STAT3 Activation in Combination with NF-KappaB Inhibition Induces Tolerogenic Dendritic Cells with High Therapeutic Potential to Attenuate Collagen-Induced Arthritis

Figure 3

Generation of a master viral vector encoding murine STAT3ca and IκBαSR (pLVX-VMmu). (a) Lentiviral genome maps for pLVX-VMmu vector containing the pLVX backbone encoding IκBαSR and STAT3ca genes separated by a P2A sequence. As a reporter gene, the green fluorescent protein (ZsGreen1) was inserted at 3 after an IRES sequence. These three genes were inserted under the control of the CMV promoter. Kozak sequence was also included where indicated. (b, c) Bone marrow-derived precursors from DBA/1J mice were cultured in conditions to differentiate to DCs in the absence of lentiviral treatment (grey) or transduced at days 4 and 5 with viral vectors (MOI 10) encoding for pLVX-VMmu (green) or with the empty control vector pLVX (black) in the presence of polybrene. After 24 h, the efficiency of lentiviral transduction was determined as the extent of ZsGreen1 expression by flow cytometry in the CD11c⁺ population. (b) Representative contour plots of CD11c versus ZsGreen1 expression. The percentage of CD11c⁺ cells expressing ZsGreen1 is indicated in the corresponding region. (c) Quantification of the efficiency of DC transduction determined as the percentage of ZsGreen1⁺ cells from the CD11c⁺-gated population. The analysis was carried out excluding dead cells by selecting only cells with negative staining for Zombie Aqua. Values are from three independent experiments carried out in triplicate. No differences were detected between the efficiency of DC transduction with pLVX and with pLVX-VMmu.

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