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DOI: 10.1002/eji.201444917
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Surrogate light chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by marginal zone B cells ¨k, Weicheng Ren ∗ , Ola Grimsholm ∗ , Angelina I. Bernardi, Nina H¨ oo Anna Stern, Nicola Cavallini and Inga-Lill M˚ artensson Department of Rheumatology and Inflammation Research, University of Gothenburg, Gothenburg, Sweden Selection of the primary antibody repertoire takes place in pro-/pre-B cells, and subsequently in immature and transitional B cells. At the first checkpoint, μ heavy (μH) chains assemble with surrogate light (SL) chain into a precursor B-cell receptor. In mice lacking SL chain, μH chain selection is impaired, and serum autoantibody levels are elevated. However, whether the development of autoantibody-producing cells is due to an inability of the resultant B-cell receptors to induce central and/or peripheral B-cell tolerance or other factors is unknown. Here, we show that receptor editing is defective, and that a higher proportion of BM immature B cells are prone to undergoing apoptosis. Furthermore, transitional B cells are also more prone to undergoing apoptosis, with a stronger selection pressure to enter the follicular B-cell pool. Those that enter the marginal zone (MZ) B-cell pool escape selection and survive, possibly due to the B-lymphopenia and elevated levels of B-cell activating factor. Moreover, the MZ B cells are responsible for the elevated IgM anti-dsDNA antibody levels detected in these mice. Thus, the SL chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by MZ B cells.
Keywords: Autoantibodies r Autoimmunity r Light chain recombination r Marginal zone B cells r Pre-BCR
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction V(D)J recombination is a stochastic process that gives rise to a vast primary repertoire of B-cell receptors (BCRs), recognizing self (auto) and nonself antigens. To purge and control autoreactive B cells, several checkpoints are in place, in both central and peripheral lymphoid organs [1, 2]. The main mechanism of central B-cell tolerance is receptor editing [3, 4] and, as some autoreactivity remains among the cells that migrate to the periphery [5], they undergo further selection as transitional B cells in the spleen
˚ Correspondence: Prof. Inga-Lill Martensson e-mail: [email protected] C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[6], mainly by clonal deletion. Extrinsic factors are also involved, for example, B lymphocyte activating factor (BAFF), also known as BLyS, a key factor that modulates survival of peripheral B cells [7]. Defective central and peripheral B-cell tolerance checkpoints, as well as elevated BAFF levels have all been associated with autoimmune diseases, for example, systemic lupus erythematosus and rheumatoid arthritis, and mouse models thereof, a hallmark of which is autoantibody production [8–10]. Selection of the primary BCR repertoire takes place also at the pro-B to pre-B-cell transition based on antibody μ heavy (μH)
∗
These authors contributed equally to this work.
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Figure 1. Reduction in λL chain expressing BM iB cells in SLC−/− mice. (A) Schematic overview of B-cell development in BM and spleen [11, 12]. Recombination status of the Ig H and L chain loci and tolerance checkpoints are indicated. Around 20% of pro-B cells express a pre-BCR [13]. (B and C) Flow cytometry analysis of BM cells from control and SLC−/− mice. (B) Expression of λ and κ on iB cells, as indicated. (C) Percentage of λ+ and κ+ iB cells. Each symbol represents an individual mouse and data are shown as mean ± SEM of three to five mice per group pooled from two experiments. (D and E) DNA rearrangement and gene expression levels in Fr.D and Fr.E cells from control and SLC−/− mice were measured by qPCR using Gapdh (DNA) or β-actin (mRNA) as internal controls. Fold difference was normalized to the levels in Fr.D cells from control mice. (D) κ Germline transcripts (κ0 ) and κ germline configuration (GL) and λ2 germline transcripts (λ20 ) and λ1,2,3 rearrangements (RE). (E) Rag-1 and Rag-2 mRNA levels. Data are shown as mean ± SEM of three to five mice per group, and are from a single experiment representative of three independent experiments. p Values were determined using a two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
chains rather than a BCR [14–19]. Functional VDJH recombination in pro-B cells results in the expression of μH chains that assemble with the invariant surrogate light (SL) chain and the signaling molecules Igα/β forming a precursor B-cell receptor (preBCR) [14, 20] (Fig. 1A). A pre-BCR is expressed also on large, cycling, but not small, resting pre-B cells [13]. Previous studies have suggested that SL chain is involved in VH repertoire selection [20], and in selection of μH chains based on the complementaritydetermining region 3 (H-CDR3) [21, 22]. The H-CDR3, encoded by the V-D-J junction, is the most hypervariable of the three CDRs, and it is dominant in antigen recognition of both pathogens and autoantigens [23–25]. Cells that express μH chains with an HCDR3 prototypic of those found in anti-DNA antibodies appear to undergo negative selection [21, 22]. Other studies have suggested that the pre-BCR itself is autoreactive, mediated by the positively charged amino acid residues in the λ5-tail, and that this drives the development of B cells [18]. In mice lacking SL chain (SLC−/− ), the levels of serum autoantibodies, for example, anti-DNA and antinuclear antibodies (ANAs) are elevated [22]. Our previous results suggested that one of the underlying mechanisms was abolished selection of μH chains at the pro-/pre-B-cell stage. However, whether the μH chains expressed in SLC−/− mice and in the context of a BCR are unable to activate mechanisms of central and/or peripheral B-cell tolerance or whether the elevated autoantibody levels can be explained by other factors is unclear. Here, we sought to investigate this.
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Results Reduction in λL chain expressing immature B cells in SLC−/− mice To understand the development of autoantibody-producing cells in SLC−/− mice, we first confirmed that B-cell development is impaired at the pro-B- to pre-B-cell transition by collecting large cell numbers (Fig. 1A) [26, 27]: the c-kit+ pro-B-cell population was enriched and the CD25+ pre-B-cell population reduced, as was the population of IgM+ immature B (iB) cells in SLC−/− mice (Supporting Information Fig. 1A). Thereafter, the potential effects on Ig L chain expression was investigated, as impaired tolerance induction affect the ratio of κ versus λ expressing cells [28], and signaling downstream of the pre-BCR has been implicated in recombination of the Ig L chain loci [29, 30]. We found a highly significant difference; the proportion of λ+ BM iB cells was lower (4% versus 9%) and that of κ+ higher in SLC−/− mice as compared to controls (Fig. 1B and C). Ig L chain recombination takes place in small pre-B (fraction D [Fr.D]) cells, with that of λ recombining ontogenetically later. Each of these is preceded by activation of germline (κ0 and λ0 , respectively) transcription [31]. In Fr.D cells from SLC−/− mice, the amount of κ alleles in germline configuration was similar to those in controls, whereas the amount of λ-recombined alleles was reduced approximately twofold (Fig. 1D, Supporting Information Fig. 2A). In Fr.E cells, germline κ alleles were increased and λ-recombined alleles reduced twofold. The
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levels of κ0 and λ0 transcripts followed to a large extent that of the recombination status. Although not distinguishing between impaired tolerance induction or L chain recombination per se, λ rearrangements and the proportion of λ+ iB cells are reduced in SLC−/− mice.
Normal RAG mRNA levels in small pre-B cells The reduction in λ recombination could be due to the RAG genes not being fully activated in SLC−/− mice, as reactivation of these genes has been linked to pre-BCR-mediated and IL-7 receptor (IL-7R) mediated signaling [29, 30]. However, RAG-1 and RAG-2 mRNA levels in Fr.D cells were not significantly altered (Fig. 1E). As expected, they were reduced in Fr.E cells, although this downregulation was not as prominent in cells from SLC−/− mice. Another possibility was an effect on the transcription factors regulating Ig recombination, for example, Irf4, E2A (E12 and E47), Ebf1, Ikzf1 (Ikaros), Ikzf3 (Aiolos), and Pax5 [29, 30]. However, we did not detect any differences in mRNA levels comparing Fr.D cells from SLC−/− and control mice (Supporting Information Fig. 3). In Fr.E cells, the levels of these factors were overall lower than in Fr.D, except for Aiolos and Pax5, and only the latter were reduced in Fr.E cells from SLC−/− mice. Thus, the reduction in λrecombined alleles in SLC−/− Fr.D cells is seemingly not due to an inability to activate genes involved in the recombination process.
Increased cell death in iB cells Another possible explanation for the reduction in λ recombination could be cell death, as survival of pre-B (and pro-B) cells has been ascribed to signaling downstream of the pre-BCR and IL-7R [30, 32]. However, the IL-7R levels on c-kit+ pro-B and CD25+ pre-B cells were similar comparing cells from SLC−/− and control mice (Fig. 2A). The mRNA levels of Stat5, encoding a signaling molecule downstream of the IL-7R involved in cell survival, were also similar (Fig. 2B). Moreover, mRNA levels of the anti-apoptotic molecules Bclx and Pim2 and the proapoptotic Bim were comparable, suggesting that survival of Fr.D cells is not altered in SLC−/− mice. By contrast, the levels of Pim2 were reduced and those of Bim were elevated in Fr.E cells from SLC−/− mice, indicating that these cells were more prone to undergoing apoptosis. Consistent with this, a higher proportion of iB cells from SLC−/− mice stained positive for activated Caspases (Fig. 2C). We also noticed a lack of iB cells expressing high surface IgM levels in SLC−/− mice (Fig. 2D). Thus, a larger fraction of iB cells apparently undergo deletion in SLC−/− mice, which could be a plausible explanation for the reduction in λ-recombined alleles.
