HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Data Supplement Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Witsch, E. J. Articles by Bettelheim, E. Search for Related Content PubMed PubMed Citation Articles by Witsch, E. J. Articles by Bettelheim, E. Pubmed/NCBI databases Gene

Allelic and Isotypic Light Chain Inclusion in Peripheral B Cells from Anti-DNA Antibody Transgenic C57BL/6 and BALB/c Mice¹

Esther J. Witsch^2,* and Eldad Bettelheim

^* Department of Pathology, Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL 60637; and Department of Physical Sciences, James Franck Institute, University of Chicago, Chicago, IL 60637

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Most mature B lymphocytes express one BCR L chain, or , but recent work has shown that there are exceptions in that some B lymphocytes express both and and some even bear two different L chains. Using the anti-DNA H chain-transgenic mouse, 56R, we find that B cells with pre-existing autoreactivity are especially subject to L chain inclusion. Specifically, we show that isotypic and allelic inclusion enables autoreactive B cells to bypass central tolerance giving rise to B cells that retain dangerous features. One receptor in dual receptor B cells is an editor L chain, i.e., neutralizes or alters self-reactivity of the 56R H chain transgene. We compare the 56R mouse when on the C57/BL/6 background, a strain prone to autoimmunity, with that of 56R when on the BALB/c background, a strain that resists autoimmunity. In the B6.56R mouse, polyreactive B cells with dual L chain move to the follicular B cell compartment. Their localization in the follicular compartment may explain the ease with which B cells in the B6.56R differentiate into autoantibody-producing plasma cells. Likewise, in the BALB/c.56R mouse, dual L chain B cells are found in the follicular B cell compartment. Yet, the lack of autoantibody-producing plasma cells in the BALB/c.56R suggests that postfollicular tolerance checkpoints are intact. The J usage in dual L chain B cells suggests increased receptor editing activity and is consistent with the expected distribution of J genes in our computational model for random selection of J.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Anti-DNA Abs are a major pathogenic factor in autoimmune diseases such as lupus erythematosus. In healthy individuals, anti-DNA Abs are regulated by deletion, anergy, and receptor editing (1, 2, 3, 4, 5, 6). All three mechanisms lead to a self-tolerant peripheral B cell repertoire with few if any remaining autoreactive B cells. Among the regulatory mechanisms, receptor editing deserves special attention because the peripheral B cell repertoire is widely shaped by receptor editing (7). Editing is based on secondary Ig gene rearrangement mainly at the L chain locus by replacing an autoreactive with a nonautoreactive receptor (8, 9). Another way of dealing with autoreactive B cells is the expression of two different L chains, and ; such isotypically and allelically included dual receptor B cells are part of the peripheral repertoire in humans and mice (10, 11, 12, 13, 14).

In our anti-DNA 56R H chain-transgenic mouse (15, 16, 17), B cells bind to dsDNA in association with most L chains, but the 56R H chain can be silenced when paired with "editor" L chains. Examples of such editor L chains are V20, V21D, and V38C (17, 18). Among L chain, Vx edits the 56R H chain transgene (19). Association of the 56R H chain with an editor L chain sufficiently alters anti-DNA reactivity allowing B cells to leave the bone marrow. Also dual L chain B cells are found in the 56R mouse (17, 20). 1 in association with the 56R is self-reactive, and self-reactive 1 B cells are thought to survive by coexpressing a L chain (21). In the 56R, dual B cells have been found in the marginal zone (MZ)³ of the spleen (20, 22, 23).

We have recently shown that failure to edit or incomplete BCR editing in the 56R strain gives rise to polyreactive autoantibodies (20). This autoantibody polyreactivity is mediated by the editor L chains V38C and V20, which incompletely edit the 56R H chain transgene. 56R with V38C binds to dsDNA and phosphatidylserine as well as a series of (self-) proteins such as myelin basic protein, thyroglobulin, cytochrome c, histone, β-galactosidase, and insulin. 56R associated with V20 binds to dsDNA and phosphatidylserine (20). Polyreactive features have also been shown for the editor L chain Vx (24). During the course of the chronic graft-vs-host reaction (cGvH), a model of induced autoimmunity, incompletely edited B cells and dual receptor B cells produce high titers of polyreactive autoantibodies that contribute to cGvH disease (20). Hence, these findings suggest that dysregulation of anti-DNA Abs can occur by incomplete L chain editing and at the level of dual receptor B cells (20). In humans, the frequency of polyreactive autoantibodies is increased in autoimmune patients (8, 9). Although, to date, a mechanistic link between polyreactive autoantibodies and disease pathogenesis is still missing.

Recent findings showed that the genetic background causes differences in the peripheral L chain repertoire in the 56R mouse: differences in the L chain repertoires correlate with differences in susceptibility to autoimmunity (25, 26). Hybridoma panels showed that the 56R-transgenic mouse on a C57BL/6 (B6) background is permissive for polyreactive Abs and mainly expresses V38C. In contrast, 56R on a BALB/c background mainly expresses V21D (17, 25, 26), an efficient editor of anti-DNA activity (17, 20). These differences in the L chain repertoires are correlated with the observation that the B6 strain is prone to autoimmunity, whereas BALB/c is more resistant (27, 28, 29, 30, 31). Thus, in lupus-susceptible mice the L chain repertoire is shifted toward incompletely edited polyreactive B cells and suggesting a role of polyreactive Abs in autoimmune disease.

To investigate the regulation of incompletely edited and dual receptor B cells in autoimmunity, we examined peripheral B cell subsets in detail by single-cell PCR. Specifically, B cells from the MZ and follicular compartment in the B6.56R and in BALB/c.56R were analyzed to define their L chain repertoires and the localization of incompletely edited B cells as well as dual L chain B cells. We determined the frequency and the type of and dual L chain B cells in the two strains and were interested to find whether receptor editing contributes to the generation of or dual L chain B cells.

The L chain repertoire was skewed toward the polyreactive L chain V38C in B6.56R, whereas in BALB/c.56R, we saw a bias toward V21D. This was in agreement with previous results (17, 25, 26). In this study, we show that the differences in the L chain repertoires between both strains were most notable in B cells of the MZ and less prominent in the follicular zone compartment. We found B cells with dual L chain in the MZ. We also demonstrate that B cells with dual L chain locate to the follicular zone B cell compartment of both strains, B6.56R and BALB/c.56R. This suggests that the lack of autoantibodies in the BALB/c.56R (25, 26, 32) is due to intact postfollicular tolerance checkpoints as compared with B6.56R. In B cells with dual L chain, one of the two L chains was the editor V21D or V38C. B cells with in-frame message always had an in-frame L chain rearrangement, hence in the 56R mouse, B cells with L chain represent a population in which isotypic exclusion is broken.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice

The generation of site-directed 3H9/56R knockin mice (56R mice) has been described previously (16, 17) and resulted in the BALB/c.3H9/56R mice. The 3H9/56R has been backcrossed onto the C57/BL/6 background for 14 generations to engender B6.3H9/56R. The 3H9/56R transgene was determined by PCR amplification of tail DNA (15, 17). All mice were maintained in our mouse colony at the University of Chicago. All animal care and procedures were conducted in accordance with the Animal Welfare Act.

