Comparative profiling of HLA-DR and HLA-DQ associated factor VIII peptides presented by monocyte-derived dendritic cells

Abstract

The development of anti-factor VIII antibodies is a major complication of the treatment of patients with hemophilia A. Generation of high affinity anti-factor VIII antibodies is dependent on help provided by CD4⁺ T cells that recognize factor VIII-derived peptides presented on class II major histocompatibility complex on the surface of antigen-presenting cells. In order to identify the immune-dominant epitopes that can be presented to CD4⁺ T cells, we previously developed a mass spectrometry-based method to identify factor VIII-derived peptides that are presented on human leukocyte antigen (HLA)-DR. In the present work, we compared the repertoire of FVIII-derived peptide presented on HLA-DR and HLA-DQ. Monocyte-derived dendritic cells from nine HLA-typed healthy donors were pulsed with recombinant factor VIII. HLA-DR and HLA-DQ molecules were purified using monoclonal antibodies. Our data show that HLA-DQ and HLA-DR present a similar repertoire of factor VIII-derived peptides. However, the number of peptides associated with HLA-DQ was lower than that with HLA-DR. We also identified a peptide, within the acidic a3 domains of factor VIII, which is presented with higher frequency on HLA-DQ. Interestingly, this peptide was found to have a higher predicted affinity for HLA-DQ than for HLA-DR. Taken together, our data suggest that HLA-DQ participates in the presentation of factor VIII peptides, thereby contributing to the development of inhibitory antibodies in a proportion of patients with severe hemophilia A.

Figure 1.

Comparative profiling of peptide presentation by HLA-DR and HLA-DQ. Five million monocyte-derived dendritic cells were pulsed with 100 nM FVIII and matured for 24 h. Peptides associated with HLA-DR and HLA-DQ were identified using mass spectrometry. (A) Panel A shows the total number of peptides identified after elution from either HLA-DR or HLA-DQ. (B) Panel B depicts the FVIII-specific peptides found on either HLA-DR or HLA-DQ. Statistical differences were determined using the non-parametric Mann-Whitney U-test. (C) Two hundred thousand immature monocyte-derived dendritic cells were incubated with 5 μg/mL of monoclonal antibody L243 (HLA-DR) or SPV-L3 (HLA-DQ). The relative binding of L243 and SPV-L3 was determined by flow cytometry using Alexa Fluor 488-conjugated anti-mouse IgG2a secondary antibody. Inset: representative histogram of the flow cytometry analysis of HLA-DR and HLA-DQ expression.

Figure 2.

Cluster analysis of proteins identified for HLA-DR and HLA-DQ. The absolute counts of unique peptide from all donors were grouped using hierarchical clustering for HLA-DR (left panel) and HLA-DQ (right panel). The top 40 proteins are displayed (black ≥ 80 peptides, blue = 40 peptides, red ≤ 10 peptides). Gene names (referred to as UniProt) are used to represent the proteins.

Figure 3.

Overview of factor VIII peptides presented by HLA-DR and HLA-DQ. FVIII-derived peptides presented on HLA-DR and HLA-DQ were identified using mass spectrometry. Core-peptide sequences and affinities were determined using NetMHCIIpan 3.1 for the different HLA-DR and HLA-DQ combinations. The core peptide with highest affinity was selected as the representative sequence for each group of overlapping peptides. In the case of overlapping discrepancies in the core-peptide sequence between HLA-DR and HLA-DQ, the core peptide determined for HLA-DR was used for the graphic representation. Core peptides were sorted based on their sequence localization. Each column represents the results for an individual donor. Green: peptide identified in the HLA-DR condition. Yellow: peptide identified in the HLA-DR and HLA-DQ condition. Red: peptide identified only in the HLA-DQ condition. Numbers within boxes indicate the total number of peptides represented by the specific core peptide. Core sequences indicated in bold correspond to previously documented T-cell epitopes: Steinitz et al., Blood 2012; Hu et al., JTH, 2004; Reding et al., JTH, 2004; Jones et al., JTH, 2005; Reding et al., JTH, 2003.

Figure 4.

Comparison of the factor VIII-derived peptide repertoire presented on HLA-DR and HLA-DQ. (A) The data obtained from the nine donors were grouped based on core-peptide sequence and their frequency (out of 9). Blue and red bars represent the frequency with which the FVIII core peptide was found in the HLA-DR and HLA-DQ conditions, respectively. Core sequences indicated in bold correspond to previously documented T-cell epitopes. (B) The affinity of the peptides found in both the HLA-DR and HLA-DQ conditions was determined using NetMHCIIpan 3.1 and plotted as paired peptides. Data points corresponding to the acidic a3 peptides are depicted in red.