Kroos Sanne, Blomberg Nienke J, Kwekkeboom Joanneke C, Hendriks Rudi W, Corneth Odilia B J, Toes René E M, Scherer Hans U
Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands.
Department of Pulmonary Medicine, Erasmus MC, University Medical Center, Rotterdam, The Netherlands.
Eur J Immunol. 2025 Jan;55(1):e202451361. doi: 10.1002/eji.202451361.
Many human autoimmune diseases (AIDs) are hallmarked by the presence and persistence of autoreactive B-cells. While autoreactive B-cells may frequently encounter antigens, the signals required to balance and maintain their activation and survival are mostly unknown. Understanding such signals may be important for strategies aimed at eliminating human B-cell autoreactivity. Here, we assessed intracellular signaling pathways in B cells targeting citrullinated protein antigens isolated from patients with rheumatoid arthritis (RA), a common and well-characterized AID. Peripheral blood mononuclear cells of 15 RA patients positive for anti-citrullinated protein antibodies (ACPA) were analyzed directly ex vivo using spectral flow cytometry and B-cell differentiation markers, citrullinated antigen-biotin-streptavidin tetramers, and intracellular (phosphoflow) markers. Tetanus toxoid (TT)-specific B cells served as antigen-specific comparators. In absence of any in vitro BCR stimulation, ACPA-expressing memory B cells (MBCs) displayed enhanced expression of Ki-67 and increased SYK-, BTK-, AKT-, and S6-phosphorylation compared with TT-specific MBCs. We demonstrate the simultaneous detection of B cell antigen-specificity and intracellular protein phosphorylation on the single-cell level. The data reveal that autoreactive B-cells in RA, in contrast to B cells against recall antigens, display enhanced phosphorylation of signaling molecules that point toward continuous, presumably antigen-mediated activation of the autoreactive B-cell compartment.
许多人类自身免疫性疾病（AIDs）的特征是自身反应性B细胞的存在和持续存在。虽然自身反应性B细胞可能经常遇到抗原，但平衡和维持其激活与存活所需的信号大多未知。了解这些信号对于旨在消除人类B细胞自身反应性的策略可能很重要。在这里，我们评估了靶向从类风湿性关节炎（RA）患者中分离出的瓜氨酸化蛋白抗原的B细胞中的细胞内信号通路，RA是一种常见且特征明确的AID。使用光谱流式细胞术、B细胞分化标志物、瓜氨酸化抗原-生物素-链霉亲和素四聚体和细胞内（磷酸化流式）标志物，直接对15名抗瓜氨酸化蛋白抗体（ACPA）阳性的RA患者的外周血单个核细胞进行体外分析。破伤风类毒素（TT）特异性B细胞用作抗原特异性对照。在没有任何体外BCR刺激的情况下，与TT特异性记忆B细胞（MBCs）相比，表达ACPA的记忆B细胞显示出Ki-67表达增强以及SYK、BTK、AKT和S6磷酸化增加。我们展示了在单细胞水平上同时检测B细胞抗原特异性和细胞内蛋白磷酸化。数据显示，与针对回忆抗原的B细胞相比，RA中的自身反应性B细胞显示出信号分子磷酸化增强，这表明自身反应性B细胞区室持续存在，可能是由抗原介导的激活。
