGL gene rearrangements more difficult.13,14 The latter would imply that IGL gene rearrangements can occur in the absence of IGK gene rearrangements and vice versa. Alt and Baltimore first postulated the ordered model of Ig gene rearrangements in mice.15,16 They suggested that the Ig gene rearrangement process starts within the Ig heavy chain gene (IGH) locus and that the rearrangement process is terminated as soon as a productive (in-frame) IgH chain is expressed on a pre-B cell, resulting in allelic exclusion of the IGH locus.16 They also postulated the ordered model for Ig light chain gene rearrangements, starting with IGK gene recombination, only followed by rearrangement in the IGL locus if no functional combination is formed.15 The ordered model assumes a feedback mechanism, which implies that the V(D)J recombinase system is down-regulated upon surface Ig expression. This feedback mechanism explains the establishment of allelic exclusion (expression of one heavy chain and one Ig light chain), including isotypic exclusion (Ig or Ig expression). In mice, monoallelic IGK demethylation ensures the ordered process of IGK rearrangements, thereby establishing allelic exclusion of the IGK alleles.17 By analogy, Engel et al described in their murine hit-and-run model that IGK and IGL are activated for recombination at consecutive developmental stages.3 Also, in humans it is believed that IGK genes rearrange prior to IGL,9,18 which is supported by recent studies in a human immature B-cell line showing that the IGK enhancer, but not the IGL enhancer, is accessible for DNaseI.19 However, it is known that an IGL gene rearrangement can occasionally be present while the IGK genes are in germline configuration.20 Until recently, allelic exclusion was generally regarded to be a safe mechanism that guarantees the expression of a single type of antigen receptor on each lymphocyte. However, during the last few years, several reports have indicated that dual receptor expression might occur in B lymphocytes as well as in T lymphocytes because of ongoing rearrangements after a functional receptor gene has been formed and expressed.21-26 It was found that a single T lymphocyte might express 2 different T-cell receptor (TCR) chains and 2 different TCR chains, indicating that both TCRB alleles as well as both TCRA alleles are functionally rearranged and expressed.24,25 By analogy, Giachino et al demonstrated that in 0.2% to 0.5% of human B lymphocytes dual expression of Ig and Ig occurs.21,22,27 This would imply that dual Ig light chain expression might be even higher owing to dual Ig/Ig and dual Ig/Ig expression, which would theoretically occur in 0.3% to 0.6% and about 0.1%, respectively. Although it will be difficult to prove the presence of dual Ig/Ig and dual Ig/Ig expression by immunophenotyping due to lack of V- and V-specific antibodies, the estimated dual Ig light chain expression might thus even be as high as 1%. Here we have studied human chronic B-cell leukemias (CBLs) for the presence of functional IGK and IGL gene rearrangements. Clonal leukemic proliferations allow a complete and extensive analysis of both IGK and both IGL alleles so that CBL can be regarded as the ideal "single-cell model" of human B cells, in which regulation of allelic and isotypic exclusion of light chain genes can be studied in detail.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Cell samples, immunophenotyping, and DNA and RNA isolation Mononuclear cells (MNCs) were obtained from peripheral blood or bone marrow samples by Ficoll-Paque (density 1.077 g/L; Pharmacia, Uppsala, Sweden) centrifugation from a series of 113 patients with CBL, including 107 B-cell chronic leukemia (B-CLL), 5 B-cell prolymphocytic leukemia, and a single hairy cell leukemia. MNCs were used for detailed immunophenotyping.28,29 In a few cases, lymph node suspensions were used. In all samples the tumor load was at least 75%. DNA and RNA were isolated from MNCs or lymph node cells, as described.30 Complementary DNA was prepared from RNA using either AMV reverse transcriptase (Promega, Madison, WI) or Superscript RT enzyme (Life Technologies, Paisley, UK) according to the manufacturer's instructions. Southern blot analysis Fifteen micrograms of DNA was digested with the appropriate restriction enzymes (Life Technologies, Rockville, MD), separated in 0.7% agarose gels, and transferred by vacuum blotting to Nytran-13N nylon membranes (Schleicher and Schuell, Dassel, Germany).30 The filters were hybridized with 32P-labeled probes. Probes specific for either J (IGKJ5), C (IGKC), and kappa-deleting element (Kde) (IGKDE) (Dako, Carpinteria, CA) were used in combination with BglII and BamHI/HindIII restriction enzyme digests to determine the IGK gene configuration.31 For the IGL alleles, isotype-specific probes (IGLC1D, IGLJ2, IGLC2D, IGLC4D, IGLJ5, IGLJ6, IGLJ7) were used in combination with HindIII, BglII, BamHI/HindIII, and EcoRI/HindIII digests.32 Reverse transciptase-polymerase chain reaction heteroduplex analysis The reverse transciptase-polymerase chain reaction (RT-PCR) mixture of 100 µL contained 0.2 mM deoxyribonucleoside triphosphate (Pharmacia), 13 pmol of each primer, 1 unit AmpliTaq Gold polymerase in Buffer II (Applied Biosystems, Foster City, CA), 1.5 mM MgCl2, and 100 ng genomic DNA or 5 µL complementary DNA (derived from 0.25 µg total RNA). The family-specific V and V primers, the J and J primers, and the C and C exon primers are listed in Table 1. PCR conditions were 10 minutes at 94°C followed by 40 cycles of 1 minute at 94°C, 1 minute at 72°C, and a final extension of 7 minutes at 72°C. PCR products were further analyzed by heteroduplex analysis to determine whether the PCR products were derived from clonal or polyclonal rearrangements.33                                View this table: [in this window] [in a new window]   Table 1. Primers for polymerase chain reaction analysis of IGK and IGL gene rearrangements Sequencing analysis Clonal RT-PCR products were directly sequenced on an ABI 377 fluorescent cycle sequencer (Applied Biosystems) with Dye Terminator mix or Big Dyes (Applied Biosystems) according to the manufacturer's instructions. V, J, V, and J segments were identified using DNAplot software (W. Müller, H-H. Althaus, University of Cologne, Germany) via VBASE and IMGT databases (http://imgt.cnusc.fr:8104).34 Subsequently, the frame of the rearrangement and the mutation status were determined. A case was classified as somatically mutated if the involved V gene segment had less than 98% homology with the most related V gene segment.     Results Top Abstract Introduction Materials and methods Results Discussion References Southern blot analysis of the CBL samples To study the complete and exact configuration of both IGK and both IGL alleles in a cohort of 113 CBLs, detailed Southern blot analysis was performed. In previous studies, the configuration of the IGK alleles (ie, germline, V-J rearrangement, or deletion of J and/or C) and the IGL alleles of most CBLs were determined.31,32 The data of the Ig+ and Ig+ CBLs are summarized in Table 2. In our series of CBL, half of the Ig+ CBLs (25 of 53) had one rearranged IGK allele, and the others had biallelic IGK gene rearrangements (11 of 53) or one rearranged and one deleted IGK allele (17 or 53). A total of 94% (50 of 53) of them showed both IGL genes in germline configuration (Table 2). On the other hand, all 60 Ig+ CBLs had at least one deleted IGK allele and 88.3% (53 of 60) of them even had either both IGK alleles deleted or one deleted IGK allele with the other in germline configuration. This supports the general idea that IGL gene rearrangements are preceded by IGK gene deletions. Table 2 demonstrates the hierarchic order in Ig light chain gene rearrangements: The light chain gene rearrangement process starts at the IGK locus, followed by IGK deletion and subsequent IGL gene rearrangement.                                