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Editing Rescues Autoreactive B Cells Destined for Deletion in Mice Transgenic for a Dual Specific Anti-Laminin Ig¹

Graham F. Brady^2,*, Kendra L. Congdon^2,*, Amy G. Clark^*, Faustina N. A. Sackey^*, Earl H. Rudolph^*, Marko Z. Radic and Mary H. Foster^3,*

^* Departments of Medicine, Duke University and Durham Veterans Administration Medical Centers, Durham, NC 27710; and Molecular Sciences, University of Tennessee, Memphis, TN 38163

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We explored mechanisms involved in B cell self-tolerance in a double- and triple-transgenic mouse model bearing the LamH-Cµ Ig H chain conventional transgene and a gene-targeted replacement for a functional V8J5 L chain gene. Whereas the H chain is known to generate anti-laminin Ig in combination with multiple L chains, the H + L Ig binds ssDNA in addition to laminin. Immune phenotyping indicates that H + L transgenic B cells are regulated by clonal deletion, receptor editing via secondary rearrangements at the nontargeted allele, and anergy. Collectively, the data suggest that multiple receptor-tolerogen interactions regulate autoreactive cells in the H + L double-transgenic mice. Generation of H + LL triple-transgenic mice homozygous for the targeted L chain to exclude secondary rearrangements resulted in profound B cell depletion with absence of mature B cells in the bone marrow. We propose that the primary tolerogen of dual reactive B cells in this model is not ssDNA, but a strongly cross-linking tolerogen, presumably basement membrane laminin, that triggers recombination-activating gene activity, L chain editing, and deletion.

	Introduction

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Autoimmune diseases afflict most organ systems and impose an enormous burden on the health care system. Autoimmunity arises when an environmental insult superimposed on genetic susceptibility disrupts normal immune regulation. Regulatory pathways thus pose obvious targets for disease intervention. Ig and TCR transgenic (Tg)⁴ models are useful tools for deciphering these pathways and determining the mechanisms that maintain or promote breakdown of immune tolerance involving disease-associated Ig.

An area of considerable interest is the capacity of structurally distinct autoantigens to act as in vivo tolerogens. Autoantigens of diverse chemical composition, cellular localization, and tissue distribution are targeted in autoimmune diseases. It is unclear, however, whether similar heterogeneity exists in the regulatory mechanisms that control autoreactivity. This issue is particularly germane to humoral autoimmunity, because B cell receptors, like soluble Ig, recognize native Ag in tertiary conformation. In this regard, it has been demonstrated that ubiquitous cell membrane-bound or multivalent self Ag are particularly likely to induce clonal deletion or receptor editing in reactive B cells, whereas soluble oligovalent tolerogen is more likely to induce a state of anergy (1, 2, 3, 4, 5, 6, 7).

To determine whether protein epitopes within basement membrane lattices can tolerize B cells, we generated mice Tg for a lupus-derived auto-Ig H chain, LamH-Cµ, reactive with basement membrane laminin (8, 9). Laminin is a target of pathogenic autoantibodies in immune nephritis and blistering dermatoses (10, 11). Phenotypic characterization of Tg mice revealed that lupus-like anti-laminin B cells are regulated in vivo by mechanisms that include clonal deletion and clonal anergy, effectively preventing recovery of anti-laminin Ig (9). Laminin is the presumed in vivo tolerogen for many of these cells, because the Tg H chain is known to pair with diverse endogenous Ig L chains to generate anti-laminin B cells (12). Notably, however, combining the Tg H chain with a lupus-derived V4J5 L chain generated a novel pathogenic Ig that bound both DNA and laminin in vitro and produced dramatic linear renal glomerular and tubular basement membrane immune deposits in vivo (13). Soluble DNA also partially inhibits laminin binding by a subset of Tg mAb derived from the autoimmune M7 LamH Tg lineage (12), raising the possibility that a cross-reactive subset of LamH-encoded B cells is regulated by nucleic acid self Ag, not laminin.

To further explore how Ag specificity directs B cell fate in this model, we generated C57BL/6 Ig double- and triple-Tg mice by crossing LamH-Cµ H chain Tg mice (H-Tg or LamH) with L-Tg mice carrying a gene-targeted replacement for a functional V8J5 L chain gene, termed V8R. This V8 gene is similar to that used by several anti-laminin LamH-Cµ Tg mAb (12) and by several murine lupus rheumatoid factor and anti-DNA mAb (14). The V8R L chain generates anti-ssDNA Ig in combination with the lupus-derived 3H9 H chain (14), and DNA-reactive B cells from 3H9 x V8R H + L double-Tg mice are anergized in vivo (15). In this study, we report that in vitro recombination of the LamH-Cµ H chain with the V8R L chain generates anti-laminin Ab cross-reactive with ssDNA, a self Ag previously shown to tolerize primarily by inducing anergy. The phenotype of LamH x V8R H + L double-Tg and LamH x V8R/V8R triple-Tg mice indicates that dual reactive H + L B cells are regulated primarily by clonal deletion and editing. This suggests that a strongly cross-linking tolerogen other than ssDNA, such as basement membrane laminin, regulates their fate.

