REFOLDING AND RECONSTITUTION OF FUNCTIONALLY ACTIVE COMPLEXES OF HUMAN-LEUKOCYTE ANTIGEN-DR2 AND MYELIN BASIC-PROTEIN PEPTIDE FROM RECOMBINANT-ALPHA AND RECOMBINANT-BETA POLYPEPTIDE-CHAINS

Major histocompatibility complex (MHC) class II molecules are cell sur face heterodimeric glycoproteins consisting of one alpha and one beta polypeptide chain of similar size. These molecules play a critical rol e in immune recognition by displaying processed antigens to CD4-positi ve T helper cells. Several attempts to express the MHC class II molecu les by recombinant methods in various systems resulted in either failu re or poor recovery of the intact heterodimer. The present study descr ibes our successful effort to refold and reconstitute HLA DR2 heterodi mer from individually expressed alpha and beta polypeptide chains lack ing the transmembrane hydrophobic regions in Escherichia coli, in the presence of an immunodominant epitope analog from human myelin basic p rotein (b-MBP(83-102)Y-83). The reconstituted DR2 heterodimer complex was selectively purified from unfolded alpha and beta chains using het erodimer-specific monoclonal antibody (L243) coupled to a solid suppor t. The detection of two polypeptide chains in the purified refolded DR 2-peptide complex preparations was accomplished by Western blot analys is and enzyme-linked immunosorbent assay using heterodimer- and chain- specific polyclonal antibodies, and the presence of equimolar amounts of both alpha chain and beta chain in the reconstituted complex prepar ation was confirmed by a double label experiment. The quantitation of the bound peptide in complex preparation was measured by incubating tw o chains in the presence of I-125-labeled peptide. An increase in the yield of refolded and reconstituted DR2-peptide complexes was observed with increasing peptide concentration in the reaction mixture. Finall y, the functional activity of the reconstituted DR2 complexes was meas ured by their ability to stimulate gamma-interferon production by SS8T cloned T cells in an antigen-specific and dose-dependent manner. Thes e results demonstrate that biologically active complexes of human DR2- b-MBP(83-102)Y-83 can be prepared by proper folding of human leukocyte antigen DR2 alpha and beta chains in the presence of antigenic peptid e, The yield of such DR2 heterodimers with bound peptide is several th ousand-fold higher over native DR2 purified from transformed B cells. Since purified MHC class II-peptide complexes have been shown to preve nt autoimmune diseases in various animal models, reconstituted heterod imer complexes may have significant clinical relevance in antigen-spec ific treatment of various autoimmune diseases. In addition, such compl exes with increased yield will provide better understanding of the tri molecular interactions between MHC-peptide and T cell receptor.