View this table: [in this window] [in a new window]   Table 2. IGK and IGL gene configurations in 113 chronic B-cell leukemias (53 immunoglobulin + and 60 immunoglobulin +) as determined by Southern blot analysis Because the presence of a rearranged band in Southern blot analysis does not necessarily imply the presence of a V-J joining, no distinction was possible between physiologic V-J rearrangements and other events like translocations. Moreover, Southern blot analysis cannot discriminate between functional and nonfunctional V-J rearrangements. Therefore, CBLs with 2 or more rearranged Ig light chain alleles were studied in more detail by PCR heteroduplex analysis and sequencing. Ig+ CBL with IGL gene rearrangements and Ig+ CBL with IGK gene rearrangements Six percent (3 of 53) of Ig+ CBLs contained IGL gene rearrangements, and 11.7% (7 of 60) of the Ig+ CBLs had IGK gene rearrangements based on Southern blot analysis. The configurations of the IGK and IGL genes of these 10 cases are summarized in Table 3. The IGK and IGL alleles of the 3 Ig+ B-CLL and 7 Ig+ B-CLL were further analyzed by PCR heteroduplex analysis and sequencing to determine the frame of the rearrangements, the involved gene segments, and the mutation status of the V genes.                                View this table: [in this window] [in a new window]   Table 3. Combined Southern blot and sequence data of the immunoglobulin + chronic B-cell leukemia with IGL gene rearrangements and immunoglobulin + chronic B-cell leukemia with IGK gene rearrangements In 2 of the 3 Ig+ CBLs (patients 1 and 3), an in-frame IGL rearrangement was detected at the DNA level. The junctional regions of all in-frame rearrangements were analyzed for the presence of stop codons. In patient 3, a functional transcript of the IGL rearrangement was detected by RT-PCR. Lack of material precluded further analysis in patient 1. In patient 2, only one out-of-frame IGL rearrangement was found. The second rearrangement could not be identified, which might be caused by the inability of the used primers to recognize the involved gene segments or by a chromosomal aberration involving chromosome 22. The 7 Ig+ B-CLL (patients 4-10) had one rearranged and one deleted IGK allele (Table 3). In patients 4, 9, and 10, the Kde was rearranged to the intron RSS (recombination signal sequence), thereby deleting the C exon but retaining the V-J rearrangement on the same allele (VJ-Kde).31 This rearrangement does not give rise to a functional transcript, because of deletion of the C exon. Therefore, in these 4 patients, 2 V-J rearrangements can be detected at the DNA level, but only the IGK rearrangement on the allele without the C deletion can be detected by RT-PCR. In patients 5 to 8, a V-Kde deletion was detected, which resulted from a rearrangement of a V segment to the Kde in which the J and C segments are deleted together with the preexisting V-J rearrangement. In patient 4, only one V-J rearrangement was found. To determine whether this rearrangement was located at the nondeleted or deleted allele, a primer was designed overlapping the junctional region. Long-range PCR analysis using the junction-specific primer and the Kde primer (and the C primer as control) showed that the identified V-J rearrangement was located on the allele with the C deletion. This rearrangement was indeed not found by RT-PCR. The identified V-J rearrangements of patients 5 to 7 were out-of-frame. Patients 8 to 10 had a functional (in-frame) IGK rearrangement on the rearranged allele, as confirmed by detection of V-C transcripts and sequencing. Somatic mutations in the V genes were determined. Patients 1, 2, 3, 4, and 10 were somatically mutated. Three of the 5 patients with 2 in-frame light chain rearrangements (patients 1, 3, and 10) carried somatic mutations either on one or both alleles against 2 of the 5 patients with a single in-frame light chain rearrangement (patients 2 and 4). In patient 3, the functional rearrangement was not found, but the IGL rearrangement was mutated although not expressed on the membrane. In patients 3, 8, 9, and 10 presenting with double in-frame IGK and IGL gene rearrangements at both the DNA and RNA level, flow cytometric analysis of surface membrane Ig light chains was repeated. No