data_sheet_1_Enhanced AKT Phosphorylation of Circulating B Cells in Patients With Activated PI3Kδ Syndrome.DOCX

Activated PI3Kδ syndrome (APDS) is a primary immunodeficiency characterized by recurrent respiratory tract infections, lymphoproliferation, and defective IgG production. Heterozygous mutations in PIK3CD, PIK3R1, or PTEN, which are related to the hyperactive phosphoinositide 3-kinase (PI3K) signaling, were recently presented to cause APDS1 or APDS2 (APDSs), or APDS-like (APDS-L) disorder. In this study, we examined the AKT phosphorylation of peripheral blood lymphocytes and monocytes in patients with APDSs and APDS-L by using flow cytometry. CD19+ B cells of peripheral blood in APDS2 patients showed the enhanced phosphorylation of AKT at Ser473 (pAKT) without any specific stimulation. The enhanced pAKT in CD19+ B cells was normalized by the addition of a p110δ inhibitor. In contrast, CD3+ T cells and CD14+ monocytes did not show the enhanced pAKT in the absence of stimulation. These findings were similarly observed in patients with APDS1 and APDS-L. Among CD19+ B cells, enhanced pAKT was prominently detected in CD10+ immature B cells compared with CD10− mature B cells. Enhanced pAKT was not observed in B cells of healthy controls, patients with common variable immunodeficiency, and hyper IgM syndrome due to CD40L deficiency. These results suggest that the enhanced pAKT in circulating B cells may be useful for the discrimination of APDS1, APDS2, and APDS-L from other antibody deficiencies.

活化PI3Kδ综合征（Activated PI3Kδ Syndrome, APDS）是一类原发性免疫缺陷病，以反复呼吸道感染、淋巴细胞增生及IgG产生缺陷为核心特征。近期研究证实，PIK3CD、PIK3R1或PTEN基因的杂合突变可诱发APDS1、APDS2（统称APDSs）或类APDS（APDS-L）病症，上述突变均与过度活化的磷酸肌醇3-激酶（phosphoinositide 3-kinase, PI3K）信号通路密切相关。本研究采用流式细胞术，检测了APDSs及APDS-L患者外周血淋巴细胞与单核细胞的AKT磷酸化水平。结果显示，APDS2患者外周血CD19+B细胞在无特异性刺激的状态下，即可出现Ser473位点AKT磷酸化（pAKT）水平升高；向该体系中加入p110δ抑制剂后，CD19+B细胞中升高的pAKT水平可恢复至正常基线。与之相对，在无外源性刺激的条件下，APDS患者的CD3+T细胞与CD14+单核细胞均未检测到pAKT水平升高。该表型在APDS1及APDS-L患者中均一致出现。进一步分析CD19+B细胞亚群发现，与CD10−成熟B细胞相比，CD10+未成熟B细胞的pAKT升高现象更为显著。健康对照个体、普通可变免疫缺陷患者以及因CD40L缺陷所致的高IgM综合征患者的B细胞中，均未观察到pAKT水平异常升高。综上，循环B细胞中升高的pAKT水平，可用于区分APDS1、APDS2及APDS-L与其他类型抗体缺陷病。