Kinetics of Foxp3 expression in the tissues after transfer of Treg and CCR2-Treg cells. (a) Five hundred thousand Treg or CCR2-Treg cells were injected into the retro-orbital vein of 14-week-old MRL/lpr mice. Then, the lungs and submandibular glands were harvested at 3 hours, 24 hours, 72 hours, 5 days, and 7 days after transfer. Quantitative real-time polymerase chain reaction analysis was performed on total RNA prepared from the above tissues. Results are calculated as a ratio of Foxp3 expression to the expression of hypoxanthine phosphoribosyl transferase 1 (HPRT1). (b) Analysis of the mononuclear cells in the lungs and submandibular glands of MRL/lpr mice after transfer of Treg and CCR2-Treg cells. The mononuclear cells in the lungs and submandibular glands were harvested at 3 hours and 24 hours after transfer as described in Materials and methods and analyzed by immunofluorescence on an Olympus IX70 inverted microscope. CCR2-Treg cells contain green fluorescent protein and red fluorescent protein (DsRed), whereas Treg cells contain only DsRed. Results are shown as a percentage of staining cells (Treg or CCR2-Treg cells) (n = 3 or 4 mice per group). CCR2-Treg cell, CD4+CD25+Foxp3+ CCR2-transfected T cell; Foxp3, forkhead box p3; MRL/lpr, MRL/MpJ-lpr/lpr; Treg cell, CD4+CD25+Foxp3+ T cell.