LYMPHOCYTE/VASCULAR INTERACTIONS IN IMMUNE RESPONSES

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There is a paucity of information about the mechanisms controlling the localizing of antigen-specific lymphocytes. This issue is of obvious importance to understanding how rare antigen-specific lymphocytes are able to locate and respond to foreign antigens or initiate autoimmune tissue injury. The focus of this grant, therefore is to examine the molecular mechanisms by which antigen-specific lymphocytes localize in sites of antigen or to the lymphoid organs draining sites of inflammation. Lymphocyte recruitment from vasculature into tissues is primarily dependent upon lymphocyte-endothelial interactions. In specific aim 1, we propose to examine the role of CD31, a putative adhesion molecule, in lymphocyte transmigration from the circulation into tissues. CD31, a member of the immunoglobulin superfamily, may play a key role in transmigration of lymphocytes through vascular endothelium. We have developed and characterized the only presently available monoclonal antibody against murine CD31, providing a unique tool for in vivo studies. In addition, we have established cultured systems employing murine endothelium and lymphoid cells that will allow us to dissect the role of this molecule in lymphocyte-endothelial interactions. With a novel immunohistochemical technique that we developed, we found that antigen-reactive lymphocytes are selectively retained in the perivascular region of stimulated lymphoid organs. In specific aim #2, we will investigate the molecules/mechanisms which mediate this perivascular tissue retention of antigen-reactive lymphocytes. We will characterize: (1) the role of accessory cells in affecting the localization of antigen-reactive lymphocytes, and (2) the potential role served by the Beta1 integrins in mediating the attachment of lymphocytes to extracellular matrices, such as basal lamina of the perivasculature. These studies will therefore correlated lymphocytic inflammation and localization in tissues and organs with the induction of antigens- specific immune responses. An important novel aspect of these studies is the use of in vivo experimental models. In specific aim 3, we will characterize unique molecules synthesized by antigen-reactive, perivascular lymphocytes. We have characterized a monoclonal antibody designated 2B6, which recognized only perivascular lymphocytes. When added in vitro during antigen-induced lymphoproliferative response, the HPLC-purified 2B6 mAb mediates a strong suppressive effect. We propose to identify this molecule by immunoprecipitation followed by SDS-PAGE, and possibly N-terminal amino acid analysis. Additionally, we will further examine the role of this molecule by attempting to block the function of this molecule in vivo using the 2B6 mAb. This approach is a more broadly focused aim that will likely yield new insights into the physiology of immune responses, since the anatomically localized perivascular subset comprises a significant portion of antigen-reactive lymphocytes in vivo.