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Background Checkpoint inhibitor therapy (CPI) has changed the landscape of cancer treatment but is associated with immune-related adverse events (irAEs), posing significant clinical challenges. Research in irAEs is scarce and has largely focused on T cell biomarkers, although emerging evidence suggests a significant role of B cells. 1–4 Understanding the role of B cells in irAE development could uncover novel therapeutic targets and improve irAE management.Methods We performed immune monitoring with flow cytometry of fresh whole blood collected from metastatic melanoma patients undergoing CPI therapy. Blood was collected at baseline, after 3 weeks, and after 6 weeks. Clinical data on irAE development were retrospectively collected from medical records. Patients were stratified into two groups based on irAE development within 7 weeks from baseline: an irAE group, which required intervention with at least 25 mg of prednisolone or hormonal replacement therapy, and a control group without irAEs. Flow cytometric data were analyzed using R software, including dimensionality reduction with UMAP after gating on CD3-CD19+ cells. B cell subsets were identified within UMAP clusters, and their dynamics were examined in relation to irAE development.Results In 41 included patients, 16 developed irAEs within approximately seven weeks following CPI initiation. In total, 114 samples were included for analysis, and ten distinct B cell subsets were identified. Evident alterations in two subsets – IgD-CD27-CD21- double negative 2 (DN2) B cells and IgD-CD27+CD21+ pre-switched memory B cells – were observed in the irAE group. Specifically, pre-switched memory B cells, which are essential for rapid antibody production and long-term immunity, significantly decreased (p=0.013) from baseline to irAE onset, while DN2 B cells, known for their autoimmunogenic potential, exhibited a non-significant increasing trend.5–7 Alteration patterns were consistent across most irAE types, most notably arthritis. The results were confirmed in absolute cell counts, as total B cell numbers remained stable during treatment. No significant changes were detected in patients without irAEs.Conclusions IrAE development during CPI is associated with distinct alterations in B cell subsets, particularly DN2 B cells and pre-switched memory B cells. These findings implicate B cells in irAE pathogenesis and highlight potential targets for irAE management.References Das R, Bar N, Ferreira M, Newman AM, Zhang L, Bailur JK, et al. Early B cell changes predict autoimmunity following combination immune checkpoint blockade. J Clin Invest. 2018 Feb;128(2):715–720.Taylor J, Gandhi A, Gray E, Zaenker P. Checkpoint inhibitor immune-related adverse events: a focused review on autoantibodies and B cells as biomarkers, advancements and future possibilities. Front Immunol. 2022;13:991433.Nishimura K, Konishi T, Ochi T, Watanabe R, Noda T, Fukumoto T, et al. CD21lo B Cells could be a potential predictor of immune-related adverse events in renal cell carcinoma. J Pers Med. 2022 May;12(6):888.Ramos-Casals M, Brahmer JR, Callahan MK, Flores-Chávez A, Keegan N, Khamashta MA, et al. Immune-related adverse events of checkpoint inhibitors. Nat Rev Dis Primers. 2020 May;6(1):38.Li Y, Li Z, Hu F. Double-negative (DN) B cells: an under-recognized effector memory B cell subset in autoimmunity. Clin Exp Immunol. 2021 Jul;205(2):119–127.Chung MKY, Gong L, Kwong DLW, Lee VHF, Lee AWM, Guan XY, et al. Functions of double-negative B cells in autoimmune diseases, infections, and cancers. EMBO Mol Med. 2023 Sep;15(9):e17341.Inoue T, Kurosaki T. Memory B cells. Nat Rev Immunol. 2024 Jan;24(1):5–17.Ethics Approval The Study was approved by the Danish Regional Ethics Committee, ref.no. H-15007985. All patients provided oral and written consent prior to inclusion.