TLR9 tolerance in pDCs. (a) Repeated treatment with CpG ODN2216 and systemic lupus erythematosus (SLE) serum reduced interferon (IFN)-α production. Plasmacytoid dendritic cells (pDCs) were purified from peripheral blood mononuclear cells (PBMCs) of healthy individuals using Diamond Plasmacytoid Dendritic Cell Isolation Kit (Miltenyi Biotec) and 2 × 104 pDCs were incubated with or without CpG ODN2216 or 30% SLE serum. After 24 hours pDCs were carefully washed in serum-free medium and then incubated again with or without CpG ODN2216 or 30% SLE serum. After 24 hours, IFN-α production was measured using ELISA. The experiments were performed in duplicate and three independent experiments were performed using PBMCs from different individuals. The data are presented in (Additional file 1 [Supplementary Figure 4]) and the averages of data are shown. (b) Experimental design to investigate the time-dependent recovery of Toll-like receptor (TLR)9 sensitivity. pDCs were purified from total PBMCs of healthy individuals, and 2 × 104 pDCs were treated with or without CpG ODN2216 or SLE sera for 1 day. The pDCs were then washed with serum-free medium and re-treated with or without CpG ODN2216 or SLE sera for 0 hours, 24 hours, and 48 hours. After 24 hours of treatment, IFN-α production was measured using ELISA. The white and black arrows represent washing and treatment with CpG ODN2216 or 30% SLE sera, respectively. The shaded areas indicate cultures undergoing stimulation. TLR9 tolerance is reversible over time. Purified pDCs were retreated with (c) CpG ODN2216 and (d) 30% SLE serum, as shown in panel b. IFN-α production and cell viability were measured 24 hours after the final stimulation. Each group was duplicated in every experiment and the values shown are the averages of duplicate samples. Three independent experiments were performed using PBMCs from different individuals. One representative case is shown here and the other data are shown in Additional file 1 (Supplementary Figure 5).