Cytotoxic activity of CD8⁺ T lymphocytes activated by MVA-B-infected MDDC.

<p>Effector cells were autologous monocyte-depleted PBMC co-cultured for 6 days with MDDC mock-treated or infected with MVA or MVA-B. After this co-culture period, CFSE staining of effector cells was performed and then were cultured for 4 h with target cells at a 10∶1 ratio. Target cells were autologous CD8-depleted T cells infected with a primary HIV-1 isolate and their corresponded to the phenotype CFSE-negative, CD3-positive CD8-negative. In (A) the flow cytometry analysis is shown. In (B), the mean (±SEM) count of target CD4⁺ T cells is a representative result of three independent experiments (performed in triplicates) is depicted. (*) p<0.05.</p

Dose-response analysis of HIV-1-specific T-cell proliferation and cytokine secretion in response to autologous MVA-B-infected MDDC.

<p>T cell proliferation and cytokine secretion (IFN-γ, IL-2 and TNF-α secretion) in response to autologous MDDC infected with different doses of MVA and MVA-B (from 0.003 to 3.3 PFU/MDDC) are depicted. A representative result of 2 independent experiments is shown. Error bars represent the SEM from the mean of the replicates (n = 2). Co-culture procedure and methods followed are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019644#pone-0019644-g004" target="_blank">Figure 4</a>.</p

HIV-1-specific T-cell proliferation and cytokine secretion induced by autologous MDDC infected with MVA-B.

<p>T cell proliferation in response to autologous MDDC infected with MVA-B at 1 PFU/MDDC in a 6-day co-culture was assessed in triplicates using the CFSE proliferation assay. In (A), a representative (out of 5 independent experiments) flow cytometry plots showing CFSE dilution in gated CD3⁺ CD8⁺ T lymphocytes after <i>in vitro</i> stimulation with MVA- and MVA-B-infected MDDC. In (B), the % of CFSE^low cells in the CD3⁺ CD8⁺ and CD3⁺ CD8⁻ (CD4⁺) gates responding to MVA-, MVA-B-infected and soluble HIV1p24-pulsed MDDC are shown. Error bars represent the SEM from the mean of 5 patients (n = 5). (***) p = 0.005, (**) p<0.01. (C) Cytokine secretion after co-culture of MVA or MVA-B-infected MDDC and CD8⁺ T lymphocytes. Concentrations of IFN-γ, IL-2, TNF-α, MIP-1β and IL-6 are represented. (***) p<0.005, (**) p<0.01, (*) p<0.05. Error bars represent the SEM from the mean of 5 patients.</p

Evaluation of MDDC maturation and viability.

<p>(A) Viability assay after MVA and MVA-B infection. Viability was expressed as the percentage of live cells in front of the total basal population. This experiment was performed in triplicates. (*) p<0.05 (B) At 16 h post-infection, in the absence of maturation cocktail, CD86 expression was measured as MDDC maturation surface marker by FACS. CD86 (measured MFI) is represented in a dose-response graphic versus PFU/MDDC at a dose range of 0.003–3.3 PFU/MDDC. This is a representative result of 2 independent experiments. (C) Expression of maturation markers and viability after MVA-B infection at 0.3 and 10 PFU/MDDC. The maturation cocktail was added during 48 h. Maturation markers and viability were measured at 48 h post-infection. HLA-DR, HLA-ABC, CD86, CD80, CD83, CD25 expression was measured as DC maturation surface markers by FACS. Viability was measured by dual Annexin V and 7AAD staining. Live cells were negative for both markers, dead cells were positive for 7AAD and apoptotic cells were positive for Annexin V and negative for 7AAD. Results are represented as the relative percentage calculated with respect to the value obtained with uninfected MDDC: (value of infected MDDC/value of uninfected MDDC)×100. The value was MFI from HLA-DR, HLA-ABC and CD86 or percentage of CD80, CD83, CD25 and viability. (*) p<0.05, (**) p<0.01, (***) p<0.005. Error bars represent the standard error (SEM) from the mean of the replicates (n = 3). (D) Correlation between relative percentage of viable cells and relative percentage of CD86.</p

Antigen presentation study.