Flow cytometry, single-cell sorting, cDNA synthesis, and PCR

Before staining, cells were preincubated with excess mouse anti FcIII/IIR (2.4G2; BD Pharmingen) to block FcRs. Dead cells were excluded by propidium iodide staining (0.33 µg/ml). The following mAb were used for staining: RA3-6B2-allophycocyanin (anti-B220), 7G6-FITC (anti-CD21), B3B4-PE (anti-CD23) (all obtained from BD Pharmingen). Single-cell PCR was done as described (8). MZ B cells, B220⁺CD21^intCD23^high, from one B6.56R mouse were sorted on a FACSVantage (BD Biosciences) into 96-well PCR plates containing 4 µl of lysis solution (0.5x PBS containing 10 mM DTT, 8 U of RNA in (Promega) and 0.4 U of 5'-3' Prime RNase inhibitor (Eppendorf) and were immediately frozen on dry ice. All samples were stored at –70°C. The purity of single-sorted B cells was 98.2% for B cells from the MZ and 98.8% for B cells from the follicular zone compartment (data not shown). cDNA was synthesized in a total volume of 14 µl in the original 96-well PCR plate. RNA from single cells was reverse-transcribed at 37°C for 55 min with 150 ng of random hexamer primer (pd(N)6; Amersham Pharmacia Biotech), 0.5 µl of dNTP mix (10 mM each), 1 µl of 0.1 M DTT, 0.5% (v/v) Nonidet P-40, RNase inhibitors (4 U of RNasin and 6 U of prime RNase inhibitor), and 50 U of Superscript III reverse transcriptase (Invitrogen Life Technologies). Ig gene rearrangement was tested using the degenerate primer V(S) (33) with C reverse primers, and specific primers for V21D, V38C with C reverse primers (for primers, see Ref. 26), and primers specific for the 56R H chain transgene and reverse primers for IgM (26), and primers for V and reverse primers for C (20). Products were amplified by nested PCR in 40-µl reactions containing 20 pM primers and 1.2 U of HotStar TaqDNA polymerase (Qiagen). The PCR conditions for were as follows: denaturation for 4 min at 94°C, then 30 s at 94°C, 30 s at 55°C (for V21D and V38C and 56R H chain) or at 50°C (for V(S) (as previously suggested in Ref. 34 and V_1/2) and 55 s (first PCR) or 45 s (nested PCR) at 72°C for 50 cycles, and elongation was allowed for 10 min at 72°C. As a template, 2 µl of cDNA were used for the first PCR and 3 µl of first PCR product were used for the nested PCR. The amount of cDNA from a single cell allowed for five PCR assays that were exhausted with the above-listed PCRs. PCR products were sequenced using primers for V20, V21D, V38C, V(S) and C, and V_1/2 and C as indicated in Results. Sense and antisense strands of each L chain sequence were aligned and the type of L chain was identified (supplemental table IB-F)⁴ using a National Center for Biotechnology Information Blast search for Ig genes. Alignments of sense and antisense strand were checked for base ambiguities using Sequencher DNA Software (Gene Codes).

Computational models

The computation of random and sequential J usage in L chain rearrangements (see Fig. 3, B and C) was conducted using The Mathematica Software Package (Wolfram Research). The calculations somewhat follow Louzoun et al. (35) and were computed under certain assumptions to get the best fit to our data. See detailed description of formulas online (www.phys.huji.ac.il/eldadb/Supplement.pdf).

Statistical analysis

Values of p were calculated using the ² test and the Student t test for small numbers (see Fig. 2B).

Sequence alignments

Analysis of the mismatch score of the degenerate primer V(S) to framework region (FR) 3 of all mouse V genes (supplemental table IA) was done using the Fuzznuc program as part of the Emboss software package. Alignment of V and V L chain genes (supplemental table I, B–F) was conducted using Sequencher Software and Clustal X.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Peripheral L chain repertoires in B6.56R vs BALB/c.56R mice and the sequestration of edited receptors

To determine the BCR repertoires of peripheral B cell subsets in the 56R mouse, single B cells from the splenic MZ (B220^posCD21^highCD23^low) and from the follicular zone compartment (B220^posCD21^intCD23^high) were sorted and analyzed by nested RT-PCR. In both strains, between 30 and 45% of all splenic peripheral B cells display the phenotype of MZ B cells (20, 23). L chain transcripts were amplified using specific primers for the editor L chains V38C and V21D, and using the degenerate primer V(S) (33). The degenerate primer V(S) binds to FR3 of most L chains and amplifies >80% of all L chains (M. Schlissel, personal communication) including V38C and V21D. We now estimate, based on our primer alignment analysis to all 98 V genes, that only very few L chains will be ignored by the V(S) primer (supplemental table IA). Sequencing analysis is necessary to determine the type of L chain amplified by this PCR. The presence of L chain was tested using specific primers for V1 and V2 L chain. We compared B cells from the MZ and follicular zone compartment in 56R mice on the B6 and on the BALB/c background.

The sum of all analyzed peripheral B cells confirmed previous results and showed that the ratio of V38C and V21D L chain was reversed in the B6.56R and the BALB/c.56R strain (25, 26, 32) (Fig. 1A). In the B6.56R, the L chain repertoire was skewed toward the polyreactive incomplete editor L chain V38C, whereas in BALB/c.56R, the B cell repertoire was biased toward the complete editor L chain V21D, a L chain that neutralizes binding to self (17, 20). Such skewing of the L chain repertoire is not found in nontransgenic B6 and BALB/c mice: V38C in the B6 and all eight V21 family members in the BALB/c are represented with 0.7 and 4%, respectively (26, 36). In the majority of B cells in B6.56R (79.1%) and in BALB/c.56R (95.7%), we could identify a L chain rearrangement amplified with the degenerate primer V(S). We also found that L chain was amplified in a similar number of B cells in B6.56R (9.9%) and in BALB/c.56R (10.3%) (Fig. 1A). The differences in the L chain repertoires held true and reached statistical significance for V21D and V38C when only B cells were analyzed in which we could identify the presence of the transgenic H chain 56R using specific primers for the IgM-56R transgene (supplemental figure 1A, data online at www.phys.huji.ac.il/eldadb/Supplement.pdf).

View larger version (18K): [in this window] [in a new window]   FIGURE 1. B cells with L chain editors are sequestered in the MZ compartment of the spleen. A, The L chain repertoire in the 56R H chain-transgenic mouse depends on the strain background. 56R on a C57/BL/6 background (B6.56R) uses mainly the polyreactive (20) V38C (black bars) and few non-anti-nuclear Ab (ANA) (20) V21D (dark gray bars) L chain. 56R on a BALB/c background (BALB/c. 56R) uses mainly V21D and less V38C L chain. The percentage of the ANA (21) 1 and 2 L chain (light gray bars) is similar in both mouse strains. Because of the overlap between V(S) with V21D and V38C, and because this representation contains B cells with dual L chain message, the bars shown here do not add up to 100%. Numbers in parentheses represent numbers of total B cells as determined by PCR product-positive cells. For the B6.56R mouse, only 184 cells are included in the representation of V21D L chain. B, The strain-specific repertoire differences of B6.56R and BALB/c. 56R are most apparent in MZ B cells. The L chain editors V21D and V38C are increased in the MZ compartment of the spleen and are found at a smaller percentage in B cells of the follicular compartment. The white bars show L chain products from a PCR with the degenerate primer V(S) which amplifies most V genes including V21D and V38C. Numbers in parentheses represent numbers of total B cells. For the MZ/B6.56R group, only 90 cells are included in the representation of V21D L chain. The graph shows all L chain PCR products prior to sequencing analysis. In-frame vs out-of-frame products are detailed in Fig. 3D and out-of-frame products did not change the L chain repertoire shift of V38C and V21D in B6.56R and BALB/c.56R mice. C, A large percentage of B cells with the editor L chains V21D or V38C are associated with the IgM-56R transgene (56R+, black bars). In some edited B cells with V21D or V38C the IgM-56R transgene could not be detected (56R–, gray bars). These 56R– B cells could have been subject to VH replacement (16) or they could be switched to IgG. The H chain status of 56R– B cells was not analyzed further. The sum of all other L chains positive with V(S) but negative for V21D and V38C also showed association with the IgM-56R transgene. The bar graph includes B cells some of which have dual L chain rearrangements and for the MZ/B6.56R group the number of cells with V38C is greater (179 cells), therefore the sum of all bars in each group may not add up to 100%.

