Early proliferation and localization of Tregs. Trafficking of allogeneic luciferase (luc⁺)–expressing CD4⁺CD25^hi (Tregs) (A) or CD4⁺CD25⁻ (B) cells at early time points following their adoptive cotransfer with donor wild-type CD4⁺CD25⁻ T cells and TCD-BMs (A) or TCD-BMs alone (B) into lethally-irradiated Balb/c hosts. Animals were imaged daily for the first week, then every other day thereafter. Images shown were chosen on specific days that indicate change in the trafficking pattern or signal intensity. Data show ventral and left lateral images of a single animal, representative of at least 3 animals per group per experiment. Data are representative of 3 independent experiments. S indicates spleen; c, cervical lymph node; i, inguinal LN; m, mesenteric LN; a, axillary LN; T, thymus; and L, liver. (C) Day-4 and day-8 ex vivo imaging of gut and spleen in animals that received luc⁺ CD4⁺CD25^hi or CD4⁺CD25⁻ cells. S indicates spleen; m, mesenteric LN. Control tissues (far right) are from animals without luc⁺ cells. Color bar represents signal intensity code over whole-body surface or organs in photons per second. (D) FACS analysis of absolute numbers of donor (H2^q) CD4⁺Foxp3⁺ cells in lymphoid tissues following transplantation showed significant reduction of cells from day 4 to day 8 in secondary lymphoid organs (*P < .05; **P < .001). One of 2 independent experiments with similar results is shown. Color bars represent signal intensity scale over whole body: (A-B) 3000 to 30 000; (C) 1000 to 8000. Error bars represent standard error of mean value.