AB0045 ULTRASOUND GUIDED BIOPSIES OF RA JOINTS AT TIME OF CLINICAL DIAGNOSIS CONTAIN PROFOUND T CELL CLONALITIES
Authors: S. Turcinov, E. Af Klint, A. De Bondt, M. S. Mia, A. Catrina, F. Stevenaert, V. Malmström
Journal: Annals of the Rheumatic Diseases (2020)

Background: Rheumatoid arthritis (RA) is a disease characterized by synovial joint inflammation, mainly affecting small joints. Histological findings in synovial biopsies ranges from inflammatory infiltration including ectopic lymphoid structures, to a cell sparse fibroid phenotype. T cells in affected joints are non-naïve and have by flow cytometry approaches been shown to have a wide TCR-beta chain gene usage. New technologies allow for analyses of paired TCR sequences and their antigen-specificities. Objectives: To study the alpha/beta-T cell receptor repertoire in single sorted T cells from synovial biopsies at time of RA-diagnosis. Meth ods: Synovial biopsies were taken, primarily using an ultrasound guided technique, from seventeen patients (12 ACPA+, 5 ACPA-) with rheumatoid arthritis. Fresh biopsies were enzymatically digested, followed by mild mechanical treatment, prior to flow cytometry cell sorting. Single cell index sorting of T cells was made into 384-well plates with PCR-buffer followed by a nested PCR and deep sequencing of the TCR amplicons. TCR-receptor sequences showing clonal expansion from four ACPA+ HLA-DRB1*0401 patients were further cloned into SKW3 cells for studies of their reactivity by in vitro stimulation with peptides of viral and citrullinated origin from the literature. A positive response, as measured by CD69-up regulation or IL-2 production, was used to define specificity. Results: Fourteen of the assessed joints were small (1 MTP, 4 MCP and 8 wrists), whereas the remaining three were large joints (2 knees and 1 ankle), table 1. Individual T cells could be isolated from all of these biopsies, with a variating CD4:CD8 ratio. Based on the flow cytometry phenotyping we could identify CD4 T cells of both Treg and T peripheral helper phenotype already at this early time point. Productive alpha/beta-TCR sequences could be retrieved from 16 out of 17 patients and clonal expansion (&gt;1 copy/TCR) was seen in all but one of these patients, with clone sizes ranging between 2 – 34 copies of each TCR. Table 1. Patient characteristics. Patients Gender (F/M ) HLA-SE alleles Joints Joint swelling prior to biopsy (months ) Stiffness specific joint (median VAS ) Pain specific joint (median VAS ) ACPA+ (n = 12) 9/3 *0401, *0404, *0408, *01 and *10 1 MTP, 4 MCP, 6 wrists, 1 knee 4 (1-12) a 46 (0-84) 45 (22-99) ACPA- (n = 5) 3/2 *0401 1 MCP, 2 wrists, 1 ankle, 1 knee 5 (0.25-7) 59 (15-73) 47 (33-81) SKW3 cell lines (patients n = 4) 4/0 * 0401/0404 n=2 *0401 n=2 1 MTP, 3 wrists 2 (1-6) 50.5 (42-84) 50 (40-99) a Data not available for one patient. One patient with prior RA-diagnosis, but after 9 months of treatment remission lasting for 20 years. Artificial T cell lines were generated from the expanded clones of HLA-DRB1*04:01 RA subjects. Our in vitro stimulation protocol identified virus specific CD4 T cells in all samples. So far, no citrulline reactivity has been found. HCMV, followed by HHV were the most commonly found viral reactivities, whereas others were found only in one donor (e.g. JCV, EBV). The majority of clones are thus “orphans”, to which we are still seeking the driving antigen. Conclusion: Clonally expanded T cells are found in the synovium of early RA patients and include virus-specific CD4+ T cells. Our data show that the local T cell repertoire is broad already at the time of RA diagnosis Disclosure of Interests: Sara Turcinov: None declared, Erik af Klint Paid instructor for: Abbvie (courses and lectures), An De Bondt Employee of: Janssen., Muhammad Sohel Mia: None declared, Anca Catrina: None declared, Frederik Stevenaert Employee of: Janssen, Vivianne Malmström Grant/research support from: VM has had research grants from Janssen Pharmaceutica