BackgroundAnti-citrullinated protein autoantibodies (ACPA) are known to associate with strong risk factors for rheumatoid arthritis (RA), as well as worse clinical prognosis[1]. Due to their high specificity in RA, ACPA are part of the classification criteria and regarded as being essential in the identification of individuals at risk of developing RA. However, not every ACPA-positive at-risk individual progresses to a clinical diagnosis[2], suggesting that the presence of ACPA per se does not dictate the clinical outcome. We have recently shown that ACPA are in fact predominantly anti-inflammatory and present different biological effects in a murine model of arthritis[3]. Having a wide diversity of ACPA clones isolated from different tissues of RA patients at our disposal, we have continued to analyze different monoclonal as well as polyclonal ACPA in their capacity to affect inflammatory processes in vivo.ObjectivesTo determine whether ACPA with pathogenic features, i.e. capable of increasing or sustaining joint inflammation, can be found among different isolated ACPA clones from patients with established RA.MethodsImmunoglobulin from single B cells from RA patients were sequenced and recombinantly expressed as monoclonal human (h)IgG1 in Expi293F cells, followed by validation of citrulline reactivity. Here, we tested 5 new monoclonal ACPA isolated based on anti-citrullinated tetramer staining of B cells[4]. Polyclonal ACPA IgG from 34 RA patients were enriched via a CCP2 affinity column. The unbound IgG fraction was used as the flow-through control. Arthritis was induced in BALB/c mice by i.v. transfer of 1.5mg of an arthritogenic cocktail of anti-type II collagen (CII) antibodies (Chondrex, USA) followed by administration of 25ug of LPS (E. colistrain O55:B5) 3 days later. Individually, 1mg of purified ACPA monomers, 2mg of ACPA-pool or 2mg flow-through IgG were transferred i.v. together with the arthritogenic antibody cocktail. An isotype control antibody and a previously identified anti-inflammatory ACPA clone (hE02 and hC03, respectively)[3]were used as experimental reference conditions. Arthritis development was monitored daily by a quantitative scoring system of inflamed joints – toes, knuckles, and wrist/ankle of front/hind paws. Statistical analysis was performed with repetitive-measure one-way ANOVA with Geisser-Greenhouse correction and Holm-Šidák’s multiple comparisons test.ResultsAdding to our previous data with 8 monoclonal ACPA[3], where 3 of them did not alter the disease course, and 4 displayed strong anti-inflammatory properties, we observed an additional 4 newly tested ACPA clones that did not influence arthritis development. Furthermore, we identified one novel monoclonal ACPA (hF2C05) that displays a pro-arthritogenic effect (p<0.01). When assessing a polyclonal ACPA sample, we observed a dominant anti-inflammatory effect of the ACPA-pool in comparison to its non-ACPA flow-through IgG fraction, displayed by a significantly reduced disease prevalence (p<0.001).ConclusionOur preliminary data focusing on distinct monoclonal ACPA, as well as a new set of patient-derived polyclonal ACPA-pool, strengthens the notion that ACPA clones differ significantly between them, and within the same individual. Although we were able to identify an ACPA clone with arthritogenic potential, the majority of ACPA display no aptitude to affect joint inflammation or are anti-inflammatory. Together, this diversity of inflammatory effects calls for a re-evaluation of the proposed role of ACPA in RA, where their heterogeneity must be considered.References[1] Hair, M. J. H.et al. Arthritis Rheumatol66, 513–522 (2014)[2] Chirivi, R. G. S.et al. Cell Mol Immunol18, 1528–1544 (2021)[3] Raposo, B.et al. Ann Rheum Disard-2022-223417 (2023) doi:10.1136/ard-2022-223417[4] Titcombe, P. J.et al. Arthritis Rheumatol70, 1933–1945 (2018)AcknowledgementsWe would like to acknowledge the following agencies for financial support of the current work: IMI project RTCure (777357), ERC consolidation grant (2017-7722209_PREVENT RA), the Swedish Research Council (VR; 2019-01664) and Ulla and Gustaf af Uggla Foundation (2020-0009).Disclosure of InterestsNone Declared.
