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The Journal of Immunology, 2003, 171: 5890-5900.
Copyright © 2003 by The American Association of Immunologists

Tolerance through Indifference: Autoreactive B Cells to the Nuclear Antigen La Show No Evidence of Tolerance in a Transgenic Model 1

Brett D. Aplin2,*, Catherine L. Keech2,*, Andrea L. de Kauwe*, Thomas P. Gordon{dagger}, Dana Cavill{dagger} and James McCluskey3,*

* Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia; and {dagger} Department of Arthritis, Allergy, and Immunology, Flinders Medical Center, Bedford Park, South Australia, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Systemic autoimmune diseases are characterized by the production of high titer autoantibodies specific for ubiquitous nuclear self-Ags such as DNA, Sm, and La (SS-B), so the normal mechanisms of B cell tolerance to disease-associated nuclear Ags have been of great interest. Mechanisms of B cell tolerance include deletion, anergy, developmental arrest, receptor editing, and B cell differentiation to the B-1 subtype. However, recent studies in our laboratory have suggested that B cell tolerance to the nuclear autoantigen La is limited in normal mice, and tolerance may reside primarily in the T cell compartment. To test this hypothesis, we created Ig transgenic mice expressing the IgM H chain from an mAb specific for a xenogeneic epitope within human La (hLa). These mice were bred with hLa-transgenic mice that constitutively express hLa in a manner comparable to endogenous mouse La. Between 5–15% of transgenic B cells developing in the absence of hLa were specific for hLa, and these cells were neither depleted nor developmentally arrested in the presence of endogenous hLa expression. Instead, these autoreactive B cells matured normally and differentiated into Ab-forming cells, capable of secreting high titer autoantibody. Additionally, the life span of autoreactive hLa-specific B cells was not reduced, and they were phenotypically and functionally indistinguishable from naive nonautoreactive hLa-specific B cells developing in the absence of hLa. Together these data suggest a lack of intrinsic B cell tolerance involving any known mechanisms indicating that these autoreactive B cells are indifferent to their autoantigen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Systemic autoimmune diseases are characterized by the production of high titer autoantibodies, many of which are specific for ubiquitous nuclear self-Ags such as La (SS-B), DNA, and Smith Ag (Sm).4 These anti-nuclear Abs mediate significant pathology in diseases such as systemic lupus erythematosus and Sjögren’s syndrome, and therefore, the normal mechanisms of B cell tolerance to disease-associated nuclear Ags are important to understand. Natural tolerance to lupus-associated nuclear autoantigens DNA and Sm has previously been examined in nonautoimmune animals using Ig-transgenic (Tg) mice. Anti-ssDNA and anti-dsDNA Tg B cells were found to be tolerized by a combination of mechanisms, including receptor editing, deletion, developmental arrest, and anergy (1, 2, 3). In contrast, anti-Sm B cells are tolerized by developmental arrest and are then thought to be either deleted or shunted into the B-1 compartment, although some Sm-specific B cells may remain autoantigen ignorant (4, 5, 6). These different mechanisms are likely to represent a continuum of possible tolerogenic responses that are dependent upon variable signaling thresholds as a result of the affinity of the B cell Ag receptor (BCR), Ag abundance and the ability of Ag to induce BCR cross-linking. However, immunization of normal mice with mouse La (mLa), or human La-transgenic mice (hLa Tg) with human La (hLa), triggers a significant anti-La humoral immune response, suggesting that autoreactive La-specific B cells may be poorly tolerized (7, 8). Therefore, tolerance to La might therefore principally reside in the T cell compartment (9, 10).

We have previously constructed hLa Tg mice that constitutively express hLa in a manner comparable to endogenous mLa in all tissues tested (7, 11). Human La shares ~77% identity with mLa (12), and its expression is partially tolerogenic for T cells in hLa Tg mice similar to the tolerance of mLa in normal mice (7). It was recently shown that cognate T cell help provided by adoptive transfer of hLa-primed T cells was sufficient to trigger anti-hLa Ab secretion in mice that constitutively express hLa (7). Significantly, hLa-primed T cells from hLa Tg mice did not trigger B cell autoimmunity following transfer into syngeneic mice, confirming that immune tolerance to hLa is primarily T cell mediated. At least a proportion of hLa-specific B cells must therefore be continuously presenting hLa peptides, rendering them constitutively set to receive Th signals given appropriate conditions. Hence, although La is primarily a nuclear-associated autoantigen, it is unlikely to be completely sequestered and may be accessible under certain circumstances, such as during apoptosis (13).

This paper describes the construction of anti-hLa Ig Tg mice in which the IgH confers specificity for a xenogeneic epitope within the LaC subfragment (aa 188–223) of hLa. These mice were bred with hLa Tg mice, allowing the fate of hLa-specific B cells to be examined in both the presence and the absence of the endogenously expressed autoantigen in (hLa x anti-hLa) double-Tg and anti-hLa single-Tg offspring, respectively. Unlike other lupus-associated nuclear autoantigens (Sm and DNA), La does not appear to be very efficient in tolerizing the B cell compartment. Autoreactive, hLa-specific B cells are not deleted despite endogenous expression of hLa and are capable of secreting autoantibodies, indicating that they are not anergic. Furthermore, the life span of these B cells was indistinguishable from that of nonautoreactive B cells in the same animals and was similar to that of hLa-specific B cells in single-Tg animals. These hLa-specific B cells also showed no sign of activation, as measured by the up-regulation of phenotypic activation markers. Human La-specific B cells appear to be functionally indifferent to autoantigen, supporting previous studies suggesting that immune tolerance to La autoantigen principally resides in the T cell compartment (7).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and Tg lines

Anti-hLa Ig Tg mice (anti-hLa Tg) were generated from a construct containing the rearranged V(D)J segments of the hLa-specific hybridoma A3 (14). The anti-hLa H and L chain variable regions were amplified from A3 cDNA using 5'-RACE PCR, and the exons were reamplified using nested oligonucleotide primers incorporating a SalI restriction site and consensus splice acceptor or donor sequences (15). These exons were then cloned into the unique SalI sites of modified pseudo-genomic, chimeric Ig-TCR gene constructs pSV2-LHV{alpha}C{gamma}2a and pSV2-L{kappa}V{beta}C{kappa}, described previously (15), replacing the V{alpha} and V{beta} TCR variable regions with VH and VL Ig regions. The 3.3-kb promoter, leader, variable, and enhancer regions of the construct were removed from the H chain construct by BamHI-EcoRI cleavage and cloned into pGEM11Zf+ (Promega, Madison, WI), and a 10-kb EcoRI-restricted IgHa µ-chain constant region excised from an anti-HEL vector (pSVGneoV10CµM, gift from C. Goodnow) was ligated into the EcoRI site downstream of the H chain enhancer. Purified inserts were coinjected into fertilized H-2bm1 x C57BL/6 mouse ova (Walter and Eliza Hall Institute, Transgenic Embryo Services, Melbourne, Australia). Seven Tg founders were obtained and backcrossed three to six generations on C57BL/6, and data from one line are presented here in detail. All transgene-positive mouse lines were subsequently found to be defective for anti-hLa L chain gene expression due to inappropriate {kappa}-chain splicing between the C{kappa} splice acceptor site, and the splice donor site of a remnant J4 region downstream of the anti-hLa VJ exon. This had no discernible bearing upon anti-hLa H chain gene expression.

