Avinash Bhandoola - US grants

DESCRIPTION (provided by applicant): T cells in thymus develop from hematopoietic stem cells (HSCs) in bone marrow (BM), but the cellular intermediates linking HSCs in BM via blood to early T lineage progenitors in thymus remain poorly defined. The long-term goal of the proposed work is to elucidate the cellular and molecular basis for early T cell development. It has recently been determined that early T lineage progenitors (ETPs) in thymus derive from a BM progenitor population distinct from previously described common lymphoid progenitors (CLPs), and that the pathways leading to T and B cells diverge earlier than had been previously appreciated. Experiments are proposed to further define and characterize the intermediate populations that lead from multipotent progenitors in BM, through a circulating progenitor cell in blood, eventually leading to ETPs and their downstream T lineage progeny within the thymus. While cytokines and Notch signals are known to be critical for early T cell development, the cellular intermediates receiving and integrating these multiple signals are largely uncharacterized. Notch signals are probably received by blood-borne hematopoietic progenitors shortly after thymic colonization, but ETP subsets within which these signals operate to result in T lineage commitment are also not characterized. Our goal is to identify a complete lineage of cells leading from HSCs in bone marrow to T lineage cells in thymus. An understanding of the key cellular intermediates in T lineage development is necessary to understand disorders in this process, and will be of basic and critical use in a wide variety of diagnostic and therapeutic endeavors. Hence understanding the T cell defects in aging, the process of malignant transformation in T lineage cells, and gene therapy to correct defects of T cell development and function all require that the steps of hematopoiesis linking HSCs to T cells be understood.

[unreadable] DESCRIPTION (provided by applicant): The molecular signals that allow progenitors to traffic to the thymus from the blood are very poorly understood. Work ongoing in our laboratory indicates that different hematopoietic progenitors differ in their ability to settle within the thymus from the blood. Hence downstream multipotent progenitors (MPPs) but not hematopoietic stem cells (HSCs) both circulate in blood, but only MPPs are able to settle within the thymus if placed in the blood. We have further fractionated MPPs, and find that only the early lymphoid progenitor subset (ELP) of MPPs, that expresses the lymphoid-specific genes RAG1 and RAG2, appears able to efficiently settle within the thymus, whereas other MPP subsets do not appear able to migrate into the thymus. This grant application is focused on understanding the molecular basis of the selective ability of ELPs to settle within the thymus. Our data thus far suggest that expression of the chemokine receptor CCR9 is regulated between ELPs that can settle within the thymus and RAG-negative MPPs and HSCs that cannot. Our data further indicate that regulated expression of CCR9 is important for the ability of ELPs to physiologically settle within the thymus. However, our preliminary data also indicate that additional molecules must be involved in the ability of ELPs to undergo regulated thymic settling. In Aim 1, we propose to identify molecules in addition to CCR9 whose expression is regulated between HSCs and MPPs. In Aim 2, we propose to perform genetic gain-of-function experiments to determine whether CCR9 and other identified molecules are sufficient to confer thymic homing on progenitors such as HSCs that normally fail to undertake this migration; and to perform loss of function experiments to determine whether such molecules physiologically play a role in the settling of circulating hematopoietic progenitors within the thymus. Together, these studies will identify the signals critically important in the physiological homing of progenitors to the thymus, and may provide new avenues for therapeutic interventions in circumstances where thymic reconstitution needs to be improved, such as improving intrathymic T cell reconstitution following bone marrow transplantation, and in aging. KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Avinash Bhandoola AVINASH University of PA P.I. OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Paul E. Love National Institutes of Health, MD Collaborator Wolfgang Weninger Wistar Institute, Philadelphia, PA Collaborator Donald A. Baldwin University of Pennsylvania, PA Collaborator The signals that direct the settling of hematopoietic progenitors within the thymus of adult mice are unknown. We propose to identify homing molecules expressed by hematopoietic progenitors that are able to settle within the thymus, and to determine the role of these molecules in the process of thymic settling. These experiments will allow a molecular definition of thymus-settling progenitors, and may enable therapeutic enhancement of T lineage reconstitution in clinical settings such as aging and bone marrow transplantation. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (11) Regenerative Medicine, and specific Challenge Topic, 1-HL-101*: Develop cell-based therapies for cardiovascular, lung, and blood diseases. Engineering hematopoietic progenitors for efficient migration to the thymus T cells develop in a specialized organ, the thymus. They derive from hematopoietic progenitors that colonize the thymus from the blood. Many different progenitors in the bone marrow (BM) possess T lineage potential and will make T cells in experimental circumstances. However, our recent work has shown that the process of thymic settling is selective and regulated. Our hypothesis is that physiological T cell progenitors differ from other progenitors with T lineage potential chiefly in their ability to migrate to the thymus. More specifically, we hypothesize that the chemokine receptors CCR7 and CCR9 cooperate to mediate recruitment of rare BM- derived progenitors to the thymus. In this challenge grant proposal, we present our preliminary data indicating CCR7 and CCR9 are selectively expressed on key hematopoietic progenitors, and are important for migration to the thymus. We propose to establish the role of CCR7 and CCR9 in thymic homing, and to identify and characterize the physiologically relevant progenitors that express these homing molecules and so contribute to T lymphopoiesis. We present data suggesting that the generation of thymic homing progenitors is deficient in aged mice. As this may contribute to the loss of thymic cellularity and immunosuppression seen with aging, we propose to determine whether ectopic expression of CCR7 and CCR9 improves T lymphopoiesis, and confers T lineage competence on aged progenitors that are otherwise incompetent to generate T cells. These studies will provide new understanding into the molecular underpinnings of early T cell development. Understanding the mechanisms of thymic settling in mice is an essential stepping-stone towards developing new clinical interventions for individuals with diminished T cell numbers. Our findings may have application for boosting T cell numbers after bone marrow transplantation. PUBLIC HEALTH RELEVANCE: T cells develop in the thymus from hematopoietic progenitors. The identity of molecules that allow progenitor cells to migrate to the thymus is not well known. We propose to identify molecules that support trafficking of progenitors to the thymus, to identify and characterize physiological T cell progenitors that express these trafficking molecules, and to determine whether the ectopic expression of these molecules will allow a broader range of blood progenitors to migrate to the thymus and improve T lymphopoiesis

