Testing the Mechanism of T lymphocyte selection in the thymus mediated by the zfp36 family of RNA binding proteins

The immune system is made up of many different types of cell. One cell type called the T cell is a sensitive sensory system that can tell the difference between our own healthy cells and microorganisms or cancerous cells. Essential functions of the T cells are to coordinate the activity of other immune cells, such as cells that produce antibodies, and to form a memory of previous infections. This property is important for the function of vaccines. The absence of T cells leads to a serious medical problem called immunodeficiency. When T cells fail to discriminate properly healthy from unhealthy cells and become "autoreactive" life-threatening and chronic diseases can develop such as MS or type I diabetes. For these reasons understanding how T cells develop throughout the lifecourse is an important question. A significant body of work from many laboratories over several decades has identified cell surface receptors, mechanisms of signalling by those receptors and gene switches (called transcription factors) that control this process. Our research now identifies a new class of regulator of T cell development and selection that acts to control gene expression by controlling a process known as RNA decay. The goals of our research are to understand how this new class or regulator functions. We will study the process of thymocyte development using state of the art genetic and molecular biology approaches. We expect to understand the molecular mechanism by which this new class or regulator acts and to be able to interpret this within the context of the body of knowledge that already exists. The knowledge gained may enable new approaches to autoimmunity or immunodeficiency to be devised. This project will also provide an excellent training opportunity for an early stage researcher in basic molecular and cellular immunology.

A significant body of unpublished data form the applicant's laboratory indicate that RNA binding proteins (RBP) of the zfp36 family play a role in thymic development and selection. We want to find the mechanism through which these RBP function. We will use well-characterised TCR transgenes which will allow lineage choice to be monitored. Importantly, the TCRs chosen will enable experiments to be performed using peptide MHC stimulation delivered by APC or tetramers and for the OT1 TCR there are a set of peptides of varying affinity which mediate positive and negative selection of thymocytes. These peptide MHCs will also allow signal transduction to be tested in a variety of assays such as imaging calcium flux or the phosphorylation of specific pathway components. The role of cell death will be examined by using a bim knockout system that will rescue cells dying from neglect and negative selection. Alternative/additional hypothesis including altered cytokine signalling can be tested using antibody based approaches for probing signal transduction (e.g. intracellular flow). The use of a GFP reporter transgene (GFP-Nur77) which acts as a reporter of TCR signal strength will allow us to examine how the signal strength of this receptor changes during development and in the absence of the RBP.

This class of RBP is able to bind to the ARE which is found in the 3'UTR of10% of mRNAs. Bioinformatics predictions based on sequence features alone have failed to identify the consequential targets of these RBP making it essential to define direct targets through empirical measurements. To do this we will use the iCLIP method which is well-established in the Turner lab. We will then discover how direct and indirect targets are changed in abundance using RNAseq data.

Overall, this project will allow the integration of an important new layer of control of gene expression into our understanding of the molecular regulation of thymocyte development.

The research we have conducted into the biology of RNA binding proteins is opening up a new area of molecular immunology. We have been taking a variety of approaches to develop the importance of this area. These include publishing high quality basic science papers; writing reviews and presenting the work at meetings and workshops whenever the opportunity will arise. Through mentoring we are developing the careers of a new generation of scientists who see the importance of this area of regulation. A pertinent example is Dr Dan Hodson who did his PhD in my lab and is now an MRC-funded clinical research fellow. This research area is starting to interest the pharmaceutical industry as it has potential to reveal drug targets in both cancer and inflammation/allergy. Through understanding the basic molecular mechanisms of differentiation we may identify new ways of manipulating these pathways.

The project provides an excellent training opportunity for Dr Katharina Vogel. This stems from the opportunity to delve deeply into the molecular mechanisms of T cell development and differentiation-a topic that is at the forefront of immunology. Furthermore, she will train in advanced molecular biology and bioinformatics approaches, skills that are essential to develop in order to further her career and to maximise her impact in science in the research. By combining her in vivo skills with molecular and bioinformatics know-how she will be well placed to undertake research in any leading university or commercial entity. Dr Vogel will also have the opportunity to further her personal and professional development, in partnership with the University of Cambridge and the Babraham Institute. Careers advice and guidance is available, in addition to on-site training courses in Bioinformatics and Flow Cytometry run by BI facility heads, as well as training in presentation making, grant writing skills and public engagement.