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Presentation of ADAMTS13 peptides on HLA-DR and HLA-DQ
Figure 3. Schematic representation of eluted peptide immunogenicity. EpiMatrix and JanusMatrix Homology Scores for each eluted peptide is depicted in the graph. In the context of nine population-spanning “supertype” HLA-DR alleles, peptides with high (>10, orange) EpiMatrix Score are predicted to be promiscuous epitopes, presented by multiple HLA-DR alleles. Additionally, peptides with elevated to high (>3) JanusMatrix Homology Scores display high homology with human proteome at the TCR face and are predicted to be tolerated or actively tolerogenic (green squares). Peptides with high EpiMatrix Score (>10) in combination with low JanusMatrix Homology Score (<3) are predicted to be immunogenic (red squares). All other peptides, with an EpiMatrix Score <10, have limited or HLA-restricted immunogenic potential (black squares).
Figure 3) showed promiscuity in binding to supertype HLA- DR alleles in combination with low cross-conservation with human proteins. These peptides could potentially ini- tiate an effector T-cell response. In contrast, four of the pep- tides (Table 3 and Figure 3) were found to be cross-con- served within the human protein repertoire and could thus be potentially tolerated or actively tolerogenic in patients with acquired TTP. The remaining 10 ADAMTS13 peptides did not register high EpiMatrix Scores for binding to HLA- DR. However, they did have significant assessments for individual HLA-DR alleles and therefore could be presented on a subset of MHC II molecules.
Discussion
In this study, we explored the repertoire of HLA-DQ- presented peptides on ADAMTS13 pulsed monocyte- derived dendritic cells. In parallel, we also assessed the HLA-DR-presented peptide repertoire. This approach allows for a direct comparison of the peptide repertoires presented on HLA-DR and HLA-DQ. For this, we used two different monoclonal antibodies, L243 and SPV-L3, that had been previously used for peptide presentation profiling on either HLA-DR or HLA-DQ.19,23,31-33 Our data revealed that the number of peptides presented on HLA- DQ is 2- to 3-fold lower when compared to the repertoire presented on HLA-DR. This is most likely explained by the higher expression of HLA-DR on dendritic cells when compared to HLA-DQ.25,34,35 Furthermore, this could also result from an overall lower binding affinity of peptides for HLA-DQ when compared to HLA-DR. Indeed, the NetMHCIIpan3.1 prediction tool suggests a higher bind- ing affinity of ADAMTS13-derived peptides to HLA-DR when compared to HLA-DQ. In a recent study, we also
observed preferential presentation of blood coagulation FVIII-derived peptides on HLA-DR when compared to HLA-DQ.25
Interestingly, using higher-energy collisional dissocia- tion fragmentation, the total number of peptides identi- fied in HLA-DR eluates ranged from 1115 to 2247. This represents a 5- to 6-fold increase when compared to a pre- vious study from our group that employed collision- induced dissociation.19 We anticipate that the use of a highly sensitive mass spectrometer (Orbitrap Fusion Tribrid) has allowed for an increased number of peptides identified on HLA-DR. Surprisingly, despite the use of a highly sensitive mass-spectrometry strategy, the number of ADAMTS13-derived peptides remained relatively low in the case of HLA-DR and even lower for HLA-DQ (an average of 7 vs. 3 ADAMTS13-derived peptides, respec- tively). In the current study, we pulsed mo-DCs with 100 nM of recombinant ADAMTS13, whereas circulating lev- els of ADAMTS13 in healthy individuals ranges from 3.5- 7 nM (740-1420 ng/mL).36 Previous work from our group has identified the mannose receptor as an endocytic recep- tor for ADAMTS13 by mo-DCs.37 More recently, we described the involvement of CD163 in the internalization of ADAMTS13 by macrophages in vitro.38 In the latter study, we observed that the uptake efficiency of CD163- positive macrophages was approximately 10 times higher when compared to that of mo-DCs (that are devoid of CD163 expression). Based on this finding, it is tempting to speculate that the limited presentation of ADAMTS13- derived peptides on mo-DCs is due to a relatively low effi- ciency to internalize ADAMTS13, thereby limiting the number of ADAMTS13-derived peptides that are present- ed. Whether the limited presentation of ADAMTS13 by dendritic cells explains the low incidence of TTP in the general population despite the high prevalence of the risk
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