Different behavior of HA-specific FoxP3⁻ cells in TCR-HAxpgk-Ha and TCR-HAxIg-HA mice. (A) Total lymph node suspensions from TCR-HA, TCR-HAxpgk-HA, or TCR-HAxIg-HA mice were stained with cell-vue Maroon and incubated in vitro with different peptide doses. Proliferation of CD4⁺6.5⁺FoxP3⁻ and FoxP3⁺ cells was determined by flow cytometry. (B) Interferon-γ present in the supernatant from the same cultures was determined by enzyme-linked immunosorbent assay. Results correspond to the pool of triplicate wells, with cells obtained from one mouse of each genotype. Two independent experiments were performed with similar results. (C) CD4⁺6.5⁺FoxP3⁺ and FoxP3⁻ cells from TCR-HAxpgk-HA (left graph) or TCR-HAxIg-HA (right graph) mice were electronically sorted and incubated in vitro with splenic-derived DCs and different peptide doses. FoxP3⁻ cells from a TCR-HA single transgenic mouse were used as control of proliferation. Thymidine was added after 48 hours of culture and left for an additional 16 hours. Experiments were performed in triplicate and 1 representative experiment of 3 is shown.