DESCRIPTION (provided by applicant): Whether age-related defects occur in hematopoietic progenitors for T cells was previously not well understood. We recently assessed the ability of bone marrow progenitors from aged mice to contribute to the T cell lineage, and established that aging significantly impacts the T cell potential of hematopoietic progenitors. Defects in T cell development were clearly evident when aged hematopoietic stem cells (HSC) and downstream multipotent progenitors (MPP) were assessed in vivo, and also in vitro using cell co-culture with OP9 stromal cells engineered to express Notch ligands (OP9- DL1) that support T cell differentiation. In the current application, we propose to exploit the OP9-DL1 cell culture system to elucidate the molecular changes underlying the loss of T lineage potential in aged hematopoietic progenitors. We hypothesize that the age-associated loss of T lineage competence in aged stem and progenitor cells is caused by specific molecular changes in gene expression. We further hypothesize that restoration of expression of these molecules will restore T lineage potential to aged hematopoietic progenitors. We will identify molecules whose expression is altered in HSC and downstream MPP isolated from aged mice, as compared to HSC and MPP from young mice. Identification of such molecules will be initially performed using DNA microarray based interrogation of sort-purified progenitors. We will confirm altered expression of identified molecules by hematopoietic progenitors using real-time PCR, coupled with additional approaches when possible. We will use retroviral gene transduction of HSC and MPP to ask whether forced expression of molecules showing reduced expression in aged progenitors is sufficient to correct defective T cell development from aged HSC and MPP in the OP9- DL1 system. These studies should provide insight into molecular mechanisms underlying the age-associated loss of T progenitor competence. PUBLIC HEALTH RELEVANCE Whether age-related defects occur in hematopoietic progenitors for T cells was previously not well understood. We recently reassessed the ability of bone marrow progenitors from aged mice to contribute to the T cell lineage, and established that aging significantly impacts the T cell potential of hematopoietic progenitors. In the current application, we propose to investigate the molecular basis of the age-related defects in T progenitor potential that we have discovered. These experiments will allow a molecular characterization of age-related defects in T cell development, and may enable therapeutic enhancement of T lineage reconstitution in clinical settings such as aging and bone marrow transplantation.

