Background Enthesitis or inflammation of the tendon/ligament anchorage points is the cardinal lesion in spondyloarthritis (SpA)[1]. Several key mediators have been shown to have a role in SpA including TNFα, IL-23, IL-17A and IL-17F [1]. There is a need for new therapeutic agents to treat SpA as current therapeutics fail to adequately target all its manifestations. Janus kinase (JAK) inhibitors have shown promise as disease modifying drugs and by targeting signaling components shared amongst several inflammatory pathways may be advantageous in treating inflammatory diseases with complex overlapping pathological mechanisms, as seen in SpA[2]. Upadacitinib is an oral selective JAK inhibitor (JAK1 over JAK2/3) which in phase II/III trials has shown efficacy for both psoriatic arthropathy and ankylosing spondylitis, suggesting specific JAK1 targeting may be superior[3, 4]. The role of JAK/STAT signaling in enthesis has not been extensively explored and given the importance of the enthesis in the development of SpA it is crucial to gain a mechanistic understanding of how JAK inhibitors affect entheseal inflammation. Objectives To determine if upadacitinib could suppress innate and adaptive immune responses in an in vitro human enthesis model and elucidate mechanisms of suppression. Methods Normal spinous process enthesis was obtained from patients undergoing spinal decompression or surgery for scoliosis correction as previously described[5]. Enthesis cells were subsequently isolated by mechanical digestion. Entheseal cells were stimulated with IFNɣ (JAK1 activator) with and without upadacitinib and STAT 1 activation measured by phos-flow cyometry. Entheseal T-cells were stimulated with anti-CD3, anti-CD3 with IL-23 and anti-CD3 with IL-1β with and without upadacitinib. IL-17A and TNFα were quantified using intracellular flow cytometry and ELISA of supernatants. Entheseal cells were also stimulated with LPS with and without upadacitinib, and IL-23 and TNFα quantified by ELISA. Results Upadacitinib inhibited phosphorylation of STAT1 in entheseal cells, following IFNɣ stimulation (Figure 1-A). Following stimulation of entheseal T cells upadacitinib inhibited both TNFα and IL-17A production as assessed by both ELISA and intracellular flow cytometry (Figure 1-B, 1-C). Upadacitinib did not attenuate LPS induced IL-23 or TNFα from entheseal myeloid cells. Conclusion Upadacitinib inhibition of entheseal T cell derived TNFα and IL-17A, therefore interrupting the IL-23/IL-17/TNFα axis so prominent in SpA, may offer a mechanistic explanation for upadaticintib’s efficacy treating enthesitis. References [1]Watad, A., et al., Enthesitis: Much More Than Focal Insertion Point Inflammation. Current rheumatology reports, 2018. 20 (7): p. 41-41. [2]Schwartz, D.M., et al., JAK inhibition as a therapeutic strategy for immune and inflammatory diseases. Nature Reviews Drug Discovery, 2017. 16 (12): p. 843-862. [3]van der Heijde, D., et al., Efficacy and safety of upadacitinib in patients with active ankylosing spondylitis (SELECT-AXIS 1): a multicentre, randomised, double-blind, placebo-controlled, phase 2/3 trial. Lancet, 2019. 394 (10214): p. 2108-2117. [4]McInnes, I.B., et al., Trial of Upadacitinib and Adalimumab for Psoriatic Arthritis. New England Journal of Medicine, 2021. 384 (13): p. 1227-1239. [5]Bridgewood, C., et al., Identification of myeloid cells in the human enthesis as the main source of local IL-23 production. Annals of the rheumatic diseases, 2019. 78 (7): p. 929-933. Disclosure of Interests Sami Giryes: None declared, Charlie Bridgewood: None declared, Chi Wong: None declared, Tom Macleod: None declared, Dennis McGonagle Grant/research support from: The study is funded by an Abbvie research grant.