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

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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.