Il-12p35在原发免疫性血小板减少症免疫失耐受中的作用、机制及临床意义

Primary immune thrombocytopenia (ITP) is a relatively common blood disorder characterized by autoimmune-mediated platelet destruction and suboptimal platelet production. The etiology of ITP is still unclear, but loss of immune tolerance to platelet surface antigens is considered as a fundamental cause of ITP. Recent researches are focused on the mechanism under its loss of immunologic self-tolerance, as well as the identification of new therapeutic targets. IL-35 is a heterodimeric cytokine including the p35 subunit and the Epstein-Barr virus (EBV)-induced gene 3 (EBI-3) subunit and classified as anti-inflammatory member of the IL-12 family. Previously, we found IL-35 contribute to the loss of immunological self-tolerance in ITP patients by modulating T cells and immunoregulatory cytokines. However, the mechanism by which it mediates its biological effects is not fully understood. Although it is implicitly assumed that immunosuppressive activities of IL-35 derive from pairing of IL-12p35 and Ebi3, it is unknown whether the single chain proteins, IL-12p35 and Ebi3, possess intrinsic biological activities. Moreover, the relative contribution of either subunit to the immunoregulatory functions of IL-35 is unclear. In this study, we aim to produce mouse recombinant IL-12p35 (rIL-12p35) and rEbi3 and to examine whether either protein can recapitulate some of the inhibitory activities of IL-35, and to elucidate the mechanism and clinical significance of IL-12p35 in ITP. We propose a new mechanism that IL-12p35 involves in the loss of immune tolerance of ITP through regulating dysfunctional T and B cells. Our study may provide evidence and new therapeutic targets for the comprehensive treatment of ITP.

原发免疫性血小板减少症（ ITP）是一种自身免疫介导的出血性疾病，因血小板破坏增加和生成不足导致外周血中血小板数量减少，免疫失耐受在其发生中扮演重要角色。其免疫失耐受机制及新的治疗靶点是目前的研究热点。IL-35由p35和Ebi3组成，是IL-12家族的抑炎因子。我们前期研究显示：IL-35通过调节自身T细胞及相关细胞因子参与ITP的免疫失耐受，但IL-35生物学活性的调节机制尚未阐明。我们推测IL-35的免疫抑制活性来源于α亚基IL-12p35和β亚基Ebi3的组合，但是单独的亚基本身是否具备生物学活性、以及每个亚基对IL-35免疫活性的贡献尚不清楚。本项目拟通过制备鼠源性重组IL-12p35和Ebi3，分别检测其生物学活性及其对IL-35活性的影响，进而阐明IL-12p35在ITP免疫失耐受中的作用及其机制，探讨IL-12p35在ITP中的临床意义，为ITP的治疗提供新的依据和靶点。

原发免疫性血小板减少症（ ITP）是一种自身免疫介导的出血性疾病，免疫失耐受在其发生中扮演重要角色。IL-35由p35和Ebi3组成，参与ITP免疫失耐受。我们推测IL-35的免疫调节活性来源于α亚基p35和β亚基Ebi3的组合，单独的亚基本身可能也具备生物学活性。本项目拟构建ITP小鼠模型，分别检测p35和Ebi3的生物学活性及其对IL-35活性的影响，进而阐明IL-12p35在ITP免疫失耐受中的作用及其机制，为ITP的治疗提供新的依据和靶点。本项目的主要研究内容：1. 获得可用于实验的具备生物学活性的鼠源性和人源性p35和EBI3。2. 构建ITP小鼠模型。3. 体内外动物实验探究p35和EBI3是否通过免疫调节作用参与ITP免疫失耐受及其机制。4. 收集ITP患者临床样本，探究p35在ITP中的表达情况及与其他免疫异常的相关性，体外实验探究p35是否参与免疫异常调节，评估其免疫调节作用是否依赖EBI3共表达。重要结果及关键数据:1. 构建ITP小鼠模型；证实p35和EBI3具备生物学活性。2. 体内外动物实验证实p35通过调节T、B细胞增殖及分泌相关细胞因子参与ITP免疫失耐受，其免疫调节作用需依赖IL-12β2受体表达。3. 收集临床ITP患者样本，活动期ITP患者IL-12p35的表达明显低于缓解组及正常对照组，与ITP多种免疫异常相关，通过调节T、B细胞增殖及分泌相关细胞因子参与免疫失耐受，它和EBI3的共表达可能是IL-35具备免疫调节作用的基础。科学意义：证实p35在ITP免疫失耐受中的免疫调节作用和机制，p35有望成为ITP治疗新靶点。