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<h4><br>FOXP3 + regulatory T cells (Treg) are a class of T lymphocyte subsets that play an immunosuppressive and regulatory function. The dysfunction of FOXP3 + Treg is closely related to the development of autoimmune diseases, such as rheumatoid arthritis. FOXP3, a transcription factor in Forkhead family, whose functional stability is regulated by post-translational modification enzymes, is a key transcription factor for Treg cells’ specific expression. The ubiquitinase USP7 was able to specifically modify the FOXP3 protein by specific ubiquitination to enhance the functional stability of FOXP3, thereby enhancing the immunosuppressive function of Treg cells. </h4> | <h4><br>FOXP3 + regulatory T cells (Treg) are a class of T lymphocyte subsets that play an immunosuppressive and regulatory function. The dysfunction of FOXP3 + Treg is closely related to the development of autoimmune diseases, such as rheumatoid arthritis. FOXP3, a transcription factor in Forkhead family, whose functional stability is regulated by post-translational modification enzymes, is a key transcription factor for Treg cells’ specific expression. The ubiquitinase USP7 was able to specifically modify the FOXP3 protein by specific ubiquitination to enhance the functional stability of FOXP3, thereby enhancing the immunosuppressive function of Treg cells. </h4> | ||
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− | <h4><br> | + | <h4><br>Firstly, we designed a SynNotch system that contains a modified Notch protein capable of specifically activating the gene expression of USP7 in inflammatory conditions with the presence of IL17A. 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. As a result, Treg cells can maintain their immunosuppressive function. Meantime, we designed a CAR system that enables Treg cells to target CD20+ B lymph Cell specifically to play an immunosuppressive function and thus play an anti-inflammatory effect. </h4> |
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<h4><br>Given our design and purpose, we call the system a Human Engineered Anti-Autoimmune-Disease Regulatory T Cells System (HEAD-Treg).</h4> | <h4><br>Given our design and purpose, we call the system a Human Engineered Anti-Autoimmune-Disease Regulatory T Cells System (HEAD-Treg).</h4> | ||
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</div> | </div> | ||
<h3 class="ar-title"><span class="dg"> </span>Summary of Mathematical Models</h3> | <h3 class="ar-title"><span class="dg"> </span>Summary of Mathematical Models</h3> | ||
− | <h4><br>In order to further evaluate the clinical value of our system and provide a theoretical reference for the next in vivo and preclinical experiments, we also establish a systematic mathematical model for the local and overall immune environment of rheumatoid arthritis (See the Model section for details). Thus, we have a theoretical explanation of the relationships among the cytokines, different cell subpopulations and rheumatoid arthritis, which lays a solid foundation for further improvement of our research ideas regarding the subsequent in vivo and preclinical experiments</h4> | + | <h4><br>In order to further evaluate the clinical value of our system and provide a theoretical reference for the next in vivo and preclinical experiments, we also establish a systematic mathematical model for the local and overall immune environment of rheumatoid arthritis (See the Model section for details). Thus, we have a theoretical explanation of the relationships among the cytokines, different cell subpopulations and rheumatoid arthritis, which lays a solid foundation for further improvement of our research ideas regarding the subsequent in vivo and preclinical experiments.</h4> |
<h3 class="ar-title"><span class="dg"> </span>RESULTS</h3> | <h3 class="ar-title"><span class="dg"> </span>RESULTS</h3> | ||
− | < | + | <h2><br>1.The construction and expressing validation of SynNotch and CAR system</h2> |
<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> | ||
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<h4 align=middle>Figure8. The expression of SynNotch and CAR system in Flag-FOXP3-Jurkat cell line introduced by lentiviral transfection and electroporation</h4> | <h4 align=middle>Figure8. The expression of SynNotch and CAR system in Flag-FOXP3-Jurkat cell line introduced by lentiviral transfection and electroporation</h4> | ||
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− | < | + | <h2 align=left><br>2.The functioning validation of SynNotch and CAR system</h2> |
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<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> | <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> | ||
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<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> | <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> | ||
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− | <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 | + | <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 |
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Latest revision as of 02:52, 2 November 2017