We analyzed the presence of the 56R-transgenic H chain in B cells from the MZ and follicular zone compartment of B6.56R and BALB/c.56R. A large percentage of B cells with the editor L chain V21D or V38C was associated with the 56R transgene (Fig. 1C). Some B cells with V21D or V38C were 56R^–. This fraction of 56R^– B cells can still carry the transgene as the PCR assay for IgM-56R does not amplify the transgene in isotype-switched IgG B cells. Another possible explanation for 56R^– B cells is that the already rearranged VDJ 56R H chain transgene can be edited by replacement with an upstream V_H gene. Such V_H replacement is possible through a recombination signal embedded in V_H genes (16, 37, 38). This gene replacement is comparable to heptamer-/nonamer-mediated rearrangement in L chain genes. Thus, 56R^– B cells can be cells that have been isotype switched to IgG or they may have been subject to V_H replacement. In both cases, cells in the IgM-56R^– fraction are likely to be derived from transgenic 56R B cells. This notion is supported by the resemblance of the L chain repertoire in IgM-56R⁺ and IgM-56R^– B cells (data not shown). The H chain status of 56R^– B cells could not be analyzed further due to a limited amount of cDNA isolated from a single cell. However, the results obtained from B cells in which the transgene could be detected (supplemental figures 1–4, data online at www.phys.huji.ac.il/eldadb/Supplement.pdf) did not differ considerably from the results that included all cells (Figs. 1–4).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Allelic inclusion at the locus depends on the self-reactivity of the L chain. A, B cells with dual L chain are found in the MZ and follicular compartment of both mice, B6.56R and BALB/c. 56R. B cell with dual L chain are decreased in the follicular compartment of BALB/c.56R. Numbers in parentheses represent numbers of total B cells analyzed. B, B cells with the polyreactive V38C L chain are more likely to have a second L chain in-frame as compared with B cells with V21D L chain. Numbers in parentheses represent numbers of total B cells with in-frame V21D and V38C. C, Frequency of B cells with message for dual L chain. B cells with a dual L chain are found in both mice B6.56R and BALB/c.56R and no difference was found between B cells from the MZ and follicular compartment. B cells with 1 L chain have rearranged a L chain. The majority of products found in the 56R mouse are dual L chain B cells.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. Experiment and computational model to explain the usage of J genes in V/J gene rearrangements. A, Experimental results of J usage in noneditor L chains, V38C and V21D L chain. Left group, J usage in a group of 64 other (noneditor) L chains consisting of 29 different V genes. All noneditor L chains were identified from dual L chain B cells. Middle group, J usage in 80 V38C L chains; 59% of the shown sequences were identified from dual L chain B cells. Right group, J usage in 106 V21D L chains; 26% of the shown sequences were identified from dual L chain B cells. In-frame products are shown in black, out-of-frame products are shown in gray. B, Computation of a random distribution of J genes under certain assumptions to best fit the experimental data in A for noneditor L chains. These are the probability for an in-frame product = one-third, the probability for another attempt being made after an out-of-frame product has been produced as = 0.5, a factor to describe the instability of an out-of-frame product as β = 0.25. C, Computation of a strictly sequential distribution under the same assumptions as in B. Computational models were inspired by previous work by Louzoun et al. (35 ) and modified to best fit our experimental data. Detailed formulas are available at www.phys.huji.ac.il/eldadb/Supplement.pdf. D, Experimental data for the frequency of and L chain in frame vs out of frame.

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Downstream J genes in B cells with dual L chain message. B cells with only one V message use mainly J2 whereas allelically - and isotypically -included B cells use more often J4 and J5. Three groups are shown, excluded B cells in which just one L chain could be identified, included B cells with dual L chain and included B cells with dual or L chain. Note that 89.6% the excluded cells with single V gene message use V21D in which J2 is the predominant. In this representation, only B cells are included of which all L chain products have been sequenced.

B cells with dual L chain message in B6.56R and BALB/c.56R

To identify B cells with dual L chain message, we further analyzed B cells showing two L chain PCR products when amplifying V21D, V38C, or L chain using the degenerate primer V(S). All PCR products from these dual / L chain B cells were sequenced using both nested primers from the 5' and from the 3' end of the L chain transcript. Sense and antisense strands were aligned and the type of L chain was identified. We screened 364 cells which included 56R⁺ and 56R^– B cells from B6.56R and BALB/c.56R mice (Table Iand Fig. 2A). Among these 356 cells, the vast majority (97.8%) had a L chain product (Table I). Two different in-frame L chain messages were found in 48 cells (12.9%) (Table I). Two PCR products were found in a larger fraction of cells, yet, in most cases, the two PCR products were identical, in that V21D L chain was often amplified using both the specific primer for V21D and the degenerate primer V(S). These cells, therefore, yielded B cells with only one L chain (Table I). The frequency of B cells with in-frame dual L chain was higher in B6.56R than in BALB/c.56R among B cells from the follicular zone (p = 0.021) but not from the MZ compartment. A significant increase of B cells with in-frame dual L chain was also found in B6.56R as compared with BALB/c.56R among B cells from the follicular zone (p = 2.3 x 10^–3) when only IgM-56R⁺ B cells were analyzed (supplemental figure 2A, data online at www.phys.huji.ac.il/eldadb/Supplement.pdf). In the B6.56R, in-frame B cells were distributed equally in the MZ (15.5%) and the follicular compartment (16%). In contrast, in the BALB/c.56R, we found a significantly (p = 0.036) higher percentage of in-frame dual B cells in the MZ (14.7%) as compared with the follicular compartment (5.4%) (Fig. 2A). This increase also held true and was statistically significant (p = 0.04) when only IgM-56R⁺ B cells were analyzed (supplemental figure 2A, data online at www.phys.huji.ac.il/eldadb/Supplement.pdf).

View this table: [in this window] [in a new window]   Table I. Single B cells with message for dual L chaina

BCRs with polyreactive L chain can result in L chain inclusion

One way by which editing functions is by inclusion of L chain genes. It has been proposed that tolerance is accomplished by "diluting" the autoreactivity of one receptor by coexpressing a second receptor (12, 13). We were interested in testing whether polyreactivity as conferred by V38C L chain leads to a breakdown of allelic exclusion in L chain genes. We focused our attention to B cells with in-frame V38C L chain and analyzed the frequency of dual L chain B cells in this group. We compared the results for dual L chain B cells with V38C to dual L chain B cells with V21D (Fig. 2B).

In the 364 analyzed B cells, which included 56R⁺ and 56R^– B cells, we found a significant difference in the frequency of allelic inclusion in B cells with V38C as compared with B cells with V21D (Fig. 2B). In all analyzed cells, 69 B cells had the V38C L chain and, among these, 41 (59%) had a second in-frame L chain rearrangement. In contrast, 100 B cells had a V21D L chain, but only 26 (26%) had a second in-frame L chain rearrangement (Fig. 2B). The same finding held true and reached statistical significance (p = 9 x 10^–4) for the fraction of 56R⁺ B cells only (supplemental figure 2B, data online at www.phys.huji.ac.il/ eldadb/Supplement.pdf). The strain-specific differences were small but there was a slightly higher percentage of dual V38C B cells in BALB/c.56R (69%) as compared with B6.56R (57%) and this difference was amplified in B cells from the MZ compartment (p = 0.124; not significant) (Fig. 2B). Interestingly, we found a large group of dual L chain B cells that had rearranged both editor L chains V38C and V21D (Table I). Of the dual L chain B cells examined, V38C together with V21D was found in 15 of 48 dual L chain B cells from both mice (31.1%) (Table I). The percentage was slightly higher in 56R BALB/c (38.9%) than in B6.56R (27.5%). Additionally, we found strain-specific differences in the type of L chain that was coexpressed with V38C in dual L chain B cells. In the B6.56R mouse, B cells with V38C mainly coexpressed a noneditor L chain: 22 of 30 (73.3%) dual V38C cells expressed a noneditor L chain and only 8 of 30 (26.6%) coexpressed V21D (data not depicted). In the BALB/c.56R, only 4 of 11 (36.3%) B cells with V38C coexpressed a noneditor L chain and 7 of 11 (63.6%) B cells coexpressed V21D L chain (data not depicted). In sum, we found that B cells with BALB/c.56R rarely use the polyreactive incomplete editor V38C, and if V38C is found, it is allelically included with the complete editor L chain V21D. In contrast, B6.56R has a more polyreactive V38C L chain but has limited ways to neutralize them because of the low frequency of V21D L chain. We interpret our findings such that efficient neutralization of polyreactive V38C BCRs by effective allelic inclusion with V21D L chain might be in part responsible for the healthy state of BALB/c.56R that is characterized by only few autoantibodies as compared with the B6.56R (25, 26, 32). In contrast, in the B6.56R, higher titers of serum autoantibody might be the result of limited L chain allelic inclusion of incompletely edited receptors with V38C.

