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T cell development requires sequential localization of thymocyte subsets to distinct thymic microenvironments. To address mechanisms governing this segregation, we used two-photon microscopy to visualize migration of purified thymocyte subsets in defined microenvironments within thymic slices. Double-negative (CD4 − 8 − ) and double-positive (CD4 + 8 + ) thymocytes...
Immature double-positive (CD4 + CD8 + ) thymocytes respond to negatively selecting peptide-MHC ligands by forming an immune synapse that sustains contact with the antigen-presenting cell (APC). Using fluorescently labeled peptides, we showed that as few as two agonist ligands could promote APC contact and subsequent apoptosis in reactive thymocytes. Furthermore, we showed that productive...
To study the spatio/temporal recruitment of lck during immunological synapse formation, we utilize high-speed time-lapse microscopy to visualize green fluorescent protein (GFP) fusions of lck and CD3ζ following agonist or altered peptide ligand (APL) stimulation. The dynamics of lck and CD3ζ recruitment are comparable; however, lck becomes excluded to the periphery of mature synapses, while most CD3ζ...
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