ERAP1-mediated immunogenicity and immune-phenotypes in HLA-B51⁺ Behcet’s and Behcet’s uveitis point to pathogenic CD8 T cell effector responses

Abstract

Purpose : HLA-B51 is a definite risk factor for Behcet’s uveitis (BU) and Behcet’s disease (BD). A coding variant of ERAP1, Hap10 – with low peptide-trimming activity – vastly potentiates this risk, but is mechanistically unclear. We hypothesized 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) T cell responses through stimulation of allogeneic donor cells with WT vs KO LCL and determined ERAP1 haplotypes in 49 untreated Turkish BD/BU subjects and healthy donors (HD) whose PBMC were profiled using 6 multicolor 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=0.0068; ELISA, p=0.0067) as was proliferation. Analysis of 133 T, B, NK and monocyte cell populations revealed predominance of CD8 T and NKT cell subsets in HLA-B51⁺/Hap10⁺ BD/BU vs HLA-B51⁺/Hap10^- BD/BU and HD, accounting for 80% of all populations reaching significance (p<0.05, Mann-Whitney). Naïve and effector memory CD8 T cell subsets were inversely correlated. Cohen’s effect sizes were large (>0.8) or very large (>1.2).

Conclusions : 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 naïve CD8 T cell subsets in affected subjects point to biologic significance of HLA-B51/Hap10 in BD/BU. Collectively, our findings suggest that an altered HLA-B51 peptidome modulates immunogenicity of CD8 effector T cells in ERAP1-Hap10 carriers with BD/BU and identify targets for future drug development.

This is a 2020 ARVO Annual Meeting abstract.