Gene expression of murine repeatedly activated Th1 cells after miR-31 knock down

Gene expression of murine repeatedly activated Th1 cells 36h, 48h and 72h after miR-31 inhibition using a specific antagomir (Antagomir-31) compared to a scrambled controll (Antagomir-SCR). CD4+CD62Lhi (naϊve) from spleens and lymph nodes of 6- to 10-weeks old OTII mice were isolated and labeled with αCD4-FITC (GK1.5, DRFZ) followed by Magnetic Cell Sorting (MACS) with αFITC microbeads (Miltenyi Biotec). Subsequent purification of CD4+CD62Lhi cells was achieved using αCD62L microbeads (Miltenyi Biotec). Cells were cultured (1:5) with irradiated (30 Gy), CD90.2 depleted APCs in the presence of OVA323-339 (0.5 mM) in RPMI supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, 0.1 mg/ml streptomycin and 10 μM β-mercaptoethanol, IL-12 (5 ng/ml, R&D Systems) and αIL-4 (10 µg/ml, 11B11). Viable cells were separated by density gradient centrifugation using Histopaque 1083 (Sigma Aldrich) and reactivated with freshly isolated APCs every six days for three times consecutively. IL-2 (10 ng/ml, R&D Systems) was added from the second stimulation on. After the fourth round of stimulation, viable cells were separated by density gradient centrifugation using Histopaque 1083 (Sigma Aldrich) and treated (5 x 10^6 cells/ml) in serum-free siRNA delivery medium (ACCELL, Dharmacon) containing antagomir-31 or antagomir-SCR (1 µM) for 1.5h at 37°C and 5% CO2. Cell suspension was diluted (1:5) with RPMI supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, 0.1 mg/ml streptomycin and 10 μM β-mercaptoethanol, IL-12 (5 ng/ml, R&D Systems), αIL-4 (10 µg/ml, 11B11) and IL-2 (10 ng/ml, R&D Systems) and reactivated with plate-bound αCD3 and αCD28 (3 µg/ml, BD). 36h, 48h, and 72h after the treatment, cells were harvested and total RNA was isolated using ZR RNA MiniPrepTM kit (Zymo Research) according to the manufacturer’s recommendations. RNA Quality control was performed with a bioanalyzer using the RNA 600 Pico Kit and 10µg RNA with a RIN > 8 was hybridized to Mouse 430_2 (Affymetrix) according to the manufacturer's recommendations.