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The Journal of Immunology, 2008, 180, 3807-3814
Copyright © 2008 by The American Association of Immunologists, Inc.
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A Lupus-Suppressor BALB/c Locus Restricts IgG2 Autoantibodies without Altering Intrinsic B Cell-Tolerance Mechanisms1

Tatyana Tarasenko, Hemanta K. Kole and Silvia Bolland2

Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Fc{gamma}R2B-deficient mice develop autoantibodies and glomerulonephritis with a pathology closely resembling human lupus when on the C57BL/6 (B6) background. The same mutation on the BALB/c background does not lead to spontaneous disease, suggesting differences in lupus susceptibility between the BALB/c and B6 strains. An F2 genetic analysis from a B6/BALB cross identified regions from the B6 chromosomes 12 and 17 with positive linkage for IgG autoantibodies. We have generated a congenic strain that contains the suppressor allele from the BALB/c chromosome 12 centromeric region (sbb2a) in an otherwise B6.Fc{gamma}R2B–/– background. None of the B6.Fc{gamma}R2B–/–sbb2a/a mice tested have developed IgG autoantibodies in the serum or autoimmune pathology. Mixed bone marrow reconstitution experiments indicate that sbb2a is expressed in non-B bone marrow-derived cells and acts in trans. sbb2a does not alter L chain editing frequencies of DNA Abs in the 3H9H/56R H chain transgenic mice, but the level of IgG2a anti-DNA Abs in the serum is reduced. Thus, sbb2a provides an example of a non-MHC lupus-suppressor locus that protects from disease by restricting the production of pathogenic IgG isotypes even in backgrounds with inefficient Ab editing checkpoints.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The development of autoimmunity in diseases like systemic lupus erythematosus requires the interaction of multiple genetic loci with environmental factors to result in the loss of tolerance, the emergence of an autoreactive repertoire, and the amplification of autoreactive cells to produce high affinity pathogenic autoantibodies (1, 2, 3). Characterization of murine models of spontaneous lupus disease, including New Zealand Black/New Zealand White, MRL/Mp-lpr/lpr, BXSB, 126/C57BL/6 (B6),3 and B6.Fc{gamma}R2B–/–, has begun to uncover specific genetic components and reveals the importance of multiple interacting genes to generate a complete disease pattern (4, 5, 6, 7, 8, 9, 10, 11). In the case of the Fc{gamma}R2B-deficient mice, the development of spontaneous autoimmune disease is dependent on specific background genes: Fc{gamma}R2B-deficient mice of the B6 background develop anti-nuclear autoantibodies (ANA) by 7 mo and lethal glomerulonephritis by 9 mo, whereas the same deficiency does not result in autoimmunity or tissue pathology when maintained on the BALB/c background (12). An analogous background effect where B6-derived mice are susceptible to lupus disease although BALB/c mice with the same genetic alteration show a different phenotype has been reported for Bcl-2 transgenic and PD1-ko mice (13, 14, 15). The general predisposition of B6 mice to develop lupus disease in a reported large number of knockout and transgenic mouse models (16) suggests that this genetic background is naturally prone to the development of antinuclear Abs, whereas the BALB/c background is not.

We have set up experiments to identify genetic variants between B6 and BALB/c that could explain this difference in lupus susceptibility in the Fc{gamma}R2B-ko mouse model. An F2 analysis of the cross between B6.Fc{gamma}R2B–/– and BALB.Fc{gamma}R2B–/– showed linkage of the autoantibody phenotype with two regions derived from the B6 genome: one on chromosome 17 closely linked to MHC genes (sbb3) and another in the centromeric portion of chromosome 12 (sbb2) (17). We have initiated the analysis of sbb2 by the generation of congenic strains that contain various regions of chromosome 12 from BALB/c in mice of the B6 background. Previous studies have successfully used the analysis of congenic strains to dissect the contribution of individual susceptibility alleles to a multigenic trait such as systemic lupus erythematosus. Individual locus contribution to susceptibility has been described using congenic strains for the NZM, BXSB, New Zealand White/B6, and 129/B6 mice (18, 19, 20, 21). Likewise, but using a reciprocal approach, we have transferred the protective BALB/c allele (sbb2a) into the lupus prone B6.Fc{gamma}R2B–/– mouse so that we can analyze its suppressor effect in the Fc{gamma}R2B-deficient lupus model. Previous analysis of the inheritance of the sbb2 locus among F2 progeny suggested that it contributes to susceptibility in an allele dose manner, intermediate for heterozygotes and complete for homozygotes (17). As predicted by the genetic analysis, we have observed that the presence of sbb2a, even as heterozygote BALB/B6, eliminates the serum ANA and the lupus pathology in B6.Fc{gamma}R2B–/– mice.

