Analysis of peripheral tolerance in vivo

描述(申请人提供)：大多数人没有遭受自身免疫，尽管产生的CD4T细胞表达T细胞受体(TCR)的自体(P)：主要组织相容性复合体II(MHCII)配体显示在胸腺。在TCR转基因小鼠模型中的许多研究表明，这是因为自身p：MHCII特异性的CD4T细胞被删除或分化为无能或抑制性调节性T(Treg)细胞。然而，关于这些机制在维持对所有自身抗原的耐受性方面的相对贡献还没有达成共识。此外，最近发现的TCR转基因模型系统的局限性使人们对这些机制中的哪些机制在完全不同的多克隆T细胞谱系中的生理条件下工作产生了怀疑。因此，基本问题仍然有待回答，如胸腺缺失的效率如何，无能T细胞是否存在，Treg细胞库是否真的富含自身pMHCII特异性细胞，以及这些机制中的哪一种在自身免疫过程中失败？我们将试图通过使用敏感的p：MHCII四聚体细胞富集法研究针对自身p：MHCII配体的多克隆内源性CD4T细胞来回答这些问题。在小鼠身上，我们将测试这样一个假设，即表达TCR的T细胞与无处不在的 自身P：MHCII配体被删除，而亲和力较低的TCR细胞存活下来，但由于忽视而不在次级淋巴器官中被触发。我们将通过测量逃脱克隆缺失的T细胞的激活表型以及其TCR的功能亲和力来检验这一假设。我们还提出，在自身免疫调节因子(AIRE)的控制下，某些p：MHCII特异性T细胞来自于胸腺中表达的外周组织特异性蛋白，因为这些p：MHCII只由一小部分髓质胸腺上皮细胞(MTEC)显示。我们进一步提出，以这种方式逃脱缺失的T细胞变得无能或分化为Treg细胞。这些假说将通过增加表达AIRE调节自身抗原的mTEC的比例，以及通过基因操作影响无能或Treg细胞发育的AIRE或分子的表达来检验。我们将尝试通过对正常血糖或1型糖尿病患者的胰岛素或谷氨酸脱羧酶p：MHCII特异性CD4T细胞的数量、功能和表型的直接体外跟踪，在人类中证实这些假设。如果成功，我们将了解胸腺克隆删除的效率，无能是否作为一种耐受机制存在，以及自我反应性T细胞群体是否丰富了Treg细胞，所有这些都在正常的多克隆谱系中，甚至在人类中。这些实验可能为未来的临床试验奠定基础，以确定基于自身p：MHCII四聚体的细胞浓缩是否可以 用作糖尿病早期诊断或监测免疫治疗效果的工具。

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.