Phagocytosis of CVB-infected Min6 cells results in upregulation of co-stimulatory markers in BDCA1⁺ mDC and BDCA3⁺ mDC, and co-inhibitory marker PD-L1 in BDCA1⁺ mDCS.

<p>DCs were stimulated o/n as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121670#pone.0121670.g002" target="_blank">Fig. 2A</a> and subsequently analyzed for expression of indicated cell surface markers using flow cytometry. Shown are averages of mean fluorescence intensity (MFI)-isotype values of >3 donors + SEM. * p<0.05, ** p<0.01 determined by ANOVA and post-hoc Tukey test.</p

BDCA1⁺ mDCs more efficiently phagocytose murine Min6 cells compared to BDCA3⁺ mDCs.

<p><b>A</b>) BDCA1⁺ mDCs or <b>B</b>) BDCA3⁺ mDCs were co-cultured overnight (o/n) with PKH67-labeled mock- or CVB-infected Min6 cells (M6/M and M6/CVB, respectively) stained for the CD11c or BDCA3 and analyzed by flow cytometry on viable, single cells (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121670#pone.0121670.s001" target="_blank">S1 Fig</a>). Percentages in upper right corner represent the percentage of DCs that has engulfed Min6 material. This was calculated as follows: percentage PKH⁺ DCs/total DCs (i.e. total CD11c positive cells or total BDCA3 positive cells) <b>C</b>) DCs were analyzed as in A) and B), shown is average + SEM for >3 donors. * p<0.05, *** p<0.001 determined by ANOVA and post-hoc Tukey test.</p

Type I and type III IFN responses are induced in mDC subsets upon encounter of CVB-infected cells.

<p><b>A</b>) DCs were co-cultured with mock- or CVB-infected Min6 cells (M6/M or M6/CVB), infected with CVB3 (MOI 50), stimulated with poly I:C, or left unstimulated (medium; Med) and after o/n incubation supernatant was harvested and analyzed for production of IFN-α2 (left panel) or IFN-λ1 (right panel). Shown are data from 4 (IFN-α2) and 3 (IFN-λ1) different donors. <b>B</b>) DCs were stimulated as in A) and after 6 hours mRNA expression was analyzed by qPCR. Shown are data from 4 donors (B, corresponding symbols represent the same donor). * p<0.05, ** p<0.01, *** p<0.001 determined by ANOVA and post-hoc Tukey test.</p

Cytokine and chemokine production in BDCA1⁺ and BDCA3⁺ mDCs upon encounter of mock- or virus-infected cells.

<p>DCs were stimulated o/n as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121670#pone.0121670.g002" target="_blank">Fig. 2A</a> and supernatant was analyzed for production of pro-inflammatory cytokines and chemokines. Data represent average of 4 independent experiments using different donors + SEM. * P<0.05, ** P<0.01, ***p<0.001 comparing M6/CVB versus M6/M or poly I:C versus medium; # p<0.05 Min6/CVB versus poly I:C; $ p<0.05 in BDCA1⁺ versus BDCA3⁺ mDCs with the same stimulus as determined by ANOVA and post-hoc Tukey test.</p

Route of CpG administration determines induction of tumor-specific CTL.

<p>(<b>A</b>) Representative dot plots of CD8⁺OVA-Kb tetramer⁺ cells of individual mice. Mice bearing B16OVA tumors (6–9 mm) were subjected to cryo ablation alone or received additional CpG injections via the indicated routes. Ten days after tumor destruction, cells from spleen and tumor draining lymph nodes were isolated and re-stimulated with IFN-γ-treated γ-irradiated B16OVA tumor cells. Cultures were cleaned by a Ficoll step after 3–4 days of culture and at day 8–9 cells were analyzed for the presence of tumor-specific CTL using APC-labeled OVA-K_b tetramers. Cells were gated on CD8⁺ T cells. Data from one representative mouse per group is shown. (<b>B</b>) Quantitative analyses of collective data as shown in (A) (mean levels of 2 separate experiments (4–6 mice per group/experiment)). * indicates significant differences (p<0.05) of the indicated group compared to all other groups.</p

Quantity and quality of lymph node DC.

