Induction of IL-10-producing CD4+CD25+ T cells in the spleen of tolerized mice. The proportion of IL-10-producing CD4+CD25+ T cells was analyzed by triple-colour fluorescence-activated cell sorting (FACS) analyses. Single-cell suspension was prepared from the spleens of normal DBA/1, and tolerized and nontolerized collagen-induced arthritis (CIA) mice at 5 weeks after primary immunization. Cells were first gated for CD4+ proportions, and subsequently analyzed for surface expression of CD25+ and for the presence of intracellular IL-10. A representative dot plot chosen from three independent measurements (summarized in Table 2) is shown.