PGE₂ exposure induced a partial activation of healthy purified MDCs and PDCs.

<p>FACS sorted MDCs and PDCs obtained from healthy donors were cultured in separate wells for 24 hours in 1% plasma medium with or without the presence of TLR3 ligand (Poly I:C) for MDCs or TLR9 ligand (C CPG) for PDCs, 10 ng/ml PGE₂, 10 ng/ml CXCL8 (IL-8), or 10 ng/ml PGE₂ in combination with 10 ng/ml CXCL8 (A). The MDCs (A) and PDCs (B) were harvested and labeled by direct conjugated antibodies against CD40, CD83, CD86, CCR5, CCR7, PDL-1, ICOSL, DCIR, and B7H3 and analyzed using multi-color flow cytometry. The data are normalized to the control medium and paired t test was performed for calculation of p values. Statistically significant differences between individuals with PDAC and healthy controls are indicated as; * = p<0.05, ** = p<0.005, *** = p<0.001.</p

Mucosal influenza HA-specific IgA in nasal wash samples.

<p>Mice were immunized with a split influenza vaccine (Vaxigrip) containing the A/H1N1/Brisbane/2007 strain with or without adjuvant. The groups were immunized three times with three-week intervals. (A) Mucosal influenza HA-specific IgA in the nasal wash samples is shown. Median and range is shown for each group and statistical significance compared to the non-adjuvanted group is indicated, **p<0.003.</p

Decreased levels of MDCs and PDCs in patients with PDAC.

<p>PBMCs isolated from individuals with pancreatic duct adenocarcinoma (PDAC) (1 week pre and 12 weeks post surgery) and healthy age matched volunteers were analyzed for DC levels by flow cytometry. The PBMCs were stained with Linage cocktail, HLA DR, CD11c, and CD123 direct conjugated mabs to distinguish all DC subsets from the rest of the cells. The DC levels were calculated from the total amount of PBMCs and compared between the different groups using nonparametric Wilcoxon signed rank test used for paired data and Mann–Whitney test for calculation of p values. Statistically significant differences between individuals with PDAC and healthy controls are indicated as; * = p<0.05, ** = p<0.005, *** = p<0.001.</p

Increased B7 family expression profile on MDCs and PDCs in patients with PDAC.

<p>Blood MDCs (HLA DR⁺CD11c⁺Lin⁻) and PDCs (HLA DR⁺CD123⁺Lin⁻) were detected in PBMCs obtained from PDAC patients pre (1 week) and post (12 weeks) surgery and healthy age matched individuals. Both DC subsets were investigated for the expression of co-stimulatory molecules from B7 family using direct conjugated antibodies against PDL-1 (A), ICOSL (B) and B7H3 (C) and analyzed using 8 color flow cytometry. Mean fluorescence intensity (MFI) values or present positive MDCs and PDCs were compared between the different groups using nonparametric Wilcoxon signed rank test used for paired data and Mann–Whitney test for calculation of p values. Statistically significant differences between individuals with PDAC and healthy controls are indicated as; * = p<0.05, ** = p<0.005, *** = p<0.001.</p

Serum IgA titers against three different influenza stimuli (rHA, Vaxigrip and vaccine against influenza) after nasal vaccination. Median, range and response frequency in each group are shown.

<p>Abbreviations: rHA =  recombinant hemagglutinin, NA  =  Not applicable.</p

Chemokine receptor expression profile on MDC and PDCs in patients with PDAC.

<p>Blood MDCs (HLA DR⁺CD11c⁺Lin⁻) and PDCs (HLA DR⁺CD123⁺Lin⁻) from PDAC patients pre (one week) and post (12 weeks) surgery were compared to age matched controls. The DC subsets from each group were stained using direct conjugated antibodies against CCR2 (A), CCR5 (B), CCR6 (C), and CCR7 (D) and detected by multi-color flow cytometry. Mean fluorescence intensity (MFI) values or the amount positive MDCs and PDCs in percentage were compared between the different groups using nonparametric Wilcoxon signed rank test used for paired data and Mann–Whitney test for calculation of p values. Statistically significant differences between individuals with PDAC and healthy controls are indicated as; * = p<0.05, ** = p<0.005, *** = p<0.001.</p

The C type lectin DCIR was down regulated on MDCs and PDCs in PDAC patients.

