Figure 1.

Inhibition of mTOR expression prolongs DC lifespan and promotes expression of costimulatory molecules CD40 and CD86. (A) DCs were pulsed with Pam2CSK4, LPS, R848, or CpG for 6 hours and then washed and cultured in complete medium. Cell viability was monitored daily by FACS analysis of 7-AAD staining of CD11c⁺ cells. (B) Western blot for mTOR and β-actin protein in Luciferase hpRNA (Luc^hp) or mTOR hpRNA (mTOR^hp) -transduced DCs. (C) DCs were stimulated with media alone, LPS, or rapamycin + LPS for 30 minutes. Cells were subsequently fixed and stained for phosphorylated S6 protein as a molecular readout for mTOR activation and analyzed by FACS. (D) Luc^hp and mTOR^hp-transduced DCs were left untreated or stimulated with LPS and monitored daily for cell viability by analysis of 7-AAD staining of CD11c⁺ cells. (E) Luc^hp or mTOR^hp DCs were cultured with or without LPS for 24 hours and analyzed by FACS for CD40 and CD86 expression. All graphs in this figure represent mean values of replicate wells; all experiments were performed at least twice with similar results.