Chemoattractant-specific T cell navigation of complex environments

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PROJECT SUMMARY/ABSTRACT T cell trafficking is crucial for development, immune surveillance, and effector function. Migration is a complex process, choreographed by a host of chemoattractants that signal through GPCRs to direct T cell cytoskeletal responses. In vivo work shows that chemokines like CCL19 and CCL21 (CCR7 ligands) mediate naïve T cell migration within lymphoid tissues, while the lipid chemoattractant S1P regulates egress. A similar process occurs in peripheral tissues, where these signals control migration of migratory effector T cells out of inflamed tissues and into afferent lymphatics. Our lab recently overcame a longstanding technical problem that made it difficult to study S1P responses in ex vivo T cells. Using this advance, we discovered that these two chemotactic signals induce distinct modes of T cell motility. CCL19 induces long-duration lamellipodial migration while S1P induces a shorter burst of bleb-based motility. This work raises several questions: How do these chemoattractants elicit such different migratory responses? What do T cells do when confronted with competing cues? Why do T cells need multiple motile mechanisms? We hypothesize that CCR7 ligands and S1P activate different cytoskeletal signaling pathways that direct distinct modes of motility, which work alone or in combination to allow T cells to navigate complex environmental obstacles like those they encounter in vivo. To test this hypothesis, we will carry out two sets of studies. In Aim 1, we will pursue our preliminary data showing that CCL19 preferentially activates a Rac1-dependent pathway leading to lamellipodial protrusion, while S1P preferentially activates a pathway involving RhoA and phospholipase activity, which directs myosin-dependent contractility and bleb formation. To verify that that these signaling events are causally linked to the migratory responses we observe, we will treat cells with pharmacological inhibitors and assess motile responses and cytoskeletal remodeling using transwell assays and live cell imaging. To ask how cells integrate signals from multiple chemoattractants, cells will be exposed to S1P and CCL19 simultaneously and sequentially, and signaling responses and cell migration will be analyzed. In Aim 2, we will test the idea that CCL19-induced lamellipodial motility is optimized for long-distance migration in relatively unconfined settings, while S1P-induced bleb-based motility permits cells to pass through small, highly confined spaces. To achieve this, we will test chemotaxis within 3D collagen gels and passage through microfluidic channels with variable geometries that mimic in vivo challenges. As part of this analysis, we will ask how actin and myosin are redistributed in the cell as a function of chemoattractant stimulus and confinement. Finally, we will analyze T cell passage across lymphatic endothelial barriers using transwell assays and tissue explants derived from mouse ears. If successful, this project will complement existing in vivo studies of T cell trafficking by providing much needed mechanistic insights into the underlying molecular and cell biological mechanisms. In the long run, our findings will reveal valuable targets for the rational design of therapeutic approaches based on modulating T cell trafficking.