Gene expression analysis of WT Th17 versus STAT4 deficient Th17 cells from EAE mice

The purpose of this study is to compare transcriptional profiles of WT and STAT4-deficient Th17 cultured cells from mice with experimental autoimmune encephalomyelitis (EAE) using RNA sequencing. WT and STAT4 deficient splenocytes from EAE immunized mice were cultured with MOG peptide under Th17 conditions for three days, and then total RNA was extracted from CD4 T cells for sequencing. Differential gene expression was determined using the DESeq2 algorithm. These data reveal a previously unrecognized role for STAT4 in Th17 gene expression and function. Age and sex matched mice between 8 and 12 weeks of age were induced for EAE by subcutaneous immunization with 50 μg MOG35−55 peptide (Biosynthesis) emulsified in CFA (150 μg Mycobacterium tuberculosis; Difco) and intraperitoneal (i.p.) administration of pertussis toxin (200 ng; List Biological Laboratories) on days 0 and 2. On day 10 post-immunization, 1x10^8 splenocytes were cultured with complete RPMI 1640 with 10ug/mL MOG35-55 peptide, 2.5ng/ml TGFb (Peprotech), 20ng/ml IL-6 (Peprotech), 10ng/mL IL-23 (Biolegend) and 10ug/mL anti-IFNγ (clone XMG1.2, Bio X Cell) and 10ug/ml anti-IL-4 (clone 11B11, Bio X Cell) for 72 hours at 37C. Following culture, live CD4 T cells were isolated by centrifugation over Histopaque 1083 (Sigma) followed by Dynabeads FlowComp Mouse CD4 kit (Invitrogen). RNA was isolated from WT and STAT4-/- CD4 cells using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocols and submitted to GENEWIZ for sequencing. Total RNA was sequenced on Illumina HiSeq 2500 (2 x 100 base pair, single-end reads) and aligned using mouse mm10 reference genome. The GI tools software along with DESeq2 software was used for differential expression analysis.