A PHOTOBLEACHING ENERGY-TRANSFER ANALYSIS OF CD8 MHC-I AND LFA-1/ICAM-1 INTERACTIONS IN CTL-TARGET CELL CONJUGATES/

The photobleaching energy transfer (pbFRET) technique is a fluorescenc e method to measure proximity relationships between molecules, especia lly cell surface proteins, labeled with fluorophore-conjugated monoclo nal antibodies, on a pixel-by-pixel base using digital imaging microsc opy. This technique enables analysis of inter- and intramolecular prox imities at cell surfaces at physiological conditions. We have develope d a pbFRET approach to measure intercellular proximities in order to a ccess spatial organization of interacting proteins in the contact regi on of two 'communicating' cells. Two examples, as possible application areas of this approach, are presented here: interaction between CD8 a nd MHC-I molecules in point contacts and interaction between LFA-1 and ICAM-1 molecules in focal contacts of CTL-target conjugates. The geom etry of these protein contacts based on our resonance energy transfer (RET) data is consistent with the observed blocking effects of monoclo nal antibodies (directed against the interacting proteins) on the cyto lytic activity of CTLs and suggest a critical role for CD8 beta-subuni t in signal transmission in peripheral T-lymphocytes. (C) 1996 Elsevie r Science B.V.