Multicoloured bimodality biophotonic imaging for in vivo non-invasive analysis of IL-17 and IFN gamma immune effector programmes

用于 IL-17 和 IFN γ 免疫效应程序体内非侵入性分析的多色双峰生物光子成像

• 批准号: BB/H005439/1
• 负责人: Rosemary Boyton
• 金额: $ 71.54万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH005439%2F1
• 关键词: Multicoloured; bimodality; biophotonic; imaging; vivo

Recent advances in microscopy have opened up new avenues for analysing biological systems. Cellular events in the body that used to be deduced from test-tube experiments or from 'snapshots' looking at tissue sections, can now be visualised directly in life in real-time. One such technique, bioluminescence, is particularly appealing because it can be used to gain information about cellular populations in a lab mouse with sensitivity and specificity, without any surgical procedure. Because the technique is rapid, non-invasive, stress-free and gives results with small numbers, it is considered an advance for laboratory animal welfare. The approach relies on the fact that there are natural proteins produced by species such as fireflies, click beetles, and some bacteria that naturally luminesce as part of a normal chemical reaction. This light is so bright, it can be detected within the cells of the living mouse, by a special camera and a digital image taken and studied. The proteins are expressed in mouse cells in the form of a transgene - that is, the DNA for a protein is incorporated into the mouse genome. Information can be attached to the gene directing it to be turned on only in one particular cell-type or another. These 'in-vivo' reporters are fundamentally different from past approaches in which a particular gene has been hooked onto a fluorescent protein that can only be seen once the mouse has been killed and its cells removed. The new approach allows images to be seen in terms of where particular cell-types are to be found in a live mouse. The specific biological question that concerns us is how the white blood cells central to regulating and orchestrating immunity, called CD4 T cells, make choices about which type of CD4 cell they will be. These effector cells make different chemicals called cytokines, associated with very different types of immune response. Two of interest are interferon-gamma and IL-17. Key producers of these are termed Th1 and Th17 cells, respectively. However, most of the information about those cell-types derives from highly manipulated systems, usually in the test tube. We would like to be able to visualise the cells directly in the live mouse in real time in the context of different immune responses. We describe in this application a number of technical steps required to achieve this goal. The major objective is to create and optimise a system that will allow us to study 2 different cytokine responses simultaneously, each labelled with a different colour. We will use a 3rd colour allowing us to image the bacteria to which the immune response is directed. In order to do this we will make different transgenic mouse lines in which the different colours are turned on, thus reporting the different cytokines. Because there is a certain amount of uncharted territory in deciding precisely which and how much DNA sequence would be needed to achieve an accurate representation of, for example, IL-17 activation, we have reduced the risk by building our strategy on the back of a proven, successful, large, construct produced by a group in Germany who were analyzing fluorescent protein (rather then luciferase) detection. We describe a series of experiments necessary to validate this system. The key point will be to apply these mice to debated aspects of immunity where we do not understand the relationship between interferon-gamma and IL-17 based responses. In some cases it is thought that Th17 cells may change into Th1 cells; using these mice we will be able to define where, when and under what conditions this occurs. In general, we can then use this system to describe cellular interactions of these immune subsets in immunity and infection. The generation of this experimental system creates a resource that can be used by the immunological community and, more widely, will reinforce the principle of multi-coloured imaging as a way for biologists to view interactions between multiple cell types.

显微镜的最新进展为分析生物系统开辟了新的途径。体内的细胞活动过去是从试管实验或观察组织切片的“快照”中推断出来的，现在可以直接在生活中实时可视化。其中一种技术，生物发光，特别有吸引力，因为它可以用于敏感性和特异性地获得有关实验室小鼠细胞群体的信息，而无需任何外科手术。由于该技术快速，无创，无压力，并且结果数量少，因此被认为是实验室动物福利的进步。这种方法依赖于这样一个事实，即萤火虫，点击甲虫和一些细菌等物种产生的天然蛋白质作为正常化学反应的一部分自然发光。这种光是如此明亮，它可以在活老鼠的细胞内被检测到，通过一个特殊的相机和一个数字图像拍摄和研究。这些蛋白质以转基因的形式在小鼠细胞中表达，也就是说，蛋白质的DNA被整合到小鼠基因组中。信息可以附着在基因上，指导它只在一种特定的细胞类型或另一种细胞类型中被打开。这些“体内”报告者与过去的方法有着根本的不同，在过去的方法中，一个特定的基因被钩在一个荧光蛋白上，只有在小鼠被杀死并取出其细胞后才能看到。这种新方法允许在活体小鼠中发现特定细胞类型的图像。我们关注的具体生物学问题是，对调节和协调免疫至关重要的白色血细胞（称为CD 4 T细胞）如何选择它们将成为哪种类型的CD 4细胞。这些效应细胞产生不同的化学物质，称为细胞因子，与非常不同类型的免疫反应有关。两个感兴趣的是干扰素-γ和IL-17。这些细胞的主要生产者分别被称为Th 1和Th 17细胞。然而，关于这些细胞类型的大多数信息来自高度操纵的系统，通常在试管中。我们希望能够在不同免疫反应的背景下，在真实的时间内直接在活小鼠中可视化细胞。我们在本申请中描述了实现该目标所需的多个技术步骤。主要目标是创建和优化一个系统，使我们能够同时研究2种不同的细胞因子反应，每种反应都用不同的颜色标记。我们将使用第三种颜色，使我们能够对免疫反应所针对的细菌进行成像。为了做到这一点，我们将制作不同的转基因小鼠品系，其中不同的颜色被打开，从而报告不同的细胞因子。因为在精确决定需要哪种DNA序列以及需要多少DNA序列来实现IL-17激活的准确表达方面存在一定的未知领域，所以我们通过建立在德国的一个小组生产的经过验证的、成功的、大型的构建体的基础上来降低风险，该小组正在分析荧光蛋白（而不是荧光素酶）检测。我们描述了一系列必要的实验来验证这个系统。关键是将这些小鼠应用于免疫性的争论方面，我们不了解干扰素-γ和IL-17反应之间的关系。在某些情况下，人们认为Th 17细胞可能会转变为Th 1细胞;使用这些小鼠，我们将能够定义这种情况在何时、何地以及在什么条件下发生。一般来说，我们可以使用这个系统来描述这些免疫亚群在免疫和感染中的细胞相互作用。这个实验系统的产生创造了一个可以被免疫学界使用的资源，更广泛地说，将加强多色成像的原理，作为生物学家观察多种细胞类型之间相互作用的一种方式。

项目成果

Post-acute COVID-19 associated with evidence of bystander T-cell activation and a recurring antibiotic-resistant bacterial pneumonia.

• DOI: 10.7554/elife.63430
• 发表时间: 2020-12-17
• 期刊: eLife
• 影响因子: 7.7
• 作者: Gregorova M;Morse D;Brignoli T;Steventon J;Hamilton F;Albur M;Arnold D;Thomas M;Halliday A;Baum H;Rice C;Avison MB;Davidson AD;Santopaolo M;Oliver E;Goenka A;Finn A;Wooldridge L;Amulic B;Boyton RJ;Altmann DM;Butler DK;McMurray C;Stockton J;Nicholls S;Cooper C;Loman N;Cox MJ;Rivino L;Massey RC
• 通讯作者: Massey RC

Elongated TCR alpha chain CDR3 favors an altered CD4 cytokine profile.

延长的 TCR α 链 CDR3 有利于改变 CD4 细胞因子谱。

• DOI: 10.1186/1741-7007-12-32
• 发表时间: 2014
• 期刊: BMC biology
• 影响因子: 5.4
• 作者: Reynolds C