Engineered immune tolerance by Stem Cell-derived thymic regeneration

In order for stem cell therapies to proceed, it will be essential that the regulation of immune responses to the stem cell derived tissues be achieved. While the function of the immune system in protection from infections is essential throughout life, some immune reactions are undesirable. Illnesses due to autoimmunity, in which the immune system attacks one’s own body, instead of only germs that cause infection, are common. Examples of autoimmune diseases that are amenable to stem cell based therapies include Type I diabetes caused by abnormal immune responses directed against the insulin producing cells and multiple sclerosis (MS) caused by immune responses that attack the nervous system. Another type of undesirable immune response is the attack on transplanted tissues, leading to rejection. Control of immune reactions is the major impediment to successful organ transplantation, and is highly likely to be an obstacle to therapeutic uses of stem cells. We are evaluating a stem cell based strategy to control the editing of the immune response to both treat autoimmunity and permit stem cell-derived tissues to be transplanted. Members of the international (California and Australia) team have collaborated on the isolation of the stem cells that give rise to specialized thymic epithelial cells (TEC), which control immunity by editing the developing T cells in the thymus before they get released into the bloodstream. Therefore, we hypothesize that by transplanting new TEC, we can control the immune response by permitting different T cells to be produced. Because TEC are always being replaced, it has become necessary for us to focus on identifying TEC stem cells, which can produce new TEC continuously. The TEC stem cells continue to make new TEC for at least 6 months. We are now comparing two ways to isolate these TEC stem cells by either making them from embryonic stem cells or by isolating them from a donor thymus. We have also developed mouse models for MS and for stem cell rejection which can be controlled by modification or suppression of the immune system. We have tested a new antibody-based approach to control autoimmune reactions against nervous system tissue in a model of MS, and for control of rejection of transplanted stem cells. The antibody-based approach improved the course of experimental MS, but by itself was unable to protect stem cells from rejection. Therefore, we are now evaluating additional therapies to augment the effect of the antibody approach to control autoimmunity or rejection.

Reporting Period:

Year 2

In order for stem cell therapies to proceed, it will be essential that the regulation of immune responses to the stem cell derived tissues be achieved. While the function of the immune system in protection from Infections Is essential throughout life, some Immune reactions are undesirable. Illnesses due to autoimmunity, in which the immune system attacks one's own body, instead of only germs that cause infection, are common. Examples of autoimmune diseases that are amenable to stem cell based therapies include Type I diabetes caused by abnormal immune responses directed against the Insulin producing cells and multiple sclerosis (MS) caused by Immune responses that attack the nervous system. Another type of undesirable immune response is the attack on transplanted tissues, leading to rejection. Control of immune reactions is the major impediment to successful organ transplantation, and is highly likely to be an obstacle to therapeutic uses of stem cells. We are evaluating a stem cell based strategy to control the editing of the immune response to both treat autoimmunity and permit stem cell-derived tissues to be transplanted. Members of the International (California and Australia) team have collaborated on the Isolation of the stem cells that give rise to specialized thymic epithelial cells (TEC), which control immunity by editing the developing T cells in the thymus before they get released into the bloodstream. Therefore, we hypothesize that by transplanting new TEC, we can control the immune response by permitting different T cells to be produced. Because TEC are always being replaced, It has become necessary for us to focus on identifying TEC stem cells, which can produce new TEC continuously. The TEC stem cells continue to make new TEC for at least 6 months. We are now comparing two ways to isolate these TEC stem cells by either making them from embryonic stem cells or by isolating them from a donor thymus. We have also developed mouse models for MS and for stem cell rejection which can be controlled by modification or suppression of the immune system. We have tested a new antibody-based approach to control autoimmune reactions against nervous system tissue in a model of MS, and for control of rejection of transplanted stem cells. The antibody-based approach improved the course of experimental MS, but by itself was unable to protect stem cells from rejection. Therefore, we are now evaluating additional therapies to augment the effect of the antibody approach to control autoimmunity or rejection.

