调节性B细胞的扩增参与妊娠免疫耐受的系统调节及其机制

Regulatory B cells (Breg) belong to B lymphocyte subsets with the immune suppressive function. Breg cells suppress the differentiation of T helper 1 cells (Th1) and the maturation of dendritic cells (DC), expand regulatory T cells (Treg) via the provision of interleukin-10 (IL-10). The suppressive capacity of regulatory B cells has been shown in several autoimmune diseases. Interleukin-35 (IL-35) induces the expansion of Breg cells and the conversion of B cells into a Breg cells population that produces IL-35 (IL-35+Breg cells). Our previous study found that human trophoblasts in maternal-fetal interface of early placentas secreted soluble IL-35 and therefore caused the higher level of IL-35 in peripheral blood. We also observed significantly augmented percentages of Breg cells in normal pregnant when compared to non-pregnant women. However, both the suppressive mechanism of Breg cells in immune tolerance during pregnancy and the effect of IL-35 or other pregnancy-associated hormones on the induction of Breg cells remain unknown. Based on the above results, we will further explore the function of Breg cells on the maintenance of peripheral tolerance during pregnancy. In the present study, we will comprehensively analyze the suppressive activity of Breg cells on effective T cells, DC cells and Treg cells and related signaling pathway. Meanwhile, the role of Breg cells in maintaining pregnancy immunotolerance will be verified in a murine model of spontaneous abortion. The results of this research will not only further improve the knowledge on biological function of Breg cells, but also provide a new therapeutic target for pathological pregnancy.

调节性B细胞（Breg）是具有免疫抑制功能的B细胞亚群，可通过分泌IL-10等方式抑制T细胞向Th1分化、抑制DC成熟、扩增调节性T细胞，在自身免疫病等多种疾病中发挥负向调控作用。IL-35对Breg有诱导扩增作用。我们的前期研究发现，人早孕外周血有Breg扩增现象，并且滋养细胞可通过分泌IL-35升高外周血IL-35水平。Breg能否在妊娠外周免疫耐受的维持中发挥作用及其机制是什么？外周Breg的扩增是否由IL-35或其它妊娠相关激素诱导产生？尚无研究报道。本研究拟在前期结果的基础上，从IL-35或妊娠相关激素对Breg的诱导作用入手，采用细胞共培养、抗体阻断实验等技术手段分析妊娠期Breg的功能及其主要信号通路；并通过小鼠易流产模型及动物实验进一步验证Breg在妊娠免疫耐受中的系统调节作用。本研究对阐明独特的母胎耐受及防御机制具有重要意义，并为病理性妊娠的防治提供新的治疗策略和靶点。

调节性B细胞（Regulatory B cell, Breg）是一群具有免疫调节功能的B细胞亚群。近期研究证实一群小鼠脾脏来源的Breg可通过分泌抑制性细胞因子IL-10发挥免疫调节作用，即IL10+Breg（B10），其分子表型为CD19+ CD5+CD1dhi。此外，抑制性细胞因子IL-35可诱导B细胞转化为一种IL-35依赖的调节性B细胞（IL-35+Breg）。我们的前期研究发现，妊娠早期绒毛滋养细胞及绒毛外滋养细胞可分泌IL-35。本课题研究证实，早孕妇女外周血CD19+CD24+CD38hiBreg细胞及CD19+IL-10+Breg细胞表达比例均显著高于健康未孕对照。与健康未孕对照相比，早孕妇女外周血中IL-35表达水平也明显增高。在此基础上，我们进一步确定了早孕妇女外周血血清可诱导健康非孕妇女外周血中的CD19+IL-10+Breg细胞比例升高。通过构建正常妊娠小鼠及自然流产小鼠模型，利用流式细胞术证实自然流产小鼠脾脏内CD19+CD5+CD1dhi Breg 细胞及CD19+IL10+Breg细胞表达比例均明显低于正常妊娠小鼠。提取小鼠脾脏内的原代CD19+B细胞，在体外用妊娠相关激素(人绒毛膜促性腺激素hCG，雌激素E2)及IL-35进行培养及诱导。将诱导后的细胞进行CCK8及流式细胞术分析，结果显示：IL-35及hCG可抑制小鼠脾脏B细胞的增殖并诱导其转化生成B10及IL-35+Breg。进一步研究显示, IL-35及hCG通过激活小鼠B细胞STAT1/STAT3转录因子发挥其增殖抑制及转化作用。E2在小鼠脾脏B细胞增殖及转化方面无显著影响。本研究证实了B10及IL-35+Breg在妊娠外周免疫耐受中的重要作用，并进一步探索了妊娠相关激素及IL-35对于外周Breg细胞增殖及分化的影响。对阐明Breg细胞在维持妊娠外周免疫耐受及防御机制中的作用，补充并建立新的免疫耐受理论具有重要意义。