dual Ig light chain expression was observed in the 4 patients (Figure 1). The Ig expression of patient 10 was lower than the Ig light chain expression in the other 3 patients (Figure 1D), but no Ig expression was found and, therefore, this CBL was concluded to be monospecific. No remaining cells were available for reanalysis of Ig light chain expression in patient 1, but the original data did not show dual Ig light chain expression in the Ig+ CBLs. View larger version (55K): [in this window] [in a new window]   Figure 1. Flow cytometric immunophenotyping of lymphocytes of 4 B-CLL patients with an in-frame IGK and IGL gene rearrangement. (A) CD19+ B lymphocytes of patient 3 express Ig but not Ig. Patient 8 (B) and patient 9 (C) express Ig on CD19+ B lymphocytes, but no Ig expression is detectable. (D) Patient 10 has low Ig expression, but Ig expression is absent. To further study whether isotypic exclusion is regulated at the translational level or by posttranslational modification, the cytoplasmic Ig expression of the 4 CBLs was analyzed on cytocentrifuge preparations.29 However, the amount of cytoplasmic Ig in CBL was too low to be detectable; even the isotype expressed on the cell surface could not be detected in the cytoplasm by fluorescence microscopy (data not shown). Analysis of CBL with biallelic IGK or biallelic IGL rearrangements In this series of CBL, 19% (10 of 53) of Ig+ CBLs had biallelic IGK gene rearrangements with IGL genes in germline configuration (Table 2), and 25% (15 of 60) of the Ig+ CBLs had biallelic IGL rearrangements with IGK genes deleted or in germline configuration (Table 2). In addition, patient 3 (Ig+ CBL) and patient 4 (Ig+ CBL) had biallelic IGK and IGL gene rearrangements, respectively, in combination with a rearrangement of the other isotype (Table 3). Sufficient cell material for RT-PCR heteroduplex analysis and sequencing was available for 10 of 11 Ig+ CBLs with 2 IGK rearrangements and 14 of 16 Ig+ CBLs with 2 IGL rearrangements. The results are shown in Tables 4 and 5, respectively.                                View this table: [in this window] [in a new window]   Table 4. Chronic B-cell leukemia with potentially functional biallelic V-J rearrangements as determined by Southern blotting                                View this table: [in this window] [in a new window]   Table 5. Chronic B-cell leukemia with potentially functional biallelic V-J rearrangements as determined by Southern blotting In none of the 10 Ig+ CBLs were 2 in-frame IGK rearrangements detected. In patient 3, no in-frame IGK rearrangement could be identified, although the CBL clearly showed Ig positivity. In patient 11, only 1 rearrangement could be detected by PCR, although 2 rearrangements seemed to be present in Southern blot analysis. By karyotyping of the clonal cells of patient 11, only 1 intact chromosome 2 was detected, which might explain why only one V-J rearrangement could be amplified. Apparently, part of the other chromosome 2 with the IGK locus was still present in the genome. Also, in patients 13 and 19, only one rearrangement could be identified, possibly due to primer mismatching or a chromosome aberration. The RT-PCR results showed that out-of-frame IGK rearrangements can be transcribed (3 of 6 cases tested). In 2 of the 6 completely analyzed cases (patients 15 and 16), the V gene segment of the in-frame rearrangement was mutated. Five of the Ig+ CBLs with biallelic IGL gene rearrangements (patients 4, 21, 23, 25, and 26) had 1 in-frame and 1 out-of-frame IGL junctional region (Table 5). In 6 other Ig+ CBLs (patients 20, 22, 24, 28, 29, and 32), 2 in-frame IGL rearrangements were found. The presence of both transcripts was demonstrated by RT-PCR in all 6 patients. All used V segments were functional except for the second allele of patient 24. This V2-5 segment is a pseudogene,35 implying that this patient has only one functional IGL gene rearrangement. In patients 27, 30, and 31, only one in-frame rearrangement could be identified by V-J PCR analysis. Somatic mutations were found in 7 of the 11 completely analyzed cases. Five of them (patients 20, 22, 28, 29, and 32) had 2 in-frame IGL gene rearrangements. Patients 20, 22, and 32 carried mutations on both alleles. The V regions of both IGL rearrangements of patient 26 were mutated, and the out-of-frame rearrangement contained a stop codon in the junctional region, which might be the result of a somatic mutation, although this cannot be proven.     