Characterization of Naturally Processed Antigen Bound to Major Histocompatibility Complex Class II Molecules

Helper T lymphocytes recognize peptide fragments of antigen bound to major histocompatibility complex (MHC) class II molecules presented on the surface of antigen-presenting cells (APCs). Previous studies showed that the MHC class II, I-Ekmolecules purified from APCs that had processed Drosophila melanogaster cytochrome c (DMc) contained functional, processed antigen-I-Ekcomplexes. This was demonstrated by the ability of purified I-Ek, incorporated into liposomes, to stimulate DMc-specific T cells in the absence of any additional antigen. Here we describe the isolation and characterization of the processed antigen bound to I-Ek. This was accomplished using DMc radiolabeled across its entire length by reductive methylation of its lysine residues, allowing an analysis of the totality of processed antigen bound to MHC class II molecules. After processing, only about 0.2% of the APC I-Ekmolecules contained processed DMc (≈ 800 per cell), yet these were sufficient to stimulate specific T cells. The DMc peptides isolated from the I-Ekmolecules showed only two predominant radioactive peaks as analyzed by reverse-phase chromatography. Less processed antigen was bound to purified I-Akmolecules, and these peptides were distinct from those bound to I-Ek. The association of processed DMc with the I-Ekand I-Akmolecules appears highly specific in that no radiolabeled peptides were isolated from purified MHC class I molecules, Kkand Dk, or from the B-cell differentiation antigen B220. The majority of processed antigen-I-Ekcomplexes migrated more slowly than the majority of the I-Ekprotein as analyzed by SDS/PAGE under nonreducing conditions without heating of the sample. This form of I-Ekmay be analogous to the earlier described "floppy" form of MHC class II molecules [Dormair, K., Rothenhausler, B. \& McConnell, H. M. (1990) Cold Spring Harbor Symp. Quant. Biol. 54, 409-416]. Since newly processed antigen binds nearly exclusively to this slow-migrating form, it may be of functional significance.