Membrane-reconstituted, purified H-2Kb specifically inhibits T-cell-target cell conjugate formation

We report the use of a sensitive microassay to detect purified H-2Kb antigens which have been functionally reconstituted into membrane vesicles of defined composition. The histocompatibility antigens have been purified by monoclonal antibody affinity chromatography. The assay utilizes inhibition of specific conjugate formation between allogeneically primed (H-2d anti- H-2b) cytotoxic T cells and H-2b target cells by the membrane-reconstituted H-2Kb antigens. Cytoskeletal proteins were added to the H-2Kb (and control H-2k) antigens. Sucrose density fractionation of reconstituted vesicles and Pronase E cleavage studies suggested that the cytoskeletal proteins aided in the incorporation and vectorial orientation of the antigens into large, cholesterol-containing membrane vesicles. As little as 6 ng purified H-2Kb plus 28 ng cytoskeletal proteins in vesicles of defined lipid composition (0.28, 0.25, 0.47 mol fraction cholesterol, dimyristoylphosphatidylcholine, and dipalmitoylphosphatidylcholine, respectively) inhibited specific conjugate formation to 50% of the maximum inhibition observed. This inhibition was shown to be specific in two ways: (i) the same H-2Kb-containing vesicles did not inhibit nonspecific conjugate formation, and (ii) control vesicles containing the same amounts of lipid, cytoskeletal proteins, and purified H-2k proteins inhibited conjugate formation but only at significantly higher H-2k concentrations, indicating the specificity of the response with the vesicles containing H-2Kb.