To test whether the presence of the sbb2a allele modifies tolerance checkpoints in B cells, we have bred B6.sbb2a congenic mice to transgenic mice expressing a BCR with dsDNA specificity. BCR-transgenic models have shown that autoreactive B cells are tolerized by mechanisms that involve deletion, anergy, and editing (1). A well studied transgenic model is the 3H9H-56R Ig H chain gene-insertion that generates B cells expressing anti-dsDNA and where tolerance is achieved by L chain editing (22). These transgenics have been characterized in the BALB/c background and in the B6 background (22, 23, 24). Anti-DNA Abs were not detected in the serum of BALB/c mice because of Ig L chain editing using silencing L chain variants, thus generating Abs that no longer bind to dsDNA. In those studies B6 and BALB/c strains show a difference in Ig L chain usage, consistent with the idea that receptor editing is the dominant pathway for the abrogation of anti-self specificities in BALB/c mice but that does not happen in B6 mice. Fc{gamma}R2B deficiency was reported to increase the level of IgG Abs to dsDNA and renal disease in B6.56R mice by allowing the persistence of anti-DNA plasma cells (25). In this manuscript, we analyze the contribution of the sbb2 locus to the background effect by introducing the 56R Vh transgene into our newly generated congenic B6.Fc{gamma}R2B–/–sbb2a/a mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

B6.Cg-Igha Thy1a Gpi1a/J and B6.Rag-1–/– mice were purchased from The Jackson Laboratory. Mice were intercrossed to produce Fc{gamma}RIIB–/–IgHa/a progeny. All animal experiments were approved by the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee.

The parental B6.Fc{gamma}RIIB–/– mice were purchased from Taconic Farms. B6.Fc{gamma}RIIB–/–sbb2a/a mice were established by repeated backcrosses (N > 12) under the control of simple sequence length polymorphism markers D12Mit37, D12Mit168, D12Mit56, D12Mit170, D12Mit83, D12Mit236, and restriction polymorphism markers designed according to the Perlegen database. Six new congenic strains were produced and used in this study. All mice were maintained under identical conditions until up to 9 mo of age. DNA was extracted from tail biopsies. Blood samples were collected by orbital sinus puncture, and the sera were stored at –20°C until use.

Flow cytometry

Single-cell suspension of spleen and lymph node cells were prepared. Analysis by flow cytometry was performed on at least 10 mice from each group. Abs were purchased from BD Biosciences except for Foxp3 Abs, which were from eBioscience. Data were acquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Immunization and serum analysis

Mice were immunized with 50 µg/mice nitrophenyl chicken gamma globulin with Imject alum (Pierce) and reimmunized after 4 wk. Sera were tested by ELISA every week for 5 wk after first immunization. Briefly, plates were coated with NP-BSA 10 µg/ml (Biosearch Technologies), bound immunoglobulins were detected by alkaline phosphotase-conjugated detection Abs to specific mouse isotypes (Southern Biotechnology Associates). Anti-dsDNA ELISA was performed as previously described (Mohan 1993).

Microsatellite genotyping

Genotypes were determined by PCR using selected simple sequence length polymorphism markers. Primer sequences and genomic localization were obtained at the Ensemble genome browser http://www.ensembl.org/index.html and are available upon request. PCR amplification was conducted with the Bioline PCR Platinum mix according to Research Genetics protocol.

Histopathology

Kidney samples were collected from 9 mo old mice, fixed in formaldehyde, and embedded in paraffin. H&E staining of tissue sections was then performed by American Histolabs and visualized via light microscopy.

