In recently generated B6.56R anti-DNA autoantibody-transgenic mice, it was noted that a substantial fraction of the B cells that had avoided DNA reactivity had done so through the rearrangement and usage of the endogenous, nontargeted H chain (HC) allele. This suggested that rearrangement at the second HC locus might be an important mechanism through which self-reactive B cells might successfully revise their initial Ag specificity. To test the importance of this mechanism in B cell tolerance, we generated B6.56R/56R mice that possessed the 56R anti-DNA H chain transgene inserted into both HC loci. These transgenic homozygotes developed higher titers of anti-DNA Abs, with an expanded population of B220lowMHC class IIlow B cells, enriched for CD21lowCD23low preplasmablasts. The analysis of hybridomas from these mice revealed that the only avenue by which these B cells could avoid DNA reactivity was through the use of the editor L chains, Vk20 or Vk21. Hence, in addition to LC editing, rearrangement and usage of the second HC locus/allele constitutes an important safety valve for B cells the primary BCR of which confers DNA reactivity. In contrast to these tolerance mechanisms, editing the first rearranged HC locus (through HC replacement) and somatic mutations appear to be less frequently used to edit/revise self-reactive B cells.

Newly generated B lymphocytes are known to undergo a meticulous censoring program through which self-reactive B cells are removed from the immune system. This is an important ritual because the persistence of potentially self-reactive B cells and their subsequent activation can lead to autoimmunity. The mechanisms through which self-reactive B cells are censored have been extensively studied, particularly through the use of BCR-transgenic (Tg)2 models. It is now apparent that several checkpoints exist to maintain B cell tolerance, as reviewed elsewhere (1).

Studies in BCR Tg models have demonstrated that B cells with strong self-reactivity are censored in the bone marrow largely through receptor editing and deletion (1, 2, 3, 4, 5). Elegant work by several investigators has shown that secondary rearrangement at the L chain (LC) locus is a particularly effective mechanism that B cells commonly use to veto self-reactivity (6, 7, 8, 9). Examples of receptor revision at the H chain (HC) locus have also been documented (10, 11, 12). B cells that have weaker degrees of self-reactivity have been shown to be rendered anergic or functionally incapacitated (1, 13, 14). Additional tolerance events in the germinal center have also been uncovered (15, 16, 17). Hence, a rich panoply of central and peripheral mechanisms exists to censor self-reactive B cells from the recirculating immune system.

It has also become apparent that the genetic background can substantially impact how effectively anti-self B cells are censored. Of relevance to this report, studies using the 3H9/56R anti-DNA Ig-Tg model have shed light on how different genetic backgrounds might impact B cell tolerance to DNA. Thus, whereas anti-DNA B cells are effectively tolerized in the normal BALB/c background, tolerance is breached when the same Tg is bred onto the MRL/lpr background (18, 19). Interestingly, on the C57BL/6 (B6) background, an intermediate degree of breach in tolerance is noted, as reported elsewhere (20, 21, 22).

Thus, when the 56R anti-DNA Ig Tg (with an arginine residue at position 56 in the CDR2 region of the 3H9 anti-DNA Ig HC) is bred onto the B6 background, these mice develop modest titers of autoantibodies to DNA as early as 4–6 mo of age. Potentially, B cells from these anti-DNA HC-only Tg mice could potentially preclude DNA reactivity in a couple of different ways: rearrangement and usage of the endogenous HC allele; pairing of the Tg HC with a LC partner that vetoes DNA binding; mutation of the Tg HC so that it loses affinity for DNA, and replacement of the targeted HC by an endogenous VH gene. When hybridomas were previously derived from B6.56R mice to determine the molecular makeup of anti-DNA and non-anti-DNA Abs, it was intriguing that a substantial faction of the B cells that had avoided DNA reactivity had done so through the rearrangement and usage of endogenous (i.e., nontargeted) HC locus (21). Most of the remaining B cells were apparently not DNA-reactive because they had paired the Tg 56R HC with a non-DNA-binding LC partner, a mechanism that has been well documented in this Tg model (6, 20).

The above findings suggested that rearrangement at the second (nontargeted) HC locus might be an important mechanism through which self-reactive B cells might successfully revise their initial Ag specificity. However, these observations did not indicate whether this particular molecular mechanism was absolutely essential for thwarting autoimmunity. Conversely, when B cells did not have the option of rearranging/using a second HC locus, it was not apparent whether LC editing alone was sufficient for revising all self-reactivity. To investigate this, we generated a mouse strain, referred to as B6.56R/56R that possessed the 56R anti-DNA HC Tg inserted into both HC loci. If these B6.56R/56R mice were to exhibit a greater degree of autoreactivity compared with Tg-hemizygous B6.56R/+ mice, this would suggest that rearrangement at the alternate HC locus and usage of the second HC allele plays an additional role in maintaining self-tolerance. In contrast, if both the hemizygous and homozygous Tg mice exhibited similar degrees of autoreactivity, this would indicate that other mechanisms (e.g., LC editing, mutation of Tg HC, etc.) may be sufficient to thwart autoimmunity, without the need for any additional contribution from rearrangement at the alternate HC locus.

