OP0307 CELLULAR PROFILING OF SYNOVIAL FLUID IN INFLAMMATORY ARTHRITIS HIGHLIGHTS SHARED AND DISEASE-SPECIFIC CELL STATES WITH BIOLOGICAL AND DIAGNOSTIC RELEVANCE
Authors: M. Christofi, S. Valma, L. Marshall, B. Mulhearn, T. Hussell, A. Barton, D. Rao, S. Viatte
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> Rheumatoid arthritis (RA) is characterised by synovial inflammation with CD4<sup>+</sup> T cells playing a central role in the autoimmune process. Conversely, spondyloarthropathies are believed to be driven by CD8<sup>+</sup> T cells. Pathogenic T cells within affected tissues usually exhibit limited T cell receptor (TCR) diversity and express activation markers. Several CD4<sup>+</sup> T cell subsets and states have been identified in RA, but their immune phenotype, pathogenic potential and disease specificity has not been fully characterised, preventing their use as diagnostic markers in a clinical setting. <h3>Objectives:</h3> Investigate the frequency, activation, TCR diversity, and disease specificity of various CD3<sup>+</sup> T cell subsets in inflammatory arthritis, including HLA-DR<sup>+</sup>CD27<sup>-</sup> CD4<sup>+</sup> T<sub>EM</sub> [1] and PD-1<sup>high</sup>CXCR5<sup>-</sup> CD4<sup>+</sup> T cells (Tph) [2] reportedly associated with RA. <h3>Methods:</h3> Using 13-colour flow cytometry and the IOTest® Beta Repertoire Kit, peripheral blood mononuclear cells and matched synovial fluid cells from patients with five different rheumatic diseases (seropositive RA, seronegative RA, psoriatic arthritis, other spondyloathropathies, non-inflammatory controls including osteoarthritis) were immunophenotyped based on the expression of T cell markers and of 24 different TCR Variable Beta chains. <h3>Results:</h3> Synovial fluid exhibits a significant enrichment of CD8<sup>+</sup> over CD4<sup>+</sup> T cells (p&lt;0.03), regardless of disease classification. The CD8<sup>+</sup> TCR repertoire is less diverse (lower Shannon diversity index, p&lt;0.01), and the frequency of CD8<sup>+</sup> T<sub>EM</sub> correlated strongly between blood and synovial fluid (p&lt;0.006), supporting a pathogenic role for CD8<sup>+</sup> T cells across all types of inflammatory arthritis. In general, immune cell types and cell states highly enriched in synovial fluid were not disease-specific but shared across diagnoses; they expressed markers of activation and differentiation; had a restricted TCR repertoire and their frequency within synovial fluid was correlated with their frequency in the blood. For example, HLA-DR<sup>+</sup>CD27<sup>-</sup> CD4<sup>+</sup> T<sub>EM</sub> [1] are 44.9 times more frequent in synovial fluid than in the blood (p&lt;0.0001), but they are not specific to RA. They are a general biological feature of synovial inflammation. Interestingly, this is also true for their CD8<sup>+</sup> counterpart (HLA-DR<sup>+</sup>CD27<sup>-</sup> CD8<sup>+</sup> T<sub>EM</sub>, relative frequency 72.6, p&lt;0.001). Both subsets display a restricted TCR repertoire (decreased Shannon diversity index, p&lt;0.01 and &lt;0.001, respectively). In HLA-DR<sup>+</sup>CD27<sup>-</sup> CD8<sup>+</sup> T<sub>EM</sub>, the decreased TCR diversity is partly driven by an expansion Vβ7.1<sup>+</sup> cells (relative frequency=5, p&lt;0.01), but this expansion is not disease-specific (<b>Figure 1</b>). In contrast, Tph were almost exclusively found in seropositive RA (Positive Predictive Value= 100%; Specificity= 100%; Negative Predictive Value= 90%; Sensitivity= 67%). The Shannon diversity index is significantly decreased in Tph cells (p&lt;0.05), with decreased frequency of Vβ1 and Vβ5.2 and expansion of Vβ9 (p&lt;0.01, p&lt;0.05 and p&lt;0.05 respectively). In addition, Tph frequencies show correlations with disease activity and autoantibody titres. Although Tph frequency is &gt;350 higher in synovial fluid (p&lt;0.001) compared to blood, frequencies in both compartments were strongly correlated (R<sup>2</sup>=0.84 and p&lt;0.0001), which implies an identical predictive accuracy in these two anatomical compartments (AUC &gt; 0.91, <b>Figure 2</b>). This is a proof of principle that blood immune cell types have a strong diagnostic potential. Based on these observations, the detection of Tph cells in one of our seronegative patient prompted a clinical reassessment, resulting in a re-classification to seropositive RA (Tph in synovial fluid pre-dated seroconversion). <h3>Conclusion:</h3> We demonstrate the potential of an experimental setting to infer immune cell pathogenicity in inflammatory arthritis and establish the proof of principle to identify diagnostic signatures from peripheral blood. <h3>REFERENCES:</h3> [1] Fonseka, Sci Transl Med, 2018; [2] Rao, Nature, 2017. <h3>Acknowledgements:</h3> <b>NIL.</b> <h3>Disclosure of Interests:</h3> <b>None declared.</b>