Two molecules believed to regulate the adhesion of lymphocytes to the specialized endothelium of lymph nodes and related lymphoid structures will be studied to define the functional domains of the molecules required for successful endothelial interaction. A saturation mutagenesis/antibody selection scheme will be used to derive monoclonal antibody epitope loss variants which will be screened for loss of function in two lymphocyte binding assays. One of these, the lymphocyte transmigration assay, relies on cultured node endothelial cells; for this and related purposes, primary cultures of rat, monkey, and human peripheral and mesenteric node endothelial cells will be established. The cultured cells will be used as targets for transfectants bearing mutated homing receptors and for soluble forms of the homing receptors created by gene fusion of the extracellular domains, or subdomains, to tag sequences derived from human or mouse immunoglobulin constant regions. An attempt will be made to biochemically define the target molecules with which the homing receptors interact; because these molecules need not be proteins, biochemical fractionation schemes are proposed to help establish whether carbohydrate recognition (suggested by the lectin or link-protein homologies of the homing receptors) or protein recognition fulfills the dominant role in determining homing specificity. However if a protein component is involved, either as a scaffolding structure or for direct interaction with the homing receptors, polyclonal or monoclonal antibodies will be generated against the protein component, and the appropriate cDNA will be isolated from a library generated from the node endothelial cells.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Allergy and Immunology Study Section (ALY)
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Massachusetts General Hospital
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