DESCRIPTION (provided by applicant): T cells develop in the thymus from hematopoietic progenitors. We have previously shown that normal migration of progenitors to the thymus uses two homing molecules, CCR7 and CCR9. However, we have now found that progenitor migration to the thymus after conditioning irradiation during bone marrow transplantation is independent of CCR7 and CCR9. Unexpectedly, we find that inhibition of other homing molecules can dramatically improve thymic reconstitution after bone marrow transplantation. We propose to identify and characterize progenitors homing both the normal thymus and after bone marrow transplantation, to identify and characterize the homing molecules that are used by progenitors for thymic homing after irradiation. Further, we will determine whether the ectopic expresion or inhibition of homing molecules will allow a broader range of blood progenitors to migrate to the thymus and improve T lymphopoiesis after bone marrow transplantation, and understand what the underlying mechanisms might be. ! PUBLIC HEALTH RELEVANCE: The identity of progenitors homing to the thymus, and the homing molecules used by cells to get to the thymus, is not well understood. In this proposal, we will identify the cells homing to the thymus normally and after bone marrow transplantation. We will identify and characterize the molecules that are for thymic homing, and determine whether the ectopic expression of homing molecules will allow a broader range of blood progenitors to migrate to the thymus and improve T lymphopoiesis after bone marrow transplantation.

DESCRIPTION (provided by applicant): The core binding factors (CBFs) are a small family of transcription factors that consist of a DNA binding subunit encoded by the Runx1, Runx2, or Runx3 genes, and a common non-DNA binding CBF2 subunit encoded by the Cbfb gene. We showed that mice with reduced (15% of normal) CBF2 levels generate all blood cell lineages with the exception of T and NK cells. T cell development in CBF2-insufficient mice aborts at the specification stage, as the very earliest markers of T cell differentiation fail to be expressed. NK cell development also fails very early, at the transition between NK progenitors and immature NK cells. We propose to further characterize the NK cell defect caused by CBF2 insufficiency by determining which DNA-binding Runx subunit (or subunits) are required for the progression of NK progenitors to immature NK cells. We will also use in vitro assays of T and NK cell development to probe the CBF's biochemical functions in these lineages. Specifically, we will identify and characterize chromatin-remodeling proteins recruited by the CBFs to their target genes in T and NK cells, and assess the relevance of these proteins for T and NK cell development. We will assess chromatin occupancy by the CBF-interacting proteins to determine whether they associate with all or a subset of genes bound by the CBFs, and if their occupancy correlates with active or inactive chromatin states. Project Narrative: These studies aim to understand how proteins that are essential for lymphocyte formation perform their functions. This is an important area of research because disruption of lymphocyte development can lead to immune disorders, leukemia, and lymphoma.

DESCRIPTION (provided by applicant): Natural Helper (NH) cells are newly discovered innate immune cells that produce T-helper type 2 (Th2) cytokines and promote protective type 2 immunity during helminth infection, or after influenza infection. The ontogeny of NH cells and their function in allergic diseases are not known. Strikingly, we find that NH cells have similar gene expression profile as T cell lineage progenitors, and that the development of NH cells is dependent on TCF-1, an essential transcription factor in early T cell development. We propose to determine the molecular pathways that underlie the development of NH cells. We will also examine the role of NH cells in the pathogenesis of allergic asthma. Studies in this proposal will improve our understanding of the generation and function of NH cells, which will inform therapeutic strategies to target NH cells for Th2 associated diseases. PUBLIC HEALTH RELEVANCE: T helper 2 (Th2) cytokines are central players in the pathogenesis of allergic asthma. Natural helper cells are newly discovered immune cells that were shown to be a major source of Th2 cytokines but neither their developmental origin nor their significance in asthma is understood. We propose to identify the cellular precursors and the signals that give rise to Natural helper cells. Using a mouse model we will also determine the role of Natural helper cells in the pathology of allergen-induced airway inflammation. Our study will provide important clues to targeting Natural helper cells as a novel therapeutic approach to Th2-driven (allergic) airway inflammation in asthma.