	Materials and Methods

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Animals

Cloning of the anti-laminin LamH-Cµ H chain construct and initial characterization of Tg mice on B6 are described elsewhere (8, 9, 13). B6 mice hemizygous for the site-directed V8J5 L chain, V8R, occupying the endogenous locus (16), generously provided by M. Weigert (Princeton University, Princeton, NJ), were crossed with LamH-Cµ conventional Tg mice of the M29 lineage. Genotyping was performed by PCR, as described for the LamH-Cµ H chain (8) and for the V8R L chain using V8 transgene-specific forward primer, V8R-5, 5'-GGTACCTGTGGGGACATTGTG-3' (17), and reverse primer specific for J5, V8R-FR4, 5'-CTTGGTCCCAGCACCGAACGT-3'. Mice hemizygous for both or either Tg and non-Tg littermates of either sex reared under conventional specific pathogen-free conditions were used. Transcriptional activity of the V8R-targeted transgene and its capacity to produce a functional L chain protein in combination with a variety of H chains were previously demonstrated (15, 17, 18). B6 breeders and BALB/c (IgMa allotype) controls were obtained from The Jackson Laboratory (Bar Harbor, ME). All studies and procedures were approved by the Animal Care and Use Committees of Duke University and the Durham Veterans Affairs Medical Center.

Production of transfectants expressing the V8R L chain and LamH-Cµ H chain

H chain-loss variant hybridoma cells 35D carrying the V8J5 L chain were cotransfected with NotI-linearized 12-kb LamH VDJ-Cµ H chain construct and the mammalian expression vector, pGEM7(KJ1)-neo, containing the aminoglycoside phosphotransferase selectable marker, using the Lipofectin method (Life Technologies, Rockville, MD), according to manufacturer’s directions. Transfected cells were selected in medium containing 500 µg/ml aminoglycoside G-418 (Life Technologies), screened for secreted IgM by ELISA, as described below, and subcloned by limiting dilution.

Generation of hybridomas

Hybridomas were derived from splenocytes of four donor H + L double-Tg mice. Following 24-h in vitro activation with 10 µg/ml Escherichia coli LPS (Sigma-Aldrich, St. Louis, MO), splenocytes were fused with Sp2 murine IL-6 myeloma cells and selected with azaserine, as described (13). Clones producing LamH-Cµ Tg H chain IgMa allotype (B6 are IgMb) were selected by ELISA (12) and subcloned by limiting dilution.

Immunofluorescence for detection of autoantibodies

Anti-dsDNA Abs were measured in supernatant or purified Ig by indirect immunofluorescence using Crithidia luciliae substrate slides (Binding Site, Birmingham, U.K.), according to manufacturer’s directions.

Flow cytometry

Single cell suspensions were prepared and stained, as described (9). FACS analysis was performed with a FACScan (BD Biosciences, San Jose, CA). List mode data were collected with live gating on small lymphocytes (by light scatter) on 10,000–30,000 cellular events and analyzed with CellQuest software (BD Biosciences). To measure cell proliferation, splenocytes were tagged with a nontoxic fluorescent dye, CFSE (Molecular Probes, Eugene, OR), that binds irreversibly to cell proteins. As cells divide, their fluorescence halves sequentially with each generation, providing a marker of proliferation. Single cell suspensions were labeled with CFSE and cultured at 1 x 10⁶/ml at 37°C with 5% CO₂ for 3.5 days in proliferation medium (defined in Ref9). Mitogens include LPS (E. coli 055:B5 and Salmonella typhosa; Sigma-Aldrich) or anti-IgM-F(ab')₂ (Pierce, Rockford, IL) in combination with submitogenic LPS. After harvest, cells were labeled with PE-tagged reagents. Proliferation of B220⁺ lymphocyte-gated cells is measured as the ratio of geometric mean fluorescence intensity (MFI; FL1 for CFSE, logarithmic data) of cells cultured with medium alone divided by geometric MFI of cells cultured with stimulant (9).

