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

拟议的跨学科PHD项目将结合定量蛋白质组学、细胞信号传递和免疫学方法，以更好地了解T细胞在增殖和静止之间状态变化的基本生物学机制，这些机制目前尚不清楚。T淋巴细胞是探索信号转导途径调节和功能的重要生理相关系统，其优点是可以使用原代未转化的人类T细胞进行所有实验。因此，这项研究将提供有关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细胞介导的针对靶肿瘤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细胞的增殖能力。刺激的人T淋巴细胞进入细胞分裂周期，克隆地扩大效应细胞群，有效清除靶细胞。抗原与T细胞抗原受体/CD3复合体的结合以及伴随的辅助受体(例如CD28)和细胞因子(例如白介素2)的结合激活了分子信号级联反应(坎特雷尔2015)，最终调节核心细胞周期调控机制的表达。T细胞的增殖受Cyclin：CDK复合体的控制，它以一种时间调节的方式磷酸化蛋白质底物，并驱动细胞周期的进展。由于缺乏对关键蛋白质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。基于质谱学(MS)的蛋白质组学的进展使蛋白质及其翻译后修饰能够在蛋白质组范围内定量，包括跨细胞分裂周期(Ly 2014)。通过开发有针对性的MS分析，报告感兴趣的途径中关键蛋白质和重要的磷酸化位点的绝对丰度和化学计量学(MatSumoto 2017)，可以获得以高定量的方式快速和可靠地测量信号通路。该博士项目旨在开发一组基于MS的有针对性的T细胞激活分析和用于分析人类T细胞的细胞周期调节网络。该分析将通过设计脚本来设计，以挖掘和整合现有的多肽质量谱大型数据集(例如，ProteomeTools、SRMAtlas、Impaqt)。稳定同位素标记的多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DCBio合作进行验证，DCBio是一家总部位于邓迪的公司，专门从事质谱学合同研究。这个博士项目将提供a)在学术和工业环境中工作的机会，b)学习原代T细胞培养、荧光激活细胞分类和流式细胞术，c)在基于定量质谱学的蛋白质组学和大型多维数据集的分析方面进行培训，以及d)在制药公司的合同研究环境中应用蛋白质组学研究方面获得经验。该项目的一个重要目标是获得T细胞增殖控制的定量分子图谱，这将提高我们对与免疫治疗相关的T细胞生物学的基本理解。

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.