Figure 6

Suppressive effect of induced CCR8+ Tregs on CTL function. Tregs were purified from healthy donors and expanded. (A) Expression of Treg-related molecules on induced CCR8+ Tregs and CCR8− Tregs was analyzed by flow cytometry (n = 8). (B) CCR8+ Tregs and CCR8− Tregs were sorted and co-cultured with CD8 T cells prepared from healthy donor PBMCs in the presence of anti-CD3 antibody and APCs for 4 to 6 days. The expressions of GZMB and perforin in CD8 T cells were detected by flow cytometry (n = 7). The values were normalized to the percentage of GZMB or perforin expression in CD8 T cells cultured without Tregs set as 100%. (C) Antigen non-specific cytotoxicity of CD8 T cells after co-culture with Tregs was evaluated. CD8 T cells were co-cultured with anti-CD3scFv-expressing BALL-1 cells (aCD3-BALL-1 cells) (E:T ratio = 1:1) for 24 h; dead target cells were detected as propidium iodide (PI) + cells by flow cytometry (n = 6). The cytotoxic activities were calculated and normalized to the value of PI+ aCD3-BALL1 cells cultured with CD8 T cells without Tregs set as 100%. (D) Antigen-specific cytotoxicity of CTLs after co-culture with Tregs was evaluated. Mart-1 tetramer–positive CTLs were co-cultured with sorted CCR8+ Tregs or CCR8− Tregs for 4 to 6 days and then co-cultured with SK-MEL-5 cells (E:T ratio = 1:1) for 24 h. After lymphocytes were removed, WST assay was performed (n = 7). The cytotoxic activities were calculated and normalized to the cell viability of SK-MEL-5 cells cultured with CTLs without Tregs set as 100%. Statistical significance was determined by paired t-test (*p ≤ 0.05; **p < 0.01). Data are shown as mean values ± SD.