AB0029 CITRULLINE REACTIVE B CELLS ARE PRESENT IN THE LUNGS OF EARLY UNTREATED RA

Abstract

Background We have previously shown that structural changes, increased tissue citrullination, signs of local inflammation and ACPA are present in the pulmonary compartment of early seropositive RA. These findings suggest a potential role for the lungs in generation of RA-associated autoimmunity. Objectives To identify citrulline reactive B cells in the lung compartment of early untreated RA patients and to generate and characterize the corresponding monoclonal antibodies. Methods Bronchoalveolar lavage (BAL) fluid cells (13 and 22.5 million respectively) were obtained from two early untreated non-smoking ACPA positive RA patients and single CD19+ B cells were sorted by flow cytometry. Immunoglobulin variable region genes were sequenced and expressed to generate recombinant monoclonal antibodies (mAbs). The citrulline reactivity was determined by in-house ELISA against different citrullinated peptides and controls. Results Single sorted CD19+ B cells (n=768) from each patient (RA.1 and RA.2) were processed and the variable region amplification and sequencing yielded 192 (RA.1) and 213 (RA.2) paired heavy and light chain sequences. Based on previously published data showing SHM-introduced N-linked glycosylation in the Fab regions of ACPA [1], 22 sequences from each patient were pre-selected with such features, and monoclonal antibodies were expressed. Two antibodies from each patient were ACPAs as determined by their reactivity against CCP2. The 4 ACPAs have varying ACPA fine specificity against citrullinated enolase, filaggrin, vimentin and fibrinogen peptides (figure 1). Sequence analysis of the heavy chain variable region revealed unique V gene usage of the ACPAs arising from different patients, V4-39 for the 2 mAbs from RA.1 and V3-49 for RA.2. Conclusion We demonstrate for the first time that citrulline-reactive B cells are present in the lung compartment of early untreated RA. Further analysis and functional characterization are needed and ongoing to understand their role in RA. Reference [1] Lloyd, K.A., et al., Variable domain N-linked glycosylation and negative surface charge are key features of monoclonal ACPA: Implications for B-cell selection. Eur J Immunol, 2018. 48(6): p.1030-1045. Disclosure of Interests Vijay Joshua: None declared, Malena Loberg-Haarhaus: None declared, Heidi Wahamaa: None declared, Aase Hensvold: None declared, Magnus Skold: None declared, Johan Grunewald: None declared, Lars Klareskog Grant/research support from: Yes, but not for the presented study., Vivianne Malmstrom: None declared, Anca Catrina Grant/research support from: Yes, but not for the presented study.