Development and function of HA-specific FoxP3⁺ and FoxP3⁻ cells in the different chimeras expressing HA in the thymus. Thymi, lymph nodes, and spleen cell suspensions from the different chimeras illustrated in panel A were analyzed for 6.5 and GFP expression within the CD4⁺ gate. Shown are representative dot plots (B) as well as the percentage of 6.5 cells in the CD4 gate, of FoxP3⁺ cells in the CD4⁺6.5⁺ gate and the absolute numbers of CD4⁺6.5⁺FoxP3⁺ cells in lymph nodes and spleen (C). Three mice per group were analyzed. (D) The mean fluorescence intensity of GFP within the CD4⁺6.5⁺GFP⁺ gate is shown. (E) CD4⁺6.5⁺GFP⁺ and GFP⁻ cells from lymph node suspensions of each type of chimera were electronically sorted and tested in vitro for their suppressive and proliferative capacity, respectively. For the suppression assay, naive 6.5⁺ cells were incubated with Balb/c splenic DCs in the absence or presence of peptide (0.1 μg/mL) and in the absence or presence of GFP⁺ cells from the different chimeras. For the proliferation assay, GFP⁻ cells from the different chimeras were coincubated with DCs and peptide. The experiment was performed in triplicate, and 2 independent experiments gave similar results.