De novo Generation of Foxp3+ iTregs

De novo generation of Foxp3⁺ iTreg cells were performed according to published protocols^{31 (link)}. Briefly, naïve CD4⁺CD25⁻ T cells were isolated from OT-II mice (or PD-1HKO OT-II mice) as previously described and labelled with 5 μM CFSE before adoptive transfer. Typically, 2 × 10⁶ cells were injected i.v. into WT B6 mice. After 24 hours and for 5 consecutive days, the drinking water in the cages was replaced with a 1.5% OVA solution (grade V; Sigma-Aldrich). On day 6, the mesenteric LN and PP were collected and the TCR-specific Foxp3⁺ Treg cells were determined using intracellular staining for Foxp3 by flow cytometry.