Reduced Vκ-RS rearrangement levels in iB cells Expression of a BCR that recognizes autoantigen leads to activation of receptor editing, a process where continued L chain C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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recombination allows the initially expressed L chain to be replaced, potentially resulting in an innocuous BCR, whereas failing to do so leads to cell death [3]. Receptor editing can be analyzed by measuring the level of recombining sequence (RS) rearrangements in the κ locus (Vκ-RS) [10]. Vκ-RS rearrangement leads to recombination of either the second κ allele or λ. Assessing Vκ-RS rearrangements showed 2.5-fold higher levels in control Fr.D compared to Fr.E cells (Fig. 2E), as expected [10], whereas in SLC−/− mice, they were approximately 75% and 30%, respectively, of those in controls. Vκ-RS rearrangements in IgM+ Fr.E cells mainly derive from κ+ cells, and comparing the levels in κ+ (IgM+ λ− ) cells from control and SLC−/− mice showed a reduction similar to those observed in IgM+ cells (Fig. 2F). No difference was observed when comparing the levels in λ+ Fr.E cells, which were higher than in κ+ cells. Thus, the reduction in Vκ-RS rearrangements in SLC−/− Fr.E cells derive from κ+ cells. Based on the lower levels of Vκ-RS rearrangements, the increase in κ germline alleles, and reduction in λ recombination, we conclude that receptor editing is defective in the absence of SL chain. In preparation for receptor editing, Fr.E cells phenotypically become Fr.D [3], which explains the reduction in Vκ-RS rearrangements and λ recombination in SLC−/− Fr.D cells.
Loss of tolerance among marginal zone B cells Because of remaining autoreactivity among BM emigrants [5], transitional B cells undergo further selection, mainly by clonal deletion, which is observed as a reduction in the proportion of λ+ splenic mature B cells [6]. As expected, in control mice we found a decrease in λ+ cells from 9% in transitional to 3–4% in marginal zone (MZ) and follicular (FO) B cells, which was also observed in BM mature B cells—most of which are recirculating FO B cells (Fig. 3A and Supporting Information Fig. 4). Moreover, the Vκ-RS rearrangement levels were two to threefold higher in MZ and FO compared to transitional B cells (Fig. 3B), suggesting that cells that have not undergone extensive receptor editing have been deleted. In SLC−/− mice, we observed several differences when compared to controls. First, the proportion of λ+ transitional B cells was 6% (Fig. 3A), representing an increase compared to BM iB cells (4%). This was not as a result of continued RAG expression, as the levels were lower than those in control transitional B cells (Fig. 3C). Second, this higher proportion (6%) of λ+ cells was retained in MZ and FO as well as in BM mature B cells (Fig. 3A and Supporting Information Fig. 4). Third, rather than increased, the Vκ-RS levels in MZ B cells were similar to those in transitional B cells (Fig. 3B), suggesting a lack of selection of these cells. Fourth, a prominent (fourfold) increase in Vκ-RS rearrangement levels was observed in FO B cells signifying a stronger selection pressure to enter the FO B-cell pool in SLC−/− mice. Fifth, the proportion of transitional B cells expressing activated Caspases was increased tenfold (Fig. 3D). These results suggest that in SLC−/− mice the proportion of autoreactive B cells entering the spleen is higher and results in a stronger selection pressure on transitional B cells, www.eji-journal.eu
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Figure 2. Increased cell death in BM iB cells and reduced Vκ-RS rearrangement levels in Fr.D and Fr.E cells in SLC−/− mice. (A) Histograms show expression of IL-7R gated on B220+ c-kit+ and B220+ CD25+ cells from control and SLC−/− mice, using B220hi B cells as a negative control. (B) mRNA levels of Stat5α, Bclx, Pim2, and Bim in Fr.D and Fr.E cells from control and SLC−/− mice were measured by qPCR, using β-actin as internal control. (C) Percentage of CaspGlow+ cells after gating on BM iB (iB) cells. (D) Histogram shows expression of IgM on iB cells with mean fluorescence intensity (MFI) in the graph. (E and F) Vκ-RS rearrangement levels were quantified by qPCR in sorted cells from control and SLC−/− mice. (E) Levels of Vκ-RS in Fr.D and Fr.E cells. The fold difference was normalized to the levels in Fr.E cells from one of the control mice. (F) Levels of Vκ-RS in sorted IgM+ λ+ and λ− (κ+ ) iB cells from control and SLC−/− mice. The fold difference was normalized to the levels in λ− iB cells from control mice. Data are shown as mean ± SEM of two to five mice per group, and are pooled from two experiments. (F) BM cells from mice (n = 6/genotype) were pooled to sort λ+ and λ− iB cells. p Values were determined using a two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
most likely those that are about to join the FO B-cell pool because of the apparent lack of selection into the MZ B-cell pool.
Elevated BAFF levels and increased signaling through the BAFF-R in MZ B cells Survival of peripheral B cells is controlled by BAFF [7], and under conditions of B-lymphopenia, decreased consumption is reflected in higher BAFF levels, which also allows the survival of autoreactive B cells [33]. Serum BAFF levels were two- to threefold higher in SLC−/− than in control mice (Fig. 3E), whereas the BAFF-receptor was similarly expressed on MZ B cells from both genotypes (Fig. 3F). Consistent with BAFF-receptor signaling in the MZ B cells in SLC−/− mice, the mRNA levels of prosurvival genes downstream of the BAFF-receptor [34], Bclx and Pim2, were increased 1.5- to twofold, whereas those of the BAFF-receptor itself were not changed compared to controls (Fig. 3G). Moreover, the levels of Bcl-2, another prosurvival factor, were also increased. However, there were no obvious signs of elevated activation status of the SLC−/− MZ B cells, at least not in terms of cellular size; cell surface levels of CD69, CD86, or MHC class II; or phosphorylation levels of the signaling molecules ERK or BLNK (SLP-65/BASH; Supporting Information Fig. 5). These data suggest that the elevated BAFF levels support the survival of MZ B cells in SLC−/− mice.
MZ and FO B-cell numbers increase with age Total splenic B-cell numbers increase until an age of 20–25 weeks in SLC−/− mice [22, 27]. In these mice, the size of the MZ C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
B-cell population was proportionally enriched already at an age of 4 weeks, representing 20% of mature B cells, and by the age of 16–22 weeks, it had increased to 35% (Fig. 4A). However, despite the expansion the MZ B-cell pool was reduced in absolute numbers in SLC−/− mice compared to controls. The major reason for the B-lymphopenic status of these mice, and at all ages, could be related to the prominent reduction in FO B cells, which was evident in both frequency and actual number (Fig. 4B). Despite the B-lymphopenia, the splenic architecture appeared normal, though we noticed more frequent IgM plasma cells in the red pulp (Supporting Information Fig. 6). Thus, the B-lymphopenia affects mostly the FO B-cell pool, whereas that of MZ B cells reaches close to normal numbers with age.
Increased frequency of IgM anti-dsDNA reactive MZ B cells MZ B cells are known to give rise to polyreactive antibodies as well as those that recognize dsDNA, which may facilitate the clearance of bacteria as well as host apoptotic cells [35]. To investigate the capacity of SLC−/− MZ B cells to produce autoantibodies, these cells were sorted and activated in vitro. Three days post stimulation with CpG or LPS about half as many cells were recovered from cultures with MZ B cells from SLC−/− mice compared to controls, whereas the supernatants contained comparable levels of IgM anti-dsDNA antibodies (Fig. 5A and B). The frequency of autoantibody-secreting cells was 1.5- to twofold higher in cells from SLC−/− mice (Fig. 5C), suggesting that the proportion of MZ www.eji-journal.eu
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Figure 4. MZ and FO B-cell numbers increase with age in SLC−/− mice. (A and B) Proportions and cell numbers of (A) MZ and (B) FO B cells in the CD19+ 93− gate from control and SLC−/− mice at indicated ages. Data are shown as mean ± SEM of three to four mice per group, and are pooled from three independent experiments. p Values were determined using a two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3. Lack of selection from transitional into the MZ B-cell pool, and elevated BAFF levels in SLC−/− mice. (A) Flow cytometry analysis of spleen cells. Proportions of λ+ cells after gating on Tr, MZ, and FO B cells. (B) Levels of Vκ-RS rearrangement were measured by qPCR in indicated B-cell populations. Vκ-RS fold difference was normalized to the levels in Tr B cells from control mice. (C) Rag-1 mRNA levels in Tr B cells from control and SLC−/− mice. Fold difference was normalized to the levels in control mice. (D) Proportions of CaspGlow+ cells after gating on Tr B cells. (E) Serum BAFF levels from control and SLC−/− mice were measured by ELISA. (F) Flow cytometry analysis of BAFF-R surface expression on MZ B cells from control and SLC−/− mice. FMO, fluorescence minus one. Plot is representative of three to four mice per group. (G) mRNA levels of Bclx, Pim2, Bcl-2, and Baff-r from sorted MZ B cells, with β-actin as an internal control. (A, B, D, and E) Data are shown as mean ± SEM of three to four mice per group, and are pooled from two independent experiments. (C and G) Spleen cells from mice (n = 5/genotype) were pooled to sort Tr and MZ B cells. p Values were determined using a two-tailed t-test. **p < 0.01, ***p < 0.001. ns, not significant.
B cells producing IgM anti-dsDNA antibodies is higher in SLC−/− mice.
Anti-dsDNA antibody levels increase with age In a cohort of SLC−/− mice aged 7–44 weeks, levels of IgM antidsDNA were elevated [22]. Investigating the kinetics showed that the IgM anti-dsDNA levels were significantly higher in SLC−/− mice compared to controls at an age of 10 weeks (Fig. 6A), and the levels increased with age comparing sera from mice aged 4–7, 9–15, and 18–22 weeks (Fig. 6B). By contrast, IgG anti-dsDNA antibody levels were not significantly higher until an age of 18–20 weeks, which was more apparent at 36–45 weeks (Fig. 6C and D). At an age of 10–12 weeks, total serum IgM as well as IgG lev C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
els were about twofold higher in SLC−/− mice compared to those in controls (Fig. 6E and F). However, total IgM did not correlate with IgM anti-dsDNA antibody levels, rather, a negative correlation was apparent (Fig. 6G). Thus, IgM and IgG anti-dsDNA antibody levels increase with age in SLC−/− mice, albeit with different kinetics.
Depletion of MZ B cells results in background IgM anti-dsDNA antibody levels The increase in MZ B-cell numbers correlated with the age-related increase in serum IgM anti-dsDNA antibodies in SLC−/− mice, with a correlation coefficient (r2 ) of 0.62 (Fig. 7A). To investigate whether the IgM anti-dsDNA antibodies in SLC−/− mice derive from MZ B cells in vivo, we selectively depleted these cells using antibodies to the α4 and LFA-1 integrins [36]. Five hours after injection, MZ B cells were found in the peripheral blood of SLC−/− mice injected with anti-α4/LFA-1, but not control antibodies (Fig. 7B), indicating that these antibodies caused the release of MZ B cells from the splenic marginal sinuses, as expected [36]. Two weeks after anti-α4/LFA-1 injection, MZ B cells were no longer detected in blood or spleen (Fig. 7C and D). By contrast, the number of FO, CD21− 23− , and B1 B cells increased, likely due to an accumulation of cells in the spleen (Fig. 7E). The depletion of MZ, but not FO B cells was corroborated by immunohistological examination of spleen sections (Fig. 7F). Hence, inhibiting the attachment to the marginal sinus selectively depletes MZ B cells from the splenic B-cell pool. The depletion of MZ B cells in SLC−/− mice was paralleled with a decrease in serum IgM anti-dsDNA antibody levels compared to before injection of anti-α4/LFA-1, but not control antibodies (Fig. 7G). In fact, the IgM anti-dsDNA antibody levels were www.eji-journal.eu
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Figure 5. Higher frequency of IgM anti-dsDNA reactive MZ B cells in SLC−/− mice. (A to C) Sorted MZ B cells from control and SLC−/− mice were stimulated in vitro with 10 mg/mL LPS, 3 mM CpG, or medium control. (A) Cell number, (B) IgM anti-dsDNA antibody levels in culture supernatants, and (C) frequency of IgM anti-dsDNA-producing cells as determined by cell counter, ELISA, and ELISPOT, respectively, after 3 days of stimulation. Data are shown as mean ± SEM of three to four mice per group, and are from a single experiment representative of three independent experiments. Med, medium; N.D., not detectable. p Values were determined using a two-tailed t-test. *p < 0.05, **p < 0.01. ns, not significant.
reduced to background levels when sacrificing the mice at day 14. Depletion of the MZ B cells also affected total IgM levels that were decreased about threefold (Fig. 7H), consistent with these cells being responsible for some of the natural antibody production [35]. Because splenic CD21− 23− , FO, and B1 B-cell numbers increased by this depletion, we conclude that the MZ B cells are indeed the major source of IgM anti-dsDNA antibody-production in SLC−/− mice.
Discussion
Figure 6. Serum IgM and IgG anti-dsDNA antibody levels increase with age in SLC−/− mice. (A to D) Anti-dsDNA antibody levels in serum from control and SLC−/− mice, of (A and B) IgM and (C and D) IgG isotypes, as determined by ELISA and at indicated ages. Dotted line in (B) indicates serum levels from a pool (n = 3) of 15-week-old control mice. (E) Total IgM and (F) IgG antibody levels in serum from control and SLC−/− mice as measured by ELISA. (G) Total serum IgM plotted versus IgM antidsDNA antibody levels in SLC−/− mice. (A to F) Each symbol represents an individual mouse and data are shown as mean ± SEM of four to ten mice per group pooled from three independent experiments. p Values were determined using a two-tailed t-test or Pearson correlation test, *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
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Our data suggest that the development of autoantibody-secreting cells in SLC−/− mice can be explained by a lack of μH chain selection in combination with compromised central and peripheral B cell tolerance, B-lymphopenia, and excess survival factors. We show here that the proportion of λ+ iB cells in SLC−/− mice is reduced, which is consistent with an inability to induce receptor editing and an increased proportion of iB cells being deleted. Some of this reduction could also be due to accelerated development, which would explain the increase in λ+ transitional B cells. A lower proportion of λ+ iB cells has been observed also in mice with defective NF-κB signaling that affects the generation of λ+ but not κ+ iB cells or editing of the κ locus [37]. This is due to a survival defect in pre-B cells, resulting in greatly reduced Pim2 mRNA levels, and this defect can be overcome by overexpressing Bcl-2. A decrease in λ+ iB cells is also observed in mice in which the κRS element has been deleted (prevents Vκ-RS rearrangements), and in this model expression of a Bcl-2 transgene rescued κ+ rather than λ+ iB cells [28]. This would be consistent with κ+ iB cells being the majority of cells that undergo Vκ-RS rearrangements [10]. In SLC−/− mice, there is no apparent survival defect in pre-B cells with normal mRNA levels of Pim2 as well as Bim and Bclx. As the reduction in λ+ iB cells in SLC−/− mice is due to compromised receptor editing, we speculate that a Bcl-2 transgene would rescue mostly κ+ iB cells also in these, by analogy to the κRS deletion model.
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Figure 7. Serum IgM anti-dsDNA antibodies are reduced to background levels after depletion of MZ B cells in vivo. (A) Correlation of MZ B-cell numbers with serum IgM anti-dsDNA antibody levels in SLC−/− mice. Data are from Figures 4 and 6. (B to H) SLC−/− mice were treated with anti-α4 and LFA-1, or isotype control antibodies. (B) Flow cytometry analysis of peripheral blood. Plots show the proportions of MZ and FO B cells in the CD19+ 93− gate, 5 h after the first injection. Plots are representative of 4–6 mice per group. (C to E) Flow cytometry analysis of (C) peripheral blood and spleen at day 14, gated as in (B). (D and E) Cell numbers of indicated splenic B-cell populations and total lymphocytes (lymph). B1 B cells were gated as CD19hi 43+ . (F) Immunofluorescence staining of splenic cryosections. MOMA-1 (green), IgM (red), and IgD (blue). Total magnification 100×. Scale bar, 200 μm. Images are representative of four to six mice per group. (G) Serum IgM anti-dsDNA antibody levels before and after treatment as measured by ELISA. Dotted line indicates serum levels from a pool (n = 3) of 15-week-old control mice. (H) Total IgM antibody levels before and after treatment as measured by ELISA. (D and E) Data are from a single experiment representative of two independent experiments. (G and H) Each line represents a single comparison and data (G) have been pooled from two experiments and (H) are from a single experiment representative of two independent experiments. p Values were determined using a two-tailed or Pearson correlation test, t-test, or paired t-test. * p < 0.05; ** p < 0.01; *** p < 0.001. ns, not significant.
BM iB cells expressing high surface IgM levels are almost absent in SLC−/− mice. One explanation could be that these cells are downregulating their BCR in preparation for receptor editing and dedifferentiation. This also suggests that some of the pre-B cells are de facto iB cells although the proportion is unknown. Attempting to estimate this, based on our previous [26] and current data (Supporting Information Fig. 1A), would suggest that at least half of CD25+ pre-B cells express intracellular μH, but no L chains, hence are pre-B cells, and could indicate that as many as half of the pre-B cells are dedifferentiated iB cells. This also supports that pro-B cells do indeed differentiate into pre-B cells. Nevertheless, the actual proportion of pre-B cells that expresses autoreactive μH chains is unclear, although our data herein indicate that this could be higher than previously estimated [22]. Expression of an autoreactive μH chain may even be a requirement for pro-B- to pre-B-cell transition, as they would mimic the autoreactive characteristics of the λ5-tail [14–19]. RAG mRNA levels are greatly downregulated as Fr.D cells differentiate into Fr.E, with only a subset of cells still expressing these genes under normal circumstances [38]. As the levels are higher in Fr.E cells from SLC−/− mice compared to those in controls, it indicates that this subset is proportionally larger in SLC−/− mice, which would be consistent with cells preparing for receptor editing. Aiolos and Pax5 mRNA levels are lower in Fr.E cells from SLC−/− mice, although the significance of this is unclear. We find it interesting as reduced Pax5 levels have been observed in iB cells from NZB mice [39], a strain characterized by defective B-cell tolerance and autoantibody production, and as Aiolos deficiency has also been shown to lead to autoantibody production [40].
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Strong selection pressure on cells entering the FO B-cell pool is evident in SLC−/− mice, however, selection could still be compromised as the proportion of λ+ cells is still high. Nevertheless, selection into the MZ B-cell pool is abolished, and results in an increased frequency of autoreactivity among these cells and elevated levels of serum IgM anti-dsDNA antibodies. The survival of the MZ B cells in SLC−/− mice is likely due to the elevated BAFF levels, presumably as a result of the B-lymphopenia and reduced consumption. This would be consistent with the rescue of autoreactive cells and their entry into the MZ B-cell pool in mice overexpressing BAFF [33]. Our previous work suggested that the CD21− 23− B-cell population is responsible for production of IgG ANAs in SLC−/− mice [22], the idea being that this population would also give rise to plasma cells secreting antibodies recognizing dsDNA, although antibodies binding nuclear antigens do not necessarily bind DNA [41]. However, depletion of the MZ B-cell pool did not affect the CD21− 23− B cells (Fig. 7E) or ANA levels (unpublished observation), suggesting that two distinct B-cell populations are responsible for autoantibody production in SLC−/− mice, MZ B cells giving rise to IgM anti-dsDNA antibodies, and CD21− 23− cells to IgG ANAs. Pre-BCR-mediated negative selection of μH chains has been described also in humans [21]. However, as B-cell development is blocked in this patient, the (auto)antibody repertoire could not be analyzed [42]. Nevertheless, the pre-BCR has been implicated in autoimmune disease, in rheumatoid arthritis where low copy number variation of the SL chain component VPREB1 correlates with clinical parameters, for example, bone erosion [43].
