Murine lupus susceptibility locus Sle2 activates DNA-reactive B cells through two sub-loci with distinct phenotypes

Abstract

The NZM2410-derived Sle2 lupus susceptibility locus induces an abnormal B-cell differentiation, which most prominently leads to the expansion of autoreactive B1a cells. We have mapped the expansion of B1a cells to three Sle2 sub-loci, Sle2a, Sle2b and Sle2c. Sle2 also enhances the breach of B-cell tolerance to nuclear antigens in the 56R anti-DNA immunoglobulin transgenic (Tg) model. This study used the Sle2 sub-congenic strains to map the activation of 56R Tg B cells. Sle2c strongly sustained the breach of tolerance and the activation of anti-DNA B cells. The production of Tg-encoded anti-DNA antibodies was more modest in Sle2a-expressing mice, but Sle2a was responsible for the recruitment for Tg B cells to the marginal zone, a phenotype that has been found for 56R Tg B cells in mice expressing the whole Sle2 interval. In addition, Sle2a promoted the production of endogenously encoded anti-DNA antibodies. Overall, this study showed that at least two Sle2 genes are involved in the activation of anti-DNA B cells, and excluded more than two-thirds of the Sle2 interval from contributing to this phenotype. This constitutes an important step toward the identification of novel genes that have a critical role in B-cell tolerance.

FIGURE 1

Genetic map of the Sle2 congenic strains used in this study. The location of the markers defining the termini of each recombinant is indicated on the top. The NZB/NZW derivation of the region is also shown. NZM2410 (NZW or NZB)-derived intervals are indicated by solid boxes, with the area of recombination between the NZM2410 and B6 genomes indicated by lines on each side. The location of each marker corresponds to the NCBIM37 built. For SNPs located within a known gene, this gene is indicated in parenthesis.

FIGURE 2

Two Sle2 sub-loci activate 56R B cells to produce anti-DNA Ab. Serum anti-ssDNA (A–C) and anti-dsDNA (D–F) IgM, with the left panels showing total IgM, the middle panels 56R Tg-encoded IgM^a, and the right panels endogenous IgM^b. G–F. Splenic anti-ssDNA IgM^a and IgM^b AFCs. Each data point represents one mouse.

FIGURE 3

In vitro Ab production in the B6.Sle2.56R congenic strains. Total Ig and anti-ssDNA IgM^a or IgM^b were measured in the supernatant of splenocytes cultured for 5 d without (A–B) or with 5 ug/ml LPS (C–G). Total IgM^a (A) and IgM^b (B) in the supernatant of unstimulated cells. Total IgM^a (C), IgM^b (D) and IgG (E) in the supernatant of LPS-stimulated cells. Anti-ssDNA IgM^a (F) and IgM^b (G) in the supernatant of LPS-stimulated cells. Graphs show means and SEM of 4–6 mice per strain.

FIGURE 4

Flow cytometric analysis of B cells in the B6.Sle2.56R congenic strains. A–B. PerC B1a/B2 cell ratios with B1a cells defined as B220^int CD5⁺ and B2 cells defined as B220^hi CD5⁻. The graph in A shows the ratios for cells gated on IgM^a and the graph in B for the cells gated on IgM^b. C–I. Analysis of splenic B cells. Percentage of total B220⁺ cells (C) and IgM^a+ B220⁺ cells (D). E. Total CD21^hi CD23⁻ MZB cells expressed as the percentage of AA4.1⁻ B220⁺ mature B cells. F. Tg MZB cells expressed as the percentage of IgM^a+ B220⁺ cells. G. Percentage of large (FSC^hi) IgM^a+ B220⁺ B cells. H. Class II MHC I-a^b expression, measured as geometric mean fluorescence intensity (mfi) on IgM^a+ B cells. I. Percentage of CD86⁺ IgM^a+ B cells.

FIGURE 5

Sle2a promotes the expansion of MZB cells in 56R Tg mice. A. Representative follicles from B6.56R, B6.Sle2a.56R, and B6.Sle2c.56R spleen sections. B220-PB (blue) and CD1d-PE (red) identify B220⁺ CD1d^hi/+ magenta MZB cells and B220⁺ CD1d^lo/⁻ blue FOB cells, and their location relative to Moma-1-FITC (green)-stained metallophilic macrophages that separate the MZ from the FO zones. Note the intensity of CD1d expression in the B6.Sle2a.56R specimen. B. Proportion of B220⁺ B cells located in the MZ outside of the Moma-1⁺ ring. C. Proportion of B220⁺ B cells that expressed CD1d^hi/+ regardless of their location. D. Proportion of B220⁺ B cells in the MZ that are CD1d^hi/+. E. Proportion of B220⁺ B cells in the MZ that are CD1d^lo/−. Each data point corresponds to a representative follicle from one mouse from the indicated strains.