HA expression in thymus induces negative selection of HA-specific T cells and an increase in HA-specific FoxP3⁺ cells, but with a different age-dependent kinetics depending on expression of the HA transgene. (A) Primary and secondary lymphoid organs from 12-week-old TCR-HA, TCR-HAxpgk-Ha, and TCR-HAxIg-HA mice were analyzed for FoxP3 and 6.5 expression among gated CD4⁺ cells. Shown are representative dot plots, with the percentage of total 6.5⁺ cells among CD4⁺ cells (value outside the parentheses) and the percentage of FoxP3⁺ cells among CD4⁺6.5⁺ cells (value in parentheses). (B) The same data are presented for a pool of 12-week-old mice, with an average of 3 mice per time point. **P < 0.01. ***P < .001. (C) Absolute numbers of 6.5⁺FoxP3⁺ cells and of total 6.5⁺ cells in periphery (lymph nodes and spleen) of the 3 different types of mice vary with the age. Each time point represents 2 to 5 mice. (D) A representative image of the mesenteric lymph nodes (mLN) and spleen of a 16-week-old TCR-HA, TCR-HAxpgk-HA, and TCR-HAxIg-HA mouse.