Regulation of the B cell receptor repertoire and self reactivity by BAFF

Abstract

The cytokine BAFF promotes B lymphocyte survival and is overexpressed in individuals with systemic lupus erythematosus and Sjögren’s Syndrome. BAFF can rescue anergic autoreactive B cells from death, but only when competition from nonautoreactive B cells is lacking. Yet high BAFF levels promote autoantibody formation in individuals possessing diverse B cells. To better understand how excess BAFF promotes autoimmunity in a polyclonal immune system, IgL-chain usage was analyzed in 3H9 site-directed IgH-chain transgenic mice, whose B cells recognize DNA and chromatin when they express certain endogenous L-chains. BAFF levels were manipulated in 3H9 mice by introducing transgenes expressing either BAFF or its natural inhibitor ΔBAFF. B cells in BAFF/3H9 mice were elevated in number, used a broad L-chain repertoire, including L-chains generating high affinity autoreactivity, and produced abundant autoantibodies. Comparison of spleen and lymph node B cells suggested that highly autoreactive B cells were expanded. By contrast, ΔBAFF/3H9 mice had reduced B cell numbers with a repertoire similar to that of 3H9 mice, but lacking usage of a subset of Vκ genes. The results suggest that limiting BAFF signaling selects against higher affinity autoreactive B cells, whereas its overexpression leads to broad tolerance escape and positive selection of autoreactive cells.

Introduction

The TNF-superfamily cytokine BAFF (TNFSF13B, BLyS) plays key roles in B cell survival and homeostasis that when dysregulated can lead to symptoms ranging from systemic autoimmunity to immunodeficiency (Mackay and Schneider, 2009). BAFF binds to three different receptors: BAFF-R (BR3, TNFRSF13C), TACI, and BCMA, whereas the related cytokine APRIL binds just to TACI and BCMA (Yu et al., 2000). BAFF is produced by diverse stromal cells and myeloid cells in which production can be increased by inflammatory stimuli such as Tlr ligands or IFN (Gorelik et al., 2003; Lesley et al., 2004; Boule et al., 2004; Yamada et al., 2010). BAFF signaling is required for survival of marginal zone (MZ) and follicular (B-2) B cells, but not B-1 cells (Thompson et al., 2000; Gross et al., 2001; Schiemann et al., 2001; Yan et al., 2001). BAFF and APRIL signaling through BCMA is important in plasma cell survival (O’Connor et al., 2004), whereas signaling through TACI is generally suppressive of most B cells, but can also promote IgA class switching and T cell independent antibody responses (Yu et al., 2000; von Bulow et al., 2001; Seshasayee et al., 2003; Mackay and Schneider, 2008). BAFF responsiveness is upregulated relatively late in B cell development, starting at the T2 transitional stage (Hsu et al., 2002; Smith and Cancro, 2003). Partial loss of BAFF or BAFF-R function leads to a reduced half-life and steady-state numbers of follicular and MZ B cells, indicating that BAFF is a homeostatic cytokine (Harless-Smith et al., 2001). By contrast, overexpression of BAFF has been linked to B cell hyperplasia, hypergammaglobulinemia, and systemic autoimmunity in both mouse models and in patients with Systemic Lupus Erythematosus (SLE) and Sjögrens Syndrome (Cheema et al., 2001; Zhang et al., 2001; Mariette et al., 2003; Collins et al., 2005; Chu et al., 2007; Mackay and Schneider, 2009; Chu et al., 2009). Suppression of BAFF levels through genetic or pharmacological means has been shown to ameliorate or delay lupus symptoms (Ramanujam et al., 2006; Kahn et al., 2008; Moisini and Davidson, 2009; Wallace et al., 2009; Furie et al., 2009; Ramanujam et al., 2010). Thus, BAFF has both beneficial and potentially toxic effects that must be tightly regulated.

One natural negative regulator of BAFF is the splice isoform ΔBAFF, which suppresses BAFF bioactivity by consuming potentially functional BAFF mRNA, “poisoning” bioactivity in BAFF/ΔBAFF heterotrimers, and preventing membrane release (Gavin et al., 2003). Transgenic mice expressing ΔBAFF under the control of the human CD68 promoter have a phenotype indicative of reduced BAFF/BAFF-R bioactivity, including subnormal B cell numbers, particularly in the marginal zone and lymph nodes (Gavin et al., 2005). Companion strains of mice expressing full length BAFF under the same control elements have a phenotype typical of BAFF overexpression, including B cell hyperplasia, elevated serum Ig levels (Gavin et al., 2005) and autoantibodies (this study).

The link between BAFF levels and autoantibody formation has prompted analyses of the effects of BAFF on B cell tolerance. Anergic autoreactive B cells have a heightened BAFF dependency compared to non autoreactive B cells owing to BCR desensitization (Lesley et al., 2004; Thien et al., 2004). This phenomenon leads to rapid, competition-dependent turnover of autoreactive B cells in the context of a polyclonal repertoire of B cells, but allows improved survival of autoreactive cells in monoclonal transgenic models and in the context of lymphopenia (Lesley et al., 2004; Thien et al., 2004; Aït-Azzouzene et al., 2006). Because in a polyclonal repertoire non autoreactive B cells are presumably in excess, it is somewhat puzzling that BAFF-overexpressing mice have elevated autoantibody levels and lupus-like disease. One explanation may be that low affinity autoreactive B cells are selectively spared (Thien et al., 2004). Another possibility is that in the context of excessive BAFF, costimulation of T cells promotes more widespread immune defects (Huard et al., 2001; Ye et al., 2004). However, recent evidence supports the contrary notion that excess BAFF may make B cells more T cell-independent in their responsiveness (Groom et al., 2007). Another possibility is that B cells reactive to nucleic acids may respond differently to BAFF overexpression compared to B cells carrying receptors recognizing other types of (auto)antigens because of their potential to internalize antigens that stimulate Toll-like receptors (Leadbetter et al., 2002; Boule et al., 2004; Lau et al., 2005).

Evidence is accumulating that therapeutic targeting of BAFF may be an effective treatment for SLE. A phase II clinical trial of belimumab, a human monoclonal antibody to BAFF, in a cohort of 449 SLE patients resulted in a response in 46% of patients at one year compared with 29% of placebo patients (Wallace et al., 2009; Furie et al., 2009). Ongoing phase III trials show promise. One of the speculations regarding the efficacy of this approach is that BAFF levels may gradually render the naïve B cell repertoire less autoreactive over time. However there is little direct evidence that BAFF levels influence the selection of the naïve autoreactive B cell repertoire.

