Flow cytometric analysis of specific binding of soluble Ia by I-region restricted alloactivated T cells

An antigen binding assay has been developed for quantitation by flow cytometry of vesicular and soluble Ia binding by alloactivated T cells. Binding of stimulator membrane vesicles was detected by anti-Ly-6.2 or anti-Ia monoclonal antibodies coupled to fluorescent latex beads. Vesicle binding by an I-A^k specific A.TH anti-A.TL T cell line occurred via I-A^k molecules, in that (a) vesicles expressing I-A^k molecules bound much more effectively than vesicles of H-2^b,q strains, and (b) inhibition of H-2^k vesicle binding occurred with anti-I-A^k, but not anti-K^k, anti-E^k, or anti-D^k antibodies. T cell receptor/Ia interactions were directly studied by inhibition of H-2^k vesicle binding by T cells with partially purified Ia glycoproteins. Inhibition of binding occurred via Ia molecules since (a) affinity column partially purified allogeneic I-A^k molecules inhibited binding much more effectively than syngeneic I-A^s molecules and (b) depletion of I-A^k but not E^k molecules in Ia^k containing glycoprotein fractions abrogated the inhibitory effect. The ability of this method to detect specific binding of soluble Ia with antigen activated T cells makes it a useful tool for studying interaction of membrane free major histocompatibility complex (MHC) products with native T cell receptor.