Spatial optimization of T cell activation at inflamed sites via cytokine/chemokine-dependent cellular clustering

项目总结/摘要-项目2 在炎症/感染部位的效应T细胞活化的时空控制是必需的，但很差。 理解过程。大多数活体成像研究已经得出结论，T细胞扫描炎症组织， 随机无方向的方式。因此，CD 4 + T细胞最终如何定位自身以获得有效的抗- 病原体免疫仍然难以捉摸。对于Th 1效应物，效应T细胞活化的最佳位置可能是 取决于分泌的效应分子如IFNγ的活性范围，估计在约80微米的范围内， 皮肤利什曼原虫感染。尽管知道一些关键的细胞和分子参与者 炎症部位的T细胞积聚所必需的，它们如何在空间和时间上定位， 释放仍然是一个关键的知识差距，阻碍了新的治疗方法， 增强对感染的免疫力并减少自身免疫性组织损伤。 使用CXCL 9/10荧光报告小鼠可视化趋化因子产生的细胞来源/位置 和IV-MPM追踪Th 1迁移，我们发现趋化因子产生细胞在空间上受到限制， 血管周围簇（PVC）富含MHC-II高抗原呈递细胞，并形成了血管周围的血管。 Th 1细胞在发炎/感染的真皮中的定位和运动性。我们的总体假设是， 外周活化发生在富含趋化因子的血管周围簇中， 扩增T细胞募集和活化以有效清除病原体。该成核步骤可以 促进有效的病原体清除，但也可能加剧免疫损伤的程度， 自身免疫性疾病该提案使用IV-MPM和光活化工具进行时空解剖， 这些趋化因子“枢纽”的组织、组成和对Th 1活化的影响，以及它们在 优化感染点的保护性免疫。 目标1。通过先天性细胞组织富含趋化因子的血管周围簇：Th 1串扰。测试 假设最初富含趋化因子的PVC用于激活进入组织的早期Th 1“先锋”， Th 1细胞因子驱动局部阳性扩增环以促进随后的Th 1细胞募集。 目标2.簇内T细胞活化的功能影响。我们假设， 趋化因子产生细胞和PVC内的抗原呈递都起到使信号成核的作用， 有效的Th 1活化。使用原位光活化，PA-GFP，我们将标记PVC内外的Th 1 并确定PVC内的活化是否赋予明显的功能优势。 目标3：血管周围簇与感染灶的关系。富含趋化因子的PNC 距离原发性感染部位100-400µm处可以发现含有Th 1细胞的细胞。我们假设早期 PVC成核之后是在感染病灶处积累的活化的Th 1细胞的局部离散。

PROJECT SUMMARY/ABSTRACT – PROJECT 2 Spatiotemporal control of effector T cell activation at sites of inflammation/infection is an essential yet poorly understood process. Most intravital imaging studies have concluded that T cells scan inflamed tissues in a random non-directional fashion. Therefore, how CD4+ T cells ultimately position themselves for effective anti- pathogen immunity remains elusive. For Th1 effectors the optimal location of effector T cell activation is likely dependent on the active range of secreted effector molecules such as IFNγ, estimated to be ~80 microns in cutaneous Leishmania major infection. Despite knowing some of the key cellular and molecular players essential for T cell accumulation at sites of inflammation, how they are spatially and temporally positioned and released remains a critical knowledge gap that hinders new approaches to therapeutic manipulation to enhance immunity to infection and to diminish autoimmune tissue damage. Using CXCL9/10 fluorescent reporter mice to visualize the cellular source/location of chemokine production and IV-MPM to track Th1 migration, we found chemokine producing cells were spatially restricted to perivascular clusters (PVC) that were enriched in MHC-IIhigh antigen presenting cells and that shaped the localization and motility of Th1 cells in the inflamed/infected dermis. Our overall hypothesis is that initial peripheral activation occurs in chemokine-rich peri-vascular clusters that serve to nucleate and amplify T cell recruitment and activation for efficient pathogen clearance. This nucleation step may facilitate efficient pathogen clearance but may also exacerbate the magnitude of immune damage in autoimmune settings. This proposal uses IV-MPM and photoactivation tools for spatiotemporal dissection of the organization, composition and impact of these chemokine `hubs' on Th1 activation and their role in optimizing protective immunity at foci of infection. Aim 1. Organization of chemokine-rich perivascular clusters via innate cell:Th1 cross-talk. To test the hypothesis that initial chemokine-rich PVCs serve to activate early Th1 `pioneers' entering the tissue and that Th1 cytokines drive a local positive amplification loop to boost subsequent Th1 cell recruitment. Aim 2. Functional impact of T cell activation within the clusters. We hypothesize that the positioning of both chemokine producing cells and antigen presentation within the PVCs serves to nucleate signals for efficient Th1 activation. Using in situ photoactivation, PA-GFP, we will mark Th1s within and outside the PVC and determine if activation within the PVC confers distinct functional advantages. Aim 3. Relationship between peri-vascular clusters and the infection foci. Chemokine-rich PNCs containing Th1 cells can be found 100-400µm from the site of primary infection. We hypothesize that early PVC nucleation is followed by local diaspora of activated Th1 cells that accumulate at infection foci.