DC-driven signaling response upon fungi, C-lectin and TLR agonists.

<p>Transcriptional analysis was performed on DCs after 4 hours of stimulation with Curdlan, Zymosan, Mannan, Sc cells and spores or without any stimuli. Clustering of Pathway Enrichment Factors (PEFs) were obtained from the FET analysis of challenged DC samples and compared with those obtained on DC stimulated with whole fungi or the publicly available dataset of LPS or R848 stimulated DCs. Colored spots indicate significant (p≤0.05) up- (red) or down- (green) regulation. The colors of the dendrogram indicate the percentages of the tree support (significance), from 50% (pink) to 100% (black).</p

Dectin-1 modular structures in response to ligand concentration determine differences in fungi recognition.

<p>(A) Clustering selection of Pathway Enrichment Factor (PEFs) obtained from the FET analysis on DC samples stimulated with Curdlan in time. Colored spots indicate significant (p, 0.05) up- (red) or down- (green) regulation. The colors of the dendrogram indicate the percentages of the tree support (significance), from 50% (pink) to 100% (black). (B) <i>CLEC7A, SYK, RAF1</i> and <i>CARD9</i> gene expression on DCs upon Curdlan stimulation. Gene expression was assessed by RT- PCR on DCs stimulated with Curdlan for 5, 15, 30, 60 and 120 minutes. (mean ± SD, N = 3). (C) DC modulation of gene expression of CLR or TLR signalling. The specificity of signals to different PRR agonists was assessed by real-time PCR on DCs stimulated with Curdlan or LPS for 5, 15, 30, 60, 120 and 240 minutes (mean ± SD, N = 3). (D) The differences at the sensing level, between <i>C. albicans</i> hyphae and Sc cells stimulation were addressed by RT-PCR. <i>CLEC7A</i> and <i>SYK</i> gene expression on DCs upon challenge with <i>C. albicans</i> hyphae and <i>S. cerevisiae</i> cells was assessed on DCs stimulated for 1, 2, 4, 8 and 12 hours (mean ±SD, N = 3): **p<0.01, ***p<0.001. (E) <i>CARD9</i> gene expression on DCs upon challenge with Sc cells. Gene expression was assessed in RT-PCR on DCs stimulated over time (mean ± SD, N = 3); FC, fold change was calculated by comparing the stimulated condition at various time points with the unstimulated control after normalization to the expression of the houskeeping gene GAPDH.</p

Microarray experiments on DCs challenged with fungi and single agonists.

<p>Microarray experiments on DCs challenged with fungi and single agonists.</p

Flowchart of DC-SIGN and Dectin-1 signalling.

<p>Following DC-ATLAS pathway structure, receptor/sensing modules, transduction modules and the outcome modules were represented for (A) Dectin-1 signaling and (B) DC-SIGN signaling. For the outcome modules the cytokines measured within the manuscript are indicated (bold). These graphs present both the vertical and horizontal resolution of DC-TLAS pathways. For the complete BCML pathways, please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042430#pone.0042430.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042430#pone.0042430.s002" target="_blank">S2</a>. Note: the TLP1-mediated signalosome upon DC-SIGN engagement recently described has not been drawn to simplify view of the modules.</p

Immune response (IR) genes activated by different cell wall components and <i>Saccharomyces cerevisiae</i> in DCs.

<p>Transcriptional analysis was performed on DCs after 4 hours of stimulation with Curdlan, Zymosan, Mannan and <i>S. cerevisiae</i> yeast or without any stimuli. (A) Venn diagram of upregulated DEGs. (B) IR differentially regulated genes (p≤0.05) commonly expressed among the four stimulation conditions. (C) IR differentially regulated genes (p≤0.05) commonly expressed among Zymosan and <i>S. cerevisiae</i> yeast stimulation condition and not affected upon single cell wall components stimulation. (D) Cytokine detection assays were performed for IL-1β, TNFα, IL-6 and IL-12p70 measurement on supernatants of DCs stimulated for 18 hours with Sc cells or zymosan; **p<0.01.</p

Immunoblot analyses of Dectin-1 and Syk upon fungal recognition.

<p>DCs were stimulated with live <i>S. cerevisiae</i> (Sc) cells, <i>Candida</i> (A) or different concentration of Curdlan (µg/ml, B, C) for the time indicated. Cell lysates were isolated and immunoblotted with the specific primary antibodies and reprobed with anti-β-tubulin antibody to control for the equal loading of cell lysates. Representative blots are shown. Quantitative analysis of protein expression is summarized in bar graphs, presented as mean integrated intensity of specific bands normalized to β-tubulin ± SD of 3–5 independent experiments.</p

Comparison of total LFA-1 binding partners (derived from mild and stringent lysis conditions) in monocytes and DCs.