Cell lines and Abs

Reagents for flow cytometry were obtained from BD PharMingen (San Diego, CA). BALB/c-derived hybridoma 35D is an L-chain-only line bearing an L chain identical with V8R (14). The origins of mAb are described previously: anti-laminin IgG H50 and anti-DNA IgM 238 (19); LamH-Cµ Tg mAb 54 and A10C (12); anti-DNA IgG H241 (20). Alkaline phosphatase-conjugated anti-isotype reagents and avidin were obtained from Pierce. MOPC 104E and MOPC 141 (Sigma-Adlrich) were used as isotype and allotype standards. Labeled flow cytometry reagents specific for the following markers were obtained, as follows: CD5, IgM, CD45R/B220, CD54 (ICAM-1), IgMa, IgMb, IgDb, CD4, CD8, CD3e, 5-bromo-2'-deoxyuridine (BrdU), CD69, CD86, CD80, CD25, CD21/CD35, CD43, 493, hamster IgG, and mouse IgG2a from BD PharMingen; mouse MHCII, CD23, CD24, CD62L, CD44, CD19, IgD, , and from Southern Biotechnology Associates (Birmingham, AL).

Nucleic acid isolation and sequence analysis

Genomic DNA was extracted from LamH-V8J5 transfectant or Tg hybridoma cell lines using Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). Total RNA was extracted using TRIzol reagent (Life Technologies), poly(A) RNA purified using Oligotex Kit (Qiagen, Valencia, CA), and double-stranded cDNA generated with Universal Riboclone Kit (Promega, Madison, WI). To verify H chain gene expression, first-strand cDNA synthesis was performed using a Cµ-specific 3' primer, 5'-CTCTCGCAGGAGACGAGGGGGAA-3', CMU3PCR. Integrity of transfectant LamH-V8J5 H and L chains as well as Tg hybridoma V8R L chain genes was confirmed by amplification of genomic DNA using primers specific for the rearranged LamH V region gene, 50HTG5PCR, 5'-GAGGTCCAGCTGCAACA-3', and CMU3PCR, or L chain primers, V8R-5, 5'-GGTACCTGTGGGGACATTGTG-3', and J5–1°, 5'-TGCCACGTCAACTGATAATGAG CCCTCTC-3' (21). Integrity of an unmutated expressed transgene H chain was confirmed by amplification and sequencing of H chain V region cDNA of Tg mAb 8 using primers 50HTG5PCR and CMU3PCR.

The presence of endogenous gene rearrangements was determined using a set of PCR for amplification of genomic DNA using the method of Prak and Weigert (18). PCR was performed with two reverse J primers complementary to intron sequences immediately downstream of the J2 and J5 gene segments, J2–1°, 5'-CAGTTTTCCCTCCTTAACACCTGATCTGAG-3' (21); J5–1° (see above) and J primer V8R-FR4, 5'-CTTGGTCCCAGCACCGAACGT-3'. Three forward V primers were designed to complement disparate Vk families: degenerate V-Vs, 5'-GGCTGCAG(C/G)TTCAGTG GCAGTGG(A/G)TC(A/T)GG(A/G)AC-3' (binds 80–90% of V genes) (22); V-L5, 5'-CCAGATGTGAGCTCGTGATGACCCAGACTCCA-3' (binds 50–60% of V genes) (18, 23); and VFR1, 5'-GACATTGTGATGACACAGTCTCCATCCTAT-3' (12). PCR products were purified using QIAQuick PCR Clean-Up Kit (Qiagen), Gel Extraction Kit (Qiagen), or GeneClean II (Qbiogene, Carlsbad, CA), and sequenced directly by the Duke University DNA Sequencing Facility using primers J2-SEQ, 5'-CTAAATCCCTGAAATCTCC-3' or J5-SEQ, 5'-CTGAACTGACTTTTAACTCC-3'.

In vitro stimulation

Differentiation into Ab-secreting cells was assessed using supernatants of 8- to 12-day cultures of 1–2.0 x 10⁶ anti-Thy-1 and rabbit complement T-depleted splenocytes plated in LPS in proliferation medium (defined in Ref. 9). Optimized LPS dose was constant within an experiment and ranged from 12.5 to 100 µg/ml, depending on source.

