Here we seek to understand how the dendritic cell (DC) actin cytoskeleton governs antigen presentation to cognate T cells and also how bidirectional communication between the two cells can rearrange cytoskeletal networks to orchestrate immune responses. DC initiates antigen presentation by imparting context specific information to responder T cells. Correct information drives T cells to dynamically reorient their actin network towards the site of DC-T cell contact and assemble actin-linked intracellular signaling components. This results in long-term sustained signaling events that induce cell behavioral and gene expression changes, thereby leading to onset of adaptive immunity. Although contribution of the actin network in T cells is well established, little is understood of C cytoskeletal roles in antigen presentation and immunity. It is also unclear how crosstalk between DC and T cells through receptor-ligand engagement at the immunological synapse (IS) could rearrange the DC cytoskeleton to influence the myriad of T cell responses. Previous studies strongly support the DC actin network playing significant roles in antigen presentation. Additionally, our preliminary work highlights the role of CD40 signaling in rearranging the actin cytoskeletal network in DC to promote efficient antigen presentation. We hypothesize that the ability to relay communication to cognate responder T cells occurs through modulation of the DC actin cytoskeleton into stiff and rigid networks. This transition of the DC network would then support in trans mobility, assembly, and activation of responder T cell signaling molecules. In this project, we will delineate the role of CD40 signaling in directing DC to upregulate and recrui the actin-bundling protein fascin to the DC IS.
In Aim 1 A we will define the role of CD40 ligation in modulating differential fascin expression, phosphorylation-activation states, and localization o fascin at the IS.
Aim 1 B will delineate the downstream intracellular signaling events of CD40 vs. the role of CD40-CD40L clustering at the IS in regulation of fascin activities. Finally, in Aim 1C we evaluate wild type vs. dominant negative mutant fascin ability to rescue CD40-defecient DC priming of responder T cells. Results will provide a better understanding of the role the DC cytoskeleton plays in antigen presentation and propose a model for a larger framework of studies describing: (a) polarized cargo transport along actin-bundled networks; (b) receptor- ligand mobility and membrane fluidity at actin-bundled sites; and (c) microtubule network dynamics in DC and respective contribution to T cell responses. Information uncovered in these studies may provide blueprints for development of tunable artificial antigen presenting material and rational design of DC-based immunotherapies.
Immunotherapies offer promising new clinical approaches. The therapies can induce strong antigen-specific immunity to combat cancer and infectious diseases or alternatively be engineered to inhibit autoimmunity or transplant rejection. Whether using dendritic cell-based, adoptive T cells, or biomimetic artificial antigen presenting cells as effective therapeutic agents, understanding the underlying mechanism that allows orderly coordination of antigen-specific T cell responses is required. This project will shed new light on the contribution of the dendritic cell actin cytoskeleton network in orchestrating effective primin of antigen-specific T cell immune responses. The knowledge gained from these studies will be important for rational development of effective immunotherapies.
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