Analysis of peripheral tolerance in vivo

描述（由申请人提供）：尽管产生CD4+ T细胞表达自身肽(p)特异性的T细胞受体（TCR）：胸腺显示的主要组织相容性复合体II （MHCII）配体，但大多数人不会患有自身免疫。许多对TCR转基因小鼠模型的研究表明，这是因为自身mhcii特异性CD4+ T细胞被删除或分化为无能或抑制性调节性T （Treg）细胞。然而，这些机制对维持对所有自身抗原的耐受性的相对贡献尚未达成共识。此外，最近揭示的TCR转基因模型系统的局限性，使人们对这些机制在完全不同的多克隆T细胞库的生理条件下运作产生了怀疑。因此，一些基本问题仍有待回答，如胸腺缺失的效率如何，无能T细胞是否存在，Treg细胞库是否真的为自身pmhcii特异性细胞富集，以及这些机制中哪些在自身免疫过程中失效？我们将尝试通过使用一种敏感的基于p:MHCII四聚体的细胞富集方法，研究自身p:MHCII配体特异性的多克隆内源性CD4+ T细胞来回答这些问题。在小鼠中，我们将测试表达TCRs亲和力最高的T细胞普遍存在的假设

DESCRIPTION (provided by applicant): Most people do not suffer from autoimmunity despite the production of CD4+ T cells expressing T cell receptors (TCR) specific for self peptide (p):major histocompatibility complex II (MHCII) ligands displayed in the thymus. Many studies in TCR transgenic mouse models have shown that this is the case because self p:MHCII-specific CD4+ T cells are deleted or differentiate into anergic or suppressive regulatory T (Treg) cells. Nevertheless, consensus on the relative contributions of these mechanisms to maintaining tolerance to all self antigens has not been reached. In addition, recent revelations of the limitations of TCR transgenic model systems have created doubt about which of these mechanisms operate under physiological conditions within fully diverse polyclonal T cell repertoires. Fundamental questions therefore remain to be answered such as how efficient is thymic deletion, do anergic T cells exist, is the Treg cell repertoire really enriched for self pMHCII-specific cells, and which of these mechanisms fails during autoimmunity? We will attempt to answer these questions by studying polyclonal endogenous CD4+ T cells specific for self p:MHCII ligands using a sensitive p:MHCII tetramer-based cell enrichment method. In mice, we will test the hypothesis that T cells expressing TCRs with the highest affinities for ubiquitous self p:MHCII ligands are deleted, while cells with lower affinity TCRs survive but are not triggered in the secondary lymphoid organs due to ignorance. We will test this hypothesis by measuring the activation phenotype of T cells that escape clonal deletion along with the functional avidities of their TCRs. We also propose that some T cells specific for p:MHCII derived from peripheral tissue-specific proteins expressed in the thymus under the control of the Autoimmune Regulator (AIRE) escape deletion because these p:MHCII are displayed by only a small subset of medullary thymic epithelial cells (mTEC). We further propose that T cells that escape deletion in this fashion become anergic or differentiate into Treg cells. These hypotheses will be tested by increasing the fraction of mTEC that express an AIRE-regulated self antigen and by genetically manipulating the expression of AIRE or molecules that influence anergy or Treg cell development. We will attempt to confirm these hypotheses in humans by direct ex vivo tracking of the number, function, and phenotype of insulin or glutamic acid decarboxylase p:MHCII-specific CD4+ T cells from normoglycemic or type 1 diabetic people. If successful, we will have learned how efficient thymic clonal deletion is, whether anergy exists as a tolerance mechanism, and if self-reactive T cell populations are enriched for Treg cells, all within the normal polyclonal repertoires, and even in humans. These experiments could set the stage for future clinical trials to determine if self p:MHCII tetramer-based cell enrichment can be used as a tool for early diagnosis of diabetes or to monitor the efficacy of immunotherapy.