Fig 1.
Transduction workflow as described in the Materials and Methods section.
Fig 2.
MDDCs can be transduced at high efficiency in the absence of Vpx.
Cells were transduced with a lentiviral vector encoding eGFP and a scrambled shRNA sequence 24 hours post-isolation from PBMCs in the absence or presence of Vpx-containing VLP, and analyzed by flow cytometry 6 days post-isolation. The top panel shows MDDC gating strategy. (A) Cells were gated based on their forward scatter (FSC) and side scatter (SSC) profile. (B) Cells gated as in (A) were subsequently gated to exclude dead cells from further analysis by setting a gate on live (propidium iodide (PI)-negative) cells. (C) Bottom dot plots show eGFP expression versus DC-SIGN-PE on MDDCs, transduced or not, as indicated. Percentages indicate fraction of eGFP expressing cells.
Fig 3.
Transduced MDDCs do not mature per se, but retain the potential to undergo maturation.
MDDCs were exposed to LPS (filled profiles) or not (open profiles) starting 4 days post-isolation. Histograms show CD80 APC, CD83 PE and CD86 APC surface staining 48h later in control non-transduced, non-transduced but exposed to Vpx VLPor shRNA-scrambled transduced cells as indicated. Numbers represent mean fluorence intensity.
Fig 4.
Vpx-independent, shRNA-mediated knock-down of SAMHD1 in MDDCs.
(A) Histogram shows intracellular SAMHD1-Alexa 660 staining at similar time point after isolation as shown in B, without ① or with ② treatment with Vpx VLP. Control histogram represents cells stained only with secondary, goat-anti rabbit Alexa 660 antibody ③. (B) Histogram shows intracellular SAMHD1-Alexa 660 staining in MDDCs, 5 days post-transduction with scrambled shRNA (sh-scrambled) ④, or sh-SAMHD1 vectors ⑤ as indicated. Histogram depicting SAMHD1 levels in not transduced cells was overlaid for comparison ①. (C) The bar graph shows SAMHD1 down-regulation in MDDCs from 5 independent donors transduced similar to panel B with or without the addition of Vpx VLP. Error bars represent standard deviation among donors, comparison to sh-scrambled **p ≤ 0.008.
Fig 5.
Vpx-independent, shRNA-mediated CD4 down-regulation in MDDCs.
(A) Histogram shows CD4 surface expression upon sh-CD4 (open profile) or sh-scrambled transduction on day 5 post-transduction (filled profile) in a representative experiment. Numbers represent mean fluorescence intensity. (B) Bar graph represents mean fluorescence intensity of anti CD4-APC surface staining of MDDCs 5 days post-transduction with respective shRNA constructs. Error bars represent standard deviation among donors *p ≤ 0.028 (N = 4).
Fig 6.
HIV infection of transduced MDDCs demonstrates functional effect on HIV infection.
Five days post-transduction with lentiviral vectors ± Vpx VLP as shown, MDDCs were infected with non-pseudotyped (A) or VSV envelope-pseudotyped (B) HIV-1 engineered to express eGFP. Graph bars represent percentage of cells expressing eGFP encoded by HIV on day 3 post-infection. Error bars represent standard deviation among 9 (A) or 4 (B) donors. p values: *p ≤ 0.0286, ***p ≤ 0.0004. (C) MDDCs transduced with scrambled shRNA or CD4 targeting shRNA (but not eGFP) expressing lentiviral vectors, were infected on day 5 post-transduction with HIV engineered to express eGFP. Graph shows day 3 infection rates in MDDCs obtained from two donors.