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Materials and methods Mice The SLC−/− mice [27], previously backcrossed for more than ten generations on the C57BL6/OlaHsd background [22], were after embryo transfer bred onto the C57BL6/NCrl background with an intact alpha-synuclein locus [44]. Mice were kept in the Gothenburg University SPF animal facility. Control C57Bl/6NCrl mice were purchased from Charles River (Germany) and Taconic (Denmark). The original backcrossed SLC−/− mice, thawed from embryos frozen in 2004, were also used with SLC+/+ mice as controls, kept as a separate line with the same phenotype. Mice were bred under project license authorization (245-2009 and 39-2010), and genotyped by PCR for targeting of VpreB2 and VpreB1/λ5 [27]. Female mice were used throughout, aged 4–45 weeks.
Molecular immunology
In vitro cultures MZ B cells were sorted from splenocytes of 6- to 11-week-old control and SLC−/− mice. In total, 5 × 104 cells were cultured for 3 days in 96-well round-bottom plates in Iscove’s complete medium with 10% FBS. The cells were stimulated with LPS (10 mg/mL) or CpG (3 mM) [45].
ELISA Serum levels of BAFF were determined according to manufacturer’s instructions (R&D Systems). Absorbance was measured at 450 nm using a microplate reader Spectra Max340PC (Molecular Devices, US). The levels of IgM and IgG anti-dsDNA antibodies and levels of total IgM and IgG were determined as previously described [22, 46].
Flow cytometry and cell sorting ELISPOT Spleen and BM cell suspensions were stained with a cocktail of antibodies (Supporting Information Table 1) following standard techniques, and analyzed on a FACSCantoIITM or FACSVerseTM (BD Biosciences). Data were analyzed using FlowJo (Treestar, Inc.). BM Fr.D (B220+ CD93+ 43− IgM− ) and Fr.E (B220+ CD93+ 43− IgM+ ) cells were sorted from individual mice (8- to 12-week-old). λ+ (B220+ CD93+ IgM+ λ+ ) and κ+ (B220+ CD93+ IgM+ λ− ) iB cells were sorted from pools of five to six mice. Spleen cells were collected from pools of five to six mice (8- to 12-week-old) followed by sorting MZ (CD19+ 93− 43− 21hi 23low ), FO (CD19+ 93− 43− 21int 23+ ), and transitional (CD19+ 93+ 24hi ) B cells. Cells were sorted on a Synergy cell sorter (Sony Biotech) with purities >92% (Supporting Information Fig. 2).
96-Well plates with a nitrocellulose membrane (Millipore) were coated with poly-L-lysine (20 mg/mL) in Tris EDTA buffer for 2– 3 h in room temperature. Plates were washed (Tris EDTA) and then coated with dsDNA at 4°C overnight. After washing, cells were added and incubated at 37°C in a humidification chamber. After 3.5 h, cells were washed (water) and incubated with APconjugated goat anti-IgM (Southern biotech) at 4°C overnight. After washing, the spots were developed with NBT-BCIP (Sigma), then washed and dried. Each well was photographed using a USB camera with the software DinoXCope and analyzed with Photoshop.
Apoptosis assay
Treatment with antibodies recognizing α4 and LFA-1 integrins
After surface staining, to detect apoptotic cells single cell suspensions were incubated with zVAD-FMK-FITC (Casp-Glow, eBioscience) in RPMI 1640 for 1 h at 37°C, according to manufacturer’s protocol and subsequently analyzed by flow cytometry.
SLC−/− mice were injected i.p. with anti-α4 (clone: M17/4; 100 μg) and anti-LFA-1 (clone: PS/2; 100 μg) or isotype control antibodies (BioXcell, USA) at days 0 and 5, and sacrificed at day 14.
Analysis of transcription factors and Ig L chain loci Immunohistochemistry RNA was isolated using RNeasy kit (Qiagen) and reversely transcribed according to the protocol of SuperScript II (Invitrogen). Levels of RS rearrangements were quantified by qPCR, using primers and probes as described [10]. Power SYBR Green (Applied Biosystems) was used to detect transcripts and DNA rearrangements (κ and λ) with previously described primers (Supporting Information Table 1). All samples were run in triplicates on a ViiA7 system (Applied Biosystems). C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Spleens from SLC−/− mice were collected and embedded in OCT compound (TissueTek, Tokyo, Japan), snap-frozen in liquid nitrogen, and stored at −80°C. Frozen sections (8 μm) were cut using a cryostat (Wetzlar, Germany), and stained (Supporting Information Table 1) as previously described [47]. Images were acquired using an LSM 700 confocal microscope and ZEN 2009 acquisition software (Zeiss, Germany). www.eji-journal.eu
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Statistics
11 Hardy, R. R. and Hayakawa, K., B cell development pathways. Annu. Rev.
Data are shown as mean ± SEM in all the plots. p Values were calculated using appropriate statistical tests: unpaired t-test or paired t-test (GraphPad Prism 6.0).
12 Osmond, D. G., Rolink, A. and Melchers, F., Murine B lymphopoiesis:
Immunol. 2001. 19: 595–621.
towards a unified model. Immunol. Today 1998. 19: 65–68. 13 Parker, M. J., Licence, S., Erlandsson, L., Galler, G. R., Chakalova, L., Osborne, C. S., Morgan, G. et al., The pre-B-cell receptor induces silencing of VpreB and lambda5 transcription. EMBO J. 2005. 24: 3895–3905. 14 Conley, M. E. and Burrows, P. D., Plugging the leaky pre-B cell receptor. J. Immunol. 2010. 184: 1127–1129. 15 Almqvist, N. and Martensson, I. L., The pre-B cell receptor; selecting for
Acknowledgments: We thank Drs. S. Cardell, I. Gjertsson, and M. Sigvardsson for critical reading of the manuscript, Dr. D. Chen for assistance with injections, and Ms. L Bergqvist for cell sorting. This work was supported by Swedish Science Research Council, the Swedish Cancer Foundation, ALF: the regional agreement on medical training and clinical research, King Gustav V Stiftelse, Lundbergs Stiftelse, Swedish Medical Society, Reumatikerf¨ orbundet, Stiftelsen Lars Hiertas minne, Stiftelsen Sigurd och Elsa Goljes Minne, Lundgrens Stiftelse, Aml¨ ovs Stiftelser, Adlerbertska stiftelsen, The Royal Society of Arts and Sciences in Gothenburg, and Sigurd och Elsa Golje’s minne, AFA insurances.
or against autoreactivity. Scand. J. Immunol. 2012. 76: 256–262. 16 Vettermann, C. and Jack, H. M., The pre-B cell receptor: turning autoreactivity into self-defense. Trends Immunol. 2010. 31: 176–183. 17 von Boehmer, H. and Melchers, F., Checkpoints in lymphocyte development and autoimmune disease. Nat. Immunol. 2010. 11: 14–20. 18 Herzog, S. and Jumaa, H., Self-recognition and clonal selection: autoreactivity drives the generation of B cells. Curr. Opin. Immunol. 2012. 24: 166–172. 19 Tussiwand, R., Bosco, N., Ceredig, R. and Rolink, A. G., Tolerance checkpoints in B-cell development: Johnny B good. Eur. J. Immunol. 2009. 39: 2317–2324. 20 Melchers, F., The pre-B-cell receptor: selector of fitting immunoglobulin heavy chains for the B-cell repertoire. Nat. Rev. Immunol. 2005. 5: 578–584. 21 Minegishi, Y. and Conley, M. E., Negative selection at the pre-BCR checkpoint elicited by human mu heavy chains with unusual CDR3 regions.
Conflict of interest: The authors declare no financial or commercial conflicts of interest.
Immunity 2001. 14: 631–641. 22 Keenan, R. A., De Riva, A., Corleis, B., Hepburn, L., Licence, S., Winkler, T. H. and Martensson, I. L., Censoring of autoreactive B cell development by the pre-B cell receptor. Science 2008. 321: 696–699.
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38 Monroe, R. J., Seidl, K. J., Gaertner, F., Han, S., Chen, F., Sekiguchi, J., Wang, J. et al., RAG2:GFP knockin mice reveal novel aspects of RAG2 expression in primary and peripheral lymphoid tissues. Immunity 1999. 11: 201–212. 39 Toda, T., Kitabatake, M., Igarashi, H. and Sakaguchi, N., The immature B-cell subpopulation with low RAG1 expression is increased in the
Abbreviations: ANA: antinuclear antibody · BAFF: B lymphocyte activating factor · FO: follicular · Fr.D: fraction D · iB: immature B · MZ: marginal zone · Pre-BCR: precursor B-cell receptor · RS: recombining sequence · SL: surrogate light
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˚ Full correspondence: Prof. Inga-Lill Martensson, Department of Rheumatology and Inflammation Research, University of Gothenburg, Box 480, SE-405 30 Gothenburg, Sweden Fax: +46-31-823925 e-mail: [email protected]
41 Jovelin, F., Mostoslavsky, G., Amoura, Z., Chabre, H., Gilbert, D., Eilat, D., Bach, J. F. et al., Early anti-nucleosome autoantibodies from a single MRL+/ +mouse: fine specificity, V gene structure and pathogenicity. Eur. J. Immunol. 1998. 28: 3411–3422. 42 Minegishi, Y., Coustan-Smith, E., Wang, Y. H., Cooper, M. D., Campana, D. and Conley, M. E., Mutations in the human lambda5/14.1 gene result
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: 5/6/2014 Revised: 8/12/2014 Accepted: 23/12/2014
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DOI: 10.1002/eji.201444917
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Surrogate light chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by marginal zone B cells ¨k, Weicheng Ren ∗ , Ola Grimsholm ∗ , Angelina I. Bernardi, Nina H¨ oo Anna Stern, Nicola Cavallini and Inga-Lill M˚ artensson Department of Rheumatology and Inflammation Research, University of Gothenburg, Gothenburg, Sweden Selection of the primary antibody repertoire takes place in pro-/pre-B cells, and subsequently in immature and transitional B cells. At the first checkpoint, μ heavy (μH) chains assemble with surrogate light (SL) chain into a precursor B-cell receptor. In mice lacking SL chain, μH chain selection is impaired, and serum autoantibody levels are elevated. However, whether the development of autoantibody-producing cells is due to an inability of the resultant B-cell receptors to induce central and/or peripheral B-cell tolerance or other factors is unknown. Here, we show that receptor editing is defective, and that a higher proportion of BM immature B cells are prone to undergoing apoptosis. Furthermore, transitional B cells are also more prone to undergoing apoptosis, with a stronger selection pressure to enter the follicular B-cell pool. Those that enter the marginal zone (MZ) B-cell pool escape selection and survive, possibly due to the B-lymphopenia and elevated levels of B-cell activating factor. Moreover, the MZ B cells are responsible for the elevated IgM anti-dsDNA antibody levels detected in these mice. Thus, the SL chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by MZ B cells.
Keywords: Autoantibodies r Autoimmunity r Light chain recombination r Marginal zone B cells r Pre-BCR
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction V(D)J recombination is a stochastic process that gives rise to a vast primary repertoire of B-cell receptors (BCRs), recognizing self (auto) and nonself antigens. To purge and control autoreactive B cells, several checkpoints are in place, in both central and peripheral lymphoid organs [1, 2]. The main mechanism of central B-cell tolerance is receptor editing [3, 4] and, as some autoreactivity remains among the cells that migrate to the periphery [5], they undergo further selection as transitional B cells in the spleen
˚ Correspondence: Prof. Inga-Lill Martensson e-mail: [email protected] C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[6], mainly by clonal deletion. Extrinsic factors are also involved, for example, B lymphocyte activating factor (BAFF), also known as BLyS, a key factor that modulates survival of peripheral B cells [7]. Defective central and peripheral B-cell tolerance checkpoints, as well as elevated BAFF levels have all been associated with autoimmune diseases, for example, systemic lupus erythematosus and rheumatoid arthritis, and mouse models thereof, a hallmark of which is autoantibody production [8–10]. Selection of the primary BCR repertoire takes place also at the pro-B to pre-B-cell transition based on antibody μ heavy (μH)
∗
These authors contributed equally to this work.
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Molecular immunology
Figure 1. Reduction in λL chain expressing BM iB cells in SLC−/− mice. (A) Schematic overview of B-cell development in BM and spleen [11, 12]. Recombination status of the Ig H and L chain loci and tolerance checkpoints are indicated. Around 20% of pro-B cells express a pre-BCR [13]. (B and C) Flow cytometry analysis of BM cells from control and SLC−/− mice. (B) Expression of λ and κ on iB cells, as indicated. (C) Percentage of λ+ and κ+ iB cells. Each symbol represents an individual mouse and data are shown as mean ± SEM of three to five mice per group pooled from two experiments. (D and E) DNA rearrangement and gene expression levels in Fr.D and Fr.E cells from control and SLC−/− mice were measured by qPCR using Gapdh (DNA) or β-actin (mRNA) as internal controls. Fold difference was normalized to the levels in Fr.D cells from control mice. (D) κ Germline transcripts (κ0 ) and κ germline configuration (GL) and λ2 germline transcripts (λ20 ) and λ1,2,3 rearrangements (RE). (E) Rag-1 and Rag-2 mRNA levels. Data are shown as mean ± SEM of three to five mice per group, and are from a single experiment representative of three independent experiments. p Values were determined using a two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
chains rather than a BCR [14–19]. Functional VDJH recombination in pro-B cells results in the expression of μH chains that assemble with the invariant surrogate light (SL) chain and the signaling molecules Igα/β forming a precursor B-cell receptor (preBCR) [14, 20] (Fig. 1A). A pre-BCR is expressed also on large, cycling, but not small, resting pre-B cells [13]. Previous studies have suggested that SL chain is involved in VH repertoire selection [20], and in selection of μH chains based on the complementaritydetermining region 3 (H-CDR3) [21, 22]. The H-CDR3, encoded by the V-D-J junction, is the most hypervariable of the three CDRs, and it is dominant in antigen recognition of both pathogens and autoantigens [23–25]. Cells that express μH chains with an HCDR3 prototypic of those found in anti-DNA antibodies appear to undergo negative selection [21, 22]. Other studies have suggested that the pre-BCR itself is autoreactive, mediated by the positively charged amino acid residues in the λ5-tail, and that this drives the development of B cells [18]. In mice lacking SL chain (SLC−/− ), the levels of serum autoantibodies, for example, anti-DNA and antinuclear antibodies (ANAs) are elevated [22]. Our previous results suggested that one of the underlying mechanisms was abolished selection of μH chains at the pro-/pre-B-cell stage. However, whether the μH chains expressed in SLC−/− mice and in the context of a BCR are unable to activate mechanisms of central and/or peripheral B-cell tolerance or whether the elevated autoantibody levels can be explained by other factors is unclear. Here, we sought to investigate this.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Results Reduction in λL chain expressing immature B cells in SLC−/− mice To understand the development of autoantibody-producing cells in SLC−/− mice, we first confirmed that B-cell development is impaired at the pro-B- to pre-B-cell transition by collecting large cell numbers (Fig. 1A) [26, 27]: the c-kit+ pro-B-cell population was enriched and the CD25+ pre-B-cell population reduced, as was the population of IgM+ immature B (iB) cells in SLC−/− mice (Supporting Information Fig. 1A). Thereafter, the potential effects on Ig L chain expression was investigated, as impaired tolerance induction affect the ratio of κ versus λ expressing cells [28], and signaling downstream of the pre-BCR has been implicated in recombination of the Ig L chain loci [29, 30]. We found a highly significant difference; the proportion of λ+ BM iB cells was lower (4% versus 9%) and that of κ+ higher in SLC−/− mice as compared to controls (Fig. 1B and C). Ig L chain recombination takes place in small pre-B (fraction D [Fr.D]) cells, with that of λ recombining ontogenetically later. Each of these is preceded by activation of germline (κ0 and λ0 , respectively) transcription [31]. In Fr.D cells from SLC−/− mice, the amount of κ alleles in germline configuration was similar to those in controls, whereas the amount of λ-recombined alleles was reduced approximately twofold (Fig. 1D, Supporting Information Fig. 2A). In Fr.E cells, germline κ alleles were increased and λ-recombined alleles reduced twofold. The
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levels of κ0 and λ0 transcripts followed to a large extent that of the recombination status. Although not distinguishing between impaired tolerance induction or L chain recombination per se, λ rearrangements and the proportion of λ+ iB cells are reduced in SLC−/− mice.
Normal RAG mRNA levels in small pre-B cells The reduction in λ recombination could be due to the RAG genes not being fully activated in SLC−/− mice, as reactivation of these genes has been linked to pre-BCR-mediated and IL-7 receptor (IL-7R) mediated signaling [29, 30]. However, RAG-1 and RAG-2 mRNA levels in Fr.D cells were not significantly altered (Fig. 1E). As expected, they were reduced in Fr.E cells, although this downregulation was not as prominent in cells from SLC−/− mice. Another possibility was an effect on the transcription factors regulating Ig recombination, for example, Irf4, E2A (E12 and E47), Ebf1, Ikzf1 (Ikaros), Ikzf3 (Aiolos), and Pax5 [29, 30]. However, we did not detect any differences in mRNA levels comparing Fr.D cells from SLC−/− and control mice (Supporting Information Fig. 3). In Fr.E cells, the levels of these factors were overall lower than in Fr.D, except for Aiolos and Pax5, and only the latter were reduced in Fr.E cells from SLC−/− mice. Thus, the reduction in λrecombined alleles in SLC−/− Fr.D cells is seemingly not due to an inability to activate genes involved in the recombination process.
Increased cell death in iB cells Another possible explanation for the reduction in λ recombination could be cell death, as survival of pre-B (and pro-B) cells has been ascribed to signaling downstream of the pre-BCR and IL-7R [30, 32]. However, the IL-7R levels on c-kit+ pro-B and CD25+ pre-B cells were similar comparing cells from SLC−/− and control mice (Fig. 2A). The mRNA levels of Stat5, encoding a signaling molecule downstream of the IL-7R involved in cell survival, were also similar (Fig. 2B). Moreover, mRNA levels of the anti-apoptotic molecules Bclx and Pim2 and the proapoptotic Bim were comparable, suggesting that survival of Fr.D cells is not altered in SLC−/− mice. By contrast, the levels of Pim2 were reduced and those of Bim were elevated in Fr.E cells from SLC−/− mice, indicating that these cells were more prone to undergoing apoptosis. Consistent with this, a higher proportion of iB cells from SLC−/− mice stained positive for activated Caspases (Fig. 2C). We also noticed a lack of iB cells expressing high surface IgM levels in SLC−/− mice (Fig. 2D). Thus, a larger fraction of iB cells apparently undergo deletion in SLC−/− mice, which could be a plausible explanation for the reduction in λ-recombined alleles.
