B-1a Cells, but Not Marginal Zone B Cells, Are Implicated in the Accumulation of Autoreactive Plasma Cells in Lyn^−/− Mice

Abstract

Mice deficient in Lyn, a tyrosine kinase that limits B cell activation, develop a lupus-like autoimmune disease characterized by the accumulation of splenic plasma cells and the production of autoantibodies. Lyn^−/− mice have reduced numbers of marginal zone (MZ) B cells, a B cell subset that is enriched in autoreactivity and prone to plasma cell differentiation. We hypothesized that this is due to unchecked terminal differentiation of this potentially pathogenic B cell subpopulation. However, impairing MZ B cell development in Lyn^−/− mice did not reduce plasma cell accumulation or autoantibodies, and preventing plasma cell differentiation did not restore MZ B cell numbers. Instead, Lyn^−/− mice accumulated B-1a cells when plasma cell differentiation was impaired. Similar to MZ B cells, B-1a cells tend to be polyreactive or weakly autoreactive and are primed for terminal differentiation. Our results implicate B-1a cells, but not MZ B cells, as contributors to the autoreactive plasma cell pool in Lyn^−/− mice.

Introduction

Pathogenic autoantibodies reactive with nucleic acid–containing Ags are a defining feature of the autoimmune disease systemic lupus erythematosus (SLE) (1). It is important to understand which B cell subsets are the source of these dangerous Abs to develop therapeutic approaches for SLE that spare protective B cell responses to infection and vaccination. Much attention has been paid to CD11c⁺T-bet⁺ B cells (age-associated B cells in mice and DN2 cells in humans), a pathogenic subset that is elevated in both murine and human lupus and produces autoantibodies in extrafollicular responses (2, 3). However, these cells are not the only source of autoantibodies in several lupus models (4–7), and they do not expand in all SLE patients (2). Similar to age-associated B cells/DN2 cells, the innate-like marginal zone (MZ) B cell and B-1a subsets are enriched in autoreactivity, highly sensitive to TLR engagement, and prone to differentiation into plasma cells (8–10). Whether these cells are pathogenic in lupus has been debated.

Under normal circumstances, MZ B cells provide rapid T-independent responses to blood-borne bacterial infections (10). Sequestration of autoreactive B cells to the MZ subset has been suggested as a means of maintaining tolerance by virtue of MZ B cells’ limited participation in T-dependent responses (11). However, in some lupus models MZ B cells are inappropriately localized to B cell follicles and are activated in a T-dependent manner (12, 13). In other lupus-prone strains, MZ B cells are dramatically reduced in number (14–17). This could imply that these cells are not involved in the disease. However, it is also possible that the loss of MZ B cells in these strains is due to excessive differentiation into plasma cells in response to autoantigen. Thus, there are several scenarios in which MZ B cells could contribute to the expansion of plasma cells and the production of autoantibodies in lupus.

B-1a cells have been suggested to have both protective and pathogenic roles in autoimmune disease. They produce natural Abs that protect against autoimmunity by clearing self-antigens and reducing inflammatory responses of innate immune cells (18, 19). However, they are increased in several murine lupus models (20–23), and many genetic manipulations that increase B-1a cell numbers also lead to lupus-like autoimmunity (24–29). B-1a cells can also express autoantibodies, including anti-dsDNA IgG (21, 30–32), and they facilitate activation of proinflammatory T cells (20, 21, 33–37).

