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ARTICLE |
allelic inclusion is a consequence of receptor editing
CORRESPONDENCE Patrick C. Wilson: wilsonp{at}omrf.ouhsc.edu
 Top ABSTRACT RESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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alleles. These cells are not the result of failure in allelic exclusion per se, but arise through receptor editing. We find that under physiological conditions, editing occurs both by deletion and by inclusion with equal probability. In addition, we demonstrate that B lymphocytes carrying two B-cell receptors are recruited to germinal center reactions, and thus fully participate in humoral immune responses. Our data measure the scope of allelic inclusion and provide a mechanism whereby autoreactive B cells might "escape" central tolerance.
, human constant region; HRP, horse-radish peroxidase; mC, mouse constant region; MZ, marginal zone; OOF, out-of-frame; YFP, yellow fluorescent protein. R. Casellas and Q. Zhang contributed equally to this paper.
B-cell antigen receptor diversity is achieved by the assembly of antibody gene segments through V(D)J recombination (1). The random nature of the recombination process leads to the unavoidable production of self-reactive specificities, which are silenced both centrally and peripherally by clonal deletion, anergy, and receptor editing (2–6). Despite these mechanisms of tolerance, autoantibodies are frequently detected in normal mice (7, 8), and recent estimates suggest that up to 20% of long-lived B cells are self-reactive in humans (9). Although the exact mechanism by which these lymphocytes escape elimination is unknown, experiments with transgenic mice carrying prerecombined autoantibodies suggest that some self-reactive specificities may persist in the B-cell compartment by coexpression of a second "innocuous" light chain on the cell surface, a phenomenon referred to as allelic inclusion (10–12). Coexpression of a non–self-reactive light chain is thought to rescue the B cell from negative selection by diluting the self-reactive receptor (13). However, the presence of B cells bearing two receptors in transgenic mice poses a challenge to the mechanism of allelic exclusion (14–17), as well as to the "one lymphocyte–one antibody" theory (18). Thus, the extent and function of light chain allelic inclusion under physiological conditions is unknown.
Until recently, the study of light chain gene recombination at the loci was hampered by a lack of natural allelic polymorphisms in humans or mice. To overcome this difficulty, we used gene targeting to replace the mouse Ig constant region (mC) with its human counterpart (hC; references 19, 20). Using mice heterozygous for the hC allele (Igm/h), we showed that 
3–5% of B lymphocytes express equal amounts of cell surface mC and hC light chains, as measured by flow cytometry (reference 19; Fig. 1 A, mC+hC+ population).
This analysis, however, failed to consider allelic inclusion within mC–hCk+ or mCk+hCk– B-cell populations (Fig. 1 A), which might conceal Ig double producers expressing predominantly one of two light chains on the cell surface. We determined the full extent of allelic inclusion in the B-cell compartment of Igm/h mice by several independent assays. We show that 10% of B lymphocytes express two cell surface receptors. Moreover, we demonstrate that these double producers arise from light chain editing, and not as a result of defects in the mechanism of allelic exclusion.
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m/h splenic B cells with anti-hC and -mC antibodies and characterized light chain transcripts in single cells by RT-PCR and sequencing (9). In agreement with our previous observations (19), VJ-mC and -hC transcripts were readily detected in mC+hC+ lymphocytes. These cells make up 3–5% of the total B-cell pool, and most express in-frame transcripts from both alleles (Fig. 1 A and Table S1, available at http://www.jem.org/cgi/content/full/jem.20061918/DC1; 183/195 in-frame). VJ-mC mRNAs were detected in 30% (37/126) of apparently single-positive mC–hC+ B cells, but only one third of these transcripts (10/37) were in-frame (Fig. 1 A). Likewise, hC+ transcripts were present at a similar frequency in mC+hC– B cells (Fig. 1 A, 35/126; 10 in-frame). Analysis of a control mixture of Igm/m and Igh/h lymphocytes demonstrated that <0.5% of the analyzed cells might be improperly scored as dual producers by this method. Taking into account the efficiency of the PCR reaction, we conclude that 11% of all B lymphocytes in Igm/h mice express in-frame light chain transcripts from both alleles (4% of the hC+mC– cells + 4% of the hC–mC+ cells + 3% of the hC+mC+ cells; Table S1).
