Analysis of peripheral tolerance in vivo

该项目的长期目标是了解CD 4 + T细胞的机制， 对次级淋巴器官而不是胸腺中呈递的抗原的耐受性。此前我们 显示在小鼠的次级淋巴器官中暴露于模型抗原的幼稚CD 4 + T细胞在小鼠的次级淋巴器官中表达。 如果没有炎症，则增殖不良，然后大多数后代死亡，幸存者进入无反应性阶段。 以淋巴因子产生不足为特征的状态。本申请的目的是确定是否 类似的一系列事件解释了外周对某些天然自身蛋白的耐受性。这已经成为一个 这是一个紧迫的问题，因为发现AIRE转录因子驱动胸腺外 胸腺中的基因产物。因此，外周耐受的生理作用仍然是可能的， 实际上是由AIRE介导的胸腺内耐受发挥作用。在这里，我们将测试假设 通过使用新的工具来识别耐受机制， 这适用于两种妊娠特异性蛋白质，一种似乎受AIRE调节，另一种 没有，还有一种精子特异性蛋白质。我们将确定这些蛋白质是否具有免疫原性 在从未在相关组织中表达它们的小鼠中（例如，非妊娠雌性小鼠），并成为 表达后在小鼠中无免疫原性（例如，怀孕的雌性小鼠）。相关的抗原肽将 鉴定并用于产生肽-MHC II多聚体。然后将多聚体与敏感的 一种新的富集方法，能够检测每只小鼠少于100个细胞，以计数肽MHC II- 特异性CD 4 + T细胞在多克隆抗体内表达相关蛋白之前、期间和之后的表达。 正常小鼠的所有基因组。这种方法应该揭示相关的CD 4 + T细胞是否被删除， 在这些发育受调节的时期或之后， 自身蛋白表达。然后将这种方法用于基因靶向小鼠，以确定是否或 而不是像Fas和Cbl-b这样的分子以及P01其他成员发现的其他分子参与了这一过程。 确定了容忍机制。成功将提供第一个明确的识别外围设备 耐受机制适用于天然自身抗原。这些信息应该有助于集中未来的研究 相关机制，并阐明其可能失败并导致自身免疫的潜在方式。

The long-term goal of this project is an understanding of the mechanisms that account for CD4+ T cell tolerance to antigens that are presented in the secondary lymphoid organs but not the thymus. Previously, we showed that naive CD4+ T cells that are exposed to model antigens in the secondary lymphoid organs in the absence of inflammation proliferate poorly, then most of the progeny die, and the survivors enter an anergic state characterized by poor lymphokine production. The goal of this application is to establish whether or not a similar series of events accounts for peripheral tolerance to certain natural self-proteins. This has become a pressing issue since the discovery that the AIRE transcription factor drives ectopic expression of extrathymic gene products in the thymus. Thus, it remains possible that the physiological role that peripheral tolerance was thought to play is really played by AIRE-mediated intrathymic tolerance. Here, we will test the hypothesis that peripheral tolerance is physiologically-relevant by using new tools to identify the tolerance mechanisms that apply to two pregnancy-specific proteins, one that appears to be regulated by AIRE and another that does not, and one sperm-specific protein. We will determine whether or not these proteins are immunogenic in mice that have never expressed them in the relevant tissue (e.g., non-pregnant female mice), and become non-immunogenic in mice after expression (e.g., pregnant female mice). The relevant antigenic peptides will be identified and used to produce peptide-MHC II multimers. The multimers will then be used with a sensitive new enrichment method capable of detecting fewer than 100 cells per mouse to enumerate peptide MHC II- specific CD4+ T cells before, during, and after expression of the relevant protein within the polyclonal repertoires of normal mice. This approach should reveal whether or not the relevant CD4+ T cells are deleted, turn into regulatory cells, or become anergic during or after the period when these developmental^ regulated self-proteins are expressed. This approach will then be used in gene-targeted mice to determine whether or not molecules such as Fas and Cbl-b, and others identified by other members of the P01, are involved in the identified tolerance mechanism. Success would provide the first definitive identification of the peripheral tolerance mechanism that applies to a natural self-antigen. This information should help focus future research on the relevant mechanism and shed light on the potential ways that it could fail and lead to autoimmunity.