Mat: Healthy adult and sJIA blood stimulation with innate immune ligands- GSE103500

The goal of this study was to characterize altered inducible immune networks in Systemic onset juvenile idiopathic arthritis (sJIA), an IL-1-driven autoinflammatory disease of unknown etiology.

A “Transcriptomic Reporter Assay” system to assess the immunogenicity of plasma from septic patients and evaluate its potential for biomarker discovery.

291 total samples, 31 donors and 17 stimuli (excluding media controls). All stimuli were not used with all donors. There is one technical replicate for donors BD1277, BD1350, BD1635 for all stimuli - the replicate was run in a later batch (larger barcode number).

Agonists for surface Toll-like receptors (CpG-C, R837, R848, TLR8L, Flagellin, LPS), a protein kinase C activator (PMA/I: phorbol myristate acetate and ionomycin), cytokines (IFNalpha, IFNgamma, IL-17A/F, IL-18, IL-1beta, TNFalpha), gram+ derived ligands (PGN, LTA), and heat-killed Staph. aureus (HKSA) as well as heat-killed Staph enteric (HKSE).

Healthy adults, pediatrics, and media controls

Ligands were diluted in medium (RPMI 1640 with GlutaMax, Thermo Fisher Scientific, Grand Island, NY, USA) and aliquoted into 2 mL polypropylene 96-well plates (Greiner Bio-One, Monroe, NC, USA). Blood was drawn in the morning into the BD Vacutainers containing ACD anticoagulant (BD, Franklin Lakes, NJ, USA). Time between blood draw and culture setup was less than 2h. Blood and appropriately diluted ligands were mixed in 1:1 ratio in total volume of 1 mL/well and incubated for 6h at 37oC/5% CO2. After the culture, blood was mixed and 100 uL aliquot set aside for activation marker phenotyping and intracellular cytokine staining (ICS) for IL-1b. The plate was then centrifuged at 12,000 x g/15 and 400 mL of plasma aliquoted and stored at -80oC until the Luminex analysis. The pellet was the lysed with Tempus solution (Tempus blood RNA tubes, Thermo Fisher Scientific) in 2:1 ratio and stored at -20oC until the RNA extraction.

The data were preprocessed using R, which included the following steps: quantile normalization, flooring all intensities <10 to 10, log2 transformation, and PALO (present at least once) filter. PALO, which selects only transcripts with detection p-values < 0.01 in at least one sample, was performed to reduce background noise.