Fig 5. TLR2-Responding Cells are CD25^- and FoxP3^- effector/memory T cells.

In order to identify the TCR/TLR2 responding populations, cells isolated from WT, FoxP3-reporter, or IL-10/FoxP3-dual reporter mice were purified by FACS into varied populations, stimulated as before for 3 days and analyzed by IL-10 ELISA (A-D) or by flow cytometry (E-G). Neither CD25⁺ (A), regulatory FoxP3⁺ (B), nor naïve CD62L⁺CD44^- (C) CD4⁺ T cells produced IL-10 in response to stimulation, whereas CD25^-, FoxP3^- and CD62L^-CD44⁺ antigen experienced CD4⁺ T cells showed IL-10 synergy in response to TCR/TLR2 co-stimulation. (D) Post-stimulation analysis at day 3 of CD4⁺ and CD4⁺FoxP3^- T cells showed a modest 10% induction of FoxP3 expression upon P3C stimulation, but only 5% of cells expressed FoxP3 under synergistic IL-10-producing conditions (αCD3+P3C). (E) After stimulation, dual reporter CD4⁺ T cells were analyzed by flow cytometry. CD4⁺ cells showed an increase in the number of IL-10⁺/GFP⁺ T cells (E, left), and among these cells, the addition of P3C selectively increased the number of IL-10⁺FoxP3^- cells (E, right, arrow). Quantitation of the percentage (F) and MFI (G) of CD4⁺FoxP3^-IL-10⁺ among all CD4⁺ T cells (n = 5) is shown. All data are n≥3, unless otherwise noted; *p<0.05; #p>0.05.