To measure the extent of allelic inclusion at the protein level, isolated Igm/h splenic B cells were stained with anti-hC (Fig. 1 B, red, Alexa Fluor 546) and anti-mC (green, Alexa Fluor 488) antibodies and characterized by confocal microscopy. The specificity of this staining was assessed in B cells isolated from Igm/m and Igh/h mice, from which 1 in 250 cells were nonspecifically recognized by both antibodies. Of 1,710 individual B cells analyzed, we found that 123 (7.2%) coexpressed detectable levels of mouse and human C light chains on the cell surface (Fig. 1 B). However, the intensity of staining varied between alleles, with some cells staining predominantly with anti-mC and others with anti-hC (Fig. 1 B, middle and right, and Fig. S1 available at http://www.jem.org/cgi/content/full/jem.20061918/DC1). Finally, we produced hybridomas from Igm/h splenic lymphocytes and measured mC and hC light chain protein production. Of 128 hybridomas screened by ELISA and Western blotting, 12% were found to be allelically included, with 4% (5/128) secreting high levels of both mC and hC light chains (Fig. 1 C, B47), whereas the remaining 8% (10/128) predominantly expressed one of two alleles (Fig. 1 C, sample A32). The divergent pattern of cell surface mC/hC expression observed in the latter population was a direct correlation of Ig transcription levels (Fig. S2), and may reflect inherent differences in V promoter strength (20). Therefore, based on single-cell PCR, confocal microscopy, and hybridoma analyses, we conclude that 10% of peripheral B lymphocytes are allelically included, indicating that B cells frequently develop expressing two B-cell surface receptors.
Allelic inclusion results from light chain receptor editing
At least two mechanisms could account for dual light chain expression. Secondary VJ rearrangements might be specifically induced to abolish self-reactivity or to rescue cells carrying less than optimal combinations of Ig heavy and light chains. Alternatively, inefficient allelic exclusion could lead to primary V–J recombination on both alleles. To discriminate between these two possibilities, we compared the developmental kinetics of bone marrow B cells carrying one or two light chains by BrdU labeling in vivo. The thymidine analogue BrdU is incorporated in the genome of cycling proB cells that give rise to the small resting preB cells in which light chains are actively rearranged (21). Edited B lymphocytes are developmentally delayed compared with unedited cells in this assay (19). In agreement with published observations (19, 22, 23), BrdU+B220low B cells expressing only mC or hC emerged first into the immature B-cell compartment, 4 h after BrdU injection (Fig. 2 A).
In contrast, allelically included lymphocytes were delayed at the preB-cell stage for at least an additional 4 h, appearing in the IgM+ immature B-cell compartment 8 h after BrdU injection (Fig. 2 A). This developmental delay suggests that dual receptor expression is not the result of simultaneous biallelic Ig recombination or failure in allelic exclusion, but instead, results from secondary light chain gene rearrangements in cells arrested at the early preB-cell stage of development.
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usage has been associated with receptor editing, as it suggests continuing V–J rearrangements (24). Our analysis of 401 antibody sequences obtained from Igm/h primary cells and hybridomas showed that J1 usage is predominant in allelically excluded cells (35% vs. 20%; 
2 test, P = 0.002; Fig. 2 B). In contrast, J5 was significantly enriched in cells carrying dual receptors (21% vs. 34%; 2 test, P = 0.005; Fig. 2 B). This observation further reinforces the conclusion that allelic inclusion occurs in cells undergoing extensive light chain editing, presumably because of self-reactivity. To examine whether allelically included lymphocytes carry autoreactive receptors, we tested individual antibodies from Ig-included (15 clones) and -excluded (38 clones) hybridomas for binding to double-stranded DNA or HEp-2 cells, which are commonly used to diagnose autoimmune diseases such as lupus (25). We found that >30% of mC+hC+ cells carried self-reactivity conferred predominantly by either the human or the mouse light chain (Fig. 2 C). Autoreactivity in these clones was confirmed using a test for mouse antinuclear antigen (ANA) reactivity (see Materials and methods). Self-reactivity in allelically excluded cells was nearly threefold less frequent (13%) than in hybridomas expressing dual receptors, although the low number of included hybridomas precluded a sufficient n-value to reach statistical significance (2 test, P = 0.09). Collectively, our data indicate that receptor editing leads to light chain allelic inclusion and retention of self-specificities in the B-cell compartment.
Allelically included B cells preferentially home to MZs and can contribute to immune responses
B cells carrying natural autoantibodies have been shown to accumulate preferentially in the marginal zone (MZ) of mice (26), but not of humans (27). To investigate the distribution of Ig-included cells in the long-lived B-cell compartment, we assessed mC and hC expression in Igm/h spleens by immunohistochemistry, flow cytometry, and single-cell PCR. We found allelically included cells both within the follicles and MZs (Fig. 3 A).
