High-Affinity Fluorescent Peptide Binding to I-Ad in Lipid Membranes

Membranes isolated from antigen-pulsed, antigen-presenting cells were extensively dialyzed and shown to retain the ability to present antigen to an I-Ad-restricted, antigen-specific T cell, 3DO-54.8. This ability to retain antigen on the membrane was duplicated in lysates of antigen-presenting cells as well as with pure I-A molecules in phospholipid vesicles. Measurement of the concentration of surface-associated fluorescent peptide on planar membranes prepared from antigen-pulsed phospholipid vesicles showed that about 1 peptide molecule was retained per 100 I-A molecules. We calculate that about 1000 I-A-associated peptide molecules are sufficient to stimulate the response of a single 3DO-54.8 cell. The association of fluorescent peptide with the surface depended on the presence of I-A and was blocked by unlabeled ovalbumin peptide or by a digest of hen egg lysozyme, added at the time of the pulse, but not after pulsing.