Effect of OX40 stimulation to T effector cells on the induction of new Foxp3⁺ Tregs.(A) CD4⁺GFP(Foxp3)⁻ T effector cells were sorted from foxp3gfpKI mice and stimulated in vitro with anti-CD3 plus wt APCs, OX40Ltg APCs or OX40L^−/− APCs in the presence or absence of TGF-β, and induction of GFP(Foxp3)⁺ cells was determined 4 days later by gating onto the CD4⁺ fraction. The FACS plot shown is the representative data of 4 individual experiments. (B) The experiments were set up as described in panel A, and the image shown was captured by confocal microscopy 4 days after the culture using a Nikon Eclipse 80i system equipped with E-max software (Nikon Instruments, Melville, NY) (40×/0.75 NA oil immersion lens). Cells were labeled with PE-antimouse CD4. (C) CD4⁺CD25⁻ T effector cells were sorted from congenic CD90.1 mice and adoptively transferred into wt C57BL/6 and OX40Ltg mice (CD90.2). The host mice were treated with DST and anti-CD154. Induction of Foxp3 expression in the CD90.1⁺ fraction in the host spleen was determined by intracellular staining for the Foxp3 protein 6 days later. Data shown are representative of 3 experiments. (A,C) Numbers are the relative percentage of cells in each region given by the flow cytometer.