Recirculating T and B lymphocytes migrate from blood into lymph nodes (LN) and Peyer's patches (PP) specifically at venules lined by high endothelium (HEV) in murine animals. We have used an in vitro lymphocyte-HEV binding assay as a means to probe the molecular mechanism of this process in rats. These studies have led to isolation of material designated High Endothelial Binding Factor (HEBF) from thoracic duct lymph. Rabbit anti-HEBF F(ab')2 binds to approximately 70 % of thoracic duct lymphocytes (TDL), and inhibits their adherence to HEV in vitro and in vivo, blocks entry of radiolabeled donor cells into LN but not accumulation into PP. Furthermore, HEBF has been isolated directly from TDL lysates by affinity chromatography on anti-HEBF Ig columns. We hypothesize that HEBF on the lymphocyte surface mediates their adherence to high endothelium of LN. To test this hypothesis, we propose to: (1) isolate HEBF+ and HEBF- TDL populations (by panning on anti-HEBF F(ab')2 plates) and examine each for its ability to interact with HEV of LN and PP and to recirculate between blood and lymph; (2) determine the specificity of 125I-HEBF for LN-HEV by overlaying the labeled factor onto LN and PP sections or by injection into recipients, followed by autoradiography; (3) use mouse monoclonal antibodies against rat HEBF to detect HEV adhesion molecules on lymphocytes.
A fourth aim i s to determine the capacity of TDL to produce surface HEBF by biosynthetic studies with 3H-Leucine, 35S-methionine or 3H-glucosamine. Finally, we plan to identify the lymphocyte class of HEBF+ and HEBF- TDL and to assess the feasibility of isolating T cells (HEBF+ and HEBF-) and B cells (HEBF+ and HEBF-) for determination of HEV binding properties. The ultimate goal is to develop means for control of lymphocyte migration and thus provide new approaches for regulating immune responsiveness. Understanding of these fundamental interactions may have an impact on health problems such as allergy, infection, transplantation, cancer and autoimmunity.
