White blood cells are the main players of human immunity in defense against infection. Defects in CD4 T white blood cells, for example, can lead to the devastating infections observed in AIDS patients and patients with a genetic immunodeficiency syndrome (“bubble boy” syndrome). A normal immune system can recognize and attack pathogens but not “self”. This is achieved by rigorous selection and “education” during development of T or B white blood cells and by regulatory T cells that suppress occasional “run-away” white blood cells. Glitches in these processes can also lead to equally devastating problems as seen in many autoimmune diseases like type I diabetes, multiple sclerosis and rheumatoid arthritis. Thus, the availability of T cells or regulatory cells could lead to therapies for many human diseases.
A major limitation in using T cells in therapy is the lack of available primary T cells generated in the laboratory. Attempts thus far to generate T cell precursors in a tissue culture dish from the existing NIH approved embryonic stem (ES) cell lines have had only limited success, perhaps due to the partial white blood cell potential of these particular lines. Efficient conversion of T cell precursors into mature T cells or regulatory T cells in a tissue culture dish have also not yet been achieved.
In this seed grant application, we propose to test a series of new (non-federally approved) human embryonic stem (ES) cells for their abilities to efficiently generate white blood cells precursors and subsequently immature T cells in a tissue culture dish. Conditions to optimize generation of white blood cell precursors and T cell precursors will be sought. We will then examine how to convert mature T cells from T cell precursors by varying the culture conditions to closely mimick the “real” situation in humans. Successful completion of this project would constitute a major first step in the long-term goal of using human T cells (that can attack pathogens, but do not react to one’s own organs) in therapy. Examples of possible clinical therapy include administration of regulatory T cells in preventing autoimmune attacks in a variety of aforementioned autoimmune diseases. Availability of T cells specific for proteins found in tumors could be used to treat cancer, and mature T cell populations could be used to restore immune function of AIDS patients and to improve the short-term survival of bone marrow transplant recipients.
Successful completion of this seed project, with the eventual goal of a comprehensive research project in T cell differentiation from human ES cells, will have a major impact in devising a new immune therapy to combat a variety of diseases. California has many cancer and AIDS patients and many of its citizens are suffering from debilitating autoimmune diseases. The availability of normal and regulatory T cells generated in the laboratory and tailored to individual person will pave a way to a novel T cell therapy to treat Californians suffering from many currently incurable diseases. At the same time, we will also significantly increase our basic understanding of how the human immune system develops, which might eventual lead to novel invention of diagnostic or therapeutic tools. Furthermore, the project could serve as a basis for biotech companies in California to exploit the therapeutic goals of this research endeavor.
SYNOPSIS: The focus of this proposal is on the development of T-lymphocytes from hESC in vitro. Aim 1: Seventeen non-approved hESC lines will be initially evaluated for their ability to become hematopoietic progentiors (HP) using conditions designed to generate embryoid bodies or upon co-culture with stromal cells. Aim 2: Simultaneously, HP will be isolated based on expression of cell surface markers and on stromal lines in various cytokines to induce differention of T-cell progenitors. The final aim will focus on attempting to induce mature T-cell development by engineering stromal cell lines to express integrin ligands or human MHC molecules. An attempt to reproduce the three dimensional structure of the thymus with collagen matrix will also be made.
SIGNIFICANCE AND INNOVATION: The generation of mature T-cells from hESCs is a potentially important step for providing cells for specific immune functions in disease and for clarifying the ontogenetic steps of lymphocyte maturation. The proposed work may also disclose whether the few NIH-approved cell lines are so inefficient in generating T cells because they have lost the ability to differentiate despite the use of different culture conditions and stromal cell support, or for more basic biological reasons. To do this, the PI will explore 17 recently reported hESC lines from the Melton lab. A particularly difficult step is the conversion of the immature CD4+, CD8+ phenotype into CD4+, CD8- and CD4-, CD8+, of the mature T cell population. The lack of CD4+ T cells should be anticipated from the lack of class II HLA ligands in the system using murine stroma; thus, the comparison between the use of embryoid bodies and OP9-DL1 assisted cultures with growth factors and cytokines. To enhance the maturation of early CD4+, CD8+ cells Dr. Robey’s solution is to transduce the HLA class I and class II MHC genes and then monitor the phenotypic expression of informative markers using CD34+ cells from umbilical cord blood. This will shorten the identification of an optimal culture system to be used with the most promising cell lines and culture conditions. In addition, she will attempt to develop effector and regulatory T cells. These are all reasonable and innovative, though laborious approaches.
The innovation in this proposal reflects the willingness to screen many (17) cell lines under many conditions in an effort to achieve the stated goals. State-of-the-art methodology will be utilized and novel, although sketchily described, strategies will be utilized in an attempt to achieve the final steps in T cell maturation. Overall, one reviewer ranks the proposal fairly high with respect to innovation. The work's significance lies in the importance of understanding the differentiation of hESCs into hematopoietic cells and the potential long term use of in vitro generated lymphocytes for therapuetic purposes.
STRENGTHS: This is submitted by a highly seasoned and well funded investigator who has made many important contributions to experimental immunology and specifically to T-cell differentiation. Her colloborator, Dr. Winoto, is also an accomplished experimental immunologist who brings expertise in molecular biology to the proposal. Overall the enviroment seems very strong for conducting this research. The proposal is generally well thought out and encompasses a diversity of approaches to improve the chances of success.
Both PI and Co-PI are well-known with solid track record in experimental immunology. Support staff is excellent, the approaches are elegant and innovative and some preliminary data is presented.
WEAKNESSES: The technical descriptions are very skimpy, although all procedures are well within the technology used by this lab with the possible exception of achieving the T-cell selection of non-self reactive and immunologically active. The proposed strategies to recreate the enviroment to support terminal T-lymphocyte development are minimally described. For example, how will human MHC be expressed to achieve both negative and positive regulation of T-cell development. Which integrins will be expressed and what is the basis for their choice? How will collagen matrix re-create the 3 diminisional structure of the thymus microenviroment?
DISCUSSION: This proposal aims to study differentiation of T cells from hESCs. The three aims of this proposal describe a three-stage protocol for generating mature T-cells that is elegant and well-constructed. Seventeen (17) cells lines will be used to test the effects of many growth conditions using FACS as a monitoring method. There was some discussion over the rationale to test 17 cell lines. 17 cell lines seems excessive but may be potentially justified based on heterogenity issues; the PI wants to screen enough to know that the conditions will work. It was noted that some of the NIH approved lines have been tested without success for differentiation to mature T lymphocytes. In the case of these lines, there is some question whether the issue is one of passage number or a more fundamental issue. The environment for completing this work is strong. From an immunological point of view, the most interesting part of this proposal is the selection of +/- (CD4,CD8)cells from hESC lines. T cell derivation has previously failed but it isn't known why. There was a certain vagueness in the proposal as to exactly what the applicant proposes to do; this aspect was not very well described in what is otherwise an elegant proposal. The proposed approach seems to be to test a lot of lines and thus figure it out.