Innate Immune Training Model for Monocyte Responses

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.