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Within the tumor immunology community, the topic of proteasomal spliced peptides (PSP) has generated a great deal of controversy. In the earliest reports, careful biological validation led to the conclusion that proteasome-catalyzed peptide splicing was a rare event. To date, six PSPs have been validated biologically. However, the advent of algorithms to identify candidate PSPs in mass spectrometry data challenged this notion, with several studies concluding that the frequency of spliced peptides binding to MHC class I was quite high. Since this time, much debate has centered around the methodologies used in these studies. Several reanalyses of data from these studies have led to questions about the validity of the conclusions. Furthermore, the biological and technical validation that should be necessary for verifying PSP assignments was often lacking. It has been suggested therefore that the research community should unite around a common set of standards for validating candidate PSPs. In this review, we propose and highlight the necessary steps for validation of proteasomal splicing at both the mass spectrometry and biological levels. We hope that these guidelines will serve as a foundation for critical assessment of results from proteasomal splicing studies.

Maintaining stable tryptophan levels is required to control neuronal and immune activity. We report that tryptophan homeostasis is largely controlled by the stability of tryptophan 2,3-dioxygenase (TDO), the hepatic enzyme responsible for tryptophan catabolism. High tryptophan levels stabilize the active tetrameric conformation of TDO through binding non-catalytic exosites, resulting in rapid catabolism of tryptophan. In low tryptophan, the lack of tryptophan binding in the exosites destabilizes the tetramer into inactive monomers and dimers, and unmasks a 4-amino-acid degron that triggers TDO polyubiquitination by SKP1-CUL1-F-box complexes, resulting in proteasome-mediated degradation of TDO and rapid interruption of tryptophan catabolism. The non-metabolizable analog alpha-methyl-tryptophan stabilizes tetrameric TDO, and thereby stably reduces tryptophanemia. Our results uncover a mechanism allowing a rapid adaptation of tryptophan catabolism to ensure quick degradation of excess tryptophan while preventing further catabolism below physiological levels. This ensures a tight control of tryptophanemia, as required for both neurological and immune homeostasis.

The proteasome is a large protease complex responsible for the selective degradation of intracellular proteins. Its basal structure, called 20S proteasome, is composed of two outer a-­rings that delimit the proteasome gates, and two inner b-rings that contain the catalytic subunits and form the catalytic chamber of the proteasome. Four main proteasome subtypes are present in mammalian cells, that differ in the composition of their catalytic subunits: the standard proteasome (b1, b2 and b5), the immunoproteasome (b1i, b2i and b5i) and the two intermediate proteasomes (b1, b2 and b5i) and (b1i, b2 and b5i). In this study we aimed to compare the degradation of ubiquitinated and oxidized proteins by these four proteasome subtypes and to evaluate how the presence of oxidized residues modifies the cleavages produced by the proteasome. First, our study on the degradation of ubiquitinated proteins suggests that the four proteasome subtypes are equally efficient to degrade ubiquitinated proteins. In contrast, degradation of oxidized proteins by the four purified 20S proteasomes showed that the 5i-containing 20S proteasomes are the most effective, and that the 5i catalytic activity is central for the degradation of oxidized proteins. Recognition of oxidized proteins seems to be dictated by their altered structure rather than increased exposure of hydrophobic patches or oxidized residues. Furthermore, studies conducted on the degradation of oxidized peptides by the proteasome showed that the same proteasome subtype differentially degrades unoxidized and oxidized peptides. Further analysis showed that these differences are due to changes in the cleaving efficiency and the cleavage sites, resulting in different fragments released. Using the spliced antigenic peptide Tyro­A24, which contains a methionine, we showed that oxidation of the methionine affected the ability of the proteasome to produce the antigenic peptide. Finally, using the Mel5­35 tumor cell line that expresses T, (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Cancer immunotherapy has been flourishing in recent years with remarkable clinical success. But as more patients are treated, a shadow is emerging that has haunted other cancer therapies: tumors develop resistance. Resistance is often caused by defects in the MHC class I Ag presentation pathway critical for CD8 T cell-mediated tumor clearance. TAP and tapasin, both key players in the pathway, are frequently downregulated in human cancers, correlating with poor patient survival. Reduced dependence on these factors may promote vaccine efficiency by limiting immune evasion. In this study, we demonstrate that PMEL209-217, a promising phase 3 trial-tested antimelanoma vaccine candidate, is robustly presented by various TAP- and/or tapasin-deficient cell lines. This striking characteristic may underlie its potency as a vaccine. Surprisingly, cytosolic proteasomes generate the peptide even for TAP-independent presentation, whereas tripeptidyl peptidase 2 (TPP2) efficiently degrades the epitope. Consequently, inhibiting TPP2 substantially boosts PMEL209-217 presentation, suggesting a possible strategy to improve the therapeutic efficacy of the vaccine.

The proteasome is the major protease responsible for the production of antigenic peptides recognized by CD8+ cytolytic T cells (CTL). These peptides, generally 8-10 amino acids long, are presented at the cell surface by major histocompatibility complex (MHC) class I molecules. Originally, these peptides were believed to be solely derived from linear fragments of proteins, but this concept was challenged several years ago by the isolation of anti-tumor CTL that recognized spliced peptides, i.e. peptides composed of fragments distant in the parental protein. The splicing process was shown to occur in the proteasome through a transpeptidation reaction involving an acyl-enzyme intermediate. Here, we review the steps that led to the discovery of spliced peptides as well as the recent advances that uncover the unexpected importance of spliced peptides in the composition of the MHC class I repertoire.

Cross-presentation enables dendritic cells to present on their MHC class I molecules antigenic peptides derived from exogenous material, through a mechanism that remains partly unclear. It is particularly efficient with long peptides, which are used in cancer vaccines. We studied the mechanism of long-peptide cross-presentation using human dendritic cells and specific CTL clones against melanoma Ags gp100 and Melan-A/MART1. We found that cross-presentation of those long peptides does not depend on the proteasome or the transporter associated with Ag processing, and therefore follows a vacuolar pathway. We also observed that it makes use of newly synthesized MHC class I molecules, through peptide exchange in vesicles distinct from the endoplasmic reticulum and classical secretory pathway, in an SEC22b- and CD74-independent manner. Our results indicate a nonclassical secretion pathway followed by nascent HLA-I molecules that are used for cross-presentation of those long melanoma peptides in the vacuolar pathway. Our results may have implications for the development of vaccines based on long peptides.

Tryptophan catabolism by indoleamine 2,3-dioxygenase (IDO1) is a physiological immunoregulatory mechanism often hijacked by tumors. Our recent extensive study of IDO1 protein expression in human tissues showed expression in mature dendritic cells and in pulmonary and placental endothelial cells. IDO1 was also expressed in 56% of tumors, either by tumoral, stromal or endothelial cells. These results and reagent will guide the clinical development of IDO1 inhibitors for cancer therapy.

With the recent developments of adoptive T cell therapies and the use of new monoclonal antibodies against the immune checkpoints, immunotherapy is at a turning point. Key players for the success of these therapies are the cytolytic T lymphocytes, which are a subset of T cells able to recognize and kill tumor cells. Here, I review the nature of the antigenic peptides recognized by these T cells and the processes involved in their presentation. I discuss the importance of understanding how each antigenic peptide is processed in the context of immunotherapy and vaccine delivery.