Reduced Vκ-RS rearrangement levels in iB cells Expression of a BCR that recognizes autoantigen leads to activation of receptor editing, a process where continued L chain C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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recombination allows the initially expressed L chain to be replaced, potentially resulting in an innocuous BCR, whereas failing to do so leads to cell death [3]. Receptor editing can be analyzed by measuring the level of recombining sequence (RS) rearrangements in the κ locus (Vκ-RS) [10]. Vκ-RS rearrangement leads to recombination of either the second κ allele or λ. Assessing Vκ-RS rearrangements showed 2.5-fold higher levels in control Fr.D compared to Fr.E cells (Fig. 2E), as expected [10], whereas in SLC−/− mice, they were approximately 75% and 30%, respectively, of those in controls. Vκ-RS rearrangements in IgM+ Fr.E cells mainly derive from κ+ cells, and comparing the levels in κ+ (IgM+ λ− ) cells from control and SLC−/− mice showed a reduction similar to those observed in IgM+ cells (Fig. 2F). No difference was observed when comparing the levels in λ+ Fr.E cells, which were higher than in κ+ cells. Thus, the reduction in Vκ-RS rearrangements in SLC−/− Fr.E cells derive from κ+ cells. Based on the lower levels of Vκ-RS rearrangements, the increase in κ germline alleles, and reduction in λ recombination, we conclude that receptor editing is defective in the absence of SL chain. In preparation for receptor editing, Fr.E cells phenotypically become Fr.D [3], which explains the reduction in Vκ-RS rearrangements and λ recombination in SLC−/− Fr.D cells.
Loss of tolerance among marginal zone B cells Because of remaining autoreactivity among BM emigrants [5], transitional B cells undergo further selection, mainly by clonal deletion, which is observed as a reduction in the proportion of λ+ splenic mature B cells [6]. As expected, in control mice we found a decrease in λ+ cells from 9% in transitional to 3–4% in marginal zone (MZ) and follicular (FO) B cells, which was also observed in BM mature B cells—most of which are recirculating FO B cells (Fig. 3A and Supporting Information Fig. 4). Moreover, the Vκ-RS rearrangement levels were two to threefold higher in MZ and FO compared to transitional B cells (Fig. 3B), suggesting that cells that have not undergone extensive receptor editing have been deleted. In SLC−/− mice, we observed several differences when compared to controls. First, the proportion of λ+ transitional B cells was 6% (Fig. 3A), representing an increase compared to BM iB cells (4%). This was not as a result of continued RAG expression, as the levels were lower than those in control transitional B cells (Fig. 3C). Second, this higher proportion (6%) of λ+ cells was retained in MZ and FO as well as in BM mature B cells (Fig. 3A and Supporting Information Fig. 4). Third, rather than increased, the Vκ-RS levels in MZ B cells were similar to those in transitional B cells (Fig. 3B), suggesting a lack of selection of these cells. Fourth, a prominent (fourfold) increase in Vκ-RS rearrangement levels was observed in FO B cells signifying a stronger selection pressure to enter the FO B-cell pool in SLC−/− mice. Fifth, the proportion of transitional B cells expressing activated Caspases was increased tenfold (Fig. 3D). These results suggest that in SLC−/− mice the proportion of autoreactive B cells entering the spleen is higher and results in a stronger selection pressure on transitional B cells, www.eji-journal.eu
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Figure 2. Increased cell death in BM iB cells and reduced Vκ-RS rearrangement levels in Fr.D and Fr.E cells in SLC−/− mice. (A) Histograms show expression of IL-7R gated on B220+ c-kit+ and B220+ CD25+ cells from control and SLC−/− mice, using B220hi B cells as a negative control. (B) mRNA levels of Stat5α, Bclx, Pim2, and Bim in Fr.D and Fr.E cells from control and SLC−/− mice were measured by qPCR, using β-actin as internal control. (C) Percentage of CaspGlow+ cells after gating on BM iB (iB) cells. (D) Histogram shows expression of IgM on iB cells with mean fluorescence intensity (MFI) in the graph. (E and F) Vκ-RS rearrangement levels were quantified by qPCR in sorted cells from control and SLC−/− mice. (E) Levels of Vκ-RS in Fr.D and Fr.E cells. The fold difference was normalized to the levels in Fr.E cells from one of the control mice. (F) Levels of Vκ-RS in sorted IgM+ λ+ and λ− (κ+ ) iB cells from control and SLC−/− mice. The fold difference was normalized to the levels in λ− iB cells from control mice. Data are shown as mean ± SEM of two to five mice per group, and are pooled from two experiments. (F) BM cells from mice (n = 6/genotype) were pooled to sort λ+ and λ− iB cells. p Values were determined using a two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
most likely those that are about to join the FO B-cell pool because of the apparent lack of selection into the MZ B-cell pool.
Elevated BAFF levels and increased signaling through the BAFF-R in MZ B cells Survival of peripheral B cells is controlled by BAFF [7], and under conditions of B-lymphopenia, decreased consumption is reflected in higher BAFF levels, which also allows the survival of autoreactive B cells [33]. Serum BAFF levels were two- to threefold higher in SLC−/− than in control mice (Fig. 3E), whereas the BAFF-receptor was similarly expressed on MZ B cells from both genotypes (Fig. 3F). Consistent with BAFF-receptor signaling in the MZ B cells in SLC−/− mice, the mRNA levels of prosurvival genes downstream of the BAFF-receptor [34], Bclx and Pim2, were increased 1.5- to twofold, whereas those of the BAFF-receptor itself were not changed compared to controls (Fig. 3G). Moreover, the levels of Bcl-2, another prosurvival factor, were also increased. However, there were no obvious signs of elevated activation status of the SLC−/− MZ B cells, at least not in terms of cellular size; cell surface levels of CD69, CD86, or MHC class II; or phosphorylation levels of the signaling molecules ERK or BLNK (SLP-65/BASH; Supporting Information Fig. 5). These data suggest that the elevated BAFF levels support the survival of MZ B cells in SLC−/− mice.
MZ and FO B-cell numbers increase with age Total splenic B-cell numbers increase until an age of 20–25 weeks in SLC−/− mice [22, 27]. In these mice, the size of the MZ C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
B-cell population was proportionally enriched already at an age of 4 weeks, representing 20% of mature B cells, and by the age of 16–22 weeks, it had increased to 35% (Fig. 4A). However, despite the expansion the MZ B-cell pool was reduced in absolute numbers in SLC−/− mice compared to controls. The major reason for the B-lymphopenic status of these mice, and at all ages, could be related to the prominent reduction in FO B cells, which was evident in both frequency and actual number (Fig. 4B). Despite the B-lymphopenia, the splenic architecture appeared normal, though we noticed more frequent IgM plasma cells in the red pulp (Supporting Information Fig. 6). Thus, the B-lymphopenia affects mostly the FO B-cell pool, whereas that of MZ B cells reaches close to normal numbers with age.
Increased frequency of IgM anti-dsDNA reactive MZ B cells MZ B cells are known to give rise to polyreactive antibodies as well as those that recognize dsDNA, which may facilitate the clearance of bacteria as well as host apoptotic cells [35]. To investigate the capacity of SLC−/− MZ B cells to produce autoantibodies, these cells were sorted and activated in vitro. Three days post stimulation with CpG or LPS about half as many cells were recovered from cultures with MZ B cells from SLC−/− mice compared to controls, whereas the supernatants contained comparable levels of IgM anti-dsDNA antibodies (Fig. 5A and B). The frequency of autoantibody-secreting cells was 1.5- to twofold higher in cells from SLC−/− mice (Fig. 5C), suggesting that the proportion of MZ www.eji-journal.eu
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Figure 4. MZ and FO B-cell numbers increase with age in SLC−/− mice. (A and B) Proportions and cell numbers of (A) MZ and (B) FO B cells in the CD19+ 93− gate from control and SLC−/− mice at indicated ages. Data are shown as mean ± SEM of three to four mice per group, and are pooled from three independent experiments. p Values were determined using a two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3. Lack of selection from transitional into the MZ B-cell pool, and elevated BAFF levels in SLC−/− mice. (A) Flow cytometry analysis of spleen cells. Proportions of λ+ cells after gating on Tr, MZ, and FO B cells. (B) Levels of Vκ-RS rearrangement were measured by qPCR in indicated B-cell populations. Vκ-RS fold difference was normalized to the levels in Tr B cells from control mice. (C) Rag-1 mRNA levels in Tr B cells from control and SLC−/− mice. Fold difference was normalized to the levels in control mice. (D) Proportions of CaspGlow+ cells after gating on Tr B cells. (E) Serum BAFF levels from control and SLC−/− mice were measured by ELISA. (F) Flow cytometry analysis of BAFF-R surface expression on MZ B cells from control and SLC−/− mice. FMO, fluorescence minus one. Plot is representative of three to four mice per group. (G) mRNA levels of Bclx, Pim2, Bcl-2, and Baff-r from sorted MZ B cells, with β-actin as an internal control. (A, B, D, and E) Data are shown as mean ± SEM of three to four mice per group, and are pooled from two independent experiments. (C and G) Spleen cells from mice (n = 5/genotype) were pooled to sort Tr and MZ B cells. p Values were determined using a two-tailed t-test. **p < 0.01, ***p < 0.001. ns, not significant.
