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. 2015 Oct 27;24(1):146–155. doi: 10.1038/mt.2015.175

Figure 5.

Figure 5

Knockdown of costimulatory molecules following injection of DEC-lipid nanoparticles (LNPs) containing 2'OMe-modified siRNAs is sufficient to inhibit a mixed lymphocyte response reaction. (a) B6 mice (3/group) were injected with siRNAs specific for CD86 (CD86) or a nontarget control (CN) followed by LPS, 6 hours later. One group was injected with LPS alone (LPS) or was left untreated (UN). One day later, splenocytes were stained for CD11c, CD8α, CD80, and CD86 and analyzed by flow cytometry. The left panel shows CD86 expression on CD11c+CD8α+ cells derived from mice that were untreated (red), CD86 siRNA (green), control siRNA (blue), or LPS only (orange). Numbers within histogram plot are mean fluorescence intensity values; each line represents one mouse. Data from all mice are quantified in the bar graphs. Significance was determined by one-way ANOVA. *P < 0.05; **P < 0.01; ns, not significant. (b) CD11c+CD8α+Dy547+ cells were isolated by FACS, total RNA extracted and CD86 mRNA levels determined by qPCR (left panel) and 5' RACE analysis was performed (right panel). PCR products were resolved by gel electrophoresis. Lane 1 = siRNA CD86; Lane 2 = siRNA control (CN); Lane 3 = 100 bp ladder. Arrowhead shows ~150 bp product. (c) B6 mice (3/group) were injected with DEC-LNPs containing a 1:1:1 mix of CD40, CD80, and CD86 siRNAs (Mix), or control siRNAs (CN), followed by LPS, 6 hrs later, or were left untreated (UN). One day later expression of CD80, CD86, and CD40 on CD11c+CD8α+ cells was determined by flow cytometry. (d) B6 mice were injected with DEC-LNPs and LPS (as in (c)). The following day CD11c+CD8α+ DCs were FACS sorted, irradiated and cocultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled T cells purified from BALB/c spleens. Two days later T-cell activation was determined by CFSE dilution using flow cytometry. The graph shows the relative stimulation index, which compares the LPS only treated group (set to 1) to untreated (UN), DEC-LNPs containing control siRNA (CN) and DEC-LNPs containing a 1:1:1 mix of CD40, CD80, and CD86 siRNAs (Mix). (c and d) Statistical significance was calculated by student's t-test. *P < 0.05; **P < 0.01; ***P < 0.001; FACS, fluorescence-activated cell sorting; NS, not significant.