Discussion Top Abstract Introduction Materials and methods Results Discussion References Ordered Ig light chain gene rearrangements The configuration of the IGK and IGL genes was investigated in a series of 113 CBLs (53 Ig+ and 60 Ig+) to study the mechanism of allelic and isotypic exclusion of human Ig light chain genes. CBLs were chosen as a "single-cell" model because large clonal cell populations allow complete analysis of both alleles of the IGK and IGL genes. Our data confirm the hypothesis of the hierarchic order in human Ig light chain gene rearrangements: The gene rearrangement process starts at the IGK locus, followed by IGK deletion and subsequent IGL gene rearrangement (Table 2).12 Because not a single CBL had 1 or 2 rearranged IGL alleles with both IGK genes in germline configuration, the data completely fit with the ordered model. However, the ordered model was not stringent in all cases, because in some cases IGL gene rearrangements apparently had started before both IGK alleles were deleted. Curiously, 3 Ig+ CBL cases had IGL rearrangements: 1 case in the group with biallelic IGK rearrangements and 2 cases in the group with 1 rearranged and 1 deleted IGK allele. One of the latter 2 cases even had biallelic IGL rearrangements. To include these cases in the ordered model, it must be slightly adapted, although the principle of the ordered Ig light chain rearrangement processes is retained: rearrangement of 1 IGK allele (R/G)  further IGK gene rearrangements (R/R)  1 IGK allele deleted (D/R) and occasionally 1 IGL allele rearranged (R/G)  both IGK alleles deleted (D/D) and 1 or 2 IGL rearrangements (R/G or R/R) (Figure 2). In other words, the original ordered model applies to IGK gene rearrangements, but IGL gene recombination processes seem to be less strictly controlled. View larger version (21K): [in this window] [in a new window]   Figure 2. Schematic diagram of Ig light chain protein expression and Ig light chain gene rearrangements according to the ordered model. Ig light chain gene rearrangements start with rearrangement of 1 IGK allele (R/G)  further IGK gene rearrangements (R/R)  1 IGK allele deleted (D/R) and occasionally 1 IGL allele rearranged (R/G)  both IGK alleles deleted (D/D) and 1 or 2 IGL rearrangements (R/G or R/R). Because the ordered rearrangement process is not fully strict, IGK and IGL rearrangements might coexist (in 6% of Ig+ cases and in 12% of Ig+ cases of the presented CBL series). G indicates germline; R, rearrangement; D, deletion. Regulation of isotypic exclusion in cases with 2 in-frame Ig light chain rearrangements of distinct isotype In about 90% of the CBLs (103 of 113), either IGK or IGL rearrangements were present, indicating that ordered Ig light chain gene rearrangements ensured isotypic exclusion in these cases. Therefore, we conclude that monospecific Ig light chain expression is primarily determined by ordered rearrangement processes. Based on a computer simulation model of murine Ig light chain rearrangements, Mehr et al also suggested that allelic exclusion in B cells is maintained if recombination occurs in an ordered rather than a random process.36 Nevertheless, our data show that the ordered rearrangement process of Ig light chain genes is not absolute and that IGK and IGL rearrangements can coexist (10 of 113 cases). In such cases a different level of regulation of monospecific Ig light chain expression (ie, allelic and isotypic exclusion) should be expected. In 5 of these 10 CBLs, both an in-frame IGK and an in-frame IGL rearrangement were detected at the DNA level (Table 6). Four of these 5 cases could be analyzed by RT-PCR, and all 4 showed bitypic