This project focuses on the adhesive interaction between human lymphoid cells and the high endothelial venules of peripheral lymph nodes (PNHEV) which initiates the migration of circulating cells into tissue. In rodents, this process contributes to the trafficking of normal lymphocytes and the dissemination of lymphoid malignancies. The principle investigator has proposed that a lectin-like structure at the lymphocyte surface with phosphomannan-binding activity (PMRad) mediates this interaction. In the murine system, PMRad and the mel 14 antigen, an independently characterized 90 kd adhesion structure, are either closely linked or identical. In humans, the relationship between PMRad and the hermes antigen, the 85-95 kd analog of the mel 14 antigen, is unknown. Furthermore, the relationship of PMRad to the well characterized phosphomannan receptors mediating delivery of acid hydrolases to the lysosome (PMRup) has not been clearly established. Comparative analyses of these recognition structures are hampered by the unavailability of purified PMRad and the lack of antibodies specific for the crucial ligand-binding domains. The PI. therefore, proposes development of monoclonal antibodies (Mabs) specific for the carbohydrate-binding domains of lymphocytic lectins. Initial studies have identified a series of cultured T-lymphoblastic malignancies with either constitutive or inducible expression of PMRad, PMRup and binding to PNHEV. These lines will be used to: (1) generate Mabs which block attachment of phosphomannan-derivatized fluorescent beads to PMRad or PMRup; (2) generate clones expressing the surface lectins over a broad range of densities; (3) determine the functional relationships between PMRad and PMRup in intact cells using multiparameter fluorescence cytometry and the direct quantitation of binding to PNHEV; (4) extract and compare the lectins structurally; (5) assess the functional linkages between PMRad, the Hermes antigen and Mel14 cross-reactive material in intact cells and (6) extract and compare structurally these independently characterized PNHEV adhesion structures. Studies (5) and (6) will also be conducted in leukemia specimens to determine whether freshly isolate hematopoetic malignancies utilize the same PNHEV adhesion receptors as normal and cultured cells, to establish the capacities of surface markers to predict and quantitate affinity for PNHEV and to enhance the likelihood of detecting either functional or structural linkages between adhesion structures.
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