Costimulatory ligand mobility effects on T cell activation

描述（由申请人提供）：共刺激信号降低T细胞活化的阈值，并调节免疫应答，将T细胞推向不同的效应物谱系，并在免疫和耐受性之间建立平衡。由于它们在形成免疫应答中的中心作用，并且由于共刺激受体-配体相互作用的保守性质，共刺激分子是强大的治疗靶标。T细胞与pMHC和共刺激配体如CD 80/86和ICAM-1的相互作用发生在免疫突触处。一些证据表明，DC细胞骨架在调节免疫突触的信号事件中起着重要作用，但其分子基础尚不清楚。我们发现，虽然pMHC在DC膜上相对自由地移动，但DC肌动蛋白细胞骨架限制了ICAM-1和CD 80的移动性，并且我们已经确定了两种肌动蛋白结合蛋白，膜突蛋白和辅肌动蛋白，它们在这一过程中发挥关键作用。我们假设细胞骨架对DC表面ICAM-1和CD 80的流动性的限制通过在受体上产生张力和调节突触T细胞侧的信号微簇的动力学来促进共刺激信号传导。这一假设将通过实现三个具体目标来检验。首先，我们将描述膜突蛋白和辅肌动蛋白与ICAM-1和CD 80胞质尾部的基本序列的相互作用，并测试干扰这些相互作用对ICAM-1和CD 80侧向扩散的影响。第二，与膜生物制药学家Tobias Baumgart合作，我们将使用新的混合流动性表面来测试不同ICAM-1流动性和模式的影响，同时保持pMHC处于移动的状态。将评估T细胞对这些表面的反应的几个方面，包括特定的信号传导事件，LFA-1构象变化，以及T细胞细胞骨架和相关信号分子的动力学。最后，我们将工程DC中的ICAM-1的流动性被改变，无论是通过干扰肌动蛋白结合蛋白或突变的相互作用的残留物在ICAM-1的尾巴，并测试T细胞活化和谱系发展的影响在体外。还将进行连续转移实验以使用活体成像测试ICAM-1迁移率对体内T细胞-DC相互作用的影响。总之，这些研究将测试一个未探索的方面的共刺激信号，并将提供重要的新的见解如何DC肌动蛋白细胞骨架调节免疫反应。这些信息将指导临床工作，以操纵DC功能的疫苗开发，移植医学和治疗过敏和自身免疫性疾病。

DESCRIPTION (provided by applicant): Costimulatory signals lower the threshold for T cell activation, and tune the immune response, driving T cells towards distinct effector lineages and establishing the balance between immunity and tolerance. Because of their central role in shaping the immune response, and because of the conserved nature of costimulatory receptor-ligand interactions, costimulatory molecules are powerful therapeutic targets. Interaction of T cells with pMHC and costimulatory ligands such CD80/86 and ICAM-1 takes place at the immunological synapse. Several pieces of evidence suggest that the DC cytoskeleton plays an important role in modulating signaling events at the immunological synapse, but the molecular basis for this is not understood. We have discovered that while pMHC moves relatively freely on the DC membrane, the DC actin cytoskeleton constrains the mobility of ICAM-1 and CD80, and we have identified two actin-binding proteins, moesin and ¿-actinin, that play a key role in this process. We hypothesize that cytoskeletal constraints to mobility of ICAM-1 and CD80 on the DC surface promote costimulatory signaling, by creating tension on receptors and modulating dynamics of signaling microclusters on the T cell side of the synapse. This hypothesis will be tested by carrying out three specific aims. First, we will characterize the interactions of moesin and ¿- actinin with basic sequences in the cytoplasmic tails of ICAM-1 and CD80, and test the effects of perturbing these interactions on lateral diffusion of ICAM-1 and CD80. Second, in collaboration with membrane biophysicist Tobias Baumgart, we will use novel mixed mobility surfaces to test the effects of varying ICAM-1 mobility and patterning while maintaining pMHC in a mobile state. Several aspects of the T cell response to these surfaces will be assessed, including specific signaling events, LFA-1 conformational change, and dynamics of the T cell cytoskeleton and associated signaling molecules. Finally, we will engineer DCs in which ICAM-1 mobility is altered, either by perturbing actin-binding proteins or by mutating interacting residue in the ICAM-1 tail, and test the effects on T cell activation and lineage development in vitro. Adoptive transfer experiments will also be performed to test the effects of ICAM-1 mobility on T cell-DC interactions in vivo using intravital imaging. Taken together, these studies will test an unexplored aspect of costimulatory signaling, and will provide important new insights into how the DC actin cytoskeleton modulates the immune response. This information will guide clinical efforts to manipulate DC function in vaccine development, transplantation medicine and treatment of allergy and autoimmune disease.