functional transcripts (ie, in-frame transcripts without stop codons because of somatic mutations). Therefore, there is no indication for regulation of isotypic exclusion at the level of transcription in these cases. Other possibilities include regulation at the level of translation, at the level of protein stability, or via preferential Ig light chain assembly with the Ig heavy chain. Although we did not find dual Ig/Ig chain expression in our CBL series (Figure 1), it has been described as occurring in normal and malignant human B lymphocytes.21-23,26                                View this table: [in this window] [in a new window]   Table 6. Summary of data of chronic B-cell leukemia with 2 complete immunoglobulin light chain rearrangements, as identified by reverse transcriptase-polymerase chain reaction analysis and sequencing Allelic exclusion in cases with biallelic IGK or biallelic IGL rearrangements The mechanism of allelic exclusion was further studied using 24 CBLs with biallelic IGK or biallelic IGL rearrangements. The allelic exclusion of biallelic IGK rearrangements was solely regulated at the DNA level because only one functional rearrangement was present in all 6 evaluable cases (Table 6). Theoretically, one third of the rearrangements on the second IGK allele in Ig+ CBLs could be in-frame. However, the murine experiments by Mostoslavsky et al showed that undermethylation of an IGK allele is required for and precedes a rearrangement.17 Moreover, they demonstrated that IGK gene demethylation takes place preferentially on only 1 allele in each cell, resulting in differential accessibility of the 2 IGK alleles for the recombinase system. If no productive rearrangement is obtained, a second rearrangement of the same allele involving an upstream V and a downstream J gene segment can take place.17 Alternatively, the second allele is demethylated and rearranged. On top of the methylation-induced differential accessibility of IGK alleles, the feedback mechanism of down-regulation of the V(D)J recombinase system probably plays a role in the maintenance of allelic exclusion. This is in line with the ordered rearrangement model of Ig light chain genes and fits with the IGK gene data of our CBL series. In 11 Ig+ CBLs, 2 V-J rearrangements could be identified. Six of them had 2 in-frame junctions (without stop codons), of which 5 were functional, and in one case a pseudo-V segment was used. So, in 5 cases 2 Ig protein chains may be expressed, but it was not possible to make a distinction between the expression of 1 or 2 Ig chains, because there are no V family-specific antibodies available. These 5 CBLs were all somatically mutated on either 1 allele (2 cases) or both alleles. Possible mechanism explaining regulation of monospecific Ig light chain expression The start of Ig light chain gene recombination seems to be strictly regulated by ordered accessibility of the IGK locus ensuring complete allelic exclusion at the DNA level. Ig+ CBL with 2 functionally rearranged IGK genes did not occur in our CBL series, suggesting that dual Ig/Ig-expressing B cells do not occur or are rare. However, when IGL rearrangements were involved, 2 in-frame (functional) rearrangements and transcripts (IGK/IGL or IGL/IGL) were occasionally detected (Table 6), implying the possibility of dual Ig light chain expression (Ig/Ig or Ig/Ig). Several mechanisms might operate to regulate ordered recombination, including demethylation of one allele, remodeling of chromatin structure, or selective accessibility of recombination machinery through differential presence of transcription factors (such as Rel/nuclear factor-B or E12/E47 bHLH factors).37 Demethylation indeed appeared to be necessary for rearrangement of Ig and TCR genes. In the mouse, monoallelic demethylation has been shown to occur at the IGK locus.17 In contrast to IGK genes, the ordered demethylation process might be less strict for the human IGL genes. The occurrence of somatic mutations and subsequent receptor editing might have contributed to the presence of 2 functional Ig light