In this paper we analyze how BAFF levels alter B cell tolerance by characterizing changes in the B cell repertoire that occur with over- or under-expression of BAFF. Our strategy involved breeding BAFF Tg or ΔBAFF Tg mice with mice transgenic for the site-directed 3H9 immunoglobulin heavy (H)-chain, whose specificity is derived from a DNA- and chromatin-specific hybridoma obtained from a diseased MRL/lpr mouse (Chen et al., 1995; Chen et al., 1997). By pairing with a wide range of immunoglobulin light (L)-chains in different B cells, the 3H9 model generates a simplified, but polyclonal, repertoire with a propensity to generate autoantibodies that are common in lupus autoimmunity. In the 3H9 model B cells retain the H-chain transgene but undergo the normal process of light chain rearrangement and expression. Many of the L-chain partners that, when paired with 3H9 H-chain, generate high and low affinity autoantibodies, or lead to innocuous antibodies, are known (Radic et al., 1991; Radic et al., 1993; Radic et al., 1995; Roark et al., 1995; Chen et al., 1997), facilitating analysis of alterations in immune repertoire. We reasoned that if BAFF sets the affinity threshold for B cell tolerance, the repertoire of allowed L-chains should be expanded in BAFF/3H9 Tg mice compared to 3H9 mice, and be reduced in the 3H9/ΔBAFF Tg background, with cells of borderline autoreactivity showing the greatest relative changes.

Results

Regulation of B cell compartment size by BAFF expression level

As BAFF levels are thought to regulate B cell survival, we first quantified B cell numbers in lymphoid tissues of non-Tg (WT), 3H9, BAFF/3H9 and ΔBAFF/3H9 (DBF/3H9) mice. Compared to WT mice, splenic B220⁺ B cell numbers in 3H9 Tg mice were significantly decreased. DBF/3H9 mice had even more severe B cell lymphopenia than 3H9 mice, especially in lymph nodes. By contrast, BAFF/3H9 mice had abnormally high B cell numbers in spleen and lymph nodes, consistent with a role for BAFF levels in regulating the size of the B cell compartment (Fig. 1A). MZ B cell numbers in spleen were increased in all 3H9 strains, but did not differ significantly regardless of BAFF levels (Fig. 1A, top right). This finding was surprising because MZ B cell numbers are most strikingly affected by altered BAFF levels in BAFF Tg and DBF Tg mice that lack the 3H9 Tg (Gavin et al., 2005). (B-1 cell numbers were low in 3H9 mice, presumably because this H-chain fails to efficiently support B-1 cell development; consistent with this, the B-1 cells that did develop lacked 3H9 H-chain (not shown).) Both κ⁺ and λ⁺ B cell numbers were increased in BAFF/3H9 mice even compared to WT mice (Fig. 1A, lower panels). Usually, λ L-chains combined with the 3H9 H-chain bind to DNA (Radic et al., 1991; Luning Prak et al., 1994; Ibrahim et al., 1995; Roark et al., 1995). In 3H9 mice λ⁺ B cell numbers were reduced compared to WT, while in BAFF/3H9 mice the proportion of λ⁺ B cells increased even in the lymph nodes. By contrast, DBF/3H9 lymph nodes had even fewer λ⁺ B cells than in 3H9 lymph nodes (Fig. 1A,B). We conclude that elevated BAFF levels regulate B cell numbers in 3H9 mice, with preferential rescue of follicular and λ⁺ cells in BAFF/3H9 mice and their preferential loss in DBF/3H9 mice.

Figure 1. Influence of BAFF bioactivity levels on the B cell compartment of 3H9 Tg mice.

A) 12 to 16-week-old mice were assessed for total numbers of B220⁺ B cells, MZ, κ⁺ and λ⁺ B cell subsets in spleen (SP) and lymph nodes (LN). Colored bars indicate genotypes as follows: WT, white; 3H9, green; BAFF/3H9, red; DBF/3H9, blue. Data for 4 mice per group (mean ± standard error (SE)). Asterisks show p values calculated by two-tailed Student’s t test. *; p<0.05, **; p <0.01, ***; p <0.001, ns; not significant. B) FACS plots of SP and LN κ⁺ and λ⁺ B cells gated on CD4⁻CD8⁻B220⁺ population. One representative experiment out of four is shown.

Regulation of anti-dsDNA antibody levels and transitional B cells by BAFF

We tested if serum anti-dsDNA titers were affected by the elevated levels of BAFF in BAFF/3H9 mice, compared to 3H9, DBF/3H9 and WT mice. In other studies, BAFF overexpression causes a lupus-like syndrome that includes activated lymphocytes and anti-self antibody production, whereas 3H9 mice on the BALB/c or C57BL/6 backgrounds lack anti-DNA antibodies (Erikson et al., 1991; Chen et al., 1997; Fukuyama et al., 2005; Liu et al., 2007). Indeed, BAFF/3H9 mice had significantly higher serum levels of IgM, IgG, IgA, and λ anti-dsDNA than did 3H9 mice. By contrast, DBF/3H9 mice had significantly lower IgM, IgG, and IgA anti-dsDNA levels compared to 3H9 controls (Fig. 2A). Low autoantibody production in DBF/3H9 mice correlated with a higher proportion of splenocytes with a CD93^hi phenotype (6.9% of B220⁺ B cells) and a T3′ phenotype (37.8%: B220⁺CD93^hiIgM^lowCD23^low) (Fig. 2B,C). These subsets have been reported as anergic self reactive B cells (Merrell et al., 2006; Kiefer et al., 2008). On the other hand, BAFF/3H9 mice had a lower proportion of CD93^hi B cells (2.0%) and 44.9% of these cells had a T2 phenotype (B220⁺CD93^hiIgM^highCD23^high) (Fig. 2B,C), which is considered to be a stage permissive for further maturation. Clearly, excess BAFF allowed maturation of autoreactive cells that were normally regulated by developmental arrest, whereas developmental arrest was apparently enhanced in DBF/3H9 mice.

Figure 2. Effects of BAFF bioactivity on serum anti-dsDNA antibodies and CD93+ splenic B cell subsets.

Mice of the indicated genotypes were analyzed by ELISA for serum anti-DNA activity and by flow cytometry for markers of B cell maturity. A) IgM, IgG, IgA and λ anti-dsDNA antibody production in sera from 12 to 16-week-old mice of the indicated genotypes. Diamonds represent values obtained in individual mice, with the mean for each group indicated by a horizontal bar. B) FACS plots of splenic CD93hi immature B cells (upper panel box) and transitional B cells (lower panel). T1; upper left, T2; upper right, T3; lower right, T3’; lower left. One representative experiment out of four is shown. C) Frequency of each B cell subset from 3H9 (green, n=5), BAFF/3H9 (red, n=5) and DBF/3H9 (blue, n=7) analyzed as in B. Shown is mean±SE. Asterisks show p values calculated by two-tailed Students t test. *; p<0.05, **; p <0.01, ***; p <0.001, ns; not significant.

