DESCRIPTION: Chronic rejection of allografts remains a major hurdle in organ transplantation and regenerative medicine. While immunosuppressive drugs can prevent graft rejection to a certain degree, their efficacies are limited and often associate with severe side effects. The underlying problem is that new T-cells reactive to alloantigens are continuously generated from the thymus. While numerous efforts have been made to modulate thymic function to induce donor-specific immune tolerance, manipulating the thymus proves to be difficult. One major challenge is to reproduce its unique extracellular matrix microenvironment that is critical for the survival and function of thymic epithelial cells (TECs), the predominant population of thymic stromal cells that are essential for the development of T-cells and for defining the "immunological self" of an individual (the capability to distinguish self from non-self molecules i the body and respond accordingly). Here, we propose an innovative bioengineering approach to modulate the thymus function. We have recently developed a thymus decellularization technique, which allows us to reconstruct a functional thymus organoid de novo with isolated TECs. Athymic mice engrafted with the bioengineered thymus are able to develop strong humoral responses against model antigen ovalbumin and promptly reject skin allografts. Conversely, tolerance to allogeneic skin grafts can be achieved by transplanting thymus organoids co-expressing both donor and recipient's major histocompatibility complex (MHC) molecules. Based on these observations, we hypothesize that the bioengineered thymus organoid can recapitulate the function of a thymus in vivo, and are able to redefine the "immunological self" of the adaptive immune system. Given that the major translational experimental focus in our group is on Type 1 diabetes (T1D), in which the insulin-secreting beta cells of the pancreas becomes targets of autoimmune destruction due to loss of self-tolerance, we will focus our investigation on whether the bioengineered thymus organoids can re-establish immune tolerance to beta-cells. Furthermore, we will investigate whether we can simultaneously induce donor-specific immune tolerance to islet allografts with the thymus bioengineering technology. Experiments in Aim 1 will optimize the construction of the thymus organoids from decellularized thymic scaffolds in vitro. Our focus in Aim 2 is to optimize the long-term survival and function of the bioengineered thymus organoids in vivo. Experiments in Aim 3 is to demonstrate that the bioengineered thymus constructed with insulin- expressing allogeneic TECs can effectively modulate the adaptive immune system to reverse insulin-autoimmunity, one of the primary driving forces for T1D progression, and to establish immune tolerance of islet allografts. The long-term goal of the research project is to translate the thymus bioengineering technique into clinical applications.

PUBLIC HEALTH RELEVANCE: Narrative: Most of the pathological conditions in human can be traced, at a certain degree, to immune dysregulation. Loss of tolerance to self-molecules of specific organ attributes to organ-specific autoimmune disorders (e.g. T1D). Chronic rejection of donor organ remains as the major unsolved obstacle in solid organ transplantation. As well, immune rejection of cells/organs of allogeneic origin is projected to be one of the major hurdles in stem cell-based regenerative medicine. The research application addresses the question: whether we can modify the self and non-self-definition at the central level (the thymus) of the adaptive immune system with bioengineering techniques? This research project is designed to develop functional bioengineered thymus organoids and, as a proof-of-principle, use them to modify the adaptive immune system for treatment of autoimmune type 1 diabetes and induction of donor-specific immune tolerance. If achieved, these approaches will have transformative impacts in transplantation medicine and treatment of autoimmune diseases.