Migration behaviour of regulatory T cells (Treg cells). (a) CD4+CD25+ and CD4+CD25- cells were purified by fluorescence-activated cell sorting (FACS) and labelled with 111In. Cells (1 × 106) were injected intravenously into antigen-induced arthritis (AIA) mice at day 7. After 24 hours radioactivity in isolated organs and the rest of the body was determined using a γ-counter. Thereafter, the total radioactivity recovered per animal was calculated by adding the counts of the organs and the rest of the body. (a) The proportion of radioactivity found in the isolated organs is shown here as a percentage of total recovered radioactivity (n = 6; mean ± standard error of the mean; one representative out of two independent experiments; **P < 0.01). (b) FACS-purified cells were labelled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and injected intravenously. After 24 hours single-cell suspensions from draining lymph node (dLN), nondraining peripheral lymph node (pLN), mesenteric lymph node (mLN), spleen and peripheral blood lymphocytes (PBL) were analyzed by FACS. The percentage of CFSE+ cells of the total CD4+ cells was measured. Histogram plots are gated on CD4+ cells after propidium–iodide exclusion of dead cells (n = 3 per group). Higher numbers of CFSEhigh cells are found in the secondary lymphoid organs in the recipients of CD4+CD25- cells.