Igκ repertoire analysis of B cells from 3H9, BAFF/3H9, and DBF/3H9 mice

In 3H9, BAFF/3H9, and DBF/3H9 strains on the C57Bl/6 background, IgH^a+ B cells virtually all express the transgenic H-chain, allowing us to analyze BAFF-dependent changes in B cell repertoire by comparing κ-chain usage. Accordingly, we sorted non activated B cells that lacked expression of IgM^b (yielding a population that was >98% IgM^a+) and PCR amplified their Igκ mRNA transcripts using a 5′-RACE approach with two invariant primers, one in Cκ and the second in a universal 5′ linker (see Materials and Methods). We then carried out conventional plasmid cloning and sequencing or direct pyrosequencing (454 sequencing) analysis of amplicons. The different approaches gave generally consistent results, but the large number of 454 samples provided better statistical power and resolution.

454 sequencing generated a total of over 117,000 high quality sequences, with 7,000 to 23,000 sequences from each tissue of each genotype tested. Each sample contained cDNA pools of four mice. Among sequences with identifiable V and J sequence, each sample included more than 270 V-J combinations. Coverage of Vκ genes was even higher: greater than 87% of all possible Vκ genes could be identified with 454 sequencing while fewer than 49.5% were found in our conventional cloning sample (Table I, Table SI). We decided to use 454 sequence data for further analysis.

Table I.

Summary of Igκ mRNA 454 pyrosequencing results.

Tissue	Spleen				Lymph node
Mouse genotype	B6	3H9	BAFF/3H9	DBF/3H9	B6	3H9	BAFF/3H9	DBF/3H9
Total reads	17,954	28,830	38,767	50,586	39,642	33,780	18,682	53,916
Identifiable VJ	8,902	12,745	15,562	21,597	15,814	12,234	7,675	23,306
Observed VJ combinations	307	304	322	313	337	299	275	328
% coverage of possible VJ combinationsa	77.5	76.8	81.3	79.0	85.1	75.5	69.4	82.8
Identifiable Vs	95	91	92	95	95	94	87	97
% Vκ coverageb	96.0	91.9	92.9	96.0	96.0	94.9	87.9	98.0

^aTheoretical total Theoretical total VJ combinations are calculated as follows; 99 functional V genes × 4 functional J genes = 396

^bThere are a total of 99 functional V genes including ORF genes in C57Bl/6 mice listed on the IMGT database (Lefranc et al., 2009) (http://imgt.cines.fr/textes/IMGTrepertoire/).

Jκ usage

Analysis of Jκ usage was of interest because previous studies of 3H9 and other autoantibody transgenics found a high frequency of downstream Jκ usage associated with receptor editing (Radic et al., 1993; Radic et al., 1995; Roark et al., 1995; Chen et al., 1997). In mice there are 4 functional Jκ elements, Jκ1, 2, 4 and 5 (Jκ3 is a pseudogene). In a control sample from non Tg C57Bl/6 mice, Jκ usage in spleen was: Jκ1, 33%; Jκ2, 23%; Jκ4, 15%; Jκ5, 30% (Fig. 3); usage in lymph nodes was similar: Jκ1, 34%; Jκ2, 20%; Jκ4, 15%; Jκ5, 31% (Fig. 3). By contrast, 3H9 and DBF/3H9 B cells had skewed repertoires with significantly reduced utilization of Jκ1 (18–22%) and increased utilization of Jκ5 (33–42%) (Fig. 3). This skewing from wild type was partly lost in BAFF/3H9 mice although Jκ2 usage in lymph nodes was elevated and Jκ1 usage somewhat reduced. The potential significance of these BAFF-regulated alterations in skewings are considered in the Discussion.

Figure 3. Jκ usage.

Frequencies of Jκ usage among κ L-chain cDNAs from B cells of the indicated mouse genotypes are shown. A) Jκ usage in spleen and B) in lymph nodes. Jκ1; white, Jκ2; green, Jκ4; red, Jκ5; blue. Asterisks show p values calculated by Chi-square test. ***; p <0.001. Data for 4 mice per group. 12 to 16-week-old mice were used.

Altered Vκ selection by BAFF overexpression, especially in lymph nodes

BAFF/3H9 B cells had a strikingly altered pattern of Vκ usage compared to 3H9 and DBF/3H9 B cells in spleen (Fig. 4A) and lymph nodes (Fig. 4B), which was even more striking when presented as absolute B cell numbers rather than percent usage (Fig. 5). These changes included higher usage of Vκs known to sustain dsDNA binding when paired with 3H9 H-chain, (Vκ1–110, Vκ1–117, Vκ9–120, Vκ4–59 [kk4, H3], Vκ8–24 and Vκ6–20) (Fig. 6A). (Note: For brevity, in this paper “Vκ” substitutes for the official prefix “IGKV”). There were also proportionate reductions in BAFF/3H9 mice in the use of Vκs that veto dsDNA binding (Vκ17-121, Vκ17-127, Vκ14-100, Vκ12-46, Vκ4-57-1 and Vκ3-4) (Ibrahim et al., 1995; Roark et al., 1995; Witsch and Bettelheim, 2008 and our hybridoma data) (Fig. 6B,C). Vκ4-62, reported as a potentially functional (open reading frame) gene, was found often in BAFF/3H9 pyrosequencing and conventional cloning samples. We have confirmed that Vκ4-62 is rarely used in wild type mice (data not shown), suggesting that there is a strong positive selection for its usage specifically in BAFF/3H9 mice. As the original 3H9 autoantibody used a mutated form of the closely related Vκ4-81 gene, Vκ4-62 may also sustain 3H9 autoreactivity. We conclude that changes in BAFF levels in vivo can cause striking alterations in the B cell repertoire that regulate the relative abundance of autoreactive cells.

Figure 4. Pyrosequence analysis of Vκ usage in mice of differing BAFF activity.

κ light chain genes observed in B cells isolated from spleen A) and lymph nodes B) in each strain. Only genes used at >0.5% frequency are shown. Colored bars indicate genotypes as follows, 3H9; green, BAFF/3H9; red, DBF/3H9; blue. Data for 4 mice per group. 12 to 16-week-old mice were used.

Figure 5. Vκ usage depicted as estimated absolute cell number.

Data shown estimate total cells in the indicated tissues that express the indicated Vκ genes. A) spleen. B) lymph nodes. Values shown are fractional usage taken from Figure 4 multiplied by total B220⁺ B cell number from Figure 1A.

Figure 6. Vκs that are significantly skewed by altered BAFF levels and analysis of corresponding hybridoma proteins for DNA reactivity.

