Immunoproteasomes preserve protein homeostasis upon interferon-induced oxidative stress.

Interferon (IFN)-induced immunoproteasomes (i-proteasomes) have been associated with improved processing of major histocompatibility complex (MHC) class I antigens. Here, we show that i-proteasomes function to protect cell viability under conditions of IFN-induced oxidative stress. IFNs trigger the production of reactive oxygen species, which induce protein oxidation and the formation of nascent, oxidant-damaged proteins. We find that the ubiquitylation machinery is concomitantly upregulated in response to IFNs, functioning to target defective ribosomal products (DRiPs) for degradation by i-proteasomes. i-proteasome-deficiency in cells and in murine inflammation models results in the formation of aggresome-like induced structures and increased sensitivity to apoptosis. Efficient clearance of these aggregates by the enhanced proteolytic activity of the i-proteasome is important for the preservation of cell viability upon IFN-induced oxidative stress. Our findings suggest that rather than having a specific role in the production of class I antigens, i-proteasomes increase the peptide supply for antigen presentation as part of a more general role in the maintenance of protein homeostasis.

Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes.

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

CD8(+) T cells of Listeria monocytogenes-infected mice recognize both linear and spliced proteasome products.

CD8(+) T cells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed peptide splicing (PCPS). To determine whether PCPS contributes to epitope processing during infection, we analyzed the fragments produced by purified proteasomes from a Listeria monocytogenes polypeptide. Mass spectrometry identified a known H-2K(b) -presented linear epitope (LLO296-304 ) in the digests, as well as four spliced peptides that were trimmed by ERAP into peptides with in silico predicted H-2K(b) binding affinity. These spliced peptides, which displayed sequence similarity with LLO296-304 , bound to H-2K(b) molecules in cellular assays and one of the peptides was recognized by CD8(+) T cells of infected mice. This spliced epitope differed by one amino acid from LLO296-304 and double staining with LLO296-304 - and spliced peptide-folded MHC multimers showed that LLO296-304 and its spliced variant were recognized by the same CD8(+) T cells. Thus, PCPS multiplies the variety of peptides that is processed from an antigen and leads to the production of epitope variants that can be recognized by cross-reacting pathogen-specific CD8(+) T cells. Such mechanism may reduce the chances for pathogen immune evasion.

The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing.

MHC class I-restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred T cell receptor targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome-mediated antigen processing has so far not been studied. Therefore, we analyzed by in vitro experiments the effect on antigen processing and recognition of a T210M exchange, which previously had been introduced into the melanoma gp100209-217 tumor epitope to improve the HLA-A*02:01 binding and its immunogenicity. A quantitative analysis of the main steps of antigen processing shows that the T210M exchange affects proteasomal cleavage site usage within the mutgp100201-230 polypeptide, leading to the generation of an unique set of cleavage products. The T210M substitution qualitatively affects the proteasome-catalyzed generation of spliced and non-spliced peptides predicted to bind HLA-A or -B complexes. The T210M substitution also induces an enhanced production of the mutgp100209-217 epitope and its N-terminally extended peptides. The T210M exchange revealed no effect on ERAP1-mediated N-terminal trimming of the precursor peptides. However, mutant N-terminally extended peptides exhibited significantly increased HLA-A*02:01 binding affinity and elicited CD8(+) T cell stimulation in vitro similar to the wtgp100209-217 epitope. Thus, our experiments demonstrate that amino acid exchanges within an epitope can result in the generation of an altered peptide pool with new antigenic peptides and in a wider CD8(+) T cell response also towards N-terminally extended versions of the minimal epitope.

Proteasome isoforms exhibit only quantitative differences in cleavage and epitope generation.

Immunoproteasomes are considered to be optimised to process Ags and to alter the peptide repertoire by generating a qualitatively different set of MHC class I epitopes. Whether the immunoproteasome at the biochemical level, influence the quality rather than the quantity of the immuno-genic peptide pool is still unclear. Here, we quantified the cleavage-site usage by human standard- and immunoproteasomes, and proteasomes from immuno-subunit-deficient mice, as well as the peptides generated from model polypeptides. We show in this study that the different proteasome isoforms can exert significant quantitative differences in the cleavage-site usage and MHC class I restricted epitope production. However, independent of the proteasome isoform and substrates studied, no evidence was obtained for the abolishment of the specific cleavage-site usage, or for differences in the quality of the peptides generated. Thus, we conclude that the observed differences in MHC class I restricted Ag presentation between standard- and immunoproteasomes are due to quantitative differences in the proteasome-generated antigenic peptides.

Role of peptide processing predictions in T cell epitope identification: contribution of different prediction programs.

Proteolysis is the general term to describe the process of protein degradation into peptides. Proteasomes are the main actors in cellular proteolysis, and their activity can be measured in in vitro digestion experiments. However, in vivo proteolysis can be different than what is measured in these experiments if other proteases participate or if proteasomal activity is different in vivo. The in vivo proteolysis can be measured only indirectly, by the analysis of peptides presented on MHC-I molecules. MHC-I presented peptides are protected from further degradation, thus enabling an indirect view on the underlying in vivo proteolysis. The ligands presented on different MHC-I molecules enable different views on this process; in combination, they might give a complete picture. Based on in vitro proteasome-only digestions and MHC-I ligand data, different proteolysis predictors have been developed. With new in vitro digestion and MHC-I ligand data sets, we benchmarked how well these predictors capture in vitro proteasome-only activity and in vivo whole-cell proteolysis, respectively. Even though the in vitro proteasome digestion patterns were best captured by methods trained on such data (ProteaSMM and NetChop 20S), the in vivo whole-cell proteolysis was best predicted by a method trained on MHC-I ligand data (NetChop Cterm). Follow-up analysis showed that the likely source of this difference is the activity from proteases other than the proteasome, such as TPPII. This non-proteasomal in vivo activity is captured by NetChop Cterm and should be taken into account in MHC-I ligand predictions.

