Visualizing regulatory T cell control of autoimmunity

越来越多的证据表明，CD4 CD25调节性T细胞(Treg)是免疫调节的重要组成部分，Treg功能的丧失可能是自身免疫的潜在原因之一。 然而，Tregs如何在体内抑制免疫反应仍不清楚。这可能是由于Tregs的频率很低，而且很难分析体内的反应。我们最近开发了一个强大的系统来恢复Tregs和控制非肥胖糖尿病小鼠的自身免疫性糖尿病，方法是在体外转移从BDC2.5TCR转基因小鼠中纯化的扩增的胰岛抗原特异性Tregs。我们假设Tregs既控制稳定状态的免疫动态平衡，又对炎症刺激做出反应，以抑制局部组织部位的免疫反应。新的成像技术提供了分析体内细胞活动的最直接和最不受操作的方法。然而，该领域面临着进入临床相关疾病模型以及建立各种成像观察与功能结果之间的因果关系的挑战。在我们的模型系统中，我们将使用新的体内成像技术直接分析Tregs在体内的稳态和炎症反应。 具体地说，我们将使用生物发光来监测在存在和不存在共转移的BDC Tregs的情况下，荧光素酶标记的糖尿病患者的CD4和CDS T细胞的淋巴归巢和组织转运。我们将使用双光子激光扫描显微镜分析糖尿病T细胞的启动和效应功能的发展，并确定BDC Tregs对LN和胰岛组织中这些活动的影响。我们将用双光子显微镜在体内直接观察Treg与抗原提呈细胞和效应T细胞的相互作用，以确定Treg的直接细胞靶点。最后，我们将使用这项新技术来研究免疫抑制分子IL-10、转化生长因子-β和CTLA-4的作用 并确定这些分子对糖尿病T细胞的归巢、运输、启动和效应器功能发展的影响。我们将结合图像分析和常规细胞免疫学方法、多色免疫荧光和免疫组织化学技术来精确定位Treg激活和功能的时间和位置。这些研究的结果将通过具体研究免疫调节和自我攻击之间的平衡，为我们理解自身免疫性疾病提供新的见解。

Accumulating evidence suggests that CD4+CD25+ regulatory T cells (Tregs) are an essential component of immune regulation, and loss of Treg functions may be one of the underlying causes of autoimmunity. However, how Tregs suppress immune responses in vivo remains unclear. This is likely due to the low frequency of Tregs and the difficulty in analyzing in vivo responses. We have recently developed a robust system to restore Tregs and control autoimmune diabetes in non-obese diabetic mice by transferring in vitro expanded islet antigen-specific Tregs purified from the BDC2.5 TCR transgenic mice. We hypothesize that Tregs both control immune homeostasis in steady state and respond to inflammatory stimuli to suppress immune responses at local tissue sites. New imaging technologies offer the most direct and unmanipulated ways to analyze in vivo cellular activities. However the field is facing the challenges of moving into clinically relevant disease models and establishing causal relationships between various imaging observations with functional outcomes. We will directly analyze both the steady-state and inflammatory responses of Tregs in vivo in our model system using novel in vivo imaging technologies. Specifically, we will use bioluminescence to monitor the lymph node homing and tissue trafficking of luciferase-tagged diabetogenic CD4 and CDS T cells in the presence and absence of co-transferred BDC Tregs. We will analyze the priming and effector function development of the diabetogenic T cells using two-photon laser scanning microscopy and determine the effect of BDC Tregs on these activities in both LN and islet tissues. We will directly visualize Treg interaction with antigen presenting cells and effector T cells in vivo with two-photon microscopy to determine the direct cellular target of the Tregs. Finally, we will examine the roles of immunosuppressive molecules IL-10, TGF- beta, and CTLA-4 using this novel technology and determine the effect of these molecules on the homing, trafficking, priming, and effector function development of the diabetogenic T cells. We will combine the imaging analysis with conventional cellular immunological approaches, multicolor immunofluorescence and immunohistochemistry techniques to pinpoint the time and location of Treg activation and function. The results of these studies will provide new insights into our understanding of autoimmune diseases by specifically studying the balance between immune regulation and auto-aggression