Increased κ light chain genes in lymph nodes from each strain are shown. Genes increased in A) BAFF/3H9, B) only in 3H9, and C) in DBF/3H9. Colored bars indicate genotypes as follows: 3H9, green; BAFF/3H9, red; DBF/3H9, blue. Data for 4 mice per group. 12 to 16-week-old mice were used. Asterisks show p values calculated by Chi-square test. ***; p <0.001.

With some notable exceptions, there was broad similarity between Vκ usages in spleen and lymph nodes of 3H9 and DBF/3H9 mice (Fig. 4, 6). Editor L-chains Vκ17-127, Vκ14-100, Vκ1-99, Vκ13-84 and Vκ3-4 were used frequently in 3H9 and DBF/3H9 mice and infrequently in BAFF/3H9 mice (Fig. 6C). However, in lymph nodes, a significant subset of Vκs was much less well represented in DBF/3H9 than in 3H9 mice, including Vκ2-109, Vκ4-58, Vκ5-45, Vκ8-18, Vκ6-14, Vκ3-9, Vκ3-7, and Vκ3-2 (Fig. 6B). As a result, the overall diversity of Vκ usage appeared to be constricted in DBF/3H9 mice, as was the case for the overall B cell number, while the opposite trend was seen in BAFF/3H9 samples (Fig. 4).

Usage of some Vκs was increased in all 3H9 strains (3H9, BAFF/3H9 and DBF/3H9) relative to WT mice (Fig. 7; B6 data not shown). These included Vκ19-93, Vκ13-85, Vκ8-28, Vκ6-23, Vκ6-17, and Vκ6-15. Vκ19-93 [gj38c] in combination with 3H9 or the related H-chain 3H9-56R is reported to have polyreactivity and to be found preferentially in the MZ compartment (Liu et al., 2007; Witsch and Bettelheim, 2008; Khan et al., 2008). Conventional sequencing analyses showed that κ sequences obtained from sorted 3H9 MZ B cell populations were almost entirely Vκ19-93 (Fig. S2), which may explain why Vκ19-93 was found more frequently in the spleen than lymph nodes in 3H9, BAFF/3H9 and DBF/3H9 samples. In a subset of cases, V-J sequencing data indicated that lower BAFF levels in 3H9 and DBF/3H9 mice correlated with increased Jκ 5 usage and reduced anti-dsDNA. Indeed, in 3H9 and DBF/3H9 B cells Vκ19-93, Vκ13-85 and Vκ6-17 were frequently joined to Jκ5, while in BAFF/3H9 B cells these Vκs were more frequently joined to Jκ1 (Fig. 7A, Fig. S3). However there was no BAFF-dependent difference of Jκ usage among L-chains carrying Vκ8-28, Vκ8-19, Vκ6-23 and Vκ6-15, which were all preferentially associated with Jκ5 (Fig. 7B).

Figure 7. Effect of BAFF activity level on Jκ usage and CDR3 amino acids frequencies of particular Vκ genes.

A) B cells with Vκ19-93 (red) or Vκ12-46 (blue) selected in each strain depends on Jκ usages in lymph nodes. Each strain has different pattern of Jκ usage. B) B cells with Vκ12-44 (black), Vκ8-28 (yellow), Vκ6-23 (green), Vκ8-19 (red) and Vκ6-15 (blue) showed similar pattern of Jκ usage in LNs. C) Analysis of CDR amino acid sequences revealed different selection of B cells with unbiased Jκ usage shown in B. Colored bars indicate genotypes as follows: 3H9, green; BAFF/3H9, red; DBF/3H9, blue.

We analyzed κ-chain CDR3 amino acid sequences in each strain to determine whether mouse strains differed in the details of their CDR3 protein sequences even among cells using the same Vκ or Vκ Jκ combination. Among Vκ8-28 sequences, 3H9, BAFF/3H9 and DBF/3H9 strains selected L-chains with CQNDHSYPLTF (Jκ5) and CQNDHSYPFTF (Jκ4) CDR3 amino acids and there were no strain-specific differences (Fig. 7C-a). However, distinct CDR3 amino acids sequences were found in L-chains carrying Vκ8-19, Vκ6-23 and Vκ6-15. In particular, B cells carrying sequences with additional asparagine (N) CDR3 amino acids were especially frequent in BAFF/3H9 mice (Fig. 7C-c,e). These cells may carry autoreactive receptors. Striking skewings of Jκ usage and CDR3 sequence were also seen among Vκ13-85 sequences (Fig. S3A,B). These findings prompted us to carry out a global analysis of amino acid preferences in the CDR3 of the different strains. We found that BAFF/3H9 samples had significantly higher usage in CDR3s of glycine, valine and histidine and significantly lower usage of leucine and asparagine (Fig. S4). Arginine has been associated with anti-DNAs in many systems, but its frequency was low and similar in all groups. Moreover, when the predicted pI of the Vκ was taken into account, there was a strong correlation between low pI and preferential usage in 3H9 and DBF/3H9 B cells, whereas κ chains with higher pIs were used preferentially in BAFF/3H9 cells.

Hybridoma analysis of dsDNA reactive clones with BAFF overexpression

To analyze in more detail the correlations between antibody specificity and L-chain sequence usage, we produced hybridomas with LPS-stimulated spleen and lymph node cells from BAFF/3H9 and DBF/3H9 mice. We randomly picked ~45 single clones derived from each tissue of each strain for further analysis. To simplify the analysis, we focused on clones clearly expressing the 3H9 H-chain. These were identified using anti-3H9 heavy chain idiotype antibody to test antibodies secreted into the supernatant. In our samples, ~70% of spleen hybridomas and ~80% of lymph node hybridomas scored strongly positive in this assay, with no significant difference between strains (Table II). (As hybridomas from 3H9 H-chain Tg mice have been characterized in many publications, we did not generate these for analysis.) Next we determined the fraction of hybridomas carrying 3H9 heavy chain that had affinity for dsDNA. Among hybrids from BAFF/3H9 spleen or lymph nodes, 72.7% and 67.0%, respectively, bound to dsDNA, while lower frequencies, 30.8% and 34.3%, respectively, were found in the DBF/3H9 samples (Table II). A similar BAFF-dependent difference in the frequency of autoreactive B cells was reflected in the λ⁺ B cell analysis (Fig. 1A). Thus, although DBF/3H9 mice produced little anti-dsDNA antibody (Fig. 2) they had significant frequencies of cells with anti-dsDNA reactivity in spleen and lymph nodes that could be captured as hybridomas by LPS activation prior to fusion (Table II). This result is generally in agreement with previous studies of the 3H9 model bred to non autoimmune-prone backgrounds (Erikson et al., 1991; Chen et al., 1994; Roark et al., 1995). B cells carrying 3H9 H-chain combined with certain κ-chains, such as Vκ8, retain a low affinity autoreactivity leading to an anergic state, which often also involves repositioning of these cells at the B-T cell interface (Seo et al., 2003).

