Costimulatory ligand mobility effects on T cell activation

描述(申请人提供)：共刺激信号降低T细胞激活的阈值，调节免疫反应，驱动T细胞走向不同的效应谱系，建立免疫和耐受之间的平衡。由于它们在形成免疫反应中的核心作用，以及由于共刺激受体-配体相互作用的保守性，共刺激分子是强大的治疗靶点。T细胞与pMHC和共刺激配体如CD80/86和ICAM-1的相互作用发生在免疫突触。一些证据表明，DC细胞骨架在调节免疫突触的信号事件中发挥着重要作用，但其分子基础尚不清楚。我们发现，虽然pMHC在DC膜上相对自由运动，但DC肌动蛋白细胞骨架限制了ICAM-1和CD80的流动性，我们已经确定了两种肌动蛋白结合蛋白，Moesin和β-Actinin，它们在这一过程中发挥了关键作用。我们假设，细胞骨架对DC表面ICAM-1和CD80的流动性的限制通过对受体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。这一假设将通过实现三个具体目标来检验。首先，我们将表征Moesin和？-Actinin与ICAM-1和CD80胞质尾部碱性序列的相互作用，并测试这些相互作用对ICAM-1和CD80横向扩散的影响。其次，与膜生物物理学家Tobias Baumgart合作，我们将使用新型混合迁移率表面来测试在保持pMHC处于移动状态的同时改变ICAM-1迁移率和图案的效果。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，我们将通过干扰肌动蛋白结合蛋白或突变ICAM-1尾部的相互作用残基来改变ICAM-1的迁移率，并在体外测试其对T细胞激活和谱系发育的影响。还将进行过继转移实验，以利用活体成像测试ICAM-1移动性对体内T细胞-DC相互作用的影响。综上所述，这些研究将测试共刺激信号的一个未知方面，并将为DC肌动蛋白细胞骨架如何调节免疫反应提供重要的新见解。这些信息将指导临床努力在疫苗开发、移植医学以及过敏和自身免疫性疾病的治疗中操纵DC功能。

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.