56R anti-DNA HC-only site-directed Tg mice were a gift from Dr. Martin Weigert (University of Chicago, Chicago, IL) (6). These mice were backcrossed onto the B6 background, as recently described (20, 21). B6.56R/+ mice are hemizygous for the 56R anti-DNA Ig HC Tg, whereas B6.56R/56R mice bear the Tg knocked into both the HC loci. Double-Tg mice were identified by PCR analysis using JH1/JH2 intron-specific PCR primers that will amplify DNA from the nontargeted locus, but not the Tg, and confirmed by flow cytometry (based on the absence on any staining for surface IgMb). B6.56R/56R mice were bred to B6.56R/+ mice to generate both the hemizygous and Tg-homozygous mice. All mice used for this study were bred and housed in a specific pathogen-free colony, at University of Texas Southwestern Medical Center Department of Animal Resources (Dallas, TX). Comparable numbers of male and female mice were pooled for all experiments, given that no significant sex differences were noted in the phenotypes studied.

Splenocytes were depleted of RBC using tris-ammonium chloride, and single-cell suspensions were prepared for culture or flow cytometric analysis, as described previously (21, 23). Sera and mAb culture supernatants were screened for Ab reactivity to ssDNA, dsDNA, and histone-DNA complexes by ELISA, as described elsewhere (21, 23). All serum samples were diluted with serum dilution buffer (2% BSA, 3 mM EDTA, 0.05% Tween 20, 0.1% gelatin). A positive control serum sample derived from a B6.Sle1z.Sle3z mouse (Ref. 23 ; which bears the IgMb/IgG2ab allotype), or a serum sample derived from a seropositive B6.Sle1z.56R mouse (which bears the IgMa/IgG2aa allotype) was included as a test standard. Sera with reactivities stronger than the test standard were diluted further and reassayed one more time. For the allotype-specific ELISA, the above assays were repeated with one modification: instead of using anti-IgM or anti-IgG second-step Abs, biotin-coupled anti-IgMa, anti-IgMb, anti-IgG2aa, or anti-IgG2ab Abs were used, followed by avidin-coupled alkaline phosphatase.

Spleens were removed from anesthetized mice. Splenocytes were depleted of RBC using ACK lysing buffer (Biosource), and single-cell suspensions were prepared as for flow cytometry. Cells were stained with anti-B220 and sorted based on the level of B220 expression, using BD FACSAria (BD Biosciences). B220high and B220int cells were collected and cultured at 1 × 106 cells/ml for 48 h in DMEM supplemented with 10% FBS, 25 mM HEPES, 2 mM l-glutamine, antibiotics, and 50 μM 2-ME, with or without LPS (20 μg/ml). Culture supernatants were analyzed for Ab levels by ELISA.

Mice were monitored at 6 mo and at 9–12 mo of age for evidence of clinical nephritis. Urine was collected over a 24-h period using metabolic cages, and the total amount of urinary protein was assayed using a Coomassie-based kit (Pierce). The level of blood urea nitrogen was measured using a commercially available assay (Sigma-Aldrich). For the analysis of kidney sections, at least 100 glomeruli were examined per section by light microscopy for evidence of hypertrophy, proliferative changes, crescent formation, hyaline deposits, fibrosis/sclerosis, and basement membrane thickening and graded semiquantitatively, as detailed previously (23).

Spleens were removed aseptically from 9- to 12-mo-old seropositive Tg mice, and the splenocytes were fused to the SP2/0 fusion partner, as described before (21, 24). All wells with single colonies were tested for Ab production 7–10 days postfusion. Cells from positive wells were subcloned once more. Ab concentration was determined using a Coomassie PLUS protein assay kit (Pierce) and an isotype-specific ELISA, as described above.

