Analysis of peripheral tolerance in vivo

描述（由申请人提供）：尽管产生了表达对自身肽（p）具有特异性的 T 细胞受体（TCR）的 CD4 T 细胞：胸腺中展示的主要组织相容性复合物 II（MHCII）配体，但大多数人并未患有自身免疫性疾病。 TCR 转基因小鼠模型的许多研究表明，出现这种情况是因为自身 p:MHCII 特异性 CD4 T 细胞被删除或分化为无能或抑制性调节 T (Treg) 细胞。然而，关于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，最近对 TCR 转基因模型系统局限性的揭露使人们对这些机制中哪些机制在完全不同的多克隆 T 细胞库中的生理条件下发挥作用产生了疑问。因此，一些基本问题仍有待回答，例如胸腺缺失的效率如何，无反应性 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，以及这些机制中哪一个在自身免疫过程中失效？我们将尝试通过使用敏感的 p:MHCII 四聚体细胞富集方法研究对自身 p:MHCII 配体特异的多克隆内源性 CD4 T 细胞来回答这些问题。在小鼠中，我们将测试以下假设：表达 TCR 的 T 细胞对普遍存在的 TCR 具有最高的亲和力。 自身 p:MHCII 配体被删除，而具有较低亲和力 TCR 的细胞存活下来，但由于无知而没有在次级淋巴器官中被触发。我们将通过测量逃脱克隆删除的 T 细胞的激活表型及其 TCR 的功能亲和力来检验这一假设。我们还提出，一些对 p:MHCII 特异的 T 细胞源自胸腺中表达的外周组织特异性蛋白，在自身免疫调节因子 (AIRE) 的控制下逃脱了缺失，因为这些 p:MHCII 仅由一小部分胸腺髓质上皮细胞 (mTEC) 展示。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。这些假设将通过增加表达 AIRE 调节的自身抗原的 mTEC 比例以及通过基因操纵 AIRE 或影响无反应性或 Treg 细胞发育的分子的表达来进行检验。我们将尝试通过直接离体跟踪血糖正常或 1 型糖尿病患者的胰岛素或谷氨酸脱羧酶 p:MHCII 特异性 CD4 T 细胞的数量、功能和表型来在人类中证实这些假设。如果成功，我们将了解到胸腺克隆删除的效率如何，无反应性是否作为一种耐受机制存在，以及自身反应性 T 细胞群是否富集 Treg 细胞，所有这些都在正常的多克隆库中，甚至在人类中也是如此。这些实验可以为未来的临床试验奠定基础，以确定基于 p:MHCII 四聚体的自我细胞富集是否可以 用作糖尿病早期诊断或监测免疫疗法疗效的工具。

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.