Figure 3.

Treg phenotypes induced by peptide NS3_358-375 and variant pools VP1 and VP2. Cell culture adn purification of CD4⁺ cells and controls were the same as described in Figure 2. Intracellular staining and four-color flow cytometry using fluorescent-labeled antibodies to human CD25, IFN-γ, TGF-β and IL-10 were performed with purified CD4⁺ cells. A pool from each test was used as an isotype control. A: Percentages of CD25⁺ gated CD4⁺ cells. Note that the percentage of CD25⁺ cells stimulated by VP2 is significantly higher. B: Percentages of Th3 and Tr1 cells. Note that 58~67% of the CD25⁺ cells were TGF-β⁺ (sum of the upper two quadrants). A majority of the CD25⁺IL-10⁺ cells were also TGF-β⁺ (the upper right quadrant). C: Percentages of CD25^- gated CD4⁺ cells. D: Percentages of Th3 and Tr1 cells. Note that the percentages of CD25^-TGF-β⁺ cells stimulated by VP1 and VP2 are significantly higher than stimulation with wild type NS3_358-375 and medium alone. Two representatives of eight experiments are shown.