Thymocyte tuning after selection: mechanisms to avoid autoreactivity

描述（由申请方提供）：人和鼠狼疮中抗核抗体的产生与自身反应性CD4+辅助T细胞的不适当激活相关。胸腺克隆缺失似乎是完整的，这表明自身反应性T细胞不仅仅是逃避负选择。我们假设，在一个后选择机制的T细胞耐受性，我们称之为发展调整的缺陷有助于狼疮的发病机制。为了避免自身免疫，阳性选择之后必须有一段时间的发育调整，此时T细胞受体（TCR）对低亲和力自身肽的反应性被抑制。我们推测，这种自我耐受的基本机制在狼疮中被破坏。我们最近利用限制性表达MHC II类分子的转基因小鼠来证明，CD4 SP胸腺细胞在被选择后不能与MHCII相互作用，保留了未成熟细胞的表型，并且对TCR信号传导具有高反应性。我们提出，发展调整是一个积极的事件，需要持续的相互作用之间的CD4 SP胸腺细胞和MHC II类（MHCII）胸腺髓质基质。我们现在发现，来自狼疮易感小鼠的CD4 SP胸腺细胞也同样过度活跃。我们现在建议确定调节发育调谐的分子机制。在具体目标I中，我们将定义调节发育调谐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相互作用以介导调谐。然后，我们将利用转基因小鼠和骨髓嵌合体，以确定哪些MHC II类阳性胸腺基质细胞-髓质上皮或造血APC-介导的CD4 SP成熟。我们的数据表明，发展调整，使T细胞活化更依赖于参与的CD4通过增加效忠的酪氨酸激酶，LCK，CD4。在具体目标II中，我们将采取生物化学方法来确定Lck和CD4的亚细胞定位和关联是如何调节的。最后，在第三个具体目标中，我们将询问在自发性抗核抗体产生和狼疮的两种小鼠模型中，发育调谐是否被破坏。这些实验将确定调节免疫系统用来避免自身反应性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.