L chain analysis from splenic cDNA

Total RNA was extracted from splenocytes of H + L Tg mice using the RNeasy Mini kit and QIAshredder spin columns (Qiagen). First-strand cDNA synthesis was performed using the ImProm-II reverse-transcription kit (Promega) and oligo(dT) primers. Four different forward primers were used for generating splenic B cell L chain cDNA libraries: VFR1, V-L5, V-Vs, and V8R-5. The former three primers amplify -chains using a wide range of V genes, while V8R-5 is specifically designed to amplify the V8J5 site-directed Tg. The reverse primer was CKAPPA3PCR (5'-CTG CTC ACT GGA TGG TGG GA-3'). Conditions for L chain amplification in GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA) were: 5 min at 94°C; 5 cycles of 94°C (30 s), 50°C (30 s), 72°C (1 min); 25 cycles of 94°C (30 s), 53°C (30 s), 72°C (1 min); final extension, 10 min at 72°C. Amplified L chain products were cloned into pCR2.1-TOPO vector with the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA) and transformed according to manufacturer’s directions. Plasmids purified using the QIAprep Spin Miniprep Kit (Qiagen) were sequenced. Sequence analysis, alignment, and assignment of V families were performed using ClustalW (www.ebi.ac.uk/clustalw), IgBLAST (www.ncbi.nlm.nih.gov), and the international ImMunoGeneTics database (imgt.cines.fr; M.-P. Lefranc, Montpellier, France, initiator and coordinator).

ELISA

Ig concentrations, Ab activity, and allotype-specific binding in serum or culture supernatants were determined by ELISA, as described (9). Results for Ag binding are reported as mean OD₄₀₅ on Ag minus mean OD₄₀₅ on sham (diluent)-coated wells. Cross-reactivity of auto-Ig with DNA was determined by competition ELISA. The dilution of serum or mAb that gave 50% of maximal binding to Ag-coated microplates was incubated with varying concentrations of ssDNA for 1 h at room temperature before plating on Ag-coated microplates. Bound Ig was detected with alkaline phosphatase-labeled goat anti-mouse IgG + IgM (Roche, Indianapolis, IN). Results are reported as percentage of Ag binding ((OD₄₀₅ with inhibitor/OD₄₀₅ without inhibitor) x 100).

In vivo BrdU labeling

Newly generated B cells were identified by BrdU incorporation by adding 0.8 mg/ml BrdU (Sigma-Aldrich) to drinking water for a defined period of time. BrdU-containing water was shielded from light and changed every third day. To identify BrdU-labeled cells, spleen cells were rinsed once with PBS and resuspended in 0.5 ml of ice-cold 0.15 M NaCl per million cells. Subsequent fixation and permeabilization were performed, as described previously (24), with slight modification. Briefly, cells were fixed first in ethanol, followed by fixation in 1% paraformaldehyde containing 0.01% Tween 20 for permeabilization. Cells were then treated with DNase I before staining with 7.8 µl of anti-BrdU FITC in the presence of a PE-labeled Ab (either B220, IgMa, or IgM) in standard FACS buffer for 30 min. Cells were then rinsed twice in PBS and maintained in 1% paraformaldehyde/PBS until analyzed by flow cytometry, as described above. Regression analysis was performed using StatView (SAS Institute, Cary, NC).

Statistical analyses

All data are shown as mean values ± SEM, unless otherwise indicated. Data analysis was performed using Statview software (SAS Institute). Comparisons between two groups were analyzed with Mann-Whitney U test. A value of p < 0.05 was considered to be significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dual specificity of LamH-Cµ anti-laminin Ig

In the original characterization of LamH-Cµ Tg lines derived from three founders, we observed that mice from the M6 and M29 lineages manifest a tolerance phenotype (9). In contrast, mice of the M7 lineage spontaneously produce nephrotropic anti-laminin Ig, presumably because defective expression of B cell membrane IgM permits escape from tolerance (8). Tg mAb derived from the aberrant M7 LamH-Cµ Tg lineage express a diverse repertoire of endogenous L chains and a subset cross-reacts with DNA (12). This led us to investigate the extent to which M7 serum auto-Ig showed similar reactivity. Because Tg Ig in M7 mice appear to arise from Ag-unselected B cells, the activity of serum Ig provides a useful gauge of the extent to which laminin and ssDNA may act as cross-tolerogens. Competition ELISA showed that soluble ssDNA at high concentrations could inhibit up to 60–80% of laminin binding by M7 serum Tg Ig (Fig. 1A). From this, we reasoned that a substantial subset of anti-laminin B cells forming in the bone marrow of Tg M6 and M29 lineage mice, and subject to tolerizing interactions, also bears receptors with dual Ag specificity.

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Dual specificity of M7 Tg serum Ig and transfectant Ig. A, Denatured DNA inhibits Tg M7 lineage serum Ig binding to laminin. The dilution of serum that gave 50% of maximal binding to laminin in ELISA was preincubated with varying concentrations of inhibitor ssDNA before incubation on laminin-coated wells. Results are shown for three Tg mice (Tg 488, 659, and 859) of the aberrant M7 lineage that fails to regulate transgene-encoded autoreactivity. B, Transfectant LamH/V8J5 (Tx H + L) binds ssDNA and laminin. Ag-coated microplates were incubated with 10 µg/ml mAb. Results are expressed as mean OD405 on Ag-coated wells minus mean OD405 on diluent-coated wells based on duplicate samples. Also shown are results for control anti-DNA IgG H241, anti-laminin IgG H50, and LamH Tg IgM A10C at 10 µg/ml. C, Soluble ssDNA inhibits transfectant binding to autoantigen. The dilution of transfectant Ig that gave 50% maximal binding to ssDNA (•) or laminin () was preincubated with varying concentrations of inhibitor ssDNA before incubation with Ag-coated wells.