chain rearrangements in our study. B-CLL can arise either from pre- or post-germinal center B cells.38 In the latter case the V regions carry somatic mutations (about 50% of cases).38 Theoretically, the somatic mutation process can result in unfavorable mutations leading to receptor editing via secondary rearrangements and light chain replacement.39,40 It is not yet clear whether the secondary rearrangements for receptor editing follow an ordered pattern comparable to primary rearrangements. In cases with 2 functional rearrangements, one should expect somatic mutations in the in-frame rearrangement that is not expressed, whereas the expressed in-frame rearrangement can be mutated depending on whether the B cell underwent a second germinal center reaction. Indeed, 8 of 10 CBLs (80%) with 2 functional IGK/IGL or IGL/IGL rearrangements contained somatic mutations, in contrast to 5 of 16 cases (about 30%) of CBL with 2 complete Ig light chain gene rearrangements, one functional and one nonfunctional (Table 6). Nevertheless, this study indicates that isotypic and allelic exclusion is regulated by ordered rearrangement of Ig light chain genes in about 90% of cases. Even in cases with 2 functional Ig light chain transcripts, monospecific Ig expression can still be maintained, probably via mechanisms at the protein level, such as differential protein stability and preferential pairing of Ig chains.     Acknowledgment The authors gratefully acknowledge Dr F. J. T. Staal for critical reading of the manuscript.     Footnotes Submitted March 10, 2000; accepted October 10, 2000. Supported by the Foundation "Vereniging Trustfonds Erasmus Universiteit Rotterdam" in The Netherlands. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: J. J. M. van Dongen, Dept of Immunology, Erasmus University Rotterdam, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.     References Top Abstract Introduction Materials and methods Results Discussion References 1. Langman RE, Cohn M. The proportion of B-cell subsets expressing kappa and lambda light chains changes following antigenic selection. Immunol Today. 1995;16:141-144[CrossRef][Medline] [Order article via Infotrieve]. 2. Ten Boekel E, Melchers F, Rolink A. The status of Ig loci rearrangements in single cells from different stages of B cell development. Int Immunol. 1995;7:1013-1019[Abstract/Free Full Text]. 3. Engel H, Rolink A, Weiss S. B cells are programmed to activate kappa and lambda for rearrangement at consecutive developmental stages. Eur J Immunol. 1999;29:2167-2176[CrossRef][Medline] [Order article via Infotrieve]. 4. Takeda S, Sonoda E, Arakawa H. The kappa:lambda ratio of immature B cells. Immunol Today. 1996;17:200-201[Medline] [Order article via Infotrieve]. 5. Miller J, Ogden S, McMullen M, Andres H, Storb U. The order and orientation of mouse lambda-genes explain lambda-rearrangement patterns. J Immunol. 1988;141:2497-2502[Abstract]. 6. Sanchez P, Marche PN, Le Guern C, Cazenave PA. Structure of a third murine immunoglobulin lambda light chain variable region that is expressed in laboratory mice. Proc Natl Acad Sci U S A. 1987;84:9185-9188[Abstract/Free Full Text]. 7. Williams SC, Frippiat JP, Tomlinson IM, Ignatovich O, Lefranc MP, Winter G. Sequence and evolution of the human germline V lambda repertoire. J Mol Biol. 1996;264:220-232[CrossRef][Medline] [Order article via Infotrieve]. 8. Vasicek TJ, Leder P. Structure and expression of the human immunoglobulin lambda genes. J Exp Med. 1990;172:609-620[Abstract/Free Full Text]. 9. Korsmeyer SJ, Hieter PA, Ravetch JV, Poplack DG, Waldmann TA, Leder P. Developmental hierarchy of immunoglobulin gene rearrangements in human leukemic pre-B-cells. Proc Natl Acad Sci U S A. 1981;78:7096-7100[Abstract/Free Full Text]. 10. Zou YR, Takeda S, Rajewsky K. Gene targeting in the Ig kappa locus: efficient generation of lambda chain-expressing B cells, independent of gene rearrangements in Ig kappa. EMBO J. 1993;12:811-820[Medline] [Order article via Infotrieve]. 