HLA-DR-presented Peptide Repertoires Derived From Human Monocyte-derived Dendritic Cells Pulsed With Blood Coagulation Factor VIII

Abstract

Activation of T-helper cells is dependent upon the appropriate presentation of antigen-derived peptides on MHC class II molecules expressed on antigen presenting cells.In the current study we explored the repertoire of peptides presented on MHC class II molecules on human monocyte derived dendritic cells (moDCs) from four HLA-typed healthy donors. MHC class II-bound peptides could be routinely recovered from small cultures containing 5 ؋ 10 6 cells. A fraction of the identified peptides were derived from proteins localized in the plasma membrane, endosomes, and lysosomes, but the majority of peptides that were presented on MHC class II originate from other organelles. Subsequently, we studied the antigen-specific peptide repertoire after endocytosis of a soluble antigen. Blood coagulation factor VIII (FVIII) was chosen as the antigen since our current knowledge on MHC class II presented peptides derived from this immunogenic therapeutic protein is limited. Analysis of the total repertoire of MHC class II-associated peptides revealed that per individual sample 20 -50 FVIII-derived peptides were presented on FVIII-pulsed moDCs. Repertoires of FVIII-derived peptides eluted from moDCs derived from a panel of four HLA typed donors revealed that some MHC class II-presented FVIII peptides were presented by multiple donors, whereas the presentation of other FVIII peptides was donor-specific. In total 32 different core peptides were presented on FVIII-pulsed moDCs from four HLAtyped donors. Together our findings provide an unbiased approach to identify peptides that are presented by MHC class II on antigen-loaded moDCs from individual donors.

von Willebrand factor binds to the surface of dendritic cells and modulates peptide presentation of factor VIII

“…38 In addition, peptides with core sequence FRN-QASPRY (A3 domain; residues 1766-1786) are presented less efficiently in the presence of VWF. Interestingly, this peptide is presented by multiple MHC class II alleles 9,10 and has also been described as a functional T-cell epitope in humanized E17 HLA-DRB1*1501 mice. 39 These observations raise the possibility that VWF modulates CD4 + Tcell responses by affecting FVIII peptide presentation by antigen-presenting cells.…”

“…9 Several studies have shown that VWF reduces the uptake of FVIII by iDCs. 13,18 However, whether VWF affects the processing and presentation of FVIII-derived peptides on MHC class II has not been addressed.…”

“…We have previously shown that only small variations in peptides were found between duplicate samples using mass spectrometry. 9 This allowed for the comparison of peptide repertories found in FVIII/VWF pulsed DCs with those found in DCs pulsed with FVIII only. Several FVIII-derived peptides were no longer detected in samples pulsed with FVIII/VWF complex (as indicated by the arrows) when compared to samples incubated with FVIII alone.…”

“…Once endocytosed, FVIII is cleaved in endo-lysosomal compartments into discrete peptides that are loaded on MHC class II. 9,10 The FVIII peptide-MHC class II complexes are then transported to the cell surface for recognition by antigen-specific CD4 + T-helper cells.…”

Mass Spectrometry-assisted Study Reveals That Lysine Residues 1967 and 1968 Have Opposite Contribution to Stability of Activated Factor VIII

“…Mass Spectrometry Analysis-FVIII peptides were separated by reverse-phase chromatography and sprayed in a LTQ OrbitrapXL mass spectrometer (Thermo Fisher Scientific) essentially as described (35). During reverse-phase chromatography, we employed a 40-min gradient from 0 to 35% (v/v) acetonitrile with 0.05% (v/v) acetic acid.…”