Rapamycin inhibits endocytosis by GM-CSF–expanded immature BM-derived DCs.

(A-E) BM-derived DCs were expanded for 7 days with GM-CSF only, as described in “Study design”; rapamycin (Rapa) was added at day 2 (± FK506) at the concentrations indicated. (A-C) FITC-albumin and FITC-dextran internalization at 37°C and 4°C (negative control), MHC class II (IA^b β-chain), and CD11c expression were analyzed by 3-color flow cytometry. CD11c⁺ MHC class II^lo cells were gated to determine endocytosis specifically in immature DCs. Regions R1 and R2 show endocytotic activity of immature MHC class II^lo and mature MHC class II^hi DCs, respectively. (A, B) Numbers indicate the percentage of CD11c⁺ MHC class II^lo cells positive for the marker indicated. (B, C) Data represent mean values (± SE) of CD11c⁺ MHC class II^lo cells after subtraction of background fluorescence (4°C). Differences between paired cultures were compared by the 2-tailed Student t test for paired samples (*P ≤ .01 vs control; ♦P < .05 vs Rapa 1). (D, E) Apoptosis of CD11c⁺ DC was determined by annexin-V/7-AAD staining (D) and the TUNEL assay (E). Numbers represent mean values (± SE). Incidences of apoptotic (annexin-V positive/7-AAD negative or TUNEL positive) or secondary necrotic (annexin-V positive/7-AAD positive) cells were consistently lower than 10%. There was a trend towarddecreased apoptosis/cell death in rapamycin-treated cultures, but the differences were not statistically significant. (A-C) Results are representative of 4 separate experiments. (D, E) Results are representative of 2 (TUNEL assay) and 3 (annexin-V/7-AAD assay) separate experiments.