Dissecting the molecular mechanisms of PI3K in Extra-Follicular Helper and Regulatory T cell differentiation

The mechanisms by which infections elicit antibody responses have been recognised as one of fundamental importance for over a century. We now understand that many different cell types collaborate to enable antibody formation, and for the generation of immunological memory - the underpinning principle of vaccine efficacy.

Antibodies are produced by specialised cells called antibody secreting plasma cells (ASC). These develop from cells called B lymphocytes. The process of development is promoted by another type of cell called the helper T lymphocyte and inhibited by a third type of cell called the regulatory T lymphocyte. Other cell types are involved, but the balance between the helper and the regulatory T cell is the ultimate determinant of the response.

During infection by salmonella bacteria a robust antibody response is generated by ASC and both helper and regulatory T cells play a role here. The frequency and potency of the helper and regulatory T cells response is mediated by signalling pathways that arise from receptors on the T lymphocyte cell surface. One component of this signalling process is an enzyme called phosphatidylinositol 3-kinase (PI3K)-specifically the gene encoding the p110d subunit. This enzyme controls many processes in cells and acts a coordinator to regulate cell number and potency.

The goal of our research project is to understand the basic molecular mechanisms by which PI3K achieves this. We have already identified one pathway that PI3K inhibits in order to promote helper T cell function and we want to gain a more detailed understanding of which components of this pathway PI3K acts on. We have identified additional molecules that are good candidates for regulation by PI3K and we want to confirm these and identify if and how they join up with the pathways we think are important. Finally, we want to test a new molecular mechanism that we believe will be important for helper T cells to function well. To do this we will employ novel technology that has never been used on T lymphocytes. This new technology will provide insight into how genes that are encoded in the DNA are converted into proteins that makes cells work.

Antibody responses elicited by salmonella infection are governed by T cells which, by analogy to the T cells that control the magnitude of the germinal centre reaction, can be called extrafollicular helper- and extra-follicular regulatory-T cells. The development, and possibly function, of these cells is governed by signalling through the enzyme phosphatidylinositol 3-kinase delta (P110d) which acts in a cell autonomous manner to control the number of these cells. We propose to use a combination of mouse genetics and molecular biology approaches to establish the mechanism through which P110d works.
We will make use of a conditional allele of the gene encoding p110d targeted to T cells to investigate the differentiation phenotype of T cells elicited by salmonella infection. This is a powerful toll for revealing the cell autonomous functions of P110d in T cell differentiation. We already have good data suggesting a hypothetical mechanism by which P110d promotes helper T cell differentiation. This hypothesis is that P110d attenuates signalling through the IL-2 pathway which is inhibitory for helper T cell function.

This will include the introduction of genetic haplo-insufficiencies in pathway components we have identified to be overactive in the absence of P110d. We will test the role of new signalling connections, novel cell surface receptors and transcription factors in helper T cell generation. In addition, we will explore the role of mRNA translation in helper T cell generation since, until recently, this has been neglected as a potentially important mechanism. This latter approach will involve the application of the cutting edge technology of ribosome profiling to primary lymphocytes. We show that we have the capability to achieve this with preliminary data. The identification of the translationally regulated mRNAs during differentiation will then lead to an investigation of their sequence features that point towards molecular mechanisms.

The research we have conducted into the biology of PI3K in general, and P110delta in particular, has been of interest to the pharmaceutical industry as it is a drug target in both cancer and inflammation/allergy. For example, recent work from colleagues at Babraham (Klaus Okkenhaug group) and their clinical collaborators has discovered mutations in PI3K that lead to a primary immunodeficiency disease, APDS, further raising the impact of research in the field. FDA approval has been granted for the small molecule delta inhibitor (Zydelig) to be used in B cell leukaemia and lymphoma. Companies in the UK that are developing PI3K inhibitors include both large mutli-nationals (GSK) and SMEs (Karus Therapeutics Ltd). These companies will be interested in understanding the biological function of PI3K in the whole animal setting, as will the regulatory authorities. Ultimately, the development of safe and efficacious medicines will benefit society as a whole. Our research on PI3K may provide new indications of where modulation of this pathway will be of use in a clinical setting. Given the progress of this field these benefits could be realised within a decade.

Our research into antibody production will provide insights into how to improve the nature of vaccines and adjuvants. We would argue from our data that one route to this goal is to increase PI3K signalling in T cells, albeit transiently and within certain (as yet unclear) limits.

Although we are using salmonella as a model antigen, the potential exists within our study to learn more about this economically important microorganism and its interactions with the host. In an era of increasing resistance to antibiotics any insights into the fundamental mechanisms of immunity could be extremely beneficial to the general public and healthcare providers, as boosting immunity is one obvious way to reduce antibiotic use.

The project provides an excellent training opportunity for Dr Ram Venigalla. This stems from the opportunity to delve deeply into the molecular mechanisms of helper T cell differentiation - a topic that is at the forefront of immunology. Furthermore, he will train in advanced molecular biology and bioinformatics approaches, skills that are essential to develop in order to further his career and to maximise his impact in science in the research. By combining his in vivo skills with molecular and bioinformatics know-how he will be well placed to undertake research in any leading university or commercial entity. Ram will also have the opportunity to further his 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.