Mice lacking the Src family kinase Lyn, which limits B and myeloid cell activation, develop lupus-like autoimmune disease (22, 23, 38–41). Polymorphisms or alterations in Lyn and its regulators are associated with SLE (42–46). Lyn-deficient B cells differentiate inappropriately into plasma cells due to reduced levels of Ets1, a transcription factor that normally prevents B cell terminal differentiation (47), and autoreactive plasma cells accumulate in the spleens of Lyn^−/− mice (22, 23, 38–41). We previously showed that T-bet–expressing B cells contribute to this autoreactive plasma cell pool (7). However, these were not the only source of plasma cells or autoantibodies in Lyn^−/− mice (7), suggesting a possible role for innate-like B cells. MZ B cells are almost absent in these animals due to a B cell–intrinsic effect of Lyn deficiency (16). They are also significantly decreased in Ets1-deficient mice (17). Because Lyn and Ets1 are components of a pathway that limits plasma cell formation (47), we hypothesized that the loss of MZ B cells in these mice is due to their unchecked differentiation into plasma cells, contributing to the production of autoantibodies. We show that although this is not the case, preventing terminal differentiation of Lyn^−/− B cells results in a significant accumulation of B-1a cells in both the spleen and peritoneal cavity.

Materials and Methods

Mice

Notch2^+/f mice (48) (The Jackson Laboratory, 010525) were crossed to mb1-cre mice (49) (The Jackson Laboratory, 020505). The resulting mb1-cre.Notch2^+/f mice were crossed to Lyn^−/− mice (22) to generate Lyn^−/− mice heterozygous for Notch2 in B cells. mb1-cre mice were crossed to IRF4^f/f mice (50) (The Jackson Laboratory, 009380) in an attempt to generate mice with a B cell–specific deletion of IRF4. However, the IRF4 locus was deleted frequently in the germline, generating IRF4^−/− mice (51). These were crossed to Lyn^−/− mice. Mice were analyzed at 4–6 mo of age. Mice were sex matched and littermate controls were used whenever possible. All mouse experiments were approved by the UT Southwestern Institutional Animal Care and Use Committee.

Flow cytometry

Single-cell suspensions of spleen and peritoneal wash were depleted of RBCs and stained extracellularly with combinations of the following Abs coupled to FITC, Alexa Fluor 488, PE, PerCP, allophycocyanin, Alexa Fluor 647, or biotin. Biotinylated Abs were detected with streptavidin-allophycocyanin (Tonbo Biosciences). For spleen, B220 (Invitrogen), CD19 (BD Pharmingen), CD21 (BD Pharmingen), CD23 (BioLegend), CD93 (Invitrogen), CD138 (BD Pharmingen), IgM (BD Pharmingen), CD5 (BD Pharmingen), and CD11b (Tonbo Biosciences) were used. For the peritoneal cavity, B220, CD19, IgM, CD5, and CD11b were used. Intracellular staining for Bim (Cell Signaling Technology) and IRF4 (Invitrogen) was performed using a Foxp3/transcription factor staining buffer kit (Tonbo Biosciences). Samples were run on a FACSCalibur (Becton Dickinson) and analyzed with FlowJo software (Tree Star).

MZ B cell purification

Pooled splenocytes from three to four mice per group were depleted of RBCs and enriched for CD23^lo/− B cells by sequential negative selection with anti-CD43 and then anti-CD23 magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions. CD23⁻ B cells were stained with Abs against B220 (Invitrogen) and CD21 (BD Pharmingen), and B220⁺CD21⁺ MZ B cells and B220⁺CD21⁻ B cells were sorted on a FACSAria.

Quantitative PCR

Cells were lysed in TRIzol (Life Technologies) and total RNA was prepared using an RNeasy mini kit (Qiagen). cDNA was subsequently generated with a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). TaqMan reagents (Thermo Fisher Scientific) for Prdm1, Dtx1, and the internal control GAPDH were used to perform real-time quantitative PCR (qPCR) with a Bio-Rad CFX96 real-time system. Results were normalized to GAPDH using the ΔCt method.

ELISAs

For anti-ssDNA and anti-dsDNA, 1:100, 1:400, and 1:1600 dilutions of serum were subjected to anti-ssDNA and anti-dsDNA IgM and IgG ELISA as described in Mayeux et al. (52). For total Ig, 1:1,000, 1:4,000, and 1:16,000 dilutions of serum were subjected to total IgM and IgG ELISA as described in (52).