Bone marrow transfer

A total of 2 x 107 bone marrow cells from 6 to 7 wk old donor mice were i.v. injected into 7–12 wk old B6.Rag–/– or B6 mice that had been irradiated (400 rads or 950 rads, respectively) 1 day before injection. All mice, donors and recipients, were male. Blood was drawn 1 mo after the transfer, and cellular fractions were stained with Abs to IgMa, IgMb, and B220 (BD Biosciences) to confirm reconstitution. Serum was collected from recipients and tested for ANA 6 mo (for B6) or 9 mo (for RAG–/–) later.

Antinuclear Ab testing

ANA was performed using 12-well microscope slides covered with Hep-2 cells (Netherlands Foundation for Biological Research). Slides were incubated with mouse serum at the indicated dilutions. After two washes of 5 min with PBS, goat anti-mouse IgG-FITC (Sigma-Aldrich) was added at 1:500 dilutions for 15 min and washed with PBS for 5 min at least three times. For detecting allotype specific autoantibodies, biotinylated goat anti-mouse IgG2aa and IgG2ab (BD Biosciences) were used as primary Ab (1/500), and streptavidin-FITC (BD Biosciences) as secondary reagent (1/1000).

Hybridoma analysis

Splenocytes were isolated from 6 mo old 56R Fc{gamma}R2B–/–sbb2b/b and 56R.Fc{gamma}R2B–/–sbb2a/a mice. All splenocytes were stimulated for 3 days with 20 µg LPS (Sigma-Aldrich) and were subsequently fused with Sp2/O-Ag14 myeloma cells. For further studies, only hybrids derived from a single colony were used. Cell culture supernatants were used for anti-dsDNA ELISA. Genomic DNA was extracted with DNeasy 96 kit (Qiagen). PCR was performed according to (25).

Single-cell sorting, cDNA syntesis, and PCR

B220+IgMa+ splenocytes from two 56R.Fc{gamma}R2B–/–sbb2b/b and two 56R.Fc{gamma}R2B–/–sbb2a/a mice were sorted on a FACS Aria (BD Biosciences) into 96-well PCR plates containing 4 µl lysis solution (Ambion) and immediately frozen on dry ice. All samples were stored in –80°C. The DNA were synthesized in a total volume of 15 µl in the original 96-well PCR plate with cDNA synthesis kit (Bio-Rad). V{kappa}21D, V{kappa}38c, and 56R Vh chain transcripts were amplified according to Fukuyama et al. (25) by two rounds of PCR.

Statistical analysis

To determine statistical significance, a paired two-tailed Student’s t test was used for all comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Congenic B6.Fc{gamma}R2B–/–sbb2a/a mice do not develop the lupus disease seen in the B6.Fc{gamma}R2B–/– parental strain

The centromeric portion of the BALB/c chromosome 12 was transferred to the B6.Fc{gamma}R2B–/– background as congenic using a marker-assisted selection protocol (26). After 7 generations of backcrosses, over 99% of the genome in these mice was determined to be of B6 origin. Six different congenic lines, shown in Fig. 1A, were created this way and maintained by brother-sister mating. From the analysis of these congenic lines, the genomic region between 15.3 and 25.6 Mb on chromosome 12 was defined as the sbb2 locus, the BALB/c allele being sbb2a and the B6 allele being sbb2b. Lines 8466 and 8948 were generally healthy and were homozygous for the BALB/c allele of sbb2 (Fig. 1, A–C). Mice originated from these two lines will be hereafter referred to as B6.Fc{gamma}R2B–/–sbb2a/a. Lines 1062, 6710, 6995, and 5389, coming from the same crosses but having the region of chromosome 12 of BALB/c allele positioned outside the boundaries of the sbb2 locus, will be referred to as B6.Fc{gamma}R2B–/–sbb2b/b. Mice originated from these last four breeding lines showed mortality rates comparable to the parental strain B6.Fc{gamma}R2B–/– and provide an appropriate control of lupus susceptible mice that are generated from the same backcrosses as the experimental sbb2a/a mice. In addition, we found that mice heterozygous for the sbb2 locus (B6.Fc{gamma}R2B–/–sbb2a/b) had better survival rates than the parental strain B6.Fc{gamma}R2B–/– (Fig. 1C).


