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 具有自身免疫特征的肠道虽然 这些疾病的发病机制知之甚少，他们的许多 这些特征表明，失调的T细胞反应是主要的，如果不是的话， 主要的致病因素对归纳事件的理解 调节肠道相关淋巴组织T细胞的发育 （GALT）在正常和炎症条件下将需要更好地 了解这些疾病的演变及其机制， 可以管理。最近的研究表明，致力于不同的 初始T细胞的细胞因子/效应子表型发生在初始T细胞的免疫应答过程中。 抗原刺激，并由APC相关分子控制， 细胞因子我们认为IBD患者的异常T细胞反应可能与 肠道中异常的表型分化。一个主要的重点将是 因此，确定抗原呈递给初始T细胞的位置， 在正常和炎症GALT中，共刺激分子的表达 这些位点中的分子和细胞因子有助于诱导 替代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），作为模型，允许控制抗原暴露于幼稚 体外和体内Tg T细胞。新的原位和免疫组织化学 分析将用于确定单细胞T细胞应答。 第一个目的是检查卵母细胞中OVA抗原呈递的位点， 正常和发炎的肠道，并确定细胞因子和增殖 这些位点中的初始T细胞的反应。不同T的演化 缺乏关键调节细胞因子IL-4时的细胞表型 将通过将OVA TCR转基因培育到携带 IL-4的纯合无效突变。第二个目标是探索 对肠内抗原耐受性由抗原启动假说 共刺激因子缺陷型肠上皮细胞的表达。初步研究将 检测分离的肠细胞的APC功能对OVA-TCR的应答 初始T细胞和Th 1和Th 2克隆。然后我们将研究 共刺激分子在这些细胞上的体内和体外表达， 在小肠上表达B7转基因的小鼠 肠脂肪酸结合蛋白（I-）控制下的上皮细胞 FABP）启动子。该转基因将被培育到OVA-TCR Tg小鼠中， 将检查对OVA的口服耐受性。第三个目标是 表征促炎性和保护性GALT T细胞的表型 根据CD 45 RB同种型的表达进行划分。通过使用T 从OVA-TCR Tg小鼠分离，我们将能够控制暴露的 在过继转移到SCID小鼠后，将单独的群体与抗原结合。这 将使我们能够剖析这两个人最早的抗原反应， 疾病进展或预防期间的人群。在平行研究中， 具有不同细胞因子表型的T细胞克隆将来源于OVA TCR Tg小鼠体外培养，并检查其产生或 预防炎症性小肠结肠炎后暴露于OVA抗原 转移到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.