The invariant chain forms complexes with class II major histocompatibility complex molecules and antigenic peptides "in vivo"

Abstract

The binding of a chicken ovalbumin peptide (residues 323-339), Ova-(323-339), to I-Ad molecules was investigated in vitro and in vivo. By using antigenic peptides labeled either with a hapten or with fluorescein, complexes formed in vitro between I-Ad and antigenic peptides were detected by Western blot analysis with an antibody recognizing the hapten 7-nitrobenzo-2-oxa-1,3-diazole and by scanning gels for fluorescence emitted by fluoresceinated peptide. Both techniques reveal that Ova-(323-339) binds not only to I-Ad α/β heterodimers and separated α and β chains but also to complexes of higher molecular mass. Additional analysis shows that one of these additional complexes contains I-Ad heterodimers, antigenic peptides, and also invariant chain. To explore the physiological role of these complexes, cells were incubated with haptenated peptide and the I-Ad-peptide complexes formed in vivo were purified by affinity chromatography using hapten-specific antibody. The complexes formed migrate with a significantly higher apparent molecular mass than the α/β heterodimers. A band at 180 kDa contained the α/β heterodimer, the antigenic peptide, and the invariant chain. These results show that in vivo high molecular mass complexes formed by the I-Ad heterodimer and the invariant chain bind antigenic peptides.