However, whereas between 8–11% of follicular B cells (B220+CD23highCD21low) were mC+hC+, 20% of MZ lymphocytes (B220+CD23lowCD21high) expressed light chains from both alleles (Fig. 3 B; 2 test, P < 0.0001). Preferential homing of Ig double producers to MZs is consistent with the reported accumulation of edited Ig+Ig
+ lymphocytes in MZs of anti-DNA transgenic mice (10). These results further suggest that included B cells often retain self-specificities in Igm/h mice, as reported in transgenic mice carrying autoantibodies (10–13).
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double producers participate in the humoral immune response, we analyzed IgG+ B cells from Igm/h mice. We readily found IgG+ lymphocytes that were mC+hC+ (2–5%; Fig. 3 B). To confirm the presence of allelically included cells in the postgerminal center B-cell compartment, we analyzed memory B cells in Igm/hAID-Cre-yellow fluorescent protein (YFP) mice. In these mice, germinal center and memory B cells are indelibly labeled with YFP (Fig. 3 C, left pseudocolor plot; unpublished data). Flow cytometry analysis showed the presence of allelically included cells within postgerminal center CD95–YFP+ cells, which carry high levels of somatic hypermutation (Fig. 3 C). These results demonstrate that B cells expressing two cell surface receptors are not excluded from the germinal center reaction and fully participate in the humoral immune response.
Editing occurs by deletion and inclusion with similar probability
Using mice carrying prerecombined light chains, we established that 25% of all developing B cells undergo light chain editing (19). To measure the extent of allelic inclusion within the edited compartment, we analyzed mice carrying a V
HEL-J2–targeted transgene and the hC allele (IgHEL/h mice). In these mice, the prerearranged light chain is normally paired to the physiological heavy chain repertoire, and V–J replacements at the hC allele are readily monitored by flow cytometry or PCR analysis (19). Single B cells were sorted from IgHEL/h mice and mC+ and hC+ transcripts were analyzed by single-cell PCR. We found that 61% of all developing B cells in these mice express the prerecombined HEL light chain (Fig. 4 A).
However, in 18% of all IgHEL/h B lymphocytes, the VHEL was edited by deletion, either through nested recombination events at the mC allele alone (HEL–mC+, 6%; Fig. 4 A), or together with recombination at the hC allele (HEL–hC+, 12%; Fig. 4 A). In close agreement with results obtained with Igm/h mice (Figs. 1 and 2), editing by inclusion occurred in 14% of IgHEL/h B cells. The majority (13%) of these lymphocytes retained the VHEL light chain and carried in-frame V–J rearrangements at the hC allele (HEL+hC+; Fig. 4 A), whereas a small number (1%) edited the VHEL by functional rearrangements in both alleles (HEL–hC+mC+; Fig. 4 A). In addition, we noted that nearly all rearrangements at the hCk allele were in-frame (Fig. 4 B; 95%), which contrasts to the 2:1 OOF/in-frame ratio expected from random recombination. This observation indicates that cells carrying successful V–J rearrangements on the human allele were selected during development. Because hC+ B cells are developmentally delayed by editing in these mice (19), these results argue that allelic inclusion in IgHEL/h mice originates from secondary gene rearrangements and not as a result of defective exclusion of light chain gene recombination. We conclude that receptor editing occurs either by deletion or by allelic inclusion with roughly equal probability (Fig. 4).
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DISCUSSION |
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TopABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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rearrangements initially target a single allele, ensuring allelic exclusion in most cells (Fig. 4; references 14–16, 31), autoreactivity and/or a 4-h developmental delay at the preB-cell stage promotes RAG accessibility to both alleles. This idea is consistent with recent findings showing that activation of both alleles is favored as development progresses from the preB to the immature B-cell stages (32).
Although natural autoantibodies were discovered >100 yr ago (33), their origin has remained obscure. In normal individuals, large numbers of self-specificities are believed to escape or bypass imperfect B-cell tolerance checkpoints (10), and this phenomenon appears to be exacerbated in patients with autoimmune diseases such as systemic lupus erythematosus (8, 9, 34). Our studies suggest that light chain receptor editing, which normally accounts for 25–50% of all antibodies (19, 35), also retains autoantibodies in the wild-type repertoire by allelic inclusion. As previously proposed (10, 11, 13), coexpression of an innocuous receptor might dampen signaling from self-reactive receptors, and thus promote development. Alternatively, light chain editors may tolerize autoreactive B cells by outcompeting self-reactive light chains for pairing with the IgH chain. This feature may, in fact, explain the higher frequency of allelic inclusion as measured by single-cell RT-PCR analysis (11%; Fig. 1 A), which detects Ig transcription, versus confocal microscopy (7.2%; Fig. 1 B), which monitors light chain cell surface expression. Another possibility is that autoreactive receptors are continuously internalized by autoantigen binding, as shown in B lymphocytes expressing anti-MHC antibodies (11) or in T cells expressing two chains (36), although whether double-producer T cells arise from a lack of chain allelic exclusion or through receptor editing is still controversial (37).
