Rhesus macaques used in challenge studies.

<p>Rhesus macaques used in challenge studies.</p

Rectally applied PICLC induces rapid local and systemic immune changes.

<p>Macaques (n = 11) were bled 4 hours (4h) and 24h after rectal PBS vs. PICLC application. Either 4h (n = 6) or 24h (n = 5) after receiving treatment, rectal biopsies were also collected. (A) Blood mDCs were characterized at the indicated times post-treatment by their frequency (%Lin^-HLA-DR⁺CD11c^high) and expression of CD80 and CCR7. (B) Blood and (C) rectal CD4⁺ T cells were characterized by their expression of CD69 and CCR7 and the frequency of α₄β₇^highCD95⁺ cells. (D) mRNA levels of the markers shown were measured in rectal tissue. (E) In a separate group of macaques biopsied 5 weeks before (Pre) and 24 hours after (Post) a single rectal application of 2 mg (filled symbols) or 4 mg (open symbols) PICLC, mRNA levels of the markers from (D) were measured in cells isolated from rectal tissue. In (A-E), statistical analyses using the Wilcoxon Signed Rank test compared the post-PICLC time points with control post-PBS time points in each animal. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Rectal CD169 and β₇ expression correlate with systemic virus replication but do not predict infection.

<p>CD169 mRNA levels were measured in (A) rectal tissues and (B) inguinal LNs from macaques infected with SIVwt and SIVΔNef at different times post-infection and in uninfected macaques (baseline of the infected and other macaques that did not become infected within the study). Correlations between rectal CD169 level and viral replication in (C) SIVwt and (D) SIVΔNef-infected macaques are shown. (E) Relationship between baseline rectal CD169 expression and infection outcome for SIVwt and SIVΔNef. (F) Correlation between baseline rectal β₇ level and peak viral loads in SIVwt and SIVΔNef-infected macaques is shown. (G) Relationship between baseline rectal β₇ level and infection outcome. (H) Correlation between rectal CD169 and β₇ levels at baseline for all animals challenged with SIVwt and SIVΔNef. In (A-H), samples from all challenged macaques were not available at every time point. In (A-B), statistical analyses used the Kruskal Wallis test and Dunns post-test. In (A), Dunns comparisons not made were SIVwt W8 vs. SIVΔNef W20 and SIVwt W28 vs. SIVΔNef W6. In (C), (D), (F), and (H), Spearman correlation coefficient was determined. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

dsRNAs mediate changes in HIV location and T cell phenotype within co-cultures.

<p>HIV-pulsed DCs were co-cultured with autologous CD4⁺ T cells in the presence or absence of dsRNAs as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161730#pone.0161730.g001" target="_blank">Fig 1</a>. After 24 hours, cells were collected, surface stained, and intracellularly stained for p24. (A) Conjugate frequency within DC-T cell co-cultures was defined as the proportion of live CD3⁺CD4⁺ large cells in the co-cultures (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161730#sec002" target="_blank">Methods</a>). (B) Frequency of p24⁺ cells within the populations of free T cells (single) and conjugated T cells (conj). (C) CD69 GMFI and (D) the percentage of α₄β₇^highCD45RO⁺ CD4⁺ T cells were monitored within the single and conjugated T cell populations. For (A-D), 5 donors and the medians are shown, and the Friedman test with Dunns post-test was used to analyze the data. Dunns post-test excluded comparisons of PIC vs. piclcDC and PICLC vs. picDC. *<i>P</i><0.05, **<i>P</i><0.01.</p

Rectal PICLC modestly decreases SIV transmission but increases SIVΔNef replication in infected animals and promotes the vaccine effect.