Figure 1
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  		FIGURE 1. Generation of congenic strains bearing the centromeric region of chromosome 12 from the BALB/c genome. A, The sbb2 locus is defined as the region between D12Mit169 and Id2 markers. Shown are the B6.Fc{gamma}R2B–/– parental strain (B6.R2–1) and six new congenic strains generated by BALB/c x B6 intercross and successive backcrosses to B6.Fc{gamma}R2B–/– until the background was >99% B6. Congenic lines 8466 and 8948 contain the BALB/c allele of sbb2 and are marked with an asterisk. Lines 1062, 6710, 6995, and 5389 contain regions in chromosome 12 from the BALB/c genome but they are outside the boundaries of sbb2. B, Candidate genes for the sbb2 locus on chromosome 12, mapped according to the UCSC genome browser. C, Survival rates of the new congenic strains. B6.R2B–/–sbb2a/a includes mice from the 8466 and 8948 lines; B6.R2B–/–sbb2b/b includes mice form the 1062, 6710, 6995, and 5389 lines; n > 12 per strain.

 
The observed low mortality rates in the newly generated B6.Fc{gamma}R2B–/–sbb2a/a mouse line correlated with absence of ANA and kidney pathology. None of the 12 B6.Fc{gamma}R2B–/–sbb2a/a mice tested developed ANA at 6 mo of age, compared with 47 of 55 (85%) for the B6.Fc{gamma}R2B–/–sbb2b/b mice and 90% for the parental B6.R2B–/– strain. None of the B6.Fc{gamma}R2B–/–sbb2a/a mice showed signs of glomerulonephritis, whereas 41 of 55 (75%) B6.Fc{gamma}R2B–/–sbb2b/b mice developed kidney disease (Fig. 2A). The reduction in ANA titer also correlated with reduced anti-dsDNA specificities in serum Abs: B6.Fc{gamma}R2B–/–sbb2a/a mice had levels of anti-dsDNA IgG2a detected by ELISA comparable to wild-type B6 mice and much lower than control B6.Fc{gamma}R2B–/–sbb2b/b mice (Fig. 2B). Total levels of serum IgG Abs were slightly lower in B6.Fc{gamma}R2B–/–sbb2a/a mice than in the parental strain but the difference was not significant (Fig. 2C).


Figure 2
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  		FIGURE 2. B6.Fc{gamma}R2B–/–sbb2a/a congenic mice do not develop autoantibodies or kidney pathology. A, ANA test on serum diluted 1/400 (top panels) and H&E staining of kidney sections at x200 magnification (bottom panels) from mice of the indicated genotypes. Data are representative of at least 12 mice per genotype. B, Anti-dsDNA ELISA test on serum from mice of the indicated genotype (n = 14). Short lines indicate average values. Dashed line is drawn at a level calculated as the median of B6 wild-type (wt) control plus two times the SD. C, Total Ig concentrations for the isotypes shown were tested by ELISA on serum diluted 1/100,000-1/500,000. Gray squares represent B6.R2–/–sbb2b/b; black triangles represent B6.R2–/–sbb2a/a.

 
Reduced spontaneous immune cell activation and lower Ag-specific IgG2 titers in sbb2a congenic mice

At 4 mo of age, the congenic B6.Fc{gamma}R2B–/–sbb2a/a mice had numbers of B cells and CD4+ and CD8+ T cells comparable to the B6.Fc{gamma}R2B–/– parental strain (Table I). The number of regulatory T cells determined by Foxp3 expression was also comparable in both strains (Table I). No difference was found in the ex vivo proliferation of naive T cells on anti-CD3 ± anti-CD28 plates or in the proliferation of purified B cells upon stimulation with anti-CD40 (data not shown). We did find differences in the number of activated lymphocytes in the spleen: CD69+ T cells were reduced by 50% in the sbb2a/a congenic mice, and the level of MHC-II surface expression (I-Ab) on B220+B cells was also reduced by half (Fig. 3 and Table I). sbb2a/a mice had reduced number of germinal center (Fas+GL7+) B cells and memory/effector (CD44low) T cells. These differences were even more pronounced at 8 mo of age (Fig. 3).