Reporting Period:

Year 3

The purpose of this grant is to regenerate and re-program the immune system by making specialized cells called thymic epithelial cells (TEC) from stem cells. TEC are supporting (stromal) cells in the thymus gland. TEC are required to 1) stimulate the growth of young T-cells and 2) select which T-cells are allowed to develop and become part of the immune system. Our hypothesis is that TEC can be generated from stem cells, transplanted into recipients, and be used to generate a new immune system that could replace an abnormal immune system. Examples of abnormal immune systems are those in which the T-cells attack cells of the body such as nerve-lining cells, resulting in multiple sclerosis. TEC turn over at a rapid rate, i.e., a high percentage of TEC are dying at any moment, necessitating their replacement by new TEC that are generated from rapidly dividing immature TEC. We have shown that the high turnover is accelerated during aging, which explains why the thymus shrinks and loses the ability to make new T-cells. Furthermore, we have shown that the high turnover rate of TEC in aging is due to the action of two genes, NFB and HNF4, which become active in response to inflammation and fat, respectively. Thus, our research has been able to causally link two common disease processes, inflammation and obesity, to the aging of the thymus. Our initial efforts were to generate new TEC from embryonic stem cells (ESC). These efforts used a complex system in which a gene called Tbx1 needed for TEC formation was introduced into ESC. This system was marked by a great deal of variability: different cultures would contain differing numbers of TEC-like cells ranging from almost none to a majority and the ability to robustly support the development of new T cells after transplantation was also inconsistent. The inability to consistently generate TEC was both a practical limit to the approach for pre-clinical studies and an anticipated problem in developing this approach further for clinical stem cell therapy. Because of the irreproducibility of the approach based on generating new TEC from ESC, we explored an alternative approach based on the isolation of adult TEC stem cells from the thymus. For many if not all organs, populations of adult stem cells exist, which can contribute to the formation of the cells of that organ. The existence of TEC stem cells can be inferred from the high turnover rates of TEC, which can only be accounted for by the existence of an adult TEC stem cell population that can continually generate new TEC to take the place of the ones that are dying. This is analogous to the systems that maintain other types of epithelial cells like those of the skin and lining of the intestine. The potential advantage of using adult TEC stem cells is that they would not require the re-programming needed to turn ESC into TEC. Previous studies had demonstrated that there were immature TEC in the fetal thymus that could regenerate a new thymus upon transplantation. However, large numbers of these fetal TEC cells were required for transplantation, suggesting that these were not a pure TEC stem cell population, Furthermore, previous efforts to isolate TEC stem cells from the adult thymus have been unsuccessful. We used a novel system to identify adult TEC stem cells, which is based on the Wnt signaling pathway, which is important for most stem cells and development. Wnt proteins are known to be important for the formation of TEC. In the genetically engineered mice that we used, TEC that were responding to Wnt signals at any time point could be switched by brief exposure to a hormone drug from expressing a protein that made them fluoresce red to expressing a different fluorescent protein that was green. When we switched the Wnt-responding cells in young or adult mice from red to green fluorescence, we found that a very small number of TEC were green while the vast majority of TEC remained red. This is consistent with the majority of TEC being Wnt-unresponsive and the rare Wnt-responsive cells being TEC stem cells. Analyses of mice for up to one year after switching showed that clusters of green TEC replaced the red TEC, consistent with the green TEC being derived from a single adult TEC stem cell that had been switched earlier in life and then developed into a group of mature TEC. Transplantation of as few as 60 of Wnt-responsive green cells into mice which lacked a thymus regenerates a robust new thymus and immune system in the recipient. To our knowledge, this is the highest level of purification of any TEC stem cell, and the first demonstration that TEC stem cells exist in the adult thymus. We have identified new markers that can be used to isolate TEC stem cells from any strain of mice, and potentially from humans. We have isolated sufficient numbers of cells to test whether the immune system of a mouse with an autoimmune disease that models MS, can be re-programmed by TEC stem cell transplantation.

