Background/Purpose: Anti-citrullinated protein antibodies (ACPA) play a role in rheumatoid arthritis (RA) pathogenesis and are associated with disease severity. Detailed analysis of ACPA^pos B cells may further clarify their role in RA. NF-κB signaling is a key regulator of B cell responses, including proliferation, differentiation, and (auto)antibody production, making NF-κB a prime candidate to target ACPA^pos B cells. Because the frequencies of ACPA^pos B cells in the peripheral blood of RA patients are low, we optimized an expansion assay for memory B cells, to study whether NF-κB inhibition is effective in targeting functional (autoreactive) responses.

Methods: Memory B cells from healthy donors and ACPA^pos B cells from RA patients were sorted and cultured with CD40L, 50 ng/ml IL-2, 10 ng/ml IL-21, and 10 ng/ml BAFF. ACPA^pos B cell clones were cultured with 2.25 µg/ml anti-CD40 and 1 ng/ml IL-21. Plasma cells differentiated from expanded memory B cells were culture with 200 ng/ml APRIL and 10 ng/ml IL-6. Canonical and non-canonical NF-κB signaling were targeted by validated small molecule inhibitors targeting Inhibitor of κB kinase β (IKKβ) and NF-κB inducing kinase (NIK), respectively. B cell responses were evaluated by flow cytometry, and antibody production was measured by ELISA.

Results: Memory B cells were expanded from 250 cells/well and differentiated into plasma cells in vitro for 8 days (mean ± SEM B cells: n=29.4×10³±2.7×10³; plasma cells: n=3.3×10³±0.35×10³); survival was analyzed at day 16 (mean ± SEM B cells: n=3.3×10³±1.1×10³; plasma cells: n=1.4×10³±0.46×10³). Both NIK (2.5 µM) and IKKβ (1.0 µM) inhibition resulted in a significant decrease in B cell expansion (NIKi=74.6%; IKKβi=63.1%) and plasma cell differentiation (NIKi=78.1%; IKKβi=45.7%) at day 8, with stronger effect of NIKi. At later timepoint, treatment of plasma cells with NF-κB inhibitors did not have a significant effect on plasma cell number (mean ± SEM DMSO=3.9×10³±1.5×10³; NIKi=3.2×10³±1.1×10³; IKKβi=2.8×10³±1.2×10³), although NIKi resulted in significant decreased survival (DMSO=83.2%; NIKi=77.6%). In ACPA^pos B cell clones from RA patients treated with these agents (12.5-50.0 µM) upon anti-CD40L and IL-21 stimulation, we observed a dose-dependent reduction in proliferation and IgG production. In addition, we succeeded in isolating ACPA^pos and ACPA^neg B cells from the peripheral blood of RA patients, and quantify ACPA production. At present the above results are being corroborated in freshly isolated ACPA^pos B lineage cells from RA patients.

Conclusion: Our data point towards a critical role of the NF-κB signaling pathways in the functional responses of ACPA-producing B cells. Consequently, targeting NF-κB signaling may have beneficial effects in limiting (autoreactive) B cell responses in RA and potentially other immune-mediated inflammatory disorders driven by B cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/in-vitro-expansion-of-acpa-positive-b-cells-from-rheumatoid-arthritis-patients-and-effect-of-small-molecule-nf-%ce%bab-inhibitors-on-differentiation-and-survival-of-autoreactive-memory-b-cells-into/