Basic Biology II
$1 447 956
The function of the immune system throughout life is essential for protection from infections and cancer. 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. The production of new T lymphocytes (“thymopoiesis”) is abnormal in some children with genetic defects in the development of the thymus (DiGeorge syndrome [DGS]), but even in healthy people, thymic function declines with age. Thymic insufficiency, the decreased ability of the thymus to make new T lymphocytes, is a serious health problem. For example, if the T lymphocytes that have been previously made were to be destroyed, for example by HIV infection, chemotherapy or radiation therapy, or hematopoietic stem cell transplantation, the restoration of immune function requires the production of new T lymphocytes to replace those that were lost. For this reason, adults with such conditions have poorer recovery of immune function than children and the elderly have increasing risk of severe infection with age. For example, 10-40% of the elderly do not respond to annual influenza vaccination and as many as 50-100,000 may die of influenza annually. Thymic insufficiency is due to injury or death of skin-like cells called thymic epithelial cells (TEC), which produce a number of proteins such as interleukin-7 (IL-7) needed by developing T lymphocytes in the thymus (“thymocytes”). Like skin cells, TEC become more fragile and easily injured with age. Also like skin cells, TEC are destroyed by chemotherapy and radiation therapy. Clinical efforts to restore thymopoiesis in patients with HIV infection by transplantation of thymic tissue from unrelated donors have not been successful because of rejection of the transplanted tissue. Experimental efforts to correct the problem of decreased thymopoiesis have included attempts to replace TEC functions by injections of IL-7 or other cells that make IL-7; or to regenerate TEC by the injection of keratinocyte growth factor (KGF), a protein that stimulates the growth of TEC. Human embryonic stem cells (hESC) are a potential source of replacement TEC that could be used to regenerate the immune system in people whose pool of T lymphocytes has been decreased, e.g., the elderly, or those with HIV or cancer. In order to implement such a strategy, research on how to control the development of TEC from hESC are necessary. The proposed studies will test how the Tbx1 gene, which is abnormal in DGS, controls the development of TEC from hESC. In addition, the studies will develop model systems in mice for testing the ability of TEC to be transplanted, a necessary scientific tool for the assessment of future therapies that will use TEC progenitors to restore immune function.
Statement of Benefit to California:
The research is aimed at understanding the generation of TEC in an effort to ultimately develop clinical strategies for thymic regeneration to treat thymic insufficiency. Thymic insufficiency occurs as both primary defects of TEC development and more commonly as acquired defects in TEC maintenance. Thymic insufficiency was first recognized in children with the rare DiGeorge syndrome (DGS), in which thymic hypoplasia occurs. More recent studies have shown that age-related thymic insufficiency is a common problem that progresses, and influences the outcome of many diseases. If an individual has a condition that results in destruction or increased turnover of mature T lymphocytes, their health will ultimately depend on the ability of the thymus to produce new T lymphocytes. An example is HIV infection, in which immunological recovery depends not just on the efficacy of anti-retroviral therapy to decrease the viral burden and T lymphocyte destruction, but also on the ability of the thymus to produce new T lymphocytes to replace those that were previously destroyed. The ability to do so is inversely related to age. Similar age-related thymic insufficiency occurs in recipients of high dose chemotherapy for cancer and in recipients of hematopoietic stem cell transplants (HSCT). Probably the largest group of individuals who are affected by thymic insufficiency are the elderly. There is evidence that the declining immune responsiveness of the elderly is a serious problem, particularly as it relates to common respiratory virus infections, such as influenza and respiratory syncytial virus, which together kill >50,000 Americans each year. In discussing the relevance of the studies to California, it must be recognized that this CIRM Basic Biology Grant is aimed at a set of basic questions that will not immediately translate into health benefits. Nevertheless, it is possible to make estimates of how many individuals have conditions that this work is directly related to. For example, DGS is thought to affect 5% of all children with congenital heart disease and 20-25% of those with severe CHD, especially those with conotruncal abnormalities. Using the estimated 500-600,000 births per year (http://cgi.rand.org) and an incidence of 0.4% of severe CHD, there are about 500-600 births of children with DGS in California per year. Based on CDC serosurveillance data, tens of thousands of Californians are HIV infected and tens of thousands others receive either intensive chemotherapy or HSCT annually. Finally, the 2000 census showed approximately 3.5 million Californians over the age of 65 (http://www.census.gov/census2000/states/ca.html). Thus, the research proposed in this grant is likely to be directly related to the health of millions of individuals in California as well as having large impact on health economics.
EXECUTIVE SUMMARY This proposal focuses on the generation of thymic epithelial cells (TECs) from human embryonic stem cells (hESCs) by recapitulating key steps in TEC development. The production of TECs could provide a model system for studying abnormal thymopoiesis and supply cells for replacement therapy for conditions of thymopoietic insufficiency. Aim 1 will be to study the clonal relationships of cells within the TEC lineage. Aim 2 involves the characterization of the mechanism by which the transcription factor Tbx1 induces generation of proto-TEC from definitive endoderm. The third specific aim will focus on the identification of mesenchyme-derived factors that induce differentiation of proto-TEC into phenotypically mature, functional TEC. Reviewers found the project described in this proposal to be significant and innovative. They believed this proposal has potential to provide novel insights into TEC development and catalyze further research in this area. Reviewers also praised the novel hESC-derived model of TEC development and the generation of TEC progenitors. They considered these aspects of the proposal to be creative and felt that the research would provide significant new information toward understanding the development of clinical strategies for thymic regeneration. However, there were some concerns that the project did not concentrate sufficiently on basic molecular or cellular mechanisms and that the proposal was overly ambitious and not sufficiently focused. Reviewers raised serious concerns regarding the project’s feasibility and experimental design. Although the proposal has a logical approach and experiments have a clear rationale, reviewers cited a number of serious deficiencies. One of these was the fact that the preliminary data was almost entirely based on mouse work, and it is unclear how much of the murine molecular detail will translate to human systems. This is particularly important, since several key markers proposed for the identification of specific cell populations have not been shown to be expressed in the appropriate human cells. A related concern was that the research team has very limited experience with hESCs. Reviewers considered the extensive use of gene and proteomic arrays as largely a “fishing expedition” where a directed study was warranted. Additionally, many experiments require an inducible Tbx1 hESC line and others rely on a unique immune deficient mouse model, both of which have yet to be generated and characterized. The applicant’s expertise and experience with thymopoiesis and related areas were recognized as particular strengths. The proposed collaborations and research environment were recognized as extremely strong and beneficial. The review panel, however, expressed serious concerns over the lack of team experience with hESC biology or culture. In summary, this application addresses the generation of TECs from hESCs through a characterization of key steps in TEC development. The proposal’s strengths include the significance of the problem, the project’s innovation, and the expertise of the applicant. Weaknesses include lack of adequate preliminary data and serious concerns about the project’s feasibility.