FIGURE 5.

Peripherally induced Treg cells are Foxp3⁺Helios⁻. A, LN cells from two adult C57BL/6 or two MHC class II-deficient mice were analyzed flow cytometry. Plots shown are gated on CD4⁺ cells and analyzed by flow cytometry for Helios and Foxp3 expression. Shown are representative plots of one mouse. B, CD4⁺GFP⁺ or CD4⁺GFP⁻ cells were purified from the LN of Foxp3-GFP mice by cell sorting. Cells were stimulated with plate-bound anti-CD3 and IL-2 in the presence or absence of TGF-β and then analyzed by flow cytometry for Foxp3 and Helios expression. Plots shown are gated on CD4⁺ cells. Shown is a representative experiment from three independent experiments. C, Human PBMCs were cell sorted for CD4⁺CD25^hiCD127⁻ (Treg) cells and CD4⁺CD25^–CD127⁺ CD45RA⁺ (Teff) cells. The cells were stimulated in the presence or absence of TGF-β and then analyzed by flow cytometry for Foxp3 and Helios expression. Plots shown are gated on CD4⁺ cells. Shown is a representative experiment from at least three independent experiments. D, CD4⁺GFP⁻ cells were sorted from OT-II × Foxp3-GFP mice, and 2 × 10⁶ cells were transferred to congenic B6.SJL recipient mice. The recipient mice were given OVA-supplemented drinking water for 6 d. LP cells were isolated and analyzed for Foxp3 and Helios expression by flow cytometry. Plots shown are gated on CD4⁺CD45.1⁺ host cells or CD4⁺CD45.2⁺ cells (OT-II). A representative result of four individual mice is shown from two independent experiments.