SAT0016 RHEUMATOID ARTHRITIS PATIENTS DISPLAY B-CELL DYSREGULATION ALREADY IN THE NAïVE REPERTOIRE
Authors: Yan Wang, Katy A. Lloyd, Ioannis Melas, Daniel Ramsköld, Diana Zhou, Radha Thyagarajan, Karin Lundberg, Lars Klareskog, Anca Catrina, Stephen Rapecki, Vivianne Malmström, Caroline Grönwall
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> Seropositive rheumatoid arthritis (RA) is associated with autoreactivity to citrullinated proteins and rheumatoid factor activity. The adaptive immune system and B cells are postulated to be central in RA pathogenesis, yet possible B-cell dysregulations have not been extensively studied. Here, we use exploratory mass cytometry and next generation sequencing to study overall B cell repertoire shifts in RA patients compared to healthy individuals. <h3>Objectives:</h3> We hypothesized that there are an underlying B cells repertoire distortion in RA that may be an important key to understanding the autoimmune pathogenesis. <h3>Methods:</h3> B cells enriched from PBMCs from nine ACPA pos., seven ACPA neg. RA patients, and six matched heathy controls were phenotyped with mass cytometry (CyTOF) using a 35-marker panel. Single cell data from all samples were pooled clustered via flowSOM in R to identify distinct cell populations, and visualized with tSNE. Cell numbers in each cluster/population were calculated to identify over-representation in patient groups or controls. ANOVA was performed to account for confounding factors such as age, sex and cell preparation date and differential expression of markers between groups was calculated via t-test. In parallel, PBMC B cell receptor (BCR) repertoires were investigated in 13 ACPA pos. RA patients and six age-matched healthy donors using the Illumina MiSeq platform and PCR multiplex amplicon libraries with a molecular barcode strategy to generate full variable region coverage. Sequences were filtered using pRESTO, annotated by IMGT and Change-O, finally generating 587 000 unique V-regions. Total serum IgM levels were screened by sandwich ELISA in 157 population controls, 193 ACPA pos., and 50 ACPA neg. RA patients. Variable gene frequency was analyzed by Chi-square with Yates correction and serum IgM levels with Kruskal-Wallis test. <h3>Results:</h3> Several B-cell phenotypes were found to be significantly different in ACPA pos. RA compared to controls including an increase in HLA-DR across subsets, CD11c in IgA memory and CD22 expression in clusters of mature naïve IgM positive B cells. Moreover, we could see lower circulating cell counts in ACPA pos. RA in IgG memory (p=0.01) and trends for elevation in an CXCR5/CCR6 high transitional B cell cluster (p=0.06), with parallel lower number of transitional B cells with lower CCR6 expression (p=0.06). Notably, ACPA neg. RA generally had an intermediate phenotype between healthy controls and ACPA pos. RA. Several significant shifts in the RA BCR repertoire could be observed, including an expected higher frequency of VH N-linked glycosylation in highly mutated BCR (p&lt;0.0001). Yet, the most striking difference was a significantly higher frequency of VH with low somatic hypermutation (SHM) levels in RA-derived B cells (&lt;5 mutations, p&lt;0.0001 14.7% vs 8,7). This was seen in all sequences, both IgM and class-switched, but was especially prominent in IgG1 rearrangements (9.6% vs 18.8% low mutation, p&lt;0.0001 OR=2.2 CI:2.0-2.35). In line with an IgM and low mutation profile in RA, we also observed that both ACPA pos. (p&lt;0.0001) and ACPA neg. (p&lt;0.001) RA patients have a significant increase in total IgM levels compared to controls (1.4 ± 0.7; 1.3 ± 0.7;1.0 ± 0.6 mg/ml, respectively). <h3>Conclusion:</h3> Previous studies have shown that anti-citrulline autoreactivity in RA is primarily originates from memory B cells and characterized by high somatic mutations and N-glycosylation sites. However, here the largest B cell distortions in ACPA positive RA are observed in the naïve B cell population that have not undergone germinal center responses. These differences could reflect baseline shifts and elevated natural autoreactivity as an underlying mechanism in RA pathogenesis. <h3>Disclosure of Interests:</h3> Yan Wang: None declared, Katy A. Lloyd: None declared, Ioannis Melas Employee of: Employed by UCB Pharma, Daniel Ramsköld: None declared, Diana Zhou: None declared, Radha Thyagarajan: None declared, Karin Lundberg: None declared, Lars Klareskog Grant/research support from: Yes, but not for the presented study., Anca Catrina Grant/research support from: Yes, but not for the presented study., Stephen Rapecki Employee of: Employed by UCB Pharma, Vivianne Malmström: None declared, Caroline Grönwall: None declared