WAS蛋白通过BCR-FcγRIIB-pSHIP通路抑制B细胞信号活化的机制研究

B cell plays critical roles in humoral autoimmune responses. B cell disorder is one of the major factors that lead to autoimmune disease. Previous studies demonstrated that WASp deficiency caused autoimmune diseases was B cell intrinsic. Our group and other groups have already proved the molecular mechanism of B cell activation and antibody deficiency in WAS. However, the negative mechanism of WASp on B cells remains elusive. To answer the question, we set up BCR-BCR or BCR- FcγRIIB lipid bilayer system to study B cell signaling inhibition by TIRF or live cell imaging. Both WAS patients’ Blood and WAS mice spleen will be used. The preliminary data showed that the level of total SHIP on naive B or GC-like was similar from mice or patients compare with controls. However, the recruitment of pSHIP from WASp deficiency B was decreased dramatically in response to BCR-FcγRIIB inhibition. Taking together, for the first time, we propose WASp is required for BCR-FcγRIIB-pSHIP inhibition by upstreaming the major inhibitory FcγRIIB signaling. As a result, B cells are out of self-tolerance, leading to autoimmune responses. This new understanding sheds light on developing new therapeutic target to WAS, and can provide novel theoretical support to other autoimmune disease or antibody treatment for cancer.

B免疫耐受打破是自身免疫性疾病发生的最主要原因之一。前期研究表明，WAS蛋白缺陷B细胞在湿疹、血小板减少免疫缺陷综合征（WAS）自身免疫并发症发生中起固有作用。我们前期已基本阐明WASp对B细胞活化的机制，但WASp缺陷对B细胞负向调控功能的影响尚不清楚。我们的前期结果提示在BCR-FcγRIIB交联时，WAS缺陷B细胞pSHIP募集水平显著下调。因此，我们首次提出WASp缺陷通过影响BCR-FcγRIIB-pSHIP抑制信号通路，打破B细胞自身免疫耐受，从而在抗体缺陷的同时发生自身免疫。本课题拟采用WAS小鼠及病人样本，运用业已建立的B细胞活化及抑制技术体系、TIRFM、活细胞成像技术，解析WASp缺陷对BCR-FcγRIIB-pSHIP介导的B细胞负调控细胞分子机制。该机制的阐明不仅可能为WAS病人自身免疫性并发症的治疗提供新靶点，也可能为其他自身免疫性疾病、肿瘤生物治疗等提供理论基础

WAS患儿高发自身免疫性疾病，是该病难以控制甚至致死的重要原因之一。B免疫耐受打破是自身免疫性疾病发生的最主要原因之一，受B细胞活化及抑制调控。WASp调节B细胞活化机制较明确，但负调控通路尚不清楚。FcγRIIB是最重要的B细胞负调控分子之一。因此，我们通过构建BCR-BCR、BCR-FcγRIIB磷脂双分子层体系，运用业已建立的B细胞活化及抑制技术体系、TIRFM、活细胞成像技术等，旨在阐明WASp调节BCR-FcγRIIB负调控的机制。结果表明，WASp缺陷B细胞通过抑制F-actin募集及其与BCR-FcγRIIB的共定位调节BCR-FcγRIIB-pSHIP负调控，可通过调节CD19去磷酸化，但不影响胞pCD19膜区募集水平，不通过肌球蛋白环形结构形成、BCR簇聚集及B细胞扩张水平调节BCR-FcγRIIB-pSHIP负调控。该机制的阐明不仅可为WAS病人自身免疫性并发症的治疗提供新靶点，也为其他自身免疫性疾病、肿瘤等生物治疗等提供理论基础。