11. Gorman JR, van der Stoep N, Monroe R, Cogne M, Davidson L, Alt FW. The Ig(kappa) enhancer influences the ratio of Ig(kappa) versus Ig(lambda) B lymphocytes. Immunity. 1996;5:241-252[CrossRef][Medline] [Order article via Infotrieve]. 12. Gorman JR, Alt FW. Regulation of immunoglobulin light chain isotype expression. Adv Immunol. 1998;69:113-181[Medline] [Order article via Infotrieve]. 13. Nadel B, Cazenave PA, Sanchez P. Murine lambda gene rearrangements: the stochastic model prevails over the ordered model. EMBO J. 1990;9:435-440[Medline] [Order article via Infotrieve]. 14. Arakawa H, Shimizu T, Takeda S. Re-evaluation of the probabilities for productive arrangements on the kappa and lambda loci. Int Immunol. 1996;8:91-99[Abstract/Free Full Text]. 15. Alt FW, Enea V, Bothwell AL, Baltimore D. Activity of multiple light chain genes in murine myeloma cells producing a single, functional light chain. Cell. 1980;21:1-12[CrossRef][Medline] [Order article via Infotrieve]. 16. Alt FW, Yancopoulos GD, Blackwell TK, et al. Ordered rearrangement of immunoglobulin heavy chain variable segments. EMBO J. 1984;3:1209-1219[Medline] [Order article via Infotrieve]. 17. Mostoslavsky R, Singh N, Kirillov A, et al. Kappa chain monoallelic demethylation and the establishment of allelic exclusion. Genes Dev. 1998;12:1801-1811[Abstract/Free Full Text]. 18. Hieter PA, Korsmeyer SJ, Waldmann TA, Leder P. Human immunoglobulin kappa light chain genes are deleted or rearranged in lambda-producing B cells. Nature. 1981;290:368-372[CrossRef][Medline] [Order article via Infotrieve]. 19. Asenbauer H, Combriato G, Klobeck HG. The immunoglobulin lambda light chain enhancer consists of three modules which synergize in activation of transcription. Eur J Immunol. 1999;29:713-724[CrossRef][Medline] [Order article via Infotrieve]. 20. Hauke G, Epplen JT, Chluba J, Hollmann A, Peter HH. IgD/lambda plasmocytoma with immunoglobulin kappa light chain genes in the germ-line configuration. J Clin Immunol. 1988;8:407-413[CrossRef][Medline] [Order article via Infotrieve]. 21. Pauza ME, Rehmann JA, LeBien TW. Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains. J Exp Med. 1993;178:139-149[Abstract/Free Full Text]. 22. Giachino C, Padovan E, Lanzavecchia A. kappa+lambda+ dual receptor B cells are present in the human peripheral repertoire. J Exp Med. 1995;181:1245-1250[Abstract/Free Full Text]. 23. Matsu Y, Nakamura S, Ariyasu T, et al. Four subclones with distinct immunoglobulin light chain phenotypes (kappa+lambda+, kappa+, lambda+ and kappa-lambda-) from acute leukemia. Leukemia. 1996;10:700-706[Medline] [Order article via Infotrieve]. 24. Padovan E, Giachino C, Cella M, Valitutti S, Acuto O, Lanzavecchia A. Normal T lymphocytes can express two different T cell receptor beta chains: implications for the mechanism of allelic exclusion. J Exp Med. 1995;181:1587-1591[Abstract/Free Full Text]. 25. Mertsching E, Wilson A, MacDonald HR, Ceredig R. T cell receptor alpha gene rearrangement and transcription in adult thymic gamma delta cells. Eur J Immunol. 1997;27:389-396[Medline] [Order article via Infotrieve]. 26. Nakano M, Kawanishi Y, Kuriyama Y, et al. Spontaneous reduction of leukemic lymphoma cells possibly by anti-tumor antibody-mediated phagocytosis: a kappa-lambda-dual-positive B cell lymphoma. Leukemia. 2000;14:278-284[CrossRef][Medline] [Order article via Infotrieve]. 27. Kawada H, Fukuda R, Yoshida M, et al. A novel variant of B-lymphoid leukemia expressing kappa/lambda light chains. Acta Haematol. 1998;100:54-56[CrossRef][Medline] [Order article via Infotrieve]. 28. Van Dongen JJM, Adriaansen HJ. Immunobiology of leukemia. In: Henderson WES,Lister TA,Greaves MF, eds. Leukemia. Philadelphia: WB Saunders; 1996:83-130. 29. Groeneveld K, Te Marvelde JG, Van den Beemd MWM, Hooijkaas H, Van Dongen JJM. Flow cytometric detection of intracellular antigens for immunophenotyping of normal and malignant leukocytes. Leukemia. 1996;10:1383-1389[Medline] [Order article via Infotrieve]. 30. van Dongen JJ, Wolvers-Tettero IL. Analysis of immunoglobulin and T cell receptor genes; Part I: basic and technical aspects. Clin Chim Acta. 1991;198