Double-Tg mice (hLa x anti-hLa Tg) expressing anti-hLa Ig and the human La protein were generated by breeding anti-hLa Tg mice with hLa Tg mice (7) (line 3) that had been backcrossed to C57BL/6 for 7–11 generations. Briefly, hLa Tg mice contain a 15.8-kb genomic fragment encoding the hLa gene and its regulatory regions. Thus, hLa is expressed in the nucleus of all cells under control of the hLa promoter.

Cell preparations

Single-cell suspensions were prepared from spleen and bone marrow. These cells or peripheral blood were subjected to RBC lysis using hypotonic Tris ammonium chloride. T cells and CD5+ cells were depleted using anti-rat Ig Dynabeads (Dynal, Oslo, Norway) according to the manufacturer’s instructions after incubation with the following mixture of mAb hybridoma supernatants: anti-CD4 (GK5.1), anti-CD8 (56-6-72), and anti-Thy-1 (T24) with or without anti-CD5 (53-7-5).

Monoclonal Abs and flow cytometry

Spleen, bone marrow, or peripheral blood cells were resuspended at 2 x 107 cells/ml in wash buffer (PBS containing 1% BSA and 0.1% sodium azide) and were incubated for 30 min with the appropriate biotinylated or directly fluoresceinated Ab or recombinant Ag. Cells were washed with wash buffer after incubations, and for cell samples that were stained with more than one fluorochrome, sequential incubations were performed. Stained cells were resuspended in wash buffer or FACS fixative (2% glucose, 1% formaldehyde, and 0.02% sodium azide), and flow cytometry was conducted using a FACS2 (BD Biosciences, Mountain View, CA) and CellQuest acquisition and analysis software according to the manufacturer’s instructions. Data were analyzed using Cell Quest or FlowJo (Treestar, Stanford, CA) analysis software. All incubations and washes were conducted at 4°C. Dead and irrelevant cell populations were excluded by setting gates on the basis of forward and side scatter profiles. Abs directed against the following molecules were used: IgMa (RS3.1 or DS-1), IgMb (AF6-78.25), I-Ak (10-2.16), B220 (RA3-6B2; BD PharMingen, San Diego, CA), CD4 (L3T4; BD PharMingen), CD5 (53-7.3; BD PharMingen), CD8 (53-6.7; BD PharMingen), CD23 (B3B4; BD PharMingen), CD24/HSA (M1/69; BD PharMingen), CD69 (H1.2F3; Cymbus, Chandlers Ford, U.K.), CD80 (16-10A1; Cymbus), CD86 (GL-1; Cymbus), and MHC class II (I-Ab, 35-4-2). In the case of biotinylated reagents, cells were also incubated with streptavidin-PE (Caltag Laboratories, Burlingame, CA). Biotinylated recombinant hLa-GST and mLa-GST were also used. The axes on histograms and contour plots depict log fluorescence intensity.

LPS stimulation

Spleen cells were harvested, and RBC were lysed in Tris ammonium chloride solution (17 mM Tris and 140 mM NH4Cl, pH 7.2). Cells were resuspended in DMEM supplemented with 10% FCS, glutamine, nonessential amino acids, 50 µM 2-ME, and 0.5 mM HEPES at 2 x 105 cells/well in flat-bottom microtiter plates (TPP, Trasadingen, Switzerland). Triplicate cultures were incubated in the presence of graded amounts of LPS (Sigma-Aldrich, St. Louis, MO) for 72 h. Proliferation was measured by uptake of [3H]thymidine (0.5 µCi/well; ICN, Irvine, CA) for the last 24 h of culture. Supernatants from triplicate cultures were analyzed by ELISA.

ELISA

Recombinant protein was diluted to 2–5 µg/ml in 0.03 M carbonate buffer, pH 9.6, and allowed to adhere to round-bottom, Polysorp microtiter plates (Nunc, Roskilde, Denmark) overnight at room temperature. Nonspecific binding sites were blocked with 1% BSA in PBS for 2 h at 37°C, plates were washed, then duplicate wells were incubated for 4 h at room temperature with serial titrations of hybridoma supernatant or sera from immunized or control mice. After further washing, the wells were incubated for 1 h at room temperature with alkaline phosphatase-labeled anti-mouse IgG (Sigma-Aldrich), biotinylated anti-IgMa (DS1), or biotinylated anti-IgMb (AF6-78.25) where appropriate. Biotinylated conjugates were further incubated with streptavidin-alkaline phosphatase (BD PharMingen) for 1 h at room temperature, and bound Ab was detected after the addition of substrate (disodium p-nitrophenyl phosphate (Sigma-Aldrich), 1 mg/ml in 90 mM diethanolamine, and 5 mM MgCl2, pH 9.6) by measuring the OD at a dual wavelength of 405/450 nm. Results were expressed in OD405/450 nm units as the mean of duplicate determinations. Supernatant, sera, and conjugates were all diluted in 0.5% BSA/0.05% Tween 20 in PBS, and all wash steps were conducted in 0.05% Tween 20 in PBS.

Immunoprecipitation and immunoblotting

Protein tyrosine phosphorylation in postnuclear extracts of purified B cells was detected following stimulation with 10 µg/ml F(ab')2 goat anti-mouse IgM (Pierce, Rockford, IL) for 3 min at 37°C. Cells were chilled quickly on ice and immediately pelleted by centrifugation. Cells were lysed at 2 x 107/ml in ice-cold lysis buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 0.4 mM EDTA, 1% Nonidet P-40, Complete protease inhibitor mixture (Roche, Mannheim, Germany), 10 mM NaF, and 100 mM sodium orthovanadate) for 15 min on ice, followed by the removal of particulate material by centrifugation at 12,000 x g for 10 min at 4°C. For Syk immunoprecipitation, 3 x 106 cell equivalents of cleared lysate were incubated with 1.5 µg of rabbit anti-Syk Ab (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4°C and precipitated with protein A-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden). Immunoprecipitates were washed three times in lysis buffer. Bound proteins were eluted by boiling in reducing SDS-sample buffer. Lysates and immunoprecipitates were then fractionated by 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes using a semidry blotting apparatus (Amersham Pharmacia Biotech), and subjected to immunoblotting. The blots were blocked with TBS containing 3% BSA, then incubated with anti-phosphotyrosine-HRP (PY99; Santa Cruz Biotechnology). Ab binding was detected using ECL (NEN, Boston, MA).