IgM termed Tx LamH/V8J5 was recovered from D35 L-chain-only hybridoma cells transfected with the LamH-Cµ H chain construct. Direct and competitive ELISA showed that the transfectant Ig bound to ssDNA as well as laminin (Fig. 1, B and C). Whereas ssDNA inhibits transfectant binding to laminin-coated plates (Fig. 1C), soluble laminin does not inhibit transfectant binding to ssDNA (data not shown), consistent with our previous observation that laminin epitopes recognized by Tg mAb are poorly displayed on soluble aggregates (12). The transfectant was not reactive with dsDNA, as shown by negative kinetoplast immunostaining of Crithidia luciliae (data not shown). Integrity of the Ig chains was confirmed by sequence analysis of PCR-amplified genomic DNA (L chain) and RT-PCR of hybridoma mRNA (H chain); there were no mutations in the transgene-coding regions to affect Ag specificity.

Characterization of LamH x V8R H + L double-Tg mice

We generated C57BL/6 H + L Ig double-Tg mice by crossing hemizygous LamH H-Tg and V8R L-Tg mice. These matings yielded viable progeny in expected Mendelian ratios, resulting in littermates of four experimental genotypes: non-Tg (wild type, WT), H-chain-only Tg (H-Tg), L-chain-only Tg (L-Tg), and H + L double-Tg (H + L Tg). Immunologic phenotyping shows that H + L Tg mice resemble their H-Tg littermates, in that they have smaller spleens and significantly fewer splenic B cells than do their WT and L-Tg littermates (Table I). The phenotype of H-Tg and WT mice is similar to that previously reported in earlier generation B6 backcrosses (9).

View larger version (63K): [in this window] [in a new window]   FIGURE 2. Flow cytometric analysis of spleen (A) and bone marrow (B) lymphocytes. Cells were stained with PE anti-B220 and either FITC anti-IgM or FITC anti-IgMa, or with PE anti-IgMa and FITC anti-IgMb and analyzed by FACS, as described in Materials and Methods. Shown are representative dot plots of log fluorescence data for stained unstimulated spleen cells gated on lymphocytes on the basis of forward and side scatter. Littermate genotypes include B6 mice homozygous for the V8R L chain (LL), mice carrying the LamH Tg H chain (H), mice carrying both the LamH and V8R transgenes (H + L), and mice carrying the LamH Tg H chain and homozygous for the V8R L chain (H + LL). Also shown are representative results from a BALB/c IgMa control mouse. The LamH Tg H chain is IgMa allotype, whereas B6 mice, including WT, L, and LL genotypes, express only endogenous IgMb. Plots derived from splenocytes of mice heterozygous for the V8R L chain (L) and WT controls were similar to those from LL mice and are not shown.

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Absence of autoreactivity among serum Ig from LamH x V8R H + L Tg mice. Transgene IgMa autoreactivity to DNA (A) and laminin (B) and total Tg IgMa allotype (C) were measured by ELISA. Autoantigen reactivity was measured as the mean OD405 on Ag-coated minus diluent-coated wells, using duplicate serum samples diluted 1/20. Genotypes as defined in the legend to Fig. 2 include WT (n = 8), L-Tg (n = 5), H-Tg (n = 5), and H + L (n = 11). Reactivity in the H-Tg mice depicted in A derives from strong serum anti-DNA IgMa detected in one of five H-Tg mice. Any IgMa detected in L-Tg and WT mice represents nonspecific background staining, as these mice express only endogenous IgMb allotype. M7 serum is an IgMa-positive control for Ag binding derived from a B6 Tg M7 lineage mouse (8 ). , Indicate values for individual mice; mean value is indicated by horizontal line.

Characterization of H-chain Tg mAb derived from LamH x V8R H + L Tg mice

Eight IgM-producing clones were obtained from two fusions generated with LPS-stimulated splenocytes from four H + L double-Tg mice. Four clones producing Tg IgMa allotype, indicating secretion of the LamH Tg H chain, were recovered by subcloning. All four mAb expressed L chains. These LamH Tg mAb were purified from culture supernatant, concentrated, and assayed for autoantigen binding. Results for Ab assayed at 5 µg/ml are shown in Fig. 4A. None bound to ssDNA or laminin, suggesting that one or both of the Ig Tg chains were altered in vivo. Sequencing of the expressed LamH Tg H chain from PCR-amplified Ig V region cDNA revealed a V region identical with LamH (data not shown), suggesting that somatic mutation or editing of the Tg H chain was not the cause for the shift in Ag specificity.

