PBMC Cytokine Response to RNA Agonists

Buffy coats, the fraction of an anti-coagulated blood sample that contains most of the white blood cells and platelets after the centrifugation of the blood (500 mL blood in 70 mL citrate phosphate dextrose coagulant), from five healthy human (consensual) blood donors, were obtained from Sanquin Blood Supply in Rotterdam, the Netherlands. PBMCs were isolated from each buffy coat within 24 h after blood collection, aliquoted, and cryopreserved. PBMCs were stimulated for 48 h with QR-1011 candidates AON32, AON44, AON59, or AON60 at a concentration of 1 μM and 10 μM; positive control R848 (1 μM); or PBS (vehicle control) at 37°C under a 5% CO₂ atmosphere. For every donor, all conditions were tested in triplicate in 96-well round-bottom microtiter plates. The total number of viable PBMCs per well was 3 × 10⁵. R848 (Resiquimod [tlrl-r848]; InvivoGen, San Diego, CA, USA), a potent Toll-like receptor (TLR)7/8 agonist, was selected as a positive control for its strong and robust immune-activating properties, inducing the production of pro-inflammatory cytokines. Also, R848 acts on the TLRs that are most likely to be involved in recognition of single-strand RNA, arguably making it the most relevant positive control for this purpose. After incubation, cell culture supernatant was isolated following centrifugation (300 relative centrifugal force for 5 min at room temperature). The viability of PBMCs after exposure to test items was assessed by resazurin reduction assay (CellTiter-Blue Reagent; Promega, Madison, WI, USA). Cytotoxicity was assessed by measurement of lactate dehydrogenase in the cell culture supernatant (CyQUANT LDH Cytotoxicity Assay; Thermo Fisher Scientific). Readout of viability and cytotoxicity assays was performed on a SpectraMax M5 Microplate reader. Cytokine levels in PBMC culture supernatants were measured using the MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel-Custom 6 Plex-Immunology Multiplex Assay (Merck KGaA, Darmstadt, Germany). Analytes included IFN-α2, IL-6, IP-10, MIP-1α, MIP-1β, and tumor necrosis factor-α. Assay plates were read on the Luminex MAGPIX platform (Luminex, San Francisco, CA, USA). Analysis of the Luminex data was performed in Bio-Plex Manager 6.1 software (Bio-Rad). Standard curves were fitted using five-parameter logistic regression. Cytokine concentrations that were outside of the detectable range of the assay were imputed for the purpose of calculation and statistical analysis. Values below the limit of detection (LOD), rendered “out of range <” by the analysis software, were imputed with a concentration value of ½ × LOD. The LOD values, which were empirically determined by the manufacturer of the Luminex kit, were derived from the technical data sheet. Conversely, cytokine concentrations that were above the upper limit of quantification, rendered “out of range >” by the analysis software, were imputed with a concentration value of two times the concentration of the highest calibrator.