L chain isotypic inclusion has been described for L chain in the 56R mouse (17, 20, 22, 23); however, the detailed analysis of these cells and their compartmental distribution is missing. To determine the scope of isotypic inclusion in B cells with L chain, we analyzed cells with L chain and determined the frequency and type of L chains that were rearranged in the same cell. In the pool of 46 cells with L chain from B6.56R and BALB/c.56R, 43 L chains were accompanied by a L chain (Table II). Sequencing analysis of all L chain identified 11 in-frame L chain products. In the group of 11 L chains, we could identify eight cells that had an in-frame product. The coexpressed L chain often was the "editor" V38C or V21D (data not shown). A large percentage of L chains was out of frame (Fig. 3D) and also these B cells had a L chain. We found an equal distribution of dual L chain B cells in the tested compartments of B6.56R and BALB/c.56R and counted the same number of in-frame dual B cells in the 56R⁺ and 56R^– fraction of cells (Table II). Hence, B cells with 1 L chain represent a population in which isotypic exclusion is broken. These dual L chain B cells were also found in the follicular compartment (Fig. 2C) and, as we have shown before, become activated and secrete autoantibodies in cGvH-induced autoimmunity (20).

View this table: [in this window] [in a new window]   Table II. Single B cells with message for dual L chaina

J gene usage in L chain products

Several works have shown that J genes 3' of the J cluster are more likely to associate with the V gene during L chain rearrangement (39) (M. Schlissel, personal communication). A computational model for such a sequential order of J genes has been described before and is shown in this work by introducing parameters that consider in-frame and out-of-frame L chain products to be formed (Fig. 3C). It has been described that L chain receptor editing leads to a bias toward downstream J genes (40, 41). Thus, we were interested in whether incompletely edited B cells and B cells with dual L chain receptor were especially subject to receptor editing. We analyzed the J genes in noneditor L chains, the V38C L chain, and the V21D L chain (Fig. 3A and supplemental table I, B–E). In the group of noneditor L chains consisting of 29 different V genes (supplemental table ID), we found an increase in the frequency from J1, J2, J4 to J5 (Fig. 3A, left). Note that in this group, all noneditor L chains were identified from dual L chain B cells. In the V38C L chain, we also found a bias to downstream J genes (Fig. 3A, middle). The majority of B cells with V38C L chain (59%) expressed an additional L chain. In contrast, V21D L chain almost exclusively used J2, very few J1 and J4, and no J5 (Fig. 3A, right). Twenty-six percent of the B cells with V21D L chain expressed an additional L chain. The usage of upstream J1/2 vs downstream J4/5 in B cells with V38C is significantly different from the usage in B cells with V21D L chain which means that B cells with V38C exhibit more distal J4/5 usage, indicative of increased receptor editing. The results held true when only IgM-56R⁺ B cells were analyzed (supplemental figure 3A, data online at www.phys.huji.ac.il/ eldadb/Supplement.pdf). We conclude that B cells with autoreactive properties such as nonedited B cells and incompletely edited B cells with V38C L chain are forced to undergo more rearrangement cycles than B cells that use the complete editor L chain V21D. Another possibility is that in B cells with dual L chain message, L chain is rearranged at random as represented in the computational model (Fig. 3B).

To exclude the possibility that certain V genes rearrange preferentially to a J, we looked at the distribution of J in the diverse group of noneditor L chains. The L chains were classified into groups of related V genes (supplemental table IE). We found an increased usage of J4 in V12 (7 V12–40/J4, 2 V12–46/J4, and 2 V12–46/J2) and a bias to J4 and J5 in V4 (2 Vaj4/J5, 1 Vad4/J4, 1 Vah4/J5, 1 Vkb4/J4, 5 Vkk4/J5, 1 Vkk4/J4, 1 Vkh4/J4, 2 Vkj4/J5, and 1 V4–57/J5), yet, in all other L chains, J usage was mixed or very few examples were found for each V gene. Hence, the data do not allow for the general conclusion that certain V genes preferentially rearrange to a particular J gene. Instead, because of the increased usage of J2, J4, J4, and J5 genes in V21D, V38C, V12, and V4 genes, respectively, we think that other processes such as selection influence the J usage of a particular V gene.

J usage in L chain-included and -excluded B cells

To test whether B cells with dual L chains behave like B cells with only one L chain with respect to J gene usage, we analyzed the usage of J genes in B cells that had a single in-frame L chain (cells with a single L chain are referred to as L chain-excluded cells) and compared these to the J usage in B cells with dual in-frame or L chain (cells with dual L chain are referred to as or L chain-included cells). We found a strong bias to J2 usage in L chain-excluded B cells and little usage of J1, J4, and J5. The distribution of J in L chain-excluded B cells strongly resembled the distribution of J genes in cells with V21D (Fig. 3A), and, indeed, 89.6% of L chain-excluded cells had rearranged V21D (see supplemental table IB). In contrast, the usage of J genes in L chain-included B cells showed less J2 and more J4 and J5 compared with L chain-excluded cells when all cells (IgM-56R^– and IgM-56R⁺) were analyzed (Fig. 4) and the same pattern was confirmed when only IgM-56R⁺ B cells were analyzed (supplemental figure 4, data online at www.phys.huji.ac.il/eldadb/Supplement.pdf). The distribution of J genes in L chain-included cells did not resemble the distribution of J genes in noneditor L chains from L chain-included B cells (Fig. 3A). Further analysis showed that L chain-included B cells are "hybrid cells" in that they carry one noneditor L chain and one editor L chain: of 56 L chain-included B cells, 25 cells (44.6%) had one V21D rearrangement that was mostly rearranged to J2, and 39 cells (69.6%) used V38C (data not depicted). In summary, we find that most L chain-excluded B cells in the 56R-transgenic mouse use the complete editor L chain V21D. In L chain-included B cells, the incomplete editor L chain V38C or a noneditor L chain is often accompanied by V21D.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Dual L chain receptor B cells

Receptor editing is a mechanism by which autoreactive receptors are replaced by nonautoreactive receptors. An example of an efficiently edited autoreactive receptor is the 56R H chain transgene paired with V21D L chain. V21D vetoes binding to dsDNA and other self-Ags (20). Yet, receptor editing has a dangerous side effect because it can be incomplete and generate polyreactive, anti-self Abs (20). An example of an incompletely edited receptor is 56R paired with V38C L chain. This combination has high relative affinity to self-proteins as well as to dsDNA (20).

