ANTIGEN HANDLING AND CYTOKINE PHENOTYPE REGULATION IN NORMAL AND INFLAMED COLON

点击翻译按钮获取中文摘要

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.