Figure 6

Wild-type Jurkat T cells shed PD-1 upon treatment with ionomycin (A) Control wild-type Jurkat T cells over-expressing a PD-1/GFP chimera were treated with 5 μM ionomycin for 15 minutes at 37 °C for 15 minutes then chilled and stained at 4 °C for expression of a variety of surface molecules and analyzed by FACS. Data shows the percentage of initial surface expression after ionomycin treatment. (B) Control wild-type Jurkat T cells over-expressing a PD-1/GFP chimera were treated with 5 μM ionomycin for 15 minutes at 37 °C then chilled and stained at 4 °C for PD-1 expression. PD-1 staining and cellular GFP expression were analyzed by FACS; data shows the percentage of initial expression after ionomycin treatment. (C) Control wild-type Jurkat T cells over-expressing a PD-1/GFP chimera, or a PD-1/GFP chimera where the PD-1 transmembrane sequence was replaced by that from HLA-A2, were treated with 5 μM ionomycin for 15 minutes at 37 °C then chilled and stained at 4 °C for PD-1 expression. PD-1 staining was analyzed by FACS; data shows the percentage of initial expression after ionomycin treatment. (D) Control wild-type Jurkat T cells over-expressing a PD-1/mCherry chimera, were imaged by SR-SIM microscopy before and after ionomycin treatment. Arrows indicate fluorescent vesicles (scale bar is 5 μm). (E) Control wild-type Jurkat T cells, or control wild-type Jurkat T cells over-expressing a PD-1/GFP chimera, were stained with an anti-PD-1 antibody, then treated with 5 μM ionomycin for 15 minutes at 37 °C in the presence of fixable viability dye eFluor780. The cell preparations were analyzed by FACS. The events shown were first gated on subcellular sized events (low forward scatter) and intact membranes (low fixable viability dye eFluor780 staining). PD-1 and GFP expression in these gated events is shown. Cellular FACS data shows the mean +/− SEM for three independent experiments. (***p < 0.001, **p < 0.01, Student t-test).