Statistical analysis

Statistical analysis was performed with GraphPad Prism. Normality was assessed using the Shapiro–Wilk test. A Student t test or one-way ANOVA was used for normally distributed groups, and Mann–Whitney or Kruskal–Wallis tests were used for nonnormally distributed groups. A p value <0.05 was considered significant.

Results

MZ B cells do not contribute to plasma cell accumulation or autoantibody production in Lyn^−/− mice

Because there is no reporter system to track MZ B cell progeny, we used several other complementary approaches to determine the degree to which this population contributes to autoantibodies in Lyn^−/− mice. We first asked whether reduced Lyn expression results in increased levels of the master plasma cell transcription factor, Blimp1, in MZ B cells. Because Lyn^−/− mice have very small numbers of MZ B cells, we used Lyn^+/− mice to obtain sufficient cell numbers. These mice have reduced MZ B cells, increased plasma cells, and increased IgM autoantibodies compared with wild-type (wt) mice, but this phenotype is milder than that of Lyn^−/− mice (39, 53). Levels of PRDM1 mRNA, which encodes Blimp, were normal in Lyn^+/− MZ B cells as measured by qPCR (Fig. 1A). To query the small number of remaining MZ B cells in Lyn^−/− mice, we performed flow cytometry for IRF4, another transcription factor required for plasma cell development (50). There was no difference in IRF4 expression among wt, Lyn^+/−, or Lyn^−/− MZ B cells (Fig. 1B). MZ B cells were similar in size among all three genotypes, suggesting that these cells are not more activated in the context of reduced Lyn dosage (Supplemental Fig. 1A). Taken together, these data indicate that Lyn^+/− and Lyn^−/− MZ B cells are not undergoing excessive differentiation into plasma cells.

FIGURE 1.

Reduced Notch2 signaling does not prevent plasma cell accumulation or autoantibody production in Lyn^−/− mice.

(A) Purified MZ B cells from wt and Lyn^+/− mice were subjected to qPCR for Prdm1. Results were normalized to GADPH using the ΔCt method. Each symbol represents a pool of three to four mice; the bar indicates the mean. (B) Splenocytes from wt (solid line/open histogram), Lyn^+/− (filled histogram), and Lyn^−/− mice (dotted line/open histogram) were stained with Abs against B220, CD21, CD23, and isotype control (black) or IRF4 (blue). Representative histograms for IRF4 expression in MZ B cells (gated as in D) are shown and IRF4 MFI is quantified as mean ± SD; n = 3. (C and D) Splenocytes from mb1-cre.Lyn^+/−, mb1-cre.Lyn^+/−.Notch2^+/f, mb1-cre.Lyn^−/−, and mb1-cre.Lyn^−/−.Notch2^+/f mice were stained with Abs against B220, CD21, and CD23. The total number of MZ B cells (CD21⁺CD23^lo/⁻) is shown in (C) as mean ± SD; n = 3–4. ****p < 0.0001 by one-way ANOVA. Representative FACS plots are shown in (D), with gating on B220⁺ cells. (E) Anti-dsDNA IgM and IgG levels in a 1:100 dilution of serum from mb1-cre, mb1-cre.Lyn^+/−, and mb1-cre.Lyn^+/−.Notch2^+/f mice. Each symbol represents an individual mouse; the bar indicates the mean. *p < 0.05 by Kruskal–Wallis test. ns, not significant. (F and G) Anti-dsDNA (F) and total (G) IgM and IgG levels in a 1:400 (F) or 1:1000 (G) dilution of serum from mb1-cre, mb1-cre.Lyn^−/−, and mb1-cre.Lyn^−/−.Notch2^+/f mice. Each symbol represents an individual mouse; the bar indicates the mean. *p < 0.05 by one-way ANOVA (F) or Kruskal-Wallis test (G). ns, not significant. (H and I) Splenocytes from mb1-cre, mb1-cre.Lyn^−/−, and mb1-cre.Lyn^−/−.Notch2^+/f mice were stained with Abs against B220 and CD138. The frequency of B220^loCD138⁺ cells (gated as in H) among splenocytes is indicated in (I). Each symbol represents an individual mouse; the bar indicates the mean. **p < 0.01 by one-way ANOVA. ns, not significant.

If MZ B cells are a source of autoantibodies, then impairing their development should reduce autoantibody levels. MZ B development (54) and subsequent “priming” for efficient plasma cell differentiation (55) require high levels of Notch2 signaling. Mice with a B cell–specific heterozygous mutation in Notch2 (mb1-cre.Notch2^+/f mice) have a significant reduction in MZ B cells (54). MZ B cell Notch2 activity appeared normal in Lyn^+/− mice as measured by qPCR for the Notch2 target Dtx1 (Supplemental Fig. 1B). We reasoned that reducing Notch2 signaling genetically would impair MZ B cell development and priming for plasma cell differentiation in Lyn^+/− and Lyn^−/− mice. This was achieved by crossing Lyn^+/− and Lyn^−/− mice to mb1-cre.Notch2^+/f mice. As expected, MZ B cell numbers were significantly reduced in mb1-cre.Lyn^+/−.Notch2^+/f mice (Fig. 1C, 1D) relative to mb1-cre.Lyn^+/− mice. Because MZ B cells are nearly absent to begin with in mice with complete Lyn deficiency, we did not observe a further reduction in this population in mb1-cre.Lyn^−/−.Notch2^+/f mice compared with mb1-cre.Lyn^−/− mice (Fig. 1C, 1D). However, although we hypothesize that Lyn^−/− MZ B cells may be lost due to inappropriate differentiation into plasma cells, Lyn^−/− MZ B cells with impaired Notch2 signaling should not develop in the first place, preventing the production of MZ B cell–derived Abs. Although it has been reported that Notch2 haploinsufficiency also reduces B-1a cells (56), this was not observed in our hands (Supplemental Fig. 1C).

Despite the dramatic reduction in MZ B cells, IgM autoantibodies were not decreased in Lyn^+/− mice (Fig. 1E, Supplemental Fig. 1D). Lyn^+/− mice did not produce IgG autoantibodies, consistent with our previous report (39) (Fig. 1E, Supplemental Fig. 1D). Plasma cell accumulation and autoantibody production were also unimpaired in mb1-cre.Notch2^+/f.Lyn^−/− mice, as anti-DNA IgM and IgG Abs (Fig. 1F), total IgM Abs (Fig. 1G), and splenic plasma cells (Fig. 1H, 1I) remained elevated these mice. Thus, impairing MZ B cell development did not prevent plasma cell accumulation or autoantibody production. However, these experiments do not rule out a contribution of B-1a cells, as they were unaffected by Notch2 haploinsufficiency in our hands.

As a third approach, we reasoned that if the loss of MZ B cells in Lyn^−/− mice is due to increased transition to the plasma cell subset, then preventing plasma cell differentiation should allow MZ B cells to accumulate. We attempted to generate Lyn^−/− mice with a B cell–specific deletion of IRF4, as IRF4 is required for plasma cell differentiation (50) and also limits MZ B cell numbers (57). However, we found that mb1-cre frequently deleted the floxed IRF4 locus in the germline (51), so we instead employed Lyn^−/−IRF4^−/− mice. MZ B cell numbers remained low in Lyn^−/−IRF4^−/− mice (Fig. 2A, 2B), indicating that their loss in Lyn^−/− mice is not due to accelerated plasma cell differentiation.

FIGURE 2.

IRF4 deficiency does not rescue MZ B cell numbers in Lyn^−/− mice.