The accumulation of allelically included B cells in MZs is intriguing. MZ B cells are commonly hyperreactive to T cell–independent type II antigens, such as phosphorylcholine, and represent the first line of defense against blood-borne pathogens (38). Conceivably, self-reactive allelically included cells are preferentially selected into the MZ because of their chronic activation by self-antigens. Diversion to this compartment might help prevent these cells from undergoing full cell differentiation (10). If recruited to germinal centers, allelically included cells could, in fact, function as Trojan horses; by introducing stop codons or otherwise disabling the innocuous editor light chain, hypermutation might unveil autoantibodies during immune responses.
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MATERIALS AND METHODS |
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TopABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(PE; BD Biosciences) and hC (FITC; previously available from SouthernBiotech). Cells were sorted using either a MoFlo (Dako Cytomation) or a FACS-Aria (Becton Dickinson) cytometer. Cytometric analyses were done using a LSR II analyzer (Becton Dickinson). Analyses and figures were done with FlowJo software.
Immunohistochemistry.
For analysis of the total frequency of allelic inclusion by immunofluorescence and confocal microscopy, total splenic B cells from Igh/m mice were enriched by magnetic bead depletion of non–B cells (Stemcell Technologies) and stained with biotin-conjugated goat anti- hCk that had been absorbed with mC protein (SouthernBiotech), followed by streptavidin-conjugated Alexa Fluor 546 (Invitrogen) and rat anti-mCk (SouthernBiotech) directly conjugated in-house to Alexa Fluor 488 (Invitrogen). The cells were adhered to microscope slides and scored using a confocal microscope (LSM-510 META; Carl Zeiss MicroImaging, Inc.). Analyses for sorted fractions of hC+ cells for surface expression of mC+ were similar, except that the cells were stained with anti-mC conjugated to alkaline phosphatase (SouthernBiotech), adhered to slides, and developed with Alkaline Phosphatase Substrate kit III (Vector Laboratories). For analysis of splenic tissue, sections from snap-frozen Igm/h, Igm/m, and Igh/h mouse spleens were stained with rat anti-mC (SouthernBiotech) and developed as per the sorted fractions (above). Sections were then stained with mouse anti–hC-HRP (SouthernBiotech) and developed with Vector NovaRED Substrate kit (Vector Laboratories). Analyses were done with a fluorescent microscope (Axioplan 2; Carl Zeiss MicroImaging, Inc.).
Single-cell RT-PCR.
Cells were presorted in bulk using a FACS-Aria cytometer, and single cells were resorted into 96-well plates containing 10 mM Tris-HCl with 40 U/µl of RNase inhibitor (Promega) with a MoFlo cytometer fitted with a cell dispenser. The plates were immediately frozen on dry ice and stored at –80°C until analysis. Each cell was amplified in a one-step RT-PCR reaction (OneStep RT-PCR kit; QIAGEN) using a cocktail of sense primers specific for the leader region (primers used were as follows: 5'-ATGGAATCACAGRCYCWGGT-3'; 5'-TTTTGCTTTTCTGGATTYCAG-3'; 5'-TCTTGTTGCTCTGGTTYCCAG-3'; 5'-ATTWTCAGCTTCCTGCTAATC-3'; 5'-TGCTGCTGCTCTGGGTTCCAG-3'; and 5'-TCGTGTTKCTSTGGTTGTCTG-3'), and antisense primers specific for either mC (5'-AGCTCTTGACGATGGGTGAAGTTG-3') or hC (5'-GTTTCTCGTAGTCTGCTTTGCTCA-3'). RT-PCR conditions were as follows: 50°C for 30 min, 95°C for 15 min, followed by 39 cycles of 95°C for 1 min, 55°C for 1 min, 72°C for 1 min, and, finally, 72°C for 10 min. 1 µl from each RT-PCR reaction was reamplified using nested PCR primers specific for FW1 regions of the various V genes (5'-GCGAAGCTTCCCTGATCGCTTCACAGGCAGTGG-3', 5'-GCGAAGCTTCCCTGCTCGCTTCAGTGGCAGTGG-3', 5'-GCGAAGCTTCCCAKCCAGGTTCAGTGGCAGTGG-3', and 5'-GCGAAGCTTSCCATCRAGGTTCAGTGGCAGTGG-3'), and antisense primers specific to either mC (5'-ATCTGGAGGTGCCTCAGTC-3') or hC (5'-GTGCTGTCCTTGCTGTCCTGCTC-3'). Nested PCR reaction conditions were as follows: 95°C for 15 min, followed by 39 cycles of 95°C for 1 min, 55°C for 1 min, 72°C for 1 min, and a final 72°C for 10 min. PCR products were resolved using 1% Agarose gels, and products were purified (Gel Extraction kit; QIAGEN) and sequenced on an ABI 3730 capillary sequencer by the Oklahoma Medical Research Foundation DNA sequencing core facility.
