P103/O14 Interleukin-17 is produced by CD4+ but not CD8+ T cells after TCR activation in synovial fluid of psoriatic arthritis patients
Authors: X Xu, N Davelaar, A-M Otten-Mus, P Asmawidjaja, J Hazes, D Baeten, M Vis, R Bisoendial, E Lubberts
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Career situation of first and presenting author</h3> Student for a master or a PhD. <h3>Introduction</h3> Interleukin-17A (IL-17A) contributes to the pathogenesis of psoriatic arthritis (PsA) as evidenced by the success of biologics targeting IL-17 pathway in PsA patients. Both CD4+ and CD8+ T cells are reportedly producers of IL-17A in PsA with IL-17A+ CD8+ T cells specifically enriched in PsA synovial fluid (SF). However, most current findings regarding IL-17A-producing cells in PsA patients are coming from intracellular staining using flow cytometry. <h3>Objectives</h3> To confirm and compare <i>ex vivo</i> production of IL-17A by CD4+ and CD8+ T cells from PsA synovial fluid using flow cytometry, enzyme-linked immunosorbent assay (ELISA) and RT-qPCR. <h3>Methods</h3> Fresh SF of established PsA patients were collected and part of the cells were directly stained intracellularly for IL-17A and IFNγ after 4 hours' (hrs) phorbol myristate acetate and ionomycin (PMA and iono) stimulation. Blood samples of early PsA patients and age/sex-matched healthy volunteers were included as controls for PsA SF. In addition, the rest of SF cells underwent density gradient separation and mononuclear cells (SFMCs) were stored till use. CD4+ and CD8+ T cells were sorted from SFMCs, and <i>ex vivo</i> cultured with soluble anti-CD3/anti-CD28 (aCD3 and 28), PMA and iono or without stimulation for 4 or 72 hours. Furthermore, SF CD4+ and CD8+ T cells were co-cultured with either allogeneic PsA fibroblast-like synoviocytes (FLS) or autologous PsA CD14+ monocytes with aCD3 and 28 activation for 72 hours. Culture supernatants were tested for ELISA and cells were analyzed with intracellular staining or RT-qPCR. <h3>Results</h3> Accumulation of IL-17A+ CD8+ T cells was significantly higher in PsA SF compared to in blood of either PsA patients or healthy volunteers. Although both CD4+ and CD8+ T cells from SFMCs are IL-17A+ with flow cytometry, only CD4+ T cells produce measurable amounts of IL-17A in culture supernatants after 72 hours of aCD3 and 28 activation. Similar results were found after 3 days' co-culture of aCD3 and CD28 stimulated CD4+ or CD8+ T cells with PsA FLS. Also, when either co-cultured with autologous CD14+ monocytes, only CD4+ T cells showed mRNA IL-17A expression and IL-17A production. In contrast, if PMA and iono were used to stimulate CD4+ or CD8+ T cells, both produce IL-17A in supernatants after 4 or 72 hours. <h3>Conclusions</h3> Although both CD4+ and CD8+ T cells show IL-17A positivity in PsA SF with flow cytometry, the use of strong stimuli such as PMA and iono during intracellular staining may account for the IL-17A positivity in CD8+ T cells. The contribution of CD8+ T cells to IL-17A production in local PsA joints might be limited as normal TCR activation, mimicked by aCD3 and 28, didn't induce IL-17A release in CD8+ T cells in contrast to CD4+ T cells. <h3>Disclosure of Interest</h3> None declared.