MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I-BINDING PEPTIDES ARE RECYCLED TO THE CELL-SURFACE AFTER INTERNALIZATION

Cytotoxic T lymphocytes (CTL) recognize target antigens as short, processed peptides bound to major histocompatibility complex class I (MHC-I) heavy and light chains (beta2-microglobulin; beta2-M).The heavy chain,which comprise the actual peptide binding alpha-1 and alpha-2 domains, can exist at the cell surface in different forms, either free, bound to beta2-M or as a ternary complex with beta2-m and peptides. MHC-1 chains are also known to internalize, and recycle to the cell surface, and this has been suggested to be important in peptide presentation. Whether MHC-1-bound peptides also can recycle is not known.We have investigated this by using both peptide transporter mutant RMA-S cells and EL4 cells loaded with D(b)-binding peptides, by two different approaches. First, peptides were covalently linked with galabiose (Galalpha4Gal) at a position which did not interfere with D(b) binding or immunogenicity, and peptide recycling tested with Gal2-specific monoclonal antibodies. By flow cytometry, a return of Gal2 epitopes to the cell surface was found, after cellular internalization and cell surface clearance by pronase treatment. This peptide recycling could be discriminated from free fluid-phase uptake and was inhibited by methylamine, chloroquine and low temperature (18-degrees-C) but not by leupeptin. Second, specific CTL were reacted with peptide-loaded target cells after complete removal of surface D(b) molecules by pronase, and after different times of incubation at 37-degrees-C to allow reexpression. By this procedure, reappearance of target cell susceptibility was confirmed. The results are in agreement with a model for optimizing peptide presentation by recycling through an intracellular compartment similar to early endosomes in certain antigen-presenting cells.