Engineering Thymic Regeneration to Induce Tolerance

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 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. A major problem with regenerating the thymus is that the TECs, which are so important for T cell growth and differentiation, tend to die during culture. Over the first year of grant support, we have developed a method to engineer one component of the thymic microenvironment (the thymic mesenchyme) to produce specific growth factors that we hope will protect TECs. We have developed specific culture conditions that allow us to grow the thymic mesenchyme separately to the TECs. We then take the mesenchyme and TECs out of culture and spin them together to form a cluster of cells called a thymic aggregate. We have shown that when we combine these “thymic aggregates” with cord blood stem cells (also known as “hematopoietic” stem cells) we can produce T cells from the cord blood. We can make T cells in the aggregates either in culture or after implantation of the aggregates into immune deficient mice. Furthermore, by genetically engineering the thymic mesenchyme to secrete the growth factor VEGF (Vascular Endothelial Growth Factor), we find that we can produce significantly more T cells and improve the architecture and function of the thymic aggregates for at least 5 weeks. We are now in the process of analyzing the function of the T cells produced in the implanted aggregates. Most of the studies in the past year have been conducted using fragments of human thymus discarded after cardiac surgery. We are now modifying the technique to use mouse thymus as this is the only way that we can test whether the T cells produced in the implants can induce tolerance. Other planned studies for the next year include tracking the survival and growth of the aggregates after implantation using an imaging technique called bioluminescence. These studies will help us understand how the initial period of culture affects long-term survival of the implants and how to optimize the number of T cells from the implants. The studies proposed during the next year will be essential for the final phase of the proposal-testing whether the implants can produce healthy, tolerant T cells.

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 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. A major problem with regenerating the thymus ex vivo is that the TECs, which are so important for T cell growth and differentiation, tend to die during culture. We have developed a method to engineer one component of the thymic microenvironment (the thymic mesenchyme) to produce specific growth factors that we propose will protect TECs. We have developed specific culture conditions that allow us to grow the thymic mesenchyme separately to the TECs. We then take the mesenchyme and TECs out of culture and spin them together to form a cluster of cells called a “thymic aggregate”. We have shown that when we combine these thymic aggregates with cord blood stem cells (also known as “hematopoietic” stem cells) we can produce T cells from the cord blood. We can make T cells in the aggregates either in culture or after implantation of the aggregates into immune deficient mice. Most of the studies in the first year of the grant were conducted using fragments of human thymus discarded after cardiac surgery. In this the second year we have tested the function of T cells produced in the implanted human aggregates and our results suggest they will be able to respond to a wide range of foreign antigens and thus protect against infection and cancer. During the past year we have also focused on modifying our methods to improve the efficiency of aggregation using mouse thymus so that we can test whether the T cells produced in the implants can induce tolerance. We have also been optimizing our culture techniques to increase TEC growth from both mouse and human thymus. These studies have been essential for the experiments in final year of the proposal-testing whether the implants can produce healthy, tolerant T cells.

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 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 T cells. A major problem with regenerating the thymus ex vivo is that the TECs, which are so important for T cell growth and differentiation, tend to die during culture. Through this grant support we have developed a method to engineer one component of the thymic microenvironment (the thymic mesenchyme) to produce specific growth factors that we propose will protect TECs. We have developed specific culture conditions that allow us to grow the thymic mesenchyme separately to the TECs. We then take the mesenchyme and TECs out of culture and spin them together to form a cluster of cells called a “thymic aggregate”. We have shown that when we combine these thymic aggregates with cord blood stem cells (also known as “hematopoietic” stem cells) we can produce T cells from the cord blood. We can make T cells in the aggregates either in culture or after implantation of the aggregates into immune deficient mice. During the past year we have focused on modifying our methods to improve the efficiency of aggregation using mouse thymus so that we can test how well the T cells produced in the implants work.

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 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 T cells. A major problem with regenerating the thymus ex vivo is that the TECs, which are so important for T cell growth and differentiation, tend to die during culture. Through this grant support we have developed a method to engineer one component of the thymic microenvironment (the thymic mesenchyme) to produce specific growth factors that we propose will protect TECs. We have developed specific culture conditions that allow us to grow the thymic mesenchyme separately to the TECs. We then take the mesenchyme and TECs out of culture and spin them together to form a cluster of cells called a thymic “aggregate” or “organoid”. We have shown that when we combine these thymic organoids with cord blood stem cells (also known as “hematopoietic” stem cells) we can produce T cells from the cord blood. We can make T cells in the organoids either in culture or after implantation of the organoids into immune deficient mice. During the final phase of support, we have focused on understanding what kind of T cells we produce in the organoids using mouse thymus. In addition we have developed a gene signature of normal thymus which can be used as a comparison for testing of our engineered organoids.