View larger version (28K): [in this window] [in a new window]   FIGURE 4. Absence of autoreactivity in -edited mAb derived from LamH x V8R H + L double-Tg mice. A, Autoantigen binding. Ag-coated microplates were incubated with 5 µg/ml of each mAb, 8, 14, 15, and 16, derived from H + L Tg mice. Results are expressed as mean OD405 on Ag-coated wells minus mean OD405 on diluent-coated wells based on duplicate samples. Controls are anti-laminin Tg mAb 54 and anti-DNA mAb 238 (12 ). B, Predicted L chain V-J junction sequences and V gene segments used by the four mAb. L chains were sequenced by PCR amplification of DNA, as described in Materials and Methods, and assigned to V gene families based upon homology to published sequences (29 ) and using the nomenclature of the International ImMunoGeneTics database http://imgt.cnusc.fr:8104 (initiator and coordinator: M.-P. Lefranc; e-mail address: lefranc{at}ligm.igh.cnrs.fr) (30 ). J genes are assigned to known BALB/c J germline gene segments (31 ). The nucleotide sequences from which these translated sequences were derived are available from GenBank under accession numbers AY148477–480.

Extensive L chain rearrangement revealed by cDNA library

To further explore the extent of editing in this model, rearranged L chains were recovered from a cDNA library generated from splenic leukocytes of an H + L Tg mouse. PCR amplification used a universal 3' C primer and a panel of four 5' primers derived from disparate V families. Amplification products were cloned, and 10 clones from each primer pair were selected at random for sequencing. Thirty-six clones yielded L chain sequences, of which 17 were the V8R Tg L chain. The other 19 L chains were endogenous in origin (Table II). At least 13 independent endogenous rearrangements are represented among these 19 L chains, which use 7 different V families and 3 J genes. A V12 family member is used by 11 of 19 clones. Frequency of use of J1 and J5 is similar, whereas only 2 of 19 clones use J2, and J4 is not represented. These findings confirm that editing at the endogenous locus is a frequent event among H + L Tg B cells.

View this table: [in this window] [in a new window]   Table II. Gene family assignments for H + L double-Tg mouse splenocyte cDNA clones bearing rearranged endogenous L chainsa

To exclude editing and to determine the default phenotype of B cells bearing dual specific receptors, we bred mice homozygous for the V8R-targeted replacement, referred to hereafter as LL, some of which also bear the LamH H chain transgene and are termed H + LL. ELISA analysis showed no Tg-encoded IgMa Ab in serum of H + LL mice (data not shown). By flow cytometric analysis, no B cells bearing the LamH H chain IgMa were detectable in the spleen (Fig. 2A). H + LL mice have very small spleens with few B cells (mean 1.2 million per spleen, Table III). Staining for the LamH Tg H chain allotype does not exceed background in LL littermates. Residual splenic B cells express endogenous IgMb H chains paired primarily with L chains. editing is limited even in the face of prohibited editing; only 17.3 ± 3.2% of the few remaining B cells express surface L chains (data not shown). There is no evidence that LamH Tg B cells are sequestered in the peritoneum (data not shown). Only rare B220⁺ cells are identified in H + LL peritoneum, and staining for Tg IgMa allotype does not exceed background in IgMb-expressing WT, L-Tg, or LL-Tg mice.

Impaired proliferation and differentiation of B cells from H + L Tg mice

The marked absence of B cells in the H + LL triple-Tg mice provides further evidence that many of the LamH Tg H chain-bearing B cells that populate the spleen in H + L Tg mice do so by exchanging their V8R L chain for an endogenous L chain, presumably altering receptor specificity. Because little LamH H chain Tg-encoded Ab and no autoreactivity are found in serum of these mice, and only rare Tg-encoded mAb are recovered by fusion from H + L spleens, we examined the functional status of residual peripheral B cells in H + L Tg mice. The ability of Tg B cells to proliferate in vitro was assessed by flow cytometry using the nontoxic dye CFSE. B cells from WT and L-Tg mice proliferate robustly in response to surface Ig cross-linking, as indicated by decreased fluorescence in a significant proportion of B cells, compared with CFSE-stained, unstimulated cells (Fig. 5). Proliferation was muted among Ig-stimulated B cells from H + L Tg mice. Labeling for Tg IgMa among unstimulated B cells cultured for 3.5 days in medium alone confirmed the persistence of H chain Tg B cells under these conditions (data not shown).

