Document Type: Research Paper
Immunology, Isfahan Medical School, IUMS
Immunology Department, Isfahan Medical School, Isfahan University of Medical Sciences, Isfahan, Iran.
Isfahan University of Medical Sciences, School of Medicine, Department of Genetics and Molecular Biology, Isfahan, Iran
department of immunology , medical university of isfahan, isfahan, iran
Immunology Department, Isfahan Medical School, Isfahan University of Medical Sciences, Isfahan, Iran
Genetics and Molecular Biology Department, Isfahan Medical School, Isfahan University of Medical Sciences, Isfahan, Iran
Isfahan university of medical sciences, Isfahan, Iran.
Background: Programmed cell death protein-1 (PD-1)/PD-L1 pathway is one of the immune checkpoint pathways involved in regulation of the immune responses and suppression of anti-tumor defense. PD-1/B7-H1-blocking antibodies improve immune responses such as cytotoxic activity of CD8+/CD4+T cells and also increase mortality of tumor cells; however their use is accompanied by adverse effects in patients.
Objectives: We aimed to produce a native blocker of human PD-1/PD-L1, for developing T cells cytotoxicity and tumor cells apoptosis.
Materials and Methods: We designed and cloned soluble human PD-1-GFP-pcDNA3.1/hygro construct in Escherichia coli strain TOP10 cells and then transfected this construct into HEK cells. Concentration of 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+/CD4+T cells and apoptosis of MDA-MB-231 cells under the influence of shPD-1 in the co-culture of T cells with MDA-MB-231 cells were evaluated using flow cytometry technique.
Results: GFP expression in the transfected cells illustrated the successful designing, transfection and production of shPD-1. Soluble human PD-1 concentration in supernatant of transfected HEK cells was significantly higher than untransfected cells. In addition, shPD-1 significantly blocked PD-L1 on the MDA-MB-231 cells, improved cytotoxicity of CD4+T cells and increased apoptosis of MDA-MB-231 cells.
Conclusions: Overall, increased CD4+T cell cytotoxicity and tumor cells apoptosis under the influence of shPD-1, confirmed effectiveness of shPD-1 as a natural blocker of PD-L1and as an augmenter of the anti-tumor immune responses.