Although the cause(s) of inflammatory bowel disease (IBD) is not yet known, the immune system is thought to mediate the tissue injury in hese disorders. Since the host cell(s) and antigens which are the putative targets of the immune assault, have not been definitively identified, it has not been possible to study specific immune effector mechanisms in IBD. Research has therefore been limited to studies of nonspecific immune functions. However, the nonspecific cytotoxicity triggered by monoclonal antibodies to the CD3 component of the T cell antigen receptor complex, can be used as an indirect marker of antigen specific, in vivo activated, cytotoxic T lymphocytes (CTL), even though the antigen to which these CTL are reacting, is not yet known. Thus, in the absence of knowing the antigen(s) inciting the immune response in IBD, important information concerning the effector CTL which are reacting to it, can still be obtained. Anti-CD3- triggered cytotoxicity is markedly increased in the blood of patients with IBD. The overall long term goal of this project is to determine the role of in vivo activated CTL in the pathogenesis of IBD. To pursue this goal in a vertical fashion, the following initial specific objectives will first be established. The phenotype of the anti- CD3-triggered CTL in the blood and mucosa will be more precisely determined using monoclonal antibody markers of cytotoxicity, lymphocyte activation, and T cell receptor category. Phenotyping will be performed positively by panning and cell sorting and negatively by complement depletion. The single cell assay technique will then be used to establish the specificity of a given phenotype for CTL effector function. The information will then be used to determine whether anti-CD3-triggered lytic activity of a particular phenotype is elevated not only in blood but also in the mucosa of patients with IBD. Whether the enhanced anti-CD3-triggered T lytic activity in IBD is mediated by additional or the same effectors as those responsible for this activity in normal subjects, and whether target specificity of the mucosal and blood anti-CD3-triggered CTL differs in IBD from that of normal subjects will then be established. In particular, the susceptibility of intestinal epithelial targets to this form of lysis will be tested, and whether such targets can directly trigger a subset of such effector CTL will be examined using colon carcinoma cell lines and cultured rabbit, canine and human (normal and IBD) colonic epithelial cells. Finally, since the CD45/T-200 molecule is a major determinant of the target specificity of anti-CD3-triggered CTL in normal subjects, the pattern of linkage of the different epitopes of CD45 to the anti- CD3-T lytic process in IBD will be examined using the inhibitory effects of a panel of monoclonal antibodies to different domains of CD45. These initial studies will provide critical information needed for the subsequent cloning of mucosal and blood CTL in IBD, so that an analysis of their antigen receptors can be used to provide valuable clues to the nature of the antigen with which they are reactive.
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