OP0199 THE HUMAN ENTHESIS HARBOURS RESIDENT ADAPTIVE CD4+ AND CD8+ T-CELLS WITH INDUCIBLE IL-17A AND TNF PROTEIN THAT IS PHARMACOLOGICALLY SUPPRESSED BY RORγT AND PDE4 INHIBITORS BUT NOT METHOTREXATE IN A NOVEL IN VITRO ENTHESITIS MODEL
Authors: Abdulla Watad, Hannah Rowe, Charlie Bridgewood, Tobias Russell, Qiao Zhou, Almas Khan, Robert Dunsmuir, Peter Loughenbury, Abhay S Rao, Peter Millner, Richard Cuthbert, Dennis Mcgonagle
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> Animal models have demonstrated that enthesitis is the primary lesion in experimental spondyloarthritis (SpA). In mice, innate lymphocytes were suggested as the major cytokine producers at the enthesis. <h3>Objectives</h3> We tested the hypothesis that the human enthesis harbours tissue resident conventional T-cells. We also assessed their ability to express SpA-related cytokines including TNF and IL-17A and if this could be blocked using psoriasis therapeutic agents (methotrexate (MTX), and phosphodiesterase type 4 inhibitor (PDE4i)) and experimental RORγt inhibitors (RORγti). <h3>Methods</h3> Entheseal spinous process was obtained from patients undergoing elective orthopedic procedures (n=20) and mechanically digested or processed for confocal staining and flow cytometry. CD4+ and CD8+ T-cells were sorted and RNA was isolated and analysed by qPCR. Magnetically isolated cells were stimulated using an anti-CD3/CD2/CD28 bead with and without the presence of MTX, RORγti and PDE4i. Following stimulation IL-17A and TNF were measured by ELISA and intracellular flow cytometry. <h3>Results</h3> CD4+ and CD8+ T-cells represent 35.7% and 23.7% of T-cells in the enthesis, respectively, with topographic confirmation by anti-CD3 immunofluorescence staining. Entheseal tissue contained a higher proportion of CD4+ and CD8+ T-cells expressing a resident memory phenotype (CD69+/CD45RA-) compared to matched blood. Sorted T-cells from entheseal tissue had a gene expression profile consistent with a tissue resident phenotype and CD4+ and CD8+ T-cells showed increased expression immunomodulatory genes including IL-10 and TGF-β compared to peripheral blood T-cells (p&lt;0.001). Following stimulation CD4+ T-cells produced more TNF than CD8+ T-cells (p&lt;0.05), IL-17A was robustly detected in CD4+ but not CD8+ T-cells. TNF and IL-17A production from CD4+ T-cells was effectively inhibited by PDE4i (p&lt;0.05), while RORγti only reduced IL-17 secretion (p&lt;0.001). MTX had no significant impact on both TNF and IL-17A production in either cell population. This pattern of inhibition was mirrored in TNF secretion from CD8+ T-cells. <h3>Conclusion</h3> This is the first description of conventional CD4+ and CD8+ enthesis resident T-cells. PDE4i was effective in abrogating induced TNF production and IL-17, whereas RORγti is highly effective for IL-17A production but not TNF. In contrast, MTX had little effect on <i>in vitro</i> enthesitis model cytokine production. These findings may have some practical implications in the treatment of subclinical enthesitis. Abbreviations: ST, entheseal soft tissue; PEB, peri-entheseal bone. <h3>Disclosure of Interests</h3> Abdulla Watad: None declared, Hannah Rowe: None declared, Charlie Bridgewood: None declared, Tobias Russell Grant/research support from: PhD Project is funded by Novartis., Qiao Zhou: None declared, Almas Khan: None declared, Robert Dunsmuir: None declared, Peter Loughenbury: None declared, Abhay S Rao: None declared, Peter Millner: None declared, Richard Cuthbert: None declared, Dennis McGonagle Consultant for: Lilly, Novartis UCB, Speakers bureau: Lilly, Novartis UCB