Viral antigens complexed with major histocompatibility complex (MHC) class I molecules are recognized by cytotoxic T lymphocytes on infected cells. Assays with synthetic peptides identify optimal MHC class I ligands often used for vaccines. However, when natural peptides are analyzed, more complex mixtures including long peptides bulging in the middle of the binding site or with carboxyl extensions are found, reflecting lack of exposure to carboxypeptidases in the antigen processing pathway. In contrast, precursor peptides are exposed to extensive cytosolic aminopeptidase activity, and fewer than 1% survive, only to be further trimmed in the endoplasmic reticulum. We show here a striking example of a nested set of at least three highly antigenic and similarly abundant natural MHC class I ligands, 15, 10, and 9 amino acids in length, derived from a single human immunodeficiency virus gp160 epitope. Antigen processing, thus, gives rise to a rich pool of possible ligands from which MHC class I molecules can choose. The natural peptide set includes a 15-residue-long peptide with unprecedented 6 N-terminal residues that most likely extend out of the MHC class I binding groove. This 15-mer is the longest natural peptide known recognized by cytotoxic T lymphocytes and is surprisingly protected from aminopeptidase trimming in living cells.Newly synthesized viral proteins are proteolytically processed before MHC 4 class I heavy chain-␤ 2 m-peptide complex formation in the lumen of the endoplasmic reticulum (ER) (1). Peptides of 8 -10 residues bind to MHC class I molecules usually by means of two major anchor residues at positions 2 and C-terminal in the antigenic peptide (2, 3). Some peptides may be directly produced by the proteasome in their final form, whereas others are generated as precursor peptides (4). These precursor peptides must display the correct C terminus of the final antigenic peptides, as the evidence suggests the absence of carboxypeptidases in the ER (1, 5). The limited number of studies on extraction and identification of natural peptides derived from a known foreign antigen shows that they constitute complex mixtures, mostly including C-terminal extensions of the minimal epitope (6 -9). On the contrary, peptides are exposed to extensive cytosolic aminopeptidase activity, and fewer than 1% of them survive and are rescued by the transporters associated with antigen processing (TAP) and translocated into the ER (10, 11).In most cases it is assumed that the natural MHC class I ligand is the one that has the canonical anchor sites, the minimal length, and the optimal antigenicity when tested as synthetic peptide. N-terminal extensions of such potential epitopes have been used to study trimming by aminopeptidases purified from the ER or microsomal fractions. There are several reports which show that the two enzymes, mouse endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP)/human ERAP1 and human leukocyte-derived arginine aminopeptidase (L-RAP)/ERAP2, can indeed trim the precurso...

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CTL recognize peptides that derive from viral protein Ags by proteolytic processing and are presented by MHC class I molecules. In this study we tested whether coexpression of viral Ags in the same cell leads to competition between them. To this end, two Ld-restricted epitopes derived from HIV-1 envelope gp160 (ENV) and from CMV pp89 phosphoprotein were coexpressed. HIV ENV strain IIIB, but not MN variant, impaired recognition by specific CTL of CMV pp89 epitope 9pp89. Susceptibility to inhibition after ENV coexpression was inversely related to the amount of antigenic 9pp89 peptide processed from different antigenic constructs. In line with it, competition decreased the yield of naturally processed antigenic 9pp89 peptide bound to MHC class I molecules in coinfected cells. Also, point mutants of the presenting MHC class I molecule differed in their competition pattern. Collectively, the data imply that competition operates at the step of MHC-peptide complex assembly or stabilization. We conclude that, although not the rule, in certain combinations there is interference between different Ags expressed in the same cell and presented by the same MHC class I allele. These studies have implications for vaccine development and for understanding immunodominance.

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