Development and function of HA-specific FoxP3⁺ and FoxP3⁻ cells in the absence of HA expression in the thymus. Thymi, lymph nodes, and spleen cells 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 (C). A total of 3 mice per group were analyzed. (D) Immunofluorescence staining for CD19 (green), Foxp3 (purple), and nuclei (blue) in spleen sections of the different chimeras. The graphs represent the quantification of Foxp3⁺ cells in CD19⁺ follicules. Bar represents 50 μm. (E) The percentage of intracellular Ki67⁺ cells within the FoxP3⁺ and FoxP3⁻ CD4⁺6.5⁺ cells was determined by flow cytometry. (F) CD4⁺6.5⁺GFP⁺ and GFP⁻ cells from lymph node and spleen suspensions of each type of chimera were electronically sorted and tested in vitro for their suppressive and proliferative capacity, respectively, as described in Figure 4.