Figure 3

Granzyme B expression in Tregs analyzed by flow cytometric gating on FOXP3-bright cells (panel A, population labeled Tr) without stimulation or with stimulation by CD3/CD28 beads and IL-2 for 4 days without inhibitors (labeled no inhibitor) or with rapamycin (10 ng/mL) or the PI3K inhibitor LY294002 (1 or 10 μM). The data is graphed as mean of the mean fluorescence intensities (MFI) of granzyme B staining in the FOXP3-bright population in triplicate samples (panel B). Statistical comparison of the indicated data sets using a t-test is shown (** denotes P < .003). In order to confirm that the decrease in granzyme B expression was not attributable to induction of cell death in the rapamycin or LY294002-treated cells, samples were stained with propidium iodide (PI) (panel C). To facilitate interpretation, we next compared FOXP3 MFIs in the samples. The level of FOXP3 expression in the gated FOXP3-bright population (see panel A) in one unstimulated sample of Tregs (panel D, far left) and in triplicate stimulated samples treated with inhibitors as indicated is shown as a bar graph (panel D). Pairwise statistical comparisons are shown (NS-not significant with P > .05; * denotes P = .02). This data is representative of more than three experiments from multiple donors.