Labeling with 5-bromo-2'deoxyuridine (BrdU)

Mice were given 0.5 mg/ml BrdU (Sigma-Aldrich) with 1 mg/ml glucose in their drinking water continuously for 7 days. BrdU-containing water was made freshly every 24 h. On day 8 mice were sacrificed, and spleens were isolated for analysis. Cell preparations and surface staining were performed as described above. Staining for BrdU was performed as previously described (16) using anti-BrdU-FITC (BD Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of expression, specificity, and estimated affinity of anti-hLa Ig in Tg mice

To investigate the mechanism of tolerance to a physiological nuclear autoantigen we generated Tg mice that express the H chain variable domain of the anti-hLa mAb A3. The A3 mAb is a mouse anti-hLa Ab that does not react with mLa (14), and this was confirmed by ELISA and immunoblot (data not shown). The expression of Tg Ig by B cells was first examined using B220 and IgMa/b allotype-specific mAbs (Fig. 1A). Mice transgenic for the anti-hLa H chain expressed a high level of IgMa on 90–95% of splenic B cells, demonstrating effective allelic exclusion of endogenous IgMb H chains. Effective allelic exclusion was stable for long periods with 85–90% of splenic B cells expressing Tg IgMa at 32 wk (data not shown).



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  		FIGURE 1. Analysis of anti-hLa Ig expression in Tg mice. A, Allelic exclusion of endogenous Ig H chain genes in anti-hLa Ig H chain. Spleen cells from anti-hLa Tg mice and non-Tg littermates were stained using mAb specific for B220 (CD45/RA3-6B2), IgMa (RS3.1), and IgMb (AF6–78.25). Three-color FACS analysis was used to assess the extent of allelic exclusion of endogenous IgMb expression by the introduced anti-hLa IgMa H chain transgene. Data are representative of 10 mice analyzed, and quadrant numbers represent the percentage of B220+ gated lymphocytes. B, Human La-specific B cells are present in spleen cells from anti-hLa Ig Tg mice. Three-color FACS analysis of Tg and non-Tg spleen cells stained with either hLa-GST (30 nM) or mLa-GST (30 nM), and Abs for B220 and either IgMa or IgMb. Contour plots are gated on B220+ lymphocytes based on forward and side scatter profiles and B220 expression. Data are representative of at least six mice per group, aged 9–14 wk. The percentage of hLa-specific B cells varied between 4–7% in the spleen. C, Spleen cells from anti-hLa Tg mice were stained with 30 nM biotinylated hLa-GST and either a 1- or 5-fold molar ratio of unlabeled hLa-GST and were analyzed by three-color FACS. Cells were also costained with Abs for B220 and IgMa. Addition of equimolar amounts of unlabeled hLa-GST (1x) markedly decreased specific binding of hLa-GST-biotin. Further increasing the concentration of unlabeled hLa-GST (5x) abrogated detectable hLa-GST biotin binding. Addition of up to 10 times the molar ratio of unlabeled mLa-GST had no effect on hLa-GST-biotin recognition by anti-hLa B cells (right panel). Contour plots are gated on B220+ lymphocytes based on forward and side scatter profiles and B220 expression. Data are representative of four mice per group, aged 9–14 wk. D, Detection of increased numbers of IgMlow and IgMhigh hLa-specific B cells with increased concentration of hLa. Spleen cells from anti-hLa Tg mice were stained with the indicated concentrations of biotinylated hLa-GST, and Abs specific for B220 and IgMa. Distinct populations of hLa binding are indicated by arrows and letters. Contour plots are gated on B220+ lymphocytes, based on forward and side scatter profiles and B220 expression. Data are representative of four mice per group, aged 9–14 wk.

 
Expression of the anti-hLa L chain was not detected in transfectant cell lines or in Tg mice due to the aberrant splicing of the L chain transgene (data not shown). This pre-mRNA splicing defect was confirmed by RT-PCR (data not shown). Therefore, surface expression of IgMa in anti-hLa Tg mice indicates that endogenous L chains assemble with the anti-hLa H chain. Anti-hLa Tg mice were then examined to determine the presence and the frequency of hLa-specific B cells. Splenocytes from anti-hLa Tg mice and non-Tg littermates were stained with either hLa-GST-biotin or mLa-GST-biotin and Abs to B220 and either IgMa or IgMb. Three-color FACS analysis showed that hLa-specific B cells were detected in the spleen of Ig Tg mice, but were not detected in non-Tg littermates, indicating that the anti-hLa H chain alone is capable of biasing the structural specificity toward hLa. The hLa-specific B cells could be divided into two populations based on surface IgMa expression: IgMa-low and IgMa-hi (Fig. 1B). This pattern is similar to the surface Ig expression in µ-H chain, anti-hen egg lysozyme (anti-HEL) Tg mice, where discrete HEL-specific B cell subpopulations expressing a range of surface IgM were detected (17, 18). This distribution probably indicates the preferential use of different L chains or L chain families within the B cell pool expressing the same IgM H chain. Anti-hLa B cells represented between 4–15% of the splenic B cell pool. Anti-hLa Tg B cells did not bind mLa (Fig. 1B, lower panels), indicating that pairing of A3 anti-hLa Tg H chains and endogenous L chains did not generate autoreactive mLa-specific B cells. Titrating in competing amounts of unlabeled hLa-GST could abrogate specific binding of biotinylated hLa-GST to hLa-specific splenic B cells, while addition of up to a 10-fold molar excess of mLa-GST had no effect (Fig. 1C). This confirmed the anti-hLa specificity in these B cells from anti-hLa Ig Tg mice.

To estimate the relative receptor affinities of each of the populations of hLa-specific B cells in anti-hLa Tg mice, the concentration of biotinylated hLa-GST used to stain splenic B cells was varied over a 2-log range. Incrementally increasing the concentration of biotinylated hLa-GST from 1.9 to 480 nM revealed at least three subpopulations within a spectrum of hLa-specific B cells, each with varying levels of surface IgM and functional avidity for hLa (Fig. 1D). The IgMlow population (arrowed population a) could be detected with as little as 1.9 nM hLa-GST-biotin, while staining with 30 nM hLa-GST revealed the IgMhigh population (arrowed, b) of hLa-specific B cells. A second IgMhigh population (arrowed, c) could be detected using >120 nM hLa-GST-biotin. This population of B cells was of higher abundance than both populations a and b. This observation suggested that the IgMhigh hLa-specific B cells (b and c) were of lower affinity than the IgMlow cells (a) and overall were more numerous than the IgMlow hLa-specific B cells.