B cells producing IgM anti-dsDNA antibodies is higher in SLC−/− mice.
Anti-dsDNA antibody levels increase with age In a cohort of SLC−/− mice aged 7–44 weeks, levels of IgM antidsDNA were elevated [22]. Investigating the kinetics showed that the IgM anti-dsDNA levels were significantly higher in SLC−/− mice compared to controls at an age of 10 weeks (Fig. 6A), and the levels increased with age comparing sera from mice aged 4–7, 9–15, and 18–22 weeks (Fig. 6B). By contrast, IgG anti-dsDNA antibody levels were not significantly higher until an age of 18–20 weeks, which was more apparent at 36–45 weeks (Fig. 6C and D). At an age of 10–12 weeks, total serum IgM as well as IgG lev C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
els were about twofold higher in SLC−/− mice compared to those in controls (Fig. 6E and F). However, total IgM did not correlate with IgM anti-dsDNA antibody levels, rather, a negative correlation was apparent (Fig. 6G). Thus, IgM and IgG anti-dsDNA antibody levels increase with age in SLC−/− mice, albeit with different kinetics.
Depletion of MZ B cells results in background IgM anti-dsDNA antibody levels The increase in MZ B-cell numbers correlated with the age-related increase in serum IgM anti-dsDNA antibodies in SLC−/− mice, with a correlation coefficient (r2 ) of 0.62 (Fig. 7A). To investigate whether the IgM anti-dsDNA antibodies in SLC−/− mice derive from MZ B cells in vivo, we selectively depleted these cells using antibodies to the α4 and LFA-1 integrins [36]. Five hours after injection, MZ B cells were found in the peripheral blood of SLC−/− mice injected with anti-α4/LFA-1, but not control antibodies (Fig. 7B), indicating that these antibodies caused the release of MZ B cells from the splenic marginal sinuses, as expected [36]. Two weeks after anti-α4/LFA-1 injection, MZ B cells were no longer detected in blood or spleen (Fig. 7C and D). By contrast, the number of FO, CD21− 23− , and B1 B cells increased, likely due to an accumulation of cells in the spleen (Fig. 7E). The depletion of MZ, but not FO B cells was corroborated by immunohistological examination of spleen sections (Fig. 7F). Hence, inhibiting the attachment to the marginal sinus selectively depletes MZ B cells from the splenic B-cell pool. The depletion of MZ B cells in SLC−/− mice was paralleled with a decrease in serum IgM anti-dsDNA antibody levels compared to before injection of anti-α4/LFA-1, but not control antibodies (Fig. 7G). In fact, the IgM anti-dsDNA antibody levels were www.eji-journal.eu
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Molecular immunology
Figure 5. Higher frequency of IgM anti-dsDNA reactive MZ B cells in SLC−/− mice. (A to C) Sorted MZ B cells from control and SLC−/− mice were stimulated in vitro with 10 mg/mL LPS, 3 mM CpG, or medium control. (A) Cell number, (B) IgM anti-dsDNA antibody levels in culture supernatants, and (C) frequency of IgM anti-dsDNA-producing cells as determined by cell counter, ELISA, and ELISPOT, respectively, after 3 days of stimulation. Data are shown as mean ± SEM of three to four mice per group, and are from a single experiment representative of three independent experiments. Med, medium; N.D., not detectable. p Values were determined using a two-tailed t-test. *p < 0.05, **p < 0.01. ns, not significant.
reduced to background levels when sacrificing the mice at day 14. Depletion of the MZ B cells also affected total IgM levels that were decreased about threefold (Fig. 7H), consistent with these cells being responsible for some of the natural antibody production [35]. Because splenic CD21− 23− , FO, and B1 B-cell numbers increased by this depletion, we conclude that the MZ B cells are indeed the major source of IgM anti-dsDNA antibody-production in SLC−/− mice.
Discussion
Figure 6. Serum IgM and IgG anti-dsDNA antibody levels increase with age in SLC−/− mice. (A to D) Anti-dsDNA antibody levels in serum from control and SLC−/− mice, of (A and B) IgM and (C and D) IgG isotypes, as determined by ELISA and at indicated ages. Dotted line in (B) indicates serum levels from a pool (n = 3) of 15-week-old control mice. (E) Total IgM and (F) IgG antibody levels in serum from control and SLC−/− mice as measured by ELISA. (G) Total serum IgM plotted versus IgM antidsDNA antibody levels in SLC−/− mice. (A to F) Each symbol represents an individual mouse and data are shown as mean ± SEM of four to ten mice per group pooled from three independent experiments. p Values were determined using a two-tailed t-test or Pearson correlation test, *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Our data suggest that the development of autoantibody-secreting cells in SLC−/− mice can be explained by a lack of μH chain selection in combination with compromised central and peripheral B cell tolerance, B-lymphopenia, and excess survival factors. We show here that the proportion of λ+ iB cells in SLC−/− mice is reduced, which is consistent with an inability to induce receptor editing and an increased proportion of iB cells being deleted. Some of this reduction could also be due to accelerated development, which would explain the increase in λ+ transitional B cells. A lower proportion of λ+ iB cells has been observed also in mice with defective NF-κB signaling that affects the generation of λ+ but not κ+ iB cells or editing of the κ locus [37]. This is due to a survival defect in pre-B cells, resulting in greatly reduced Pim2 mRNA levels, and this defect can be overcome by overexpressing Bcl-2. A decrease in λ+ iB cells is also observed in mice in which the κRS element has been deleted (prevents Vκ-RS rearrangements), and in this model expression of a Bcl-2 transgene rescued κ+ rather than λ+ iB cells [28]. This would be consistent with κ+ iB cells being the majority of cells that undergo Vκ-RS rearrangements [10]. In SLC−/− mice, there is no apparent survival defect in pre-B cells with normal mRNA levels of Pim2 as well as Bim and Bclx. As the reduction in λ+ iB cells in SLC−/− mice is due to compromised receptor editing, we speculate that a Bcl-2 transgene would rescue mostly κ+ iB cells also in these, by analogy to the κRS deletion model.
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Figure 7. Serum IgM anti-dsDNA antibodies are reduced to background levels after depletion of MZ B cells in vivo. (A) Correlation of MZ B-cell numbers with serum IgM anti-dsDNA antibody levels in SLC−/− mice. Data are from Figures 4 and 6. (B to H) SLC−/− mice were treated with anti-α4 and LFA-1, or isotype control antibodies. (B) Flow cytometry analysis of peripheral blood. Plots show the proportions of MZ and FO B cells in the CD19+ 93− gate, 5 h after the first injection. Plots are representative of 4–6 mice per group. (C to E) Flow cytometry analysis of (C) peripheral blood and spleen at day 14, gated as in (B). (D and E) Cell numbers of indicated splenic B-cell populations and total lymphocytes (lymph). B1 B cells were gated as CD19hi 43+ . (F) Immunofluorescence staining of splenic cryosections. MOMA-1 (green), IgM (red), and IgD (blue). Total magnification 100×. Scale bar, 200 μm. Images are representative of four to six mice per group. (G) Serum IgM anti-dsDNA antibody levels before and after treatment as measured by ELISA. Dotted line indicates serum levels from a pool (n = 3) of 15-week-old control mice. (H) Total IgM antibody levels before and after treatment as measured by ELISA. (D and E) Data are from a single experiment representative of two independent experiments. (G and H) Each line represents a single comparison and data (G) have been pooled from two experiments and (H) are from a single experiment representative of two independent experiments. p Values were determined using a two-tailed or Pearson correlation test, t-test, or paired t-test. * p < 0.05; ** p < 0.01; *** p < 0.001. ns, not significant.
BM iB cells expressing high surface IgM levels are almost absent in SLC−/− mice. One explanation could be that these cells are downregulating their BCR in preparation for receptor editing and dedifferentiation. This also suggests that some of the pre-B cells are de facto iB cells although the proportion is unknown. Attempting to estimate this, based on our previous [26] and current data (Supporting Information Fig. 1A), would suggest that at least half of CD25+ pre-B cells express intracellular μH, but no L chains, hence are pre-B cells, and could indicate that as many as half of the pre-B cells are dedifferentiated iB cells. This also supports that pro-B cells do indeed differentiate into pre-B cells. Nevertheless, the actual proportion of pre-B cells that expresses autoreactive μH chains is unclear, although our data herein indicate that this could be higher than previously estimated [22]. Expression of an autoreactive μH chain may even be a requirement for pro-B- to pre-B-cell transition, as they would mimic the autoreactive characteristics of the λ5-tail [14–19]. RAG mRNA levels are greatly downregulated as Fr.D cells differentiate into Fr.E, with only a subset of cells still expressing these genes under normal circumstances [38]. As the levels are higher in Fr.E cells from SLC−/− mice compared to those in controls, it indicates that this subset is proportionally larger in SLC−/− mice, which would be consistent with cells preparing for receptor editing. Aiolos and Pax5 mRNA levels are lower in Fr.E cells from SLC−/− mice, although the significance of this is unclear. We find it interesting as reduced Pax5 levels have been observed in iB cells from NZB mice [39], a strain characterized by defective B-cell tolerance and autoantibody production, and as Aiolos deficiency has also been shown to lead to autoantibody production [40].
