GENE REGULATORY EVENTS IN ESTABLISHING MATURE T CELL TOLERANCE

现代免疫学的基本问题之一是控制CD 4+辅助性T细胞分化为具有离散效应子功能的亚群。 特别感兴趣的是这些亚群如何相互作用以促进强免疫力和终身稳定的免疫耐受。 各种研究表明，目前至少有五个定义明确的子集：Th 0，Th 1，Th 2，Treg，Th 17和T滤泡辅助细胞，它们可以促进和抑制彼此的行为。 最近，大量的实验注意力已经指向表达高水平的翼叉头转录因子FoxP 3的CD 4+、CD 25 + T细胞亚群。 该子集严格限制其自身增殖细胞因子的表达，并且通常无法产生生长细胞因子，例如IL-2和IL-4。 这些细胞已被证明会损害其他常规CD 4 + T细胞的激活，当细胞非常接近时，这在自身免疫反应中可能很重要;因此这些细胞被称为T调节（Treg）细胞。 尽管进行了大量的研究，但仍不清楚Treg细胞能够抑制其他T细胞亚群的分子机制以及它们在正常免疫反应中发挥的作用。为了研究Tcl 4抑制作用的分子基础，我们研究了共培养系统，其中Tcl 4用于抑制响应于T细胞受体激动剂的常规CD 4 T细胞。 我们发现，1：1混合的细胞可以观察到有效的抑制。 在这些条件下，我们观察到常规T细胞发生凋亡。 此外，我们发现抑制IL-2基因转录的流行模型是不正确的。应答细胞的死亡可以定量地解释T细胞应答的丧失。我们发现各种常见的γ链细胞因子能够完全逆转由于Treg抑制引起的死亡。 这似乎是由于T细胞消耗但不能产生细胞因子，从而剥夺了常规T细胞的细胞因子。 结合这些实验，我们发现Bim基因的缺陷完全拯救了Treg抑制的T细胞。我们还证明了在炎症性肠病模型中可以在体内观察到类似的凋亡和T细胞缺失效应。 综上所述，这些数据表明Treg细胞通过多克隆缺失的形式而不是通过抑制应答常规T细胞中的细胞因子转录来发挥其抑制作用。 我们还发现Treg细胞表达γ链细胞因子的受体，并且依赖于这些细胞因子的外源性供应来克服体外细胞因子戒断凋亡。该结果通过Treg细胞在Bim-/-和Bcl-2 tg小鼠中的积累在体内得到验证，所述Treg细胞已经阻止了细胞因子剥夺凋亡。我们还发现，CD 25和Foxp 3的表达下调，在这些细胞因子的情况下。来自Scurfy小鼠的CD 25+细胞的存活不依赖于细胞因子，这表明Foxp 3通过抑制Treg细胞中的细胞因子产生来增加它们对细胞因子的依赖性。最近，我们一直在研究Treg细胞如何与Th 17相互作用。 我们已经发现，这些相互作用与Treg与Th 1或Th 2细胞之间的相互作用之间存在根本差异。 我们的研究表明，Treg细胞的存活严格依赖于细胞因子和细胞因子产生细胞，因为它们不产生细胞因子。 在另一系列研究中，我们研究了基因调控如何控制无反应性的免疫耐受机制。 我们已经开始了我们的调查研究，在一个关键的调节途径，涉及活化T细胞的核因子（NF-AT），是负责无反应诱导基因的表达：grail和caspase-3。 我们偶然发现，在免疫应答过程中，有一种主要的支架蛋白协调NF-AT的核转位。有趣的是，这种相同的支架蛋白与某些神经退行性疾病有关。因此，我们的工作可能会产生意想不到的结果，将异常免疫反应与某些神经系统疾病的耐受失败联系起来。此外，这些实验可能提供深入了解的分子调控事件，是至关重要的耐受性，特别是无反应性。 我们的观察为T细胞的这种调节过程提供了新的线索，我们现在正试图开发一种T细胞亚群如何实现耐受性和免疫性的通用模型。 这些研究可能对自身免疫性疾病、感染性疾病和肿瘤免疫具有重要意义。 我们还通过参与来自随机乙基亚硝基脲项目的小鼠的分析研究了T淋巴细胞的发育和成熟。 在合作研究中，我们发现Themis蛋白在CD 4淋巴细胞发育中的作用和DOCK 8蛋白在免疫能力中的作用。 这些研究显示了随机诱变研究对于检测淋巴细胞中表达的基因的新功能的价值。

One of the fundamental questions of modern immunology is the control of CD4+ helper T-cell differentiation into subsets with discrete effector functions. Of special interest is how the subsets interact with each other to promote strong immunity and lifelong stable immunological tolerance. A variety of investigations show that there are at this time at least five well-defined subsets: Th0, Th1, Th2, Treg, Th17, and T follicular helper that can both promote and inhibit the behavior of each other. Recently, a great deal of experimental attention has been directed at the subset of CD4+,CD25+ T cells that express high levels of the winged fork head transcription factor, FoxP3. This subset strictly limits their own expression of proliferative cytokines and is generally incapable of producing growth cytokines such as IL-2 and IL-4. These cells have been shown to impair the activation of other conventional CD4+ T cells when the cells are in close proximity which could be important during autoimmune reactions; hence these cells are called T regulatory (Treg) cells. Despite a great deal of investigation, it is still unclear by what molecular mechanisms Treg cells are capable of suppressing other subsets of T cells and what role they play in normal immune reactions. In order to examine the molecular basis of the suppressive effects of Tregs, we investigated a co-cultivation system in which Tregs were used to suppress conventional CD4 T cells responding to T cell receptor agonists. We found that potent suppression could be observed with 1:1 mix of cells. Under these conditions, we observed that the conventional T cells underwent apoptosis. Furthermore, we found that the prevailing model of suppression of IL-2 gene transcription was incorrect. Death