CD96 a new immunoregulatory molecule - molecular mechanisms

背景特殊的白色血细胞，称为调节性T细胞（T细胞），通常控制免疫系统：过多的控制会导致癌症的发生，而过少的调节会导致自身免疫。细胞表面的特殊分子，称为辅助受体，可以介导和改变Treg功能。共享相同配体的辅助受体可以触发免疫应答的增加或减少。这些辅助受体似乎是阻止癌症和/或自身免疫的良好药物靶标，其中一些已经在临床上使用（CTLA-4，PD 1），其他正在开发中。其中一个调节免疫系统的辅助受体是CD 96，我们想知道它是如何工作的。CD 96与共同受体CD 226和TIGIT共享其配体CD 155，共同受体CD 226提高免疫应答，TIGIT降低免疫应答。T细胞表达所有三种辅助受体，但我们还不知道它们如何决定Treg功能以及CD 96如何确切地调节整个途径。假设和目的我们认为CD 96通过控制同一家族中其他辅助受体的配体CD 155来调节免疫应答，我们认为这影响了T细胞的工作方式。我们将在我们的目的中用三个主要问题来测试这个整体假设：目的1：CD 96是如何主动将CD 155带入细胞的？目的2：细胞内还有哪些分子与CD 96共同发挥作用？目的3：CD 96如何改变Treg功能？实验计划我们将使用持续生长的细胞系模型以及来自健康人血液的细胞，我们可以修改这些细胞以仅表达特定的辅助受体。我们有一些数据表明，CD 96通过从其他细胞中获取CD 155来发挥作用。我们将产生表达带有荧光标记的CD 96和CD 155的细胞系，当特定的光照射在它们上时，它们会发光。这样，我们就可以看到并跟踪CD 96和CD 155如何相互作用，并通过使用强大的共聚焦显微镜，流式细胞术来同时评估每个细胞的许多参数，以及我们可以提取CD 96和与其结合的蛋白质的生化测定，然后测量它们。通过在功能测试之前改变CD 96的部分，我们可以发现CD 96的哪个部分对其功能很重要。我们还将研究CD 96如何与CD 155竞争结合和/或与其他共受体家族成员CD 226/TIGIT相互作用。我们将寻找在细胞内与CD 96结合后与其结合的蛋白质，以使CD 96能够发挥作用。我们有一些可能与细胞内的CD 96结合的候选蛋白，但我们也将在细胞内与其配体CD 155结合后筛选与CD 96相互作用的新蛋白。我们将使用CRISPR-Cas9等尖端技术从健康的人类T细胞中去除CD 96（CD 96敲除），以找出CD 96对细胞功能的必要性。我们将测试这种敲除是如何改变TcB的激活、增殖和信号传导的。Treg可以抑制其他T细胞分裂，因此我们还将测试CD 96是否影响这种关键的Treg功能。由于效应T细胞也可以表达CD 96，我们将平行测试它们，以寻找Treg特异性的CD 96作用和表达CD 96的细胞的通用作用。免疫系统在健康状态下受到严格的调节，但过多的控制会导致癌症，而过少的控制会导致自身免疫。通过了解辅助受体CD 96如何调节免疫系统以及它如何影响Treg功能，我们将能够在未来利用这些知识来设计靶向辅助受体的新药来治疗癌症和/或自身免疫。

Background Special white blood cells, called regulatory T cells (Tregs) normally keep the immune system in check: too much control lets cancer arise, while too little regulation leads to autoimmunity. Special molecules on the surface of cells, called co-receptors, can mediate and alter Treg function. Co-receptors that share the same ligand can trigger either an increase or decrease in the immune response. These co-receptors appear to be good drug targets to stop cancer and/or autoimmunity with some already in use clinically (CTLA-4, PD1), and others being developed. One such co-receptor that does regulate the immune system is CD96, and we want to find out how it works. CD96 shares its ligand CD155 with the co-receptors CD226, which turns the immune response up, and TIGIT, which lowers the immune response. Tregs do express all three co-receptors, but we do not yet know how they dictate Treg function and how exactly CD96 regulates the whole pathway.Hypothesis and AimsWe believe CD96 regulates the immune responses by controlling the ligand CD155 for the other co-receptors in the same family and we think this affects how Tregs work.We will test this overall hypothesis with three main questions in our aims:Aim 1: How does CD96 actively take CD155 into the cell?Aim 2: What other molecules inside the cell work with CD96 for its action?Aim 3: How does CD96 change Treg functions?Experimental PlanWe will use continuously growing cell line models as well as cells from the blood of healthy people that we can modify to only express specific co-receptors. We have some data that suggests that CD96 works by taking CD155 from other cells. We will generate cell lines that express CD96 and CD155 with fluorescent tags that glow when specific light is shone on them. That way we can see and follow how CD96 and CD155 interact and move around inside the cells by using a powerful confocal microscope, flow cytometry to evaluate many parameters on each cell at once and biochemical assays where we can pull out CD96 and proteins bound to it and then measure them. By changing parts of CD96 before functional tests, we can find out which part of CD96 is important for its functions. We will also look at how CD96 competes for CD155 binding and/or interacts with the other co-receptor family members CD226/TIGIT.We will look for the proteins that bind to CD96 inside the cell after it has bound ligand to enable CD96 actions. We have some candidates that likely bind to CD96 inside the cell, but we will also screen for new proteins interacting with CD96 inside the cell after it has seen its ligand CD155. We will use cutting edge techniques like CRISPR-Cas9 to take away CD96 (CD96 knockout) from healthy human Tregs to find out for what cell functions CD96 is necessary. We will test how this knockout changes the activation, proliferation and signalling of Tregs. Tregs can supress other T cells from dividing, thus we will test also whether CD96 influences this crucial Treg function. Since effector T cells can also express CD96, we will test them in parallel to look for CD96 actions that are Treg-specific and ones that are universal for cells expressing CD96. Importance The immune system is tightly regulated in health, but too much control leads to cancer and too little to autoimmunity. By understanding how the co-receptor CD96 regulates the immune system and how it affects Treg functions, we will be able to harness that knowledge in the future to design new drugs targeting the co-receptor to treat cancer and/or autoimmunity.