T-cell activation involves interactions between a number of different receptors on the T-cell with their respective ligands on the antigen presenting cell. One approach to studying the contributions of the individual receptors is the use of purified ligands, alone or in combination, to stimulate the cells. However, effective T-cell recognition requires that the ligands be displayed on a surface having dimensions similar to those of a cell. Methods are described for the rapid and efficient immobilization of purified membrane proteins, including class I MHC proteins, B7.1 and ICAM-1, on 5μm diameter latex microspheres in a manner that preserves biological activity. Non-membrane proteins, such as anti-receptor antibodies, can be immobilized in a similar manner and can be coimmobilized along with membrane proteins. These artificial cell surface constructs can be handled in the same way as cells, including characterization by flow cytometry using antibodies specific for the immobilized proteins and stimulation of T-cell responses. The density of ligand on the surface of the microspheres can be easily varied and controlled and multiple proteins can be immobilized on the same surface. Thus, the described methods provide a great deal of flexibility in assessing the contributions of the various receptors to T-cell signaling and activation, and should be applicable to the study of other cell-cell interactions.
Bibliographical noteFunding Information:
Supported by grants AI36950, AI34824 and AI35296 from the National Institutes of Health. M.J.D. was supported in part by National Institutes of Health grant IT32-AI07421. The authors are grateful for the expert technical assistance of Paul Champoux and Debra Lins.
- Cell surface
- Lymphocyte activation
- T- lymphocytes