The construction and expressing validation of SynNotch and CAR system
To engineer our regulatory T cells, we designed a three-plasmid expressing system for genes of SynNotch and CAR. We chose PLVX-IRES-Puro, PLVX-IRES-Neo and pcDNA3.1 as backbones for the plasmid vectors that carry the SynNotch fusion protein gene, the CAR-CD20 fusion protein gene and the UAS-USP7-promoter-USP7 sequence individually (these three genes were synthesized and connected to their vectors by Genscript). Find more details about the design of the fusion protein and the plasmid vector in Parts section.
To test the feasibility of transfecting multiple plasmids into Treg cells, we acquired the Flag-FOXP3-Jurkat cell line from Shanghai Institute of Immunology, Medical College, Shanghai Jiao Tong University. As a stable transfection strain with high expression of Flag-FOXP3 obtained by transfecting Flag-FOXP3 fusion protein into Jurkat T cells, it is a decent model to simulate the Treg status in human body. In our experiment, we introduced our three-plasmid expressing system into the Flag-FOXP3-Jurkat cells by lentiviral transfection and electroporation respectively. The expression of both SynNotch and CAR system in Flag-FOXP3-Jurkat cells were confirmed by western blot and quantitative real-time PCR (Figure 1).
Figure1. The expression of SynNotch and CAR system in Flag-FOXP3-Jurkat cell line introduced by lentiviral transfection and electroporation
The functioning validation of SynNotch and CAR system
To test SynNotch’s stabilization function on FOXP3 in Treg cells under inflammatory conditions, inflammatory factor IL-6 was added into the culture medium to simulate the microenvironment in RA patients, then western blot and quantitative real-time PCR were performed. Without IL-17A, the expression of FOXP3 was significantly reduced compared to normal one due to the inactivation of SynNotch. However, with the addition of IL-17A, the FOXP3 level was greatly uplifted (Figure 2), indicating that the SynNotch system stabilized FOXP3 in Treg cells with the presence of IL-6.
To explore the effect of IL-17A concentration on the function of SynNotch with the presence of IL-6, various concentrations of IL-17A were given and the expression of USP7 and FOXP3 was tested. With a higher concentration of IL-17A, a higher expression of USP7 and FOXP3 was detected, indicating that the concentration of IL-17A played an important role in the expression level of SynNotch.
Figure2. The SynNotch system stabilizing FOXP3 in Treg under inflammatory conditions
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