Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai 20032, China.
Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai 20032, China.
Cell Immunol. 2014 Jul;290(1):169-77. doi: 10.1016/j.cellimm.2014.04.005. Epub 2014 Apr 24.
To detect the effects of DCs sensitized by mPD-L1-Ig fusion protein in heart transplantation in mice as well as its mechanisms.
The mPD-L1-IgG1 construct was used to build a yeast expression system, and the fusion protein was expressed by secretion after the transfection of the GS115 yeast strain, purified by affinity chromatography and ion exchange chromatography, and assayed by SDS-PAGE and Western blot. The ability of the fusion protein to bind to the acceptor PD-1 was tested by ELISA, and the ability of the fusion protein to inhibit the function of T cells was tested by mixed lymphocyte reaction (MLR).
We used the new PD-L1-IgG1 fusion protein to sensitize imDCs and maintained the immature state of DCs, so as to induce stable and effective immune tolerance to heart transplantation. After the treatment of DCs by mPD-L1-Ig in vitro, the levels of CD80, CD40 and I-Ab expression on DCs are relatively weaker, the ability of DCs to stimulates the proliferation of allogeneic spleen T cells was significantly decreased (P<0.01), and the levels of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) secreted by induced allogeneic T cells were significantly decreased (P<0.01). An in vivo experiment also revealed that DCs sensitized by mPD-L1-IgG1 could prolong the survival time of a transplanted heart to 17.8±1.12days, and alleviate the pathological change of the cardiac allografts compared with other three groups.
DCs sensitized by the yeast-expressed mPD-L1-Ig fusion protein are shown to alleviate the cardiac allograft rejection in mice.
检测 mPD-L1-Ig 融合蛋白致敏的树突状细胞（DCs）在小鼠心脏移植中的作用及其机制。
构建 mPD-L1-IgG1 构建体，用于酵母表达系统，转染 GS115 酵母株后通过分泌表达融合蛋白，亲和层析和离子交换层析纯化，SDS-PAGE 和 Western blot 检测。通过 ELISA 检测融合蛋白与受体 PD-1 的结合能力，通过混合淋巴细胞反应（MLR）检测融合蛋白抑制 T 细胞功能的能力。
我们使用新型 PD-L1-IgG1 融合蛋白致敏未成熟 DCs，使其保持未成熟状态，从而诱导心脏移植的稳定和有效免疫耐受。体外 mPD-L1-Ig 处理 DCs 后，DCs 上 CD80、CD40 和 I-Ab 的表达水平较弱，DCs 刺激同种异体脾 T 细胞增殖的能力显著降低（P<0.01），诱导的同种异体 T 细胞分泌的 Th1（IL-2、IFN-γ）和 Th2（IL-4、IL-10）水平也显著降低（P<0.01）。体内实验还表明，mPD-L1-IgG1 致敏的 DCs 可将移植心脏的存活时间延长至 17.8±1.12 天，并减轻心脏移植物的病理变化，与其他三组相比。
酵母表达的 mPD-L1-Ig 融合蛋白致敏的 DCs 可减轻小鼠心脏移植排斥反应。