The HC and LC sequences were amplified and sequenced from the cDNA of the hybridomas. The primers and conditions for HC and LC RT-PCR were determined as described previously (21, 24). Specifically, the HC was amplified using a 5′-primer that hybridizes to all VH genes (5′-AGGT(G/C)(A/C)A(A/G)CTGCAG(G/C)AGTC(A/T)GG-3′) and 3′ primers to Cμ (5′-CAGGGGGCTCTCGCAGGAGACGAGG-3′) and Cγ (5′-GGACAGGGATCCAGAGTTCC-3′), as described previously (24). This should allow the amplification of Tg as well as endogenous sequences, including those with nonproductive rearrangements and edited sequences with VH replacements or D invasions. The Ig-κ LC primers used have the potential to amplify most Ig-κ gene families including Vκ1, Vκ2, Vκ4, Vκ9, Vκ19, Vκ20, Vκ21, Vκ23, and Vκ38c, based on our previous work (Refs. 21 and 24 and data not shown). All Ab sequences were blasted against the public IgBlast database of mouse Ig sequences at National Institutes of Health/National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/igblast/), to determine the closest germline gene of origin. Comparing these sequences with those in the deposited repertoire of germline genes allowed us to identify residues that were potentially somatically mutated. The CDR position and numbering scheme adopted matched that used by the National Center for Biotechnology Information IgBlast database. The usage frequencies of individual genes were compared using χ2 tests. Where applicable, Fisher's exact test was applied. Intergroup comparisons of phenotypes between the two Tg strains were conducted using a one-tailed Student t test (SigmaStat; Jandel Scientific). Complete sequence information is available from the authors.

Interestingly, B6.56R/56R homozygous mice exhibited elevated levels of IgMa anti-ssDNA and anti-dsDNA Abs, relative to B6.56R/+ hemizygous controls, as displayed in Fig. 1. This serological difference was apparent as early as 3–6 mo of age and became more pronounced with age. However, there were no significant differences in the titers of IgG (IgG2Aa) Tg-encoded anti-DNA Abs between the hemizygous and homozygous Tg mice (Fig. 1). There were also no gender differences in these phenotypes (data not shown). Finally, there was no evidence of renal disease in these strains, reproducing earlier results in B6.56R/+ mice (Ref. 21 and data not shown).

FIGURE 1.

Serum levels of IgM and IgG Abs to nuclear Ags in B6.56R/+ and B6.56R/56R Tg mice. Allotype-specific IgM and IgG Abs to ssDNA, dsDNA were measured by ELISA. Both 56R homozygous and hemizygous Tg male and female mice (pooled) were analyzed at the ages of 3–6 and 9–12 mo. Each dot represents a single mouse. The horizontal lines represent the mean levels of serum autoantibodies in each group of mice. p values are the result of a Student t test comparison of B.56R/56R vs B6.56R/+ mice. OD405 readings for the positive control sera ranged from 1.2 to 1.5.

FIGURE 1.

Serum levels of IgM and IgG Abs to nuclear Ags in B6.56R/+ and B6.56R/56R Tg mice. Allotype-specific IgM and IgG Abs to ssDNA, dsDNA were measured by ELISA. Both 56R homozygous and hemizygous Tg male and female mice (pooled) were analyzed at the ages of 3–6 and 9–12 mo. Each dot represents a single mouse. The horizontal lines represent the mean levels of serum autoantibodies in each group of mice. p values are the result of a Student t test comparison of B.56R/56R vs B6.56R/+ mice. OD405 readings for the positive control sera ranged from 1.2 to 1.5.

Close modal

Next, we examined the cellular makeup of the lymphoid organs in B6.56R/56R and B6.56R/+ mice. Both strains exhibited similar splenic weights and cell numbers (Table I). Likewise, both strains exhibited similar numbers of CD4 and CD8 T cells, as well as B cells (Table I). Most of the B cells in B6.56R/+ spleens exhibited high surface levels of IgMa (i.e., with high mean fluorescent intensity), and also exhibited low grade staining for the endogenous HC, IgMb (Fig. 2 and Table I). In contrast, almost all the B cells in B6.56R/56R-Tg mice expressed higher staining intensity for IgMa with the absence of staining for endogenous IgMb, as one might expect (Fig. 2). If the Tg B cells were segregate based on their surface levels of IgMa into IgMa-high, IgMa-int and IgMa-low B cells, as demonstrated in Fig. 2, B6.56R/56R mice possessed significantly higher percentages of IgMa-high B cells but lower percentages of IgMa-low B cells compared with B6.56R/+ mice (Table I and Fig. 2).

Table I.

