POS0007 LOSS OF BALANCE BETWEEN PROTECTIVE AND PRO-INFLAMMATORY SYNOVIAL TISSUE T CELL POLYFUNCTIONALITY PREDATES CLINICAL ONSET OF RHEUMATOID ARTHRITIS
Authors: A. Floudas, N. Neto, M. Canavan, T. Mcgarry, V. Krishna, S. Nagpal, M. Monaghan, D. Veale, U. Fearon
Journal: Annals of the Rheumatic Diseases (2021)

Background: Effective treatment of Rheumatoid arthritis (RA) patients is achievable within a short window of opportunity following diagnosis. T-cells are early drivers of synovial inflammation of RA, therefore, identification of pathogenic T-cell subsets at the synovial tissue of pre-RA, arthralgia subjects, would greatly improve our understanding of disease pathogenesis. Comparative analysis of healthy control, arthralgia subject and RA-patient derived synovial tissue T-cell responses will lead to the identification of pathogenic as well as protective cytokine milieu, thus enabling the identification of early therapeutic targets to help steer the immune response towards resolution. Objectives: Characterization of T-cell polyfunctionality in the periphery and synovial tissue of ’at-risk; subjects (Arthralgia) RA-patients and healthy controls (HC). Identification of specific, pathogenic, synovial tissue T-cell subsets. Methods: Synovial biopsies from RA, AR and HC were obtained by arthroscopic surgery followed by RNAseq analysis (Guo et al., PLoS One, 2018). Single cell synovial tissue cell suspensions from RA, AR and HC and paired PBMC were stimulated in vitro and polyfunctional synovial T-cell subsets examined by flow cytometric analysis, SPICE visualization and FlowSom clustering. Flow-Imaging, was utilised to confirm specific T-cell cluster identification. Fluorescent Lifetime Imaging Microscopy (FLIM) was used to visualise metabolic status of specific T-cell populations. Results: T-cell associated pro-inflammatory gene pathways were increased in RNAseq analysis of RA-patient and arthralgia subject compared to HC synovial tissue biopsies. Flow cytometric analysis of pro-inflammatory cytokine (TNF-α, IFN-γ, IL-2, GM-CSF, IL-17A, IL-22) production and SPICE analysis of ex vivo stimulated T-cells revealed marked polyfunctionality of arthralgia subject synovial T-cells, thus providing evidence for a dysregulated synovial T-cell response that pre-dates clinical onset of disease. Importantly, HC synovial tissue harbours a small, albeit surprisingly polyfunctional, CD4 T-cell population characterised by significantly increased IL-4 and GM-CSF cytokine production compared to arthralgia subject (P&lt;0.001 and P=0.01) and RA-patient (P&lt;0.001 and P=0.004) synovial tissue. However, not all polyfunctional T-cells are equal in their pathogenic potential. Therefore, in order to identify highly pathogenic synovial T-cells, cluster analysis of flow cytometric data using the unsupervised algorithm FlowSom was performed and led to the identification of specific T-cell clusters with unique polyfunctionality characteristics. Specifically a cluster of CD4 + CD8 + double positive (DP) T-cells with high polyfunctionality scores was identified. Hybrid flow cytometry and imaging technique confirmed the co-expression of CD4 and CD8 by a synovial T-cell population. DP T-cells are enriched in RA-patient synovial fluid and synovial tissue and arthralgia subject synovial tissue, but are absent from HC synovial tissue. Importantly, DP T-cell synovial accumulation strongly (P=0.002) correlates with DAS28(CRP) of RA-patients. Initial studies utilising the novel, non-invasive FLIM technique for visualisation of cellular NAD, revealed that DP T-cells have a metabolic profile indicative of activated memory T-cells. Conclusion: These data highlight a key early loss of balance between protective and pathogenic synovial T-cell polyfunctionality and the emergence of specific, highly polyfunctional and pathogenic T-cell clusters in RA. Figure 1. Identification of highly polyfunctional and pro-inflammatory synovial DP T-cells . A. Cluster analysis of RA-patient synovial tissue T-cells (asterisks indicate DP T-cell clusters). B. Flow imaging of CD4 + , CD8 + and DP synovial T-cells. C. SPICE flow cytometric data visualization of DP arthralgia subject and RA-patient synovial T-cells. D. Correlation between the frequency of RA-patient synovial DP T-cells and disease severity. Disclosure of Interests: Achilleas Floudas: None declared, Nuno Neto: None declared, Mary Canavan: None declared, Trudy McGarry Employee of: Novartis, Vinod Krishna Employee of: Janssen, Sunil Nagpal Employee of: Janssen, GSK, Michael Monaghan: None declared, Douglas Veale Speakers bureau: Abbvie, Janssen, Novartis, MSD, Pfizer, UCB, Consultant of: Abbvie, Janssen, Novartis, MSD, Pfizer, UCB, Grant/research support from: Janssen, Abbvie, Pfizer, UCB, Ursula Fearon Speakers bureau: Abbvie, Grant/research support from: Janssen, Abbvie, Pfizer, UCB.