(A and B) Splenocytes from wt, Lyn^−/−, and Lyn^−/−IRF4^−/− mice were stained with Abs against B220, CD21, and CD23 and B220⁺ cells analyzed for follicular (CD23⁺CD21⁺) (FO) and MZ (CD21^hiCD23^lo/−) cells as in (A). Total numbers are shown in (B) as mean ± SEM; n = 6–8. ****p < 0.0001 by unpaired Student t test. ns, not significant by Mann–Whitney U test. (C) Splenocytes from wt (solid line), Lyn^−/− (light gray filled), and Lyn^−/−IRF4^−/− (dotted line) mice were stained with Abs against B220, CD23, and Bim. Bim levels in B220⁺CD23⁺ are shown. Results are representative of two independent experiments. (D and E) Splenocytes from wt, Lyn^−/−, and Lyn^−/−IRF4^−/− mice were stained with Abs against B220, CD93/AA4.1, and CD23 and gated as in (D). Total numbers of T1 (B220⁺CD93⁺CD23⁻) and T2/T3 (B220⁺CD93⁺CD23⁺) cells are shown in (E) as mean ± SEM; n = 7–8. ***p < 0.001 by unpaired Student t test. ns, not significant.

Taken together, these results suggest that the reduction in MZ B cells in Lyn^−/− mice is not due to either excessive plasma cell differentiation or impaired Notch2 signaling, and that MZ B cells are not a major contributor to the accumulation of plasma cells and autoantibodies in these animals.

Reduced follicular B cells in Lyn^−/−IRF4^−/− mice

Whereas MZ B cell numbers were unchanged in Lyn^−/−IRF4^−/− mice, a number of other B cell populations were significantly affected. Follicular B cells (CD23⁺CD21⁺), already low in Lyn^−/− mice, were further reduced by IRF4 deficiency (Fig. 2A, 2B). Lyn^−/− follicular B cells have reduced survival due to increased expression of the proapoptotic molecule Bim (58). IRF4 has been reported to repress Bim in multiple myeloma cells (59), so we asked whether the further loss of follicular B cells in Lyn^−/−IRF4^−/− mice could be explained by a further increase in Bim expression as a result of IRF4 deficiency. Bim expression was not affected by loss of IRF4, however (Fig. 2C). We next asked whether the loss of follicular B cells in Lyn^−/−IRF4^−/− mice could result from a block at earlier stages of B cell maturation. New emigrants from the bone marrow, T1 cells, have a B220⁺CD93⁺CD23⁻ phenotype. These were reduced in Lyn^−/− mice as previously described (58), but not further affected by loss of IRF4 (Fig. 2D, 2E). However, Lyn^−/−IRF4^−/− mice did have a reduction in B220⁺CD93⁺CD23⁺ T2/T3 cells compared with Lyn^−/− mice (Fig. 2D, 2E), consistent with the known skewing of IRF4^−/− transitional B cells toward T1 cells (51). The ratio of follicular to T2/T3 B cells was not significantly different (p = 0.2457) between Lyn^−/− (6.0 ± 2.8) and Lyn^−/−IRF4^−/− (8.6 ± 4.8) mice, indicating no further effect of IRF4 deficiency on the T2/T3 to follicular B cell transition. Thus, the reduction in follicular B cells in Lyn^−/−IRF4^−/− mice is likely due to the combined effects of Lyn deficiency on T1 cells and IRF4 deficiency on T2/T3 cells.