of the responding cells could quantitatively account for the loss of T cell response. We found that various common gamma chain cytokines were able to completely reverse the death due to Treg suppression. This appeared to be due to the fact that Tregs consumed, but could not produce, the cytokines and thereby deprived the conventional T cells of cytokines. In conjunction with these experiments, we found that a deficiency of the Bim gene completely rescued T cells from Treg suppression. We also demonstrated that similar apoptosis and T cell deletion effects could be observed in vivo in an inflammatory bowel disease model. Taken together, these data suggest that Treg cells exert their suppressive effect by a form of polyclonal deletion rather than suppression of cytokine transcription in the responding conventional T cells. We also discovered that Treg cells express receptors for gamma chain cytokines and are dependent on an exogenous supply of these cytokines to overcome cytokine withdrawal apoptosis in vitro. This result was validated in vivo by the accumulation of Treg cells in Bim-/- and Bcl-2 tg mice which have arrested cytokine deprivation apoptosis. We also found that CD25 and Foxp3 expression were down-regulated in the absence of these cytokines. CD25+ cells from Scurfy mice do not depend on cytokines for survival demonstrating that Foxp3 increases their dependence on cytokines by suppressing cytokine production in Treg cells. More recently, we have been examining how Treg cells interact with Th17. We have found that there are fundamental differences between these interactions and those between Treg and Th1 or Th2 cells. Our study reveals that the survival of Treg cells is strictly dependent on cytokines and cytokine producing cells because they do not produce cytokines. In another series of investigations, we have examined how gene regulation controls the immune tolerance mechanism of anergy. We have begun our investigation by studyin a key regulatory pathway involving the Nuclear Factor of Activated T cells (NF-AT) that is responsible for the expression of anergy inducing genes: grail and caspase-3. We have made the serendipitous observation that there is a major scaffold protein that coordinates the nuclear translocation of NF-AT during an immune response. Interestingly, this same scaffold protein has been implicated in certain neurodegenerative diseases. Thus our work may have the unexpected outcome of linking abnormal immune responses due to the failure of tolerance in certain neurological conditions. Moreover, these experiments may provide insights into the molecular regulatory events that are critical for tolerance especially anergy. Our observations shed new light on this regulatory process of T cells and we are now attempting to develop a general model of how T cell subsets achieve tolerance and immunity. These investigations may have importance for autoimmune disorders, infectious diseases, and tumor immunity. We have also studied the development and maturation of T lymphocytes by participating in the analyses of mice derived from a random ethylnitrosourea project. In collaborative studies we have uncovered a role for the Themis protein in the development of CD4 lymphocytes and the DOCK8 protein in immune competency. These studies show the value of random mutagenesis studies for detecting new functions for genes expressed in lymphocytes.