Incomplete editors such as V38C raise the question of whether it is appropriate to call V38C an "editor" L chain. However, the tolerance threshold for incomplete editor L chains is defined by genetic predisposition as seen by the L chain repertoire shift in BALB/c.56R and B6.56R and the "lupus-resistant" strain background, BALB/c, is stringent whereas the more "lupus-susceptible" strain background, B6, is less efficient (27, 28, 29, 30, 31). In BALB/c.56R, lupus resistance concurs with a high percentage of B cells with the complete editor L chain V21D. In contrast, in the lupus-susceptible strain, B6.56R, a high percentage of B cells with the incomplete editor V38C L chain is found (Fig. 1). The shift in L chain usage is not a natural property of the B6 and BALB/c mouse but is caused by the 56R H chain transgene and the genetic differences between the strains B6 and BALB/c subsequently lead to an L chain repertoire shift. Analysis of V38C in B6 and V21D L chain in BALB/c nontransgenic mice showed no overrepresentation of these L chains as compared with other V genes. In the nontransgenic B6, V38C is found in 0.7% of peripheral B cells (26), and in the BALB/c mouse, the whole of the V21 family (eight members) is represented with no more than 4% among splenic B cells (36). Further evidence for the genetic influence on the L chain repertoire comes from B6.56R mice which lack the Fcr2b. In this mouse, the peripheral L chain repertoire is further shifted toward the usage of polyreactive V38C L chain as compared with the B6.56R mouse. Interestingly, B6.56R/Fcr2b^–/– mice show IgG and complement C3 deposition in the kidney glomeruli and succumb to autoimmune disease (26). Increased numbers of polyreactive B cells have also been observed in human lupus erythematosus as compared with healthy individuals. It has been shown that checkpoints to eliminate polyreactive B cells upon emigration from the bone marrow and maturation of B cells in the periphery are defect (8, 9). Even though coincidence of polyreactive B cells with autoimmune diseases is shown, proof is missing for a direct involvement of polyreactive B cells in autoimmunity or in disease onset. Some work even suggests the contrary in that polyreactive B cells prevent the onset of experimental autoimmune disease (42). Polyreactive or natural (auto) Abs are considered to have beneficial functions and are important in the first line of defense against pathogens through the activation of immune complement and the formation of immune complexes which are delivered to follicular dendritic cells (reviewed in Ref. 43). Natural autoantibodies are also involved in the clearance of apoptotic cells, thereby limiting the circulation of autoantigens and reducing the risk of autoimmunity (44). Thus, the concurrence of polyreactive autoantibodies and autoimmunity does not show polyreactivity to be causal for the onset of disease. Yet, deregulation and overexpression of polyreactive anti-DNA autoantibodies may bear dangerous features and promote autoimmune phenotypes.

A way of creating tolerance is the coexpression of two L chains (11, 13), and we show here that at least one of these L chains is an editor L chain. Coexpression of two L chains leads to a partially edited B cell. Incompletely edited receptors can be further regulated by allelic inclusion and, here again, the genetic background of the mouse is important: in the BALB/c.56R mouse, the presence of V21D L chain may facilitate the silencing of autoreactive/polyreactive receptors. In contrast, in the B6.56R mouse, the main editor L chain V38C has polyreactive/self-reactive properties itself.

How do these partially edited (or included) B cells come about? Do both alleles rearrange simultaneously or do they rearrange sequentially? BrdU labeling experiments showed that allelically included B cells take longer to develop and appear later in the immature B cell compartment than allelically excluded B cells (11). The time the first dual L chain B cell appears in the immature B cell pool takes twice as long as a B cell needs to edit a single receptor (45, 46, 47). This favors subsequent rearrangement of alleles and suggests that dual receptor B cells arise by receptor editing. Simultaneous rearrangement is unlikely. It has been shown that locus activation by Ig enhancers is quite low (around 10%), and therefore the probability that a cell has two active loci is 1%. Only one-third of these rearrangements are in frame, which results in a frequency of cells with two in-frame gene rearrangements of 0.3% (48). Therefore, it is unlikely that dual L chain receptor B cells occur simply by accident. In our study, the frequency of dual L chain receptor B cells in the 56R mouse is 12% (Table I).

We find that the generation of dual L chain receptor B cells is directed in that it occurs primarily when receptor editing is incomplete as is the case in 56R/V38C B cells. We hypothesize that receptor inclusion may be a self-Ag-driven tolerance mechanism because B cells with V38C L chain often coexpress a second L chain, and two explanatory models are available for the generation of dual L chain B cells (1). The B cell could have first rearranged a noneditor L chain in an attempt to silence the 56R H chain thereby using up all available J genes; to escape deletion the B cell rearranges an editor L chain on the other allele resulting in the generation of a second receptor (2). Alternatively, the B cell first rearranged an editor L chain, V21D or V38C, to silence the 56R H chain. This can result in the successful neutralization of 56R as is the case in V21D B cells, yet this first editing attempt can also be incomplete and result in the incompletely edited polyreactive receptor 56R/V38C. In case of incomplete editing, rearrangement of a second L chain rescues the cell from clonal deletion. We favor the latter. First, the frequency of dual L chain B cells is higher in B cells with V38C than in B cells with V21D, and we interpret this to be the result of autoreactivity of B cells with V38C (Fig. 2B). Second, B cells with V38C are biased to downstream J genes such as if V38C B cells already muddled through several attempts in search of a complete editor L chain (Fig. 3A). The bias to downstream J genes has been shown to be a property of receptor editing (40, 41). Caution is warranted in interpreting the finding that polyreactivity infers L chain inclusion in the cell, because the degenerate primer V(S) might not amplify all L chains amplified equally well (supplemental table IA). However, a competition effect would skew the relative frequencies uniformly across all compartments in both mice, B6.56R and BALB/c.56R. Consequently, differences observed between different compartments and between both mice would be retained even after compensating for this skewing. Therefore, we argue that L chain inclusion is truly enhanced in incompletely edited B cells.

Another way of creating dual L chain receptor B cells is by isotype inclusion as recognized by the coexpression of and L chain on the cell surface. It has been shown that 56R with V1 has high relative affinity for dsDNA (21), and the self-reactivity of this combination was thought to lead to expression of a L chain (22, 23). In the 56R mouse, such dual receptors are expressed on the cell surface (17, 20, 22, 23), and hybridoma studies showed that V38C was a common L chain accompanying V1 (17). In light of our new findings, we think that V1 rearrangement has taken place after rearrangement of the incomplete editor V38C. In support of this idea, the rearrangement of L chain is the ultimate bias in strongly edited B cells (4, 49, 50). Even though L chain lacks the ability of secondary rearrangement due to the lack of rearrangement signals, receptor editing may be acting in parallel on the locus and could explain the abundant expression of "editor" L chains such as V21D and V38C with V1.

Coexpressed, autoreactive, and a nonautoreactive receptor both deliver signals into the cell, one Ag-induced and one tonic signal (31) and these B cells undergo differentiation as well as receptor editing (13). This also true for dual receptor B cells in the 56R mouse, except that probably both receptors deliver Ag-induced signals. The existence of B cells with 1 and V38C therefore demonstrates that B cells with two self-reactive receptors can become part of the splenic B cell compartment.

Our work suggests that partially edited B cells "dilute" anti-self-specificity of one receptor by coexpressing a second L chain, for example, if a V38C B cell rearranges V21D as the second L chain. Expression of two L chains can be dangerous for the immune system and help self-reactive B cells to bypass central tolerance mechanisms. As discussed above, dual receptor B cells can have two self-reactive receptors. In this case, the differences in the specificities of the two receptors may lead to sufficiently weak avidity for a given (self-) Ag so that the B cell can leave the bone marrow. Each receptor contributes to selection and reduces the selective pressure as is the case if the B cell had just one receptor. Beneficial effects of dual receptors have been shown in T cells in which dual -chain expression extends the available repertoire of TCR specific for foreign Ags (51), a process suggested to contribute to allorecognition (52). The second TCR rescues the cell from death by neglect (53).

Self-reactive B cells that reach the spleen can be rendered harmless for the immune system in other ways: unresponsiveness to autoantigens by ignorance and anergy (1, 6) have been well-documented mechanisms and prevent autoimmunity in newly generated B cells (1, 6). Another path to tolerance is peripheral sequestration of potentially pathogenic autoreactive B cells into immunological niches such as the MZ. Weakly self-reactive B cells are found in the MZ and sequestration in the MZ was suggested as a mechanism of peripheral tolerance (54, 55, 56). We demonstrate here that incompletely edited B cells are also regulated by sequestration into the MZ (Fig. 1B).

