MOLECULAR EVASION OF ORAL TOLERANCE FOR MUCOSAL VACCINES

口服可溶性药物引起全身无反应 蛋白质和其他抗原，称为口服耐受性，最近出现了 对于在临床应用中预防可能的使用增加了重要意义 自身免疫性疾病。然而，当我们考虑口服疫苗是否有效时 通过共同粘膜免疫系统的免疫，诱导口腔 耐受性不会有益，因为理想的粘膜疫苗应该 产生粘膜免疫和全身免疫。这样一来，人们只能 实现对自身抗原的口服耐受与对外来抗原的免疫 当更好地了解基本机制时疫苗蛋白 阐述了口服耐受性。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。然而，CD4 与 CD8 的确切作用 T细胞尚不清楚。 CD4 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， IFN-γ 和 TNF-β 诱导细胞介导的免疫并有助于 用于 IgG 亚类反应。相反，Th2细胞合成IL-4、IL-5、 IL-6 和 IL-10 调节 IgG1、IgA 和 IgE 反应。我们有 假设口服可溶性抗原，例如 白喉类毒素 (DT) 会导致患者无反应（包括无反应） CD4 T 细胞亚群。我们进一步假设口服 DT 会诱发无反应性 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 的子集。这种联合压制 解释了 DT 特异性的系统性无反应。相比之下，DT- 产生 IL-5 和 IL-6 的特定 Th2 型细胞支持 IgA 抗 DT 粘膜效应部位的反应。为了检验这个假设， 提出以下具体目标。 l) DT特异性Th1检查 小鼠派耶氏淋巴结 (PP) 和脾脏 (SP) 中的 Th2 细胞反应 通过使用抗原诱导的增殖反应来口服 DT 耐受， 以及细胞因子特异性 ELISPOT、ELISA 和反转录 (RT)-PCR 测定。 2) 使用抗细胞因子治疗，即抗-IFN-γ， 抗IL-4或抗IL-10，以及IFN-γ和IL-4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 细胞参与诱导口服耐受 最近的研究表明，该子集包含 TGF-β 产生细胞 从而引起旁观者的压制。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（类别 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/δ TCR 子集，用于 维持口腔耐受状态下的粘膜 IgA 反应。 5）测试 粘膜佐剂霍乱毒素（CT）是否可以逆转现有的 通过增强 DT 特异性 Th2 细胞来调节口服耐受状态。前 3 个 具体目标集中于参与的细胞和分子机制 全身无反应，而后 2 个更关注粘膜如何 在全身无反应的情况下仍能维持 IgA 反应。 此外，我们将检查粘膜辅助 CT 以确定是否全身 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，包括特异性敲除和 转基因动物可进行 T 细胞介导的口腔体内研究 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.