Transcription profiling by array of in vitro grass pollen extract (allergen) challenged CD4+T-cells against paired unchallenged controls from Seasonal Allergic Rhinitis patients

In this work we present an analytical strategy to systematically identify early regulators by combining gene regulatory networks (GRN) with GWAS. We hypothesized that early regulators in T-cell associated diseases could be found by defining upstream transcription factors (TFs) in T-cell differentiation. Time series expression and DNA methylation profiling of T-cell differentiation identified several upstream TFs, of which TFs involved in Th1/2 differentiation were most enriched for disease associated SNPs identified by GWAS. Peripheral blood mononuclear cells (PBMCs) were prepared from fresh blood from 10 patients with seasonal allergic rhinitis and 10 healthy controls using Lymphoprep (Axis-Shield PoC, Oslo, Norway) according to the manufacturer’s protocol. PBMCs were stimulated with allergen extract (ALK-Abelló A/S; 100 μg/ml) or diluent (PBS) in RPMI 1640 supplemented with 2 mM L-glutamine (PAA Laboratories, Linz, Austria), 5% human AB serum (Lonza, Switzerland), 5 µM beta-mercaptoethanol (Sigma-Aldrich, St. Louis, Missouri, USA) and 50 µg/mL gentamicin (Sigma-Aldrich, St. Louis, Missouri, USA). After 17 hours of incubation, total CD4+ T cells were enriched from PBMCs by MACS negative sorting. Total RNA was extracted using a miRneasy Mini Kit (Qiagen, Valencia, CA, USA). The cRNA was prepared using a Low Input QuickAmp Labeling Kit. The expression microarray analyses were performed using Agilent SurePrint G3 Human Exon 4x180K Microarrays according to the manufacturer's instructions. Complementary microRNA data have been deposited in ArrayExpress under accession number E-MTAB-4900 (