Background: Anti-citrullinated protein antibodies (ACPA) are implicated in the pathogenesis of rheumatoid arthritis (RA) and are associated with disease severity. Detailed analysis of ACPA-positive (ACPA ^pos ) B cells may further clarify their role in RA. NF-κB signalling 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. However, the frequency of ACPA ^pos B cells in the peripheral blood of RA patients is low, prompting us to optimize an expansion assay for memory B cells, to study whether NF-κB inhibition is effective in targeting functional (autoreactive) responses.

Objectives: To study the effect of small molecule inhibitors of NF-κB on the functional responses of ACPA ^pos B cells.

Methods: Memory B cells from healthy donors, and ACPA ^pos and ACPA ^neg memory 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. Plasma cells derived from expanded memory B cells were cultured with 200 ng/ml APRIL and 10 ng/ml IL-6. ACPA ^pos and ACPA-negative (ACPA ^neg ) B cell clones were cultured with 2.25 µg/ml anti-CD40 and 1.0 ng/ml IL-21. Canonical and non-canonical NF-κB signalling were targeted using previously 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 and differentiated into plasma cells in vitro for 8 days (mean cell number ± SD B cells: 29.4x10 ³ ±6.6x10 ³ ; Plasma cells: 3.3x10 ³ ±0.86x10 ³ ). Both NIK (2.5 µM) and IKKβ (1.0 µM) inhibition led to 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 a stronger effect observed after NIKi. A slight increase in apoptosis was seen upon NIKi and IKKβi (Annexin V ^pos B cells mean ± SD: Control=14.31±3.8%; NIKi=22.33±3.9%; IKKβi=33.2±4.2%). A significant decrease in IgG levels was also observed upon NIKi treatment. Extending the culture to 14 days showed that the NF-κB inhibitors exhibited a trend toward a decrease in plasma cell numbers (mean ± SD Control=3.9x10 ³ ±3.6x10 ³ ; NIKi=3.2x10 ³ ±2.7x10 ³ ; IKKβi=2.8x10 ³ ±2.8x10 ³ ), which was accompanied which was accompanied by a slightly increased proportion of apoptotic plasma cells after NF-κB inhibition (Control=8.8%; NIKi=12.6%, IKKβi=10.4%). In ACPA ^pos B cell clones from RA patients treated with these agents upon anti-CD40L and IL-21 stimulation, we observed a dose-dependent reduction in proliferation and IgG production. Additionally, we successfully isolated ACPA ^pos and ACPA ^neg B cells from the peripheral blood of RA patients and quantified ACPA production. Treatment with NIKi and IKKβi inhibited the expansion of ACPA ^pos memory B cells, with more pronounced effect compared to ACPA ^neg memory B cells.

Conclusion: Our data point towards a critical role of the NF-κB signalling pathway in the functional responses of ACPA-producing memory B cells. Targeting NF-κB signalling may therefore offer beneficial effects in limiting (autoreactive) B cell responses in RA and potentially other immune-mediated inflammatory disorders driven by B cells.

REFERENCES: NIL.

Acknowledgements: NIL.

Disclosure of Interests: Giulia Frazzei: None declared, Ana Merino-Vico: None declared, Jeroen Christiaans: None declared, Jan Piet Van Hamburg: None declared, Ronald F. van Vollenhoven AbbVie, AstraZeneca, Biogen, BMS, Galapagos, GSK, Janssen, Pfizer, RemeGen, UCB, AbbVie, AstraZeneca, Biogen, BMS, Galapagos, GSK, Janssen, Pfizer, RemeGen, UCB, Sander W. Tas NovoNordisk, Pfizer, AbbVie, UCB (all payments made to institution), Galvani bioelectronics, Lilly (all payments made to institution), GSK, Lilly, Celgene, Pfizer, Roche, AstraZeneca, Galapagos, Citryll, Galvani bioelectronics (all payments made to institution).

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.