Gardiquimod

The following primers were used for quantitative PCR (Eurofins MWG Operon): Actb (5′-ctaaggccaaccgtgaaaag, 5′-accagaggcatacagggaca). Tnf (5′-tcttctcattcctgcttgtgg 5′-ggtctgggccatagaactga), Il6 (5′-gctaccaaactggatataatcagga, 5′-ccaggtagctatggtactccagaa), Il12 (5′-ccatcagcagatcattctagacaa, 5′-cgccattatgattcagagactg), and Bmx (5′-gagcagcttcgcttcacc, 5′-gatttactctccatattgtcgtcca). The following compounds were used: CC-292(23 (link)) and Compound1. The following antibodies were used: Trem2(24 (link)), pY-100 (Cell Signaling Technologies, 9411), Mapk1/3 (Cell Signaling Technologies, 4695), pMapk1/3 (Cell Signaling Technologies, 4377), PT66 (Sigma, P3300), 4G10 (Millipore, 05-321), Tec (Millipore, 05-551), Bmx (BD Biosciences, 610792), Btk (BD Biosciences, 558528), and IRDye (LI-COR, 800CW and 680RD). The following additives and TLR agonists were used: Lipopolysaccharide (List Biological Labs, 434), CpG DNA (Invitrogen, tlrl-1826), Pam3CSK4 (Invitrogen, tlrl-pms), Gardiquimod (Invitrogen, tlrl-gdgs) and Polymyxin B (Sigma, P4932).

B cells were purified from the peripheral blood by positive selection using CD20 magnetic beads (Miltenyi Biotec) and plated for 48 hours at 150,000-200,000 cells per well in a 96-well plate in RPMI containing 10% FBS alone or RPMI+10% FBS and either 2 μg/ml polyclonal F(ab')2 rabbit anti-human IgM (Jackson ImmunoResearch, West Grove, PA), multimeric, soluble, 0.05 μg/ml recombinant-human CD40L (Alexis Biochemicals, San Diego, CA), 2 μg/ml gardiquimod (TLR7 agonist; Invitrogen, Waltham, MA), or 0.5 μg/ml CpG (TLR9 agonist; Invitrogen).