Fig 2: Non-random distribution of the ERAP2-protein coding haplotype across common ERAP1 haplotypes. Ten missense variants in ERAP1 encode eight discrete and common haplotypes, while ERAP2 encodes a common protein-coding haplotype (HapA tagged by the A allele of rs2248374) and non-coding haplotypes (tagged by the G allele). Each chromosome 5 harbors one ERAP1 haplotype and one ERAP2 haplotype. Using phased ERAP1-ERAP2 haplotype data of 890 Dutch controls (1780 phased haplotypes) and 2103 Spanish controls (4206 phased haplotypes), we outlined the percentage of HapA per ERAP1 haplotype on the same chromosome. Error bars indicate the 95% confidence interval based upon 20 000 bootstrap samples. Frequency of HapA of ERAP2 for each of the ERAP1 haplotypes was considered non-random if the observed frequency deviated from an expected 50% using an exact binominal test with [A or G rs2248374 for 8 ERAP1 haplotypes = 0.05/16] P < 2.5 × 10-3 in both the Dutch and Spanish populations with consistent direction of effect. ERAP1 haplotypes that fulfilled these criteria are highlighted by asterisks (for summary statistics see Supplementary Table 6). As a reference, we plotted the ERAP1-ERAP2 haplotype data for the 360 samples from the CEU, FIN, GBR and TSI populations from the 1000 Genomes sample collection used for functional investigation in this study (48).

Fig 3: Study design and sample preparation. (A) Design of the patient-derived model for antigen processing by ERAP2. (B) Western blot analysis of the protein expression of ERAP1, ERAP2, and Tubulin as a control in the HLA-A*29:02-positive Birdshot uveitis model cell lines in ERAP2-wild type cells (WT) and cells after CRISPR-Cas9 mediated knock-out (KO) of ERAP2. The relative amount of protein (in microgram) used for each lane is indicated. M, marker. (C) Overview of cultured stable isotope labeling by amino acids in cell culture (SILAC) labeled WT lymphoblastoid cells (LCLs) and unlabeled ERAP2 KO LCLs followed by combining the differentially labeled conditions for lysis and immunoprecipitation of HLA-A29 and, subsequently other human leukocyte antigen (HLA) class I molecules, respectively. HLA-bound peptides were eluted, followed by LC/MS analysis. All steps in c were conducted in two separate experiments to generate biological replicates. (D) Schematic overview of filtering steps of the identified peptides in this study. All peptides identified in both biological replicates with high confidence were filtered for limma analysis (see methods). After differential expression analysis, 8-11 mers were used to deconvolute and assign peptides to HLA alleles using HLAthena. The venn diagrams indicate the overlap from data sets and subsetting for subsequent analysis.

Fig 4: Cell surface expression of WT and A-pocket variant SCT. HeLa and HeLa ERAP1 KO (E1KO) cells were transfected with WT-SCT, A-pocket variants E63A-, K66A-, or W167A-SCT and cell surface expression assessed using the SIINFEKL/Kb-specific antibody 25-D1.16 or anti-mouse ß2m. A, 25-D1.16 and anti-mouse ß2m staining of HeLa cells transfected with either WT-SCT or IINFEKL (IL7)-SCT. B, CRISPR/Cas9 knockout of ERAP1 in HeLa cells. C, 25-D1.16 staining of HeLa E1KO cells transfected with WT or A-pocket variant SCT.

Fig 5: Activity of the phosphinic inhibitor DG011A. Panel (A), titration of DG011A inhibits ERAP2 with a 70-fold higher potency than ERAP1. Panel (B), DG011A shows no toxicity versus MOLT-4 cells up to 100 µM as measured by the MTT assay. Panel (C), representative FACS traces used for the quantitation of the presence of HLA molecules (stained by the W6/32 antibody) on the surface of MOLT-4 cells incubated with 1 µM DG011A. Panel (D), quantitation of the geometric mean of the signal from the FACS experiments.