Use of conjugates made between a cytolytic T cell clone and target cells to study the redistribution of membrane molecules in cell contact areas

Abstract

In many models of cell-cell adhesion, it was reported that some cell membrane molecules might be redistributed into contact areas. However, this phenomenon was not subjected to precise quantification. In the present work, fluorescence microscopy, immunolabelling and digital image processing were combined to analyse quantitatively the spatial organization of specific or nonspecific conjugates made with a cytolytic T (CTL) lymphocyte clone (BM3.3) and target cells (EL4 or RDM4). Binding was achieved under calcium-free conditions to study the earliest steps of cell interaction, preceding CTL activation. Fluorescent antibodies were used to label class I histocompatibility molecules on both killer and target cells, and T cell receptor, CD3, CD8 and LFA-1 (CD18/CD11a) on the killer cells. Membrane bilayers were stained with a fluorescent phospholipid, glycoconjugates were labelled with periodic oxidation and Lucifer Yellow uptake, and polymerized actin was revealed with a fluorescent phallacidin derivative. Also, the fine geometry of killer-target interaction area was studied with electron microscopy and computer-assisted contour analysis. It is concluded that: (1) qualitative examination of fluorescence photomicrographs cannot permit accurate comparison between different fluorescence densities. (2) The cell-cell contact area was about fourfold higher in specific conjugates than in non-specific ones. (3) The surface density of adhesion molecules exhibited similar increases (between 30 and 80%) in the contact areas of both specific and nonspecific conjugates. (4) However, the amount of redistributed surface molecules was higher when cell-cell interaction was enhanced either by specific immunological recognition (in specific conjugates) or periodate oxidation. (5) Since redistribution did not require extracellular calcium and it was detected on nonspecific conjugates, this did not require full lymphocyte activation. Spatial reorganization of cell surface molecules may thus be a general consequence of adhesion, cell surface mobility and intermolecular forces.