(BFH) Development of robust, quantitative mass spectrometry assays to investigate regulation of human T cell growth and proliferation

拟议的跨学科博士项目将结合定量蛋白质组学，细胞信号传导和免疫学方法，以更好地了解目前尚不清楚的T细胞增殖和静止之间状态变化的基本生物学机制。T淋巴细胞是探索信号转导通路调控和功能的重要生理相关系统，其优势在于可以使用原代未转化的人T细胞进行所有实验。因此，这项研究将提供关于调节人类细胞增殖和分化过程的基本信息。此外，这些数据将引起制药行业的兴趣，因为重塑T细胞介导的针对癌症新抗原的免疫反应已经彻底改变了癌症治疗，而任何T细胞治疗的关键挑战是维持T细胞增殖能力。受刺激的人T淋巴细胞进入细胞分裂周期，克隆扩增效应细胞群，有效清除靶细胞。抗原与t细胞抗原受体/CD3复合物结合，并伴随共受体（如CD28）和细胞因子（如白细胞介素2）的结合，激活分子信号级联反应（Cantrell 2015），最终调节核心细胞周期调节机制的表达。T细胞增殖受细胞周期蛋白：CDK复合物控制，该复合物以暂时调节的方式磷酸化蛋白底物并驱动细胞周期进程。由于缺乏关键蛋白和磷酸化事件的定量测量，对细胞周期进入的分子阈值的理解一直受到阻碍。基于质谱（MS）的蛋白质组学的进步使蛋白质组及其翻译后修饰（包括整个细胞分裂周期）的蛋白质组级定量成为可能（Ly 2014）。通过开发靶向质谱分析，可以以高度定量的方式快速可靠地测量信号通路，报告感兴趣通路中关键蛋白质和重要磷酸化位点的绝对丰度和化学计量学（Matsumoto 2017）。该博士项目旨在开发一组针对T细胞活化和细胞周期调控网络的靶向，基于ms的分析人类T细胞。该分析将通过设计脚本来设计，以挖掘和整合现有的大型肽质谱数据集（例如proteomeTools， SRMAtlas, iMPAQT）。稳定同位素标记的肽将被用作关键信号事件绝对定量的内部标准和保留时间校准标准，并与位于邓迪的DCBio公司合作进行验证，DCBio公司专门从事质谱合同研究。检测将以最先进的质谱仪器为基准。该博士项目将为以下人员提供机会：a)在学术和行业环境中工作；b)学习原代T细胞培养、荧光活化细胞分选和流式细胞术；c)在基于定量质谱的蛋白质组学和大型多维数据集分析方面进行培训；d)获得在制药合同研究环境中应用蛋白质组学研究的经验。该项目的一个重要目标将是获得T细胞增殖控制的定量分子图谱，这将提高我们对免疫治疗相关T细胞生物学的基本认识。

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.