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  		Table I. FACS analysis of splenic cell populations in 4-mo-old sbb2 congenic mice

 

Figure 3
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  		FIGURE 3. Flow cytometric analysis of splenocytes from sbb2 congenic mice. Top panels, The gate and percentage of germinal center B cells (Fas+GL7+). Middle panels, The percentage of activated CD69+ CD4 T cells. Histograms show level of MHC-II surface expression on B cells (I-Ab staining) and the proportion of effector/memory T cells (CD44 staining). B6.Fc{gamma}R2B–/–sbb2a/a samples are shown as black lines, whereas B6.Fc{gamma}R2B–/–sbb2b/b samples are shown as gray histograms. Note that mice with the sbb2a allotype express lower levels of MHC-II on B cells and have lower numbers of effector/memory (CD44high) T cells.

 
We next tested exogenous Ag-specific responses in the new congenic strains using various immunization protocols to determine whether the presence of the lupus suppressor locus sbb2a could generally alter the humoral response. IgM responses were equivalent in these mice when immunized with the T-independent Ag 2,4,6-trinitrophenyl-Ficoll (Fig. 4A). In contrast, B6.Fc{gamma}R2B–/–sbb2a/a mice responded less efficiently in T-dependent immunizations, particularly in IgG2 switched isotypes: NP-specific IgG2a and IgG2b were reduced in B6.Fc{gamma}R2B–/–sbb2a/a mice when immunized with NP-CGG alum, whereas there was no difference in the IgG1 or IgM isotypes (Fig. 4B).


Figure 4
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  		FIGURE 4. B6.Fc{gamma}R2B–/–sbb2a/a congenic mice show lower IgG2 responses in T-dependent immunization. A, B6.Fc{gamma}R2B–/–sbb2a/a and B6.Fc{gamma}R2B–/–sbb2b/b mice show equal response to T-independent immunization with TNP-Ficoll, as determined by ELISA measurements of TNP-specific IgM levels in serum diluted 1/1000. B, ELISA measurements of NP-specific Abs of the isotypes shown in mice immunized with NP-CGG plus alum. Arrows indicate time of injection. Samples from B6.Fc{gamma}R2B–/–sbb2a/a mice are indicated as gray triangles and samples from B6.Fc{gamma}R2B–/–sbb2b/b mice are indicated as black diamonds. Serum was diluted 1/105 for IgG1 and 1/104 for all other isotypes.

 
The suppression conferred by sbb2a is provided in trans by non-B bone marrow cells

To begin to assess cellular components that contribute to the protection from autoimmune disease in sbb2a/a congenic mice, we performed bone marrow transfers from asymptomatic 6-wk-old B6.Fc{gamma}R2B–/–sbb2a/a or B6.Fc{gamma}R2B–/–sbb2b/b into lethally irradiated B6.Fc{gamma}R2B–/– mice. While mice reconstituted with B6.Fc{gamma}R2B–/–sbb2b/b bone marrow developed autoantibodies and kidney disease, animals reconstituted with B6.Fc{gamma}R2B–/–sbb2a/a bone marrow did not develop ANA or glomerulonephritis even 9 mo after the transfer (Fig. 5 and Table II). This result indicates that the protection from disease conferred by sbb2a is fully transferable and dependent on bone marrow cells.


Figure 5
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  		FIGURE 5. Mixed bone marrow transfer show that sbb2 is expressed in non-B bone marrow cells and it acts in trans to suppress anti-nuclear IgG2 production. A, Bone marrow from the indicated genotype was transferred to lethally irradiated B6.Fc{gamma}R2B–/–mice. Serum ANA was tested 6 mo after the transfer at the dilution indicated. B, Bone marrow transfers were performed with an equal mix of the indicated donor genotypes into lethally irradiated B6 recipients. ANA was tested 6 mo after the transfer at the indicated dilutions. Allotype-specific Abs were detected by using biotinylated allotype-specific anti-mIgG2a plus FITC-streptavidin. C, Anti-dsDNA IgG2a ELISA test on serum from the reconstituted mice described in B (B6 recipient, gray bars) or from transfers into RAG-ko recipients (black bars). Serum was diluted 1/100 in all cases.

