重组可溶性PD-1免疫抑制性受体增强抗小鼠H22肝癌的免疫效应

目的:探讨真核表达PD-1的重组可溶性分子(sPD-1)增强肿瘤局部免疫效应的作用机制.方法:采用半定量RT-PCR方法检测PD-1的配体PD-L1和PD-L2在小鼠H22肝癌细胞和癌组织中的表达水平,流式细胞仪检测其在激活T细胞表面的表达;体外细胞杀伤实验检测sPD-1作用于肿瘤细胞或脾细胞对Hsp70-H22抗原肽复合物激活的脾细胞杀伤H22肝癌细胞的影响;体内抑瘤实验评价局部转染表达sPD-1的抗瘤作用.结果:H22肿瘤细胞本身表达PD-L1基因,PD-L1和PD-L2基因在癌组织中的表达高于正常肌肉组织和单纯癌细胞;PD-L1亦表达于激活的T细胞表面;sPD-1可增强抗原特异性激活的淋巴细胞对肿瘤细胞的杀伤效应;在肿瘤接种部位肌注转染表达sPD-1可显著抑制H22肿瘤生长.结论:在肿瘤局部表达可溶性受体sPD-1阻抑PD-L/PD-1通路,既可作用于免疫细胞来提高其正向免疫力,同时也可拮抗H22细胞通过PD-L对免疫细胞的抑制作用,可望成为提高肿瘤基因治疗疗效的一种新手段.

Augmentation of Antitumor Immune Effect on Mouse H22 Hepatocarcinoma by Recombinant Soluble PD-1 (sPD-1) Inhibitory Immunoreceptor

Objective: To evaluate the mechanism of the enhanced antitumor immune effect of the locally expressed soluble molecule sPD-1 on mouse H22 hepatoma. Methods: The mRNA expression level of PD-L1 and PD-L2, the lig ands of PD-1, were investigated in mouse H22 cells as well as H22 tumor tissues by using semi-quantitative RT-PCR method. The cytotoxicity assay in vitro was used to evaluate the lysis activity of HSP70-peptides complex-stimulated spleen cells on H22 cells when the tumor cells or spleen cells were pretreated with sPD-1. The antitumor effect of sPD-1 on H22 hepatoma was investigated by experiment in vivo after mice were inoculated with H22 tumor cells. Results: PD-L1 but not PD-L2 mRNA was expressed in H22 hepatoma cells. Both PD-L1 and PD-L2 mRNAs were expressed in tumor tissues of tumor-bearing mice and upregulated as compared with muscle tissues in normal mice. Blocking PD-Ls on either tumor cells or spleen cells by sPD-1 mediated enhanced lysis of H22 cells by HSP70-peptides complex-stimulated spleen cells. sPD 1 also mediated strong antitumor immune effects on mouse H22 tumor model in vivo. Conclusion: The results provide a novel antitumor method of expression of soluble receptor of PD-1 in tumor sites by local gene therapy, which could block the action of PD-Ls on both immune cells and H22 tumor cells, and then increase the antitumor immune activity.