Fig. 3
Innate stimuli expand and activate dual-κ B cells. a Relative expression of genes in dual-κ and single-κ FO B cells in the Toll-like Receptor (TLR, left; differential expression of 21 out of 76 genes) or Interferon (IFN, right; differential expression of 14 out of 36 genes) signaling pathways identified in the RNAseq data described in Fig. 2 (FDR ≤ 0.1). The heat maps colors are explained in Fig. 2a. Data are from three independent biological replicates analyzed in one experiment. b, c Mean frequency of dual-κ B cells (b) and fold change of CD69 expression (MFI) on dual-κ relative to single-κ cells (c) within CD43– B cells cultured for 48 h in media or with CpG for TLR9, R848 for TLR7, LPS for TLR4. Gating strategy for this analysis and subtraction of background events are described in Supplementary Fig. 3a, b. Data are combined from two independent experiments using 14–15-wk-old MRL/lpr-Igkm/h mice (N = 7 total). Data in c are not significant. d, e Untouched (CD43–) B cells were stimulated for 24 h with IFNα or IFNγ. Bar graphs show MFI of CD69 on dual-κ cells as a fold change relative to amounts on single-κ cells (d), or the mean frequency of dual-κ B cells (e), within the CD19+Igκ+ cell population gated as in Supplementary Fig. 3a. Data are combined from two independent experiments (N = 6 total) using 12-wk-old MRL/lpr-Igkm/h mice. f, g Untouched naive B cells (magnetic selection of CD43–CD80–CD86– cells, Supplementary Fig. 4c) were stimulated for 48 h with CpG, R848, or IFNα. Shown are the mean frequencies of dual-κ B cells (f) or the MFI of CD69 (g) within the CD19+Igκ+ cells gated as in Supplementary Fig. 3a. Data were obtained using 12-wk-old MRL/lpr-Igkm/h mice (N = 4) in one experiment. In panels (c, d–g), the CD69 MFI values of cells cultured in media were subtracted from those in stimulated samples. *P < 0.05, **P < 0.01, ***P < 0.001; n.s. not significant. Significance was assessed by Student’s t- or Mann–Whitney tests. In all bar graphs the standard error bars represent SEM and each symbol an individual mouse
