Activation of DC is partially dependent on reverse transcription. Manipulation of day 7 DC was performed as described in the legend of Fig. 1. Lentiviral vectors contained either an active reverse transcriptase (RT+) or inactive reverse transcriptase (RT−) (equal amount of p24). (a) Graphs representing the mean fluorescent intensity of the evaluated markers as obtained by flow cytometric analysis. The expression of Thy1.1, CD40, CD86, and MHC class II on immature DC (no), LPS-activated DC (LPS), and active and inactive reverse transcriptase-transduced DC was evaluated. The numbers in the graph represent the MFI of the CD11c⁺ population. (b) Production of TNF-α and IFN-β by the respective DC. Each experiment is represented by a dot. The horizontal lines show the means.

Lentiviral transduction of DC results in the upregulation of NF-κB, AP-1, and IRF. At day 4, DC were plated at 10⁶ DC/ml medium and transduced with lentiviral vectors encoding cherry fluorescent protein or GFP under the transcriptional control of NF-κB or the latter together with AP-1 and IRF3 (IFN-β promoter) (n = 3). (a and c) Graphs representing the flow cytometry data of a representative experiment demonstrating the relative upregulation of reporter expression (fold increase compared to background) driven by the NF-κB and the IFN-β promoters, respectively. Stimulation with LPS (white bars), active reverse transcriptase (black bars), and inactive reverse transcriptase (gray bars) lentiviral vectors is depicted. (b and d) Confocal images representative of the data obtained with the NF-κB (b) and IFN-β (d) promoters.

TLR3 and TLR7 are involved in the activation of DC by lentiviral vectors. DC of wild-type, MyD88^−/−, TRIF^−/−, TLR3^−/−, and TLR7^−/− mice were transduced as described in the text. As a comparison, MyD88^−/− and TRIF^−/− DC were stimulated with poly(I:C) (pIC) or ssRNA40/Lyovec (ssRNA). Flow cytometry evaluating surface marker expression was performed 24 h after manipulation. (a) Graph showing the upregulation of CD86 upon stimulation with LPS (light gray bars), poly(I:C) (white bars), and ssRNA40/Lyovec (dark gray bars) or transduction with lentiviral vectors (black bars) of wild-type, MyD88^−/−, and TRIF^−/− DC. The data shown represent the means ± standard deviations from two independent experiments. (b) Graph showing the upregulation of CD86 upon stimulation with LPS (black bars) and transduction with lentiviral vectors (white bars) and reverse transcriptase mutant lentiviral vectors (gray bar) of wild-type, TLR3^−/−, and TLR7^−/− DC. The data shown are representative of data from three independent experiments. (c) Graphs showing the secretion of TNF-α and IFN-β by wild-type, TLR3^−/−, and TLR7^−/− DC. The data are a summary of the collected data. (d) Flow cytometry graphs representing the Thy1.1 positivity of wild-type, TLR3^−/−, and TLR7^−/− DC transduced with lentiviral vectors expressing IiOVA and Thy1.1. (e) Representative graphs from pentamer staining (top) and histograms from the in vivo CTL assay (bottom). (f and g) Graphs showing the percentage of CD8/pentamer H-2K^b SIINFEKL-positive cells (f) and percent specific lysis (g) in wild-type, TLR3^−/−, and TLR7^−/− DC. Each dot represents the data for one mouse, and the number of mice per group was 5. The horizontal lines indicate the means.

Activation of DC following in vitro lentiviral transduction. At day 7 of culture, DC were plated at 10⁶ DC/ml medium. These DC were not manipulated (no), incubated with 100 ng/ml LPS for 24 h (LPS), or transduced with lentiviral vectors encoding Thy1.1 at an MOI of 15 (LV) (n = 5). (a) Flow cytometric analysis performed 24 h after transduction. The expression of CD11c, Thy1.1, CD40, CD80, CD86, and MHC class II on the immature (black), mature (red), and transduced (blue) DC was evaluated. The gray line represents isotype control staining. The numbers in the graph represent the mean fluorescence intensities (MFI). Data from one representative experiment out of five are shown. (b) Levels of cytokines in supernatants of immature (no), mature (LPS), and transduced (LV) DC. Each experiment is represented by a dot, and the horizontal line indicates the mean. These DC were used as stimulators in a mixed-lymphocyte reaction at a stimulator-to-responder ratio of 1 to 100. The proliferation of allogeneic CD90⁺ T cells was evaluated 4 days after the start of the coculture as a measure for DC functionality. (c) Proliferation was measured by [³H]thymidine incorporation. The data shown are representative of data from one experiment out of five independent experiments.

Tracking and evaluation of in vivo-transduced cells. (a) In vivo bioluminescence imaging was performed 6, 24, and 48 h after footpad injection of 10⁷ TU of lentiviral vectors encoding firefly luciferase. (b) Thirty-six hours after the subcutaneous administration of lentivectors encoding Thy1.1, lymph nodes were isolated, and CD11c⁺ cells were sorted and stained for Thy1.1 (n = 3). (c) Isolated cells were stained for CD40, CD86, and MHC class II. Cells were gated as CD11c⁺/Thy1.1⁻ or CD11c⁺/Thy1.1⁺. The graphs in b and c are representative of data from three independent experiments and show the percentages and MFI of marker-positive cells. (d) Production of TNF-α by the DC. Each experiment is represented as a dot, and the horizontal line shows the mean. The CD11c⁺ cells were loaded with the MHC class I-restricted ovalbumin peptide SIINFEKL or an irrelevant peptide (Trp2 [SVYDFFVWL]) and cultured at a stimulator-to-responder ratio of 1 to 100 with ovalbumin TCR-transgenic CD8⁺ T cells (n = 2). (e and f) Proliferation of T cells (e) and production of IFN-γ (f) by these T cells. The results are representative of data from two independent experiments.

DC activation by lentiviral vectors is dependent on both TRIF and MyD88. DC of wild-type, TRIF^−/−, MyD88^−/−, or TRIF/MyD88^−/− mice were manipulated as described in the legend of Fig. 1 (n = 3). (a) Graph showing the upregulation of CD86 upon stimulation with LPS (black bars) or transduction with lentiviral vectors (white bars) of wild-type (WT), TRIF^−/−, and MyD88^−/− DC. The data shown are representative of data from three independent experiments. (b) Flow cytometry graphs representing TRIF/MyD88^−/− DC, which were not stimulated (black lines) or stimulated with lentiviral vectors (red lines) or TNF-α (blue lines). The percentage of marker-positive cells is indicated. The data are representative of data from three independent experiments. (c) Graphs showing a summary of the TNF-α and IFN-β secretion by wild-type (white bars), MyD88^−/− (black bars), TRIF^−/− (dark gray bars), and TRIF/MyD88^−/− (light gray bars) DC (n = 3). KO, knockout; DKO, double knockout.