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RE-ENGINEERING THE MICROENVIRONMENT OF MICE THYMUS TO PROMOTE TOLERANCE TO A VCA TRANSPLANT by Jialu Wang A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Science in Engineering Baltimore, Maryland May 2019 Abstract Nowadays, patients with a solid organ transplant or a vascularized composite allotransplant (VCA) transplant rely on life-long immunosuppression or a highly controversial bone marrow transplant to induce immune tolerance and to maintain their grafts. With only a few novel methods that attempt to induce donor-specific tolerance such as the splenocyte induced donor-specific tolerance, such methods target the peripheral tolerance mechanism instead of central tolerance.1 This study searches for a novel tolerance induction method that does not compromise the immune system with long-term immunosuppression, focuses on the practicality of inducing donor-specific central tolerance and aims to reduce graft-versus-host-disease seen in VCA. We hypothesize that through intrathymic injection of donor thymic epithelial cells after a hindlimb transplant, a possible donor-recipient hybrid thymus may achieve immunotolerance without the risk of graft-versus-host-disease induced by a bone marrow transplant. Some possible mechanisms that are impacted by the hybrid thymus are investigated. Although with many obstacles before this hybrid thymus idea could be translated into clinical treatment, the concept remains a potential option for the induction of VCA transplant tolerance. ii Acknowledgment I would like to thank the entire Vascularized Composite Allotransplantation Lab. The PIs, Dr. Giorgio Raimondi and Dr. Gerald Brandacher, are tremendously resourceful, guided me patiently through all the trouble-shooting and pointed me in the right direction when encountered obstacles. The postdoctoral fellow, Dr. Marcos Iglesias, has accommodated my requests no matter how busy he is or how late at night. I am grateful for all the hours Dr. Yinan Guo put into microsurgeries so I could complete this project. The lab has a dynamic and supportive environment, without which I would not accomplish what I have accomplished. I would like to thank every member of the VCA lab for making my stay in the lab so enjoyable. I would like to thank the Biomedical Engineering department faculty and staff, all of whom are extremely helpful and encouraging. I hold great gratitude to Sam Bourne, the BME Master’s Program Manager, who has given me invaluable advice in the past two years and supported all of my decisions along the journey. I would like to thank my fiancé, Alexander Samuel Kaplitz, and his family, who have provided me a home away from home here in the US. Alex knows me better than myself and offered me the emotional support that I needed to complete my study. Last but not least, I would like to thank my parents, who have financially and emotionally supported me all these years. They had never doubted my potential even in situations when I had no faith in myself. iii Table of Content Title i Abstract ii Acknowledgment iii List of Figures vi 1. Introduction 1-14 1.1 Current Treatment Challenges in Transplantation 1-4 1.2 T cell development 4-9 1.2.1 Early Stage T Cell Development 5-6 1.2.2 Late Stage T Cell Development 6-7 1.2.3 T Cell Activation 7-8 1.2.4 Regulatory T Cells 8-9 1.3 Thymic Epithelial Cells 9-12 1.3.1 cTEC 10-11 1.3.2 mTEC 11-12 1.4 Antigen Presenting Cells 12-13 1.5 Purpose of the Study 13-14 2. Optimal Donor TEC Processing 15-25 2.1 Animals Used 15 2.2 Method 15-17 2.2.1 Digestion 15-16 2.2.2 Negative Selection 16-17 2.2.3 Density Gradient Selection 17 iv 2.2.4 Positive Selection 18 2.2.5 FACS Sorting 18 2.3 Material and Reagents 19 2.4 Results 20-25 3. Donor TEC Engraftment 26-31 3.1 Animals Used 26 3.2 Method 26-27 3.3 Material and Reagents 27 3.4 Results 27-31 4. Donor TEC’s Interaction with the Recipient 32-35 4.1 Animals Used 32 4.2 Method 32 4.3 Material and Reagents 32 4.4 Results 33-35 5. Allogeneic TEC Survival in a Hindlimb Recipient 36-37 5.1 Animals Used 36 5.2 Method 36 5.3 Material and Reagents 36 5.4 Results 36-37 6. Discussion 38-42 7. Bibliography 43-47 8. Curriculum Vitae 48 v List of Figures Figure 1. A model for cTEC contributions to T cell development 5 Figure 2. A flow cytometry analysis of the effect of different digestion buffer compositions 20 Figure 3. A flow cytometry analysis of the effect of different medium 20 Figure 4. Cell aggregates formed due to thermic shock 21 Figure 5. Comparison between different panning strategies 22 Figure 6. TEC yield after using different density solutions 23 Figure 7. The positive enrichment consistently yields around 10% TEC in the population 24 Figure 8. The two-step positive enrichment yields satisfactory percentage of TEC 24 Figure 9. Conditions for optimal