MOLECULAR EVASION OF ORAL TOLERANCE FOR MUCOSAL VACCINES

粘膜疫苗口服耐受性的分子规避

• 批准号: 2071908
• 负责人: HIROSHI KIYONO
• 金额: $ 11.2万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2071908
• 关键词: Peyer's patches; anergy; cytotoxic T lymphocyte; diphtheria toxoid; enzyme linked immunosorbent assay; genetically modified animals; helper T lymphocyte; immune tolerance /unresponsiveness; immunoglobulin A; interferons; interleukin 4; interleukin 5; interleukin 6; laboratory mouse; lymphocyte proliferation; mucosa; oral administration; polymerase chain reaction; secretory immune system; spleen; vaccines

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.

诱导口服可溶性药物的全身无反应性 蛋白质和其他抗原，称为口服耐受，最近采取了 增加了可能用于临床应用的意义，以防止 自身免疫性疾病然而，当我们考虑口服疫苗有效时， 免疫通过共同粘液免疫系统，诱导口服 因为理想的粘膜疫苗应该 导致粘膜和全身免疫。因此，只能 实现对自身抗原的口服耐受性与对外来抗原的免疫性 当更好地了解疫苗蛋白的基本机制时， 口服耐受性的影响。迄今为止最引人注目的研究表明， T淋巴细胞是主要的调节细胞类型， 诱导口服耐受。然而，CD 4+与CD 8+的确切作用 T细胞目前还不清楚。CD 4 + T细胞可分为两大类。 基于产生的细胞因子的模式的子集。Th 1细胞分泌IL-2， IFN-γ和TNF-β，诱导细胞介导的免疫，以及帮助 IgG亚类应答。相反，Th 2细胞合成IL-4，IL-5， IL-6和IL-10并调节IgG 1、伊加和IgE应答。我们有 假设口服可溶性抗原， 白喉类毒素（DT）诱导无反应性（包括无反应性）， CD 4 + T细胞亚群。我们进一步假设，口服DT诱导无能， Th 1细胞和DT特异性Th 2细胞，进一步抑制前者 亚群通过产生IL-4和IL-10。这种联合抑制 解释了DT特异性全身无反应性。相比之下，DT- 产生IL-5和IL-6的特异性Th 2型细胞支持伊加抗DT 粘膜效应部位的反应。为了验证这一假设， 提出了以下具体目标。l）DT特异性Thl的检查 小鼠派尔集合淋巴结（PP）和脾脏（SP）中的Th 2和Th 3细胞反应 通过使用抗原诱导的增殖反应口服DT耐受， 以及丝氨酸特异性ELISPOT、ELISA和逆转录 （RT）-PCR测定。2)抗细胞因子处理的用途，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Th 1和Th 2细胞在口服耐受中的确切作用。 3)CD 8 + T细胞参与诱导口服耐受， 最近的研究表明，这一亚群含有TGF-β产生细胞 从而引起旁观者抑制。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）将在这些研究中使用敲除小鼠。4)上皮内的作用 淋巴细胞（IEL），尤其是γ/δ TCR+亚群， 在口服耐受状态下维持粘膜伊加应答。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Th 2细胞来改善口服耐受状态。前3 具体的目标集中在细胞和分子机制参与 全身无反应性，而最后2个重点是如何粘膜 伊加反应在全身无反应性的情况下维持。 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 转基因动物允许体内研究T细胞介导的口服 宽容