RESULTS

√ The construction and the expressing validation of SynNotch and CAR system

To engineer our regulatory T cells, we designed a three-plasmid expressing system for SynNotch and CAR. We choose PLVX-IRES-Puro, PLVX-IRES-Neo and pcDNA3.1 as backbones for the plasmid vectors, each carrying the SynNotch fusion protein gene, the CAR-CD20 fusion protein gene and the UAS-USP7 promoter-USP7 sequence (these three genes were synthesized and connected to their vectors by Genscript). Please find more details about the design of the fusion protein and the plasmid vector in Parts.

To test the feasibility of transfecting multiple plasmids into Treg, we acquired a Flag-FOXP3-Jurkat cell line from Shanghai Institute of Immunology, Medical College, Shanghai Jiaotong University. This is stable transfection strain with high expression of Flag-FOXP3, which is a decent model simulating the Treg status in human body constructed by transfecting Flag-FOXP3 fusion protein into Jurkat T cells. 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: The expression of SynNotch and CAR system in Flag-FOXP3-Jurkat cell line

Next, to test the feasibility of transfecting these plasmids into primary Treg, we used the flow cytometer (BD FACS AriaIII) from天然药物活性组分与药效国家重点实验室, China Pharmaceutical University to separate CD4+CD25+CD127low natural Treg (nTreg) and CD4+CD45RA+ naïve T cells (naïve T) from peripheral blood (Figure 2A). Naïve T cells differentiated into induced Treg under the stimulus of cytokine TGF-β and IL-2, with the expression level of FOXP3 as the judging criteria for the success of induced differentiation by flow cytometer (Figure 2B).

Figure 2: Separation of naïve T cells and nTreg from human peripheral blood and testing of induced level of iTreg

After harvesting Treg from human, we transfected our three-plasmid expressing system into Treg by electroporation, and later the expression of our SynNotch and CAR system in primary Treg were also confirmed by western blot, quantitative real-time PCR and fluorescence microscope (Figure 3A).

Figure 3: The expression of SynNotch and CAR system in Treg

√ The functioning validation of SynNotch and CAR system

To test SynNotch’s stabilization function on FOXP3 in Treg 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 4A, 4B), indicating that the SynNotch system stabilized FOXP3 in Treg 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.

Figure 4: The SynNotch system stabilizing FOXP3 in Treg under inflammatory conditions

To further demonstrate that the SynNotch system deubiquitinated FOXP3 by activating USP7, we designed an immunoprecipitation experiment (Figure 5). We noticed that with addition of IL-6 and IL-17A, the Treg expressing the SynNotch system showed an upregulation of USP7 expression as well as a significant drop in FOXP3 deubiquitination level compared to wild type Treg, indicating that the SynNotch system did lower the FOXP3 ubiquitination level by activating USP7.

Figure 5: The SynNotch system downregulates the ubiquitination level of FOXP3 by activating USP7

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