The importance of immunoproteasomes to antigen presentation has been unclear because animals totally lacking immunoproteasomes have not been previously developed. Here we show that dendritic cells from mice lacking the three immunoproteasome catalytic subunits display defects in presenting multiple major histocompatability (MHC) class I epitopes. During viral infection in vivo, the presentation of a majority of MHC class I epitopes is markedly reduced in immunoproteasome-deficient animals, while presentation of MHC class II peptides is unaffected. By mass spectrometry the repertoire of MHC class I-presented peptides is ~50% different and these differences are sufficient to stimulate robust transplant rejection of wild type cells in mutant mice. These results indicate that immunoproteasomes play a much more important role in antigen presentation than previously thought.

The MHC class I (MHC-I) molecules ferry a cargo of peptides to the cell surface as potential ligands for CD8+ cytotoxic T cells. For nearly 20 years, the cargo has been described as a collection of short 8-9 mer peptides, whose length and sequences were believed to be primarily determined by the peptide-binding groove of MHC-I molecules. Yet the mechanisms for producing peptides of such optimal length and composition have remained unclear. In this study, using mass spectrometry, we determined the amino acid sequences of a large number of naturally processed peptides in mice lacking the endoplasmic reticulum aminopeptidase associated with Ag processing (ERAAP). We find that ERAAP-deficiency changed the oeuvre and caused a marked increase in the length of peptides normally presented by MHC-I. Furthermore, we observed similar changes in the length of viral peptides recognized by CD8+ T cells in mouse CMV-infected ERAAP-deficient mice. In these mice, a distinct CD8+ T cell population was elicited with specificity for an N-terminally extended epitope. Thus, the characteristic length, as well as the composition of MHC-I peptide cargo, is determined not only by the MHC-I peptide-binding groove but also by ERAAP proteolysis in the endoplasmic reticulum.

Adoptive cell therapy (ACT) using tumor-reactive T lymphocytes is a promising approach for treating advanced cancer. Successful tumor eradication depends primarily on the expansion and survival of the adoptively transferred T cells. Lymphodepletion using total body irradiation (TBI) and administering high dose IL2 have been used with ACT to promote T cell expansion and survival to achieve maximal therapeutic effects. However, TBI and high dose IL2 increase the risk for major complications that impact overall survival. Here we describe an alternative approach to TBI and high dose IL2 for optimizing ACT, resulting in dramatic therapeutic effects against established melanomas in mice. Administration of a potent, non-infectious peptide vaccine after ACT dramatically increased antigen-specific T cell numbers leading to enhancement in the survival of melanoma-bearing mice. Furthermore, combinations of peptide vaccination with PD1 blockade or IL2/anti-IL2 antibody complexes led to complete disease eradication and long-term survival in mice with large tumors receiving ACT. Our results indicate that PD1 blockade and IL2/anti-IL2 complexes enhance both the quantitative and qualitative aspects of the T cell responses induced by peptide vaccination after ACT. These findings could be useful for the optimization of ACT in cancer patients without the need of toxic adjunct procedures.