Molecular mechanisms of miRNA-mediated control of T cell regeneration after Hematopoietic Stem Cell Transplantation
Hematopoietic stem cell transplantation (HSCT) is applied as therapy for a number of malignant and non-malignant diseases mostly of the hematopoietic system. However, HSCT is generally accompanied with a prolonged phase of immunodeficiency resulting from slow recovery of the T cell arm of the adaptive immune system, the consequences of which are reduced control of opportunistic infections as well as an increased risk of malignant relapse. Developing strategies to improve T lineage reconstitution after HSCT depends on a thorough understanding of the mechanisms governing hematopoietic lineage decisions. Such lineage decisions occur as progressive loss of alternate lineage fates as well as loss of self-renewal potential of hematopoietic stem cells. They are induced by both instructive events, such as extrinsic signals, and stochastic events, such as fluctuations in expression of lineage-specific transcription factors. The role of miRNAs in these decisions remains ill understood. We have previously investigated the interdependence of transcription factor controlled gene regulatory programs and miRNAs downstream of these programs. We have identified 2 families of miRNAs, miR-17~92 and miR-181, with profound impact on T cell development and, thus, with the potential to serve as candidate targets to develop strategies for improved T lineage regeneration. However, our understanding of the role of miRNAs suffers from the complex biology of miRNA-mediated gene regulation. Thus, miRNAs frequently target hundreds of potentially relevant genes, many of which are modulated to a small extent only. Consequently, it remains a difficult but equally important task to identify functionally relevant miRNA targets. In order to be able to translate findings from murine models into strategies for improved T lineage regeneration robust and flexible experimental tools to study human T cell differentiation are required. However, to date in vitro models rely on primary cells with limited availability and humanized mouse models do not reliably recapitulate the human system. Here we will dissect miRNA-mRNA target relationships relevant for both physiological T cell development and T lineage regeneration after HSCT. The proposed project will provide important insight into the integration of miRNAs in gene regulatory networks governing physiological T cell development and T lineage regeneration after HSCT in both mouse and human. Furthermore, the technological advances envisioned for the project are likely to impact future studies of miRNA biology and hematopoiesis in general.
Mapping the thymic progenitor niche
Malignant diseases of the blood system, such as leukemias, can be successfully treated by hematopoietic stem cell transplantation (HSCT) following destruction of the patient’s tumor cells but also his hematopoietic system. However, HSCT is frequently accompanied by a prolonged phase of immunodeficiency, which results in an increased danger of acute bacterial and fungal infections and reactivation of latent viruses. This phase of immunodeficiency is largely due to slow recovery of the adaptive immune system and T cells in particular. Therefore, it is critical to better understand the molecular and cellular mechanisms of T cell development and regeneration to devise strategies to improve T cell reconstitution after HSCT.
T cell development occurs in the thymus, but this process is strictly dependent on the recruitment of progenitor cells from the bone marrow. We have previously characterized multiple progenitor cell populations that are able to enter the thymus and give rise to T cells. Furthermore, we have identified the chemokine receptors CCR7 and CCR9 as critical mediators of progenitor entry. Recruitment of progenitor cells to the thymus is tightly regulated, but molecular and cellular feedback mechanisms that restrict entry into the thymus remain poorly understood. Here, we plan to characterize the earliest phase of progenitor entry using a novel mouse model in combination with cellular bar coding to track the offspring of individual progenitor cells. These tools will allow us to directly determine the number of progenitors entering the thymus under conditions of health and disease, to separate entry into thymic tissue from early expansion phases, and to identify cellular and molecular feedback loops restricting progenitor entry. Furthermore, experiments proposed here will allow us to shed light on the highly controversial question, whether the thymic microenvironment is permissive for non-T cells to develop.
Taken together, the proposed experiments will foster our understanding of the earliest events of T cell development under both, physiologic and pathophysiologic conditions, and, thus, point toward new directions of improving T cell reconstitution after HSCT.
iRNA-mediated control of regulatory T cell development and function – a role for miR-181
The adaptive immune system is finely balanced to efficiently fight infectious pathogens on the one hand and to tolerate innocuous antigen as well as self-antigen on the other hand. Disruption of this balance may result in allergy or autoimmune disease. Regulatory T cells (Treg cells) constitute a major tolerogenic T population capable of suppressing an unwanted immune response, whose loss is accompanied by fatal autoimmunity. However, excessive action of Treg cells may also constitute an underlying cause of inefficient immune responses against cancer. Treg cells can be generated in the thymus (nTreg cells) or from naïve cells in the periphery (iTreg cells). The mechanisms controlling development of nTreg cells are only incompletely understood. It has been suggested that nTreg cells are essentially auto-reactive and, therefore, receive TCR signals of different strength and possibly duration. MicroRNAs (miRNAs) are small regulatory RNAs, which function primarily through post-transcriptional repression of a wide variety of target genes. Development and function of Treg cells critically depend on the presence of miRNA. However, the role of individual miRNAs in these processes remains elusive. We have recently shown that miR-181a/b-1 critically controls the generation of invariant natural killer T cells, which follow a very similar developmental pathway as Treg cells. Thus, we hypothesize that miR-181a/b-1 regulates development of nTreg cells. In order to test this hypothesis, we have generated mice deficient in miR-181a/b-1. Employing this novel mouse model we plan to address the following questions in this project: What are the mechanisms by which miR-181a/b-1 controls development of Treg cells? How does miR-181a/b-1 control Treg cell function? Finally, we will complement this proposal by an unbiased approach to identify novel miR-181 target genes. Taken together, this project will shed light on the molecular mechanisms of Treg cell development and function controlled by miRNA. In consequence, our studies might open up new avenues for targeted manipulation of Treg cells to restore the balance between tolerance and autoimmunity in disease.