B-cell receptor (BCR)-activated B cells contribute to pathogenesis in chronic graft-versus-host disease (cGVHD), a condition manifested by both B-cell autoreactivity and immune deficiency. We hypothesized that constitutive BCR activation precluded functional B-cell maturation in cGVHD. To address this, we examined BCR-NOTCH2 synergy because NOTCH has been shown to increase BCR responsiveness in normal mouse B cells. We conducted ex vivo activation and signaling assays of 30 primary samples from hematopoietic stem cell transplantation patients with and without cGVHD. Consistent with a molecular link between pathways, we found that BCR-NOTCH activation significantly increased the proximal BCR adapter protein BLNK. BCR-NOTCH activation also enabled persistent NOTCH2 surface expression, suggesting a positive feedback loop. Specific NOTCH2 blockade eliminated NOTCH-BCR activation and significantly altered NOTCH downstream targets and B-cell maturation/effector molecules. Examination of the molecular underpinnings of this “NOTCH2-BCR axis” in cGVHD revealed imbalanced expression of the transcription factors IRF4 and IRF8, each critical to B-cell differentiation and fate. All-trans retinoic acid (ATRA) increased IRF4 expression, restored the IRF4-to-IRF8 ratio, abrogated BCR-NOTCH hyperactivation, and reduced NOTCH2 expression in cGVHD B cells without compromising viability. ATRA-treated cGVHD B cells had elevated TLR9 and PAX5, but not BLIMP1 (a gene-expression pattern associated with mature follicular B cells) and also attained increased cytosine guanine dinucleotide responsiveness. Together, we reveal a mechanistic link between NOTCH2 activation and robust BCR responses to otherwise suboptimal amounts of surrogate antigen. Our findings suggest that peripheral B cells in cGVHD patients can be pharmacologically directed from hyperactivation toward maturity.

B细胞受体（BCR）激活的B细胞促成慢性移植物抗宿主病（cGVHD）的发病机理，这种疾病既表现为B细胞自身反应性，又表现为免疫缺陷。我们假设本构性BCR激活排除了cGVHD中功能性B细胞的成熟。为了解决这个问题，我们检查了BCR-NOTCH2的协同作用，因为已证明NOTCH可增加正常小鼠B细胞的BCR反应性。我们进行了来自有和没有cGVHD的造血干细胞移植患者的30个主要样本的离体激活和信号传导测定。与途径之间的分子联系一致，我们发现BCR-NOTCH激活显着增加了近端BCR衔接子蛋白BLNK。BCR-NOTCH激活还启用了持续的NOTCH2表面表达，表明存在正反馈回路。特定的NOTCH2封锁消除了NOTCH-BCR激活，并显着改变了NOTCH下游靶标和B细胞成熟/效应分子。在cGVHD中对该“ NOTCH2-BCR轴”的分子基础进行的检查显示出转录因子的表达失衡IRF4和IRF8分别对B细胞分化和命运至关重要。清一色反式维甲酸（ATRA）增加IRF4表达，恢复了IRF4 -到- IRF8比，废除BCR-NOTCH过度活化，并减少在慢性GVHD B细胞表达NOTCH2而不损害生存能力。经ATRA处理的cGVHD B细胞的TLR9和PAX5升高，但BLIMP1没有（与成熟的滤泡性B细胞相关的基因表达模式），并且还提高了胞嘧啶鸟嘌呤二核苷酸的响应能力。在一起，我们揭示了NOTCH2激活和对其他次优量的替代抗原的强大BCR反应之间的机制联系。我们的发现表明，cGVHD患者的外周血B细胞可以在药理学上从过度激活走向成熟。