Figure 1.
Presentation of FVIII peptides on immature and mature DCs.
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.
Figure 2.
Persistent presentation on FVIII peptides on mature DCs.
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.
Figure 3.
Graphical representation of all FVIII peptides presented at each time point.
Figure 4.
Presentation of FVIII peptides by moDCs and macrophages.
A. Endocytosis of different concentrations of FVIII by either moDCs or M2 macrophages was measured by flow cytometry. The highest MFI measured in DCs was set to 100%. All other data points are relative to that MFI (expressed as %MFI) B. Cell lysates from FVIII-treated moDCs or macrophages were used to purify HLA-DRB1-presented peptides using an anti-MHC class II antibody. Graph shows quantification of individual MHC class II-bound FVIII peptides. C. Schematic representation of FVIII core peptides presented by dendritic cells and macrophages generated from monocytes of donor F.
Figure 5.
Presentation of OVA peptides by moDCs.
A. 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. 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.
Figure 6.
Distribution of FVIII core peptides in eight different donors.
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.
Figure 7.
Promiscuity plot of FVIII peptides presented on HLA-DRB1.
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.