Novel mechanisms of regulatory T cell mediated suppression: a fundamental role for VPS34

免疫系统为我们提供了终身的保护，使我们免受传染病的侵袭。被称为T细胞的特殊细胞既可以协调其他免疫细胞的活动，也可以直接杀死受感染的细胞。值得注意的是，需要一小部分T细胞，称为调节性T细胞(Tregs)来控制所有其他T细胞(常规T细胞，或Tcon)。虽然Tregs只占所有白细胞的1%左右，但没有它们我们就不能生存。因此，缺乏一种名为FOXP3的基因的功能性版本的儿童不会活超过两年，在这一点上，他们发育中的免疫系统对他们不利，导致一种被称为IPEX的致命综合症。FOXP3是Tregs的关键基因。缺乏FOXP3功能的小鼠也会在6周内死亡，因为它们的器官和皮肤和肠道中的微生物受到了不受限制的免疫攻击。仍然令人困惑的是，如此少的免疫细胞群如何对维持健康具有如此巨大的重要性。Tregs如何控制数量更多的Tcon的活动？我们发现，一种最初在酵母中发现的名为Vps34的酶对Tregs的功能是必不可少的。这是与Foxp3的重要区别，Foxp3是Tregs开发所必需的。因此，FOXP3基因缺陷的小鼠完全缺乏Tregs，而如果我们删除编码Vps34的基因，并且只在Tregs中，我们发现免疫相关器官中Tregs的数量正常，但小鼠仍然无法存活超过4-6周。正常情况下，小鼠可以存活2-3年。因此我们假设，通过更好地了解Vps34在Tregs中的作用，我们也可以了解更多关于Tregs是如何工作的，并回答哪些功能对于它们抑制Tcons的能力是绝对必要的这个问题。我们将使用几种不同的实验方法来解决这个问题。大部分工作将利用转基因小鼠模型，在该模型中，Vps34的基因只在Tregs中缺失。Tregs的作用机制一度被认为是通过消耗某些刺激性蛋白质分子，使它们无法用于Tcon。我们已经证实，缺乏Vps34的Treg仍然能够结合这些蛋白质并将它们内化。我们现在将关注这些蛋白质一旦被运送到特雷格体内后会发生什么。正常情况下，它们会被消化和降解，但我们怀疑这一过程在缺乏VPS34的Tregs中被破坏。我们也将使用只有大约一半Tregs缺乏VPS34的小鼠。这样的老鼠过着正常的寿命。我们将通过在皮肤下植入肿瘤细胞或将细菌感染这些小鼠来挑战这些小鼠。这将刺激Tcon和Tregs繁殖，并从静止状态变为高度激活状态。然后，我们可以在同一只小鼠中比较带有或不带有Vps34的Treg，并问：缺乏Vps34是否会干扰Treg被激活的能力，或许会达到它们的全部免疫抑制潜力。每个T细胞大约有6000-8000种不同的蛋白质。我们已经能够在正常和Vps34缺陷的Treg中测量这些蛋白质。因此，我们可以确定，如果没有Vps34，你最终会得到更多的某些蛋白质，而不是其他蛋白质。许多在缺乏Vps34的情况下发生变化的蛋白质参与了细胞新陈代谢(即调节Treg如何吸收和使用营养的过程)。我们将确定其中一些代谢活动是否会影响Treg的功能和抑制。我们相信，这些互补的方法将引导我们发现Treg功能的新方面，有朝一日可能会被用于治疗目的，例如通过设计增强或抑制Treg功能的药物，以及可以用于癌症或自身免疫性疾病的药物。

The immune system provides us with life-long protection against infectious agents. Specialised cells called T cells can both orchestrate the activities of other immune cells and kill infected cells directly. Remarkably, a small subset of T cells, called regulatory T cells (Tregs) are required to keep all the other T cells (conventional T cells, or Tcon) in check. Although Tregs comprise only about 1% of all the white blood cells, we cannot survive without them. Hence, children that lack a functional version of a gene called FOXP3 which is essential for Tregs do not live beyond two years, at which point their developing immune system turns against them, leading to a lethal syndrome known as IPEX. Mice that lack functional FOXP3 also die between 6 weeks because of an unrestrained immune attack against their organs and microorganisms in their skin and gut.What remains a puzzle is how such a small immune cell population can have such enormous importance for maintaining health. How do Tregs control the activity of the much more abundant Tcon? We have discovered that an enzyme called VPS34, originally identified in yeast, is essential for the function of Tregs. This is an important distinction from Foxp3, which is required for the development of Tregs. Therefore, FOXP3-deficient mice lack completely Tregs, whereas if we deleted the gene encoding VPS34, and that only in Tregs, we find normal numbers of Tregs in immune-related organs, but the mice nonetheless do not survive beyond 4-6 weeks. Normally, mice can live for 2-3 years. We therefore hypothesise that by gaining a better understanding of what VPS34 does in Tregs, we can also learn more about how Tregs work and answer the question about which function is absolutely essential for their ability to restrain Tcon.We will use several different experimental approaches to address this question. Most of the work will take advantage of a genetically modified mouse model in which the gene for VPS34 is deleted only in Tregs. Once mechanism through which Tregs are thought to work is by consuming certain stimulatory protein molecules so that they become unavailable for Tcon. We have already established that VPS34-deficient Tregs are still able to bind these proteins and internalise them. We will now follow what happens to these proteins once they have been transported inside the Tregs. Normally, they would be digested and degraded, but we suspect that this process is disrupted in Tregs lacking VPS34.We will also use mice in which only about half the Tregs lack VPS34. Such mice live a normal life span. We will challenge such mice by either implanting tumour cells under the skin or by infecting them with bacteria. This will stimulate both Tcon and Tregs to multiply and change from a resting state to a highly activated state. We can then compare Treg with or without VPS34 in the same mouse and ask: does whether lack of VPS34 interfere with the ability of Tregs to be activated and perhaps reach their full immune suppressive potential. Each T cell has about 6000-8000 different kinds of proteins. We have been able to measure these proteins in normal and VPS34-deficient Tregs. We can therefore determine whether without VPS34, you end up with more of some proteins, and less of others. Many of the proteins that are altered in absence of VPS34 are involved in cellular metabolism (i.e. processes that regulate how Tregs take up and use nutrients). We will determine if some of these metabolic activities affect Treg function and suppression. We believe that these complementary approaches will lead us to discover novel aspects of Treg function that might one day can be exploited for therapeutic purposes, for instance by designing drugs that either enhance or inhibit Treg function, and that could be used in the context of cancer or autoimmune diseases.