 

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  		Table II. FACS analysis of splenic cells populations in 6-mo-old 56R transgenic mice

 
To determine whether B cells need to intrinsically express the suppressor allele of sbb2 to be tolerized, we performed bone marrow reconstitutions with an equal mix of B6.Fc{gamma}R2B–/–sbb2a/a and B6.Fc{gamma}R2B–/–sbb2b/b bone marrow cells. Control transfers used wild-type bone marrow (B6.Fc{gamma}R2B+/+sbb2b/b) in the mix. For these experiments, B6.Fc{gamma}R2B–/–sbb2b/b mice were bred into a congenic B6.IgHa/a strain so that Abs produced by sbb2b/b cells (Iga allotype) could be distinguished from Abs derived from sbb2a/a B cells (Igb allotype) by the use of allotype-specific immunofluorescence staining. An additional control experiment used a mix of bone marrow from B6.Fc{gamma}R2B–/–sbb2b/b mice with both IgH a/a and IgHb/b allotypes to account for the effect of the Ig allotype change. Recipient mice were either low irradiated RAG-1–/–mice or lethally irradiated B6 mice. In all experiments, we observed that reconstituted mice harbored equivalent numbers of IgMb + and IgMa + B cells in the spleen and peripheral blood, so that the mixed transfer had resulted in 50% of the reconstituted B cells coming from each genotype (data not shown). Serum Abs were tested 6 mo (for B6 recipients, shown in Fig. 5B) and 9 mo (for RAG-1–/–recipients, Table II) after the transfer. We observed that in mice reconstituted with mixed bone marrow, sbb2a/a B cells produced ANAs as efficiently as sbb2b/b cells (Fig. 5B, top), implying that B cells from sbb2a/a congenic mice are not irreversibly tolerized and can produce autoantibodies in the context of a lupus-prone mouse. Wild-type B6 cells in the same mix do not produce detectable ANAs (Fig. 5B, middle panels). This experiment suggests that the suppressor effect of sbb2a is not B cell intrinsic.

Remarkably, titers of autoantibodies were much higher in reconstituted mice that had received sbb2b/b bone marrow alone (Fig. 5B, bottom), compared with the sbb2a/a plus sbb2b/b mix reconstitution (Fig. 5B, top). This result suggests that the presence of sbb2a/a in the mix reduces the level of Abs produced by sbb2b/b cells, because it has a suppressor effect on autoreactive B cells in trans. A quantitative assessment of this effect was obtained by ELISA on dsDNA plates (Fig. 5C). As McGaha et al. (27) has previously described, the presence of wild-type cells in the bone marrow mix reduces the level of anti-dsDNA Abs in serum to a certain extent, yet we see a further reduction by the presence of sbb2a/a bone marrow (which, in this case, includes Fc{gamma}R2B-deficient cells).

sbb2a reduces serum anti-dsDNA IgG in the 56R transgenic model, but it does not alter editing patterns

To study mechanisms of tolerance in the newly generated congenic strain, we have analyzed the effect of the sbb2a locus on the production of autoantibodies in mice that express the 3H9H-56R H chain site-directed transgene, which gives rise to anti-DNA specificity with high frequency (22). In the BALB/c background, the 56R H chain pairs mainly with the {kappa} L chain V{kappa}21D, an efficient editor of anti-dsDNA activity. BALB/c.56R B cells also become anergic when expressing dual {lambda} and {kappa} L chains with high frequency (28, 29). As a result, BALB/c 56R mice have low levels of anti-dsDNA IgG in the serum. On a B6 background, less efficient editor V{kappa}38c and V{kappa}20 light chains are used and high titers of anti-DNA IgG are found in serum. Deletion of Fc{gamma}R2B in these mice was shown to promote the survival and expansion of IgG+ plasma cells and results in elevated titers of anti-DNA IgG in the serum (25). We bred our newly congenic B6.Fc{gamma}R2B–/–sbb2a/a and B6.Fc{gamma}R2B–/–sbb2b/b mice to 56R transgenic mice and tested for serum dsDNA Abs in the progeny. We observed that mice with the sbb2a allele had significantly less IgG2a dsDNA Abs in the serum, but similar titers of IgM and IgG1 dsDNA Abs compared with mice with the sbb2b allele (Fig. 6A). When carrying the sbb2a allele, 56R Fc{gamma}R2B–/– mice had smaller spleen (0.07 g vs 0.14 g; p < 0.002), less germinal center (GL7+) B cells, less plasma cells (measured as IgM–CD138+B220int cells), less activated CD69+CD4 T cells and a lower level of I-Ab on B cells. 56R Fc{gamma}R2B–/– –sbb2a/a mice also accumulated less effector/memory (CD44low, CD45RBhigh) T cells with age. The marginal zone B cell population, which in 56R mice harbors the majority of B cells expressing dual L chains, did not change in mice with the sbb2a allele. Comparable levels of {kappa} and {lambda} L chain-surface expression were detected by FACS analysis (data not shown). No difference was observed on IgMa surface expression levels, which detects the transgenic Ab but not endogenous IgM in the B6 background (Fig. 6, B and C, Table III).


