Be0054

24 well plates were coated with 10 μg/ml anti-CD3ε (BioXCell BE0001–1) overnight at 4°C. Plates were washed out with PBS, and plated with 5×10⁵ to 1.5×10⁶ naïve (CD4⁺Foxp3⁻CD25⁻CD44^loCD62L^hi) T cells FACS sorted from TCli TCRαβ Foxp3^IRES-GFPRag1^+/− Tg or TCli TCRβ Foxp3^Thy1.1 IFNγ^YFP IL-17A^GFPTcra^+/− mice in D10 in Th0 conditions (1 μg/ml anti-CD28 (BioXcell #BE0015–1), 10 μg/ml anti-TGFβ (BioXcell #BE0057), 5 μg/ml anti-IL-12 (BioXCell #BE0052), 5 μg/ml anti-IFNγ (BioXCell #BE0054), 5 mg/ml anti-IL4 (BioXCell #BE0045)). TCRα chain IRES-Thy1.1 retroviral transduction was performed as described (Hsieh et al., 2004 (link)) using TransIT-293 (Thermo Fisher #MIR2700). Transduced cultures were left to rest in Th0 media for ~66 hours. Foxp3⁻ TCRα-transduced Thy1.1+ cells were sorted by FACS and 2×10⁴ sorted cells were retro-orbitally injected into each host for in vivo analysis unless otherwise specified. Cells were stained with CTV (Thermo Fisher #C34571) in some experiments. Transferred cells were identified as CD4⁺ Vβ6⁺ Thy1.1⁺. For transcription factor staining experiments, 10⁵ cells were injected per mouse to increase recovery. Transcription factors were stained using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher #00–5523-00).