$1 357 398
A healthy immune system produces T cells that can recognize and react against foreign molecules (antigens) to protect against infection, while leaving normal host cells with “self antigens” undamaged. All T cells are produced in the thymus from blood stem cells that migrate from the bone marrow. “Tolerant” T cells are those that have been “educated” to not react against self antigen on host cells. The key cells in the thymic microenvironment that control T cell production and tolerance are the thymic epithelial cells (TECs). When TECs are lost or become dysfunctional, T cell production is poor and patients are at risk for a wide range of infections. When tolerance is lost, T cells react to host tissues as if they were foreign, producing inflammation and damage and causing autoimmune diseases such as Type I Diabetes, multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus. The ability of a patient to accept cells or an organ transplant from another person also requires tolerance to occur, or the graft will be rejected. The goal of our studies is to develop a method for engineering and transplanting new, healthy thymus tissue into patients, thus creating a way to generate healthy, tolerant T cells. We have developed a method to engineer one component of the thymic microenvironment (thymic mesenchyme aka Tmes) to produce specific growth factors in a regulated fashion to help TECs grow, and are able to combine TECs and Tmes to form a functional thymus which can then be implanted into mice. This method can be applied to either mouse thymus or human thymus, and in each case the thymic implants allow T cells to develop. We now propose to engineer the thymic implants to produce specific growth factors that we have identified as critical for the rapid thymic growth seen in the fetal and neonatal periods. The ability of engineered thymic aggregates to support the production of mouse and human T cells in cell culture and after transplants in mice will be determined. Importantly, the ability of the thymic implants to educate T cells to become immune tolerant will be assessed. The success of this proposal will have wide-ranging applications for stem cell and regenerative medicine. Re-generation of new thymic tissue for transplantation will be useful for patients who have poor thymic function e.g. during aging, after chemotherapy and bone marrow transplantation and in certain conditions such as AIDS. Induction of immune tolerance with regenerated thymus will be a critical component of the treatment of autoimmune diseases and graft versus host disease of bone marrow transplantation and to prevent rejection of organs and stem cells from mis-matched donors.
Statement of Benefit to California:
The ability to regenerate and control the immune system is of critical importance in the treatment of a wide-range of life-threatening diseases. A healthy immune system is required to control infections and prevent autoimmune diseases. The thymus gland, which is central in the regulation of the immune system, is damaged by infections such as HIV-1, by chemotherapy and by aging. Each year, tens of thousands of new patients are diagnosed with autoimmune diseases such as Type I Diabetes, Multiple Sclerosis, Systemic Lupus Erythematosus and rheumatoid arthritis. Over 21,000 Californians and 100,000 Americans are estimated to be in need of organ donation for life threatening diseases [Donate Life, California, Organ and Tissue Donor Registry]. Heavy suppression of the immune system is required to prevent rejection of transplanted organs resulting in many serious side effects for patients. Each year, thousands of patients undergo bone marrow transplantation for leukemia and genetic diseases. The main problem for patients after bone marrow transplantation is immune-mediated graft versus host disease and infection from poor thymic function. Thus, understanding how to regenerate and control the function of the thymus would have an enormous potential impact on the treatment of many common and debilitating diseases that affect Californians.
The overall goal of this proposal is to engineer a functional thymic unit that could be transplanted to improve self-tolerance in autoimmune disease as well as acceptance of stem-cell derived grafts. The applicant proposes to co-culture two populations of cells, thymic epithelial cells (TECs) and thymic mesenchymal cells (Tmes) to generate TEC/Tmes aggregates. These aggregates will be engineered to conditionally produce various factors that are important in thymic development with the goal of generating a functional unit prior to transplantation. The applicant proposes three Specific Aims: (1) to optimize ex vivo growth and function of TECs through signals presented from engineered Tmes, beginning with mouse tissue and transitioning to human; (2) to optimize in vivo survival and function of engineered TEC/Tmes aggregates after implantation into a mouse model; and (3) to test the ability of engineered TEC/Tmes aggregates to generate tolerant T cells and functional regulatory T cells (Tregs) after implantation. Reviewers found this proposal to be highly innovative and appreciated its novel approach to thymic regeneration. The strategy of isolation, engineering, and recombination of the cellular components of the adult thymus to form aggregates is creative, especially in contrast with prevailing approaches. Reviewers agreed that this proposal could have a major impact, as the successful generation of an implantable thymus that could provide true thymopoiesis could provide a means of treating autoimmune diseases and pave the road for achieving tolerance to stem-cell derived tissue grafts. The reviewers described the scientific rationale for this proposal as strong and the research plan as well-organized, with logical Aims and reasonable timelines. However, they did raise a number of concerns with the experimental design and described the project as high-risk. Among reviewer concerns were that only two cell types are included in the thymic aggregates, which may not be sufficient for thymic reconstitution. Reviewers noted that the aggregates are devoid of thymic endothelial cells, as well as other populations that may be present during thymic development, such as dendritic cells and lymphoid tissue inducer cells, that may be critical for the generation of thymic architecture. There were also questions as to whether adult human thymus, proposed as the source of TECs, contains the necessary progenitors to sustain TEC proliferation. Reviewers were slightly concerned by the minimal preliminary data presented describing the TEC/Tmes aggregates. Specifically, they would have appreciated data demonstrating some semblance of normal thymic architecture in implanted aggregates. Finally, reviewers noted that while alternative plans are discussed, in many cases they assume success of initial experiments and don’t acknowledge potential pitfalls. Reviewers described the Principal Investigator as an expert in hematopoiesis and lymphopoiesis with extensive research and clinical experience in stem cell biology and immune reconstitution. They appreciated the contributions of the Co-Investigator and primary post-doctoral fellow, who are experts in Tregs, construction of thymic organoids and transplantation assays. In general, the reviewers found the assembled team to be highly qualified to carry out the proposed research. Overall, while reviewers acknowledged that this proposal is high-risk, they praised its innovative approach and agreed that it could have a major impact on strategies for tolerance induction. They raised concerns about the experimental design and preliminary data, but were ultimately convinced that the excellent research team could address and overcome these concerns. PROGRAMMATIC REVIEW A motion was made to move this application into Tier 1, Recommended for Funding. Reviewers briefly summarized the proposal and their critiques. They argued that thymic reconstitution studies are of critical importance in understanding mechanisms of tolerance. The motion carried.