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Presentation of ADAMTS13 peptides on HLA-DR and HLA-DQ
Table 1. MHC-II genotype of healthy donors included in the study. The donors included in this study were typed for HLA-DRB1/DQB1 using PCR- SBT and HLA-DQA1 using next generation sequencing workflow.
Donor
1 07:01
2 04:04
3 01:01
4 01:01
5 01:01
6 01:01
7 01:01
8 04:01
9 03:01
HLA-DRB1
14:54 15:01 15:01 08:01 07:01 11:01 11:01 11:01 13:01
HLA-DQA1
HLA-DQB1
05:03 06:02 06:02 05:01 05:01 05:01 05:01 03:02 06:03
01:04 01:02 01:01 01:01 01:01 01:01 01:01 03:01 01:01
02:01 03:03 03:01 03:02 04:01 05:01 04:01 04:02 02:01 02:02 05:05 03:01 05:05 03:01 05:05 03:01 05:01 02:01
database using Proteome Discover 1.4 (Thermo Scientific) with a 20 ppm tolerance for precursor mass and 10 ppm tolerance for fragment mass. Oxidation (+15.995 Da) on methionine was select- ed as dynamic modification. A decoy database comprising the reverse protein sequences from the same database was used to obtain a false discovery rate (FDR). Only peptides with a high or medium confidence (FDR threshold 0.05%) were considered for protein scoring.
Results
Presentation of ADAMTS13-derived peptides on HLA-DQ
To assess the contribution of HLA-DQ to the presenta- tion of ADAMTS13-derived peptides, we pulsed mo-DCs from a panel of 9 HLA-typed healthy donors with 100 nM ADAMTS13. The HLA type of the donors included in this study is shown in Table 1. Mo-DCs from the same panel of donors were recently analyzed for the presentation of FVIII-derived peptides on HLA-DQ.25 Mo-DCs were incu- bated with 1 mg/mL LPS and 1% fetal calf serum to allow for their maturation. HLA-DR and HLA-DQ molecules were purified employing monoclonal antibodies directed against HLA-DR (L243) and HLA-DQ (SPV-L3),22 and the eluates were analyzed by mass-spectrometry. In agree- ment with our previous reports,19,23,25 peptides derived from endogenously expressed as well as internalized pro- teins were presented on HLA-DR and HLA-DQ (Online Supplementary Table S1A and B, respectively). A subset of peptides was derived from proteins that are found within the endolysosomal compartment; these include HLA-DR itself, several proteases (such as cathepsin B) and endocyt- ic receptors (such as the macrophage mannose receptor and prolow-density lipoprotein receptor-related protein 1).
The total number of unique peptides that was identified in the pulldowns of HLA-DR and HLA-DQ is shown in Figure 1A and Online Supplementary Figure S1. The total number of peptides presented on HLA-DQ was approxi- mately 3-fold lower when compared to HLA-DR (median of 566 unique peptides on HLA-DQ compared to 1521 on HLA-DR). Similarly, although not statistically significant, the number of ADAMTS13-derived peptides presented on HLA-DQ was 3-fold lower when compared to HLA-DR (Figure 1B). HLA-DR-presented ADAMTS13-derived pep- tides were identified for all donors whereas ADAMTS13- derived peptides presented on HLA-DQ were only found
in 4 out of 9 donors (Table 2A and B, and Online Supplementary Figure S1B).
Repertoire of ADAMTS13-derived peptides presented on HLA-DR
In order to compare the repertoire of ADAMTS13- derived peptides presented on HLA-DR and HLA-DQ, the binding cores of the ADAMTS13-derived unique peptides were predicted using the NetMHCIIpan 3.1 software (Table 2A and B).26 ADAMTS13-derived peptides that shared the same core sequence were grouped (Figure 2). Previously, we have shown that ADAMTS13-derived pep- tides containing the sequence FINVAPHAR were present- ed on HLA-DRB1*11.19 Peptides sharing the same sequence were also presented on non-HLA-DRB1*11 pos- itive donors when mo-DCs were pulsed with 500 nM of ADAMTS13.19 In the current study, peptides containing the FINVAPHAR sequence were presented on mo-DCs of HLA-DRB1*11 positive donors 7 and 8. Peptides with core-sequence FINVAPHAR were also presented by mo- DCs derived of HLA-DRB1*11 negative donors 1 and 9 (Table 2A). However, no peptides containing the FIN- VAPHAR sequence were identified in HLA-DRB1*11 pos- itive donor 6 (Table 2A). Peptides derived from the sequence IHALATNMG which is located adjacent to the FINVAPHAR peptide were found in DRB1*11 negative donor 2 (Table 2A). Peptides derived from sequence LIRDTHSLR were identified in DRB1*03 positive donor 9. In a previous study, LIRDTHSLR-derived peptides were also presented by mo-DCs derived of DRB1*03 positive donors.19 It is noteworthy that donors 6 and 7 with the same HLA-DRB1 haplotype (DRB1*01:01/DRB1*11:01) both presented peptides derived of core sequence LKTLP- PARC originating from the TSP3 domain of ADAMTS13 (Table 2A). The majority of the HLA-DR-presented pep- tides identified in this study were derived from the CUB domains (Table 2A and Figure 2).
HLA-DQ contributes to presentation of ADAMTS13-derived peptides
We found ADAMTS13-derived peptides associated with HLA-DQ in 4 out of 9 donors (Table 2B and Figure 2). We identified 4 sets of peptides that were exclusively presented by HLA-DQ that originated from the cysteine- rich, TSP2, and TSP2-linker 1 of ADAMTS13 (Figure 2). This suggests that the repertoire of peptides presented on HLA-DQ differs from that presented on HLA-DR. We also identified peptides that were presented both on HLA-DR
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