MOLECULAR EVASION OF ORAL TOLERANCE FOR MUCOSAL VACCINES

口服可溶性药物诱导全身无反应性 蛋白质和其他抗原，被称为口服耐受，最近表现出 增加了在临床应用中可能使用的重要意义 自身免疫性疾病。然而，当我们考虑口服疫苗是否有效时 免疫通过普通粘膜免疫系统，诱导口服 耐受性不会有好处，因为理想的粘膜疫苗应该 导致粘膜免疫和全身免疫。因此，人们只能 实现口服对自身抗原的耐受与对外来抗原的免疫 当更好地理解疫苗蛋白质的基本机制时 对口服耐受性进行了阐述。迄今为止最引人注目的工作表明 T淋巴细胞是主要的调节细胞类型 诱导口服耐受性。然而，CD4和CD8的确切作用 T细胞尚不清楚。T细胞可分为两大类 基于产生的细胞因子模式的子集。Th1细胞分泌IL-2， 干扰素-γ和肿瘤坏死因子-β诱导细胞免疫以及帮助 用于免疫球蛋白亚类反应。相反，Th2细胞合成IL-4，IL-5， IL-6和IL-10，并调节IgG1、IgA和IgE反应。我们有 假设口服可溶抗原，如 白喉类毒素(DT)可引起小鼠的无反应(包括无能) CD4T细胞亚群。我们进一步假设口服DT可诱导无能 Th1细胞和DT特异性Th2细胞进一步抑制前者 通过产生IL-4和IL-10的亚群。这种联合压制 解释了DT特有的全身性无反应。相比之下，DT- 产生IL-5和IL-6的特异性Th2型细胞支持IgA抗DT 粘膜效应部位的反应。为了检验这一假设， 提出了以下具体目标。L)DT特异性Th1的检测 和Th2细胞在小鼠Peyers斑块(PP)和脾(SP)的反应 通过使用抗原诱导的增殖反应口服DT， 以及细胞因子特异性ELISPOT、ELISAT和逆转录 (RT)-PCR检测。2)使用抗细胞因子治疗，即抗-干扰素-γ， 抗IL-4或抗IL-10，以及干扰素-γ和IL-4基因敲除小鼠 确定体内Th1和Th2细胞在口服耐受中的确切作用。 3)CD8 T细胞参与诱导口服免疫耐受 最近的研究表明，这个亚群含有产生转化生长因子-β的细胞 这会导致旁观者的压制。为了直接解决这个问题， 转基因抗Lyt-2或抗Lyt-3以及β-2微球蛋白(类 I)基因敲除小鼠将用于这些研究。4)上皮内的作用 淋巴细胞(IEL)，尤其是γ/βTCR亚群，用于 维持口腔耐受状态下的粘膜免疫球蛋白A反应。5)测试 粘膜佐剂霍乱毒素(CT)能否逆转现有的 通过增强DT特异性Th2细胞的口服耐受状态。前3名 具体的目标集中在细胞和分子机制上。 全身性无反应，而最后两个更关注粘膜如何 免疫球蛋白A反应是在全身无反应的情况下维持的。 此外，我们将检查粘膜佐剂CT以确定全身性 反应迟钝是可以逆转的。为了实现这些目标，我们将使用 最现代的分子方法，包括特定的基因敲除和 允许在体内进行T细胞介导的口腔研究的转基因动物 宽容。

The induction of systemic unresponsiveness to orally administered soluble proteins and other antigens, termed oral tolerance, has recently taken on added significance for possible use in clinical applications to prevent autoimmune disease. However, when we consider oral vaccines for effective immunity through the Common Mucosal Immune System, the induction of oral tolerance would not be beneficial since ideal mucosal vaccines should result in both mucosal and systemic immunity. Thus, one can only accomplish oral tolerance to self antigens versus immunity to foreign vaccine proteins when a better understanding of the fundamental mechanisms of oral tolerance are set forth. The most compelling work to date suggests that T lymphocytes are the major regulatory cell type responsible for induction of oral tolerance. However, the precise role of CD4+ versus CD8+ T cells is not yet clear. CD4+ T cells can be divided into two broad subsets based upon patterns of cytokines produced. Th1 cells secrete IL-2, IFN-gamma and TNF-beta and induce cell-mediated immunity as well as help for IgG-subclass responses. In contrast, Th2 cells synthesize IL-4, IL-5, IL-6 and IL-10 and regulate IgG1, IgA and IgE responses. We have hypothesized that oral administration of soluble antigens such as diphtheria toxoid (DT) induces unresponsiveness (including anergy) in the CD4+ T cell subset. We further hypothesize that oral DT induces anergic Th1 cells and DT-specific Th2 cells which further suppress the former subset via production of IL-4 and IL-10. This combined suppression accounts for DT-specific systemic unresponsiveness. In contrast, DT- specific Th2-type cells producing IL-5 and IL-6 support IgA anti-DT responses in mucosal effector sites. To test this hypothesis, the following specific aims are proposed. l) Examination of DT-specific Th1 and Th2 cell responses in Peyers patches (PP) and spleen (SP) of mice orally tolerized with DT by using antigen-induced proliferative responses, as well as cytokine-specific ELISPOT, ELISA and reverse transcription (RT)-PCR assays. 2) Usage of anti-cytokine-treated i.e., anti-IFN-gamma, anti-IL-4 or anti-IL-10, as well as IFN-gamma and IL-4 knockout mice to determine the exact role of in vivo Th1 and Th2 cells in oral tolerance. 3) Involvement of CD8+ T cells for the induction of oral tolerance since recent work has shown that this subset contains TGF-beta producing cells which induce bystander suppression. To directly address this issue, transgenic anti-Lyt-2 or anti-Lyt-3 as well as beta-2 microglobulin (class I) knockout mice will be used in these studies. 4) Role of intraepithelial lymphocytes (IELs), especially the gamma/delta TCR+ subset, for maintenance of mucosal IgA responses in a state of oral tolerance. 5) Test whether the mucosal adjuvant cholera toxin (CT) can reverse an existing state of oral tolerance by enhancing DT-specific Th2 cells. The first 3 specific aims are focused on cellular and molecular mechanisms involved in systemic unresponsiveness, while the last 2 focus more on how the mucosal IgA response is maintained in the presence of systemic unresponsiveness. Further, we will examine the mucosal adjuvant CT to determine if systemic unresponsiveness can be reversed. To accomplish these goals, we will use the most modern molecular approaches including specific knockout and transgenic animals which allow in vivo studies of T cell mediated oral tolerance.