Alterations in the organization of the phospholipids in the lymphocyte plasma membrane may affect several lymphocyte functions. This proposal is designed to assess the role of phospholipid packing in the interactions of lymphocytes with reticuloendothelial cells and with the targets of cytolytic T lymphocytes (CTL). Immature and activated lymphocytes isolated from tissues bind merocyanine 540 (MC540), a fluorescent lipophilic probe for loosely-packed lipids, whereas lymphocytes isolated from the peripheral circulation do not. Whether this difference in phospholipid packing is responsible for differences in the interactions of these lymphocytes with other cells will be tested in several ways. Using centrifugation to produce a controlled and reproducible force to distinguish adherent from nonadherent cells, the strength of the interactions of peripheral blood lymphocytes, thymocytes and splenocytes with macrophages and endothelial cells will be measured. Using flow cytometry, thymocytes and splenocytes stained with MC540 will be fractionated according to fluorescence intensity and the adherence properties of the fractions determined. The dependence of cellular interactions on lipid organization will be examined in vivo by following clearance from the circulation of reinfused lymphocytes which differ in phospholipid packing. Two biophysical approaches will be used to investigate the mechanisms by which altered phospholipid organization could affect adherence. Fluorescence recovery after photobleaching will be employed to assess whether increases agglutinability of thymocytes with loosely-packed lipids results from increased mobility of membrane receptors. Fluorescence transfer methods will be used to determine whether the loose packing of lipids in activated lymphocytes is accompanied by local aggregation of a lymphocyte adhesion-related membrane protein, LFA-1. Finally, phospholipid organization in CTL and their targets will be assessed by MC540 staining to evaluate the potential role of this parameter in the resistance of CTL to self-lysis and in the efficiency of killing of target cells.
