Regulation of Immune Responses in Humans and Non-Human Primates

人类和非人类灵长类动物免疫反应的调节

• 批准号: 6431566
• 负责人: WARREN STROBER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6431566
• 关键词: B lymphocyte; CD2 molecule; CD3 molecule; CD95 molecule; T cell receptor; T lymphocyte; animal tissue; apoptosis; autoimmunity; biological signal transduction; cell cell interaction; cytokine; cytotoxic T lymphocyte; helper T lymphocyte; human tissue; immunoregulation; interferon gamma; interleukin 2; laboratory mouse; leukocyte activation /transformation; lymphocyte proliferation; tissue /cell culture

Project 1: In previous studies we have defined a potentially important set of human regulatory T cells that recognize self MHC and are therefore called self-MHC reactive (autoreactive) T cells. In essence, we showed that such cells exert their regulation (suppression) via interactions with activated B cells and the production of TGF-beta and IL-10. Furthermore, we showed that TGF-beta secretion required interaction between Beta7 costimulatory molecules on the B cell and CTLA-4 on the self-MHC reactive T cell. In the current studies we explored the interactions between self-MHC reactive regulatory T cells and stimulatory B cells necessary for the secretion of IL-10. We found that regulatory T cell lines stimulated by activated B cells produced increased amounts of IL-10 when cultured with IL-12. In addition, we found that secretion of IL-10 by such cells were inhibited if the latter are cultured in the presence of ICOS-Fc, a reagent that blocks interaction between ICOS on the T cell surface and ICOS ligand on the stimulatory B cell surface. In further studies we showed that patients with active SLE who have been previously shown to be prone to disturbances in regulatory cell function, have cells expressing increased expression of ICOS and suppression of autologous MLR's which was reversed by addition of ICOS-Fc. These studies thus link IL-10 production in regulatory T cells to a particular co-stimulatory molecule interaction and may imply that autoimmunity is, at least in part, controlled by regulatory T cells employing this mechanism. Project 2: In previous studies, we observed that treatment of mice with TNBS-colitis, a Th1 T cell-mediated colitis resembling human Crohn's disease, with anti-IL-12 induce apoptosis of Th1 T cells in the inflamed colon. This effect is reduced in mice carrying the lpr gene and in SJL/J mice treated with Fas-Fc suggesting that the induction of apoptosis by anti-IL-12 is mediated by the Fas signaling pathway, i.e., IL-12 normally prevents Fas-mediated apoptosis of Th1 T cells bearing the IL-12R. In the present studies, we investigated the biochemical basis of the anti-apoptotic effect of IL-12 using a human cell line, Kit225 cells, bearing an IL-12R and having susceptibility to Fas-mediated apoptosis. In initial studies we showed that IL-12 substantially reduced apoptosis induced by anti-Fas antibody; importantly, this effect was not due to IL-12 down-regulation of surface Fas expression. In addition, we showed that IL-12 inhibited apoptosis mediated by Trail, a TNF-receptor superfamily molecule that induces apoptosis via DR4 and DR5 (but not Fas). In further studies we analyzed discrete steps of the Fas apoptotic pathway to determine the mechanism of the anti-apoptotic effects of IL-12. First we showed that IL-12 does not interfere with the formation of the DISC (death-inducing signaling complex) which forms as a result of Fas-signaling; this was done by showing via immunoprecipitation (IP) studiesthat cells cultured with IL-12 contains IP's containing FLICE, FADD, and Fas. In further studies, we showed that the anti-apoptotic effect of IL-12 does not involve mitochondrial apoptotic mechanisms since IL-12 does not suppress UV irradiation-induced apoptosis and IL-12 did not affect Bcl-2 or Bcl-Lx levels as determined by Western blot. In a final series of studies designed to localize the effect of IL-12, we showed that IL-12 treatment of Kit225 cells results in the major down-regulation of caspase 8 activity as well as some down-regulation of caspase 3 activity, suggesting that the anti-apoptotic effect is specific to a caspase that functions early in the caspase cascade. One possible mechanism of this effect on caspase 8 activity is the activation of an IAP protein specific for its caspase. Indeed, we showed that IL-12 treatment resulted in upregulation of IAP-2 protein and we are provisionally considering this effect to be the mechanism of IL-12 anti-apoptosis.

