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 HLA-typed 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.

Fig. 1.

Cell surface markers of immature and mature moDCs. Cells were analyzed at the immature (A) and mature (B) state for the presence of cell surface markers CD80, CD83, CD86, and MHC class II. Gray histograms indicate isotype controls. A representative culture is depicted. C, Endocytosis and presentation of FVIII by moDCs. Histogram shows the fluorescence intensity for a FITC-labeled anti FVIII antibody (CLB-CAg-117). Binding of CLB-CAg-117-FITC to untreated cells is shown in gray. Cells stained in the absence of saponin show the membrane binding of FVIII (dotted curve). Increase of fluorescence in cells stained in the presence of saponin indicates that endocytosis of FVIII has taken place. A representative graph of one of the four donors is depicted.

Fig. 2.

Identification and relative quantification of MHC class II-bound peptides. A, Volcano plot representation showing reproducibly detected peptide ions across duplicate analyses. MoDCs from donor A (DRB1*0101, 1301) were used. FVIII-treated cells were immunoprecipitated using either an anti-MHC class II antibody or an isotype control antibody. Experiments were performed in duplicate. SIEVE was used to compare intensities of individual peptides eluted from L243 or isotype-control Sepharose. Peptide clusters highlighted in the box on the right hand side were considered as MHC class II-bound peptides (fold change ≥5). Only peptides that were sequenced by MS/MS are depicted. FVIII-derived peptides are indicated in red. The sequence and ratio of all FVIII peptides are shown in panel (B).

Fig. 3.

Identification and relative quantification of MHC class II-bound FVIII peptides. A, Volcano plot representation showing reproducibly detected peptide ions across duplicate analyses. MoDCs from donor A (DRB1*0101, 1301) were used. Cells were treated with either 100 nm FVIII or with PBS. All samples were immunoprecipitated using an anti-MHC class II antibody. Experiments were performed in duplicate. SIEVE was used to compare intensities of individual peptides obtained from FVIII and PBS treated cells. Only peptides that were derived from MS2 spectra are depicted. Peptides identified as FVIII-derived peptides are indicated in red. B, Peak intensities of all identified FVIII peptides are shown for experimental and control samples. It should be noted that the sets of FVIII peptides that were identified in the analysis presented in Fig. 3B when compared with Fig. 2B are not completely identical. The reason for the observed differences between Fig. 2B and 3B is that these particular peptides were filtered out by the algorithms employed by SIEVE during the comparison, due to local spectral differences. Manual analysis of the raw data files revealed that all peptides depicted in Fig. 2B and 3B were indeed present in the duplicate samples prepared from FVIII-treated cells using L243 (MHC class II specific) Sepharose.

Fig. 4.

Subcellular and functional and categorization of source proteins. All proteins of which peptides with a ratio ≥5 were identified in Fig. 2A (specific MHC class II-bound peptides) were annotated based on subcellular localization or function using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resource. A, A pie chart shows the percentages of proteins found presented on MHC class II according to their subcellular localization. B, A pie chart shows the functional association of these proteins.

Fig. 5.

FVIII-derived MHC class II ligands identified from donor. A The first column shows residue numbers and corresponding domain of the FVIII molecule. The following columns show amino acid sequence, charge, XCorr value as provided by Sequest, retention time (RT), mass-to-charge ratio (m/z) and mass tolerance (ΔM). The two last columns show predicted binding scores to the MHC class II alleles of the donor. Binding scores to corresponding MHC class II alleles of the donor were calculated using Propred and NetMHCIIpan epitope prediction programs. Binding thresholds were defined as ≤500 nm for NetMHCIIpan and as top 3% natural binders for Propred (≥0.023 for DRB1*0101 and ≥0.295 for DRB1*1301). The binding motif of peptides that are predicted to bind above the binding threshold are underlined.

Fig. 6.

Presentation of FVIII peptides in duplicate experiments from donor B. Table of FVIII-derived MHC class II ligands identified from donor B. The first column shows residue numbers and corresponding domain of the FVIII molecule. The following columns show amino acid sequences of the first and second experiment. The two last columns show predicted binding scores to the MHC class II alleles of the donor. Binding scores to corresponding MHC class II alleles of the donor were calculated using Propred and NetMHCIIpan epitope prediction programs. Binding thresholds were defined as ≤500 nm for NetMHCIIpan and as top 3% natural binders for Propred (≥0.353 for DRB1*0701 and ≥0.329 for DRB1*1501). The binding motif of peptides that are predicted to bind above the binding threshold are underlined.

Fig. 7.

Distribution of FVIII core peptides in four different donors. Different core peptides are presented by different donors. Results displayed in this figure. were obtained from single experiments for all donors. FVIII-derived MHC class II peptides are represented as rectangles for each individual donor. Indicated in yellow are sequences that are common between two donors. Indicated in orange are sequences common between three donors. Indicated in red is a sequence that is common in all four donors. The different domains and the location of the peptides are depicted schematically. Some sequences have been marked with a reference number, indicating that they have been identified previously as CD4⁺ T-cell epitope in the corresponding publication.