Inhibition of T-cell proliferation and IFN-γ secretion by TGFβ and PD-1. (A) Freshly isolated primary human CD4⁺ T cells were labeled with CFSE and left unstimulated or were stimulated with the indicated magnetic beads (artificial antigen-presenting cells, or CD3/CD28/MHC-I or CD3/CD28/PD-1) in the absence or presence of 30 ng/mL TGFβ. After 4 days, CFSE dilution was analyzed by flow cytometry. The overall percentage of dividing cells is displayed inside the corresponding dot plot. (B) CD4⁺ T cells were stimulated as in panel A. After 4 days of incubation, the concentration of IFN-γ was determined using flow cytometric bead arrays. The presented data are representative for at least 3 independent experiments; error bars in panel B represent 1 representative experiment performed in triplicate.