<p>(A) Phagocytosis assay. Immature MDDC were labeled with CFSE and infected with MVA at a MOI of 10 PFU/MDDC for 1 hour. They were then extensively washed and mixed with uninfected immature MDDC (ratio 1∶2) and maturation was induced for 48 h. Phagocytosis of apoptotic bodies resulting from MVA-infected MDDC by uninfected MDDC were detected by flow cytometry. Density plots showing infected MDDC, detected as CFSE⁺ CD80⁻ (<i>left box</i>), uninfected MDDC, detected as CFSE⁻ CD80⁺ (<i>middle box</i>) and CD80⁺ MDDC that ingested apoptotic CFSE-labeled MDDC during the incubation period, detected as CFSE⁺ CD80⁺ (<i>right box</i>). These data are representative of two individual experiments. (B) Phagocytosis assessed by fluorescence microscopy. Phagocytosis of apoptotic bodies resulting from MVA-infected MDDC by uninfected MDDC were visualized by fluorescence microscopy. MVA-B infected MDDC were CFSE⁺ CD80⁻ (green colour), uninfected matured MDDC were stained CFSE⁻ CD80⁺ antibody (red colour), nucleus was visualized with Hoechst staining (blue colour) and phagocytosis was observed as CFSE stained vacuoles (green) inside the MDDC CD80 positive cells (red). As negative controls of phagocytosis, uninfected MDDC and infected MDDC cultured separately were used. These data are representative of 2 independent experiments. (C) Cross-presentation induced proliferation of CD8⁺ T cells. Representative dot plots showing the proliferation rate of CD8⁺ T cells after co-culture between infected MDDC with autologous lymphocytes. The white plots correspond to a MVA-B infection model at 0.3 PFU/MDDC. The black plots correspond to infection of immature MDDC (two thirds of the initial culture; cultured during 72 hours) with apoptotic bodies (obtained after infection of MDDC –one third of the initial culture- with MVA-B at a dose of 10 PFU/MDDC and during a period of 72 hours). The grey plots correspond to infection between immature MDDC and apoptotic bodies obtained as described above and subsequently heat-inactivated at 55°C for one hour. Subsequently, infected MDDC were collected and co-cultured with CFSE labeled autologous lymphocytes in triplicates. Six days after, proliferation was tested by flow cytometry. These data are representative of 3 individual experiments.</p

MDDC infected with MVA-B expressed intracellular Gag protein in a dose-dependent manner.

<p>MDDC were infected with serial three-fold dilutions of a stock of MVA-B, from 0.003 to 10 PFU/MDDC. After MVA-B infection of MDDC, Gag and CD86 expression was measured by flow cytometry. (A) Intracellular viral protein expression in MDDC observed by fluorescence microscopy. MDDC at 16 h post-infection not infected (Mock) or infected with MVA-B (at 10 PFU/MDDC) were fixed-permeabilized and viral proteins were stained indirectly with FITC (green) and nucleus was visualized using Hoechst (blue). These data are representative of two individual experiments. (B) Density plots showing the percentage of Gag and CD86 double positive MDDC from 0.003 to 3.3 doses (PFU/DC). (C) Linear regression of percentage of double positive CD86 Gag cells versus viral dose (PFU/MDDC). A representative result of 3 independent experiments is shown. (D) Intracellular Gag expression in MDDC observed by fluorescence microscopy. MDDC were infected with MVA-B and MVA (at 10 PFU/MDDC) and at 16 h post-infection were stained with PE-CD86 (red) and fixed-permeabilized and stained with FITC-anti-HIV-Gag (green) antibody and nucleus was stained with Hoechst (blue). These data are representative of two individual experiments.</p

Levels of α-defensins 1-3 produced by imMDDC from HIV-infected individuals.

