ANERGY AND SIGNALLING IN MURINE T CELL SUBSETS

无反应性，定义为长期不能分泌IL-2， 增殖反应抗原刺激，可诱导IL-2- 产生鼠T细胞克隆。 易感细胞包括CD 4+细胞， 辅助性T细胞-1（Th 1）亚群和非辅助性CD 8+细胞溶解性T细胞 淋巴细胞（CTL）。 无反应性是由T细胞的刺激引起的 - 在不存在“共刺激”信号的情况下，针对抗原的TCR; 伴刀豆球蛋白A（Con A），固定的抗TCR单克隆抗体（mAb）， 抗原脉冲的抗原呈递细胞不表达共 刺激分子（因为APC已经被固定，或者它们 不能表达这样的分子），并固定II类主要 用抗原脉冲组织相容性复合体（MHC）提供了这样的 刺激. 然而，无反应性也可以通过以下治疗引入： 在不存在TCR刺激的情况下的钙离子载体。 显然， 不能在常规CTL的Th 2中诱导;这些细胞亚群不 分泌IL-2。 CD 4 + T细胞中的基本生化事件 导致无反应性的诱导，也没有信号缺陷， 无反应性细胞不能产生IL-2的原因是 特征充分。 此外，还讨论了共- 防止无反应性诱导的刺激分子尚未被 完全定义。 这些将使用现有的Th 1、Th 2和Th 0进行探索 克隆以及来源于不表达p59 fyn的小鼠的克隆 以及来自表达转基因TCR的小鼠。 在项目2中，我们 主要集中在近端信号事件，包括蛋白质 磷酸化和细胞内钙（[Ca 2 +]）的变化，但将 在研究更远端的信号事件时与项目1互动 无反应 尽管在CD 8+鼠T细胞克隆中诱导无反应性 虽然已经描述了，但尚未研究CD 8 + T细胞中的无反应性 彻底 常规CTL（其中无反应性显然不能被 诱导的）和Th 1细胞（可以被无反应）似乎至少共享 一些信号通路。 我们已经获得了大量的CD 8+小鼠T细胞， 分泌IL-2或IL-4的克隆以及两者都不分泌的克隆 这些淋巴因子。 这些克隆体和其他 来自表达转基因TCR的小鼠的克隆 将被确定，与诱导相关的生化事件 将CD 8 + T细胞中无反应性的比例与那些被发现 在诱导或维持CD 4 + T细胞无反应性状态中起重要作用 细胞 项目3中产生的转基因和“敲除”小鼠， 转染的肿瘤和表达各种细胞的转化细胞系 项目1中产生的表面分子已被研究得最多 广泛使用来自常规的Th 1克隆， 小鼠 然而，与体内无反应性相关的情况可能涉及 在初始T细胞中诱导无反应性。 来自小鼠的T细胞表达 转基因TCR不显示组成性的功能活性;它们 需要刺激以分泌淋巴因子，增殖， 表达细胞溶解活性。 诱发无反应性的必要条件 在克隆的T细胞中，将与那些需要诱导无反应性的T细胞进行比较。 初始CD 4+和CD 8 + T细胞。 逃逸细胞的特征 将确定无反应性的诱导，以及这种诱导之间的关系。 将测定细胞建立的CD 4+和CD 8 + T细胞亚群。 这些 这些研究将与研究无能源的项目4协调进行 体内诱导。

Anergy, as defined by the long-lived inability to secrete IL-2 and proliferate in response to antigenic stimulation, can be induced in IL-2- producing murine T cell clones. Susceptible cells include CD4+ cells of the T helper-1 (Th1) subset and helper-independent CD8+ cytolytic T lymphocytes (CTL). Anergy is induced by the stimulation of the T cell receptor (TCR) for antigen in the absence of "co-stimulatory" signals; concanavalin A (Con A), immobilized anti-TCR monoclonal antibodies (mAb), antigen-pulsed antigen-presenting cells that do not express co- stimulatory molecules (either because the APC have been fixed or they fail to express such molecules), and immobilized class II major histocompatibility complex (MHC) pulsed with antigen provide such stimulation. However, anergy also can be introduced by treatment with calcium ionophores in the absence of TCR stimulation. Anergy apparently cannot be induced in Th2 of conventional CTL; these cell subsets do not secrete IL-2. Neither the essential biochemical events in CD4+ T cells that lead to the induction of anergy nor the signalling defects that account for the failure of anergic cells to produce IL-2 have been characterized adequately. Also, the essential events induced by co- stimulatory molecules that prevent the induction of anergy have not been fully defined. These will be explored using existing Th1, Th2, and Th0 clones as well as clones derived from mice which do not express p59fyn and from mice that express transgenic TCR. In Project 2, we will concentrate mainly on proximal signalling events including protein phosphorylation and changes in intracellular calcium ([Ca2+]) but will interact with Project 1 in investigating the more distal signaling events in anergy. Although induction of anergy in a CD8+ murine T cell clone has been described, anergy in CD8+ T cells has not been investigated thoroughly. Conventional CTL (in which anergy apparently cannot be induced) and Th1 cells (which can be anergized) appear to share at least some signaling pathways. We have derived a number of CD8+ murine T cell clones that secrete IL-2 or IL-4 as well as clones that secrete neither of these lymphokines. The susceptibility of these clones and other clones that are being derived from mice that express a transgenic TCR will be determined, and the biochemical events associated with induction of anergy in CD8+ T cells will be compared with those found to be important in induction or maintenance of the anergic state in CD4+ T cells. Transgenic and "knockout" mice produced in Project 3 and transfected tumor and transformed cell lines expressing various cell surface molecules produced in Project 1 have been studied most extensively using Th1 clones that have been derived from conventional mice. However, situations associated with anergy in vivo may involve induction of anergy in naive T cells. T cells from mice expressing transgenic TCR do not display functional activity constitutively; they require stimulation in order to secrete lymphokines, proliferate, and express cytolytic activity. The conditions necessary to induce anergy in cloned T cells will be compared with those needed to induce anergy in naive CD4+ and CD8+ T cells. Characteristics of cells that escape induction of anergy will be determined and the relationship between such cells established CD4+ and CD8+ T cell subsets will be determined. These studies will be coordinated with Project 4 which is investigating anergy induction in vivo.