<h3>Background</h3> Antiphospholipid syndrome (APS) is an autoimmune disease defined by arterial or venous events or pregnancy complications in association with autoantibodies against phospholipids. Despite these autoreactive antibodies, B lineage cells remain poorly studied in APS.[1] Besides triple-positivity of autoantibodies and the occurrence of lupus anticoagulant, there are no biomarkers for risk assessment in APS.[2] Characterization of autoantigen-specific B cells hold promise for better understanding APS pathogenesis which may guide targeted immune therapies including identification of biomarkers. <h3>Objectives</h3> The study characterized B cell subsets among patients with primary (pAPS) and secondary APS (sAPS) compared to healthy controls. In particular, we analyzed phosphatidylcholine (PtC)-specific B cells for their frequency of occurrence and immunophenotype related to disease characteristics. <h3>Methods</h3> We analyzed PBMCs from 20 HCs, 25 pAPS and 16 sAPS patients. Using multi-dimensional flow cytometry, we analyzed the expression of 15 surface markers on B cells including the frequency of PtC-specific B lineage cells. We established a novel assay to detect antigen-specific B cells against PtC as a potential correlate of antiphospholipid antibodies as previously reported for autoreactive B1 cells in mice.[3] The obtained cellular subsets and autoreactive cells were subjected to the FlowSOM algorithm in R to identify B cell clusters through an unbiased strategy. <h3>Results</h3> pAPS and sAPS patients showed increased frequencies of atypical CD21low as well as CD21low/CD11c+ B cells, most prominent within the IgD+/CD27+ memory compartment (CD21low: p&lt;0.01, CD21low/CD11c+: p&lt;0.001). We found higher frequencies of total PtC-specific B cells compared to HCs among pAPS in contrast to sAPS patients. In HCs, PtC-specific B cells were found mainly among naïve B cells, while they were significantly enriched within IgD+/CD27+ pre-switch memory B cells in pAPS patients (p&lt;0.01). Most notably, high frequencies of PtC-specific IgD+/CD27+ pre-switch memory B cells were associated with a high-risk APS profile according to the EULAR classification criteria (p&lt;0.05). Unsupervised FlowSOM clustering identified eight distinct B cell clusters. Remarkably, PtC-reactive memory B cells mainly resided in a unique cluster of IgD+/CD27+/CD21low/CD11c+ memory B cells (<b>Figure 1</b>). <h3>Conclusion</h3> Although APS patients carry an overall comparable B cell subset distribution as HCs, increased frequencies of atypical B cells are characteristic of pAPS and sAPS patients. PtC-specific B cells are substantially enriched among memory- and atypical CD21low/ CD11c+ memory B cells in pAPS. The data indicate that there is ongoing induction of autoantigen-specific atypical memory B cells apparently induced outside the germinal centers escaping negative selection. <h3>References</h3> [1]Dieudonné Y, Guffroy A, Poindron V, et al. B cells in primary antiphospholipid syndrome: Review and remaining challenges. <i>Autoimmun Rev</i>. May 2021;20(5):102798. doi:10.1016/j.autrev.2021.102798 [2]Tektonidou MG, Andreoli L, Limper M, et al. EULAR recommendations for the management of antiphospholipid syndrome in adults. <i>Ann Rheum Dis</i>. Oct 2019;78(10):1296-1304. doi:10.1136/annrheumdis-2019-215213 [3]Lino AC, Dang VD, Lampropoulou V, et al. LAG-3 Inhibitory Receptor Expression Identifies Immunosuppressive Natural Regulatory Plasma Cells. <i>Immunity</i>. Jul 17 2018;49(1):120-133.e9. doi:10.1016/j.immuni.2018.06.007 <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.