Figure 1. The structure of SynNotch fusion protein.
SynNotch is a system that is capable of specifically activating the expression of the USP7 gene in the inflammatory environment, and thereby maintaining the activity of Treg cells by stabilizing the FOXP3 protein.
In our SynNotch system, we retained the functional sequence of the transmembrane domain and the cleavage site of the Notch 1 protein. At the N-terminus, we fused the extracellular domain of IL17RA with Notch 1 to specifically recognize IL-17A secreted by Th17 cells, so that our regulatory T cells to obtain the ability to response to IL-17A. We also connected the gene of Gal4-vp64 (a fusion protein) in the downstream of Notch 1. In the presence of inflammatory cytokines IL17A, SynNotch protein is cleaved, and thus Gal4-VP64 fusion protein is detached from the cell membrane.
The released Gal4-VP64 will recognize UAS sequence in the upstream of promoter USP7 (in our another part BBa_K2506004) and then these two proteins combine together, which enable USP7 gene express with high efficiency. USP7 proteins will deubiquitinate the ubiquitinated FOXP3, so that to enhance the stability of FOXP3 protein in the inflammation environment by protecting FOXP3 from degradation via ubiquitination. In this way, Treg cells can survive and play a role of immunosuppressor.
Flag-FOXP3-Jurkat cell line is a stably transfected cell line that highly expresses Flag-FOXP3. It is established by transfecting Flag-FOXP3 fusion protein gene in Jurkat T cells, and is a good model to simulate the state of human regulatory T cells. We obtained it from the molecular immunology research group of Shanghai Institute of Immunology, School of Medicine in Shanghai Jiao Tong University. In our experiment, we transfected three- plasmid expression system into Flag-FOXP3-Jurkat cells through the lentivirus-mediated gene transfer system and electroporation respectively. The expression of the SynNotch system in Flag-FOXP3-Jurkat cells was confirmed by immunoblotting and real-time quantitative PCR (Figure 3).
Figure 2. The plasmid of the SynNotch system
Figure 3. The expression of the SynNotch system in Flag-FOXP3-Jurkat cell transfected by lentivirus transfection and electroporation
In order to investigate the relationship between the concentration of IL-17A ad the level of activity of the SynNotch system in the presence of inflammatory cytokine IL-6, we administered different concentrations of IL-17A and detected the expression levels of USP7 and FOXP3 (Figure 4). The result showed that the expression of USP7 and FOXP3 protein increased with the increase of IL-17A, which indicates that IL-17A concentration is an important factor affecting the expression level of SynNotch system. This verifies that our SynNotch system has worked well.
Figure 4. The expression of Flag-FOXP3 by activating the expression of the USP7 gene in SynNotch system in inflammatory condition
Figure 5. The design of UUpromU and its application.
UUpromU is a system specailly designed for the SynNotch system (BBa_K2506001) that is capable of specifically expressing the target gene in the case of SynNotch. UUpromU consists of three parts: the promotor sequence of USP7 in the middle and three repeats of UAS gene in both 5’ and 3’ terminus. In order to activate the expression of USP7, we synthesized the UUpromU gene and the USP7 gene, and put it into pcDNA3.1 (+) plasmid (with the assistance from GenScript), where UUpromU replaced the CMV promoter in the pcDNA3.1(+). Therefore, in the case that the SynNotch system is stimulated by IL-17A, the Gal4-vp64 fusion protein that is detached from the cell membrane can specifically recognize and bind to the UAS sequence on UUpromU. Then, the transcription of USP7 gene can be motivated by VP-64, a transcription activator, which leads to the high expression of USP7. USP7 proteins can lead to de-ubiquitination of the FOXP3 protein, so that enhance the stability of FOXP3 protein in the inflammation environment by protecting FOXP3 from degradation via ubiquitination. Finally, the goal of regulating the stability of T cells can be achieved.
UUpromU can synergize with SynNotch (BBa_K2506001), activate the USP7 gene by the action of the inflammatory cytokine IL-17A and stabilize the expression of FOXP3. To verify that, we transfected three-plasmid expression system into Flag-FOXP3-Jurkat cells by lentivirus transfection and electroporation. In the presence of inflammatory cytokine IL-6, we administered IL-17A with different concentrations and detected the expression levels of USP7 and FOXP3 by immunoblotting (Figure 6). The result showed that the expression of USP7 and FOXP3 protein increased with the increase of IL-17A. It means that UUpromU can synergize with SynNotch (BBa_K2506001) and activate USP7 gene under the action of inflammatory cytokine IL-17A, thus stabilizing the effect of FOXP3.
Figure 6. The expression of USP 7 under the synergy of UUpromU and SynNotch system in inflammatory conditions.
CAR-CD20 is a module that enables engineered modified T cells to specifically recognize the surface antigen CD20 in B lymphocytes to play anti-inflammatory function. Our CAR-CD20 system is made up of the gene sequences of several proteins.
At the N terminus, we chose variable region of CD20 monoclonal antibody as the scFv fragment, so that it can accurately identify and bind to B lymphocyte surface antigen CD20. Then, we used two 4-1-BB sequences as a co-stimulatory signal and a CD3Z sequence as a stimulatory signal that allowed the signal to be delivered to the cell at a high level, thereby activating Treg cells in the presence of CD20 B lymphocytes.
In addition, we linked the fluorescent protein mCherry at the C-terminus of the CAR system to facilitate the detection of gene expression. In order to accomplish this goal, we optimized the codon on BBa_K106004 and connected it to the C-terminus of the CAR system.
Flag-FOXP3-Jurkat cell line is a stably transfected cell line that highly expresses Flag-FOXP3. It is established by transfecting Flag-FOXP3 fusion protein gene in Jurkat T cells, and is a good model to simulate the state of human regulatory T cells. We obtained it from the molecular immunology research group of Shanghai Institute of Immunology, School of Medicine in Shanghai Jiao Tong University. In our experiment, we transfected three- plasmid expression system into Flag-FOXP3-Jurkat cells by lentiviral transfection and electroporation respectively. The expression of the CAR system in Flag-FOXP3-Jurkat cells was confirmed by immunoblotting and real-time quantitative PCR (Figure 9). We also observed a red fluorescence under a fluorescence microscope (Figure 10).
Figure 8. The plasmid of the CAR system
Figure 9. The expression of the CAR system in Flag-FOXP3-Jurkat cell transfected by lentivirus transfection and electroporation
Figure 10. The expression of fluorescence in CAR system observed by fluorescence microscope
For more experimental data details, please refer to our RESULTS section.
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