Anti-hLa B cells develop in the presence and the absence of endogenously expressed hLa Ag

To examine B cell tolerance to the nuclear autoantigen La, anti-hLa Ig Tg mice were mated with mice expressing human La under the control of the endogenous hLa promoter (hLa Tg) to create hLa x anti-hLa double-Tg offspring (hLa x anti-hLa Tg). This allowed the fate of anti-hLa B cells to be examined in the presence and the absence of constitutive hLa expression. The number of B cells in the spleen and bone marrow of double-Tg mice was the same as that in anti-hLa single-Tg littermates, and was approximately one-third of that in hLa Tg mice or non-Tg littermates (Table I). The reduced number of total spleen cells observed in both anti-hLa single- and double-Tg mice was due to a 50% reduction in the number of B cells and was also reflected in the bone marrow and peripheral blood. This is a common observation in Ig Tg animals and probably reflects the constraints imposed by the expression of a limited Ig repertoire (reviewed in Ref.19). The proportions and numbers of CD4+ and CD8+ T cells were unchanged in anti-hLa Tg mice, indicating that B and T cell numbers were properly maintained in the presence of the hLa autoantigen.


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  		Table I. Lymphocyte populations in anti-hLa Tg micea

 
Allelic exclusion of endogenous IgH genes remained at ~95% efficiency, and the pattern of surface IgM (sIgM) staining of B cells from double-Tg mice was identical with that in anti-hLa single-Tg littermates (Fig. 2A). Pre-B cell (B220low IgM-) and mature B cell (B220high, IgM+) B cell populations within the bone marrow each comprised between 10–12% of gated lymphocytes in anti-hLa single- and double-Tg mice (Fig. 2B), suggesting that the kinetics of B cell maturation were unchanged in double-Tg animals. Given that an increase in immature B cell (B220low, IgM+) and total bone marrow B cell numbers has been associated with the editing of autoreactive B cells (20, 21), these data suggest that receptor editing of hLa-specific B cells is unlikely to be occurring significantly in hLa x anti-hLa double-Tg mice.



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  		FIGURE 2. Mature anti-hLa B cells develop in the presence of endogenous hLa Ag expression. A, Allelic exclusion of endogenous IgMb and surface IgM levels were examined using three-color FACS analysis of splenic lymphocytes from anti-hLa and hLa x anti-hLa Tg mice. Cells were stained with Abs specific for B220, IgMa, and IgMb, and contour plots were gated on B220+ lymphocytes. B, Bone marrow from anti-hLa and hLa x anti-hLa Tg mice were stained with Abs specific for B220 and IgMa and were analyzed using two-color FACS. Relative levels of pre-B cell (B220low IgM-ve; upper left quadrants) and mature B cell (B220high, IgM+ve; upper right quadrants) populations were not significantly different between anti-hLa and hLa x anti-hLa Tg mice tested. Dot plots are representative of at least four mice from each group, aged 9–14 wk. Quadrant data are expressed as a percentage of the gated lymphocytes.

 
Anti-hLa B cells mature and populate the periphery normally despite ubiquitous expression of the autoantigen hLa

To determine whether the frequency of hLa-specific B cells in hLa x anti-hLa double-Tg mice was altered compared with that in anti-hLa single-Tg littermates, bone marrow and spleen cells were stained and analyzed by three-color FACS. Autoreactive hLa-specific B cells were detected in the spleen and bone marrow of double-Tg mice at levels comparable to those seen in anti-hLa single-Tg mice (4–7% spleen and 1–4% bone marrow; Fig. 3). The ratios of the IgMlow and IgMhigh populations of splenic and bone marrow autoreactive B cells were unchanged despite the constitutive expression of the hLa autoantigen.



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  		FIGURE 3. Autoreactive hLa-specific B cells can be detected in the spleen and bone marrow of hLa x anti-hLa double-Tg mice. Splenocytes (A) and bone marrow cells (B) were stained with biotinylated hLa-GST or mLa-GST, and Abs for B220 and IgMa. Contour plots and percentages represent B220+ gated lymphocytes. The percentage of hLa-specific B cells varied between 4–7% in the spleen and 1–4% in the bone marrow in at least six mice, aged 9–14 wk, from each group.

 
The maturation of autoreactive hLa-specific B cells was determined by the expression of phenotypic markers CD23 and CD24/HSA and compared with that of hLa-specific B cells from anti-hLa single-Tg mice lacking endogenous expression of the hLa autoantigen. Most hLa-specific splenic B cells from anti-hLa and double-Tg mice were CD23+ (Fig. 4A, upper right quadrants) and CD24low (Fig. 4B, upper left quadrants), reflecting a mature B cell phenotype. The data suggest that the maturation of hLa-specific B cells is comparable in the presence and the absence of endogenous expression of hLa autoantigen. This finding is in contrast to the anti-Sm/anti-ssDNA Tg model, where the majority of peripheral autoreactive B cells appeared to be arrested at the T-1 transitional B cell stage (4, 6). Overall, therefore, the CD23 and CD24 phenotype data suggest that the presence of the hLa autoantigen in hLa x anti-hLa double-Tg mice does not significantly alter the maturation of hLa-specific B cells in these animals.



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  		FIGURE 4. The majority of autoreactive hLa-specific B cells from hLa x anti-hLa Tg mice are mature. Spleen cells were stained with biotinylated hLa-GST and Abs specific for B220, IgMa, and the maturation markers CD23 (A) or CD24 (B). Analysis was by four-color FACS, and percentages of B220+ lymphocytes shown in each quadrant are representative of at least four mice from each group, aged 9–14 wk. C, Peritoneal cells were stained with biotinylated hLa-GST and Abs specific for B220 and CD5. The B220+ population was gated, and the percentages of B220+, hLa-specific (top) and B220+, non-hLa-binding (bottom) cells expressing CD5 are shown. Data were obtained from four mice, aged 6–12 wk, pooled in each group and are representative of three separate experiments.

 
Recent work (4, 22) has suggested that differentiation of B cells to the B-1 linage may help maintain ignorance by increasing the activation threshold of autoreactive B cells. However, this does not seem to be the case for hLa-specific B cells, which were predominantly of the B-2 lineage in the peritoneum. CD5+ B-1 B cells made up 30–50% of B220+ cells regardless of their hLa specificity or whether they were transgenic (Fig. 4C).