项目1：在先前的研究中，我们定义了一组潜在的人类调节性T细胞，这些T细胞识别自我MHC，因此称为自我MHC反应性（自动反应性）T细胞。 从本质上讲，我们表明这种细胞通过与活化的B细胞的相互作用以及TGF-β和IL-10的产生来发挥其调节（抑制）。此外，我们表明TGF-β分泌需要在B细胞上的beta7共刺激分子与自我MHC反应性T细胞上的CTLA-4之间的相互作用。 在当前的研究中，我们探讨了自我MHC反应性调节T细胞与分泌IL-10所需的刺激B细胞之间的相互作用。 我们发现，当用IL-12培养时，被激活的B细胞刺激的调节T细胞系增加了IL-10的增加。 此外，我们发现，如果在ICOS-FC的存在下培养后者，则抑制此类细胞的IL-10分泌，ICOS-FC存在，该试剂阻止了ICOS在T细胞表面上的ICOS与刺激B细胞表面上ICOS配体之间的相互作用。 在进一步的研究中，我们表明活跃的SLE患者先前被证明容易受到调节细胞功能的干扰，具有表达ICOS表达增加的细胞和自体MLR的抑制，而自体MLR的抑制是通过添加ICOS-FC逆转的。 因此，这些研究将调节性T细胞中的IL-10产生与特定的共刺激分子相互作用联系起来，并且可能暗示自身免疫至少部分受使用该机制的调节T细胞控制。项目2：在先前的研究中，我们观察到用TNBS-肺炎治疗小鼠，一种类似于人克罗恩氏病的Th1 T细胞介导的结肠炎，抗IL-12在发炎的结肠中诱导Th1 T细胞的细胞凋亡。 在携带LPR基因和用FAS-FC处理的SJL/J小鼠的小鼠中，这种作用降低，这表明抗IL-12诱导凋亡是由FAS信号传导途径介导的，即IL-12，即IL-12，通常会导致FAS介导的TH1 T细胞的TH1 T细胞具有IL-12R的TH1介导细胞。 在本研究中，我们使用人类细胞系KIT225细胞，具有IL-12R，具有IL-12R并具有FAS介导的细胞凋亡的敏感性。在最初的研究中，我们表明IL-12大大降低了抗FAS抗体诱导的凋亡。重要的是，这种作用不是由于IL-12表面表达的IL-12下调。 此外，我们表明IL-12抑制了TRAIL介导的细胞凋亡，TRAIL是一种TNF受体超家族分子，可通过DR4和DR5诱导凋亡（但未FAS）。在进一步的研究中，我们分析了FAS凋亡途径的离散步骤，以确定IL-12的抗凋亡作用的机理。 首先，我们表明IL-12不会干扰由于FAS信号而形成的椎间盘的形成（诱发死亡的信号传导复合物）。这是通过通过IL-12培养的免疫沉淀（IP）StudiestHat细胞显示的，其中包含IP的FLICE，FADD和FAS来完成。 在进一步的研究中，我们表明IL-12的抗凋亡作用不涉及线粒体凋亡机制，因为IL-12不抑制紫外线辐射诱导的凋亡，而IL-12不会影响由Western Blot确定的BCL-2或BCL-LX水平。 在旨在定位IL-12效果的最后一系列研究中，我们表明IL-12处理KIT225细胞会导致CASPase 8活性的主要下调以及caspase 3活性的某些下调，这表明抗凋亡效应是caspase在caspase cascade中在caspase cascade中起作用的特异性。 这种影响对caspase 8活性的一种可能机制是激活对其caspase特异性的IAP蛋白。 实际上，我们表明IL-12治疗导致IAP-2蛋白上调，我们在临时将这种作用视为IL-12抗凋亡的机理。