Tlrl r848

The ability of generated ICs to enhance monocyte responses was evaluated in an in vitro innate immune training model [43 (link)]. PBMCs were isolated, and CD14⁺ monocytes were negatively selected and trained as described elsewhere [33 (link),34 (link)]. RPMI 1640 medium (Gibco, #A1049101, Cincinnati, OH, USA) and 100 µg/mL of whole glucan particle (WGP) (InvivoGen, #tlrl-wgp, San Diego, CA, USA) were applied as the experiment negative and positive controls, respectively. The training was done with preF protein only (50 µg/mL), bIgG only (10 µg/mL), and their corresponding immune complexes (ICs) comprised of bIgG: preF as described earlier. In addition, α-bIgG only (5 µg/mL), bIgG only (3 µg/mL), and bIgG: α-bIgG immune complexes (ICs) were also used separately as the training compounds. After training and resting, the cells were stimulated with either 10 pg/mL of LPS (TLR4 ligand) (Sigma-Aldrich, #L2880, St. Louis, MO, USA) or 5 ng/mL of R848 (TLR7/8 ligand) (InvivoGen, #tlrl-r848, San Diego, CA, USA). Cytometric bead array (CBA) and individual cytokines Flex Sets were used for measuring IL-6 (BD, #558276, Franklin Lakes, NJ, USA) and TNF-α (BD, #558273, Franklin Lakes, NJ, USA) in the culture supernatant of the cells (supplementary Figure S3). The experiments were performed with the PBMCs isolated from 7–10 donors.

Spleen cells were stained with a cocktail of biotinylated Abs for CD4, CD8, CD43, CD49b, Ter119, and Streptavidin Particles Plus DM, from which naive B cells were purified by magnetic negative sorting using the IMag system (BD Biosciences) and MACS system (Miltenyi Biotec), as described previously (Nojima et al., 2011 (link)). B cells were cultured in RPMI-1640 medium (Wako) supplemented with 10% heat-inactivated fetal bovine serum, 10 mM HEPES pH 7.5, 1 mM sodium pyruvate, 50 mM 2-mercaptoethanol, 100 U/ml penicillin, and 100 mg/ml streptomycin (GIBCO). Typically, B cells were cultured at 2 × 10⁵ /ml in the presence of the following stimuli at the indicated doses, unless otherwise noted: NP₄₆-Ficoll (10 ng/ml), NP₄₀-CGG (10 ng/ml), LPS (1 µg/ml, L2880; Sigma), R-848 (1 µg/ml, tlrl-r848; InvivoGen), CpG ODN 1826 (1 µg/ml, tlrl-1826; InvivoGen), IL-1α (1 ng/ml, 211–11 A; Pepro Tech), IL-1β (1 ng/ml, 211-11B; Pepro Tech), or IFNα (100 ng/ml, 752802; Biolegend). Concentrations of cytokines used in Figure 1—figure supplement 1D are shown in Supplementary file 1.

pDC-Gen2.2 cells were cultured at 1–2 10^6 cells/ml in complete RPMI 1640 on the MS-5 feeder cells. Before the experiment, the non-adherent fraction of the culture was harvested, pelleted at 1500 rpm and re-suspended in a fresh medium. 50,000 cells were plated in 96-well round bottom plates and stimulated with 0.25μM CpGA (Invivogen, tlrl-2216), 0.25μM CpGB (Invivogen, tlrl-2006), 3.75μg/ml R848 (Invivogen, tlrl-r848), 0.5μg/ml LPS (Invivogen, tlrl-b5lps) or were left unstimulated (Mock) in total volume of 150μl. The stimulation assay was performed with and without 5μM RBV (1221 ng/ml) (Sigma, R9644). At time points (indicated in figures), the culture supernatants were analyzed for IFNα by ELISA and cells were harvested for RNA isolation and qRT-PCR analysis of gene expression. Where total PBMCs were used, 50,000 cells were stimulated with 0.25μM CpGA +/- RBV and analyzed as above.