Analysis of Ig proteins from hybridomas (ELISA, Western blot, and ANA).
Hybridomas were produced and subcloned by the Monoclonal Antibody Core Facility at the Memorial Sloan-Kettering Cancer Center. Ig splenic B cells from 4-wk-old mice were stimulated for 3 d in fetal calf serum–supplemented RPMI with 25 µg/ml of lipopolysacharide (Sigma-Aldrich) and fused to the SP2 cell line. Two separate fusions with different mice produced similar results and were combined herein (58 and 70 clones for 128 total Igm/h hybridomas). Each clone expressing protein or transcripts from both Ig alleles was subcloned in triplicate by limiting dilution to verify monoclonality. Clones from which no Ig expression was detectable were discarded. Antibodies were expressed in serum-free RPMI media supplemented with Nutridoma (Roche) and purified from supernatant using Agarose-bead–conjugated L-protein (Pierce Chemical Co.). Capture ELISA assays were performed using purified anti-hC or -mC (Jackson ImmunoResearch Laboratories) and horse-radish peroxidase (HRP)–conjugated anti-hC or anti-mC (Jackson ImmunoResearch Laboratories). For the anti-DNA ELISAs, purified antibodies were captured on microtiter plates coated with 10 g/ml of salmon-sperm double-stranded DNA (Roche). The HRP was developed using a HRP detection kit (Bio-Rad Laboratories). All ELISA assays were analyzed using a SpectraMax plus microtiter plate reader (Invitrogen). Western blots were performed using HRP-conjugated antibodies (Jackson ImmunoResearch Laboratories). Purified antibodies were screened for ANA reactivity using commercially prepared HEp-2 slides (BION Enterprises, Ltd.) and the aforementioned FITC-conjugated anti-mC or -hC antibodies. The HEp-2–reactive clones were verified using a commercial ANA assay kit designed to test mouse autoantibodies (The Binding Site).
Ribonuclease-protection assays.
RNA was isolated from 1 million cells for each hybridoma clone using the RNAwiz reagent (Ambion) and treated with RNase-free DNase for 15 min at 37°C. The reaction was stopped with addition of one twentieth volume of 0.5 M EDTA, followed by heat inactivation of the DNase I for 10 min at 90°C, phenol/chloroform extraction, and ETOH precipitation. 5 µg of RNA were used in each of three replicate RPA reactions for each hybridoma analyzed. RPA probes were designed as described in Fig. S2 and cloned in the PcR2.1 vector containing both T3 and T7 RNA polymerase promoters. The [32P]UTP-labeled RNA probes were generated using the MAXIscript In Vitro Transcription kit (Ambion). To ensure full-length probes, each probe was purified from a 5% polyacrylamide/8 M urea gel. RNA probes were eluted overnight at room temperature from excised bands of the appropriate length with NH4-acetate/1 mM EDTA/0.2% SDS, followed by ETOH precipitation and resuspension in diethylpyrocarbonate-treated water at a concentration of 5 x 104 cpm in 10 µl. RPA reactions were performed using the RPA III kit (Ambion). All antisense probes were hybridized simultaneously to each sample. Protected fragments were resolved using a 5% denaturing polyacrylamide DNA sequencing gel and quantified using a phosphorimager (Storm 840; Molecular Dynamics). Protected bands were normalized relative to the Sp2 cell line fusion partner OOF mC transcript that is produced by each hybridoma to account for variations in RNA sample quality.
Online supplemental material.
Fig. S1 provides an analysis of the abundance of either allele expressed as measured by immunohistochemistry. Fig. S2 is an analysis of transcript expression levels from either Ig allele. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20061918/DC1.
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Acknowledgments |
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This research was supported in part by National Institutes of Health (NIH) grants P20RR018758-01 and P20RR15577-02 (to P.C. Wilson), and by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH (to R. Casellas).
The authors have no conflicting financial interests.
Submitted: 6 September 2006
Accepted: 6 December 2006
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