Anti-hLa B cells are not activated in vivo

To discriminate between B cell anergy vs ignorance, the expression levels of a number of surface markers were examined on hLa-specific B cells that developed in the presence and the absence of endogenously expressed hLa autoantigen. The expression levels of MHC class II, which is up-regulated upon exposure to cognate Ag, and the activation markers CD69, CD80, and CD86 were equivalent on hLa-specific B cells in hLa Tg and non-Tg mice (Fig. 5). Furthermore, the expression of these markers was equivalent to those observed on nonspecific B cells from naive non-Tg mice as well as on nonspecific B cells from anti-hLa and hLa x anti-hLa-Tg mice (data not shown). These data suggest that hLa-specific B cells from both single- and double-Tg mice do not have an activated phenotype. In contrast, activation of anti-hLa B cells by culture in the presence of LPS for 16 h resulted in increased surface expression of MHC class II (2-fold) and CD69 (7-fold) as well as the induction of CD80 and CD86 expression in B cells from anti-hLa Tg and hLa x anti-hLa Tg mice (Fig. 5, right panels).



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  		FIGURE 5. The phenotype of anti-hLa B cells developing in the presence of endogenous hLa is identical with that of naive anti-hLa B cells, suggesting Ag ignorance. Spleen cells from hLa x anti-hLa (bold line) and anti-hLa (filled) Tg mice were gated for staining with B220 and biotinylated hLa-GST. Human La-specific B220+ B cells were analyzed for expression levels of B220, CD69, CD80, CD86, and MHC class II immediately ex vivo (left panels) or following 16-h activation in the presence of 50 µg/ml LPS (right panels). Histograms represent the log intensity of fluorescence.

 
Autoreactive serum titers are unaltered in the presence of endogenous hLa Ag

Basal serum IgMa levels were measured in Tg mice and non-Tg littermates to determine whether Tg IgMa-positive B cells could spontaneously differentiate into Ab-secreting cells. IgMa was not detectable in non-Tg mice, as expected, but anti-hLa single- and double-Tg mice had significant levels of serum IgMa, averaging 189 ± 53 and 163 ± 54 µg/ml, respectively, similar to those reported in naive anti-HEL (23), and anti-Sm (5) Ig Tg mice. The spontaneous differentiation of naive Tg B cells into Ab-secreting cells, presumably as a result of polyclonal activation by exogenous Ag, is a common feature of Ig Tg mice and suggests that B cell maturation and function are normal.

The serum titer of specific anti-hLa Abs was also measured in anti-hLa single- and double-Tg mice. Double-Tg mice produced anti-hLa IgMa Abs at levels comparable to those found in anti-hLa single-Tg mice, whereas anti-mLa IgMb Abs were not detectable in either Tg group (Fig. 6A). Pooled sera from non-Tg, hLa Tg, single- and double-Tg littermates were also analyzed for their specificity by immunoblot. While positive control sera from hLa-hexa-histidine (hLa-6xHis)-immunized BALB/c and C57BL/6 mice both reacted with mLa-GST and hLa-GST, the sera from anti-hLa single- and hLa x anti-hLa double-Tg mice only reacted with hLa-GST (data not shown). Pooled sera from young (9–14 wk) and older (25–29 wk) mice did not react with a panel of human autoantigens, including Sm (SmB and SmD), RNP (RNP-70k, RNP-A, RNP-C), Ro52 and Ro60, centromere (Cenp-B), Scl-70 (DNA topoisomerase I), Jo-1, and ribosomal P (INNO-LIA Anti-Nuclear Ab kit; Innogenetics, Ghent, Belgium; results not shown). Unlike the findings in HEL x anti-HEL double-Tg mice (24, 25), anti-H-2Kk mice (26), and anti-Sm Tg mice (5), these data suggest that autoreactive hLa-specific B cells in anti-hLa double-Tg mice are apparently normal, with no evidence of peripheral regulation despite endogenous expression of the hLa autoantigen.



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  		FIGURE 6. Detection of anti-hLa Abs in anti-hLa Tg mice in the presence and the absence of endogenous hLa. A, Sera from individual anti-hLa and hLa x anti-hLa Tg mice were measured for basal IgMa hLa-specific Abs by ELISA. ELISA plates were coated with 5 µg/ml of recombinant mLa-GST or hLa-GST, and sera were diluted 1/250. Bound Abs were detected using a biotinylated anti-IgMa (DS1) Ab. B, BALB/c, anti-hLa and hLa x anti-hLa Tg mice were immunized s.c. with 50 µg of recombinant hLa-6xHis in CFA and boosted with 20 µg of hLa-6xHis in IFA on day 21. Preimmune (day 0), day 14, and day 28 sera were collected. ELISA plates were coated with 5 µg/ml of recombinant hLa-GST, and individual sera were titrated from 1/125. Bound Abs were detected using a biotinylated anti-IgMa (DS1) Ab. End-point titers were determined by the mean ± 3 SD of the preimmune BALB/c sera. C, IgH Tg mice can generate Abs to a nominal Ag. Sera from individual hLa x anti-hLa, anti-hLa, and non-Tg mice were measured for induced IgMa (left) and IgMb (right) anti-OVA Abs following immunization with OVA. Mice were immunized with 100 µg of OVA in CFA and were boosted twice with OVA in IFA at 14-day intervals. Plates were coated with 5 µg/ml of OVA, and sera were tested at 1/125. Bound Abs were detected using biotinylated anti-IgMa (DS-1) or anti-IgMb (AF6-78.25) Ab. Positive sera were determined by the mean ± 3 SD of the preimmune sera.

 
To determine whether basal anti-hLa production could be boosted in the Tg mice, Tg and control mice were immunized with hLa-6xHis, with a secondary challenge on day 21. Anti-hLa IgMa Ab titers were then determined in preimmune and day 14 and 28 immune sera. Control BALB/c mice developed anti-hLa IgMa and IgG serum Ab after primary immunization, and these responses were boosted following secondary immunizations. In contrast, neither single- nor double-Tg mice developed autoantibody levels above basal anti-hLa IgMa Ab titers (Fig. 6B). There are two explanations for the lack of boosting of anti-hLa autoantibody levels in Ig Tg mice. Firstly, constitutive anti-hLa Abs in the serum of Ig Tg mice are likely to mop up the autoantigen, preventing efficient stimulation of resting B cells. Secondly, maximum secretion with saturation levels of anti-hLa Abs may have already been reached in these mice, given that their basal Ab levels are equivalent to the highest titers seen in BALB/c immunized and boosted animals. Another possibility is that B cells from Tg mice are poorly responsive to any Ag challenge.

Because the Tg H chain must pair with endogenous L chains, only a proportion of the B cells in these Tg mice have anti-hLa specificity. We therefore examined the capacity the transgenic mice to generate an IgMa response to the nominal Ag OVA. Mice were immunized with OVA in CFA, and their sera were examined for both IgMa and IgMb OVA-specific Abs (Fig. 6C). Both Tg and non-Tg mice made a substantial IgMb anti-OVA response, even though in the Tg mice the endogenous IgMb-expressing B cells make up <10% of the total B cells. Anti-hLa Tg and hLa x anti-hLa Tg mice were both capable of making only a very weak, variable IgMa anti-OVA response. Thus, the Tg IgMa H chain is capable, albeit in a severely constrained manner, of pairing with endogenous L chains to form BCRs with other specificities, then signal through the transgenic H chain and secrete Ab.

Stimulation of splenocytes with LPS in vitro results in production of high titer autoantibody

Despite the variety of phenotypes associated with B cell anergy, a reduced capacity to differentiate into Ab-secreting cells remains the fundamental property that defines an anergic B cell. Previous studies have shown that while LPS triggers the proliferation of anergic B cells, Ab secretion is markedly reduced (27). To assess whether autoreactive anti-hLa B cells from double-Tg mice respond to activation by secreting Ab, splenocytes were stimulated with LPS for 3 days in vitro, and culture supernatants were examined for the presence of anti-hLa Abs by ELISA (Fig. 7). Stimulation with LPS induced a dose-dependent proliferation (data not shown) and secretion of anti-hLa Ab in splenocytes from both anti-hLa single-Tg and hLa x anti-hLa double-Tg mice (Fig. 7). Secreted Ab was specific for hLa, as the resulting Abs did not react with mLa by ELISA, and Ab binding to hLa was inhibited by hLa-GST protein, but not by mLa-GST or GST alone (data not shown). Importantly, there was no significant difference in the levels of secreted hLa-specific Ab by single- and double-Tg mice. In addition, depletion of CD5+ B cells from the splenic B cell populations had no effect on the LPS-induced secretion of anti-La IgMa Ab (data not shown). Interestingly, peritoneal B cells could only be induced to secrete low levels of anti-hLa IgMa, and this also was not altered by depletion of CD5+ B cells. This indicates that autoreactive hLa-specific B cells from mice expressing endogenous hLa were equally responsive to varying concentrations of LPS as the naive hLa-specific B cells from anti-hLa single-Tg mice, suggesting that the autoreactive B cells are not anergic. Moreover, this finding suggests that the inability to boost anti-hLa Ab titers by immunization of anti-hLa Tg mice is most likely due to sequestration of immunizing Ag by the basal level of anti-hLa autoantibodies.



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  		FIGURE 7. Anti-hLa B cells can be stimulated to secrete Ab in vitro. Splenocytes from anti-hLa (•), hLa x anti-hLa ({circ}), and hLa ({blacksquare}) mice and non-Tg littermates ({square}) were cultured for 3 days in the presence of graded amounts of LPS. Culture supernatants were harvested, diluted 1/5, and tested by ELISA for reactivity to hLa-GST or mLa-GST. Spleen cells from five mice of each genotype were cultured in triplicate.

 
Anti-La Tg B cells developing in the presence of hLa retain their capacity to signal via the BCR

In addition to the failure to secrete Ab in response to LPS, anergic B cells have been shown to be refractory to BCR stimulation. To assess the capacity of the transgenic IgM H chain to signal, we have examined the tyrosine phosphorylation capacity of splenic B cells from non-Tg, anti-hLa Tg, and hLa x anti-hLa Tg mice upon membrane IgM cross-linking. Splenic B cells were stimulated with anti-IgM and lysed, and lysates or Syk immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine. No difference in intracellular protein tyrosine phosphorylation (Fig. 8A) or Syk phosphorylation (Fig. 8B) was observed between non-Tg B cells and anti-hLa Tg B cells developing in the presence or the absence of the hLa Ag.



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  		FIGURE 8. B cells expressing the anti-hLa IgMa H chain are competent at inducing tyrosine phosphorylation following IgM cross-linking. Splenic B cells were isolated from anti-hLa, anti-La x hLa, and their non-Tg littermates. Splenic B cells (3 x 107 cells/ml) were then stimulated with 10 µg/ml F(ab')2 goat anti-mouse IgM at 37°C for 3 min. Cells were lysed, and 2 x 105 cell equivalents of whole lysate (A) or 1.5 x 106 cell equivalents of Syk immunoprecipitates (B) were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine Ab.

 
Anti-hLa B cells do not have a reduced life span when developing in the presence of hLa autoantigen

Anergic B cells are reported to have a reduced life span of 3–4 days compared with nontolerant B cells that survive up to 4 or 5 wk (3, 17, 28, 29). The in vivo life span can be estimated by the incorporation of the thymidine analog BrdU into dividing cells. An increased proportion of cells incorporating BrdU reflects the increased rate of cell renewal required to homeostatically maintain B cell numbers. The effect of endogenous hLa expression on the life span of B cells in vivo was estimated by continuous labeling of anti-hLa, hLa x anti-hLa, and non-Tg mice with BrdU for 8 days. BrdU incorporation into hLa-specific or nonspecific splenic B cells was determined by flow cytometry (Fig. 9), and the mean incorporation of each gated population is shown in Table II. In all mice expressing the IgMa transgene the proportion of B cells incorporating BrdU was approximately two-thirds of that observed in non-Tg littermates, reflecting differences in life span intrinsic to IgM transgene expression. The proportion of BrdU-labeled cells was the same in both hLa-specific and nonspecific B cell populations in Tg mice. Moreover, there was no difference in BrdU incorporation of hLa-specific B cells developing in the presence or the absence of endogenous hLa Ag. Together these data revealed no difference in the life span of anti-hLa and nonspecific B cells from Tg mice regardless of the presence or the absence of autoantigen.



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  		FIGURE 9. Anti-hLa-specific B cells have the same in vitro turnover rate in the presence and the absence of endogenously expressed hLa Ag. BrdU was continuously administered to mice in the drinking water for 8 days. Splenocytes from anti-hLa, hLa x anti-hLa, and non-Tg mice were stained with biotinyated hLa-GST and Abs for B220, fixed, and permeabilized, and then incorporated BrdU was detected with anti-BrdU. Contour plots show binding to hLa (left) and histograms show BrdU incorporation within the hLa-specific or nonspecific B220+ populations (right). Percentages are given for BrdU+ cells. Contour plots and histograms are representative of six mice of each phenotype.