Table II.

Anti-dsDNA specificity and 3H9 H-chain analysis of hybridomas from BAFF/3H9 and DBF/3H9 mice.

Tissue	Genotype	total hybridomas	3H9 H id+a (%)	dsDNA+(%b)	dsDNA−(%b)	p
Spleen	BAFF/3H9	47	33 (70.2)	24 (72.7)	9 (27.3)	.0017c
	DBF/3H9	38	26 (68.4)	8 (30.8)	18 (69.2)
LN	BAFF/3H9	37	29 (78.4)	20 (67.0)	9 (31.3)	.0114c
	DBF/3H9	42	34 (80.9)	12 (35.2)	22 (64.7)

^a3H9 H-chain positive as measured by strong binding to anti-iditoype antibody 1.209 (Gay et al., 1993).

^bpercent of 3H9 id+ hybridomas.

^cp value, Fisher exact test.

We carried out sequencing of L-chains expressed by all 3H9 H-chain positive hybridomas to analyze their Vκ usage and to correlate that usage with antibody autoreactivity. BAFF/3H9 hybridomas had higher diversity of Vκ usage among dsDNA-reactive clones, while DBF/3H9 hybrids had more diverse Vκ usages in non-dsDNA reactive clones (Fig. 8). Positive clones from BAFF/3H9 were paired with more varied κ L-chains compared to clones from DBF/3H9 and V-J combinations of these clones were also varied. Vκ families that tend to confer affinity against dsDNA when combined with 3H9 H-chain, such as Vκ1 (Vκ1-117), Vκ4 (Vκ4-68, Vκ4-91, Vκ4-80, Vκ4-55), Vκ9/10 (Vκ13-85) and Vκ8 (Vκ8-19, Vκ8-24, Vκ8-27, Vκ8-28 and Vκ8-30) were well selected in BAFF/3H9 lymph nodes (Fig. 8A). There were fewer dsDNA positive clones from DBF/3H9 spleen, but these clones did not show any skewed usage or rearrangement compared to those from BAFF/3H9 spleen. By contrast, among lymph node-derived hybridomas 8 of 12 dsDNA-reactive clones from DBF/3H9 were paired with Vκ8 (Vκ8-24, Vκ8-27 and Vκ8-28) and 7 out of 8 were rearranged to Jκ5 (Fig. 8A). It has been reported that B cells carrying 3H9 H-chain paired with Vκ8 are anergic in the periphery and that 3H9/Vκ8 double Tg mice had no anti-dsDNA antibody in the sera (Erikson et al., 1991; Roark et al., 1995). These similarities found in DBF/3H9 mice suggest that these clones are anergic.

Figure 8. Hybridoma analysis.

Shown are κ L-chain family usages of hybridomas produced from spleen and lymph nodes previously stimulated for 3 days with LPS. Upper left, SP from BAFF/3H9; upper right, SP from DBF/3H9; lower left, lymph nodes from BAFF/3H9; and lower right, lymph nodes from DBF/3H9. Colored bars indicate J usage as follows, Jκ1; yellow, Jκ2; green, Jκ4; red and Jκ5; blue. A) κ L-chain family usage observed in dsDNA reactive clones. Asterisks show p values calculated by Chi-square tests between Vk8 family usage in lymph nodes from BAFF/3H9 and DBF/3H9. *; p<0.05. B) κ light chain family usages observed in clones without binding affinity against dsDNA.

Previous studies showed that Vκ21D (Vκ3-4), Vκ20 and Vκ12/13 gene family members render the 3H9 antigen receptor non-autoreactive to chromatin and DNA. Sequence analysis of all anti-dsDNA negative clones confirmed that Vκ17-127 (bw20), Vκ14-100 (cf9) and Vκ3-4 (Vκ21D) were used often in all samples and efficiently suppressed affinity against dsDNA (Fig. 8B). Vκ12/13, Vκ12-46 and Vκ19-93 could lower autoreactivity, but not invariably because there were some dsDNA binding clones paired with these L-chains (Fig. 8A, B). Other Vκ12/13 family members, for example, Vκ12-44 and Vκ12-98 could not edit autoreactivity (Fig. 2A).

To further determine the relationship between BAFF related effects on B cell repertoire and self reactivity, we analyzed DNA reactivity of selected hybridomas expressing Vκ genes whose expression was markedly correlated with BAFF bioactivity (Fig. 6D). Antibodies in which 3H9 H-chain was paired with L-chains whose usage was specifically elevated in BAFF/3H9 mice had relatively high affinity to DNA. These included Vκ1-117 and Vκ8-30 (Fig. 6D red lines). Hybridoma antibodies using Vκs that were reduced in usage in DBF/3H9 mice, but allowed in 3H9 mice, were clearly reactive to DNA, but more weakly. These included Vκ2-209 and Vκ8-19 (green lines). Under these conditions, antibody expressing 3H9 H paired with Vκ19-93/Jκ5, which was common in DBF/3H9 mice failed to bind to DNA (blue line), whereas Vκ19-93/Jκ2, which was counterselected, scored positive (data not shown).

Discussion

The data presented here support the notion that elevated BAFF levels not only promote B cell survival in general, but also affect B cell tolerance, rescuing cells with affinity for self antigens and promoting autoantibody secretion. In agreement with recent studies (Groom et al., 2007), T cells do not appear to be required in our model system for BAFF-induced autoreactive B cell escape and autoantibody formation (data not shown), though they likely contribute when present. Excess BAFF led to much more striking autoantibody formation than several other genetic modifications that have been studied in the context of the 3H9 model, such as deficiency of FcRγIIb (Bolland and Ravetch, 2000), CD21/35 (Seo et al., 2003), or lyn (Seo et al., 2001), or introgression of the Sle2^z allele (Liu et al., 2007). If autoreactive B cells require higher levels of BAFF for survival or development than non autoreactive cells then, in a polyclonal repertoire, equilibrium BAFF levels might have been expected to affect only overall cell numbers rather than the repertoire as a whole. However, this clearly was not the case. In BAFF/3H9 mice, there appeared to be elevation of total B cell numbers along with a broadening of the B cell repertoire to include many L-chain usages that are normally counterselected in 3H9 mice. The resulting κ repertoire was more similar to wild type cells unconstrained by the transgenic H-chain specificity. In BAFF/3H9 mice, there was both broad rescue of many autoreactive specificities and apparent skewing favoring highly autoreactive cells. Thus these findings do not clearly support the simplest possible model for the tolerance-altering effects of BAFF elevation, namely that low affinity self reactive cells are selectively rescued (Thien et al., 2004).

