Background/Purpose: Structural changes, increased tissue citrullination, signs of local inflammation and anti-citrullinated protein autoantibodies (ACPA) are present in the pulmonary compartment of early untreated seropositive rheumatoid arthritis (RA). These findings provide evidence of a potential role for the lungs in generation and initiation of RA-associated autoimmunity. Here we aimed to investigate the presence of citrulline-reactive B cells in the lung compartment of early untreated RA patients by single-cell cloning of monoclonal antibodies (mAbs) from lung-derived B cells.

Methods: Bronchoalveolar lavage (BAL) fluid cells were obtained from ACPA positive individuals with arthralgia (n=4), early untreated ACPA positive RA (n=3) and ACPA negative RA (n=5) patients at the time of diagnosis. CD19+ B cells were single cell sorted by flow cytometry. Immunoglobulin heavy and light chain variable regions were PCR amplified, sequenced, and analyzed by V-Quest and IgBLAST towards the IMGT database to annotate variable gene usage. Sequences having high somatic hypermutations (SHM) and Fab N-glycosylation sites were selected to be cloned and expressed as human IgG1 recombinant mAbs. Citrulline reactivity was determined by CCP2 assay and in-house ELISA against citrullinated and native peptides from vimentin, a-enolase, fibrinogen and filaggrin. Polyreactivity was determined by reactivity against double stranded DNA, bacterial lipopolysaccharides (LPS) and insulin. The functional properties of mAbs were analyzed in osteoclastogenesis assay, fibroblast migration assay and neutrophil binding assay.

Results: Significant higher proportion of CD19+ B cells were found in ACPA positive compared to ACPA negative BAL (median 6.7% vs 0.79%). PCR amplification and subsequent BCR sequencing from 8 individuals yielded 581 paired heavy and light chain sequences. 49 monoclonal antibodies (from 5 individuals) were selected for expression based on high number of SHM and predicted Fab N-linked glycosylation. Notably, four of these selected mAbs (8%; 2 antibodies from each patient, one risk RA and one early untreated RA) were determined to be ACPAs by CCP2 ELISA. The 4 ACPAs had varying ACPA fine-specificity against citrullinated a-enolase, filaggrin, vimentin and fibrinogen peptides without any native peptide binding. All the 4 monoclonal ACPAs were negative in polyreactivity test. Clone L204:01A01 and L201:11C11 but not clone L201:10D07 and L204:05E10 bound to activated neutrophils (49.75% for clone A01 and 22.3% for clone C11). Clone L204:01A01 and L204:05E10 but not L201:10D07 and L201:11C11 induced osteoclastogenesis with a fold increase of 1.5 and 1.9 respectively. Clone L204:01A01 and L201:10D07 but not L201:11C11 and L204:05E10 promoted fibroblasts migration with a fold increase of 1.4 and 1.5 respectively.

Conclusion: We demonstrate for the first time that citrulline-reactive B cells producing pathogenic ACPAs are present in the lung compartment of seropositive individuals at risk for or having early untreated RA. This provides a strong link between the lung and the adaptive autoreactive response in RA, and support the hypothesis of the lung as one of the key sites of initiation and propagation of disease in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/citrulline-reactive-b-cells-are-present-in-the-lungs-of-risk-ra-and-early-untreated-ra/