Diversity of intraepithelial CD8aa T cells that recognize antignes from commensal flora

? DESCRIPTION (provided by applicant): The small intestine contains a significant number of intraepithelial T cells (IELs) that express CD8?? homodimer and ??TCRs. Currenlty these cells function and antigenic specificities remain enigmatic. Our long term goal is to understand how these cells contribute to homeostatic balance in the intestine, and how their TCR repertoires respond to microbial flora. Our central hypothesis is that clonal expansions and selective trophism of these IELs depends on microbial antigens that these cells recognize in vivo. These interactions may also contribute to sustain intestinal equilibrium. To test our hypothesis we propose two specific aims. First we will show how these IELs ??TCR repertoire changes in response to particular microbial species. We will examine this issue in a mouse model mice where T cells express semi diverse repertoire of TCRs and in vivo activated IELs are labeled with fluorescent protein (GFP). Limited diversity of TCRs allows to track individual IEL clones in response to different microbial species and organs using TCR CDR3 regions as clone-specific tags, whereas GFP allows to isolate IELs activated in their native environment. We expect to show that prime repertoire of IELs is broad, but become more oligoclonal following contacts with specific commensal antigens. In our Specific Aim 2 we propose to refine our new method to retrieve original pairs of ??TCRs sequences expressed by individual IELs for high-throughput sequencing (HTS), and use this approach to determine natural diversity of ??TCRs on IELs from wild type mice. This protocol is universal and in the future can be used to analyze native ??TCRs from humans.