By contrast, reduction of BAFF levels in DBF/3H9 mice lowered total B cell numbers, among which a higher frequency carried L-chains that disallowed dsDNA binding. Vκ genes that were frequently used in 3H9 mice tended to be used even more often in DBF/3H9 mice with the notable exception of a subset found in 3H9 but not in DBF/3H9 lymph nodes (Fig. 7B). Our interpretation of this subset of L-chains is that it confers on 3H9 Tg B cells a low affinity self reactivity which allows cells to be efficiently counterselected only under conditions of moderately low levels of BAFF. In support of this notion, two hybridomas expressing members of this group (Vκ2-109 and Vκ8-19) were captured among BAFF/3H9 hybrids and were found to be weakly DNA reactive. The reduction in BAFF bioactivity in DBF/3H9 mice is likely to be less than that observed in BAFF heterozygous-deficient mice (Gavin et al., 2005) (i.e., 50% of normal), suggesting that a small reduction in BAFF levels can have profound effects on B cell repertoire. Although it is an attractive notion that limiting BAFF levels might preferentially eliminate the highest affinity autoreactive B cells in the population, consistent with the notion of Thien et al (Thien et al., 2004), this model fits well only to our data with DBF/3H9 cells. By contrast, excess BAFF in BAFF/3H9 mice leads to escape of both high and low affinity autoreactive cells.

Despite these selections, even in DBF/3H9 mice many autoreactive B cells were found in the spleen and lymph nodes, as revealed most directly in the specificity of hybridoma antibodies. About 30% of DBF/3H9 hybridomas retained some apparent self reactivity as measured in dsDNA ELISA, but these cells apparently did not contribute to serum antibodies, reminiscent of 3H9/Vκ8 double transgenic B cells that have been shown to be anergic and of relatively low affinity for DNA (Erikson et al., 1991; Chen et al., 1997; Erikson et al., 1998). Indeed, most of the dsDNA-reactive B cells remaining in DBF/3H9 lymph nodes appeared to use a subset of Vκ8 family members.

One possible explanation for the broad escape of autoreactive B cells in BAFF/3H9 mice is that the BAFF transgene leads to expression in such excess that all autoreactive B cells are rescued. However, we know that BAFF is limiting in this model because in the same BAFF transgenic mice expressing low levels of a high affinity Igκ-reactive superantigen (BAFF/pUliκ^low), the survival of κ⁺ B cells only occurred when competing non “autoreactive” B cells were in the minority (Aït-Azzouzene et al., 2006). We do not have a definitive explanation for the differences between BAFF/3H9 and BAFF/pUliκ^low mice, though several factors could contribute. In BAFF/3H9 mice the nucleic acid-containing autoantigens are likely to be present in vivo only intermittently and are able to stimulate toll-like receptors (Leadbetter et al., 2002; Boule et al., 2004; Lau et al., 2005), whereas in BAFF/pUliκ^low mice “self” antigen was constantly available and associated with cell surfaces that may be especially tolerogenic (Russell et al., 1991; Duong et al., 2010).

The B cell repertoire of BAFF/3H9 mice is consistent with possible BAFF-regulated positive selection or expansion of certain highly autoreactive clones, such as those expressing 3H9 H with λ-chain or κ-chains encoded by Vκ4 and Vκ1 family genes. Several lines of evidence support this possibility. First, the autoantibody formation in BAFF/3H9 mice indicates that tolerance is not intact and that autoreactive B cells are not well regulated. Second, usage of many L-chains known to promote high affinity autoreactivity, such as λ-chain, is not only higher than 3H9 controls, but often elevated beyond even the normal levels found in non transgenic mice. Elevation of λ usage in BAFF/3H9 mice was confirmed by flow cytometry analyses. Finally, analyses of the CDR3 sequences of L-chain clones suggested elevations of rare sequences that were inefficiently created by V/J recombination.

Positive selection among B cells has been recognized to occur by different mechanisms. Continuous signals through BCRs (known as tonic signals) are required for mature B cell survival (Lam et al., 1997; Srinivasan et al., 2009). Signals from BCR ligands appear to be important in B-1 cell development (Hayakawa et al., 1999). Cancro and colleagues recently proposed a model that involves crosstalk between BAFF and BCR signaling through the NFκB pathway that regulates B cell maturation at the transitional stages (Stadanlick et al., 2008). Our data from BAFF/3H9 mice showed apparent positive selection of B cells carrying Vκ1-117 and Vκ1-110, which from previous studies and our hybridoma analysis are likely to have high affinity for DNA and are normally counterselected by receptor editing or deletion. However, we find that in the presence of excess BAFF these B cells not only avoid deletion but also appear to selectively survive or expand and secrete autoantibody. Autoreactive B cells require more BAFF for survival than innocuous cells and in their presence turn over rapidly (Lesley et al., 2004; Thien et al., 2004; Aït-Azzouzene et al., 2006). However in autoimmune disease, autoreactive B cells must survive and produce autoantibody despite an initially polyclonal repertoire. Our data suggest that autoreactive B cells with certain specificities or autoantigen properties might be positively selected if excess BAFF is available.

In this study we analyzed the κ L-chain repertoires of nonactivated B cells in spleen and lymph nodes separately. Splenic B cells are more heterogeneous than lymph node B cells in part because they include semimature B220⁺CD93^high transitional B cells emigrating from bone marrow, which turn over rapidly and are thought to be enriched in autoreactive B cells (Allman et al., 1992; Allman et al., 1993; Carsetti et al., 1995; Allman et al., 2001; Merrell et al., 2006). After selection at the transitional stage, B cells differentiate into follicular or MZ B cells. MZ B cells are thought to be long-lived, self renewing cells with a distinct repertoire and function, but unlike follicular B cells appear to remain in the spleen (Allman and Pillai, 2008; Carey et al., 2008). Lymph node B cells by contrast are mostly long-lived recirculating cells and so, using our sorting strategy which eliminated plasma cells, may reflect the output of preimmune B cell repertoire selection more clearly. These distinctions might explain why BAFF-regulated repertoire differences were more clearly seen in lymph nodes.

In BAFF/3H9 mice B cells carrying Vκ1-110 and Vκ1-117 were increased in lymph nodes compared to spleen suggesting ongoing positive selection or expansions in lymph nodes after negative and positive selection in spleen. By contrast, reduction of BAFF reactivity in DBF/3H9 mice resulted in comparable Vκ (and Jκ) repertoires in spleen and lymph nodes and low absolute B cell numbers in lymph nodes, suggesting minimal positive selection.

