FRI0168 Phenotypic, functional and molecular characterisation of il-17+cd8+ t cells in human health and psoriatic arthritis

Abstract

Background Psoriatic arthritis (PsA) is a spondyloarthritis affecting the joint and skin. The genetic association with HLA Class I and the clinical efficacy of IL-17A inhibitors in the treatment of PsA suggest a potential role for IL-17 +CD8+T cells in PsA pathogenesis. This concept is supported by our previous data showing that IL-17 +CD8+T cell frequencies are increased in the synovial fluid (SF) vs the peripheral blood (PB) of patients with PsA1. We hypothesise that IL-17 +CD8+T cells are pro-inflammatory contributors to PsA pathogenesis. Objectives To perform molecular, phenotypic and functional characterisation of IL-17 +CD8+T cells. Methods Healthy donor PB CD8 +T cells were cultured for 3 days with anti-CD3/CD28 mAb, IL-1β and IL-23 to induce IL-17 +CD8+T cells in vitro, whilst PB and SF mononuclear cells were isolated from patients with PsA and stimulated ex vivo with PMA/ionomycin. Cytokine secretion assays were used to sort IL-17 +CD8+T cells, from which RNA was extracted for RNA-sequencing of PsA PB and SF cytokine-secreting T cells. 24 hour supernatants were generated from cultured sorted cells for cytokine analysis (Luminex) or culture with fibroblasts (IL-6 and IL-8 secretion measured by ELISA). Results In vitro-generated IL-17 +CD8+T cells produced significant levels of IL-17A, IL-17F, IFN-γ, TNF-α, IL-22 and GM-CSF (10–9000 pg/ml range), but little IL-10. Flow cytometry showed that in vitro-generated IL-17 +CD8+T cells co-expressed IFN-γ (median 80%), TNF-α (40%) and GM-CSF (35%) at comparable frequencies to ex vivo PsA synovial IL-17 +CD8+T cells (70%, 50%, 55% respectively). Whilst only 5% of PsA SF IL-17 +CD8+T cells co-expressed the MAIT cell marker Vα7.2, 50% of in vitro-generated IL-17 +CD8+T cells co-expressed Vα7.2. The cytokine profile of in vitro-generated Vα7.2+and Vα7.2- IL-17 +CD8+T cell supernatants was however comparable, sharing the cytokine profile of total IL-17 +CD8+T cells. Functionally, in vitro-generated sorted IL-17 +CD8+T cell culture supernatants enhanced IL-6 and IL-8 production by synovial tissue fibroblasts from patients with PsA compared to IL-17- counterparts, thus exhibiting pro-inflammatory capacity; we will also determine if this response is IL-17 and/or TNF-mediated. Additionally, the majority of synovial IL-17 +CD8+T cells co-expressed cytotoxic molecule Granzyme B, which may contribute to PsA pathogenesis. Finally, RNA-sequencing revealed that IL-17 +CD8+T cells from PsA synovial fluid displayed a distinct transcriptomic signature compared to PB IL-17 +CD8+T cells as well as to synovial Th17 or Tc1 cells. Conclusions In vitro-generated and ex vivo-derived synovial IL-17 +CD8+T cells display a type 17 profile, as evidenced by flow cytometry and Luminex. In contrast to ex vivo PsA synovial IL-17 +CD8+T cells, 50% of in vitro IL-17 +CD8+T cells co-express Vα7.2; however, both Vα7.2+and Vα7.2- subsets share a similar cytokine profile. Functionally, IL-17 +CD8+T cells exhibit pro-inflammatory potential, upregulating IL-6 and IL-8 production via fibroblasts. Analysis of our RNA-sequencing data will further reveal the molecular profile of human IL-17 +CD8+T cells, and how they may contribute to joint inflammation in PsA. Reference [1] Menon, et al. Arthritis Rheum2014;66(5):1272–1281. Acknowledgements Funded by King’s Health Schools (MRC DTP), King’s Health Partners R and D challenge award, Novartis and NIHR BRC. Disclosure of Interest U. Srenathan: None declared, K. Steel: None declared, M. Ridley: None declared, B. Kirkham Grant/research support from: Abbvie, Novartis, Roche, UCB, Speakers bureau: Eli Lilly and Co, Janssen, Novartis, L. Taams Grant/research support from: UCB, Novartis, GSK and Novo Nordisk A/S, Speakers bureau: UCB, Novartis