Effect of blocking PD-1 and PD-L1 on immune checkpoint expression in CD4+CD25+FoxP3+Helios+ Tregs. Activated PBMCs co-cultured with breast cancer cells were treated or untreated with 2 µg/mL of anti-PD-1, 0.5 µg/mL of anti-PD-L1 or with both mAbs. At 72 h post-mAb treatment cells were stained for immune checkpoints and FoxP3/Helios expression, and analyzed by flow cytometry. Representative flow cytometric plots show the expression of PD-1, CTLA-4, TIM-3 and LAG-3 in Tregs from activated PBMC and MDA-MB-231 co-culture treated or untreated with mAb(s) (A). Bar plots show the differences in IC expression on Tregs in the presence or absence of MDA-MB-231 (B), MDA-MB-468 (C) and MCF-7 (D) cells. Data represent the mean + SEM of four independent experiments.