Fig. 5.

Expansion of antigen-induced FOXP3^high Tregs from naive CD4⁺CD25⁻ T-cell pool. PBMCs were obtained from MS patients that had significant response to MBP_83–99 peptide stimulation as evidenced by proliferation assay. Purified CD4⁺CD25⁻ T cells or CD4⁺CD25⁺ Treg cells (Miltenyi) from PBMC were pre-treated with 5 μM CFSE and cultured with 10 μg ml⁻¹ MBP_83–99 peptide plus irradiated APC in the presence of anti-IL-6 neutralizing antibody or isotype-matched control antibody. At day 7 and 10 after initial stimulation, cells were harvested and stained with anti-FOXP3 antibody. Cell division was analyzed by flow cytometry. The upper left quadrant is used to determine the frequency of FOXP3 up-regulated cells in CD4⁺ T cells.