Lymphocyte composition of spleen and peritoneal cavitya

B6.56R/+ (N = 11)B6.56R/56R (N = 13)p
Spleen    
 Weight (mg) 109 ± 15.9 104.5 ± 27.6 NS 
 Total cell nos. (×10668 ± 20.9 66.3 ± 14.5 NS 
 Total CD4+ (×10618.7 ± 2.4 20.9 ± 4.5 NS 
  % CD4+ of all splenocytes 12.7 ± 0.6 13.6 ± 0.9 NS 
 Total CD8+ (×10618.4 ± 1.9 14.9 ± 3.9 0.017 
  % CD8+ of all splenocytes 12.5 ± 0.6 10.1 ± 0.8 0.015 
 CD4:CD8 ratio 1.0 1.4 NS 
 CD4:%CD69+ 17.2 ± 3.4 17.5 ± 7.3 NS 
 CD8:%CD69+ 18.4 ± 1.9 14.9 ± 3.9 0.003 
 Total B cells (×10641.9 ± 0.5 44.6 ± 1.9 NS 
  % of all splenocytes 28.5 ± 3.1 29.4 ± 2 NS 
 IgMa (MFI) 218.4 ± 23.9 215 ± 23.7 NS 
  %IgMa+ 69.1 ± 6.2 95.4 ± 1.7 0.0001 
   %IgMa-high 48.7 ± 4.6 56.6 ± 4.9 0.0009 
   %IgMa-int 21.6 ± 2.7 26.9 ± 2.8 0.0003 
   %IgMa-low 19.7 ± 2.9 11.9 ± 2.4 0.0001 
  %IgMb+ 14.2 ± 2.7 0.8 ± 0.7 0.0001 
  % B1a cells 3.9 ± 1.9 4.7 ± 2.2 NS 
 Among IgMa cells    
  % follicular B cells 42.5 ± 4 39.8 ± 1.5 NS 
  % MZ B cells 44.3 ± 4.2 48.2 ± 1.6 NS 
  % CD21CD23 B cells 17 ± 4 10.8 ± 1.3 NS 
  % Igλ+ 8.2 ± 1.2 10.4 ± 1.3 NS 
  I-Ab (MFI) 803 ± 100.6 687.9 ± 60.7 NS 
   CD86 (MFI) 46.3 ± 2.7 143.8 ± 12.9 0.0001 
Peritoneal cavity    
 Among B220+ cells    
  % B1a 31.9 ± 11.8 8.9 ± 6.4 0.0001 
  % B1b 37.1 ± 8.2 44.41 ± 17.7 NS 
  % B2 31 ± 7.4 46.8 ± 19.8 0.023 
B6.56R/+ (N = 11)B6.56R/56R (N = 13)p
Spleen    
 Weight (mg) 109 ± 15.9 104.5 ± 27.6 NS 
 Total cell nos. (×10668 ± 20.9 66.3 ± 14.5 NS 
 Total CD4+ (×10618.7 ± 2.4 20.9 ± 4.5 NS 
  % CD4+ of all splenocytes 12.7 ± 0.6 13.6 ± 0.9 NS 
 Total CD8+ (×10618.4 ± 1.9 14.9 ± 3.9 0.017 
  % CD8+ of all splenocytes 12.5 ± 0.6 10.1 ± 0.8 0.015 
 CD4:CD8 ratio 1.0 1.4 NS 
 CD4:%CD69+ 17.2 ± 3.4 17.5 ± 7.3 NS 
 CD8:%CD69+ 18.4 ± 1.9 14.9 ± 3.9 0.003 
 Total B cells (×10641.9 ± 0.5 44.6 ± 1.9 NS 
  % of all splenocytes 28.5 ± 3.1 29.4 ± 2 NS 
 IgMa (MFI) 218.4 ± 23.9 215 ± 23.7 NS 
  %IgMa+ 69.1 ± 6.2 95.4 ± 1.7 0.0001 
   %IgMa-high 48.7 ± 4.6 56.6 ± 4.9 0.0009 
   %IgMa-int 21.6 ± 2.7 26.9 ± 2.8 0.0003 
   %IgMa-low 19.7 ± 2.9 11.9 ± 2.4 0.0001 
  %IgMb+ 14.2 ± 2.7 0.8 ± 0.7 0.0001 
  % B1a cells 3.9 ± 1.9 4.7 ± 2.2 NS 
 Among IgMa cells    
  % follicular B cells 42.5 ± 4 39.8 ± 1.5 NS 
  % MZ B cells 44.3 ± 4.2 48.2 ± 1.6 NS 
  % CD21CD23 B cells 17 ± 4 10.8 ± 1.3 NS 
  % Igλ+ 8.2 ± 1.2 10.4 ± 1.3 NS 
  I-Ab (MFI) 803 ± 100.6 687.9 ± 60.7 NS 
   CD86 (MFI) 46.3 ± 2.7 143.8 ± 12.9 0.0001 
Peritoneal cavity    
 Among B220+ cells    
  % B1a 31.9 ± 11.8 8.9 ± 6.4 0.0001 
  % B1b 37.1 ± 8.2 44.41 ± 17.7 NS 
  % B2 31 ± 7.4 46.8 ± 19.8 0.023 
a

Mice of each strain were examined at 9–12 mo of age. Total cell numbers and percentage of splenocyte subsets are means ± SEM. p values pertain to one-tailed t test comparisons of the two strains. MFI, Geometric mean fluorescence intensity; MZ, marginal zone.

