ANERGY AND SIGNALLING IN MURINE T CELL SUBSETS

无能，定义为长期无法分泌IL-2和 在抗原刺激下增殖，可在IL-2- 生产小鼠T细胞克隆。易感细胞包括 辅助性T细胞-1(Th1)亚群和非辅助性CD8+细胞溶解T细胞 淋巴细胞(CTL)。无能是由T细胞的刺激引起的 在没有“共刺激”信号的情况下，抗原的受体(TCR)； 刀豆蛋白A(ConA)，固定化抗TCR单抗(MAb)， 抗原冲击的抗原提呈细胞不表达co- 刺激分子(要么是因为APC已经固定，要么是因为他们 不能表达这种分子)，以及固定的II类主要 用抗原冲击的组织相容性复合体(MHC)提供了这样的 刺激。然而，无能也可以通过以下治疗而引入 钙离子载体在没有TCR刺激的情况下。显然无能为力 不能在常规CTL的Th2中诱导；这些细胞亚群不能 分泌IL-2。既不是CD4+T细胞中的基本生化事件 这会导致无能的诱导，也不会有信号缺陷 无能细胞产生IL-2失败的原因一直被认为是 具有充分的特点。此外，由共同- 防止诱导无能的刺激性分子尚未被 完全定义好了。我们将使用现有的Th1、Th2和Th0来探索这些问题 克隆以及不表达p59fyn的小鼠克隆 以及表达转基因TCR的小鼠。在项目2中，我们将 主要集中在包括蛋白质在内的近端信号事件 磷酸化和细胞内钙([Ca+])的变化，但会 在调查更远端的信令事件时与项目1互动 处于无能为力状态。尽管在CD8+小鼠T细胞克隆中诱导无能 已被描述，CD8+T细胞的无能尚未被研究 彻底地。传统的CTL(其中无能显然不能 诱导)和Th1细胞(可被激活)似乎至少共享 一些信号通路。我们已经获得了一些CD8+小鼠T细胞 分泌IL-2或IL-4的克隆以及既不分泌IL-2或IL-4的克隆 这些淋巴因子。这些克隆和其他克隆的易感性 从表达转基因TCR的小鼠那里获得的克隆 将被确定，以及与诱导相关的生化事件 CD8+T细胞中无能的比例将与发现的 在诱导或维持CD4+T细胞无能状态中的重要作用 细胞。项目3和项目中培育的转基因和“基因敲除”小鼠 表达多种细胞的转染瘤和转化细胞系 项目1中产生的表面分子被研究得最多 广泛使用从传统克隆派生的Th1克隆 老鼠。然而，与体内无能相关的情况可能涉及 诱导幼稚T细胞无能。小鼠T细胞表达 转基因TCR不具有结构性的功能活性；它们 需要刺激才能分泌淋巴因子、增殖和 表达细胞溶解活性。诱发无能的必要条件 将克隆的T细胞与诱导无能的T细胞进行比较 幼稚的CD4+和CD8+T细胞。逃逸细胞的特征 无能的诱导将被确定，以及这样的关系 细胞建立的CD4+和CD8+T细胞亚群将被测定。这些 研究将与项目4协调，该项目正在调查无能 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.