The proposed interdisciplinary PhD project will combine quantitative proteomics, cell signalling, and immunological methods to better understand basic biological mechanisms underlying the state change between proliferation and quiescence in T cells which are currently not well understood. T lymphocytes are an important physiological relevant system for exploring the regulation and function of signal transduction pathways and have the advantage that it is possible to use primary non-transformed human T cells for all experiments. This study will thus provide fundamental information about the processes that regulate proliferation and differentiation in human cells. Moreover, the data will be of interest to the pharmaceutical industry as reshaping T cell mediated immune responses to target cancer neoantigens has revolutionised cancer treatment and a key challenge in any T cell therapy is the maintenance of T cell proliferative competency.
Stimulated human T lymphocytes enter the cell division cycle to clonally expand effector cell populations for effective clearance of target cells. Antigen binding to the T-cell antigen receptor/CD3 complex and concomitant engagement of co-receptors (e.g. CD28) and cytokines (e.g. Interleukin 2) activates molecular signalling cascades (Cantrell 2015) that ultimately regulates expression of the core cell cycle regulatory machinery. T cell proliferation is controlled by Cyclin:CDK complexes which phosphorylate protein substrates in a temporally regulated manner and drive cell cycle progression. Understanding the molecular thresholds for cell cycle entry has been stymied by lack of quantitative measurements of key proteins and phosphorylation events.
Advances in mass spectrometry-based (MS) proteomics has enabled the proteome-wide quantitation of proteins and their post-translational modifications, including across the cell division cycle (Ly 2014). Rapid and robust measurement of signalling pathways in a highly quantitative manner can be obtained by developing targeted MS assays that report on the absolute abundance and stoichiometries of key proteins and important sites of phosphorylation in a pathway of interest (Matsumoto 2017).
This PhD project aims to develop a panel of targeted, MS-based assays for T cell activation and the cell cycle regulatory network for the analysis of human T cells. The assay will be designed by designing scripts to mine and integrate existing large datasets of peptide mass spectra (e.g. proteomeTools, SRMAtlas, iMPAQT). Stable isotope labelled peptides will be employed as internal standards for absolute quantitation of key signalling events and as retention time calibration standards and validated in collaboration with DCBio, a Dundee based company that specialises in mass spectrometry contract research. The assays will be benchmarked on state of the art MS instrumentation.
This PhD project will provide opportunities to a) work in both academic and industry settings, b) learn primary T cell culture, fluorescence-activated cell sorting, and flow cytometry, and c) train in quantitative mass spectrometry-based proteomics and in the analysis of large, multidimensional datasets, and d) gain experiences in applying proteomics research in a contract research environment for pharma. An important aim of the project will be to obtain quantitative molecular map of T cell proliferation control, which will improve our basic understanding of T cell biology relevant to immunotherapy.