FIGURE 2.

Splenic B cell phenotypes in B6.56R/+ and B6.56R/56R Tg mice. Splenocytes from 9- to 12-mo-old B6.56R/+ (top) and B6.56R/56R mice (bottom) were analyzed by flow cytometry for the expression of B220 vs IgMa, IgMb, CD86, and I-Ab, respectively. IgMa B cells were further divided arbitrarily into three groups as demarcated by the dotted lines: IgMa-high B cells, IgMa-int B cells and IgMa-low B cells; the frequencies of B cells with different surface IgMa levels are summarized in Table I. Boxes indicate the expanded population of B cells in B6.56R/56R-Tg mice that were B220int, I-Abint, CD86high, relative to the B6.56R/+ controls. Profiles are representative of data obtained from eight mice per stain.

FIGURE 2.

Splenic B cell phenotypes in B6.56R/+ and B6.56R/56R Tg mice. Splenocytes from 9- to 12-mo-old B6.56R/+ (top) and B6.56R/56R mice (bottom) were analyzed by flow cytometry for the expression of B220 vs IgMa, IgMb, CD86, and I-Ab, respectively. IgMa B cells were further divided arbitrarily into three groups as demarcated by the dotted lines: IgMa-high B cells, IgMa-int B cells and IgMa-low B cells; the frequencies of B cells with different surface IgMa levels are summarized in Table I. Boxes indicate the expanded population of B cells in B6.56R/56R-Tg mice that were B220int, I-Abint, CD86high, relative to the B6.56R/+ controls. Profiles are representative of data obtained from eight mice per stain.

Close modal

When activation markers were examined, it was interesting that B6.56R/56R B cells expressed significantly higher levels of CD86 (B7-2), as illustrated in Fig. 2, and quantitated in Table I. Two clusters of B cells were discerned based on their surface levels of B220 and I-Ab: B220intI-Ab-int B cells (bearing intermediate levels of both surface molecules); and B220high I-Ab-high B cells, with the former being boxed in Fig. 2. Interestingly, B220intI-Ab-int B cells were significantly expanded in B6.56R/56R mice (Table II). When these two populations of B cells were further subgated, it was apparent that the expanded population of B220intI-Ab-int B cells in B6.56R/56R spleens were skewed toward being CD21CD23 in surface phenotype, compared with B220highI-Ab-high B cells from the same spleens (Table II and Fig. 3). The possibility that the expanded population of B cells in the Tg homozygous mice might include preplasmablasts is supported by their increased production of Abs in culture (Fig. 4). When all B cells were pooled, the B6.56R/56R and B6.56R/+ spleens did not differ significantly in their overall fractions of B cells that were follicular, marginal zone, or B1a in phenotype (Table I). When peritoneal B cells were examined, almost all B6.56R/56R B cells lacked CD5; in contrast, one-third of the peritoneal B cells in B6.56R/+ mice were CD5+, i.e., B1a in phenotype (Table I), suggesting that endogenous IgMb expression was required for selection into the B1a pool, as noted in our earlier work (21).

Table II.

Comparison of B220high and B220int B cellsa

B220highB220intp
B6.56R/56R (n = 12)    
 % of all B cells 57 ± 4.3 42.9 ± 4.3b  
 Mean size 72.5 ± 1.0 64.5 ± 1.3 0.0001 
 % FO 39.0 ± 1.2 21 ± 2.2 0.0001 
 % MZ 56.2 ± 1.3 36.7 ± 3.3 0.0001 
 % CD21CD23 4.1 ± 0.7 41.4 ± 4.7 0.0001 
 I-Ab (MFI) 883.1 ± 48.9 405.5 ± 39.6 0.0001 
 CD86 (MFI) 133.9 ± 12 122.3 ± 9.9 NS 
B6.56R/+ (n = 11)    
 % of all B cells 65.2 ± 3.7 34 ± 3.8b  
 mean size 71.7 ± 6.6 71.4 ± 0.4 NS 
 % FO 39.8 ± 2.2 21.7 ± 2.8 0.0001 
 % MZ 46.9 ± 2.0 37 ± 2.0 0.0012 
 % CD21CD23 11.8 ± 0.9 38.5 ± 3.8 0.0001 
 I-Ab (MFI) 1050 ± 78.4 407.4 ± 39.6 0.0001 
 CD86 (MFI) 24.9 ± 4.9 37.5 ± 8 NS 
B220highB220intp
B6.56R/56R (n = 12)    
 % of all B cells 57 ± 4.3 42.9 ± 4.3b  
 Mean size 72.5 ± 1.0 64.5 ± 1.3 0.0001 
 % FO 39.0 ± 1.2 21 ± 2.2 0.0001 
 % MZ 56.2 ± 1.3 36.7 ± 3.3 0.0001 
 % CD21CD23 4.1 ± 0.7 41.4 ± 4.7 0.0001 
 I-Ab (MFI) 883.1 ± 48.9 405.5 ± 39.6 0.0001 
 CD86 (MFI) 133.9 ± 12 122.3 ± 9.9 NS 
B6.56R/+ (n = 11)    
 % of all B cells 65.2 ± 3.7 34 ± 3.8b  
 mean size 71.7 ± 6.6 71.4 ± 0.4 NS 
 % FO 39.8 ± 2.2 21.7 ± 2.8 0.0001 
 % MZ 46.9 ± 2.0 37 ± 2.0 0.0012 
 % CD21CD23 11.8 ± 0.9 38.5 ± 3.8 0.0001 
 I-Ab (MFI) 1050 ± 78.4 407.4 ± 39.6 0.0001 
 CD86 (MFI) 24.9 ± 4.9 37.5 ± 8 NS 
a