<p>(A) Macaques were rectally challenged with 3000 TCID₅₀ SIVmac239 (SIVwt) coincident with (n = 7, “Coincident”) or 24 hours after (n = 7, “24h pre”) rectal PICLC or 24 hours after rectal PBS (n = 8). The fraction of PBS vs. PICLC-treated macaques that became infected is shown as a percent, and the number of animals infected is above each bar. (B) SIV RNA copies/ml were measured over time in each animal shown in (A). (C) Plasma viremia in infected animals in each group is shown at peak (highest observed viremia, 2–4 weeks post-challenge in all macaques, left) and 16 weeks post-challenge (middle), and as the area under the curve (AUC) of viremia over the whole observation period (right). The timing of PICLC administration is denoted by the symbols used in (B). (D) Macaques were rectally challenged with 3000 TCID₅₀ SIVmac239ΔNef (SIVΔNef) 24 hours after rectal PICLC (n = 7) or PBS (n = 7). 12 weeks after SIVΔNef challenge, all animals were rectally challenged with 3000 TCID₅₀ SIVwt. The fraction of PBS vs. PICLC-treated animals that became infected with SIVΔNef is shown as a percent, and the number of SIVΔNef-infected macaques is above each bar. (E) SIV RNA copies/ml of SIVΔNef (open symbols) and SIVwt (filled symbols) were measured over time in each animal. The two SIVΔNef-infected macaques not protected from SIVwt are shown in red. (F) SIVΔNef plasma viremia in each group is shown at peak (2–4 weeks post-challenge, left) and 16wks post-challenge (4 weeks post-SIVwt challenge, middle), and as AUC (right). The two macaques not protected from SIVwt are shown in red. (G) Fraction of SIVΔNef-infected animals that subsequently also became infected with SIVwt is shown by treatment group. In (C) and (F), P values were derived from Mann Whitney test comparisons of the control group with each of the treated groups.</p

Role of HIV capture by CD169 in dsRNA-mediated effects on HIV infection in DCs and DC-T cell co-cultures.

<p>HIV p24 expression was analyzed in DCs after pulsing (A) immediately (8–15 donors, median) or (B) after 24 hours of culture in media or in 10 μg/ml PIC or PICLC (6 donors, median). In a separate set of donors, CD169 surface expression was evaluated by flow cytometry as the (C) geometric mean fluorescence intensity (GMFI) on total DCs (17 donors, median) and (D) the percent of CD169^high DCs (17 donors, median). (E) CD169 mRNA expression was evaluated in DCs by RT-qPCR (8 donors, median). (F) The GMFI of CD169 on DCs was compared immediately before and after HIV pulsing (9 donors). In (A-E), statistical analyses that derived the P values shown on the panels were performed using the Friedman test in with post-tests performed using Dunns (significance shown by asterisks). In (B), Dunns post-test did not include PIC vs. piclcDC or PICLC vs. picDC comparisons. In (F) analyses were done using Wilcoxon Signed Rank test. *<i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p

PAU promotes CD169-independent HIV replication in DCs and DC-T cell mixtures.

<p>(A) picDCs and pauDCs were generated and pulsed with HIV and 7 day cultures were established as described for picDCs and piclcDCs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161730#pone.0161730.g001" target="_blank">Fig 1</a>. Results from HIV gag qPCR are shown for each condition as a percent of the infection in the iDC (left) or iDC-T cell (right) control. More than 9 donors are shown with the median for each condition. (B) GMFI of CD169 is shown on the differently matured vs. immature DCs for 10 donors (shown with the median). In (A-C), the statistical analyses used the Friedman test with Dunns post-test. In (A), Dunns post-test did not include PIC vs. pauDC or PAU vs. picDC comparisons. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Synthetic dsRNAs induce varying levels of DC maturation, HIV-capture molecules, and antiviral factors.

