Line 94: | Line 94: | ||
<h4><br>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.</h4> | <h4><br>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.</h4> | ||
<br> | <br> | ||
− | <h4><br>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 | + | <h4><br>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 8).</h4> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/3/ | + | <center><img src="https://static.igem.org/mediawiki/2017/f/f4/T--CPU_CHINA--results_fig1.png" width = "800"></center> |
− | + | <h4 align=middle>Figure8. The expression of SynNotch and CAR system in Flag-FOXP3-Jurkat cell line introduced by lentiviral transfection and electroporation</h4> | |
+ | </div> | ||
+ | <h4><br>2.The functioning validation of SynNotch and CAR system</h4> | ||
+ | <br> | ||
+ | <h4><br>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 9), indicating that the SynNotch system stabilized FOXP3 in Treg cells with the presence of IL-6.</h4> | ||
+ | <br> | ||
+ | <h4><br>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.</h4> | ||
+ | <div> | ||
+ | <center><img src="https://static.igem.org/mediawiki/2017/f/f4/T--CPU_CHINA--results_fig1.png" width = "800"></center> | ||
+ | <h4 align=middle>Figure9. The SynNotch system stabilizing FOXP3 in Treg under inflammatory conditions</h4> | ||
+ | </div> | ||
+ | <h3 class="ar-title"><span class="dg"> </span>PROSPECT</h3> | ||
+ | <h4><br>Although we demonstrated the function of the SynNotch system and the CAR system in the Flag-FOXP3-Jurkat cell line, the results obtained from our experiments still have some limitations: Although Flag-FOXP3-Jurkat cells are able to stimulate the functional characteristics of T cells on the core transcription factors and its posttranslational modification, Flag-FOXP3-Jurkat cell line, as a simplified Treg model, does not fully exhibit the functional characteristics of Treg cells in other respects, such as cell surface receptor types and cytokine types. Therefore, it is only suitable for studying the mechanism of transcription, translation and post-translational modification on the upstream genes of Treg cells. But we can prove that with the cytokines IL-17A and IL-6 stimulation, our SynNotch system and CAR system can work effectively based on our experimental data. Specifically, the SynNotch system activated the expression of the USP7 gene under the stimulation of IL-17A and IL-6 and enhanced the stability of FOXP3 protein. And its intensity increased with the increase of IL-17A concentration. FOXP3 protein stability level can be equivalent to Treg function level to a large extent, so we can initially confirm that our overall idea and experimental design was correct.</h4> | ||
+ | <br> | ||
+ | <h4><br>In order to further determine the reliability of our experimental design, we also need to conduct a series of experiments. First of all, for the CAR system, we need to further verify its inhibition of CD20 + B lymphocytes, and the completion of these experiments requires primary T lymphocytes. The reconstructed HEAD-Tregs need to be cultivated together with primary CD20 + B lymphocytes and we also need to verify their inhibitory function by flow cytometry and ELISA. The next main problems we need to solve are the induction of primary Treg cell differentiation and primary Treg cell transfection. We will gradually optimize the experimental conditions to carry out the experiments of the isolation, induction, transfection and functional validation of human primary T lymphocytes, thus to confirm the reliability of the HEAD-Treg system in human primary Treg cells. After that, we will further develop animal experiments to verify the viability and effect of HEAT-Treg cells in the intact immune system.</h4> | ||
+ | <br> | ||
+ | <h4><br>In addition, in order to prepare for further clinical trials, we designed a complete HEAD-Treg treatment strategy. As shown in Figure 3, there are many bottlenecks in clinical treatment that we need to overcome, such as minimizing the minimum amount of cells required for cell therapy, optimizing the conditions of sorting by flow cytometry and inducing differentiation for nTreg and iTreg cells, confirming parameters of efficient electroporation or lentiviral transfection, optimizing in vitro amplification techniques for high efficiency Treg cells and so forth. How to cultivate nTreg and iTreg cells in serum-free medium conditions is also a difficulty that we cannot ignore. We will continue to do in-depth study on the pre-clinical experiments of HEAD-Treg system, relying on GLP level laboratory of the New Drug Safety Evaluation Research Center in China Pharmaceutical University. We will definitely spare no effort to make contributions to the development and mature of our HEAD-Treg system–SynNotch CAR-Tregs. </h4> | ||
+ | <div> | ||
+ | <center><img src="https://static.igem.org/mediawiki/2017/f/f4/T--CPU_CHINA--results_fig1.png" width = "800"></center> | ||
+ | <h4 align=middle>Figure10. The SynNotch system stabilizing FOXP3 in Treg under inflammatory conditions</h4> | ||
+ | </div> | ||
− | + | ||
− | + | ||
<h3 class="ar-title">Reference:</h3> | <h3 class="ar-title">Reference:</h3> |
Revision as of 18:04, 1 November 2017