Splenic cells from both strains were assayed by flow cytometry, and B cells were distinguished based on surface B220 levels. Tabulated are the surface phenotype and subset distribution data pertaining to these two B cell types. p values pertain to comparisons of these two B cell types using Student's t test. FO, Follicular; MZ, marginal zone; MFI, geometric mean fluorescence intensity.

b

The percentage of B cells that typed as B220intI-Ab-int were significantly different in the two strains (p < 0.01).

FIGURE 3.

Surface phenotypes of B220intI-Ab-int, and B220highI-Ab-high B cells from B6.56R/56R and control spleens. Splenic B cells from 56R Tg mice were first gated based on surface levels of B220 and I-Ab, as indicated in Fig. 2, and then analyzed from their surface CD21-CD23 profiles. Plots are representative of data obtained from 11–12 mice each, as detailed in Table III. hi, High.

FIGURE 3.

Surface phenotypes of B220intI-Ab-int, and B220highI-Ab-high B cells from B6.56R/56R and control spleens. Splenic B cells from 56R Tg mice were first gated based on surface levels of B220 and I-Ab, as indicated in Fig. 2, and then analyzed from their surface CD21-CD23 profiles. Plots are representative of data obtained from 11–12 mice each, as detailed in Table III. hi, High.

Close modal
FIGURE 4.

In vitro Ab production by B220int and B220high B cells from B6.56R/56R spleens. Splenic B cells were sorted based on surface B220 levels from B6.56R/56R mice and cultured at indicated cell numbers (per well) with LPS (20 μg/ml) for 48 h, after which culture supernatants were assayed for Ab levels by ELISA. Each bar represents the mean ± SEM of three experiments.

FIGURE 4.

In vitro Ab production by B220int and B220high B cells from B6.56R/56R spleens. Splenic B cells were sorted based on surface B220 levels from B6.56R/56R mice and cultured at indicated cell numbers (per well) with LPS (20 μg/ml) for 48 h, after which culture supernatants were assayed for Ab levels by ELISA. Each bar represents the mean ± SEM of three experiments.

Close modal

Shown in Table III are the HC and LC gene compositions noted in B6.56R/56R B cells, juxtaposed to previously reported data from B6.56R/+ control (21). As is evident from Table III, DNA-binding B cells from both the B6.56R/+ and B6.56R/56R mice used the Tg HC, predominantly or exclusively, paired with a spectrum of LC partners, notably Vκ20, Vκ21, and Vκ38c. When non-antinuclear autoantibody (ANA) B cells were next examined (Table III, bottom), interesting molecular differences were noted. More than 80% of the non-DNA-binding B cells from B6.56R/56R spleens possessed BCRs composed of the 56R HC-Tg paired with either Vκ20 or Vκ21, both of which have previously been shown to be effective at vetoing DNA binding (6). In contrast, ∼70% of the non-ANA-binding B cells from B6.56R/+ spleens apparently avoided DNA binding through the use of endogenous HC genes (Ref. 21 and Table III). Hence, in the absence of an alternative (nontargeted) HC locus, B cells expressing a potentially self-reactive BCR HC appear to avoid self-reactivity exclusively through the use of DNA-vetoing LC partners. Very few of these B cells showed evidence of mutations in the 56R Tg or evidence of HC replacement or D invasion into the Tg (Table III).

Table III.

