Figure 3: Effect of rapamycin (Rapa) on the phenotype and function of H1-DC. DCs were either untreated, matured in response to the maturation cocktail or treated with Rapa for 3 days prior to maturation. (a) H1-DC stained for the expression of the maturation marker CD83, the costimulatory molecules CD86 and CD40, as well as the inhibitory receptor PD-L1. Dead cells were excluded from analysis using 7-AAD. Open histograms represent the level of background staining using appropriate isotype-matched controls. Data from one of 3 independent experiments are shown. (b) Phenotypic analysis of control populations of moDC treated and stained in parallel with rapamycin. (c) Effect of rapamycin on the allostimulatory capacity of DC in the allogeneic MLR. DCs were mitotically-inactivated using mitomycin C and plated in triplicate at a top dose of 104 cells per well of a 96-well round-bottomed plate; naïve CD4+ T cells were plated at 5 × 104 cells/well. Cells were incubated for 5 days before pulsing with 3H-thymidine overnight. Graphs show the mean of triplicate cultures ± S.D. Data are shown from one experiment, representative of 3 independent experiments.