Binding of low concentration of peptide to H-2Kd produced in insect cells requires mouse β2-microglobulin co-expression

Abstract

A recombinant baculovirus expressing the murine class I MHC heavy chain H-2K^d cDNA under the transcriptional control of Autografa californica nuclear polyhedrosis virus (AcNPV) polyhedrin promoter has been isolated and used to infect Sf9 lepidopteran cells either alone or in association with a previously isolated virus expressing mouse β2-microglobulin^b (β₂-m^a). When infected with the heavy chain-encoding virus alone, H-2K^d was produced in a β2-m-free conformation detected on the surface of infected cells by conformation-independent antibodies. When Sf9 cells were co-infected with both viruses, ∼10% of the heavy chain pool was engaged in the formation of native heterodimeric MHC class I molecules, which were glycosylated and transported to the cell surface as demonstrated by radio-binding experiments and flow cytometry. The assembly of the recombinant class I molecule was dependent on peptide, since heterodimer formation was brought about by H-2K^d-specific peptide ligands both in vivo, upon incubation with dually infected cells, and in vitro. In cell-free detergent extracts. In addition, a change in heavy chain conformation was brought about upon incubation with high concentrations (100 μM) of an H-2K^d-restricted octapeptide epitope from Plasmodlum berghel. Furthermore, using low concentrations (3 nM) of a photoaffinity label derivative of this peptide, we show direct binding to cells co-expressing class I heavy chain and mouse β₂-m but not to cells expressing free heavy chain only.