Induction and analysis of CD4+25+ Tregs induced by peripheral administration of βgal in B10.A mice. (A-C) FACS analysis of CD4+25- (A), CD4+25+ (B), and Thy-1 depleted cells (C) for use in the functional assays. The cells in A and B were depleted with anti-CD19, -CD11b, -CD11c, and -CD8 followed by selection for CD4+25- or CD4+25+ cells. (D) Ag-stimulated cytokine production by naive CD4+25- βgalTCR T cells in co-culture with populations of CD4+CD25+ T cells from normal B10.A mice or βgal-inoculated B10.A mice. Irradiated, Thy1-depleted splenocytes (3 × 105/well) were added as APCs and βgal Ag was added at 100 μg/mL. Results are given as the mean ± SD of duplicate assays (3 wells/sample in each assay) of 72 h supernatants. BD = below detection. Cytokine production in unstimulated cultures was at background levels, and is not shown.