CD47 Regulation of Leukocyte Integrins during Leukocyte Trafficking

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 DESCRIPTION (provided by applicant): Inflammation is a significant contributor to cardiovascular disease and related pathophysiological processes including stroke, hypertension, atherosclerosis, diabetes-metabolic syndrome. The activation and recruitment of effector T cell subsets into tissues is a highly regulated process that is dependent upon adhesive interactions between T cell subsets and the endothelium lining the vascularture. Therefore, our overall goal is to gain a better understanding of the mechanisms that regulate CD4+ T cell subset recruitment to sites of immune-mediated inflammation. Based on our exciting preliminary data and recent publication, we hypothesize that CD47 plays an important role in leukocyte β1 and β2 integrin adhesive functions by regulating the expression of high affinity conformations. By virtue of its effect on LFA-1 and VLA-4 integrin adhesive function and its well-documented role in immune cell homeostasis, CD47 and its ligand SIRPα play a critical role in multiple steps (arrest and migration) in the inflammatory response. The specific aims will determine the molecular mechanisms and underlying structural basis of CD47 regulation of T cell LFA-1 and VLA-4 adhesive function and its role in T effector cell functions. We propose complementary and well-characterized in vitro cell culture systems and novel CyTOF mass spectroscopy technology to dissect the biochemical and molecular signaling pathways used by CD47 in human and murine immune cells to regulate integrin function. Specifically, Specific Aim 1 will employ recently developed single cell mass spectrometry (CyTOF mass spectrometry) and standard biochemical assays to interrogate intracellular signaling pathways in CD47-/- T cells to identify defects that cause impaired integrin activation in CD47-/- leukocytes. Up to 40 cell-surface molecules, intracellular signaling molecules and mediators (cytokines) can be measured in a single sample using CyTOF to provide much more information about cell phenotypes at a single-cell level. Specific Aim 2 will address whether in trans CD47 ligands, Signal Regulatory Protein (SIRP)α, and SIRPγ, and the secreted extracellular matrix protein, thrombospondin-1, affect CD47 in cis regulation of LFA-1 and VLA4 function during T cell recruitment. In Specific Aim 3 we propose to determine the residues in CD47 that interact with β2 integrins, which may provide guidance for designing novel and innovative therapeutic target(s), and identify biological reagents that impair these interactions. These approaches and our collaborations with established investigators will generate novel insights into CD47 regulation of integrin activation and adhesive functions in T cell trafficking.