H chain and L chain usage analysis in hybridomasa

B6.56R/+B6.56R/56Rp2)
ANA    
 Total cells (N62 69  
 H chain amplified    
  Endogenous HC (%) 29 0.001 
  56R Tg HC (%) 71 100 0.001 
  % of 56R HC Tg with mutations NS 
  Tg HC invasion (NNS 
  IgG in isotype (%) 13 0.001 
 LC usage among B cells with Tg HC    
  %Vκ4 NS 
  %Vκ9 21 0.002 
  %Vκ19 20 0.004 
  %Vκ20 36 0.001 
  %Vκ21 18 0.003 
  %Vκ38c 28 20 NS 
  % other Vκ 20 13 NS 
Jκ usage among B cells with Tg HC    
  %Jκ1, Jκ2 40 28 NS 
  %Jκ4, Jκ5 60 72 NS 
Non-ANA    
 Total cells (N27 21  
 H chain amplified    
  Endogenous HC (%) 70 0.001 
  56R Tg HC (%) 30 100 0.001 
  % of 56R HC Tg with mutations NS 
  Tg HC invasion (NNS 
  IgG in isotype (%) 11 NS 
 LC usage among B cells with Tg HC    
  %Vκ9 19 NS 
  %Vκ20 52 31 NS 
  %Vκ21 56 0.001 
  %Vκ38c NS 
  % other Vκ 23 NS 
Jκ usage among B cells with Tg HC    
  %Jκ1, Jκ2 25 63 0.009 
  %Jκ4, Jκ5 75 37 0.009 
B6.56R/+B6.56R/56Rp2)
ANA    
 Total cells (N62 69  
 H chain amplified    
  Endogenous HC (%) 29 0.001 
  56R Tg HC (%) 71 100 0.001 
  % of 56R HC Tg with mutations NS 
  Tg HC invasion (NNS 
  IgG in isotype (%) 13 0.001 
 LC usage among B cells with Tg HC    
  %Vκ4 NS 
  %Vκ9 21 0.002 
  %Vκ19 20 0.004 
  %Vκ20 36 0.001 
  %Vκ21 18 0.003 
  %Vκ38c 28 20 NS 
  % other Vκ 20 13 NS 
Jκ usage among B cells with Tg HC    
  %Jκ1, Jκ2 40 28 NS 
  %Jκ4, Jκ5 60 72 NS 
Non-ANA    
 Total cells (N27 21  
 H chain amplified    
  Endogenous HC (%) 70 0.001 
  56R Tg HC (%) 30 100 0.001 
  % of 56R HC Tg with mutations NS 
  Tg HC invasion (NNS 
  IgG in isotype (%) 11 NS 
 LC usage among B cells with Tg HC    
  %Vκ9 19 NS 
  %Vκ20 52 31 NS 
  %Vκ21 56 0.001 
  %Vκ38c NS 
  % other Vκ 23 NS 
Jκ usage among B cells with Tg HC    
  %Jκ1, Jκ2 25 63 0.009 
  %Jκ4, Jκ5 75 37 0.009 
a

Whereas the fusion data for the B6.56R/56R spleen were generated in this study, the data shown for the B6.56R/+ mouse has been recently reported elsewhere (21 ).

It was somewhat perplexing that whereas some B cells expressing Vκ20 paired with the Tg HC were DNA reactive, others were not (Table III). When the bt20 Vκ20 germline gene was recombined to Jκ4, it was associated with DNA reactivity (when paired with the 56R HC); when the same Vκ germline gene was recombined to Jk2, it was apparently not associated with DNA reactivity (Fisher's exact test; p < 0.002). However, this difference alone is unlikely to account for the differential DNA reactivity, based on previous reports (20, 21). Likewise, it was intriguing that whereas some of the B cells bearing the 56R HC-Tg paired with the Vκ21 LC gene did not bind DNA, others clearly did (Table III). When we compared these two specificities of B cells, there were no significant differences in Jκ or Vκ germline gene usage between the DNA-binding and non-DNA-binding B cells. The specificity differences were not due to any somatic mutations because all Vκ21-expressing clones exhibited no mutations in the Tg HC or the LC partners (data not shown). Also, we could not amplify any Igλ from these B cells. Currently, we do not know whether these Vκ21-expressing B cells differ in the expression of any additional LC partners that our PCR primers might have failed to amplify.

Developing pre-B cells first rearrange their HC locus during early B cell development. In the event that the first VH gene randomly selected for rearrangement confers self-reactivity when paired with an LC partner, several molecular events occurring at different stages of B cell development have the potential to revise the molecular makeup of the initial BCR so that it is no longer self-reactive. These nonmutually exclusive options include: 1) rearrangement at the alternate HC locus and usage of the alternate HC allele; 2) pairing of the HC with an appropriate LC partner (generated either through primary recombination or through LC editing), so that DNA reactivity is precluded, as discussed previously (6, 19); 3) editing the first HC allele so that it is no longer self-reactive (e.g., through VH replacement), as reported by Weigert et al. (10); and 4) mutation of the HC or the paired LC gene so that DNA reactivity is abrogated.

