Mohammadzadeh Samane, Khanahmad Hossein, Esmaeil Nafiseh, Eskandari Nahid, Rahimmanesh Ilnaz, Rezaei Abbas, Andalib Alireza
Immunology Department, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Genetics and Molecular Biology Department, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Iran J Biotechnol. 2019 Dec 1;17(4):e2104. doi: 10.30498/IJB.2019.85180. eCollection 2019 Dec.
Programmed cell death protein-1 (PD-1)/PD-L1 pathway is one of the immune checkpoint pathways involved in the regulation of the immune responses and the suppression of anti-tumor defense. PD-1/PD-L1 blocking antibodies improve immune responses such as cytotoxic activity of CD8/CD4T cells and increase mortality of tumor cells as well; however, their use is accompanied by adverse side effects.
We aimed to produce a native blocker of human PD-1/PD-L1, for developing T cells cytotoxicity and tumor cells apoptosis.
We designed and cloned soluble human PD-1-GFP-pcDNA3.1/hygro construct in strain TOP10 cells and then transfected this construct into the HEK cells. The concentration of the secreted shPD-1 in the supernatant was measured and the supernatant was used for blocking PD-L1 on the MDA-MB-231 cells. The cytotoxicity of CD8/CD4T cells and the apoptosis of MDA-MB-231 cells, under the influence of shPD-1 in the co-culture of T cells with the MDA-MB-231 cells, were evaluated using flow cytometry technique.
The GFP expression in the transfected cells illustrated the successful designing, transfection, and production of shPD-1. Soluble human PD-1 concentration in the supernatant of the transfected HEK cells was significantly higher than the untransfected cells. In addition, shPD-1 significantly blocked PD-L1 on the MDA- MB-231 cells, improved the cytotoxicity of CD4T cells, and increased the apoptosis of MDA-MB-231 cells.
Overall, increased CD4T cell cytotoxicity and tumor cells apoptosis under the influence of shPD-1, confirmed the effectiveness of shPD-1 as a natural blocker of PD-L1and as an augmenter of the anti-tumorimmune responses.
程序性细胞死亡蛋白1（PD-1）/程序性死亡受体配体1（PD-L1）通路是参与免疫反应调节和抗肿瘤防御抑制的免疫检查点通路之一。PD-1/PD-L1阻断抗体可改善免疫反应，如CD8/CD4 T细胞的细胞毒性活性，同时也增加肿瘤细胞的死亡率；然而，其使用会伴随不良副作用。
我们旨在制备一种人PD-1/PD-L1的天然阻断剂，以增强T细胞的细胞毒性和肿瘤细胞的凋亡。
我们在TOP10菌株细胞中设计并克隆了可溶性人PD-1-GFP-pcDNA3.1/ hygro构建体，然后将该构建体转染到HEK细胞中。测量上清液中分泌的短发夹结构RNA干扰的人PD-1（shPD-1）浓度，并将上清液用于阻断MDA-MB-231细胞上的PD-L1。使用流式细胞术技术评估在T细胞与MDA-MB-231细胞共培养中shPD-1影响下CD8/CD4 T细胞的细胞毒性和MDA-MB-231细胞的凋亡。
转染细胞中的绿色荧光蛋白（GFP）表达说明了shPD-1的成功设计、转染和产生。转染的HEK细胞上清液中的可溶性人PD-1浓度显著高于未转染细胞。此外，shPD-1显著阻断MDA-MB-231细胞上的PD-L1，提高CD4 T细胞的细胞毒性，并增加MDA-MB-231细胞的凋亡。
总体而言，在shPD-1影响下CD4 T细胞细胞毒性增加和肿瘤细胞凋亡，证实了shPD-1作为PD-L1天然阻断剂和抗肿瘤免疫反应增强剂的有效性。
