Efficacy, specificity, and duration of siRNA-mediated PD-L1 and PD-L2 silencing on DCs. Immature DCs were electroporated with 0.25 nmol PD-L1, PD-L2, or negative control (MED GC vs LOW GC) siRNA duplexes and subsequently cultured in maturation medium containing IL-1β, IL-6, TNF-α, and PGE2 for 2 days (A-D) or 1-5 days (E). (A) PD-L1 and PD-L2 mRNA expression was measured and subsequently normalized for HMBS expression using RT Q-PCR. PD-L mRNA expression in DCs electroporated without siRNA was set to 1. (B) PD-L1 and PD-L2 protein expression (black lines), compared with isotype control (gray histograms), was analyzed using flow cytometry. The numbers in the plots represent the mean fluorescence intensity (MFI). The data of 1 representative donor of 6 is shown. (C) Percentage of PD-L1⁺ or PD-L2⁺ DCs was determined for 3-6 donors by flow cytometry. (D) Expression of maturation and costimulatory molecules by electroporated DCs was analyzed using flow cytometry. The bars represent DCs electroporated without siRNA (black), with MED GC control siRNA (white), PD-L1 siRNA 2 (dark gray), or PD-L2 siRNA 2 (light gray). Data are expressed as mean ± SEM of 3-6 donors. (E) Percentage of PD-L1⁺ or PD-L2⁺ DCs over time was determined by flow cytometry. The percentage of PD-L⁺ DCs electroporated without siRNA was set to 100%. The lines represent DCs electroporated without siRNA (dotted line, ●), with MED GC control siRNA (dotted line, ○), PD-L1 siRNA 2 (solid dark gray line), or PD-L2 siRNA 2 (solid light gray line). The data are representative of 2 independent experiments on 2 different donors.