ObjectiveHuman leucocyte antigen (HLA)-B27 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly associated with ankylosing spondylitis (AS). ERAP1 is a key aminopeptidase in HLA class I presentation and can potentially alter surface expression of HLA-B27 free heavy chains (FHCs). We studied the effects of ERAP1 silencing/inhibition/variations on HLA-B27 FHC expression and Th17 responses in AS.MethodsFlow cytometry was used to measure surface expression of HLA class I in peripheral blood mononuclear cells (PBMCs) from patients with AS carrying different ERAP1 genotypes (rs2287987, rs30187 and rs27044) and in ERAP1-silenced/inhibited/mutated HLA-B27-expressing antigen presenting cells (APCs). ERAP1-silenced/inhibited APCs were cocultured with KIR3DL2CD3ε-reporter cells or AS CD4+ T cells. Th17 responses of AS CD4+ T cells were measured by interleukin (IL)-17A ELISA and Th17 intracellular cytokine staining. FHC cell surface expression and Th17 responses were also measured in AS PBMCs following ERAP1 inhibition.ResultsThe AS-protective ERAP1 variants, K528R and Q730E, were associated with reduced surface FHC expression by monocytes from patients with AS and HLA-B27-expressing APCs. ERAP1 silencing or inhibition in APCs downregulated HLA-B27 FHC surface expression, reduced IL-2 production by KIR3DL2CD3ε-reporter cells and suppressed the Th17 expansion and IL-17A secretion by AS CD4+ T cells. ERAP1 inhibition of AS PBMCs reduced HLA class I FHC surface expression by monocytes and B cells, and suppressed Th17 expansion.ConclusionsERAP1 activity determines surface expression of HLA-B27 FHCs and potentially promotes Th17 responses in AS through binding of HLA-B27 FHCs to KIR3DL2. Our data suggest that ERAP1 inhibition has potential for AS treatment.

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Background Increasing evidence implicates the type 17 immune axis in the pathogenesis of inflammatory arthritidies including ankylosing spondylitis (AS), and targeting of the pathway in AS has shown benefit in early clinical trials1. Cells driving inflammation in this axis express the transcription factor retinoid-related orphan nuclear receptor gamma T (ROR-gt) and make the signature pro-inflammatory cytokine interleukin-17A (IL-17A)2. Digoxin, a small-molecule inhibitors of ROR-gt has proved successful in the treatment of mouse experimental autoimmune encephalomyelitis3 but the dose needed to achieve inhibition in human cells is over 100 fold greater than the toxic threshold. New non-digoxin small molecule ROR-gt inhibitors have now been developed and we explore their role in the human inflammatory arthritidies. Objectives To determine the effects of novel small molecule ROR-gT inhibitors on in-vitro peripheral blood derived Type 17 T cell responses of patients with inflammatory arthritis. Methods Blood samples were obtained from patients with AS, rheumatoid arthritis (RA) and psoriatic arthritis (PsA). CD4 positive type 17 cells were expanded from peripheral blood monocuclear cells (PBMCs) of patients with inflammatory arthritis using low strength anti-CD2/3/28 stimulation and recombinant IL-2 in a 7 day culture system. Two different ROR-gt inhibiting compounds (A & B) were added at the start of the culture system and their effects on multiple cytokine expression (IL-17A, IFN-gamma, IL-22, GM-CSF, TNF-alpha) was determined by flow cytometry using intracellular staining. IL-17A production was validated using ELISA. Results Both ROR-gt small molecule inhibitors resulted in a consistent decrease in the percentage of patient-derived IL-17A-producing CD4 T cells. Overall there was approximately a 50% reduction in IL-17A production in AS (n=24, p<0.0001. see fig). The inhibition of IL-17A was confirmed on ELISA. There was a similar level of inhibition observed in IL-17F production. IL-22/IL-17A double producing cells were also inhibited but there was no significant effect on IL-22 single producers (Th22 cells). The effects of these inhibitors seem to be specific to type-17 cytokines (IL-17A, IL-17F) since we did not observe significant effects on the total amounts of other measured cytokines (IFN-gamma, TNF-alpha, GM-CSF). We saw inhibition of IL-17A production in RA and PsA. ROR-gt compounds did not adversely affect cell viability or proliferation in our culture system. Figure 1. Effects of ROR-gt inhibitor (compound A) on PBMC-derived Th17 cells from AS, RA and PsA patients. Conclusions Our results demonstrate a consistent and specific inhibition of IL-17A responses from patient derived PBMCs using small molecule ROR-gt inhibitors. We believe these results provide a solid rationale for the testing of these compounds in clinical trials particularly in AS where the treatment options remain limited. References Baeten, D. et al. Anti-interleukin-17A monoclonal antibody secukinumab in treatment of ank...