BAFF/3H9 mice had an increased total B cell number with a skewing not only of Vκ usage but also of Jκ usage, involving an increase in the percentage of cells carrying Jκ1 and a relative reduction in Jκ5 usage. Although Jκbias has been interpreted in the context of receptor editing, it is unclear if BAFF can act directly on editing-competent B cells because they respond poorly to BAFF and have little BAFF-R expression (Rolink et al., 2002; Hsu et al., 2002). Bosma and colleagues working with the 3H9-56R model have suggested that a small splenic B cell subset that they call T3′ might include autoreactive cells in the process of editing (Kiefer et al., 2008). T3′ cells had markers reminiscent of putative natural anergic B cells (T3 cells), though they lacked expression of CD23. T3 cells are believed to be at least in part BAFF responsive (Lesley et al., 2004; Thien et al., 2004; Merrell et al., 2006), but the possible BAFF responsiveness of T3′ cells is unknown. Thus it is not excluded that in BAFF/3H9 mice BAFF overexpression might suppress editing in T3′ cells, thus contributing to the Jκ skewing. Eilat and colleagues have also argued for peripheral editing in another anti-DNA transgenic model, but in that case it was considered a possible contributor to autoimmunity, not a hindrance (Yachimovich-Cohen et al., 2003). Arguing against a role for suppressed editing in response to excess BAFF in the BAFF/3H9 mice is the fact that λ expression, which is also associated with editing, was enhanced rather than reduced in BAFF/3H9 mice. We favor instead the interpretation that post-editing autoreactive cells of many kinds may be selectively expanded or rescued from cell death in BAFF/3H9 mice. Autoreactive cells that fail to edit but are able to mature to BAFF responsiveness because of the intermittent presence of cognate autoantigen should be rescued from peripheral deletion in the presence of excess BAFF. These cells may be dominated by usage of Jκ 1 and Jκ 2 because they rearrange first and most efficiently (Wood and Coleclough, 1984). In the context of reduced BAFF, the B cells may have to have previously reduced autoreactivity through editing to survive BAFF dependent selection at a later stage.

Two studies have taken an approach with similarities to ours, but with somewhat different results. Erikson and colleagues (Hondowicz et al., 2007) studied conventional 3H9 μ-chain Tg mice that were given exogenous BAFF for 9-21 days, focussing on λ-chain cells known to have high affinity for chromatin (Radic et al., 1991). Although BAFF injection increased total and CD93⁻λ⁺ B cell numbers, λ⁺ autoantibodies were not elevated, even when the mice were bred to a Igκ^−/− background where there was less competition with B cells carrying innocuous specificities. The quantity, quality, or duration of exposure to excess BAFF may have contributed to the differences between the results of that study and ours. Alternatively, the inability of the H-chain transgene to undergo class switch or the analysis of mice on the BALB/c genetic background may have affected the results. The B6 background, which was used in the present study, has been suggested to be more autoimmune prone than BALB/c owing to differences in B cell tolerance (Bolland and Ravetch, 2000; Sekiguchi et al., 2006; Witsch and Bettelheim, 2008). In a second study, Spatz and colleagues (Thorn et al., 2010) studied BAFF Tg mice carrying a conventional IgM transgene encoding an anti-DNA antibody called R4A. In the R4A model (in the absence of excess BAFF) B cell numbers were reduced ~90% from normal and the mice produce little anti-dsDNA antibody, suggesting strong negative selection and minimal competition from non autoreactive B cells. BAFF transgene expression promoted autoantibody production in a subset of animals and promoted the development of more B cells from early transitional to later developmental stages, but it was unclear if BAFF had a selective B cell survival effect depending upon self specificity or affinity.

A significant technical feature of the present study was the use of pyrosequencing analysis of L-chains along with fixation of H-chain to facilitate a broad repertoire analysis. A major disadvantage of conventional sequencing techniques is that it is laborious to analyze more than 200 clones per sample. In any case, we found that it was necessary to obtain more samples than was practical using conventional sequencing methods. Among 1511 clones obtained using conventional techniques, each sample contained fewer than 86 out of 396 possible V-J combinations (99 Vκ × 4 Jκ functional gene segments), suggesting a relatively poor sampling of the possible repertoire (Suppl. Table I). 454 technology allowed us to obtain more sequences from 5′-RACE κ L-chain amplicons. The present studies have generated more mouse Igκ sequences than are presently in the IMGT database (Lefranc et al., 2009) by a factor of >20-fold, providing an unprecedented view of the mouse Igκ repertoire. The technology is ideal to study the repertoire of more complex immune systems (Weinstein et al., 2009). We are currently characterizing Igκ usage in unmanipulated C57Bl/6 mice.

Materials and methods

Animals

All mice were bred and maintained in The Scripps Research Institute Animal Resources facility according to the Institutional Animal Care and Use guidelines. C57BL/6J mice were purchased from Jackson Laboratories and bred in the TSRI custom breeding colony. Site-directed 3H9 heavy chain transgenic mice (Chen et al., 1995; Chen et al., 1997) on a C57BL/6 background were kindly provided by Dr. M. Weigert (University of Chicago). BAFF and DBF Tg mice on a C57BL/6 background have been previously described (Gavin et al., 2005). BAFF/3H9 and DBF/3H9 double Tg mice were generated by breeding homozygous BAFF or DBF Tg and 3H9 mice.

ELISA

Assay for dsDNA autoantibodies was carried out as follows: 2 μg/ml dsDNA from salmon sperm was coated to Nunc Maxisorp 96-well plates in Re- acti-Bind DNA coating solution (Pierce). After overnight coating, wells were blocked for 1 hr in Tris-buffered saline containing 5% bovine serum albumin (BSA). Mouse sera or hybridoma supernatant diluted in blocking solution were applied and incubated for 90 min at room temperature. After extensive washing, bound antibodies were detected with 1:3000 diluted horseradish- peroxidase-conjugated goat anti-mouse IgM or goat anti-mouse IgG (Jackson Immunoresearch) and developed with 1-Step-Ultra TMB colorimetric sub- strate (Pierce). OD450nm was measured with a Versamax plate reader (Molecular Devices). For IgA and lambda detection we used biotin conjugated rat anti-mouse IgA (C10-1, BD) or biotin conjugated rat anti-mouse Ig, λ1, λ2, and λ3 light chain (R26–46, BD) for secondary antibody and bound antibodies were detected with 1:5000 diluted Streptavidin-Horseradish Peroxidase (BD).

