POS0170 CIRCULATING PATHOGENIC LIKE LYMPHOCYTES ARE MECHANISTIC TARGETS FOR THE TOLEROGENIC RE-EDUCATION IN RHEUMATOID ARTHRITIC PATIENTS
Authors: J. Y. Leong, A. Lajam, C. T. Ng, S. Albani
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> We have previously discovered a subset of pathogenic-like T cells (CPL) that (1) possess inflammatory function; (2) has systemic migratory properties; (3) overlaps with synovial clonotypes; and (4) is correlating with active disease activity in rheumatoid arthritis (RA)[1]. The autoimmune antigenic targets of CPLs remain uncharacterised. Separately, we have demonstrated that mucosal tolerance re-education with the heat shock protein derived peptide, dnaJP1, has shown promising synergistic potentiation with hydroxycholorquine (P = 0.009 in ACR20 score) in a phase 2 trial with rheumatoid arthritic (RA) patients[2]. Consequently, whether CPLs reacts to the dnaJP1 peptide remains unknown. <h3>Objectives:</h3> Here we seek to investigate in a preliminary cohort of treatment naive RA patients, with the application of high parametric platform, mass cytometry, whether CPLs react to the dnaJP1 peptide. <h3>Methods:</h3> A cohort of adult RA patients (n=10 PBMC) and healthy controls (n=8 PBMC) were stimulated for 72hrs with dnaJP1 (10ug/ml). PBMCs were stained with a cocktail of metal conjugated antibodies and interrogated with mass cytometry (CyToF). The CyToF data is pre-processed with FlowJO and analysed with the EPIC analytics suite[3]. <h3>Results:</h3> We assessed the CD4 memory landscape in RA patients and healthy controls with mass cytometry. Unsupervised UMAP projections of total pooled CD4<sup>+</sup> memory T cells reveal the presence of CPLs (CD4<sup>+</sup>HLADR<sup>+</sup> T effectors). FlowSOM clustering revealed two clusters of CPLs expressing inflammatory cytokines (TNFa with IL-17A or IFNg) that are enriched in RA patients. Elevation of CPLs in RA patients as compared to controls were shown to be statistically significant (p &lt; 0.001) in manual gating. Memory frequencies of CPLs (p &lt; 0.01), CPLs CD69<sup>+</sup> (p &lt;0.05), CPLs Ki67<sup>+</sup> (p &lt; 0.05) and CPLs IFNg<sup>+</sup> (p &lt; 0.01) T cells, increased in the presence of dnaJP1 peptide (p &lt; 0.05). Intriguingly, within the regulatory memory landscape, we have also detected analogous presence inflammation associated Tregs (iaTregs) that were elevated (p &lt; 0.05) in RA patients, and responded to dnaJP1 stimulation (p &lt; 0.01). <h3>Conclusion:</h3> Overall, through the application of the high parametric technological platform, CyToF, we have demonstrated the enriched memory presence of a systemic population of inflammatory pathogenic T cells, CPLs. More importantly, CPLs elicited antigenic memory response in the presence of dnaJP1, unveiling a new mechanism through which aberrant self recognition could occur. This study identifies, CPLs, as an accessible pool of pathogenic-like T cells that could be used to advance our understanding of autoimmunity, and aid in designing diagnostic and therapeutic tools for RA. <h3>REFERENCES:</h3> [1] Spreafico R, et al. Ann Rheum Dis. 2016 Feb;75(2):459-65. [2] Koffeman EC, et al. Arthritis Rheum. 2009 Nov;60(11):3207-16. [3] Yeo JG, et al. Nat Biotechnol. 2020 Jun;38(6):679-684. <h3>Acknowledgements:</h3> <b>NIL.</b> <h3>Disclosure of Interests:</h3> <b>None declared.</b>