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. 2017 Aug 16;8(8):1732–1743. doi: 10.1080/21505594.2017.1356535

Figure 4.

Figure 4.

Co-cultured C11 cells mature iDCs through CD40-CD40L signaling pathway. (A) CD40 expression on MDDCs. Immature MDDCs or LPS-treated MDDCs were co-cultured with or without C11 cells for 24 h, and MDDCs were distinguished with CD11c+ immunostaining and the expression of CD40 was detected with flow cytometry, and the positive percentage was shown. (B) CD40 expression detected at mRNA level. MDDCs were co-cultured with or without C11 cells for 6 h, and C11 cells stimulated with or without TNF-α (20 ng/ml) for 6 h were also prepared. CD40 expression was detected at mRNA level by qRT-PCR and normalized with β-actin. Data were presented as mean ± SD. (C, D, E) CD40 knockdown impairs iMDDCs-purged HIV-1 reactivation from latency. IMDDCs were transfected with specific CD40 siRNAs or off-target control for 48 h, and CD40 expression was detected by Western blotting (upper panel) and flow cytometry (lower panel, MFI for CD40-positive staining was calculated) at protein level (C), and by mRNA quantification at transcription level (D). These transfected MDDCs were further co-cultured with C11 cells for additional 24 h and HIV-1 reactivation was assessed with flow cytometry (E). (F, G, H) CD40L knockdown on C11 diminishes the response to MDDCs stimulation for HIV-1 reactivation. C11 cells were nucleofected with CD40L specific siRNAs or off-target control for 48 h, and CD40L expression was detected by Western blotting at protein level (F) and mRNA quantification at transcription level (G), and these nucleofected C11 cells were co-cultured with immature MDDCs for additional 24 h, and HIV-1 reactivation was assessed with flow cytometry (H). (I, J) The supply with recombinant CD40Ligand protein complements HIV-1 reactivation from C11 cells. Recombinant CD40 ligand protein (1 μg/ml) was supplied during transwell-culture (pore size, 0.4 μm) of C11 cells with iDCs for 24 h, and C11 cells were harvested and GFP expression was detected with flow cytometry (I), and the supernatant was collected for detecting TNF-α with ELISA (J). One representative result from at least 3 independent repeats was shown. ** P < 0.01 and *** P < 0.001 were considered as significant difference in ANOVA analysis.