DMS/NIGMS 1: An Experimental Mathematical Framework for understanding and Controlling Regulatory Delay in Self- Nonself Determination in the Immune System

免疫系统如何区分自我和非我？我们的假设基于电路模拟，并得到数学模型的支持，即辅助细胞(Th)和调节性T细胞(Treg)反应之间的延迟允许免疫系统对Ag增加的速度做出反应，正是这种信号触发了更广泛的免疫反应。因此，这个项目的目标是提供确凿的实验证据来验证这一假设。具体地说，我们提出了一个创新的体内实验框架来分离和控制免疫反应的延迟，并使用这个框架来证明通过改变这种延迟，我们可以改变自我非自我决定。 该项目提出了一种新的实验框架，用于分离和控制免疫反应延迟的影响。显而易见的挑战是，实际上，不可能消除体内的延迟。因此，我们的方法不是消除Treg延迟，而是在Th反应中诱导延迟。不幸的是，这也是不切实际的，因为Th反应涉及无数的途径，并且无法在不改变Th反应的其他部分的情况下减缓这些途径。为了解决这个问题，我们分离了Th和Treg群体，并证明在提供给Th电路而不是Treg电路的疫苗计划(VS)中引入延迟可以用来否定甚至逆转PD反应-这意味着免疫决定可以逆转。这就产生了如何延迟Th电路的VS而不是Treg电路的VS的问题。我们的解决方案是在体内将这些电路去耦合(E2)。具体来说，我们使用的是免疫系统正常的供体小鼠(M0)。然后我们从M0中去除免疫系统(类似于骨髓移植)，使用流式细胞术从TH中分离Tregs，然后将Th细胞移植到小鼠H(MH)中，并将Treg细胞移植到小鼠R(MR)中，其中MH和MR都没有对Ag的预先存在的免疫反应。然后，我们使用由合作者Pi Acharya开发的微粒将VSS应用于MR和MH，但延迟了MH的VS。然后我们的电路模型预测，如果Th延迟足够大，VS对响应的影响将被否定或颠倒-验证了我们的假设。E2的一个关键部分是选择标记来对M0免疫系统进行排序。为了识别这些标记，我们使用了E1，它复制了E2，但只有一只小鼠。然后，我们使用PI Peet开发的流式细胞术和特征选择算法来识别要分类的目标。

How does the immune system differentiate self from nonself? Our hypothesis, based on circuit analogues, and supported by mathematical models, is that the delay between helper (Th) and regulatory T cell (Treg) response allows the immune system to respond to the rate of increase in Ag, and that it is this signal which triggers a broader immune response. The goal of this project, then, is to provide definitive experimental evidence verifying this hypothesis. Specifically, we propose an innovative in vivo experimental framework for isolating and controlling delay in immune response and use this framework to show that by changing this delay, we can alter the self-nonself determination. This project proposes a novel experimental framework for isolating and controlling the effect of delay in immune response. The obvious challenge is that, practically, it is not possible to eliminate delay in vivo. Our approach, then, is not to eliminate Treg delay, but rather to induce delay in the Th response. Unfortunately, this too is impractical, due to the myriad of pathways involved in Th response and inability to slow down these pathways without altering other parts of the Th response. To address this, we isolate the Th and Treg populations, and prove that introducing delay in the Vaccine Schedule (VS) presented to the Th circuit, but not to the Treg circuit, can be used to negate or even reverse the PD response - implying that immune decisions can be reversed. This presents the problem of how to delay the VS for the Th circuit, but not the Treg circuit. Our solution is to decouple these circuits in vivo (E2). Specifically, we use a donor mouse (M0) with a normal immune system. We then remove the immune system from M0 (similar to a bone-marrow transplant), use flow cytometry to separate Tregs from Ths, and then transplant the Th cells to Mouse H (MH) and the Treg cells to mouse R (MR), where neither MH nor MR have a pre-existing immune response to Ag. We then use microparticles developed by co-PI Acharya to apply the VSs to MR and MH, but delaying the VS for MH. Our circuit model then predicts that the effect of VS on response will be negated or reversed if Th delay is sufficiently large - verifying our hypothesis. A critical part of E2 is selecting markers by which to sort the M0 immune system. To identify these markers, we use E1, which replicates E2, but with a single mouse. We then use flow cytometry and feature selection algorithms developed by PI Peet to identify targets for sorting.