Modulating Th17 Cell miRNA Dynamics

Th17-polarized human or mouse primary CD4⁺ T cells were transfected with miRNA mimics, siRNAs or inhibitors at 48 h of cell culture with the Neon Transfection System (Invitrogen) as previously described (41 (link)). miRIDIAN miRNA mimics (Dharmacon) or siGENOME SmartPools (Dharmacon) were used at 500nM and miRCURY LNA microRNA Power family inhibitors (Exiqon) were used at 5μM or 20μM with appropriate controls. MSCV-PGK-hCD25 miRNA sensors for miR-17, miR-18a, miR-92a, and miR-19b (Simpson et al., 2014 (link)) were constructed to express EGFP with four perfectly complementary binding sites for the miRNA of interest in the 3′UTR as previously described (41 (link)). Cells were transduced by spin infection early on day 2 of Th17 cultures and transfected with miRNA mimics or inhibitors later on day 2 of Th17 cultures. hCD25⁺ CD4⁺ T cells were analyzed on day 3.5 for EGFP expression.