Major histocompatibility complex (MHC) class II molecules are cell surface heterodimeric glycoproteins consisting of one alpha and one beta polypeptide chain of similar size. These molecules play a critical role in immune recognition by displaying processed antigens to CD4-positive T helper cells. Several attempts to express the MHC class II molecules by recombinant methods in various systems resulted in either failure or poor recovery of the intact heterodimer. The present study describes our successful effort to refold and reconstitute HLA DR2 heterodimer from individually expressed alpha and beta polypeptide chains lacking the transmembrane hydrophobic regions in Escherichia coli, in the presence of an immunodominant epitope analog from human myelin basic protein (b-MBP(83-102)Y83). The reconstituted DR2 heterodimer complex was selectively purified from unfolded alpha and beta chains using heterodimer-specific monoclonal antibody (L243) coupled to a solid support. The detection of two polypeptide chains in the purified refolded DR2-peptide complex preparations was accomplished by Western blot analysis 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 preparation was confirmed by a double label experiment. The quantitation of the bound peptide in complex preparation was measured by incubating two chains in the presence of 125I-labeled peptide. An increase in the yield of refolded and reconstituted DR2-peptide complexes was observed with increasing peptide concentration in the reaction mixture. Finally, the functional activity of the reconstituted DR2 complexes was measured by their ability to stimulate gamma-interferon production by SS8T cloned T cells in an antigen-specific and dose-dependent manner. These results demonstrate that biologically active complexes of human DR2.b-MBP (83-102)Y83 can be prepared by proper folding of human leukocyte antigen DR2 alpha and beta chains in the presence of antigenic peptide. The yield of such DR2 heterodimers with bound peptide is several thousand-fold higher over native DR2 purified from transformed B cells. Since purified MHC class II-peptide complexes have been shown to prevent autoimmune diseases in various animal models, reconstituted heterodimer complexes may have significant clinical relevance in antigen-specific treatment of various autoimmune diseases. In addition, such complexes with increased yield will provide better understanding of the trimolecular interactions between MHC-peptide and T cell receptor.
Major histocompatibility (MHC) class II molecules are cell surface heterodimeric (alphabeta) glycoproteins that display processed antigens to T cell receptors (TCRs) of CD4-positive T cells. The present study describes that individual recombinant alpha and beta chains of human MHC class II molecules lacking the transmembrane region (alpha-Tm and beta-Tm) are capable of binding antigenic peptide and that these complexes of chain-peptide are recognized by TCRs to induce antigen-specific apoptosis in restricted T cells. The alpha-Tm and the beta-Tm of human HLA-DR2 (DRB5*0101) were cloned, expressed in Escherichia coli, and purified in large scale by conventional chromatographic methods. The in vitro binding of an immunodominant epitope from the myelin basic protein (MBP-(83-102)Y83) to purified DR2 alpha-Tm and DR2 beta-Tm was demonstrated with biotinylated and fluoresceinated MBP-(83-102)Y83 peptide. The specificity of the MBP-(83-102)Y83 peptide binding to both DR2 alpha-Tm and DR2 beta-Tm was demonstrated in a competitive peptide binding assay. When exposed to a transformed T cell clone (SS8T) restricted to DR2(DRB5*0101) and MBP-(84-102) peptide, complexes of DR2 alpha-Tm and DR2 beta-Tm with MBP-(83-102)Y83 peptide were able to specifically recognize TCRs as measured by the increase in gamma-interferon (gamma-IFN) cytokine. Such recognition of TCRs by soluble alpha-MBP-(83-102)Y83 and beta-MBP-(83-102)Y83 complexes led to the induction of antigen-specific apoptosis in SS8T cells as measured by double fluorescence flow cytometry and electron microscopy. These results provide the first evidence that soluble complexes of antigenic peptide and individual chains of human MHC class II molecules lacking the transmembrane region can recognize TCRs and induce antigen-specific apoptosis in T cells. Since activated CD4-positive T cells are involved in pathogenesis of various autoimmune diseases, the apoptosis triggered by individual soluble chain-peptide complexes has significant potential for eliminating autoreactive T cells.
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