In the B6.56R, the majority of B cells with V38C was found in the MZ (Fig. 1, B and C) and serum autoantibody titers to dsDNA and histone in the B6.56R (32) may be due to the location and easy activation of MZ B cells. Yet, in our study, we observe that a fraction of 56R/V38C B cells also locate to the follicular B cell compartment (Fig. 1, B and C). Even though a direct transition to the follicle was previously suggested for MZ B cells upon activation (57), proof of this hypothesis is still missing. The location of 56R B cells in the follicular zone compartment could explain the ease with which 56R B cells differentiate into plasma cells and produce autoantibodies of the IgM and the IgG isotype (25, 26, 32). In sum, susceptibility to develop an autoimmune phenotype in the B6.56R is accompanied by at least three findings: 1) inefficient ability to control the expression of V38C with the resulting poor ability to silence unedited or incompletely edited receptors by coexpression of the complete editor V21D (Fig. 1 and Table I); 2) the location of 56R/V38C B cells to the follicular B cell compartment (Fig. 1, B and C); and 3) plasma cell differentiation with the production of autoantibodies (25, 26, 32).

In the BALB/c.56R mouse, we find that B cells with V21D reside in the MZ B cell compartment (Fig. 1B). This could be due to the following reasons: a large percentage of MZ B cells in the 56R mouse are not self-reactive. The MZ has been shown to serve as a bottleneck container in many transgenic mouse models in which B cell development is impaired due to a transgenic receptor (as reviewed in Ref. 58). In these transgenic models, the enlargement of the MZ compartment is not related to self-specificity of the BCR transgene. An example is the anti-HEL H chain-transgenic mouse (6). Another possibility is that 56R B cells with V21D are weakly self-reactive to an unknown autoantigens. If 56R/V21D has weak self-reactive features, the distribution of BCRs in the BALB/c.56R could also be explained by the signal-strength-hypothesis. According to this theory, B cells with weak self-reactive properties are selected into the MZ, whereas B cells with strong self-reactivity are selected into the follicle (6, 59, 60). We indeed see noneditor L chain in the follicular compartment of BALB/c.56R mice (Fig. 1C). Yet, the lack of autoantibodies in the serum of BALB/c.56R mice suggests that postfollicular tolerance checkpoints are intact and do not allow autoreactive B cells to differentiate into Ab-producing plasma cells (61, 62).

Another way to bypass central tolerance in the bone marrow is intracellular retention of the incompletely edited receptor 56R/V38C. We found recently that 56R/V38C bound to an intracellular Ag in the Golgi and therefore surface expression was delayed. Yet, 56R/V38C was eventually expressed on the cell surface. B cells with 56R/V38C were sequestered in the MZ (S. N. Khan, E. J. Witsch, N. G. Goodman, A. K. Panigrahi, C. Chen, Y. Yang, A. M. Cline, J. Erikson, M. Weigert, E. T. L. Prak, and M. Radic, submitted for publication). Also other work has shown that B cells reactive to an ubiquitously present self-Ag hide their autoreactivity by intracellular retention of the autoreactive receptor. Upon emigration from the bone marrow these receptors are invisible to the immune system. The autoreactive receptor is internalized but seems to be ready to go as these mice show autoantibody titers with increased age (13).

J usage in L chain rearrangements

A large fraction of newly rearranged Ig genes are nonfunctional and the necessity to shape the peripheral B cell repertoire by receptor editing is obvious, assuming that two-thirds of the rearrangements are out of frame and a considerable number of pseudogenes lead to nonfunctional L chain rearrangements. In the 56R mouse, the anti-DNA H chain transgene adds to the low frequency of functional rearrangements, and it has been estimated that at least 50% of the B cells are subject to L chain receptor editing (63, 64). Usage of downstream J genes is thought to be a general sign of receptor editing (40, 41).

Some studies addressed the question of whether DNA rearrangement occurs with equal frequency at each of the J genes and have shown that the proximal J gene 5' of the J cluster is more likely to rearrange than the 3' J gene. In B cells that were unperturbed by antigenic selection, the usage of J genes followed a sequential order, starting with J1. The data sets analyzed in this work, however, were rather small (39). Confirmation for a sequential usage of J genes comes from experiments in which rearrangement was induced in a bulk of B cells that are germline at both alleles. This resulted in a striking bias of dsDNA recombination signal sequence breaks at J1 (M. Schlissel, personal communication). In none of these experiments did pre-existing autoreactivity or Ag specificity influence the usage of J. A bias to upstream J could also be observed in the 56R mouse. This upstream bias was found in hybridoma panels from BALB/c.56R mice. Yet, most of the hybridomas used the editor L chain V21D which almost always rearranged to J2 (N. Prak, unpublished observation). To explain the distribution of J genes in the BCR repertoire in BALB/c.56R, a mathematical model was suggested previously (35). According to this model, V and J genes are rearranged in a "semisequential" order, and by this model the strong bias of J2 in the L chain repertoire of the BALB/c.56R was explained. Generally, in a sequential process downstream J genes are less likely to participate in a rearrangement because they are excluded once a J gene upstream has rearranged in frame (Fig. 3C). Yet, as a consequence of receptor editing the bias to upstream J genes can be reversed and shifted to downstream J genes because downstream J genes are still available during secondary rearrangement. In this study, we find a shift to downstream J4 and J5 in incompletely edited B cells with V38C as well as in B cells with noneditor L chains. We conclude that B cells with V38C and with noneditor L chains are preferentially subject to L chain receptor editing (Fig. 3A, left and middle group). The same results were obtained when only 56R⁺ B cells were analyzed (supplemental figure 3A, data online at www.phys.huji.ac.il/eldadb/Supplement.pdf).

Yet, we would like to suggest another explanation for the observed downstream usage of J genes. The observed bias to downstream J genes in V38C B cells and in B cells with a noneditor L chain (Fig. 3A) is consistent with random rearrangement as it is represented by a computational model (Fig. 3B). In the random process, J genes upstream from a J gene, which has participated in a rearrangement, are excluded from participating again and making them less likely to form products (Fig. 3B). It is possible that B cells with V38C B cells and B cells with a noneditor L chain have a restricted time window in which they have to seclude the rearrangement process of a second L chain. As discussed above, rearrangement of a second allele takes time, and we propose that this time restriction can lead to random rearrangement, as computed in Fig. 3B.

A possible explanation for the J2 skewing in V21D L chain is the rate of B cell generation. Cells that successfully express an editor on the first try of rearrangement are rare, but because they are generated in a single step, they are produced at a higher rate than cells that must muddle through several rounds of editing rearrangement. In the case of V21D, rearrangement to downstream J may be even less frequent because of the placement of the gene proximal to the J cluster (65).

	Acknowledgments

 
We thank Drs. Martin Weigert, Chandra Mohan, Gudrun Debes, Hidehiro Fukuyama, Yoram Louzoun, and Salar Khan for critical reading of the manuscript and helpful discussions. We thank the Sequencing Facility of the University of Chicago (Chicago, IL) for sequencing and the Bioinformatics Unit of the Weizmann Institute (Rehovot, Israel).

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

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

¹ This work was supported by National Institutes of Health Grant GM02964-32, by a grant from the Lupus Research Institute, and by the Israeli Science Foundation.

² Address correspondence and reprint requests to Dr. Esther J. Witsch at the current address: Department of Biological Regulation, Weizmann Institute of Science, Candiotty Building, POB 26, Rehovot 76100, Israel. E-mail address: esther.witsch{at}weizmann.ac.il

³ Abbreviations used in this paper: MZ, marginal zone; cGvH, chronic graft-vs-host reaction; FR, framework region.

⁴ The online version of this article contains supplemental material.

