RNA-seq of human memory Th1 (mTh1), mTh2 and mTh17 cells treated with tapinarof against untreated control
RNA-seq of human memory Th1 (mTh1), mTh2 and mTh17 cells treated with tapinarof against untreated control
Peripheral Blood Mononuclear Cells (PBMC) were isolated by Ficoll-Plaque Plus (GE Healthcare, UK) density gradient centrifugation. CD4+ T cells were isolated from PBMCs using the EasySep positive selection kit (Cat. #17852, Stemcell Technologies) as per the manufacturer's instructions. Positively selected CD4+ T cells were stained for the subsequent sorting of the TH cell subset. Memory TH cell subsets were sorted with a purity of > 90% according to the expression of chemokine receptors from CD45RA-CD25-CD8-CD3+ cells: TH1 (CXCR3+CCR6-CCR4-), TH2 (CXCR3-CCR6-CCR4+) and TH17 (CXCR3-CCR6+CCR4+).  Culture medium consisted of PRMI-1640 with Hepes (GIBCO) supplemented with 5% of heat-inactivated human serum (Swiss Red Cross, Basel, Switzerland), 2mM L-Glutamine (Biochrom), 50U/ml penicillin and 50μg/ml streptomycin (Bioconcept) and 50 IU/ml IL-2 (Roche). T cells were cultured at a density of 0.25-1x105cells/96 well plate in a total volume of 200μL cell culture medium mTh1, mTh2 and Th17 cells were incubated with Tapinarof for 24 hours either in the resting state or activated with aCD3/2/28. Total RNA was isolated with RNAeasy kit from Qiagen as per the manufacturer's instructions Illumina TruSeq Stranded mRNA protocol (Directional, first strand)