Producing Soluble Human Programmed Cell Death Protein-1: A Natural Supporter for CD4+T Cell Cytotoxicity and Tumor
Samane Mohammadzadeh1, Hossein Khanahmad2, Nafiseh Esmaeil1
1Immunology Department, Faculty of Medicine, 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 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.
Insights
Researchers developed a soluble human PD-1 (shPD-1) protein to block the PD-1/PD-L1 pathway, enhancing anti-tumor immune responses. This novel blocker effectively increased T cell cytotoxicity and tumor cell apoptosis, offering a potential new cancer therapy.
Area of Science:
- Immunology
- Molecular Biology
- Cancer Research
Background:
- The Programmed Cell Death protein-1 (PD-1)/PD-L1 pathway is a key immune checkpoint that suppresses anti-tumor immune responses.
- Blocking antibodies targeting PD-1/PD-L1 can enhance anti-tumor immunity but are associated with adverse side effects.
Purpose of the Study:
- To produce a native blocker of the human PD-1/PD-L1 pathway.
- To enhance T cell cytotoxicity and induce tumor cell apoptosis.
Main Methods:
- Designed and cloned a soluble human PD-1-GFP construct.
- Transfected HEK cells and collected secreted shPD-1 from the supernatant.
- Evaluated shPD-1's ability to block PD-L1 on MDA-MB-231 cells and its effect on T cell cytotoxicity and tumor cell apoptosis via co-culture and flow cytometry.
Main Results:
- Successful design, transfection, and production of soluble human PD-1 (shPD-1) confirmed by GFP expression.
- Significantly higher shPD-1 concentration in the supernatant of transfected HEK cells.
- shPD-1 effectively blocked PD-L1, enhanced CD4+ T cell cytotoxicity, and increased MDA-MB-231 tumor cell apoptosis.
Conclusions:
- Soluble human PD-1 (shPD-1) acts as a natural blocker of PD-L1.
- shPD-1 effectively augments anti-tumor immune responses by increasing CD4+ T cell cytotoxicity and tumor cell apoptosis.
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