Thymocyte tuning after selection: mechanisms to avoid autoreactivity

描述(由申请人提供)：人类和小鼠狼疮中抗核抗体的产生与自身反应性CD4+辅助T细胞的不适当激活有关。胸腺克隆性删除似乎完好无损，这表明自我反应性T细胞不仅仅是逃避负选择。我们假设，T细胞耐受的选择后机制的缺陷，我们称之为发育调节，有助于狼疮的发病。为了避免自身免疫，阳性选择之后必须有一段发育调整期，此时T细胞受体(TCR)对低亲和力自体多肽的反应性被抑制。我们假设这种自我耐受的基本机制在狼疮中被破坏了。我们最近利用限制表达MHCⅡ类基因的转基因小鼠来证明，经筛选后不能与MHCII相互作用的CD4SP胸腺细胞保留了未成熟细胞的表型，并对TCR信号具有高反应性。我们认为，发育调节是一个活跃的事件，需要CD4SP胸腺细胞和MHC II类(MHCII)胸腺髓质基质之间的持续相互作用。我们现在发现，狼疮易感小鼠的CD4SP胸腺细胞也有类似的过度活跃。我们现在建议确定调控发育调节的分子机制。在特定的目标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相互作用以调节调节。然后，我们将利用转基因小鼠和骨髓嵌合体来确定哪种MHC 11阳性胸腺基质细胞-骨髓上皮或造血祖细胞-介导CD4SP成熟。我们的数据表明，发育调节通过增加酪氨酸激酶Lck对CD4的忠诚度，使T细胞的激活更依赖于CD4的参与。在特定的目标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调节的。最后，在第三个特定目标中，我们将询问自发产生抗核抗体和狼疮的两种小鼠模型的发育调节是否受到干扰。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途径的机制。

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.