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Strong selection pressure on cells entering the FO B-cell pool is evident in SLC−/− mice, however, selection could still be compromised as the proportion of λ+ cells is still high. Nevertheless, selection into the MZ B-cell pool is abolished, and results in an increased frequency of autoreactivity among these cells and elevated levels of serum IgM anti-dsDNA antibodies. The survival of the MZ B cells in SLC−/− mice is likely due to the elevated BAFF levels, presumably as a result of the B-lymphopenia and reduced consumption. This would be consistent with the rescue of autoreactive cells and their entry into the MZ B-cell pool in mice overexpressing BAFF [33]. Our previous work suggested that the CD21− 23− B-cell population is responsible for production of IgG ANAs in SLC−/− mice [22], the idea being that this population would also give rise to plasma cells secreting antibodies recognizing dsDNA, although antibodies binding nuclear antigens do not necessarily bind DNA [41]. However, depletion of the MZ B-cell pool did not affect the CD21− 23− B cells (Fig. 7E) or ANA levels (unpublished observation), suggesting that two distinct B-cell populations are responsible for autoantibody production in SLC−/− mice, MZ B cells giving rise to IgM anti-dsDNA antibodies, and CD21− 23− cells to IgG ANAs. Pre-BCR-mediated negative selection of μH chains has been described also in humans [21]. However, as B-cell development is blocked in this patient, the (auto)antibody repertoire could not be analyzed [42]. Nevertheless, the pre-BCR has been implicated in autoimmune disease, in rheumatoid arthritis where low copy number variation of the SL chain component VPREB1 correlates with clinical parameters, for example, bone erosion [43].
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Materials and methods Mice The SLC−/− mice [27], previously backcrossed for more than ten generations on the C57BL6/OlaHsd background [22], were after embryo transfer bred onto the C57BL6/NCrl background with an intact alpha-synuclein locus [44]. Mice were kept in the Gothenburg University SPF animal facility. Control C57Bl/6NCrl mice were purchased from Charles River (Germany) and Taconic (Denmark). The original backcrossed SLC−/− mice, thawed from embryos frozen in 2004, were also used with SLC+/+ mice as controls, kept as a separate line with the same phenotype. Mice were bred under project license authorization (245-2009 and 39-2010), and genotyped by PCR for targeting of VpreB2 and VpreB1/λ5 [27]. Female mice were used throughout, aged 4–45 weeks.
Molecular immunology
In vitro cultures MZ B cells were sorted from splenocytes of 6- to 11-week-old control and SLC−/− mice. In total, 5 × 104 cells were cultured for 3 days in 96-well round-bottom plates in Iscove’s complete medium with 10% FBS. The cells were stimulated with LPS (10 mg/mL) or CpG (3 mM) [45].
ELISA Serum levels of BAFF were determined according to manufacturer’s instructions (R&D Systems). Absorbance was measured at 450 nm using a microplate reader Spectra Max340PC (Molecular Devices, US). The levels of IgM and IgG anti-dsDNA antibodies and levels of total IgM and IgG were determined as previously described [22, 46].
Flow cytometry and cell sorting ELISPOT Spleen and BM cell suspensions were stained with a cocktail of antibodies (Supporting Information Table 1) following standard techniques, and analyzed on a FACSCantoIITM or FACSVerseTM (BD Biosciences). Data were analyzed using FlowJo (Treestar, Inc.). BM Fr.D (B220+ CD93+ 43− IgM− ) and Fr.E (B220+ CD93+ 43− IgM+ ) cells were sorted from individual mice (8- to 12-week-old). λ+ (B220+ CD93+ IgM+ λ+ ) and κ+ (B220+ CD93+ IgM+ λ− ) iB cells were sorted from pools of five to six mice. Spleen cells were collected from pools of five to six mice (8- to 12-week-old) followed by sorting MZ (CD19+ 93− 43− 21hi 23low ), FO (CD19+ 93− 43− 21int 23+ ), and transitional (CD19+ 93+ 24hi ) B cells. Cells were sorted on a Synergy cell sorter (Sony Biotech) with purities >92% (Supporting Information Fig. 2).
96-Well plates with a nitrocellulose membrane (Millipore) were coated with poly-L-lysine (20 mg/mL) in Tris EDTA buffer for 2– 3 h in room temperature. Plates were washed (Tris EDTA) and then coated with dsDNA at 4°C overnight. After washing, cells were added and incubated at 37°C in a humidification chamber. After 3.5 h, cells were washed (water) and incubated with APconjugated goat anti-IgM (Southern biotech) at 4°C overnight. After washing, the spots were developed with NBT-BCIP (Sigma), then washed and dried. Each well was photographed using a USB camera with the software DinoXCope and analyzed with Photoshop.
Apoptosis assay
Treatment with antibodies recognizing α4 and LFA-1 integrins
After surface staining, to detect apoptotic cells single cell suspensions were incubated with zVAD-FMK-FITC (Casp-Glow, eBioscience) in RPMI 1640 for 1 h at 37°C, according to manufacturer’s protocol and subsequently analyzed by flow cytometry.
SLC−/− mice were injected i.p. with anti-α4 (clone: M17/4; 100 μg) and anti-LFA-1 (clone: PS/2; 100 μg) or isotype control antibodies (BioXcell, USA) at days 0 and 5, and sacrificed at day 14.
Analysis of transcription factors and Ig L chain loci Immunohistochemistry RNA was isolated using RNeasy kit (Qiagen) and reversely transcribed according to the protocol of SuperScript II (Invitrogen). Levels of RS rearrangements were quantified by qPCR, using primers and probes as described [10]. Power SYBR Green (Applied Biosystems) was used to detect transcripts and DNA rearrangements (κ and λ) with previously described primers (Supporting Information Table 1). All samples were run in triplicates on a ViiA7 system (Applied Biosystems). C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Spleens from SLC−/− mice were collected and embedded in OCT compound (TissueTek, Tokyo, Japan), snap-frozen in liquid nitrogen, and stored at −80°C. Frozen sections (8 μm) were cut using a cryostat (Wetzlar, Germany), and stained (Supporting Information Table 1) as previously described [47]. Images were acquired using an LSM 700 confocal microscope and ZEN 2009 acquisition software (Zeiss, Germany). www.eji-journal.eu
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Statistics
11 Hardy, R. R. and Hayakawa, K., B cell development pathways. Annu. Rev.
Data are shown as mean ± SEM in all the plots. p Values were calculated using appropriate statistical tests: unpaired t-test or paired t-test (GraphPad Prism 6.0).
12 Osmond, D. G., Rolink, A. and Melchers, F., Murine B lymphopoiesis:
Immunol. 2001. 19: 595–621.
towards a unified model. Immunol. Today 1998. 19: 65–68. 13 Parker, M. J., Licence, S., Erlandsson, L., Galler, G. R., Chakalova, L., Osborne, C. S., Morgan, G. et al., The pre-B-cell receptor induces silencing of VpreB and lambda5 transcription. EMBO J. 2005. 24: 3895–3905. 14 Conley, M. E. and Burrows, P. D., Plugging the leaky pre-B cell receptor. J. Immunol. 2010. 184: 1127–1129. 15 Almqvist, N. and Martensson, I. L., The pre-B cell receptor; selecting for
Acknowledgments: We thank Drs. S. Cardell, I. Gjertsson, and M. Sigvardsson for critical reading of the manuscript, Dr. D. Chen for assistance with injections, and Ms. L Bergqvist for cell sorting. This work was supported by Swedish Science Research Council, the Swedish Cancer Foundation, ALF: the regional agreement on medical training and clinical research, King Gustav V Stiftelse, Lundbergs Stiftelse, Swedish Medical Society, Reumatikerf¨ orbundet, Stiftelsen Lars Hiertas minne, Stiftelsen Sigurd och Elsa Goljes Minne, Lundgrens Stiftelse, Aml¨ ovs Stiftelser, Adlerbertska stiftelsen, The Royal Society of Arts and Sciences in Gothenburg, and Sigurd och Elsa Golje’s minne, AFA insurances.
or against autoreactivity. Scand. J. Immunol. 2012. 76: 256–262. 16 Vettermann, C. and Jack, H. M., The pre-B cell receptor: turning autoreactivity into self-defense. Trends Immunol. 2010. 31: 176–183. 17 von Boehmer, H. and Melchers, F., Checkpoints in lymphocyte development and autoimmune disease. Nat. Immunol. 2010. 11: 14–20. 18 Herzog, S. and Jumaa, H., Self-recognition and clonal selection: autoreactivity drives the generation of B cells. Curr. Opin. Immunol. 2012. 24: 166–172. 19 Tussiwand, R., Bosco, N., Ceredig, R. and Rolink, A. G., Tolerance checkpoints in B-cell development: Johnny B good. Eur. J. Immunol. 2009. 39: 2317–2324. 20 Melchers, F., The pre-B-cell receptor: selector of fitting immunoglobulin heavy chains for the B-cell repertoire. Nat. Rev. Immunol. 2005. 5: 578–584. 21 Minegishi, Y. and Conley, M. E., Negative selection at the pre-BCR checkpoint elicited by human mu heavy chains with unusual CDR3 regions.
Conflict of interest: The authors declare no financial or commercial conflicts of interest.
Immunity 2001. 14: 631–641. 22 Keenan, R. A., De Riva, A., Corleis, B., Hepburn, L., Licence, S., Winkler, T. H. and Martensson, I. L., Censoring of autoreactive B cell development by the pre-B cell receptor. Science 2008. 321: 696–699.
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Abbreviations: ANA: antinuclear antibody · BAFF: B lymphocyte activating factor · FO: follicular · Fr.D: fraction D · iB: immature B · MZ: marginal zone · Pre-BCR: precursor B-cell receptor · RS: recombining sequence · SL: surrogate light
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C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: 5/6/2014 Revised: 8/12/2014 Accepted: 23/12/2014
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