ObjectivesThe endoplasmic reticulum aminopeptidase (ERAP1) haplotype Hap10 encodes for a variant allotype of the endoplasmic reticulum (ER)-resident peptide-trimming aminopeptidase ERAP1 with low enzymatic activity. This haplotype recessively confers the highest risk for Behçet’s diseases (BD) currently known, but only in carriers of HLA-B*51, the classical risk factor for the disease. The mechanistic implications and biological consequences of this epistatic relationship are unknown. Here, we aimed to determine its biological relevance and functional impact.MethodsWe genotyped and immune phenotyped a cohort of 26 untreated Turkish BD subjects and 22 healthy donors, generated CRISPR-Cas9 ERAP1 KOs from HLA-B*51+ LCL, analysed the HLA class I-bound peptidome for peptide length differences and assessed immunogenicity of genome-edited cells in CD8 T cell co-culture systems.ResultsAllele frequencies of ERAP1-Hap10 were similar to previous studies. There were frequency shifts between antigen-experienced and naïve CD8 T cell populations of carriers and non-carriers of ERAP1-Hap10 in an HLA-B*51 background. ERAP1 KO cells showed peptidomes with longer peptides above 9mer and significant differences in their ability to stimulate alloreactive CD8 T cells compared with wild-type control cells.ConclusionsWe demonstrate that hypoactive ERAP1 changes immunogenicity to CD8 T cells, mediated by an HLA class I peptidome with undertrimmed peptides. Naïve/effector CD8 T cell shifts in affected carriers provide evidence of the biological relevance of ERAP1-Hap10/HLA-B*51 at the cellular level and point to an HLA-B51-restricted process. Our findings suggest that variant ERAP1-Hap10 partakes in BD pathogenesis by generating HLA-B51-restricted peptides, causing a change in immunodominance of the ensuing CD8 T cell response.

Background:HLA-B51 is a definite risk factor for Behçet’s disease (BD). A coding variant of ERAP1, Hap10 – with low peptide-trimming activity – vastly potentiates this risk, but is mechanistically unclear1,2).Objectives:To test the hypothesis that low or absent ERAP1 activity alters CD8 T cell immunogenicity through changes in the HLA-B51 peptidome and shapes the CD8 T cell immune response in affected subjects.Methods:We generated HLA-B51+ERAP1 KO LCL clones using CRISPR-Cas9, performed mass spectrometry of the immunoprecipitated MHC-class I peptidome with subsequent computational deconvolution for HLA-B51-binding peptides. We then assessed single cell (ICS), bulk (ELISA) and proliferative (CFSE) CD8 effector (IFNg, granzyme B, perforin) T cell responses through stimulation of allogeneic donor cells with WT vs KO LCL and determined ERAP1 haplotypes in 49 untreated Turkish BD subjects with ocular and/ or major vascular involvement as well as healthy donors (HD) whose PBMC were profiled using 6 multicolour flow cytometry panels.Results:WT and KO peptidomes differed significantly (p<0.0005 Fisher’s exact test) with a distinctive shift of peptide length frequencies exceeding 9-mer (binding optimum) in the KO vs WT. This held true for computationally deconvoluted HLA-B51 binders. IFNg secretion from CD8 T cells stimulated with KO LCL was significantly different from WT (ICS, p=0.0006; ELISA, p=0.0059) as were CD8 T cell proliferation and ICS of perforin/granzyme B+CD8 T cells. Analysis of 133 T, B, NK and monocyte cell populations revealed predominance of CD8 T and NKT cell subset in HLA-B51+/Hap10+ BD vs HLA-B51+/Hap10- BD and HD, accounting for 80% of all populations reaching significance (p<0.05, Mann-Whitney). Naive and effector memory CD8 T cell subsets were inversely correlated. Cohen’s effect sizes were large (>0.8) or very large (>1.2).Conclusion:We show that absence of functional ERAP1 alters human CD8 T cell immunogenicity. This is mediated by an HLA-class I peptidome with propensity for longer peptides above 9mer and suggests loss or de-novo presentation of peptide-HLA-B51 