The mechanochemical control of T-cell directional migration under flow Daniel A. Hammer (PI) and Janis K. Burkhardt (co-Investigator) Project Summary T-lymphocytes are key players in the adaptive immune response, and motility is critical to their function. T- cells are equipped with multiple different adhesion molecules that interact with ligands that are expressed differentially throughout the immune system. Furthermore, T-cells often must act under an imposed flow field as they traffic through the vasculature and lymphic system. Our goal is to understand how T-cells respond to the different adhesion ligands and shear rates they encounter to effectively migrate to sites of inflammation and immune communication. Understanding this process at the molecular level is important for development of therapeutic strategies to treat inflammatory and infectious diseases, and cancer Recently, we have discovered that directional T-cell migration varies as a function of the type of ligand they encounter and the shear rate to which they are exposed. When placed on a surface bearing vascular cell adhesion molecule-1 (VCAM-1), which engages the ?1-integrin receptor VLA-4, T-cells crawl downstream under flow (in the direction of flow). However, when placed on a surface bearing intercellular adhesion molecule-1 (ICAM-1), which engages the ?2-receptor LFA-1, T-cells crawl against the direction of flow, like a salmon swims upstream. The magnitude of upstream migration depends on shear rate, with T-cells more committed to upstream migration as the shear rate increases. On surfaces in which adhesion molecules are mixed, any amount of ICAM-1 supports upstream migration. When the flow is removed, T-cells exhibit migrational memory, but only if they have been exposed to both ICAM-1 and VCAM-1. This observation points to a novel mechanism of crosstalk between two distinct integrin receptors. We propose to investigate the mechanisms that drive the upstream migration of T-cells under flow on ICAM-1, and the origins of migrational memory. We hypothesize that upstream migration is caused by ?2 integrin forming a catch bond, which holds the cell in place while signals generated by integrin ligation strengthen adhesive interactions and spur the polymerization of actin at the leading edge, driving forward migration. To test this, we will use molecular engineering, flow chambers, micropatterned surfaces, and microfabricated post array detectors (mPADs) to measure forces exerted by the migrating cell. We have preliminary evidence that other motile amoeboid cells such as the immortalized KG1a cell line display the same phenomenon, facilitating our use of molecular engineering tools and imaging methods to identify the relevant molecules. By dissecting the mechanisms that underlie this fascinating phenomenon, we expect to elucidate key features of integrin-dependent T cell trafficking.
Our aims i n this work are to: 1. Measure the dynamics of T-cell and KG1a directional motion and migrational memory; 2. Identify the signals and clutch molecules responsible for the differential migration under flow in response to ?1 and ?2 integrin ligands; and 3. Measure the mechanisms of force generation when T-cells spread and crawl directionally on integrin ligands.

Public Health Relevance

The mechanochemical control of T-cell directional migration under flow Daniel A. Hammer (PI), Janis K. Burkhardt (co-I) Relevance T-lymphocytes are key players in the adaptive immune response, and developing a fundamental understanding of T-cell motility would lead to new therapeutic strategies for treating inflammatory and infectious diseases, and improved methods for cell therapy for treating cancer. It has been found that T-lymphocytes can crawl in different directions under flow, depending on the adhesion molecules they encounter. We will use biomolecular and biophysical tools to understand why T-lymphocytes display this bidirectionality.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Sammak, Paul J
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University of Pennsylvania
Biomedical Engineering
Biomed Engr/Col Engr/Engr Sta
United States
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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:
Anderson, Nicholas R; Lee, Dooyoung; Hammer, Daniel A (2018) An Experimentally Determined State Diagram for Human CD4+ T Lymphocyte CXCR4-Stimulated Adhesion Under Shear Flow. Cell Mol Bioeng 11:91-98
Buffone Jr, Alexander; Anderson, Nicholas R; Hammer, Daniel A (2018) Migration against the direction of flow is LFA-1-dependent in human hematopoietic stem and progenitor cells. J Cell Sci 131: