Team:CPU CHINA/Demonstrate

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.



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.



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


FOXP3 is the most important transcription factor in Treg, taking part in most of the transcription and expression of Treg structural genes. We can assume that the expression and stability of FOXP3 protein determines the function level of Treg. Under inflammation environment, a series of cytokines, such as IL-6, will activate a ubiquitinase Stub1 which will ubiquitinate FOXP3, thus leading to its deactivation and degradation. Consequently, Treg loses its immune inhibitory function, which is mainly why Treg cannot function properly in RA patients. In this way, how to engineer Treg cell to maintain its FOXP3 stability under inflammation conditions and how to direct Treg to target RA specific inflammatory cells to exert its immune inhibitory function becomes a new strategy against RA.

Figure4. Post-translational modification and interference targets of FOXP3 protein

Mathematical Model Analysis


In different tissues, the proportion of different types of CD4+ lymphocytes, to a large extent, is determined by various cytokines that can induce the differentiation of CD4+ CD45RA+ naive T cells. For example, IL-12 leads to Th1 cells, Il-4 leads to Th2 cells and TGF-β leads to Treg cells. Many studies have also shown that subgroups of helper T cells such as Th1, Th2, Th17, and Treg, which have been differentiated into functional subpopulations, can also be transdifferentiated into other subgroups of helper T cells under the action of certain cytokines. Their original functions may decline if transdifferentiation does not happen in some conditions.



In order to theoretically demonstrate that regulatory T cells are dysfunctional or transdifferentiated in the cell microenvironment of rheumatoid arthritis patients imbued with a large number of inflammatory factors, and to describe the necessity of intervening the corresponding regulatory pathway of Treg cells in the clinical treatment of rheumatoid arthritis, we established the mathematical model of T cell differentiation and transdifferentiation (see more from Model section).



Based on our mathematical model analysis, we can theoretically come to the conclusion that in patients with rheumatoid arthritis, due to the secretion of inflammatory cytokines, the T cell differentiation to functional effector T cells will be promoted and meanwhile the function of regulatory T cells will be further weakened. We can also learn that regulatory T cells cannot survive in too intensive inflammatory environment in the bodies of rheumatoid arthritis patients. In the light of all conclusions of the mathematical model analysis, our design ideas for the treatment of rheumatoid arthritis has become clearer: enhance the stability of regulatory T cells in the presence of inflammatory cytokines by inserting our new system, thereby blocking inflammation the occurrence of dysfunction and transdifferentiation of Treg cells. Finally, we established the following intervention strategy (Figure 5).

Figure5. The ubiquitination mechanism of FOXP3 in regulatory T cells under inflammatory conditions and our intervention strategy

Syn-Notch System


In order to deal with RA, we need to arm Treg with a sharp spear as well as a solid shield. With no doubt, a solid shield is the premise of survival in a difficult situation. That is why we try to activate a pathway to protect FOXP3 under inflammatory conditions in our design. We choose to focus on USP7, a protein that can promote FOXP3 stability by deubiquitinating FOXP3 specifically. An up-regulation in USP7 expression can result in an enhanced immune inhibitory function of Treg. Thus, we decide to express a SynNotch system in Treg, which can specifically activate the transcription of USP7 after receiving an inflammatory stimulus.



SynNotch, as you can tell from its name, is a synthetic biologically engineered Notch protein. Notch protein is a transmembrane protein, which can be divided into three domains: extracellular domain, transmembrane domain and intracellular domain. There is a cleavage site within its intracellular domain at which the Notch protein will be cleaved under stimulus, releasing its C-terminal peptide from the membrane to bind with a downstream protein and carrying out the signal transduction. Apparently, this is a non-specific signal transduction system. In our SynNotch system, we retain the transmembrane domain as well as its cleavage site, while on the N-terminal we fuse the extracellular domain of IL-17AR with Notch1 so that it can specifically recognize IL-17A, a cytokine secreted by Th17. In this way, our Treg is capable of sensing the presence of IL-17A. The next question is how to enable the protein cleaved by SynNotch to recognize and activate the transcription of USP7. That is where we introduce our next system, the Gal4-UAS system in yeast.



In yeast cells, Gal4 protein can specifically recognize the UAS sequence in its genome. If we attach an artificial transcription factor VP64 to Gal4 and insert UAS sequence before the promoter of target gene, we can presume that the transcription of target gene will be specifically activated. Thus, we add a Gal4-VP64 fusion protein sequence after the cleavage site in SynNotch system, meanwhile we add the UAS sequence before USP7 promoter. Now with the presence of the inflammatory cytokine IL-17A, SynNotch system will be activated and the cleavage will take place. Gal4-VP64 will be freed from cell membrane before entering the cell nucleus and binding with the UAS sequence before USP7 promoter. Then USP7 protein will be expressed and will deubiquitinate FOXP3, stabilizing FOXP3 under inflammation conditions, making it possible for Treg to survive the inflammatory environment and to exert its immune inhibitory function. Above is the shield we equip Treg with.

Figure6. Design of SynNotch system and CAR system

CAR System


Next, we design a sharp spear for Treg. We choose the B lymphocyte with CD20 high expression as our target for Treg, so we introduce a CAR system into Treg to let it specifically recognize the CD20 antigen at the surface of B lymphocytes in order to achieve anti-inflammatory function. As for the design of our CAR system, we use the scFv fragment of CD20 monoclonal antibody as the extracellular fraction because it can recognize and bind with CD20 on B lymphocyte’s surface accurately. We then choose a CD3Z sequence as the stimulatory signal and two 4-1-BB sequence as the co-stimulatory signal to make sure the signal can be delivered into the cell at a high level, thus activating an effective response of Treg. We attach a red fluorescent protein mCherry to the end of the CAR fusion protein as a report signal for a more convenient detection. By designing this CAR system we equip our Treg with a spear, which can reduce the inflammation level in RA patients.



Based on our design, we name our engineered Treg as “Human Engineered Anti-Autoimmune-Diseases Regulatory T cell (HEAD-Treg) System”. We hope it can serve as a mighty warrior, leading the immune system to rebalance itself, to conquer the disease and to reestablish the patient’s health.

Figure7. HEAD-Treg System

Summary of Mathematical Models


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.

RESULTS


1.The construction and expressing validation of SynNotch and CAR system


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.



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).

Figure8. The expression of SynNotch and CAR system in Flag-FOXP3-Jurkat cell line introduced by lentiviral transfection and electroporation


2.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 9), 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.

Figure9. The SynNotch system stabilizing FOXP3 in Treg under inflammatory conditions

PROSPECT


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.



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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