<p>(<b>A</b>) Absolute numbers of CD11c⁺ cells in the draining lymph nodes 2 days after ablation. Tumor-bearing mice were left untreated (NT) or were subjected to cryo ablation (cryo). In addition, some groups of mice received CpG via the indicated routes. Brackets indicate significant differences (p<0.05) between indicated groups (n = 4–6/group, 2 similar experiments). (<b>B</b>) Uptake of antigen after cryo ablation. OVA-Alexa488 was injected in B16OVA tumors (6–9 mm) just prior to cryo ablation to monitor the fate of antigen after in situ tumor destruction. Mice were additionally treated with CpG via the indicated routes. Two days after ablation, the uptake of antigen was analyzed in CD11c⁺ cells enriched from pools of tumor-draining lymph nodes. Numbers in the panels indicate the percentage of OVA⁺ of all CD11c⁺ cells. Data from one representative mouse is shown per group. (<b>C</b>) Quantitative analyses of replicates of experiments as shown in (B) (5 mice/group, representative of 2 experiments). (<b>D</b>) Uptake of antigen and CpG by CD11c⁺ cells monitored after p.t. and i.v. CpG-Cy5 administration and intra-tumoral injection of OVA-Alexa488. Data from one representative mouse is shown (n = 4/group). (<b>E</b>) The expression of CD80 on CD11c⁺ antigen-loaded cells. * indicates significantly different (p<0.05) from all other groups (n = 5/group, 2 similar experiments).</p

Route of CpG administration determines efficacy of antitumor immunity.

<p>(<b>A</b>) Kaplan-Meier survival curves of naïve mice versus mice that have been treated with cryo ablation alone, or in combination with concurrent CpG administration via the indicated routes. Established B16OVA tumors on the right femur were treated with cryo ablation alone or in combination with CpG administrations via different routes: p.t., i.v., or s.c. contra lateral of the tumor. Forty days later, naïve and tumor-free mice (8–13 mice per group) received a s.c. re-challenge with tumor cells (25.000 B16OVA cells). (<b>B</b>) Tumor size determined every 2–3 days after treatment. Data is representative of two independent experiments.</p

Replication of human enteroviruses in primary human myeloid BDCA1⁺ dendritic cells.

<p>A) Freshly isolated BDCA1⁺ mDCs were infected with indicated viruses at an MOI of 2 and replication was assessed at indicated time points by endpoint titration. B) Freshly isolated mDCs were stained with indicated antibodies or corresponding isotypes and analyzed using flowcytometry. C) mDCs were infected as indicated (MOI 50) and after 18 h infection 3D polymerase protein expression was assessed by western blot analysis. D) mDCs infected as indicated were harvested 18 h after infection using cold PBS and amount of dsRNA was analyzed using flowcytometry. E) mDCs infected as in D) were tested for cell viability using Annexin V/7-AAD double staining. Statistical significance was determined using ANOVA and post-hoc Tukey test. Shown are representative experiments of more than 3 independent experiments using different donors (A–D) or average of 3 independent experiment (E) (mean values+ SEM). * p<0.05; ** p<0.001.</p

EV, but not CVB infection impairs TLR-induced responses in BDCA1+ mDCs.

<p>A) Freshly isolated BDCA1⁺ mDCs were infected as indicated (MOI 50) and after 16 h all cells were stimulated with poly I:C (20 µg/ml). After an additional 24 h expression of cell surface markers was determined using flowcytometry. Data is shown as mean fluorescent intensity minus isotype control. B) Supernatant taken from mDCs infected and stimulated as in A) was analyzed for production of IL6 and TNF-α. Shown are averages of 2 experiments using different donors. Statistical significance determined by Students T-test, * p<0.05. **p<0.01.</p

EV1 infection results in phenotypic maturation and production of IL6 and TNF-α.

<p>A) Freshly isolated BDCA1⁺ mDCs were infected as indicated (MOI 50) and after 18 h expression of cell surface markers was determined using flowcytometry. B) Supernatant taken from mDCs infected as in A) was analyzed for production of IL6 and TNF-α. Data shown (mean+SEM) are averages of at least 8 different experiments using different donors. Statistical significance determined by Students T-test, * p<0.05; **p<0.01. MFI; mean fluorescence intensity.</p