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

Abstract

Background: 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⁺/CD4⁺T cells and increase mortality of tumor cells as well; however, their use is accompanied by adverse side effects.

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 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⁺/CD4⁺T 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.

Results: 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 CD4⁺T cells, and increased the apoptosis of MDA-MB-231 cells.

Conclusion: Overall, increased CD4⁺T 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.

Keywords: Apoptosis; PD-L1; Soluble Human PD-1; T Cell Cytotoxicity.