<p>(A) Venn diagrams of proteins identified in monocytes (blue) and DCs (yellow). Numbers of identified proteins, as well as common proteins are indicated. (B) PPI network of directly interacting LFA-1 (heterodimer formed by and αL (ITGAL) and β2 (ITGB2) chain) binding partners derived from monocytes in stringent lysis and mild lysis conditions. A network was generated by uploading the protein names to the database of functional protein interactions (STRING v9.05) and retrieving experimental proven direct protein-protein interactions. The resulting network was drawn by the authors. Based on our MS data, we could retrieve 3 high confidence networks (score 0.6), with a maximum of 19 directly interconnected nodes. Blue nodes represent proteins identified in mild lysis conditions, and red nodes represent proteins identified in stringent lysis conditions.</p

Protein-protein interaction network of directly interacting LFA-1 binding candidates derived from DCs.

<p>(A) Comparison of immunoprecipitated LFA-1 from day 6 imDCs in mild and stringent lysis conditions. Venn diagrams of proteins identified in DCs in stringent (red) and mild (blue) IP conditions. Numbers of identified proteins, as well as common proteins (yellow) are indicated. (B) A network of LFA-1 (heterodimer formed by an αL (ITGAL) and β2 (ITGB2) chain) binding partners was generated by fusing the data sets derived from mild and stringent IP conditions in DCs, uploading the protein names to the database of functional protein interactions (STRING v9.05) and retrieving experimental proven direct protein-protein interactions. (ribosomal and histone complexes were removed for better visualization of proteins involved in integrin function). The resulting network was redrawn by the authors. Based on our MS data, we could construct a high confidence network (score 0.6) containing 78 nodes and 154 connections. Blue nodes represent proteins identified in mild lysis conditions, and red nodes represent proteins identified in stringent lysis conditions. Green nodes represent proteins identified both in mild and stringent IP condition. * indicates an interaction that was not present in the STRING database (version 9.1) with experimental support, but this node and interactions were added by the authors based on the current literature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149637#pone.0149637.ref027" target="_blank">27</a>].</p

Validation of MS results by WB and proximity study by confocal microscopy for selected proteins.

<p>(A) Protein complexes of LFA-1, thrombospondin-1, talin-1, CD13 (top) and galectin-3 (bottom) in imDCs (day6) were co-immunoprecipitated with LFA-1. LFA-1 was enriched using mAb (clone SPV-L7) directed against αL (CD11a). mIgG1 coated beads were included as control IP. PNS: post nuclear supernatant. Samples were analysed in non-reducing conditions. (B) Confocal microscopy analysis of co-capping of LFA-1 and galectin-3, CD44 and CD71 on imDCs (day6). Receptor co-capping and staining were performed as described in <i>Material and Methods</i>. Antibodies against LFA-1 (clones: NKI-L15 and TS2/4) and CD71 are positive and negative markers for co-localization, respectively. Results are representatives of multiple cells per condition (n>10.) in two independent experiments. (C) To quantify the degree of co-localization between LFA-1 and binding candidates, Pearson´s coefficient was calculated. The values can vary between 0 and 1 (1 = 100% colocalization). P-values were compared to co-capping of LFA-1 with CD71 by two-tailed t-test, *** <0.001. Co-capping and staining were performed as described in Materials and Methods.</p

pH and ROS Responsiveness of Polymersome Nanovaccines for Antigen and Adjuvant Codelivery: An In Vitro and In Vivo Comparison

The antitumor immunity can be enhanced through the synchronized codelivery of antigens and immunostimulatory adjuvants to antigen-presenting cells, particularly dendritic cells (DCs), using nanovaccines (NVs). To study the influence of intracellular vaccine cargo release kinetics on the T cell activating capacities of DCs, we compared stimuli-responsive to nonresponsive polymersome NVs. To do so, we employed “AND gate” multiresponsive (MR) amphiphilic block copolymers that decompose only in response to the combination of chemical cues present in the environment of the intracellular compartments in antigen cross-presenting DCs: low pH and high reactive oxygen species (ROS) levels. After being unmasked by ROS, pH-responsive side chains are exposed and can undergo a charge shift within a relevant pH window of the intracellular compartments in antigen cross-presenting DCs. NVs containing the model antigen Ovalbumin (OVA) and the iNKT cell activating adjuvant α-Galactosylceramide (α-Galcer) were fabricated using microfluidics self-assembly. The MR NVs outperformed the nonresponsive NV in vitro, inducing enhanced classical- and cross-presentation of the OVA by DCs, effectively activating CD8+, CD4+ T cells, and iNKT cells. Interestingly, in vivo, the nonresponsive NVs outperformed the responsive vaccines. These differences in polymersome vaccine performance are likely linked to the kinetics of cargo release, highlighting the crucial chemical requirements for successful cancer nanovaccines