Difference between revisions of "Team:CPU CHINA/Demonstrate"
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<div class="body-container"> | <div class="body-container"> | ||
<h5>Demonstrate</h5> | <h5>Demonstrate</h5> | ||
| − | <h3 class="ar-title"><span class="dg"> </span> | + | <h3 class="ar-title"><span class="dg"> </span>Abstract</h3> |
| − | <h4><br> | + | <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> |
<br> | <br> | ||
| − | <h4><br> | + | <h4><br>In our design, first, 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> |
| + | <br> | ||
| + | <h4><br>Given our design and purpose, we call the system a Human Engineered Anti-Autoimmune-Disease Regulatory T Cells System (HEAD-Treg).</h4> | ||
| + | <h3 class="ar-title"><span class="dg"> </span>Introduction and Background</h3> | ||
| + | <h4><br>People are exposed to billions of pathogens every day. In the meantime, billions of cells within our body undergo a complicated process from generation, aging to apoptosis, together with injuries and mutations. Our immune system identifies and eliminates the threat within and from outside of our body with precision and efficacy, just like an elaborate network.</h4> | ||
| + | <br> | ||
| + | <h4><br>In accordance with the Chinese Yin-Yang theory and the western critical thinking, our immune system is under a dynamic equilibrium at all times. The immune network consisting immune organs, immune tissues and immune cells rely on each other and promote each other, which is a both unified and varying state that we call as immune homeostasis, with immune response and immune tolerance as two most critical features. On one hand, immune response helps us get rid of antigens, eradicating harmful factors against human body; while on the other hand, immune tolerance helps us distinguishing the enemies from our own, avoiding any harm from overreaction and indiscriminate destruction. Thus, the immune homeostasis is a prerequisite for our healthy life, once the balance is no longer maintained a series of diseases will occur and physical functions may fail. If immune tolerance shows more potency than immune response, the human body will be in an immune inhibited state, where pathogens and cancer cells are left uncontrolled leading to the onset of severe infection and malignancy. If immune response gains the upper hand over immune tolerance, normal cells will be targeted for elimination leading to diseases we call as autoimmune diseases, for example rheumatoid arthritis (RA), systematic lupus erythromatosis, autoimmune type I diabetes, etc. In this way, finding a way to recover the immune homeostasis becomes an essential strategy in dealing with these two types of diseases.</h4> | ||
| + | <br> | ||
<div> | <div> | ||
<center><img src="https://static.igem.org/mediawiki/2017/f/f4/T--CPU_CHINA--results_fig1.png" width = "800"></center> | <center><img src="https://static.igem.org/mediawiki/2017/f/f4/T--CPU_CHINA--results_fig1.png" width = "800"></center> | ||
| − | <h4 align=middle>Figure1. | + | <h4 align=middle>Figure1. Therapeutic targets and interference strategies of rheumatoid arthritis</h4> |
</div> | </div> | ||
| + | <h4><br>An imbalance in immune cell’s function is often found in them. Sometime the immune system cannot recognize the target cell, sometime a certain type of immune cell may proliferate. Thus, remodeling the function of a crucial type of immune cell turns into a key point. Cytotherapy has, therefore, become a rising star against these diseases, with CAR-T and TCR-T as the representatives in this technology. The trick in these therapies lies in transforming T cells with synthetic biology approaches, inserting and expressing artificial genes of chimeric antigen receptor into T cell genome and enabling them to recognize and eradicate target cells precisely. Obviously, such strategy has achieved a certain success. This year, the CAR-T product from Novartis was officially approved by FDA to treat acute lymphoblastic leukemia(ALL) in enfants and young adults, which has shown high efficacy in the treatment of non-solid tumors.</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>Figure2. The design of CAR-T system</h4> | ||
| + | </div> | ||
| + | <h4><br>Two subtypes of immune cells are of great significance with respect to the onset of autoimmune diseases like RA, one is the incendiary of inflammation which is called Th17 cell, and one is the firefighter of inflammation which is called regulatory T cell(Treg). In normal human body, the ratio of Th17 to Treg is in a dynamic equilibrium, while in rheumatoid arthritis patients Th17 greatly outnumbers Treg in the focus of a lesion. Inflammatory cytokines secreted by Th17, including IL-17, IL-6, TNF-α, will trigger a cascade of reaction causing severe inflammation locally together with B lymphocytes which may further lead to organ dysfunction and could be fatal.</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>Figure3. The dynamic balance and mutual transformation between Regulatory T cells and Th17 cells</h4> | ||
| + | </div> | ||
| + | |||
| + | |||
<h3 class="ar-title"><span class="dg"> </span>The functioning validation of SynNotch and CAR system</h3> | <h3 class="ar-title"><span class="dg"> </span>The functioning validation of SynNotch and CAR system</h3> | ||
<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 2), 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 2), indicating that the SynNotch system stabilized FOXP3 in Treg cells with the presence of IL-6. </h4> | ||
Revision as of 17:50, 1 November 2017
Demonstrate
Abstract
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.
In our design, first, 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.
Given our design and purpose, we call the system a Human Engineered Anti-Autoimmune-Disease Regulatory T Cells System (HEAD-Treg).
Introduction and Background
People are exposed to billions of pathogens every day. In the meantime, billions of cells within our body undergo a complicated process from generation, aging to apoptosis, together with injuries and mutations. Our immune system identifies and eliminates the threat within and from outside of our body with precision and efficacy, just like an elaborate network.
In accordance with the Chinese Yin-Yang theory and the western critical thinking, our immune system is under a dynamic equilibrium at all times. The immune network consisting immune organs, immune tissues and immune cells rely on each other and promote each other, which is a both unified and varying state that we call as immune homeostasis, with immune response and immune tolerance as two most critical features. On one hand, immune response helps us get rid of antigens, eradicating harmful factors against human body; while on the other hand, immune tolerance helps us distinguishing the enemies from our own, avoiding any harm from overreaction and indiscriminate destruction. Thus, the immune homeostasis is a prerequisite for our healthy life, once the balance is no longer maintained a series of diseases will occur and physical functions may fail. If immune tolerance shows more potency than immune response, the human body will be in an immune inhibited state, where pathogens and cancer cells are left uncontrolled leading to the onset of severe infection and malignancy. If immune response gains the upper hand over immune tolerance, normal cells will be targeted for elimination leading to diseases we call as autoimmune diseases, for example rheumatoid arthritis (RA), systematic lupus erythromatosis, autoimmune type I diabetes, etc. In this way, finding a way to recover the immune homeostasis becomes an essential strategy in dealing with these two types of diseases.
Figure1. Therapeutic targets and interference strategies of rheumatoid arthritis
An imbalance in immune cell’s function is often found in them. Sometime the immune system cannot recognize the target cell, sometime a certain type of immune cell may proliferate. Thus, remodeling the function of a crucial type of immune cell turns into a key point. Cytotherapy has, therefore, become a rising star against these diseases, with CAR-T and TCR-T as the representatives in this technology. The trick in these therapies lies in transforming T cells with synthetic biology approaches, inserting and expressing artificial genes of chimeric antigen receptor into T cell genome and enabling them to recognize and eradicate target cells precisely. Obviously, such strategy has achieved a certain success. This year, the CAR-T product from Novartis was officially approved by FDA to treat acute lymphoblastic leukemia(ALL) in enfants and young adults, which has shown high efficacy in the treatment of non-solid tumors.
Figure2. The design of CAR-T system
Two subtypes of immune cells are of great significance with respect to the onset of autoimmune diseases like RA, one is the incendiary of inflammation which is called Th17 cell, and one is the firefighter of inflammation which is called regulatory T cell(Treg). In normal human body, the ratio of Th17 to Treg is in a dynamic equilibrium, while in rheumatoid arthritis patients Th17 greatly outnumbers Treg in the focus of a lesion. Inflammatory cytokines secreted by Th17, including IL-17, IL-6, TNF-α, will trigger a cascade of reaction causing severe inflammation locally together with B lymphocytes which may further lead to organ dysfunction and could be fatal.
Figure3. The dynamic balance and mutual transformation between Regulatory T cells and Th17 cells
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.
