Thymocyte tuning after selection: mechanisms to avoid autoreactivity

描述（由申请人提供）：人类和小鼠狼疮的抗核抗体产生与自身反应性CD4+辅助性T细胞的不适当激活有关。胸腺克隆缺失似乎是完整的，这表明自反应性T细胞不仅仅是逃避负选择。我们假设T细胞耐受的后选择机制中的缺陷，我们称之为发育调节，有助于狼疮的发病机制。为了避免自身免疫，阳性选择之后必须经过一段发育调节期，此时T细胞受体（TCR）对低亲和力自身肽的反应性被抑制。我们假设这种自我耐受的基本机制在狼疮中被破坏了。我们最近利用限制MHCII类表达的转基因小鼠来证明CD4 SP胸腺细胞在被选中后不能与MHCII相互作用，保留了未成熟细胞的表型，并对TCR信号反应过度。我们提出发育调节是一个活跃的事件，需要CD4 SP胸腺细胞和MHCII类（MHCII）胸腺髓质间质之间持续的相互作用。我们现在发现来自狼疮易感小鼠的CD4 SP胸腺细胞也同样亢进。我们现在建议确定调节发育调节的分子机制。在Specific Aim I中，我们将定义调节发育调节的分子和细胞相互作用。首先，我们将确定MHC II类分子是否与T细胞受体或CD4相互作用以介导调节。然后，我们将利用转基因小鼠和骨髓嵌合来确定MHC类型的all阳性胸腺基质细胞-髓上皮细胞或造血apc介导CD4 SP成熟。我们的数据表明，通过增加酪氨酸激酶（Lck）对CD4的忠诚，发育调节使T细胞激活更依赖于CD4的参与。在Specific Aim II中，我们将采用生化方法来确定Lck和CD4的亚细胞定位和关联是如何被调节的。最后，在第三个特定目标中，我们将询问自发抗核抗体产生和狼疮的两种小鼠模型中发育调节是否被破坏。这些实验将确定调节免疫系统用来避免自身反应的关键胸腺通路的机制。

DESCRIPTION (provided by applicant): Anti-nuclear antibody production in human and murine lupus is associated with inappropriate activation of self-reactive CD4+ helper T cells. Thymic clonal deletion appears intact, suggesting that self-reactive T cells do not merely escape negative selection. We hypothesize that defects in a post-selection mechanism of T cell tolerance that we call developmental tuning contribute to the pathogenesis of lupus. To avoid autoimmunity, positive selection must be followed by a period of developmental tuning when T cell receptor (TCR) responsiveness to low affinity self-peptides is inhibited. We hypothesize that this fundamental mechanism of self-tolerance is disrupted in lupus. We recently utilized transgenic mice with restricted expression of MHC class II to demonstrate that CD4 SP thymocytes that cannot interact with MHCII after they have been selected retain the phenotype of immature cells and are hyperresponsive to TCR signaling. We proposed that developmental tuning is an active event requiring ongoing interactions between the CD4 SP thymocyte and MHC class II (MHCII) thymic medullary stroma. We now find that CD4 SP thymocytes from lupus prone mice are similarly hyperactive. We now propose to identify the molecular mechanisms regulating developmental tuning. In Specific Aim I, we will define the molecular and cellular interactions which regulate developmental tuning. First, we will determine whether MHC class II molecules are interacting with the T cell receptor or CD4 to mediate tuning. Then, we will utilize transgenic mice and bone marrow chimeraes to determine which MHC class ll-positive thymic stromal cells-medullary epithelium or hematopoietic APC-mediate CD4 SP maturation. Our data suggest that developmental tuning makes T cell activation more dependent on engagement of CD4 by increasing the allegiance of the tyrosine kinase, Lck, for CD4. In Specific Aim II, we will take a biochemical approach to determine how the subcellular localization and associations of Lck and CD4 are regulated. Finally, in the third Specific Aim we will ask if developmental tuning is disrupted in two murine models of spontaneous anti-nuclear antibody production and lupus. These experiments will define the mechanisms which regulate a critical thymic pathway that the immune system utilizes to avoid autoreactivity.