POS0017 ACTIVATED B CELLS ARE ENRICHED FOR RANKL AND PROINFLAMMATORY CYTOKINE PRODUCTION PRIOR TO ONSET OF CLINICAL RHEUMATOID ARTHRITIS

Background

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by inflammation of the synovial tissue lining the joint, leading to bone damage. Anti-citrullinated protein autoantibodies (ACPAs) play an important role in the pathogenesis of RA and can be detected before the onset of classifiable or clinical RA. ACPA⁺ individuals without clinical RA, or ‘ACPA⁺ At-Risk’, are being studied to identify effective preventive interventions. B cells also contribute to disease through autoantibody-independent mechanisms, including production of RANKL and inflammatory cytokines. RANKL-producing B cell in clinical RA have been linked to the CD11c⁺ CD21^lo B cell phenotype and FcRL4 expression^[1,2]. Notably, activated memory B cells also promote osteoclastogenesis in vitro in a RANKL-dependent manner, which may be enhanced by IL-6 and TNFa^[3].

Objectives

We sought to determine if activated peripheral B cells were predisposed to production of RANKL and proinflammatory cytokines in ACPA⁺ donors without clinical RA (termed ‘At-Risk’) when compared to ACPA^- controls. Further, we wanted to understand the functional B cell subset identity, FcRL4 expression status, and cytokine co-expression profiles of RANKL⁺ B cells in the ACPA⁺ and control groups.

Methods

We employed spectral flow cytometry to achieve high-dimensional characterization of activated B cells, with simultaneous quantification of 23 cell-identity and state-defining molecules, from age-matched ACPA^- control and ACPA⁺ At-Risk individuals (Table 1). TLR9 ligand (CpG) and CD40 stimulation was applied to generate RANKL⁺ B cells among total PBMCs. We evaluated differences in frequencies of RANKL⁺ cells as well as RANKL/FcRL4 and RANKL/cytokine co-expression profiles between ACPA^- and ACPA⁺ individuals. Single-cell ATACseq analysis was applied to unstimulated total B cells from ACPA⁺ and ACPA^- individuals to determine the accessibility at the RANKL promoter and enhancer sites.

Results

Here, we observed that B cells are primed for RANKL production in ACPA⁺ At-Risk donors. A substantially higher frequency of B cells, particularly IgM/IgD⁺ naïve and memory cells, produce RANKL (p=0.06) upon in vitro activation in ACPA⁺ At-Risk donors as compared to ACPA^- controls (Figure 1). RANKL production was not restricted to any single B cell identity. RANKL⁺ B cells exhibited a highly polyfunctional state, defined by proinflammatory cytokine production, as well as elevated surface activation molecule expression. Among ACPA⁺ donors, 34% of RANKL⁺ activated B cells co-expressed TNFa and IL-6 and 55.3% co-expressed FcRL4. Additionally, we found that resting peripheral B cells from ACPA⁺ At-Risk individuals exhibited an open chromatin state at the RANKL gene promoter and upstream regulatory sites by ATACseq analysis.

Conclusion

These results demonstrate that peripheral B cells are poised for proinflammatory responses and RANKL production upon activation in ACPA⁺ individuals. Our work suggests that loss of immune tolerance predates detectable synovial joint inflammation and highlights autoantibody-independent contributions of B cells to immune dysregulation. Future studies will evaluate these changes in relationship to the development of clinical RA.

References

[1]Yeo, L. et al. Expression of FcRL4 defines a pro-inflammatory, RANKL-producing B cell subset in rheumatoid arthritis. Ann. Rheum. Dis.74, 928–935 (2015). [2]Amara, K. et al. B cells expressing the IgA receptor FcRL4 participate in the autoimmune response in patients with rheumatoid arthritis. J. Autoimmun.81, 34–43 (2017). [3]Ota, Y. et al. Generation mechanism of RANKL⁺ effector memory B cells: relevance to the pathogenesis of rheumatoid arthritis. Arthritis Res. Ther.18, 67 (2016).

Acknowledgements:

NIL.

Disclosure of Interests

Marla Glass: None declared, Mark-Phillip Pebworth: None declared, Adam Savage: None declared, Mark Gillespie: None declared, Elisabeth Dornisch: None declared, Nicholas Moss: None declared, Morgan Weiss: None declared, Peter Skene: None declared, Julian Reading: None declared, Lynne Becker: None declared, Thomas Bumol: None declared, Cate Speake: None declared, Jane Buckner Consultant of: GentiBio, BMS, Kristine A. Kuhn: None declared, Kristen Demoruelle: None declared, V.Michael Holers: None declared, Kevin Deane Consultant of: Werfen, ThermoFisher, BMS, BI, Grant/research support from: Scipher Medicine, Gilead, Gary Firestein Grant/research support from: Eli Lilly, Troy Torgerson: None declared.