Producing Soluble Human Programmed Cell Death Protein-1: A Natural supporter for CD4+T cell Cytotoxicity and Tumor Cells Apoptosis

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.