Generation of Induced Regulatory T Cells

Induced Tregs (iTregs) were generated as described previously.⁷ (link) Briefly, CD4⁺ cells were isolated from B6 spleen and lymph nodes by magnetic bead sorting (L3T4 Microbeads, Miltenyi Biotec), and cultivated in plates coated with 10 µg/ml anti-CD3 (145-2C11), 1 µg/ml anti-CD28 (37.51) (BD Pharmingen) in the presence of 100 U/ml interleukin-2 (IL-2; Sigma) and 5 ng/ml recombinant human transforming growth factor-beta (rhTGF-β; R&D Systems) for 5 days. At the end of culture, the Treg-enriched cell population was used for therapeutic administration without additional sorting steps. FoxP3 expression was usually >80%. Then 3 × 10⁶ cells were injected intravenously per mouse.