FIG 5.

The neutral-risk allele resulted in significant induction of FoxP3-negative LAP-expressing Tregs in vivo and in vitro. (A) A total of 7 to 10 mice expressing either the HLA-II protective or neutral-risk allele were infected subcutaneously with 1 × 10⁸ to 10 × 10⁸ CFU/mouse of M1T1 GAS isolate 2006 for 72 h. Splenocytes were isolated and stained with antibodies against surface CD3, CD4, CD25, GARP, and LAP, as well as intracellular FoxP3, and levels were measured by flow cytometry. Data shown represent the frequencies of FoxP3⁻ LAP⁺ cells within activated CD4⁺ CD25⁺ T cells gated on viable cells. Each mouse is represented by a symbol. Error bars represent the SEM. (B) Splenocytes from HLA-II tg mice expressing either the protective or neutral-risk allele were either left unstimulated or stimulated with 10, 1, and 0.1 ng/ml of SmeZ. Cells were cultured for 72 h at 37°C and 5% CO₂. After 72 h, the cells were washed and incubated with antibodies to surface CD3, CD4, CD25, GARP, and LAP, as well as intracellular FoxP3, and subjected to flow cytometry. Results are shown as bar graphs and indicate the mean frequencies of FoxP3⁻ LAP⁺ cells within activated CD4⁺ CD25⁺ T cells gated on viable cells. Error bars represent the SEM from the means of results of at least three biological replicates. (C and D) Representative flow cytometry data as a contour plot of FoxP3⁻ LAP⁺ cells in vivo (C) and in vitro (D). *, P ≤ 0.05.