Basic Biology I
$1 320 303
T cells (or T lymphocytes) are necessary for normal immune surveillance systems, and their dysfunction leads to development of fatal diseases, such as Acquired Immune Deficiency Syndrome (AIDS), congenital T cell deficiency and cancer. These diseases are life threatening, because T cells, key effectors eradicating pathogens within the body, are severely reduced and medications (e.g. anti-viral treatment) cannot fully compensate for such fundamental defects. Thus, in these circumstances, providing T cells to the patient, so-called “T cell replacement therapy”, could be the sole therapeutic option to cure the disease. However, such therapy has not been successfully established because of the following reasons: 1) T cells are difficult to expand in culture; 2) even if the patient’s T cells can be expanded in culture, they may not work normally when they are returned to the patient (e.g. T cells will be again infected with HIV virus) and 3) T cells from others (donor) could recognize the patient (recipient) as "foreign" and mount an immunologic attack. Human pluripotent stem cells, such as human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs), have tremendous potential in T cell replacement therapy, as: 1) they are virtually unlimited; 2) patient-specific hiPSCs can overcome immunological barriers between donors and recipients and 3) hESCs/hiPSCs could be safely manipulated for T cells to be functional. While T cells have been successfully derived from mouse embryonic stem cells (mESCs), hESCs demonstrated little lymphoid potential, preventing the use of hESCs for T cell replacement therapy. Considering clinical implications, there is a critical need to better understand the mechanisms underlying hESCs/hiPSCs differentiation toward T cells. Our goal is to develop a system by which hESCs/hiPSCs efficiently give rise to T cells in culture. The objective of this application is to determine the role of the Leukemia/lymphoma Related Factor (LRF) gene in human T cell differentiation from hESCs/hiPSCs. Guided by our preliminary studies, we hypothesize that LRF inactivation promotes efficient T cell development from hESCs/hiPSCs. We will test this hypothesis employing molecular biological approaches as well as a series of genetically engineered mouse models. We expect that the combination of work proposed will provide further understanding of how hESCs/hiPSCs develop T cells in culture. This contribution is significant because it will lead to the development of new therapeutic strategies for T cell replacement therapy.
Statement of Benefit to California:
T cells are essential for human immune system and the lack of functional T cells results in development of diseases such as AIDS, sever infectious diseases and cancer. These diseases are one of the major health issues in California. It has been estimated that nearly 130,000 people in California live with human immunodeficiency virus (HIV) infection, among which 70,000 are diagnosed as AIDS (HIV Prevalence Estimates of California, CDC, 2008). For these patients, providing healthy and functional T cells, so-called T cell replacement therapy, could be a new therapeutic strategy, as current therapies (e.g. anti-viral drag for AIDS, chemotherapy for cancer) cannot cure the disease, and cost of the treatment is high. Human pluripotent stem cells (hPSCs) are a very promising source for such T cells, as they are virtually unlimited and relatively easy to be manipulated in culture (e.g. gene therapy for AIDS and cancer). However, it is not fully understood how hPSCs differentiate to T cells, preventing their use in the clinic. Our goal is to develop a new way of creating T cells from hPSCs in culture by elucidating the molecular mechanisms of T cell development from hPSCs. If successful, it will provide the people of California with tremendous benefits, potentially leading to new therapeutic strategies for life-threatening diseases such as cancer and AIDS. It may also lead to the significant reduction of therapeutic costs, thus benefiting the State economy.
This proposal seeks to achieve robust differentiation of T cells from human pluripotent stem cells. Based on preliminary data in the murine system, the applicant hypothesizes that inactivation of Leukemia/lymphoma Related Factor (LRF), a zinc finger transcription factor, will enhance T cell differentiation. The applicant proposes in the first aim to utilize knock down technologies and a series of genetically engineered reporter mice to investigate the role of LRF in lymphoid lineage fate determination in human hematopoietic stem cells (HSCs). In the second aim, the applicant proposes to use mouse genetic models to identify the molecular mechanisms by which LRF inactivation effects differentiation. Finally, in the third aim, the applicant will determine the effect of LRF inactivation on human embryonic stem cell (hESC) or induced pluripotent stem cell (iPSC) differentiation toward T cells. The reviewers agreed that if successful, this proposal would have significant impact on the development of T cell replacement therapies for treatment of immunodeficiency disorders and cancer. Currently, there are not robust and reproducible protocols to differentiate T cells from human ESC or iPSC sources. This proposal addresses this deficiency with the innovative hypothesis that LRF plays a key role in specifying the balance between B and T cell development from progenitor hematopoietic stem cells (HSCs) and its inactivation will result in a bias toward T cell differentiation. The reviewers considered the experimental approach to be based upon sound rationale and the specific aims generally well designed, albeit not especially innovative. The reviewers appreciated the well-described experimental plan that clearly listed timelines and milestone and addressed potential pitfalls and suggested alternative approaches. However, the reviewers did raise significant concerns about the proposal’s feasibility. While the proposal includes significant and strong preliminary data from the murine system to support Aims 1 and 2, there is little to indicate that this data is informative of the molecular mechanism of action of LRF in human cells or that the methodologies utilized to promote HSC induction from pluripotent cells in murine systems will be successful with human cells. The reviewers commented that Aim 3, which was considered to be the crux of the proposal, was particularly weak and unsupported by preliminary data. There was no evidence presented to support the overexpression approach utilized in this aim to generate HSCs, and this experiment was considered confusing and not coherent with the rest of the proposal. One reviewer commented that it was unlikely the investigators would be able to successfully differentiate T cells from hPSCs using this approach. The PI was considered well suited for the project with a strong track record, but the reviewers were concerned about the PI’s lack of experience in working with human pluripotent cells. One reviewer was particularly concerned that this deficiency decreased the overall feasibility that aim 3 would be successful. Overall, the reviewers agreed that the investigator proposes an innovative hypothesis to address an issue critical to developing T cell replacement therapies, but felt there was insufficient preliminary data from human cells to support the proposal and that Aim 3, the crux of the proposal, lacked sufficient feasibility that T cells could be successfully derived from hPSCs.