View larger version (30K): [in this window] [in a new window]   FIGURE 5. Impaired in vitro proliferation of LamH x V8R H + L Tg B cells in response to Ig cross-linking. Single cell suspensions of RBC-depleted splenocytes were labeled with CFSE, as described in Materials and Methods, and cultured 3.5 days with or without anti-IgM-F(ab')2 and submitogenic LPS. Cells were harvested and labeled with PE-conjugated reagents for the B cell surface marker B220. A, Shown are representative dot plots of log fluorescence data gated on lymphocytes on the basis of forward and side scatter. B, Proliferation index, based on B220-gated cells only, is calculated, as described previously (9 ). Numbers of mice appear in parentheses; error bars indicate SD. As a single control H-Tg mouse was studied in parallel for reference, and WT and L-Tg B cells were functionally indistinguishable (p = 0.7213); WT and L-Tg were pooled for analysis. *, p < 0.05, H + L vs WT and L-Tg, Mann-Whitney U test.

Characterization of H + L splenic B cell life span and surface marker expression

The absence of significant spontaneous or induced Tg autoantibody production and hypoproliferation of H + L Tg B cells suggests that these cells are functionally impaired. To determine whether H + L splenic Tg B cells have a life span characteristic of immature or mature B cells, rate of B cell replenishment was determined using in vivo long-term BrdU labeling. Mice were fed BrdU in drinking water for up to 48 days before sacrifice, and extent of BrdU labeling was determined by flow cytometry. Incorporation of BrdU into DNA indicates cell division. As shown in Fig. 6, Tg mice have a higher percentage of recently generated splenic B cells than non-Tg mice. The kinetics of BrdU labeling by splenic B cells for H Tg and H + L Tg mice was similar, as were the kinetics of labeling for WT and L Tg mice. Regression analysis predicted that 50% BrdU labeling of B cells is achieved in H and H + L Tg mice at 28.1 and 27.2 days, respectively, and in WT and L Tg mice at 48.3 and 47.3 days, respectively.

View larger version (17K): [in this window] [in a new window]   FIGURE 6. Kinetics of BrdU labeling of splenic B cells. Ten adult mice of each genotype were fed BrdU for up to 48 days. At the indicated time points, splenic B cells from two mice of each genotype were analyzed for BrdU content.

View larger version (30K): [in this window] [in a new window]   FIGURE 7. Surface marker expression. A, Splenocytes were dual labeled with Abs reactive with B220 and the indicated cell surface Ag and analyzed by flow cytometry. Shown are representative histograms of log fluorescence data, gated on B220+ small lymphocytes, from H + L Tg (filled plots) and L-Tg (open plots) mice. B, Data shown are mean ± SEM (n = 7–8 per genotype) of percentage of positive staining cells for each marker, based on background staining of non-B or nonlymphoid cells. Statistically significant differences are indicated with asterisks, comparing H vs WT, and H + L vs L (*, p < 0.05; #, p < 0.005; +, p < 0.001, Mann-Whitney U test).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We explored mechanisms involved in B cell self-tolerance in an Ig multiple Tg mouse model bearing the LamH-Cµ Ig H chain and a targeted V8J5 L chain. The LamH x V8R H + L Ig binds both ssDNA and laminin. When the H and L chain Tg are coexpressed in vivo, neither anti-DNA nor anti-laminin Tg Ig is recovered in serum or among hybridoma derived from mitogen-stimulated splenocytes. Phenotypic characterization suggests that H + L Tg B cells are tolerized primarily by clonal deletion and receptor editing. H + L Tg mice have 5- to 7-fold fewer splenic B cells than their L-Tg or WT littermates, mirroring the B cell hypocellularity observed in H-Tg mice bearing the anti-laminin LamH-Cµ Tg H chain (this study) (9). Analysis of splenic B cells, Tg IgMa mAb, and a cDNA library reveals limited editing and extensive secondary V-J rearrangements on the nontargeted allele. B cells that populate the spleen in H + L mice share features with anergic B cells. They hypoproliferate, have shortened life spans, and express surface markers suggesting prior Ag engagement. Generation of H + LL Tg mice homozygous for the targeted allele, to preclude editing, reveals profound B cell depletion and absence of splenic B cells expressing the H chain Tg. Collectively, the data are consistent with a scenario wherein engagement of tolerogen in the bone marrow triggers developmental arrest in H + L B cells, providing an opportunity for L chain editing that rescues a subset of LamH H chain Tg-bearing B cells. The anergic phenotype of surviving splenic B cells in H + L mice further suggests that many of these surviving B cells retain an as yet unidentified autospecificity.

