ANTIGEN HANDLING AND CYTOKINE PHENOTYPE REGULATION IN NORMAL AND INFLAMED COLON

炎症性肠病是一种慢性炎症性疾病。 具有自身免疫特性的肠道。尽管 这些疾病的发病机制还知之甚少，他们的许多 特征表明，如果不是的话，T细胞反应失调是主要的 主导性致病因素。对归纳事件的理解 肠道相关淋巴组织中T细胞的发育调控 (GALT)在正常和炎症条件下将需要更好的 了解这些疾病的演变以及它们的发病机制 是可以管理的。最近的研究表明，对不同的承诺 初始T细胞的细胞因子/效应表型发生在 抗原刺激，受APC相关分子和 细胞因子。我们认为IBD的异常T细胞反应可能如下 来自肠道中异常的表型分化。一个主要的焦点将是 因此，要确定抗原呈递给初始T细胞的位置 正常和炎症GALT中共刺激分子的表达 这些部位的分子和细胞因子有助于诱导 可选择的T细胞分化途径。实验方法 将使用具有特异性的α-β-TCR转基因(TG)小鼠 卵清蛋白(OVA)，作为允许控制幼稚抗原暴露的模型 甘油三酯T细胞体外和体内实验。新的原位和免疫组织化学 分析将用于确定单个细胞T细胞的反应。 第一个目的是检查OVA抗原提呈的部位 正常和炎症肠道组织中细胞因子和增殖活性的测定 幼稚T细胞在这些部位的反应。不同T的进化 关键调节细胞因子IL-4缺失时的细胞表型 将通过将OVA TCR转基因培育成携带OVA TCR的小鼠进行检测 IL-4纯合子零突变。第二个目标是探索 对肠道抗原的耐受性是由抗原启动的假说 共刺激分子缺陷型肠细胞的研究进展。初步研究将 检测分离的肠上皮细胞的APC功能对OVA-TCR的反应 幼稚T细胞和Th1、Th2克隆。然后，我们将研究 共刺激分子在体内和体外这些细胞上的表达，通过产生 在小肠上表达B7转基因的小鼠 肠道脂肪酸结合蛋白(I-FBP)控制下的上皮细胞 FABP)启动子。这一转基因将被培育成OVA-TCR转基因小鼠并 将检查口服对OVA的耐受性。第三个目标将是 促炎性和保护性Galt T细胞表型的特征 根据CD45RB亚型的表达情况进行划分。通过使用T 从OVA-TCRTG小鼠中分离出来，我们将能够控制暴露于 过继转移给SCID小鼠后，将种群分离为抗原。这 将使我们能够剖析这两种病毒最早的抗原反应 在疾病进展或预防期间的人口。在平行研究中， 不同细胞因子表型的T细胞克隆将来自OVA TCR 转基因小鼠的体外实验，并将检测它们产生或 预防暴露于OVA抗原后的炎性小肠结肠炎 转接至SCID主机。

The inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the intestine with autoimmune characteristics. Although the pathogenesis of these disorders is poorly understood, many of their features point to a dysregulated T cell response as a major, if not dominant, etiologic factor. An understanding of the inductive events that regulate development of T cells of the gut-associated lymphoid tissues (GALT) under normal and inflammatory conditions will be required to better understand the evolution of these diseases and mechanisms by which they can be managed. Recent studies indicate that commitment to different cytokine/effector phenotypes by naive T cells occurs during the initial antigenic stimulation and is controlled by APC-associated molecules and cytokines. We propose that the abnormal T cell responses in IBD may follow from aberrant phenotype differentiation in the gut. A major focus will therefore be to define where antigen presentation to naive T cells occurs in the normal and inflamed GALT and how expression of costimulator molecules and cytokines in these sites contribute to induction of alternative T cell differentiation pathways. The experimental approach will be to use an alpha-beta-TCR transgenic (Tg)mouse with specificity for ovalbumin (OVA), as a model to allow control of antigen exposure to naive Tg T cells in vitro and in vivo. Novel in situ and immunohistochemical analyses will be used to determine single-cell T cell responses. The first aim is to examine the sites of OVA antigen presentation in the normal and inflamed gut and to determine the cytokine and proliferative responses of naive T cells in these sites. The evolution of distinct T cell phenotypes in the absence of the critical regulatory cytokine IL-4 will be examined by breeding the OVA TCR transgenes into mice carrying homozygous null mutations for IL-4. The second aim is to explore the hypothesis that tolerance to enteral antigens is initiated by antigen presentation on costimulator-deficient enterocytes. Initial studies will examine the APC function of isolated enterocytes for responses of OVA-TCR naive T cells and Th1 and Th2 clones. We will then examine the effects of costimulator expression on these cells in vivo and in vitro, by generating mice in which a B7 transgene is expressed on the small intestinal epithelium under control of the intestinal fatty acid binding protein (I- FABP) promoter. This transgene will be bred into the OVA-TCR Tg mouse and oral tolerance to OVA will be examined. The third aim will be to characterize the phenotypes of proinflammatory and protective GALT T cells divided on the basis of expression of the CD45RB isoform. By using T isolated from OVA-TCR Tg mice, we will be able to control exposure of the separate populations to antigen after adoptive transfer to SCID mice. This will allow us to dissect the earliest antigenic responses of these two populations during disease progression or prevention. In parallel studies, T cell clones of distinct cytokine phenotypes will be derived from OVA TCR Tg mice in vitro and will be examined for their capacity to produce or prevent inflammatory enterocolitis upon exposure to OVA antigen after transfer into SCID hosts.