Platinum e packaging cells

For gain-of-function experiments, we used pMXs retroviral expression system as previously described³⁹ . The sequences used for shRNA-mediated loss-of-function experiments were: shNFIA #1, 5′-CCUUCUCAACUCUGUAACA-3′; shNFIA #2, 5′-GUCAGCAGUUACAUACAUA-3′; shPPARγ, 5′-CAAGAGAUCACAGAGUAUG-3′; shPRDM16, 5′-GAAGAGCGUGAGUACAAAU-3′. The corresponding double-stranded DNA sequences were subcloned into pLMP retroviral vector (Open Biosystems) using XhoI and EcoRI restriction enzyme sites. For retrovirus production, Platinum E packaging cells (Cell Biolabs) were transfected with the vector using lipofectamine 2000 (Invitrogen). Two days afterwards, conditioned medium was centrifuged at 2,000 rpm for 5 minutes, and the supernatant was supplemented with 10 mg/ml polybrene and used for overnight infection. Subsequently, infected cells were selected using appropriate antibiotics.

Retroviral expression and transduction of T cells were performed essentially as in the reference (25 (link)). Briefly, retroviral expression plasmids were transfected into Platinum-E packaging cells (Cell Biolabs, Inc., San Diego, CA) using Lipofectamine 2000 (Invitrogen Life Technologies, CA, USA). After 48 h, viral supernatants were collected, filtered through 0.4-μm syringe filters, and stored at -80°C until use. For transduction, CD4⁺ T cells were first activated with 0.5 μg/ml hamster anti-CD3 (145-2C11; BioLegend, CA, USA), 0.5μg/ml hamster anti-CD28 (37.51; BioLegend, CA, USA) in a 24-well plate precoated with goat anti-hamster Ab (0855397; MP Biomedicals) for 24 h, then spin-infected with viral supernatants (2500 rpm, 30°C for 2 h) in the presence of 8 μg/ml polybrene (TR-1003-G; EMD Millipore Corporation, CA, USA). After spin infection, appropriate cytokines were added to the culture media to induce Th17 differentiation.