When we recently derived B cell hybridomas from B6 and B6.Sle2z congenics bearing the 56R anti-DNA Tg, the first two of the above mechanisms were evidently more commonly used, compared with the latter options (21). Surprisingly, in that study, the vast majority of the B6.56R-derived non-DNA-binding B cells had apparently avoided DNA reactivity through the rearrangement of the nontargeted HC locus (i.e., by adopting the first mechanism listed above). Although it was clear that this mechanism was commonly used to maintain B cell tolerance (at least on the B6 background), it was not apparent whether this mechanism was absolutely essential. This study, designed to directly test this, illustrates that B cells that are engineered to lack a nonrearranged alternate HC locus are more likely to remain autoreactive, presumably because the only available mechanism through which they can revise their initial BCR is through LC editing.

Previous studies using Tg-hemizygous 3H9/56R/76R mice (with progressively increasing avidity of the Tg for DNA) have shown that 1) increasing the B cell avidity for DNA has a profound influence on the degree and nature of B cell editing and 2) the availability of a nontargeted HC locus is not sufficient to prevent autoimmunity in there mice (25, 26). Although the latter mechanism of tolerance by itself may not be sufficient at re-establishing tolerance, it appears that it may contribute incrementally in this direction, based on findings from the present work. When operative, the non-DNA-binding HC substitute appears to originate predominantly from de novo rearrangement of the nontargeted locus, rather than through VH replacement at the targeted HC locus (21), and this difference may relate to the relative efficiencies of these two processes.

The phenotype of the B cells observed in the Tg-homozygous mice points to some interesting observations. First, the copresence of the prearranged HC Tg at both loci did not alter total B cell numbers, consistent with previous reports in HC Tg-homozygous mice (27). Also, the total IgM levels (i.e., IgMa + IgMb) on B cells did not differ between the Tg-hemizygous and Tg-homozygous mice (Fig. 2 and Table I), again in keeping with previous reports in the literature (27). Presently, we do not know whether the increased numbers of IgMa-high B cells in Tg-homozygous mice might arise in part from expression of HC from both alleles, but this appears to be unlikely based on earlier reports in other Ab-Tg-homozygous mice (27).

Though Tg-hemizygous and Tg-homozygous mice did not differ significantly in B cell numbers or surface levels of total IgM, it was interesting that the Tg-homozygous mice exhibited an expanded population of B220int I-Ab-int B cells. Because these B cells were skewed toward being CD21CD23 (Table II), they may be composed of memory B cells and/or preplasmablasts. Expansions of similar populations of B cells have been noted in other autoimmune mouse models (28, 29). Consistent with this, sorted B220int I-Ab-int B cells from Tg-homozygous mice tended to elaborate more ANAs in culture (Fig. 4). On the basis of these findings, we envisage the following model. Precluding B cells from rearranging and using the alternate HC allele may augment the pool of autoreactive B cells, as observed in the Tg-homozygous mice. Continuous stimulation of these B cells by self DNA might skew these cells toward plasmacytoid and memory B cell differentiation, a scenario consistent with the B cell phenotypes and elevated autoantibody levels seen in these mice.

On the basis of these observations and previous reports in the literature, we propose that newly developing B cells (in a non-Tg, physiological setting) may use two main mechanisms to revise their BCR whether the initial HC-LC combination turned out to be self-reactive: rearrangement at the second HC locus or LC editing. In a context where anti-self B cells have the luxury of rearranging a second HC locus, as well as editing the LC partner, as is the case in B6.56R/+ mice, the former mechanism is used by 70% of the B cells that might have potentially been DNA reactive (Ref. 21 and Table III). Precluding the former tolerance mechanism from operating (as engineered in the B6.56R/56R double-knock-in mice) may amplify the degree of autoimmunity that ensues, as demonstrated in the present study. In these mice, editing at the LC locus using strong DNA-vetoing LCs such as Vκ20 and Vκ21 appear to be the only mechanism available for precluding DNA reactivity, a finding that resonates well with previous reports in Tg-hemizygous mice by Weigert et al. (6). Hence, in addition to LC editing, rearrangement and usage of the second HC locus constitutes an important safety valve for B cells whose primary BCR confers DNA reactivity. In contrast to these mechanisms, editing the first rearranged HC locus (through HC replacement) and somatic mutation do not appear to be commonly used for editing/revising self-reactive B cells.

The authors have no financial conflict of interest.

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.

2

Abbreviations used in this paper: Tg. transgenic; HC, H chain; LC, L chain; ANA, antinuclear autoantibody; int, intermediate.

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