SEPARATION OF COMPLEXES OF MAJOR HISTOCOMPATIBILITY CLASS-II MOLECULES AND KNOWN ANTIGENIC PEPTIDE BY METAL CHELATE AFFINITY-CHROMATOGRAPHY

A small fraction of affinity-purified MHC class II molecules are known to bind antigenic peptides in vitro. No simple method with acceptable recovery exists for separation of complexes of a known antigenic epit ope and MHC class II from empty MHC class II and complexes of MHC clas s II and endogenously bound peptide. Here we describe an one step meta l chelate affinity chromatography method to purifiy complexes of MHC c lass II and antigenic peptide of known composition. Complexes of human HLA-DR2 (DRB11501/DRB5*0101) and a peptide analog from human myelin basic protein MBP(84-102) containing a 6 histidine tag (6 X His) and a tyrosine residue at the N-terminus end [6 x His-MBP(83-102)Y-83] were prepared and purified. The absence of residual free 6 x His-MBP pepti de in the complex preparations were confirmed by gel filtration and TL C analyses. The purified complexes were applied onto Ni2+.nitrilotriac etic acid (Ni2+.NTA)-agarose affinity support and 6 x His-tagged pepti de class II complexes were selectively eluted with imidazole-containin g buffer. The quantitation of bound peptide in the eluted complexes sh owed 100% occupancy of HLA-DR2 (DRB11501/DRB5*0101) with [6 X His-MBP (83-102)Y-83] peptide with a recovery of 50-75%. The presence of a sin gle peptide entity in the eluted complexes was confirmed by reverse-ph ase narrowbore HPLC analysis of the acid-extracted supernatant and by amino acid sequencing analyses. As expected, no endogenous polypeptide was detected in the Ni2+.NTA eluted complexes when analyzed by two-di mensional IEF gel electrophoresis. Finally, we demonstrate that both M BP(84-102) and [6 x His-MBP(83-102)Y-83] peptides were equally capable of stimulating restricted T cell line in the presence of autologous a ntigen presenting cells (APCs). These results demonstrate that metal c helate affinity chromatography can be used to prepare MHC class II-pep tide complexes containing single peptide. Such complexes of class II m olecules containing known peptide have significant clinical relevance for antigen-specific therapy of various autoimmune diseases and may pr ovide better understanding of the trimolecular interaction between MHC class II, antigenic peptide and T cell receptor (TCR).