complexes to cognate CD8 TCR. The reciprocal changes in antigen- experienced vs naive CD8 T cell subsets in affected subjects point to biologic significance of HLA-B51/Hap10 in BD. Collectively, our findings suggest that an altered HLA-B51 peptidome modulates immunogenicity of CD8 effector T cells in ERAP1-Hap10 carriers with BD and identify targets for future drug development.References:[1]Kirino, Y., G. Bertsias, Y. Ishigatsubo, N. Mizuki, I. Tugal-Tutkun, E. Seyahi, Y. Ozyazgan, F. S. Sacli, B. Erer, H. Inoko, Z. Emrence, A. Cakar, N. Abaci, D. Ustek, C. Satorius, A. Ueda, M. Takeno, Y. Kim, G. M. Wood, M. J. Ombrello, A. Meguro, A. Gul, E. F. Remmers, and D. L. Kastner. 2013. ‘Genome-wide association analysis identifies new susceptibility loci for Behcet’s disease and epistasis between HLA-B*51 and ERAP1’,Nat Genet, 45: 202-7.[2]Takeuchi, M., M. J. Ombrello, Y. Kirino, B. Erer, I. Tugal-Tutkun, E. Seyahi, Y. Ozyazgan, N. R. Watts, A. Gul, D. L. Kastner, and E. F. Remmers. 2016. ‘A single endoplasmic reticulum aminopeptidase-1 protein allotype is a strong risk factor for Behcet’s disease in HLA-B*51 carriers’,Ann Rheum Dis, 75: 2208-11.Disclosure of Interests:Arshed F. Al-Obeidi: None declared, Ann Cavers: None declared, Yesim Ozguler: None declared, Olivier Manches: None declared, Hua Zhong: None declared, Berna Yurttas: None declared, Beatrix Ueberheide: None declared, Gulen Hatemi Grant/research support from: BMS, Celgene Corporation, Silk Road Therapeutics – grant/research support, Consultant of: Bayer, Eli Lilly – consultant, Speakers bureau: AbbVie, Mustafa Nevzat, Novartis, UCB – speaker, Matthias Kugler: None declared, Johannes Nowatzky: None declared

BackgroundCellular immunity of Behçet’s Disease (BD) remains poorly understood. Previous work has provided clues pointing to most innate and adaptive immune cell types in BD, but strong signals from non-immunogenetic studies are rare and often inconclusive.ObjectivesHere we aimed to identify BD/HD discriminant single immune cell profiles in semi-biased and targeted approaches and determine their significance at a BD relevant effector site.MethodsWe utilized multi-parametric flow cytometry to dissect cellular phenotypes in PBMC of untreated BD patients (n=27) and HD (n=22) consisting predominantly of active ocular and major vascular BD subjects. Data were subjected to supervised machine learning (CITRUS) and results verified with targeted gating. We also analyzed anterior chamber (AC) fluid cells and autologous PBMC from BD uveitis subjects with scRNA seq.ResultsCITRUS identified CD16+, CD14low, CD4low, CD3-, CD19- cells as a BD/HD distinguishing cellular expression pattern at an FDR of >0.05. Targeted gating confirmed highly significant differences with large effect sizes in PBMC of BD vs HD for “non-classical” (CD14lowCD16hi) and “intermediate” (CD14+CD16+) monocytes at decreased frequencies compared to peripheral blood. “Classical” (CD14++CD16-) monocytes were more abundant in BD PBMC than in HD. CD16+ dendritic cells (DC) were significantly decreased in BD PBMC. CD14+ cells showed high abundance in the AC during BD uveitis and co-expressed CD16 far more frequently than CD14+ cells in autologous peripheral blood.ConclusionSignificantly lower frequencies of CD16+ monocyte and DC subsets in PBMC of untreated active BD vs HD strongly point to their importance in BD. The high abundance of CD14+ cells with CD16 co-expression in the eye during uveitis relative to their frequency in autologous peripheral blood, suggests their transmigration or post-migrational interconversion within the eye during BD uveitis rather than a stochastic process.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsYesim Ozguler: None declared, Ziyan Lin: None declared, Gulen Hatemi: None declared, Ann Cavers: None declared, Johannes Nowatzky Grant/research support from: NIH-NEI R01EY031383 (Nowatzky), NIH-NEI R01EY033495 (Nowatzky)