120. ERAP1 Mediates Immunogenicity in HLA-B51+ Behçet's Disease Pointing to Pathogenic CD8 T Cell Effectors
Creators: name: Cavers1, Ann
name: Arshed Al-Obeidi1
name: Yeşim Özgüler2
name: Überheide1, Beatrix
name: Zhong1, Hua
name: Hatemi2, Gülen
name: Kugler1, Matthias Kugler Matthias
name: Nowatzky1, Johannes
Publisher: name: Zenodo
Year: 2022
Subjects: Vasculitis
ANCA
Abstracts
Giant Cell Arthritis
MPA
GPA
EGPA
Takayasu
IgA vasculitis

description: <strong>Background:</strong> 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 unclear. Here, we aimed 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. <strong>Methods:</strong> We generated HLA-B51<sup>+</sup>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 vascular involvement as well as healthy donors (HD) whose PBMC were profiled using multicolor flow cytometry panels. To assess cellular composition and clonotypes at a disease-relevant effector site, we performed single cell RNA sequencing with TCR analyses of intraocular cells in BD uveitis. <strong>Results:</strong> WT and KO peptidomes differed significantly (p&lt; 0.0005 Fisher’s exact test) with a distinct 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<sup>+ </sup>CD8 T cells. Analysis of 133 T, B, NK, and monocyte cell populations revealed a 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&lt; 0.05, Mann-Whitney). Naive and effector memory CD8 T cell subsets were inversely correlated. Cohen’s effect sizes were large ( &gt;0.8) or very large ( &gt;1.2). CD8 T cells in BD uveitis showed differential subset distribution in between the eye and peripheral blood as well as oligoclonal expansions. <strong>Conclusions:</strong> We show that the absence of functional ERAP1 alters human CD8 T cell immunogenicity. This is mediated by an HLA-class I peptidome with a propensity for longer peptides above 9mer and suggests a 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 the biologic significance of HLA-B51/Hap10 in BD. CD8 T cell subset analyses in between peripheral blood and the eye during uveitis point to the non-random localization of CD8 T cells into that compartment. Collectively, our findings suggest that an altered HLA-B51 peptidome modulates the immunogenicity of CD8 effector T cells in ERAP1-Hap10 carriers with BD and underlines their potential importance in BD pathogenesis. <strong>Disclosures:</strong> None