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

 描述(申请人提供)：小肠含有相当数量的上皮内T细胞(IEL)，表达CD8、αα同源二聚体和αβTCR。目前，这些细胞的功能和抗原特异性仍是个谜。我们的长期目标是了解这些细胞如何促进肠道内的动态平衡，以及它们的TCR谱系如何对微生物菌群做出反应。我们的中心假设是，这些IELs的克隆扩张和选择性营养依赖于这些细胞在体内识别的微生物抗原。这些相互作用也可能有助于维持肠道平衡。为了检验我们的假设，我们提出了两个具体目标。首先，我们将展示这些IELαβTCR谱是如何随着特定微生物物种的变化而变化的。我们将在小鼠模型小鼠中研究这个问题，在小鼠模型中，T细胞表达半多样化的TCRs，体内激活的IEL用荧光蛋白(GFP)标记。TCR的有限多样性允许使用TCR CDR3区域作为克隆特异性标签来跟踪单个IEL克隆，以响应不同的微生物物种和器官，而GFP允许分离在其自然环境中激活的IEL。我们希望展示IEL的主要谱系是广泛的，但在接触特定的共生抗原后变得更具寡克隆性。在我们的具体目标2中，我们建议改进我们的新方法，以检索由单个IEL表达的原始αβTCR序列对，用于高通量测序，并使用该方法来确定野生型小鼠IEL上αβTCR的自然多样性。该协议是通用的，未来可以用于分析来自人类的本地αβTCR。

 DESCRIPTION (provided by applicant): The small intestine contains a significant number of intraepithelial T cells (IELs) that express CD8αα homodimer and αβTCRs. 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 αβTCR 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 αβTCRs sequences expressed by individual IELs for high-throughput sequencing (HTS), and use this approach to determine natural diversity of αβTCRs on IELs from wild type mice. This protocol is universal and in the future can be used to analyze native αβTCRs from humans.