Figure 6
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  		FIGURE 6. sbb2a reduces the level of anti-DNA IgG2 in the serum but does not alter L chain editing in 56R transgenic mice. A, Anti-DNA Abs detected by ELISA on serum diluted 1/100. Note that animals of the sbb2a/a allotype have less IgG2a anti-DNA in the serum but similar levels of IgM and IgG1 anti-DNA, compared with sbb2b/b mice. Short lines indicate average values. B, Flow cytometric analysis shows that sbb2a/a mice have slightly less germinal center (GL7+) B cells and less CD69+ activated T cells. C, Histograms show flow cytometric analysis of B (B220 gated) and T (CD4 gated) splenic cells from B6.Fc{gamma}R2B–/–sbb2a/a mice (black lines) and B6.Fc{gamma}R2B–/–sbb2b/b mice (gray histograms). Note that mice of the sbb2a allotype have equal levels of IgMa (56R allotype) but lower MHC-II expression (I-Ab staining) and less memory/effector T cells (CD45RBlow and CD44high). D, Single-cell PCR analysis of the L chain usage in mice of the indicated genotype. Similar frequencies were obtained for the editor light chains V{kappa}38c and V{kappa}21D in both strains. For this experiment, B220+IgMhigh cells were sorted on 96-well plates, and PCR was performed on cDNA using primers described by Fukuyama et al. (25 ). Two mice per genotype and at least 100 single-cell PCRs for each were used in this analysis. E, Analysis of hybridomas obtained from LPS/stimulated B cells of the indicated genotype. Most hybridomas were positive for the 56R H chain measured by PCR. A total of 75% of the hybridomas were positive for dsDNA ELISA, independent of genotype or the type of L chain. L chain usage was determined for 37 clones in 56R.R2–/–sbb2a/a and 28 clones in 56R.R2–/–sbb2a/a and was detected by specific PCR primers as described in Materials and Methods.

 

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  		Table III. FACS analysis of splenic cell populations in bone marrow reconstituted mice