<p>(A) Comparison between the secreted levels of α-defensins 1-3 by imMDDC from HIV-controllers (elite controllers and viremic controllers; n = 19) and HIV non-controllers (viremic non-controllers and patients with HAART; n = 20). (B) Secreted levels of α-defensins 1-3 by imMDDC from healthy non-infected (NI; n = 15), elite controllers (ELITE; n = 4), viremic controllers (VC; n = 15), viremic non-controllers (VNC; n = 11) and patients receiving HAART (HAART; n = 9). Dots indicate each patient and lines represent median ± interquartile ranges.</p

Correlation between CD86 expression on MVA-B-infected MDDC and HIV-1-specific CD8^<b>+</b> T-cell proliferation and cytokine secretion.

<p>MDDC were infected with MVA-B at serial three-fold dilutions (from 0.003 to 3.3 PFU/MDDC) and expression of the maturation surface marker of MDDC, CD86, measured by flow cytometry at 16 h post-infection in the absence of maturation cocktail was assessed. Subsequently, MVA-B-infected MDDC matured with cytokine cocktail were co-cultured for 6 days with autologous lymphocytes and HIV-1-specific CD8⁺ T proliferation and cytokine secretion were measured. A representative result of 2 independent experiments is shown.</p

Chemotaxis assay.

<p>Chemokine-induced migration of infected mature MDDC was tested 48 h and 72 h after infection in a chemotaxis assay (n = 6). Entire populations that migrated toward CCL9 and CCL21 were collected after incubation and counted during one minute by flow cytometry. The number of migrated DC is depicted in percent of the initial input. Subsequently, infected (MVA and MVA-B) and uninfected MDDC that migrated in the chemotaxis assay were collected and co-cultured with CFSE labeled autologous lymphocytes at a 1∶40 ratio (5×10³ MDDC: 2×10⁵ lymphocytes/well). After a period of 6 days, proliferation was tested by flow cytometry. (A) Percentage of CCL19 specific migration of MVA-B-infected MDDC 48 hours after infection. Error bars represent the mean (±SEM) from 6 independent experiments with 6 different HIV-1 patients (n = 6) performed in triplicates. (B) Percentage of CCL19 specific migration of MVA and MVA-B-infected and uninfected MDDC (Mock) 48 and 72 h post-infection and maturation. Error bars represent the mean (±SEM) from 3 independent experiments with 3 different HIV-1 patients (n = 3) performed in triplicates. (C) Representative flow cytometric plots showing CFSE dilution in gated CD3⁺ CD8⁺ lymphocytes after co-culture with MVA and MVA-B infected MDDC that were able to migrate in the chemotaxis assay. One representative result of 2 independent experiments is shown. Error bars represent the mean (±SEM) (n = 3). (D) Percentage of CFSE^low cells in the CD3⁺ CD8⁺ gate, observed in one sample. Error bars represent the SEM from the mean triplicates (***) p = 0.005.</p

Cytokine, chemokine and IFN-α production by MDDC upon infection with MVA and MVA-B.

<p>MDDC were infected with MVA or MVA-B at high doses (3.3 PFU/MDDC) at 16 h post-infection (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019644#pone-0019644-g003" target="_blank">Figure 3A</a>) and at low doses (0.33 PFU/MDDC) at 48 h post-infection (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019644#pone-0019644-g003" target="_blank">Figure 3B</a>) and at 48 h post-infection in the presence of maturation cocktail (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019644#pone-0019644-g003" target="_blank">Figure 3C</a>) and secreted mediators were assessed by Luminex technology. Results are expressed as mean ± SD from 4 independent experiments. Error bars represent the SEM from the mean of the replicates (n = 4). (*) p<0.05, (**) p<0.01, (***) p<0.005.</p