Major histocompatibility class I antigenic peptides derived from translation of pre-mRNAs generate immune tolerance.

Antigenic peptides derived from introns are presented on major histocompatibility (MHC) class I molecules, but how these peptides are produced is poorly understood. Here, we show that an MHC class I epitope (SL8) sequence inserted in the second intron of the ß-globin gene in a C57BL/6 mouse (HBB) generates immune tolerance. Introduction of SL8-specific CD8+ T cells derived from OT-1 transgenic mice resulted in a threefold increase in OT-1 T cell proliferation in HBB animals, as compared to wild-type animals. The growth of MCA sarcoma cells expressing the intron-derived SL8 epitope was suppressed in wild-type animals compared to HBB mice. The ß-globin pre-mRNA was detected in the light polysomal fraction, and introducing stop codons identified a non-AUG initiation site between +228 and +255 nts upstream of the SL8. Isolation of ribosome footprints confirmed translation initiation within this 27 nt sequence. Furthermore, treatment with splicing inhibitor shifts the translation of the pre-mRNA to monosomal fractions and results in an increase of intron-derived peptide substrate as shown by polysome profiling and cell imaging. These results show that non-AUG-initiated translation of pre-mRNAs generates peptides for MHC class I immune tolerance and helps explain why alternative tissue-specific splicing is tolerated by the immune system.

Substrate-specific presentation of MHC class I-restricted antigens via autophagy pathway.

The accumulation of protein aggregates is toxic and linked to different diseases such as neurodegenerative disorders, but the role of the immune system to target and destroy aggregate-carrying cells is still relatively unknown. Here we show a substrate-specific presentation of antigenic peptides to the direct MHC class I pathway via autophagy. We observed no difference in presentation of peptides derived from the viral EBNA1 protein following suppression of autophagy by knocking down Atg5 and Atg12. However, the same knock down treatment suppressed the presentation from ovalbumin. Fusing the aggregate-prone poly-glutamine (PolyQ) to the ovalbumin had no effect on antigen presentation via autophagy. Interestingly, fusing the EBNA1-derived gly-ala repeat (GAr) sequence to ovalbumin rendered the presentation Atg5/12 independent. We also demonstrate that the relative levels of protein expression did not affect autophagy-mediated antigen presentation. These data suggest a substrate-dependent presentation of antigenic peptides for the MHC class I pathway via autophagy and indicate that the GAr of the EBNA1 illustrates a novel virus-mediated mechanism for immune evasion of autophagy-dependent antigen presentation.

In Cellulo Protein-mRNA Interaction Assay to Determine the Action of G-Quadruplex-Binding Molecules.

Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.

Sneaking Out for Happy Hour: Yeast-Based Approaches to Explore and Modulate Immune Response and Immune Evasion.

Many pathogens (virus, bacteria, fungi, or parasites) have developed a wide variety of mechanisms to evade their host immune system. The budding yeast Saccharomyces cerevisiae has successfully been used to decipher some of these immune evasion strategies. This includes the cis-acting mechanism that limits the expression of the oncogenic Epstein-Barr virus (EBV)-encoded EBNA1 and thus of antigenic peptides derived from this essential but highly antigenic viral protein. Studies based on budding yeast have also revealed the molecular bases of epigenetic switching or recombination underlying the silencing of all except one members of extended families of genes that encode closely related and highly antigenic surface proteins. This mechanism is exploited by several parasites (that include pathogens such as Plasmodium, Trypanosoma, Candida, or Pneumocystis) to alternate their surface antigens, thereby evading the immune system. Yeast can itself be a pathogen, and pathogenic fungi such as Candida albicans, which is phylogenetically very close to S. cerevisiae, have developed stealthiness strategies that include changes in their cell wall composition, or epitope-masking, to control production or exposure of highly antigenic but essential polysaccharides in their cell wall. Finally, due to the high antigenicity of its cell wall, yeast has been opportunistically exploited to create adjuvants and vectors for vaccination.

p53-mediated suppression of BiP triggers BIK-induced apoptosis during prolonged endoplasmic reticulum stress.

Physiological and pathological conditions that affect the folding capacity of the endoplasmic reticulum (ER) provoke ER stress and trigger the unfolded protein response (UPR). The UPR aims to either restore the balance between newly synthesized and misfolded proteins or if the damage is severe, to trigger cell death. However, the molecular events underlying the switch between repair and cell death are not well understood. The ER-resident chaperone BiP governs the UPR by sensing misfolded proteins and thereby releasing and activating the three mediators of the UPR: PERK, IRE1 and ATF6. PERK promotes G2 cell cycle arrest and cellular repair by inducing the alternative translated p53 isoform p53ΔN40 (p53/47), which activates 14-3-3σ via suppression of p21CDKN1A. Here we show that prolonged ER stress promotes apoptosis via a p53-dependent inhibition of BiP expression. This leads to the release of the pro-apoptotic BH3-only BIK from BiP and activation of apoptosis. Suppression of bip mRNA translation is mediated via the specific binding of p53 to the first 346-nt of the bip mRNA and via a p53 trans-suppression domain located within the first seven N-terminal amino acids of p53ΔN40. This work shows how p53 targets BiP to promote apoptosis during severe ER stress and further illustrates how regulation of mRNA translation has a key role in p53-mediated regulation of gene expression during the UPR.

The source of MHC class I presented peptides and its implications.

The source of peptides that enter the major histocompatibility class I (MHCI) pathway has been intensively debated over the last two decades. The initial assumption that peptides are derived from degradation of full length proteins was challenged by a model in which alternative translation products are a source of peptides. This model has been tested and supported by scientific data. We now need new hypotheses on the physiological implications of different sources of peptides for the MHCI pathway. The aim of this overview is to give an up-to-date account of the source of antigenic peptide material for the MHCI pathway and to incorporate the more recent observations of alternative mRNA translation products into existing models of the direct and cross-presentation pathways.

Exosome-driven transfer of tumor-associated Pioneer Translation Products (TA-PTPs) for the MHC class I cross-presentation pathway.

Cellular immune reactions against non-self-epitopes require activation of cytotoxic CD8+ T-cells via cross-presentation of MHC class I-restricted peptides by professional antigen presenting cells (pAPCs), with the consequent detection and elimination of cells expressing the same antigens via the endogenous (direct) pathway. The source of peptides for the endogenous pathway is constituted of alternative mRNA translation products; however, it is still unclear which source of peptides is used for cross-presentation. Furthermore, the presentation of non-canonical translation products, produced during a non-conventional translation event, on class I molecules of tumor cells has been reported but how these peptides are generated, presented to pAPCs, and their capacity to stimulate CD8+ T cells is still not known. Here, we report that pioneer translation peptides (PTPs) derived from intron or exon pre-mRNAs can serve as tumor-associated antigens (TA-PTPs) and are delivered from the producing tumor cells to pAPCs via exosomes where they are processed by the cytosolic pathway. Injection of TA-PTPs and tumor-derived exosomes efficiently induce CD8+ T-cell proliferation and prevent tumor growth in mice. Our results show that TA-PTPs represent an efficient source of antigenic peptides for CD8+ T cell activation and that full-length proteins are not required for cross-presentation. These findings can have interesting implications for generating tolerance and for designing vectors to generate vaccines.