 
We determined L chain usage in the sbb2 congenic mice by single-cell analysis of sorted B220+IgMhigh splenocytes (Fig. 6D). We observed that Abs originated from 56R.Fc{gamma}R2B–/– mice and, including the 56R H chain, preferentially used the V{kappa}38C L chain, and that was independent of the sbb2 allele. Frequencies of the V{kappa}21D L chain, which is the most efficient editor of dsDNA specificities in BALBc mice, were comparable in sbb2a vs sbb2b mice. Vk38C was also the most abundant L chain pairing in both genotypes in the analysis of hybridomas obtained from LPS-stimulated B cells (Fig. 6E). In both 56R Fc{gamma}R2B–/– –sbb2a/a and 56R Fc{gamma}R2B–/– –sbb2b/b mice, the percentage of clones positive for dsDNA ELISA was around 75%. In addition, most clones that used the Vk38c, Vk20, and Vk21D light chains were DNA positive in both sbb2a/a and sbb2b/b mice. Thus, the disparity in sbb2 alleles between B6 and BALB/c does not explain the previously reported differences in L chain editing patterns of the dsDNA Abs in these two strains.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Background genes have been shown to be a very relevant factor in the development of various autoimmune diseases (30, 31, 32, 33, 34). In the context of the Fc{gamma}R2B-deficient mice, we have shown that the B6 background is conducive to lupus disease while the BALB/c background is not (12). These two strains exhibit a good number of differences in their immune function that could explain the disparity in lupus susceptibility, the most obvious being the difference in MHC genes. Our genetic analysis determined that a chromosome 17 locus near the MHC genes had important contributions to susceptibility in this model, but that it was not sufficient to convert the BALB/c strain into a lupus-susceptible strain (17). Thus, we have started the analysis of the other susceptibility locus that was uncovered in the same analysis and that is localized in a region of the genome on chromosome 12 (sbb2) with no obvious candidate genes. Our approach has been to eliminate lupus disease by including the suppressor allele of sbb2 (sbb2a) into a lupus-prone mouse (B6.Fc{gamma}R2B–/–). The sbb2a-mediated lupus protection, measured as absence of IgG ANA and kidney pathology, was particularly effective in homozygous mice, although the heterozygous sbb2a/b allele also resulted in partial protection (Fig. 1 and data not shown). This result suggests that the sbb2 suppressor effect is additive and most likely due to a gain of function. The presence of sbb2a reduces not only the levels of spontaneous IgG2a autoantibodies in lupus prone mice, but also the IgG2a and IgG2b response to exogenous Ags in T-dependent immunizations. Other isotypes (IgM and IgG1) or T-independent responses were not altered.

Our experiments point toward a bone marrow-derived cell that, by expressing the sbb2a allele, can alter the number of IgG2-producing B cells in a trans effect. Our data on mixed bone marrow reconstitution also suggest that the protective effect of sbb2a is most likely B cell extrinsic. We exclude myeloid cells as likely candidates because we have gotten the same results in bone marrow transfers that use RAG-ko or B6 recipients. In our experience, RAG-host reconstitution maintains the host CD11b+ and CD11c+ cells whereas lethal irradiation of the B6 recipient eliminates most of the host myeloid cell, and after the transfer these are primarily of donor origin (data not shown). T cells seem to be a good candidate because the most noticeable difference between sbb2a and sbb2b strains is the disparity in the number of effector/memory T cells. Because this phenotype is apparent in mice before the inflammatory pathology is evident, it is most likely a primary result from the different sbb2 alleles. A reduction in the number of activated T cells could have an impact in the serum IgG levels and be specific to certain isotypes, either by limiting the formation of germinal centers, by changing the cytokine environment, or by reducing the amount of tissue destruction and concomitant exposure to new Ags. A similar correlation between a reduction in the number of effector/memory T cells and a lupus suppressor phenotype has been described for the BXSB model of autoimmune disease in the absence of p21 cyclin kinase (35).

Although the presence of sbb2a does not change the frequencies of L chain usage in anti-dsDNA 56R transgenic mice, it does reduce the level of IgG2 anti-dsDNA Abs in the serum of these mice. The primary effect of the sbb2a-mediated lupus suppression seems to be to counterbalance the increase in the number of IgG-producing cells due to Fc{gamma}R2B deletion. B cell editing mechanisms, such as dual L chain expression or the use of DNA-silencing light chains, are not required for the lupus protection observed in sbb2a mice. Thus, even though inefficient Ab editing mechanisms have been shown in B6.Fc{gamma}R2B–/– and other lupus-prone strains to be relevant in the onset of disease (25, 36, 37, 38), our experiments show that reverting this condition is not necessary to abolish autoimmune disease and it implies that complete elimination of autoreactive B cells is not strictly required for effective treatment. Overall, our experiments uncover a protective mechanism for lupus disease that reduces certain autoantibody isotypes and reduces the lupus pathology even when B cell tolerance checkpoints are inefficient.


   Acknowledgments
 
We thank B. Scott for help managing the mouse colony and M. Weigert, University of Chicago, for providing the 3H9H/56R transgenic mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Silvia Bolland, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852. E-mail address: sbolland{at}nih.gov Back

3 Abbreviations used in this paper: B6, C57BL/6; R2, Fc{gamma}R2B; ANA, anti-nuclear autoantibodies. Back

Received for publication October 24, 2007. Accepted for publication January 17, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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