De novo virus production in IM-MDDCs is affected by TLR2 and 4 engagement. (A) IM-MDDCs were either left untreated (mock) or stimulated for 2 hours with the listed TLR agonists. Thereafter, cells were washed twice and pulsed with NL4-3/Balenv. IM-MDDCs were either left untreated (left panel) or treated with EFV (right panel) before addition of TLR agonists. Supernatants were harvested at 3, 6 and 9 days post-infection (dpi) and the viral content was monitored by a p24 test. Data depicted represent the mean ± standard deviations of quadruplicate samples from a single donor and are representative of 3 different donors. (B) A similar experimental strategy was used except that cells were either left untreated or exposed to the listed TLR2 ligands. Data shown represent the mean ± standard deviations of quadruplicate samples from two different donors (3 dpi). (C) Cells were either left untreated (mock) or stimulated for 2 hours with the listed TLR2 agonists. Thereafter, cells were washed twice and pulsed with the clinical HIV-1 isolate 93TH054. Supernatants were harvested at 5 dpi and the viral content evaluated by a p24 test. The data shown represent the mean of quadruplicate samples from 2 different donors.

Some cytokines and chemokines are secreted following engagement of TLRs. IM-MDDCs were either left untreated or stimulated for 8 and 24 hours with the listed TLR ligands. Cell-free supernatants were harvested and analyzed with a Bio-Plex assay that can detect all the indicated soluble factors. The results shown are representative of two separate experiments performed with two distinct donors.

TLR2 and 4 triggering increases viral entry in IM-MDDCs. A) IM-MDDCs were either left untreated (mock) or treated with TLR2 and 4 ligands for 2 hours and washed twice. Then, cells were pulsed with NL4-3/Balenv for 15, 30 and 60 min at 37°C. Next, the virus-cell mixture was washed extensively with PBS and trypsinized to remove uninternalized virus. Finally, cells were lysed and the p24 contents were measured by ELISA. Numbers depicted above bars represent fold increase relative to p24 levels in untreated control cells (considered as 1). The data shown represent the mean ± standard deviations of triplicate samples from 3 different donors.

TLR4-mediated decrease in de novo virus production involves type-I IFNs. A) IM-MDDCs were either left untreated (mock) or stimulated for 6 hours with the listed TLR ligands. Cell-free supernatants were harvested and the levels of IFNα/β were quantified through the use of HEK-Blue™ IFNα/β cells. The data shown represent the mean ± standard deviations of quadruplicate samples from a single donor. B) IM-MDDCs were either left untreated (mock) or stimulated for 2 hours with the TLR4 ligand LPS. Cells were then washed twice, pulsed with NL4-3/Balenv and cultured in absence or presence of B18R (0.1 μg/ml). Supernatants were harvested at 72 hours post-infection and the viral content was evaluated by a p24 test. The data shown represent the mean ± standard deviations of quadruplicate samples from a single donor and are representative of 4 different donors.

TLR2 and 4 triggering modulates HIV-1 transfer between IM-MDDCs and CD4⁺ T cells. A) IM-MDDCs were either left untreated (mock) or stimulated for 2 hours with the following TLR agonists: Pam3Csk4 (5 μg/ml), LPS (0.1 μg/ml), flagellin (5 μg/ml), R837 (5 μg/ml) and unmethylated DNA (5 μg/ml). Cells were then pulsed with NL4-3/Balenv and co-cultured with autologous CD4⁺ T cells. Finally cell-free supernatants were harvested at 2, 3 and 6 days post-coculture (dpcc) and the viral content was assessed by a p24 assay. Data depicted in the left panel represent the mean ± standard deviations of quadruplicate samples from a representative single donor at 2 dpcc, whereas the kinetics of virus production for the same donor are displayed in the small insert. Results from multiple different donors are illustrated in the right panel (2 dpcc) (**: P < 0.01; ***: P < 0.001). B) IM-MDDCs were initially either left untreated or treated with EFV. Thereafter, cells were either left untreated or treated with Pam3Csk4. Data shown represent the mean ± standard deviations of quadruplicate samples from a single donor at 2 dpcc and are representative of 8 distinct donors. C) A similar experimental approach was used except that transfer studies were carried out with the X4-tropic strain NL4-3. Data shown represent the mean ± standard deviations of quadruplicate samples from a single donor at 2 dpcc and are representative of 3 different donors.

NF-κB is activated in IM-MDDCs following TLR2, 4 and 5 triggering. Cells were either left untreated (mock) or stimulated for 0, 2, 5, 15 and 30 min with the listed TLR ligands. Cells were then lysed and proteins were loaded on a 12% SDS-polyacrylamide gel, transferred to a membrane, and revealed by anti-phospho-IκBα, anti-IκBα, or anti-actin. Data from a single donor representative of 4 different donors are displayed.

TLR2 stimulation influences an early step in HIV-1 life cycle. A)IM-MDDCs were either left untreated (mock) or stimulated for 2 hours with the TLR2 agonist Pam3Csk4 (5 μg/ml) before or after exposure for 1 hour to NL4-3/Balenv. Supernatants were harvested at 72 hours post-infection and the viral content was evaluated by a p24 test. B) Cells were infected with VSV-G pseudotyped reporter viruses for 2 hours, washed twice and put in culture for 48 hours. Next, IM-MDDCs were either left untreated (mock) or stimulated for 2 hours with Pam3Csk4. Cells were then washed twice, cultured for 48 hours and lysed to monitor luciferase activity (expressed in relative light units/RLU). The data shown represent the mean ± standard deviations of quadruplicate samples from a single donor and are representative of 3 distinct donors.

Early steps in HIV-1 replication are modulated by TLR2 and 4 agonists. IM-MDDCs were either left untreated or stimulated for 2 hours with the TLR2 or 4 agonist. Cells were next pulsed with NL4-3/Balenv for 1 hour, washed twice and incubated at 37°C. Total DNA was extracted at 6, 24 or 48 hours post-infection and used for the detection and quantification of early reverse transcripts (A), late reverse transcripts (B) and integrated viral DNA (C) using a real-time PCR method. The number of HIV-1 copies was determined by a standard curve prepared with the NL4-3/Balenv vector. Data depicted in panels (A) and (B) represent the mean ± standard deviations of duplicate samples representative of two distinct donors whereas those illustrated in panel (C) represent the mean ± standard deviations of duplicate samples from 5 (Pam3Csk4) or 3 separate donors (LPS) (*: P < 0.05).