<p>The surface phenotype of DCs generated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161730#pone.0161730.g001" target="_blank">Fig 1</a> was assessed. (A) The GMFI of the indicated markers was measured on the total DC population (21–43 donors with median except for MAdCAM-1 and CD4 with 5–6 donors). (B) The frequency of CCR5^high DCs within the total DC population (41 donors with median). (C) mRNA levels of IFNα, A3A, A3G, and CD317 in DC lysates (8 donors with median). In (A-C), statistical analyses that derived the P values shown on the panels were performed using the Friedman test in with post-tests performed using Dunns (significance shown by asterisks). All Dunns comparisons were performed. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Synthetic dsRNAs block HIV replication in DC-T cell mixtures dependent on timing of DC stimulation and virus capture.

<p>Immature DCs (iDCs) were exposed to 10 μg/ml PIC or PICLC for 48 hours to produce picDCs and piclcDCs, respectively, or were maintained as iDCs by 48 hours of culture in medium. (A) iDCs were pulsed with HIV, washed, and re-cultured in the presence of medium (iDC) or 10 μg/ml PIC (PIC) or PICLC (PICLC). picDCs and piclcDCs were similarly pulsed and re-cultured in medium (picDC, piclcDC). After 7 days, the cells were lysed, and HIV DNA was measured by <i>gag</i> qPCR (left). Pulsed DCs were cultured with autologous CD4⁺ T cells for 7 days before HIV DNA was measured (right). iDC-T cell co-cultures were left in medium or had PIC/PICLC added as for iDCs alone. For DCs and DC-T cell co-cultures, ≥9 donors are shown with the medians. (B) Responsiveness of picDCs and picDC-T cell co-cultures to exogenous PIC was determined by re-culturing pulsed picDCs (picDC) or picDCs and autologous CD4⁺ T cells (picDC-T) in the presence of 10 μg/ml PIC (+ PIC) vs. medium (- PIC). Four donors and the medians are shown. (C) iDCs (in the presence/absence of 10 μg/ml PIC or PICLC), picDCs, and piclcDCs were infected directly in the plates (not pre-pulsed) with HIV in the absence (left) or presence (right) of autologous CD4⁺ T cells, and HIV DNA was measured in cell lysates after 7 days (7–8 donors and the median). (D) CD4⁺ T cells were infected (not pulsed) with HIV in the presence of 50 U/ml IL-2 and the absence (med) or presence of 10 μg/ml PIC or PICLC before HIV DNA was measured in cell lysates after 7 days (6–8 donors and the median). Statistical analyses that derived the P values shown on the panels were the Kruskal Wallis test in (A) and the Friedman test in (B-D). In all cases, the Dunns test was used for pairwise comparisons, shown as asterisks. All Dunns comparisons were made except in the following cases: In (A) and (C), we did not compare PIC vs. piclcDC or PICLC vs. picDC. In (B), we did not compare picDC-PIC vs. picDC-T+PIC or picDC+PIC vs. picDC-T-PIC. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Additional file 1: Figure S1. of Vaccination with poly(IC:LC) and peptide-pulsed autologous dendritic cells in patients with pancreatic cancer

Cytokine secretion by DCs. Frozen DCs obtained from patients were thawed and cultured with GM-CSF/IL-4 overnight before adding poly (IC:LC) for maturation overnight. The supernatant was collected after 16 hrs. and evaluated for cytokines IL12 (upper panel), and IL10 (lower panel) as per the manufacturer’s protocol. Figure S2. PFS and OS details. The raw data showing details of progression free survival (PFS) and the median overall survival (OS) is presented in tabular form. Figure S3. Gating scheme for flow cytometry analysis. PBMC was obtained from patients post vaccination and cryopreserved cells were stained using the multiple fluorochrome-conjugated antibodies. Cells were gated based on singlets (FSC-A vs FSC-H), size (SSC vs FSC-H), a live-dead stain (L/D), and subsequently markers to determine specific cell phenotypes. A) CD3+ T cells were phenotyped for CD4 and CD8. B) CD19 B cells were identified. C) NK cells were identified based on their CD56 and CD16 expression. The data was acquired using BD FACS Aria and analyzed using FlowJo software. (PDF 351 kb