Accumulation of B-1a cells in Lyn^−/−IRF4^−/− mice

Despite the significant loss of follicular B cells in Lyn^−/−IRF4^−/− mice, the total number of splenic CD19⁺ B cells did not differ between Lyn^−/− and Lyn^−/−IRF4^−/− mice (Fig. 3A, 3B). This was due to the expansion of a population of CD19^hiB220⁻ cells that expressed CD43 and intermediate levels of CD21 (Fig. 3A, 3B). These cells also have an IgM^hiCD23⁻CD5⁺CD11b⁺ phenotype, characteristic of B-1a cells (Fig. 3C). The frequency and number of B-1a cells were also significantly increased in the peritoneal cavity of Lyn^−/−IRF4^−/− mice, where this cell population usually resides (Fig. 4A–C). Although IRF4-deficient mice have impaired plasma cell differentiation (50), it has been reported that in some contexts B-1a cells can secrete Abs in a manner independent of Blimp1 and IRF4 (60, 61). This suggested that the accumulating B-1a cells in Lyn^−/−IRF4^−/− mice may still be able to produce Ig, allowing for straightforward analysis of their autoreactivity. However, we were unable to detect IgM, IgG, or IgA in the serum of Lyn^−/−IRF4^−/− mice (Fig. 4D).

FIGURE 3.

B-1a cells accumulate in the spleens of Lyn^−/−IRF4^−/− mice.

Splenocytes from wt, Lyn^−/−, and Lyn^−/−IRF4^−/− mice were stained with combinations of Abs against B220, CD19, CD43, CD21, CD23, IgM, CD5, and CD11b. (A and B) Representative FACS plots of lymphocyte-gated cells are shown in (A) and quantified in (B). (B) The percentage (left) and total number (right) of CD19⁺, CD19⁺CD43⁺CD23⁻, and CD19^hiCD21^int cells are shown. Each symbol represents a mouse; the bar indicates the mean. *p < 0.05, ****p < 0.0001 by one-way ANOVA; ^#p < 0.05, ^##p < 0.01 by Kruskal–Wallis test. (C) Levels of CD23, IgM, CD5, and CD11b (open histogram) compared with fluorescence minus one control (light gray filled histogram) are shown for CD19^hiCD21^int cells from Lyn^−/−IRF4^−/− mice (gated as in A). Data are representative of five mice.

FIGURE 4.

B-1a cells accumulate in the peritoneal cavity of Lyn^−/−IRF4^−/− mice.

(A–C) Peritoneal wash cells from wt, Lyn^−/−, and Lyn^−/− IRF4 mice were stained with Abs against CD19, IgM, and CD5 or CD11b. (A) A representative dot plot, gated on lymphocytes, is shown for CD19 versus CD5. (B) Frequency and total number of CD19⁺CD5⁺ peritoneal cells, gated as in (A). Each symbol represents an individual mouse; the bar indicates the mean. *p < 0.05 by Kruskal–Wallis test; **p < 0.01 by one-way ANOVA. ns, not significant. (C) Staining for CD11b and IgM is shown on CD19⁺ peritoneal lymphocytes from a representative Lyn^−/−IRF4^−/− mouse. (D) Total IgM, IgG, and IgA ELISAs were performed with the indicated dilutions of serum from Lyn^−/− (open symbols, dotted line) and Lyn^−/−IRF4^−/− mice (filled symbols, solid line); n = 3.

Discussion

The contribution of MZ B cells to autoantibody production has been debated, in part because some lupus models have an expanded MZ B cell population whereas others, including Lyn^−/− mice, lack these cells. We reasoned that the loss of MZ B cells in the latter case may be due their inappropriate differentiation into plasma cells in response to self-antigen. However, three lines of evidence suggest that this is not the case, at least for Lyn^−/− mice. First, a reduced dosage of Lyn did not increase expression of the plasma cell transcription factors Prdm1 or IRF4 in MZ B cells. Second, impairment of MZ B cell development did not impair autoantibody production or prevent the increase in total IgM in Lyn^−/− mice. Finally, impairment of plasma cell differentiation did not allow Lyn^−/− MZ B cells to accumulate. Although our data suggest that MZ B cells are not a major source of accumulating plasma cells or autoantibodies in Lyn^−/− mice, they may still contribute to or protect from autoimmune pathology via Ab-independent mechanisms such as Ag presentation or cytokine secretion (62).