 

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  		Table II. Anti-hL-specific B cells have the same in vivo turnover rate in the presence or absence of endogenously expressed hLa Ag

 
Anti-hLa autoantibodies are not pathogenic

In human systemic autoimmune diseases such as systemic lupus erythematosus and Sjögren’s syndrome, immune complexes of autoantibody and autoantigens can induce tissue damage by depositing in basement membranes of endothelial cells. Moreover, in primary Sjögren’s syndrome anti-La autoimmunity is associated with salivary gland infiltration and cellular damage. Therefore, kidney and submandibular salivary gland samples were taken from 8- to 9-mo-old non-Tg, hLa, anti-hLa, and hLa x anti-hLa Tg mice and were examined for tissue pathology in a double-blind analysis of multiple sections by two independent pathologists. No tissue destruction or lymphocytic infiltrate above that expected of animals of this age was observed (data not shown), indicating that anti-hLa B cells chronically exposed to endogenous hLa autoantigen in 8- to 9-mo-old mice do not provoke systemic lupus erythematosus-like autoimmunity in this context despite the high basal titer of serum hLa-specific Abs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have examined the fate of B cells specific for human La Ag developing in both the presence and the absence of endogenously expressed cognate Ag in a double-Tg model. There are a number of advantages of this model over other Tg models investigating B cells tolerance. Firstly, the Ag is an authentic nuclear protein target of the autoimmune diseases systemic lupus erythematosus and primary Sjögren’s syndrome. Secondly, by exploiting Tg mice that only express the IgM H chain it has been possible to follow the development of specific B cells in the presence of polyclonal competitor B cells. Thirdly, the use of double-Tg mice, which exploits the difference between the mouse and human homologues of La, allows examination of the functional state of B cell tolerance in both the presence and the absence of endogenous cognate Ag. The most extensively studied lupus-associated nuclear autoantigen is DNA, and anti-DNA Ig Tg models have made important contributions to the tolerance paradigm underlying systemic autoimmune disease. However, DNA is a high abundance nucleic acid and so is likely to elicit tolerogenic responses that rely on mechanisms different from those that may operate for low abundance protein autoantigens such as La. While anti-Sm B cell anergy or differentiation to B-1 cells (4, 30) may be more likely to reflect the fate of autoreactive La-specific B cells, in both anti-Sm and anti-DNA Tg models it has not been possible to monitor the fate of autoreactive B cells in the absence of the autoantigen. Without this important control, any functional or phenotypic B cell changes that result from transgene insertion and/or expression may incorrectly be interpreted as tolerance-related. The development of tolerance in the presence or the absence of ligand has been extensively studied with soluble (23, 31, 32) or membrane-associated Ags (18); however, this is unlikely to reflect the effect of a nuclear Ag on developing B cells.

Autoreactive hLa-specific B cells mature and populate the periphery

Although La is a relatively low abundance protein sequestered primarily in cell nuclei, it is likely to be accessible to autoreactive lymphocytes under certain circumstances, such as apoptosis of cells (13, 33, 34), since hLa peptides appear to be constitutively presented by B cells in hLa Tg mice (7). Despite this Ag exposure, splenic autoreactive hLa-specific B cells were not deleted in hLa x anti-hLa double-Tg mice, but instead were present in numbers equivalent to those of naive hLa-specific B cells from single-Tg anti-hLa littermates. Anti-hLa B cells developing on either an hLa+ or hLa- background were both predominantly B220high, CD23+, and CD24low, indistinguishable from the majority of other Hµ transgene-positive B cells or mature follicular B cells from non-Tg littermates. This indicated that hLa-specific B cells mature and populate the spleen regardless of whether the hLa autoantigen is endogenously expressed.

Human La-specific B cells were also detected in the bone marrow of single- (anti-hLa) and double- (hLa x anti-hLa) Tg mice in equivalent numbers, confirming that autoreactive hLa-specific B cells were not deleted or developmentally arrested. CD23 and CD24 expression was not examined in the bone marrow, since expression levels on pro- and pre-B cells can mimic those seen on immature, transitional, or mature B cells, making CD23 and CD24 unreliable as markers of B cell maturation in the bone marrow. However, the majority of IgM+ bone marrow B cells were mature B220high cells, detectable in the presence and the absence of endogenous hLa autoantigen. The immature and T-1 transitional bone marrow B cell pool in both single- and double-Tg mice was markedly reduced compared with that in non-Tg littermates, probably due to accelerated maturation prompting an earlier exit of transgene-positive B cells from the bone marrow (data not shown). This phenomenon is commonly observed in Ig Tg mice (reviewed in Ref.19). Despite this observation, the proportion of mature recirculating B220high B cells relative to developing IgM- B220low pro- and pre-B cells remained the same in the bone marrow of single-, double-, and non-Tg littermates, suggesting that B cell development was otherwise normal in the absence or the presence of endogenous hLa. While our data cannot exclude the possibility that a subset of high affinity hLa-specific B cells are generated and deleted in hLa x anti-hLa Tg mice, this seems unlikely for a number of reasons. Firstly, there is no significant difference between the number of bone marrow hLa-specific B cells developing both in the presence or the absence of the hLa autoantigen. Secondly, clonal deletion of autoreactive B cells in the bone marrow is usually preceded by the developmental arrest of immature B cells and the rearrangement of Ig endogenous L chain genes characteristic of receptor editing (1). These data suggest that, in contrast to data from anti-DNA and anti-Sm Ig Tg mice (1, 4, 5, 35), clonal deletion or developmental arrest are unlikely to be mechanisms of B cell tolerance to endogenous La. While we have not formally ruled out receptor editing in Tg bone marrow B220+ cells, the absence of a population of arrested sIgMlow immature B cells in the bone marrow makes this type of tolerance unlikely.

The maintenance of peripheral ignorance by differentiation of autoreactive B cells to B-1 cells has been described in several systems (4, 22, 36). Generation of B-1 B cells appears to depend upon the BCR specificity from which it originated, with B-1-derived transgenes generating B cells of a CD5+ B-1 phenotype, and B-2-derived Ig transgenes generating predominantly B-2 cells (reviewed in Ref.37). Human La-specific B cells were derived from a B-2 hybridoma, and hLa-specific transgenic B cells do not appear to be over-represented in the B-1 compartment of peritoneal B cells as is observed in the anti-Sm Tg model (4). Splenic B cells depleted of CD5+ B-1 cells are capable of secreting anti-hLa Ab upon in vitro LPS stimulation and signal following IgM cross-linking. These findings further reduce the likelihood that hLa-specific B cells have encountered and responded to an endogenous tolerogen.

