Experimental allergic encephalomyelitis is a T-cell-mediated, major histocompatibility complex (MHC) MATERIALS AND METHODS Mice. Female SJL/J mice were purchased from The Jackson Laboratory and were used between 8 and 12 weeks of age.Antigens. Peptides of MBP and PLP corresponding to amino acids 1-14, 91-103, and 139-151 were synthesized by solid-phase 9-fluorenylmethoxycarbonyl (FMOC) procedures. Peptides were purified by reverse-phase HPLC and were characterized by both HPLC and mass spectroscopy.Purification of dass H MHC. I-Al protein was purified from a Nonidet P-40 extract of spleen cell membranes from SJL/J mice by affinity chromatography using the monoclonal antibody 10-2.16 (specific for I-Ak and I-Al), coupled to Sepharose 4B beads. Extracted lysate from the high-speed (100,000x g) membrane fraction in a buffer of 10 mM Tris HCI, pH 8.3/0.5% Nonidet P-40/0.1 M sodium chloride/5 mM EDTA/0.02% sodium azide/1 mM phenylmethylsulfonyl fluoride was recycled over the preequilibrated column at 40C for 16 hr. The column was washed first with 10 bed volumes of deoxycholate buffer/10 mM Tris-HCl, pH 8.3/0.5% deoxycholate/0.1 M sodium chloride/5 mM EDTA/0.02% sodium azide/1 mM phenylmethylsulfonyl fluoride and then by 5 bed volumes of phosphate-buffered saline (PBS)/1% 1-octyl P-Dglycopyranoside buffer. Finally, I-AS was eluted with 20 mM phosphate buffer, pH 11/0.1 M sodium chloride/1% octyl glucoside/0.02% sodium azide/1 mM phenylmethylsulfonyl fluoride. Fractions were neutralized with acetic acid to a final Abbreviations: EAE, experimental allergic encephalomyelitis; MBP, myelin basic protein; PLP, proteolipoprotein; MHC, major histocompatibility complex. tTo whom reprint requests should be addressed. 11465The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

In autoimmune disorders, inactivation of pathogenic antigenspecific T cells, rather than global immunosuppression, would be highly desirable. One way to achieve this would be to deliver the first antigen-specific signal to the T cell in the absence of the second costimulatory signal. Myasthenia gravis (MG) is a well-characterized autoimmune disease in which T cell-dependent autoantibodies are directed against the acetylcholine receptor (AChR) at the neuromuscular junction. AChR-specific T cells have been cloned from MG patients, and in this study, we have induced long-lasting tolerance in vitro in one particular clone (PM-Al) with a known peptide epitope (al44-163) and MHC class II restriction (DR4 Dwl4.2 or 4.2) by using soluble MHC-class II peptide complexes.Preincubation of PM-Al T cells with such complexes induced death by apoptosis in < 40-50% of the AChR-specific cells. Surviving cells remained refractory to stimulation with AChR-derived synthetic peptides or recombinant polypeptides for < 38 d after complex treatment. These effects were highly specific, dose-dependent and required > 2 h preincubation. The T cells could be protected from the tolerizing effects of complex by coincubation with DR-matched or -mismatched antigen-presenting cells.This work shows that antigen-specific T cells can be selectively killed or anergized using soluble MHC class II:peptide complexes. Such an antigen-specific therapy offers a rational approach to the immunotherapy of autoimmune or allergic disease in vivo. (J. Clin. Invest. 1994. 93:1361-1369

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.

No abstract

No abstract