Cpg oligodeoxynucleotide odn 2006

TLR7, TLR8 and TLR9 reporter gene assays were performed in HEK293 cells as described previously.23 The TLR7/TLR8 agonist, R-848, or the TLR9 agonist, CpG oligodeoxynucleotide (ODN) 2006 (Invivogen), were used as positive controls.

1 × 10⁵ CD27⁺ or CD27⁻ B-cells were incubated with agonistic anti-CD40 mAb (1 μg/ml, CP-870,893; kindly provided by Pfizer, New London, CT), the dsRNA complex polyinosinic:polycytidylic acid (10 μg/ml, poly(I:C); Sigma), lipopolysaccharide (10 μg/ml, LPS; Sigma), resiquimod (10 μg/ml, R848; Sigma) or CpG oligodeoxynucleotide (ODN) 2006 (1 μg/ml, InvivoGen, San Diego, CA). After 48 hours, cells were washed in complete medium and were then cultured in complete medium containing agonistic anti-CD95 mAb (2 μg/ml, CH11; MBL International, Woburn, MA) or human rTRAIL (1 μg/ml; R&D Systems, Minneapolis, MN) for an additional 18 hours. In confirmatory experiments, an alternative anti-CD95 mAb (SM1/1, eBioscience, San Diego, CA) or a sFasL (R&D Systems, Minneapolis, MN) incubated with anti-His Tag (R&D Systems, Minneapolis, MN) to allow cross-linking of Fas receptors. Cells were then washed in PBS and resuspended in Annexin-V binding buffer with Annexin-V-FITC and propidium iodide (PI; BioLegend, San Diego, CA). The apoptosis induced by adding agonistic anti-CD95 mAb or rTRAIL was defined as the change in % Annexin-V⁺ and calculated by subtracting the value for percentage of Annexin-V-positive cells in culture medium alone (background apoptosis) from the value for percentage of apoptosis in a replicate culture containing agonist anti-Fas mAb or rTRAIL.