Significance of the two major populations of hLa-specific B cells in anti-hLa and hLa x anti-hLa Tg mice

Anti-hLa B cells in both single- and double-Tg mice were distributed into two predominant populations based on surface IgMa expression. Given that anti-hLa Ig Tg mice do not express a functional {kappa}-chain transgene, multiple hLa-specific B cell subpopulations may result from the preferential pairing of different L chains or L chain family members with the fixed anti-hLa µ-chain (17, 18). While the association of endogenous L chains with fixed Tg H chains can produce B cells of unpredictable specificity (38), the H chain often provides most of the structural specificity for Ags (39). If endogenous L chain usage is restricted to a few V{kappa}/{lambda} families, then differential compatibility for the Tg H chain might lead to differential cell surface IgM expression. Endogenous V{kappa}/{lambda}-chain usage may also influence Ag affinity, and this might explain the difference in hLa affinity seen for each hLa-specific B cell population. This was observed in the anti-HEL Ig H chain Tg mice, in which the expression of the H chain alone resulted in four discrete populations of B cells with different affinities for HEL and different surface IgM expression levels in the absence of Ag (17, 18). In the presence of membrane-bound HEL, these lower affinity B cells are efficiently deleted. The down-regulation of sIgM has been associated with B cell anergy when B cells are exposed to self-Ag during development (23). However, in the model described here, the IgMlow hLa-specific B cell population is also present in mice that do not express endogenous hLa. Theoretically these B cells could cross-react with an unknown endogenous tolerogen, as has been observed in anti-p-azophenylarsonate Ig Tg mice (38). Benschop et al. (38) found that B cells cross-reacting with an unidentified autoantigen demonstrated many of the hallmarks of anergy (down-regulation of IgM, activated phenotype, refractory to BCR-mediated induction of tyrosine phosphorylation, and Ab production). However, we have not detected reactivity with a panel of known autoantigens including mLa, nor do anti-hLa B cells show any evidence of an anergic phenotype. In this study we have been careful to compare the fate of the hLa-specific Tg B cells, Tg B cells unable to bind hLa, and non-Tg littermates, and in all instances we have been unable to observe any evidence of an anergic phenotype.

Anti-hLa B cells and clonal ignorance

The definition of anergy has become increasingly complex as more numerous phenotypic and functional measures of clonal inactivation are defined. In its broadest sense, anergy is an active Ag-driven modification of autoreactive B cell activation thresholds resulting in an impaired proliferative and secretory response following subsequent Ag encounter. In this study the constitutive expression of the nuclear autoantigen hLa failed to trigger the elimination or inactivation of autoreactive hLa-specific B cells in hLa x anti-hLa double-Tg mice. High titer secreted anti-hLa Abs were detected in the sera of both anti-hLa single-Tg and hLa x anti-hLa double-Tg mice, indicating that hLa-specific B cells in double-Tg mice are likely to be maturationally and functionally normal (23, 29, 40). This is also consistent with their nonactivated phenotype, life span in vivo, ability to vigorously secrete Ab in response to in vitro stimulation, and unaltered capacity to signal through surface IgM cross-linking, similar to the findings of Keoning-Marrony et al. (41), who observed that multireactive, low affinity, natural autoantibody Tg B cells were ignorant of their Ag in vivo and in vitro, but were readily activated through both BCR-dependent and independent mechanisms. Thus, we have not observed any difference in the presence, development, signaling, or activation status of anti-hLa B cells in the presence or the absence of the hLa Ag.

Clonal ignorance has also often been linked to subthreshold Ag affinity (reviewed in Refs. 19 and 41). The anti-hLa Tg H chain variable region of mAb A3 was derived from class-switched, affinity-matured splenic B cells and is of high affinity, as reflected in its activity in indirect immunofluorescence, FACS, immunoprecipitation, and immunoblot assays (14). However, the sIgM anti-hLa Abs in Tg mice assemble with endogenous Ig L chains to produce a spectrum of high (2 nM staining) and low (500 nM staining) avidity interactions with hLa Ag in FACS analysis. Therefore, we consider it unlikely that the failure to delete or inactivate anti-hLa B cells is due to low avidity for hLa autoantigen. Moreover, low affinity BCR interactions alone do not always lead to ignorance (18, 36). Ag valency may play a significant role in autoreactive B cell tolerance, given evidence that tolerance appears to require BCR cross-linking (reviewed in Ref.42). The lack of observable tolerance to hLa and similar lupus-associated autoantigens might be linked to the concentration of specific Ag (9, 10) or the fact that La is not accessible in multimeric form.

Ironically, hLa-specific autoreactive B cells encounter Ag in quantities sufficient to be activated in the presence of T cell help (7). This supports the idea that the incomplete B cell tolerance to endogenous La observed in normal and hLa Tg mice (8, 11) depends upon failure to activate the T cell compartment. A similar finding was seen in mice expressing the neo-self Ag HEL in the thymus, where HEL-specific B cells were not tolerized, yet partial tolerance to HEL existed in the T cell compartment (43). In contrast to the significant B cell tolerance that exists to the lupus-associated autoantigens DNA and Sm (6, 44, 45), this finding suggests that even though hLa- or HEL-specific autoreactive B cells are not directly tolerized, the development of humoral autoimmunity is constrained by a deficiency of functional autoreactive Th cells. Under these circumstances, self-reactive B cells are not strictly ignorant, in that they appear to take up endogenous autoantigen constitutively and are capable of Ag presentation to T cells. Therefore, we prefer to think of these B cells as being "indifferent" to Ag encounter until Th signals are delivered and autoimmunity can develop. This indifference is not associated with any discernible phenotypic change in these B cells.


   Acknowledgments
 
We express appreciation to Prof. C. Goodnow for the expression constructs, Dr. E. K. L. Chan for the A3 anti-hLa-specific hybridoma, Dr. R. Brink for the anti-IgMa (RS3.1) and IgMb (AF6-78.25) mAbs, Dr. N. Baumgarth for the gift of biotinylated anti-IgMa (DS-1) and biotinylated anti-IgMb (AF6-78.25), Prof. P. Waring for histological examination of tissue sections, and Dr. D. Tarlington for technical advice and support.


   Footnotes
 
1 This work was supported by the National Health and Medical Research Council of Australia, the Arthritis Foundation of Australia, and the Victorian Lupus Association. Back

2 B.D.A. and C.L.K. contributed equally to this report. Back

3 Address correspondence and reprint requests to Prof. James McCluskey, Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria 3010, Australia. E-mail address: jamesm1{at}unimelb.edu.au Back

4 Abbreviations used in this paper: Sm, Smith Ag; BCR, B cell Ag receptor; BrdU, 5-bromo-2'deoxyuridine; HEL, hen egg lysozyme; 6xHis, hexa-histidine; hLa, human La Ag; mLa, mouse La Ag; RNP, ribonucleoprotein; sIg, surface Ig; Tg, transgenic. Back

Received for publication October 24, 2002. Accepted for publication September 26, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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