Our results also imply that impaired Notch2 signaling does not underlie the reduction in MZ B cells. In addition to impairing plasma cell differentiation (50), IRF4 deficiency results in increased Notch2 signaling in B cells (57). However, this was not sufficient to restore MZ B cell numbers in Lyn^−/− mice. Furthermore, Lyn^+/− MZ B cells had normal expression of the Notch2 target Dtx1. Elevated BCR signaling (41) may instead be responsible for the decrease in Lyn^−/− MZ B cells, as has been suggested by an inverse correlation between BCR signal strength and MZ frequency in several knockouts of signaling molecules (63).

In contrast to the lack of an effect on MZ B cells, IRF4 deficiency resulted in a significant increase in B-1a cells in Lyn^−/− mice. Although we cannot rule out that this is due to a role for IRF4 distinct from its effect on plasma cell differentiation, these results support a scenario in which inappropriate terminal differentiation of B-1a cells may contribute to autoimmunity in Lyn^−/− mice. Other lines of evidence provide further support for this model. Similar to MZ B cells, B-1a cells are enriched in autoreactivity, prone to differentiation into Ab-secreting cells, and sensitive to TLR engagement (8, 9). We have previously shown that a reduced dosage of the tyrosine kinase Btk both prevents autoimmunity and reduces B-1a cell numbers in Lyn^−/− mice (64). Lyn has also been shown to limit BCR-induced proliferation and Ca²⁺ flux in B-1a cells (65, 66). Finally, Lyn deficiency exacerbates disease in a BCR transgenic model of autoimmune hemolytic anemia in which B-1a cells are the source of pathogenic autoantibodies (67).

The accumulation of CD21^loCD23⁻ cells with a B-1a phenotype in the spleens of Lyn^−/−IRF4^−/− mice is reminiscent of that observed in mice lacking both PTEN and Blimp-1 in B cells (68). PTEN counteracts signaling by PI3K by dephosphorylating its product PIP3, and the PI3K pathway is inappropriately activated in its absence. Blimp-1 is required for plasma cell differentiation. It is thought that Blimp-1–mediated terminal differentiation normally limits the expansion of autoreactive B-1a cells whose activation is driven by increased PI3K activity (68). Loss of the anergy-associated genes Egr2 and Egr3 also drives the expansion of a splenic B-1a population with a similar phenotype (69). Similar mechanisms may be at work in Lyn^−/−IRF4^−/− mice. Lyn limits PI3K signaling by promoting the activation of SHIP (41), another inositol phosphatase that targets PIP3 and maintains anergy under normal circumstances (70). IRF4 is upstream of Blimp-1 in the transcription factor network that initiates plasma cell differentiation (71). Thus, loss of Lyn-mediated inhibition of PI3K signaling may promote a breach in tolerance and the inappropriate activation of B-1a cells. With further loss of IRF4, these cells accumulate due to their failure to differentiate into plasma cells. Increased Notch2 signaling in the absence of IRF4 may also contribute to the expansion of B-1a cells in Lyn^−/−IRF4^−/− mice (72), although Lyn^−/− B-1a cells do not seem particularly sensitive to changes in Notch2 dosage, as they are unchanged with Notch2 heterozygosity.

Taken together, our results suggest a possible role for B-1a cells, but not MZ B cells, in the autoimmune phenotype of Lyn^−/− mice. However, because B-1a cells have been suggested to have both protective and pathogenic roles in autoimmune disease, further study of this population is warranted. It will be interesting to determine the degree of autoreactivity of the accumulating Lyn^−/−IRF4^−/− B-1a cells using BCR expression cloning. Furthermore, the recent development of mouse strains that target cre to B-1 cells (73) and allow for cre-driven tagging of Ab molecules (74) will facilitate future studies of B-1 cell contribution to lupus pathogenesis.

Disclosures

The authors have no financial conflicts of interest.