Presentation of FVIII peptides on immature and mature DCs.

<p>The presentation of FVIII peptides was compared between immature DCs and LPS-matured DCs. Cells were homogenized and PNS was fractionated on a sucrose density-gradient. Fractions from the sucrose gradient were analyzed by ELISA for MHC class II molecules and divided into a plasma membrane pool (PM) and an intracellular pool (IC) (see Figure S2 in File S1). Peptides were purified from the pools using L243-sepharose and analyzed by mass spectrometry. On the left y-axis the total number of FVIII peptides recovered from MHC class II is indicated (Peptide #). On the right y-axis number of “core peptides” (Core peptide #) is indicated. Core peptides are defined as a set of peptides with an overlapping sequence harboring an MHC class II binding motif. Heterogeneity in peptide length is due to amino-terminal and/or carboxy-terminal trimming of the presented peptides. </p

OPG content of mock- or KLF2-transduced BOECs.

<p>(A) Immunofluorescence image showing co-localization of OPG (green) and VWF (red) in both mock- and KLF2-tranduced BOECs. Nuclei were stained using DAPI (blue). Scale bars: 10 µm.(B) Western blot analysis for VWF, KLF2, IL-8 and IL-6 expression in lysates of mock- and KLF2-transduced BOECs; α-tubulin was shown as a loading control.</p

IL-6 and IL-8 content of KLF2-transduced BOECs.

<p>(A) Immunofluorescence image showing co-localization of IL-6 (green) and VWF (red) in IL-1β-treated KLF2- and mock-transduced BOECs. Nuclei were visualized with DAPI (blue). Scale bars: 10 µm. (B) Western blot analysis for VWF, KLF2, IL-8 and IL-6 expression in lysates of mock- and KLF2-transduced BOECs; α-tubulin was shown as a loading control. (C) Immunofluorescence image showing co-localization of IL-8 (green) and VWF (red) in IL-1β-treated KLF2- and mock-transduced BOECs. Nuclei were visualized with DAPI (blue). Scale bars: 10 µm. (D) Release of VWF from PMA-stimulated KLF2 (black bars)- and mock (white bars)-transduced cells (IL-1β-treated), measured by determining the concentration of VWF in the conditioned medium by ELISA. **P<0.001; ***P<0.0001 by Students t-test (E-F) Release of IL-6 and IL-8 from PMA-stimulated KLF2 (black bars)- and mock (white bars)-transduced cells (IL-1β-treated), measured by determining the concentration of IL-6 and IL-8 in the conditioned medium by ELISA. The amount of IL-6 released without stimulation was slightly reduced in KLF2 expressing cells when compared to mock-transduced cells. NS: non-significant; *P<0.01; ***P<0.0001 by Students t-test.</p

Angiopoietin-2 content of WPBs in mock- and KLF2-transduced BOECs.

<p>(A) Immunofluorescence image showing the co-localization of Ang2 (green) and P-selectin (red) in WPBs of mock-transduced BOECs. Nuclei were visualized with DAPI (blue). Scale bars: 10 µm. (B) Western blot analysis of VWF, KLF2 and Ang2 expression in lysates of mock- and KLF2-transduced BOECs; α-tubulin is shown as a loading control. (C) Mock- and KLF2-transduced BOECs stained for VWF (red) and Ang2 (green). Nuclei were stained using DAPI (blue). Scale bars: 10 µm. (D-E) Release of Ang2 and VWF from PMA-stimulated KLF2 (black bars)- and mock (white bars)-transduced BOECs measured by determining the concentration of Ang2 in the medium by ELISA. **P<0.001; ***P<0.0001 by Students t-test. (F) Time course of regulated VWF and Ang2 secretion after PMA stimulation of mock- and KLF2-transduced BOECs.</p

Reduced average length of WPBs in KLF2-transduced BOECs.

<p>(A) Confocal microscopy analysis of WPBs in mock- and KLF2-transduced BOECs stained for VWF (red) and DAPI (blue). Scale bars: 10 µm. (B) Average amount of WPBs per cell in unstimulated and stimulated mock-transduced BOECs and KLF2-transduced BOECs. ***P<0.0001 using Student’s t- test (C) The average length of the WPBs in individual mock- or KLF2-transduced BOECs. WPBs from 20 randomly selected cells were analyzed. ***P<0.0005 by Student’s t-test (D) Release of VWF from forskolin/IBMX- and epinephrine/IBMX-stimulated KLF2 (black bars)- and mock (white bars)-transduced BOECs. ***P<0.0001 by Students t-test. Error bars represent SEM.</p

Expression of KLF2 in BOECs.

<p>(A) Immunofluorescent analysis of mock- and KLF2-transduced BOECs. Cells were immunostained for CD31 (red) and KLF2 (green); nuclei were stained using DAPI (blue). Scale bars: 20 µm; (B) Western blot analysis of KLF2 expression in lysates of KLF2-transduced and mock-transduced BOECs; α-tubulin is shown as a loading control.</p

Persistent presentation on FVIII peptides on mature DCs.

<p>Endocytosis of FVIII by immature DCs was followed by maturation with LPS for either 12h, 24h, 48h, 72h or 96h. HLA-DRB1 bound peptides were purified from L243-sepharose and analyzed by mass spectrometry. Bar diagrams indicate the total amount of DRB1-bound peptides identified under each condition as well as the amount of FVIII peptides and core peptides. Core peptides are defined as a set of peptides with an overlapping sequence harboring an MHC class II binding motif. Heterogeneity in peptide length is due to amino-terminal and/or carboxy-terminal trimming of the presented peptides. </p

Presentation of OVA peptides by moDCs.

<p><b>A</b>. Ovalbumin-derived peptides were identified by mass spectrometry after purification of HLA-DRB1 from lysates of ovalbumin-pulsed cells with either an anti-HLA-DR antibody or an isotype antibody. SIEVE was used to compare intensities of individual peptides and average intensities of each identified peptide are plotted. The diagonal line indicates an equal intensity under each conditions and the dotted lines indicate 5-fold differences in intensity. Peptide clusters with a fold change of 5 or higher were considered as true MHC class II-bound peptides. Only peptides that were sequenced by MS/MS are depicted. <b>B</b>. Ovalbumin peptides identified in this experiment are listed with sequence, charge, mass/charge ratio (m/z), retention time (RT), Xcorr value and HLA-DR/isotype ratio. This is the ratio of intensity with which the peptide was detected in the anti-MHC class II pulldown (L243) over the intensity with which it was detected in the pulldown with the isotype control antibody. Peptides excluded due to a ratio lower than 5 are marked in red.</p

Distribution of FVIII core peptides in eight different donors.

<p>Different core peptides are presented by different donors. FVIII-derived HLA-DRB1-presented peptides are represented as rectangles for each individual donor. Depicted in color coding are sequences that are common between two or more donors. Yellow: 2 donors, orange: 3 donors, red: 4 donors, violet: 5 donors and purple: 7 donors. The different FVIII domains and the amino acid sequence of the peptides are depicted schematically. </p

Promiscuity plot of FVIII peptides presented on HLA-DRB1.

<p>The number of peptides presented in single or multiple (2-7) donors is indicated. The number of peptides is plotted on the y-axis. The number of donors positive for an individual peptide is displayed on the x-axis. </p