<p>Dendritic cell subsets, MDCs (HLA DR⁺CD11c⁺Lin⁻) and PDCs (HLA DR⁺CD123⁺Lin⁻), were distinguished form PBMCs obtained from PDAC patients pre (1 week) and post (12 weeks) surgery and age matched control individuals by flow cytometry. Changes in DC phenotype were detected using direct conjugated antibody for dendritic cell immunoreceptor (DCIR). Mean fluorescence intensity (MFI) values from the patients included in the different groups were compared using nonparametric Wilcoxon signed rank test, used for paired data and Mann–Whitney test for calculation of p values. Statistically significant differences between individuals with PDAC and healthy controls are indicated as; * = p<0.05, ** = p<0.005, *** = p<0.001.</p

IL-2 release from splenocytes, stimulated with different antigens.

<p>Mice were immunized with a split influenza vaccine (Vaxigrip) containing the A/H1N1/Brisbane/2007 strain with or without adjuvant. The groups were immunized three times with three-week intervals. Splenocytes were analyzed regarding IL-2 release after influenza stimuli. (A) IL-2 responses against influenza A/H1N1/Brisbane/59/2007 (whole virus). (B) IL-2 responses against influenza A/H1N1/ California/04/2009 (whole virus). (C) IL-2 responses against influenza nucleoprotein-peptides from A/H1N1/Brisbane. Median and range is shown for each group and statistical significance compared to the non-adjuvanted group are indicated, *p<0.05, **p<0.01 and ***p<0, 001.</p

Exposure to plasma from PDAC patients induced activation of MDCs and PDCs from healthy individuals.

<p>MDCs and PDCs from healthy blood donors were freshly isolated from PBMCs using direct conjugated antibodies followed by FACS sorting. (<b>A</b>) The DC subsets were cultured in separate wells for 24 hours in 1:4 diluted pooled human plasma medium obtained from PDAC patients or healthy controls. The cells were harvested and stained using direct conjugated antibodies against CD40, CD83, CD86, CCR5, CCR7, PDL-1, ICOSL, DCIR and B7H3 and analyzed using multi-color flow cytometry. (<b>B</b>) MDCs were cultured in separate wells for 24 hours in 25% single human plasma medium obtained from PDAC patients (PC013, PC021, PC045, or PC065) and one representative age matched healthy control (C2). The cells were harvested and labeled by direct conjugated antibodies against CD40, CD83, CD86, CCR5, CCR7, PDL-1, ICOSL, DCIR, and B7H3 (presented as non normalized data as the control plasma diminished the levels in some experiments (C) and analyzed by multi-color flow cytometry. Mean fluorescence intensity (MFI) or present positive DCs were normalized to controls and paired t-test was used for calculation of p values. Statistically significant differences between individuals with PDAC and controls are indicated as; * = p<0.05, ** = p<0.005, *** = p<0.001.</p

HAI and NT-antibody titers against influenza A/H1N1/Brisbane in serum after the final immunization.

<p>Mice were immunized with a split influenza vaccine (Vaxigrip) containing the A/H1N1/Brisbane/2007 strain with or without adjuvant. The groups were immunized three times with three-week intervals. The HAI and NT-antibody reactivity against influenza A/H1N1/Brisbane in serum after the final immunization are shown. Median and range is shown for each group. Values <10 in HAI was set as 5 and values <100 in the NT-assay was set as 20. Statistical significances compared to the non-adjuvanted group are indicated, *p<0.017 and **p<0.003.</p