Development of a Human IFN-ß Expression System using Chinese Hamster Ovarian Cells

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Todd Cameron Hylton (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Two human IFN-ß expression systems were derived based on the pIRES2-AcGFP1 plasmid backbone. One expression plasmid encoded human IFN-ß fused to a C-terminal linker and an 8-histidine affinity chromatography tag. A second expression plasmid encoded human IFN-ß without the C-terminal additions to determine if the addition of the 8-his tag alters IFN-ß function. Both expression vectors encoded the native signal sequence to direct secretion of IFN-ß as a glycosylated soluble protein. These plasmids were then transfected into Chinese Hamster Ovary (CHO) cells. Stable transfected CHO cells were selected based on plasmid-encoded resistance to the antibiotic Geneticin. IFN-ß-producing cells were selected by Fluorescence-Activated Cell Sorting of the brightest 10% fraction of GFP+ cells. Expression supernatants from each cell line exhibited similar amounts of cytotoxic activity in the IFN-ß reactive TF-1 erythroleukemia cell line. These results provided suggestive evidence that the C-terminal affinity tag did not adversely affect the activity of the N-terminal IFN-ß cytokine domain. This IFN-ß-8his recombinant protein was purified by Ni-NTA affinity chromatography and was shown to exhibit potent activity in the in vitro TF-1 cytotoxicity assay. Human peripheral blood mononuclear cells (PBMCs) were activated with Con-A , IL-2 , and either IFN-ß , TGF-ß , IFN-ß + TGF-ß , or no additional cytokine. Cell numbers were counted at each passage. The main finding was that IFN-ß caused the induction of T cell anergy. Human T cells (90% CD8+) were activated with RS4 (11) cells (acute lymphoblastic leukemia cell line) , Con-A , and IL-2 in the presence or absence of IFN-ß , TGF-ß , IFN-ß + TGF-ß. T cells were cultured for eight days , and then reactivated. Supernatants were collected from reactivation cultures to measure IL-2 production as a measure of T cell responsiveness. Human T cells activated in the presence of IFN-ß and TGF-ß produced less IL-2 compared to T cells activated in the absence of TGF-ß alone. This expression system will be used to reveal whether IFN-ß elicits differentiation of human FOXP3+ Tregs.

Additional Information

Publication

Thesis

Language: English

Date: 2019

Keywords

Multiple Sclerosis, IFN-ß, expression system

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