engraftment 28 Figure 10. TECs from donors older than 20 days old do not survive in the recipients 28 Figure 11. Allogeneic TEC survival in BALB/c animals 29 Figure 12. Donor TEC percentage in congenic animals 31 Figure 13. Long-term effect of donor TECs on the recipient TEC population 33 Figure 14. Short-term effect of donor TECs on the recipient TEC population 34 vi Introduction Current Treatment Challenges in Transplantation Transplantation, such as solid organ transplantation, has long been an accepted standard therapeutic method to patients with end-stage organ failures and has saved countless lives. Although not life-saving, the vascular composite allotransplantation (VCA) has also increasingly become a valid option for patients who seek to restore normality after devastating injuries. VCA refers to a form of transplant with multiple tissue types and fills the gap where conventional reconstruction would be unable to regain the form and function.2 Until the end of 2017, 61 VCA programs were approved in the United States with 50 patients completing the surgery before 2014, over 100 upper extremity transplants and 30 face transplants have been performed worldwide.3,4 With the ever-increasing rates of both solid organ and VCA transplant, how to suppress the allograft rejection remains the central problem. The most common treatment after kidney transplantation, for example, is to use a life-long immunosuppressive regimen. Immunosuppression is a crucial component in the prevention of immune-related allograft rejection, and it dramatically decreases acute rejection after a transplant.5 Common immunosuppression can be achieved in three ways: lymphocytes depletion, diverting lymphocyte traffic and blocking the lymphocyte response pathways.6 The introduction of immunosuppressants in the 1990s such as cyclosporine Neoral, tacrolimus, and mycophenolate mofetil has reduced the incidence of acute rejection episodes during the first year after solid organ transplantation. Unfortunately, these agents not only fail to demonstrate efficacy on long-term graft survival or the prevalence of chronic rejection, 1 but also fail to prevent acute rejections. Acute rejection is common in solid organ transplants, with 50-70% patients experience at least one episode, and due to the complex tissue types in a VCA transplant, as high as 85% of all VCA patients experience one or more episodes of acute rejection in the first year.7,8,9,10 The commonly used immunosuppressants are all non-specific, and each has its agent-specific side effects, for instance, studies have identified the usage of immunosuppressants as a risk factor for high cardiovascular morbidity, diabetes onset, nephrotoxicity, hypertension, increased infectious, and cancer risks following a liver transplant.11,12 An alternative treatment to increase graft survival is through bone marrow transplantation. The original purpose for bone marrow transplantation was to provide the recipient with a functional stem cell population to aid a defected immune system.13 However, a broader application of donor bone marrow was adopted in order to accompany a solid organ or VCA transplant; the donor bone marrow is transplanted into the recipient with the expectation to derive donor-specific immune cells and thus induce tolerance to the graft. Mixed chimerism, a phenomenon observed in bone marrow recipients, refers to the co-existence of cells from two different subjects in one body is called chimerism.14 Induction of mixed chimerism includes but not limited to the use of cyclophosphamide, anti-CD2 mAb, TBI, and hATG.15 In a kidney transplant model, transient mixed chimerism could promote solid organ allograft tolerance, although durable mixed chimerism can be observed in almost half of HLA-matched bone marrow transplants, where the grafts are tolerated without any immunosuppressants.16,15 Nonetheless, bone marrow transplantation has its problem: the graft-versus-host disease (GVHD). GVHD can be observed in 30-70 percent of bone marrow recipients when the 2 donor lymphoid cells actively conduct an immunologic assault against host target organs, and the consequences of GVHD can result in infection and affect graft function.17 In an effort to induce peripheral tolerance, several mechanisms were extensively studied. Fas ligand (FasL)’s interaction with Fas receptor on T cells is the main mechanism for cell apoptosis. Since naïve T cells do not express Fas receptors, activated T cells with upregulated Fas receptors are extremely susceptible to FasL-induced apoptosis, therefore administering FasL together with alloantigen in nanoparticles can induce systematic cell death in the alloreactive T cell population.18 This peripheral tolerance induction was proven effective with a single protein antigen and induced total depletion of reactive CD8+ T cells.