Grant Application Details

Application Title:

“Engineered immune tolerance by Stem Cell-derived thymic regeneration”

Public Abstract:

Stem cell therapies have the potential to transform medicine by allowing the regeneration of tissues or organs damaged by disease or trauma. In order for stem cell therapies to proceed, it will be essential that the regulation of immune responses to the stem cell derived tissues be achieved. While the function of the immune system in protection from infections is essential throughout life, some immune reactions are undesirable. Illnesses due to autoimmunity, in which the immune system attacks one’s own body, instead of only germs that cause infection, are common. Examples of autoimmune diseases that are amenable to stem cell based therapies include Type I diabetes caused by abnormal immune responses directed against the insulin producing cells and multiple sclerosis (MS) caused by immune responses that attack the nervous system. Another type of undesirable immune response is the attack on transplanted tissues, leading to rejection. Control of immune reactions is the major impediment to successful organ transplantation, and is highly likely to be an obstacle to therapeutic uses of stem cells.

T lymphocytes are white blood cells that choreograph the multiple responses that the body uses to control infection. T lymphocytes are produced in the thymus, a specialized organ located in the chest in front of the heart. A function of the thymus is to “edit” the immune response by selecting desirable T cells that are allowed to grow up, and removing young T cells that would cause autoimmunity if they were released into the body. Abnormal selection in the thymus, due to genetic variation, disease or errors in editing leads to autoimmune disease. On the other hand, replacement of the immune system by bone marrow transplantation can be used to engineer the immune system so that the body will accept organ transplants if they came from the same donor as the bone marrow.

We are evaluating a stem cell based strategy to control the editing of the immune response to both treat autoimmunity and permit stem cell-derived tissues to be transplanted. Members of the international collaborative team have developed techniques for directing embryonic stem cells (ESC) into specialized thymic epithelial cells (TEC), which can be transplanted into recipient mice to support the development of new T cells. We have also developed mouse models for MS and for stem cell rejection which can be controlled by modification or suppression of the immune system. We will evaluate the ability of TEC made from ESC to 1) edit the immune system to prevent or treat MS, or 2) allow ESC to be transplanted without rejection. The studies will test TEC to support the production of new T cells that can respond normally to germs, but will not attack either nervous system cells or transplanted ESC. Success of the studies will advance our understanding of how to regulate the immune system for the goal of using stem cells therapies to treat disease and regenerate tissues.

Statement of Benefit to California:

The research is aimed at understanding the how ESC can be used to re-engineer immune responses so that autoimmune diseases can be controlled and other stem cell derived tissues can be transplanted. The studies are based on our team’s demonstration that functional TEC can be generated from ESC. Since TEC are very important for selection in the thymus of which T cells will be allowed to mature and become part of the immune system. The studies will explore two models for the use of such ESC-derived TEC – 1) an autoimmune disease of mice that mimics many aspects of human multiple sclerosis; and 2) transplantation of ESC from between genetically non-identical donors and recipients. Autoimmunity is a major cause of disease in California. A recent study of the Kaiser Northern California patient population estimated an overall incidence for 11 autoimmune diseases of 160 new cases per 100,000 person years (PY)(Klein NP et al, Vaccine [2009] 28:1062-68). With an estimated California population of 37 million, this translates to nearly 60,000 new cases of autoimmune disease yearly. Furthermore, many of these diseases are disproportionately found in younger individuals, thereby amplifying the potential effects of disability on the population. The autoimmune disease that is being approached in this grant, multiple sclerosis (MS), has the fourth highest incidence in the Kaiser study at 14.2 cases/100,000 PY overall, and 22.9 cases/100,000 PY for women 25-62 years of age. The proposed studies include a novel use of ESC to control autoimmune responses in mouse models of MS as well as a novel approach to immune suppression, which could have a significant impact on MS and autoimmune diseases in general. The second proposed application of ESC-derived TEC is the control of rejection of transplanted ESC. Since one of the limitations of all ESC-based therapies is likely to be immune responses leading to rejection of the transplanted tissues, a strategy to make recipients tolerant to the ESC has the potential to benefit all Californians who might benefit from stem cell based therapies. Examples would include patients who could benefit from regenerated nervous system, heart, liver or kidney tissue, as well islet cells for treating diabetes. The proposed studies include the innovative use of ESC-derived TEC to re-engineer the immune system to allow it to accept (become tolerant of) cells derived from the same ESC, an approach that could significantly advance the development of treatments for all Californians likely to benefit from stem cell therapies.