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

Iranian Journal of Biotechnology
Samane MohammadzadehAlireza Andalib

Abstract

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. 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 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. 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 s...Continue Reading

Methods Mentioned

BETA
transfection
PCR
fluorescence
flow
ELISA
Assay
flow cytometry

Software Mentioned

FlowJo
signalP
SPSS
signalP3
TargetP
signalP4
target

Related Concepts

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Apoptosis

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis