Updated project metadata. Whether the presentation levels of the major histocompatibility complex (MHC, called the human leukocytes antigens, HLA, in humans) is limited by the availability of peptide ligands for loading or by the supply of empty MHC molecules, is a yet unresolved issue. This study aims to tackle this dilemma using large-scale immunopeptidome analyses. First, cultured cells (MCF-7) were treated with interferons, which dramatically increased presentation levels of their HLA-B molecules, much more than HLA-A and HLA-C. The differential increase in the HLA-B molecules with their bound peptides, was driven by elevated HLA synthesis levels and not by supply of peptides, since all three HLA allotypes draw peptides from the same peptide pool, which should have been affected similarly by the interferons treatment. In addition, artificial competition for peptides was created by recombinant high levels expression of soluble HLA-A*02:01 molecules, in the same MCF-7 cells and on top of their endogenous membranal HLA-A*02:01. This high level expression of the soluble HLA did not affect the endogenous membranal HLA-A*02:01 peptidomes or its cell surface presentation levels. We therefore concluded that a surplus supply of peptides is available for loading and that presentation levels are more likely limited by the supply of HLA molecules.