Flow cytometry analysis

Cell-surface marker stains for FACS analyses were performed using standard protocol. All of the following antibodies were used at 1:200 dilution in FACS buffer (HBSS containing 1% BSA, 2 mM EDTA, and 0.1% NaN₃), with 3 × 10⁶ cells/stain: FITC or Alexa Fluor 647 anti-IgM^a (DS-1, BD or self conjugated), Alexa Fluor 647 anti-IgM (clone M41), FITC anti-CD21 (7G6, BioLegend), PE anti-CD1d (1B1, eBiosciense), PE anti-CD23 (B3B4, BD), APC anti-CD93 (AA4.1, eBioscience), FITC anti-Kappa (187.1, BD), APC anti-Lambda (RML-42, Biolegend), Pacific Blue anti-CD45R/B220 (RA3-6B2, BD), and PerCP-Cy5.5 anti-CD4 (GK1.5, BD), PerCP-Cy5.5 anti-CD8 (53-6.7, BD). For intracellular Ig κ and λ staining, cells incubated during surface staining with unlabeled anti-κ (187.1, BD) and unlabeled anti-λ (RML-42, Biolegend); after fixation and permeabilization using a kit (Cytofix/Cytoperm, BD) cells were stained with FITC anti-κ and APC anti-λ. All flow cytometric data were acquired on a LSR II (BD) and were analyzed using the FlowJo program (Tree Star, Inc.).

κ chain variable region 5′-RACE (conventional method)

B cells were isolated from spleen or lymph node cells of three month old mice using a “no touch” B cell isolation kit (Miltenyi Biotec) supplemented with bio-anti-IgM^b (AF6-78, BD) for samples carrying the 3H9 gene or bio-anti-IgM^a (DS-1, BD) for control C57BL/6 cells. Antibody bound cells were identified with phycoerythrin streptavidin. Selected cells were >98% IgM^a+. Total RNA was obtained from purified B cells using RNeazy Plus kit (QIAGEN). κ chain variable sequence was reverse transcribed and amplified using 5′-RACE kit (Ambion) according to the manufacture’s protocol. PCR was performed with inner primer and mIgKr: 5′-ctgctcactggatggtgggaagatgg-3′ using Phusion Hot Start (NEB). PCR products were purified with agarose gel and cloned using Zero Blunt® TOPO® PCR Cloning Kits (Invitrogen). Ligated product was transformed into Top10 chemically competent cells (Invitrogen). Single colonies were directly amplified by PCR with T7 and M13 reverse primer and sequenced. Obtained sequences were analyzed at the IMGT website (www.imgt.org) (Lefranc et al., 2009).

454 sequencing

B cells were isolated from four 3-month old mice, which included two females and two males. Spleen and lymph node cells were harvested, then depleted of erythrocytes using ACK buffer prior to B cell isolation. For splenic B cell isolation, 50 million spleen cells from each gender were combined and IgM^a B cells isolated using the “no touch” B cells isolation kit (Miltenyi Biotec) supplemented with bio-anti-IgM^b. Total RNA was obtained from purified B cells using TRIZOL^@ reagents (Invitrogen). κ chain variable from each sample was obtained using a 5′ RACE kit (Ambion) according to the manufacturer’s protocol. 1.0μg of total RNA per sample was used. For RT or PCR, Transcriptor High Fidelity (Roche) and Phusion Hot Start (NEB) was used, respectively. A bar code strategy was used to distinguish female and male samples in later analysis. The following oligonucleotides were used to amplify κ chain sequences. K-R1 (Male) 5′-TTGACTGCTCACTGGATGGTGGGAAGATGG-3′, K-R2 (Female) 5′-TTATCTGCTCACTGGATGGTGGGAAGATGG-3′, RACE-1 (Male) 5′-TTGACGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG-3′, RACE-2 (Female) 5′-TTGTCGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG). Each PCR product was purified on agarose gel and 2.5μg each of female and male amplicons were combined and used for 454 sequence analysis. GS FLX Titanium sequencing kit XLR70 (Roche) was used for sample preparation and 8 region gasket were used for processing. Data was collected at GeneChip^™ Microarray Core (UCSD) and analyzed at Scripps Translational Science Institute. Data was analyzed using stand-alone BLAST program (Altschul et al., 1990) downloaded from NCBI. κ chain sequences were obtained from the ImMunoGeneTics (IMGT) website (www.imgt.org) (Lefranc et al., 2009).

Sequence analysis of reads obtained from 454 sequencing

κ-chain sequences were analyzed by BLAST (Altschul et al., 1990) downloaded from NCBI and compared to germline Vκ and Jκ sequences obtained from IMGT (Lefranc et al., 2009) (listed in Supplementary Table III). We analyzed clones without unread bases using the MEGABLAST algorithm. Average read length was 318 bp. We ran MEGABLAST requiring 90% sequence identity and using a 28 bp word size setting to match Vκ-regions. Reads were assigned to genes based upon a hierarchy of BLAST result parameters where the next parameter in the hierarchy was considered only if a read matched multiple genes with the previous parameter. The hierarchy, ordered to be more permissive to gaps in the BLAST alignments because of known homopolymer sequencing errors using 454 technology, was as follows: highest bit-score, highest percent-identity, longest alignment length, least number of mismatches, and finally least number of gaps. For a small percentage (~3%) of reads for which the score matched multiple V-genes exactly the read was distributed according to the % of uniquely mapping reads for each gene. Jκ genes were assigned similarly, requiring 80% sequence identity, using a 7 bp word size, and requiring the read to span at least 23 bases of the Jκ genes.

For CDR sequence and Jκ usage analysis, in-frame sequences for which Vκ and Jκ were identified were used to define CDR3, starting with the conserved cysteine residue and ending with the conserved phenylalanine in the third Jκ codon. The number of clones we analyzed is summarized on Table I. All programs used in this analysis are available on request.

Hybridoma generation and analysis

Lymphocytes from spleen and pooled lymph nodes from 12- to 16-wk-old mice were cultured for 72 hrs in RPMI supplemented with 10 % fetal calf serum (FCS) and lipoplysaccharide (LPS) (50 μg/ml) and fused with the SP2/0 myeloma line with polyethelene glycol. Cells from each fusion were then plated into 96-well plates and hybrids selected with hypoxanthine-aminopterin-thymidine (HAT) medium. Any wells with two distinct colonies were excluded. Cell supernatants were then screened for 3H9 heavy chain idiotype. Reactivity against dsDNA was measured by ELISA. Light chain sequences were amplified and sequenced from the cDNA from hybridomas. The κ L-chain sequence was amplified using mixed 5′ primers for Vκ gene amplification and Cκ primer (5′-CTGCTCACTGGATGGTGGGAAGATGG-3′) to obtain V-J recombination sequences. The PCR products were electrophoresed on 1.5 % agarose gels, purified with a kit (QIAGEN) and cloned in pSMART-HCKan vector (Lucigen^® Corporation), and the insert were sequenced (Retrogen). Sequence analysis was carried out with the IMGT/V-QUEST program (http://imgt.cines.fr/).