Spleen and lymph nodes from Foxp3.IRES-DTR/GFP animals were harvested. Treg cells were pre-enriched with CD25 bead-based positive selection, while Tconv cells were pre-enriched with CD4 bead-based positive selection (autoMACS Pro Separator, Miltenyi Biotec). Pre-enriched Treg and Tconv cells were sorted by FACS to isolate pure KLRG1–ST2– populations from both cell types. Cells were then seeded at 100,000 cells per well with anti-CD3/CD28 beads (Life technologies), IL-12-23p40 blocking mAb, IFNγ blocking mAb, murine IL-2 (Peprotech) and escalating doses of murine IL-4 (Peprotech). Cells were incubated for six days at 37°C and afterwards lysed for immediate RNA isolation, cDNA synthesis and real-time PCR, as described previously.
To generate in vitro induced Treg cells (iTreg cells), Tconv cells were pre-enriched by negative selection with CD8, CD11b, CD11c, CD19, CD25, and CD49b antibodies and magnetic beads. Afterwards, cells were treated with murine TGF-β (Peprotech) and incubated with CD3/28 microbeads for six days at 37°C.
To generate in vitro induced Treg cells (iTreg cells), Tconv cells were pre-enriched by negative selection with CD8, CD11b, CD11c, CD19, CD25, and CD49b antibodies and magnetic beads. Afterwards, cells were treated with murine TGF-β (Peprotech) and incubated with CD3/28 microbeads for six days at 37°C.