Protective immune responses rely on the ability of leukocytes to cross endothelial barriers in response to inflammatory stimuli. However, inflammation is a double-edged sword; immune cells attack healthy tissues in situations such as asthma, rheumatoid arthritis, multiple sclerosis, and transplantation. T cell migration into sites f inflammation is tightly regulated by localized chemokine signals, which activate integrin dependent adhesion and cytoskeletal changes needed for migration across endothelial barriers. Transendothelial migration (TEM) is a multi-step process involving tethering and rolling, firm adhesion, crawling, and finally transmigration. TEM represents an important checkpoint in the inflammatory response, and reagents that block this process are therapeutically valuable. We have found that Crk family proteins function in the chemokine pathway leading to integrin activation and cytoskeletal remodeling, and we find that T cells lacking these proteins have defects in adhesion and TEM. Strikingly, we find that these T cells traffic normally to lymphoid organs, but show defects in migration to sites of inflammation. Moreover, these cells can carry out an efficient graft vs leukemia (GvL) response, with minimal graft vs host disease (GvHD). We hypothesize that Crk proteins are required for chemokine-induced integrin activation and cytoskeletal remodeling in T cells, and for corresponding changes in the endothelium that allow transendothelial migration and T cell trafficking into inflamed tissue. This hypothesis will be tested by carrying out following three Aims: First, we will define the mechanisms through which Crk protein signaling in T cells promotes TEM. Using ligand-coated surfaces and endothelial monolayers under shear flow conditions, we will test the ability of Crk/L deficient T cells to undergo individual steps in the TEM process, and test hypothesis that Crk proteins function through both Rap1 and Cdc42 in this context. Second, we will assess endothelial cell responses to Crk/L-deficient T cells. We will test the hypothesis that defects in Crk-dependent integrin activation within the T cell result in a failure to appropriately activate one or more endothelial ell signaling pathways that are required for efficient TEM. Three pathways that function together to weaken endothelial junctional complexes and generate a specialized conduit for T cell passage will be investigated. Third, we will analyze Crk protein dependent T cell trafficking in acute inflammation and GvHD/GvL models. Consistent with their role in controlling TEM, we find that Crk/L deficient T cells show defects in migration into inflamed tissues. Moreover, these cells cause minimal GvHD, even under conditions where they can efficiently eliminate lymphoma. In this aim, we will use animal models to explore Crk protein function in T cell trafficking during an in vivo immune response. We will conduct histological analysis and intravital imaging to study T cell-vessel interactions in inflamed skin. In addition, we will use GvHD and GvHD/GvL models to study T cell trafficking to target organs, and to test our ability to target Crk protein function i a therapeutic setting.
T cell recirculation and migration into sites of inflammation is highly orchestrated by molecules that control adhesion and migration through endothelial barriers. This proposal tests the role of Crk family proteins, which differentially control discret aspects of T cell trafficking. The knowledge gained from these studies will be important for modulating T cell function, to augment their ability to kill leukemic cells while minimizing graft-s- host disease and other pathological inflammatory responses.
| Roy, Nathan H; MacKay, Joanna L; Robertson, Tanner F et al. (2018) Crk adaptor proteins mediate actin-dependent T cell migration and mechanosensing induced by the integrin LFA-1. Sci Signal 11: |
| Du, Jing; Flynn, Ryan; Paz, Katelyn et al. (2018) Murine chronic graft-versus-host disease proteome profiling discovers CCL15 as a novel biomarker in patients. Blood 131:1743-1754 |
| Jankowska, Katarzyna I; Burkhardt, Janis K (2017) Analyzing Actin Dynamics at the Immunological Synapse. Methods Mol Biol 1584:7-29 |