Deletion is not the phenotype predicted for B cells regulated primarily by soluble ssDNA. Specificity to ssDNA is regulated predominantly by anergy in normal mice (1). This includes Tg anti-ssDNA B cells formed by intentional pairing of the lupus-derived 3H9 Ig H chain with the V8J5 L chain (15, 17, 25). Moreover, anergized anti-ssDNA B cells formed by the 3H9 x V8J5 union express near normal levels of surface IgM and have near normal numbers of B cells. In contrast, clonal elimination and mechanistically linked receptor editing are described primarily in B cells reactive with cell membrane-bound or multivalent self Ag, such as dsDNA, capable of extensively cross-linking Ig receptors (2, 7, 26). Clonal deletion has been described as proceeding in two distinct stages: Ag initially triggers developmental arrest at the transition from the pre-B to immature B cell stage. Arrested B cells then enjoy a window of opportunity during which persistently activated or reactivated recombination-activating gene enzymes catalyze secondary rearrangements to replace the functional, but autoreactive H or L chain with a nonautoreactive chain and rescue the cell from subsequent apoptotic deletion (4, 5, 6, 27, 28). Thus, demonstration of editing and deletion as dominant immune phenotypes in LamH x V8R H + L Tg mice suggests one of two scenarios: either the primary in vivo tolerogen for H + L Tg cells is a high valency cross-reactive self Ag, and not soluble ssDNA, or the dual specificity of binding sends a tolerizing signal that is quantitatively or qualitatively different from that transduced by binding to either Ag alone.

We propose that the in vivo fate of LamH x V8R H + L-Tg B cells is determined by engagement of basement membrane laminin, not soluble ssDNA. According to this hypothesis, receptor interaction with the matrix tolerogen is of sufficient strength and quality to trigger persistence or reactivation of recombination-activating gene activity with subsequent L chain editing and deletion of B cells that fail to generate a functional secondary L chain. In the LamH Tg model, L chain editing is likely to generate novel productive, but persistently laminin-reactive Ig, because the LamH Tg H chain is dominant, generating laminin specificity in combination with many different L chains (12). Such newly generated anti-laminin B cells presumably would also be subject to tolerizing interactions. The presumed tolerogen in this setting is an epitope on the 1 or 1 chain of laminin. Anti-laminin Ab bearing the LamH Tg H chain bind to the laminin1 isoform, an 111 heterotrimer. Whereas the 1 chain is highly tissue restricted and not expressed in adult bone marrow, the 1 and 1 chains are highly expressed in murine bone marrow assembled with the 4 and 5 chains as constituents of laminin8 and laminin10 (32). Alternatively, the anti-laminin B cells may be tolerized by engaging a cross-reactive epitope shared with the 4 and 5 chains, reminiscent of the promiscuous binding described for integrin receptors that bind multiple laminin isoforms and epitopes formed by discontinuous segments of matrix proteins (33). A novel Tg model in development will address the role of ssDNA cross-reactivity in defining the tolerance phenotype of H + LL mice. It is unknown whether LamH Tg H chain and endogenous L chain combinations can generate specificity for DNA independent of anti-laminin activity.

Immune phenotyping indicates that a subset of nondeleted LamH-Cµ Tg B cells is functionally inactivated. The absence of B cells expressing the LamH Tg in H + LL spleens suggests that only L chain-edited LamH⁺ B cells survive in H + L mice. The specificity of these H + L B cells is unknown, including whether this subset includes residual anti-laminin B cells with altered affinity for laminin. A small residual population of splenic B cells in H + LL mice bears endogenous IgMb H chain on their surface, suggesting that their escape from tolerance depends on replacement of or coexpression with the LamH H chain Tg.

	Acknowledgments

 
We thank Dr. Martin Weigert for providing mutant mice carrying the site-directed V8J5 L chain. We thank Gabriel Mixon and Erica Ungewitter for expert technical assistance. We thank the Duke University DNA Analysis and Flow Cytometry Facilities for assistance.

	Footnotes

 
¹ This work was supported by the National Institutes of Health Grant DK47424. These experiments comply with the current laws of the U.S.

² G.F.B. and K.L.C. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Mary H. Foster, Department of Medicine, Division of Nephrology, Box 3014, Duke University Medical Center, Durham, NC 27710. E-mail address: mhfoster{at}duke.edu

⁴ Abbreviations used in this paper: Tg, transgenic; BrdU, 5-bromo-2'-deoxyuridine; MFI, mean fluorescence intensity; WT, wild type.

Received for publication December 12, 2003. Accepted for publication February 26, 2004.

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