Received for publication August 13, 2007. Accepted for publication January 9, 2008.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Nossal, G. J., B. L. Pike. 1980. Clonal anergy: persistence in tolerant mice of antigen-binding B lymphocytes incapable of responding to antigen or mitogen. Proc. Natl. Acad. Sci. USA 77: 1602-1606. [Abstract/Free Full Text]
2. Adams, E., A. Basten, C. C. Goodnow. 1990. Intrinsic B-cell hyporesponsiveness accounts for self-tolerance in lysozyme/anti-lysozyme double-transgenic mice. Proc. Natl. Acad. Sci. USA 87: 5687-5691. [Abstract/Free Full Text]
3. Gay, D., T. Saunders, S. Camper, M. Weigert. 1993. Receptor editing: an approach by autoreactive B cells to escape tolerance. J. Exp. Med. 177: 999-1008. [Abstract/Free Full Text]
4. Tiegs, S. L., D. M. Russell, D. Nemazee. 1993. Receptor editing in self-reactive bone marrow B cells. J. Exp. Med. 177: 1009-1020. [Abstract/Free Full Text]
5. Nemazee, D., K. Buerki. 1989. Clonal deletion of autoreactive B lymphocytes in bone marrow chimeras. Proc. Natl. Acad. Sci. USA 86: 8039-8043. [Abstract/Free Full Text]
6. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael, et al 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334: 676-682. [Medline]
7. Nemazee, D.. 2006. Receptor editing in lymphocyte development and central tolerance. Nat. Rev. Immunol. 6: 728-740. [Medline]
8. Wardemann, H., S. Yurasov, A. Schaefer, J. W. Young, E. Meffre, M. C. Nussenzweig. 2003. Predominant autoantibody production by early human B cell precursors. Science 301: 1374-1377. [Abstract/Free Full Text]
9. Yurasov, S., H. Wardemann, J. Hammersen, M. Tsuiji, E. Meffre, V. Pascual, M. C. Nussenzweig. 2005. Defective B cell tolerance checkpoints in systemic lupus erythematosus. J. Exp. Med. 201: 703-711. [Abstract/Free Full Text]
10. Giachino, C., E. Padovan, A. Lanzavecchia. 1995. ⁺⁺ dual receptor B cells are present in the human peripheral repertoire. J. Exp. Med. 181: 1245-1250. [Abstract/Free Full Text]
11. Casellas, R., Q. Zhang, N. Y. Zheng, M. D. Mathias, K. Smith, P. C. Wilson. 2007. Ig allelic inclusion is a consequence of receptor editing. J. Exp. Med. 204: 153-160. [Abstract/Free Full Text]
12. Huang, H., J. F. Kearney, M. J. Grusby, C. Benoist, D. Mathis. 2006. Induction of tolerance in arthritogenic B cells with receptors of differing affinity for self-antigen. Proc. Natl. Acad. Sci. USA 103: 3734-3739. [Abstract/Free Full Text]
13. Liu, S., M. G. Velez, J. Humann, S. Rowland, F. J. Conrad, R. Halverson, R. M. Torres, R. Pelanda. 2005. Receptor editing can lead to allelic inclusion and development of B cells that retain antibodies reacting with high avidity autoantigens. J. Immunol. 175: 5067-5076. [Abstract/Free Full Text]
14. Sirac, C., C. Carrion, S. Duchez, I. Comte, M. Cogne. 2006. Light chain inclusion permits terminal B cell differentiation and does not necessarily result in autoreactivity. Proc. Natl. Acad. Sci. USA 103: 7747-7752. [Abstract/Free Full Text]
15. Erikson, J., M. Z. Radic, S. A. Camper, R. R. Hardy, C. Carmack, M. Weigert. 1991. Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature 349: 331-334. [Medline]
16. Chen, C., Z. Nagy, E. L. Prak, M. Weigert. 1995. Immunoglobulin heavy chain gene replacement: a mechanism of receptor editing. Immunity 3: 747-755. [Medline]
17. Li, H., Y. Jiang, E. L. Prak, M. Radic, M. Weigert. 2001. Editors and editing of anti-DNA receptors. Immunity 15: 947-957. [Medline]
18. Chen, C., M. Z. Radic, J. Erikson, S. A. Camper, S. Litwin, R. R. Hardy, M. Weigert. 1994. Deletion and editing of B cells that express antibodies to DNA. J. Immunol. 152: 1970-1982. [Abstract]
19. Li, Y., Y. Louzoun, M. Weigert. 2004. Editing anti-DNA B cells by Vx. J. Exp. Med. 199: 337-346. [Abstract/Free Full Text]
20. Witsch, E. J., H. Cao, H. Fukuyama, M. Weigert. 2006. Light chain editing generates polyreactive antibodies in chronic graft-versus-host reaction. J. Exp. Med. 203: 1761-1772. [Abstract/Free Full Text]
21. Radic, M. Z., J. Mackle, J. Erikson, C. Mol, W. F. Anderson, M. Weigert. 1993. Residues that mediate DNA binding of autoimmune antibodies. J. Immunol. 150: 4966-4977. [Abstract]
22. Li, Y., H. Li, D. Ni, M. Weigert. 2002. Anti-DNA B cells in MRL/lpr mice show altered differentiation and editing pattern. J. Exp. Med. 196: 1543-1552. [Abstract/Free Full Text]
23. Li, Y., H. Li, M. Weigert. 2002. Autoreactive B cells in the marginal zone that express dual receptors. J. Exp. Med. 195: 181-188. [Abstract/Free Full Text]
24. Doyle, C. M., J. Han, M. G. Weigert, E. T. Prak. 2006. Consequences of receptor editing at the lambda locus: multireactivity and light chain secretion. Proc. Natl. Acad. Sci. USA 103: 11264-11269. [Abstract/Free Full Text]
25. Sekiguchi, D. R., L. Yunk, D. Gary, D. Charan, B. Srivastava, D. Allman, M. G. Weigert, E. T. Prak. 2006. Development and selection of edited B cells in B6.56R mice. J. Immunol. 176: 6879-6887. [Abstract/Free Full Text]
26. Fukuyama, H., F. Nimmerjahn, J. V. Ravetch. 2005. The inhibitory Fc receptor modulates autoimmunity by limiting the accumulation of immunoglobulin G⁺ anti-DNA plasma cells. Nat. Immunol. 6: 99-106. [Medline]
27. Rozzo, S. J., T. J. Vyse, C. G. Drake, B. L. Kotzin. 1996. Effect of genetic background on the contribution of New Zealand Black loci to autoimmune lupus nephritis. Proc. Natl. Acad. Sci. USA 93: 15164-15168. [Abstract/Free Full Text]
28. Vidal, S., D. H. Kono, A. N. Theofilopoulos. 1998. Loci predisposing to autoimmunity in MRL-Fas lpr and C57BL/6-Fas^lpr mice. J. Clin. Invest. 101: 696-702. [Medline]
29. Santiago, M. L., C. Mary, D. Parzy, C. Jacquet, X. Montagutelli, R. M. Parkhouse, R. Lemoine, S. Izui, L. Reininger. 1998. Linkage of a major quantitative trait locus to Yaa gene-induced lupus-like nephritis in (NZW x C57BL/6)F₁ mice. Eur. J. Immunol. 28: 4257-4267. [Medline]
30. Morel, L., X. H. Tian, B. P. Croker, E. K. Wakeland. 1999. Epistatic modifiers of autoimmunity in a murine model of lupus nephritis. Immunity 11: 131-139. [Medline]
31. Bolland, S., Y. S. Yim, K. Tus, E. K. Wakeland, J. V. Ravetch. 2002. Genetic modifiers of systemic lupus erythematosus in FcRIIB^–/– mice. J. Exp. Med. 195: 1167-1174. [Abstract/Free Full Text]
32. Liu, Y., L. Li, K. R. Kumar, C. Xie, S. Lightfoot, X. J. Zhou, J. F. Kearney, M. Weigert, C. Mohan. 2007. Lupus susceptibility genes may breach tolerance to DNA by impairing receptor editing of nuclear antigen reactive B cells. J. Immunol. 179: 1340-1352. [Abstract/Free Full Text]
33. Schlissel, M. S., D. Baltimore. 1989. Activation of immunoglobulin gene rearrangement correlates with induction of germline gene transcription. Cell 58: 1001-1007. [Medline]
34. Paul, E., J. Lutz, J. Erikson, M. C. Carroll. 2004. Germinal center checkpoints in B cell tolerance in 3H9 transgenic mice. Int. Immunol. 16: 377-384. [Abstract/Free Full Text]
35. Louzoun, Y., T. Friedman, E. Luning Prak, S. Litwin, M. Weigert. 2002. Analysis of B cell receptor production and rearrangement. Part I. Light chain rearrangement. Semin. Immunol. 14: 169-190. discussion 221–122. [Medline]
36. Kalled, S. L., P. H. Brodeur. 1991. Utilization of V families and V exons: implications for the available B cell repertoire. J. Immunol. 147: 3194-3200. [Abstract]
37. Reth, M., P. Gehrmann, E. Petrac, P. Wiese. 1986. A novel V_H to V_HDJ_H joining mechanism in heavy-chain-negative (null) pre-B cells results in heavy-chain production. Nature 322: 840-842. [Medline]
38. Reth, M. G., S. Jackson, F. W. Alt. 1986. V_HDJ_H formation and DJ_H replacement during pre-B differentiation: non-random usage of gene segments. EMBO J. 5: 2131-2138. [Medline]
39. Wood, D. L., C. Coleclough. 1984. Different joining region J elements of the murine immunoglobulin light chain locus are used at markedly different frequencies. Proc. Natl. Acad. Sci. USA 81: 4756-4760. [Abstract/Free Full Text]
40. Radic, M. Z., J. Erikson, S. Litwin, M. Weigert. 1993. B lymphocytes may escape tolerance by revising their antigen receptors. J. Exp. Med. 177: 1165-1173. [Abstract/Free Full Text]
41. Nemazee, D., M. Weigert. 2000. Revising B cell receptors. J. Exp. Med. 191: 1813-1817. [Abstract/Free Full Text]
42. Bruley-Rosset, M., L. Mouthon, Y. Chanseaud, F. Dhainaut, J. Lirochon, D. Bourel. 2003. Polyreactive autoantibodies purified from human intravenous immunoglobulins prevent the development of experimental autoimmune diseases. Lab. Invest. 83: 1013-1023. [Medline]
43. Ochsenbein, A. F., R. M. Zinkernagel. 2000. Natural antibodies and complement link innate and acquired immunity. Immunol. Today 21: 624-630. [Medline]
44. Logtenberg, T., F. M. Young, J. H. Van Es, F. H. Gmelig-Meyling, F. W. Alt. 1989. Autoantibodies encoded by the most J_H-proximal human immunoglobulin heavy chain variable region gene. J. Exp. Med. 170: 1347-1355. [Abstract/Free Full Text]
45. Casellas, R., T. A. Shih, M. Kleinewietfeld, J. Rakonjac, D. Nemazee, K. Rajewsky, M. C. Nussenzweig. 2001. Contribution of receptor editing to the antibody repertoire. Science 291: 1541-1544. [Abstract/Free Full Text]
46. Allman, D. M., S. E. Ferguson, V. M. Lentz, M. P. Cancro. 1993. Peripheral B cell maturation. II. Heat-stable antigen^high splenic B cells are an immature developmental intermediate in the production of long-lived marrow-derived B cells. J. Immunol. 151: 4431-4444. [Abstract]
47. Rocha, B., C. Penit, C. Baron, F. Vasseur, N. Dautigny, A. A. Freitas. 1990. Accumulation of bromodeoxyuridine-labeled cells in central and peripheral lymphoid organs: minimal estimates of production and turnover rates of mature lymphocytes. Eur. J. Immunol. 20: 1697-1708. [Medline]
48. Schlissel, M.. 2002. Allelic exclusion of immunoglobulin gene rearrangement and expression: why and how?. Semin. Immunol. 14: 207-212;. discussion 225–206. [Medline]
49. Retter, M. W., D. Nemazee. 1998. Receptor editing occurs frequently during normal B cell development. J. Exp. Med. 188: 1231-1238. [Abstract/Free Full Text]
50. Hertz, M., D. Nemazee. 1997. BCR ligation induces receptor editing in IgM⁺IgD^– bone marrow B cells in vitro. Immunity 6: 429-436. [Medline]
51. He, X., C. A. Janeway, Jr, M. Levine, E. Robinson, P. Preston-Hurlburt, C. Viret, K. Bottomly. 2002. Dual receptor T cells extend the immune repertoire for foreign antigens. Nat. Immunol. 3: 127-134. [Medline]
52. Padovan, E., G. Casorati, P. Dellabona, S. Meyer, M. Brockhaus, A. Lanzavecchia. 1993. Expression of two T cell receptor chains: dual receptor T cells. Science 262: 422-424. [Abstract/Free Full Text]
53. Hardardottir, F., J. L. Baron, C. A. Janeway, Jr. 1995. T cells with two functional antigen-specific receptors. Proc. Natl. Acad. Sci. USA 92: 354-358. [Abstract/Free Full Text]
54. Qian, Y., H. Wang, S. H. Clarke. 2004. Impaired clearance of apoptotic cells induces the activation of autoreactive anti-Sm marginal zone and B-1 B cells. J. Immunol. 172: 625-635. [Abstract/Free Full Text]
55. Chen, X., F. Martin, K. A. Forbush, R. M. Perlmutter, J. F. Kearney. 1997. Evidence for selection of a population of multi-reactive B cells into the splenic marginal zone. Int. Immunol. 9: 27-41. [Abstract/Free Full Text]
56. Fulcher, D. A., A. B. Lyons, S. L. Korn, M. C. Cook, C. Koleda, C. Parish, B. Fazekas de St. Groth, A. Basten. 1996. The fate of self-reactive B cells depends primarily on the degree of antigen receptor engagement and availability of T cell help. J. Exp. Med. 183: 2313-2328. [Abstract/Free Full Text]
57. Cinamon, G., M. Matloubian, M. J. Lesneski, Y. Xu, C. Low, T. Lu, R. L. Proia, J. G. Cyster. 2004. Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone. Nat. Immunol. 5: 713-720. [Medline]
58. Martin, F., J. F. Kearney. 2002. Marginal-zone B cells. Nat. Rev. Immunol. 2: 323-335. [Medline]
59. Cariappa, A., M. Tang, C. Parng, E. Nebelitskiy, M. Carroll, K. Georgopoulos, S. Pillai. 2001. The follicular versus marginal zone B lymphocyte cell fate decision is regulated by Aiolos, Btk, and CD21. Immunity 14: 603-615. [Medline]
60. Pillai, S.. 1999. The chosen few: positive selection and the generation of naive B lymphocytes. Immunity 10: 493-502. [Medline]
61. Ekland, E. H., R. Forster, M. Lipp, J. G. Cyster. 2004. Requirements for follicular exclusion and competitive elimination of autoantigen-binding B cells. J. Immunol. 172: 4700-4708. [Abstract/Free Full Text]
62. Mandik-Nayak, L., S. Seo, A. Eaton-Bassiri, D. Allman, R. R. Hardy, J. Erikson. 2000. Functional consequences of the developmental arrest and follicular exclusion of anti-double-stranded DNA B cells. J. Immunol. 164: 1161-1168. [Abstract/Free Full Text]
63. Novobrantseva, T., S. Xu, J. E. Tan, M. Maruyama, S. Schwers, R. Pelanda, K. P. Lam. 2005. Stochastic pairing of Ig heavy and light chains frequently generates B cell antigen receptors that are subject to editing in vivo. Int. Immunol. 17: 343-350. [Abstract/Free Full Text]
64. Nemazee, D.. 1996. Antigen receptor "capacity" and the sensitivity of self-tolerance. Immunol. Today 17: 25-29. [Medline]
65. Brekke, K. M., W. T. Garrard. 2004. Assembly and analysis of the mouse immunoglobulin gene sequence. Immunogenetics 56: 490-505. [Medline]

This article has been cited by other articles:

				E. Makdasi, R. Fischel, I. Kat, and D. Eilat Autoreactive Anti-DNA Transgenic B Cells in Lupus-Prone New Zealand Black/New Zealand White Mice Show Near Perfect L Chain Allelic Exclusion J. Immunol., May 15, 2009; 182(10): 6143 - 6148. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Witsch, E. J. Articles by Bettelheim, E. Search for Related Content PubMed PubMed Citation Articles by Witsch, E. J. Articles by Bettelheim, E. Pubmed/NCBI databases Gene