The immunoproteasome ß5i subunit is a key contributor to ictogenesis in a rat model of chronic epilepsy.

The proteasome is the core of the ubiquitin-proteasome system and is involved in synaptic protein metabolism. The incorporation of three inducible immuno-subunits into the proteasome results in the generation of the so-called immunoproteasome, which is endowed of pathophysiological functions related to immunity and inflammation. In healthy human brain, the expression of the key catalytic ß5i subunit of the immunoproteasome is almost absent, while it is induced in the epileptogenic foci surgically resected from patients with pharmaco-resistant seizures, including temporal lobe epilepsy. We show here that the ß5i immuno-subunit is induced in experimental epilepsy, and its selective pharmacological inhibition significantly prevents, or delays, 4-aminopyridine-induced seizure-like events in acute rat hippocampal/entorhinal cortex slices. These effects are stronger in slices from epileptic vs normal rats, likely due to the more prominent ß5i subunit expression in neurons and glia cells of diseased tissue. ß5i subunit is transcriptionally induced in epileptogenic tissue likely by Toll-like receptor 4 signaling activation, and independently on promoter methylation. The recent availability of selective ß5i subunit inhibitors opens up novel therapeutic opportunities for seizure inhibition in drug-resistant epilepsies.

Impairment of immunoproteasome function by ß5i/LMP7 subunit deficiency results in severe enterovirus myocarditis.

Proteasomes recognize and degrade poly-ubiquitinylated proteins. In infectious disease, cells activated by interferons (IFNs) express three unique catalytic subunits ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7 forming an alternative proteasome isoform, the immunoproteasome (IP). The in vivo function of IPs in pathogen-induced inflammation is still a matter of controversy. IPs were mainly associated with MHC class I antigen processing. However, recent findings pointed to a more general function of IPs in response to cytokine stress. Here, we report on the role of IPs in acute coxsackievirus B3 (CVB3) myocarditis reflecting one of the most common viral disease entities among young people. Despite identical viral load in both control and IP-deficient mice, IP-deficiency was associated with severe acute heart muscle injury reflected by large foci of inflammatory lesions and severe myocardial tissue damage. Exacerbation of acute heart muscle injury in this host was ascribed to disequilibrium in protein homeostasis in viral heart disease as indicated by the detection of increased proteotoxic stress in cytokine-challenged cardiomyocytes and inflammatory cells from IP-deficient mice. In fact, due to IP-dependent removal of poly-ubiquitinylated protein aggregates in the injured myocardium IPs protected CVB3-challenged mice from oxidant-protein damage. Impaired NFκB activation in IP-deficient cardiomyocytes and inflammatory cells and proteotoxic stress in combination with severe inflammation in CVB3-challenged hearts from IP-deficient mice potentiated apoptotic cell death in this host, thus exacerbating acute tissue damage. Adoptive T cell transfer studies in IP-deficient mice are in agreement with data pointing towards an effective CD8 T cell immune. This study therefore demonstrates that IP formation primarily protects the target organ of CVB3 infection from excessive inflammatory tissue damage in a virus-induced proinflammatory cytokine milieu.

CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope.

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.

Antigen-presentation capacity of dendritic cells is impaired in ongoing enterovirus myocarditis.

Coxsackievirus B3 (CVB3)-infection is a frequent cause of acute myocarditis, which may result in chronic myocarditis and virus persistence. Investigation of the initial immune responses to CVB3 may shed light on the mechanisms that contribute to ongoing disease. DCs, as key professional APCs, were investigated in two MHC-matched hosts: while C57BL/6 mice are resistant to chronic CVB3-myocarditis, the A.BY/SnJ mouse strain exhibits susceptibility. DC maturation and activation were critically impaired in A.BY/SnJ mice, as reflected by the failure of DCs to induce co-stimulatory molecules and cytokine/chemokine responses. MHC class I-restricted antigen presentation via the cross-presentation pathway was also affected in DCs from A.BY/SnJ mice. DC maturation involves the accumulation of DC aggresome-like induced structures (DALISs) and the transient storage of defective ribosomal products (DRiPs). DCs from A.BY/SnJ mice showed permanent DALIS accumulation; the detection of poly-ubiquitinylated CVB3 proteins in these DALISs suggested a limitation in the MHC class I antigenic supply in this host. In conclusion, ongoing chronic disease in A.BY/SnJ mice due to a failure to clear the virus may be attributed to defects in DC maturation/activation and DC MHC class I antigen processing.

Neo-Splicetopes in Tumor Therapy: A Lost Case?

Proteasome generates spliced peptides by ligating two distant cleavage products in a reverse proteolysis reaction. The observation that CD8+ T cells recognizing a spliced peptide induced T cell rejection in a melanoma patient following adoptive T cell transfer (ATT), raised some hopes with regard to the general therapeutic and immune relevance of spliced peptides. Concomitantly, the identification of spliced peptides was also the start of a controversy with respect to their frequency, abundancy and their therapeutic applicability. Here I review some of the recent evidence favoring or disfavoring an immune relevance of splicetopes and discuss from a theoretical point of view the potential usefulness of tumor specific splicetopes and why against all odds it still may seem worth trying to identify such tumor and patient-specific neosplicetopes for application in ATT.

In vitro proteasome processing of neo-splicetopes does not predict their presentation in vivo.

Proteasome-catalyzed peptide splicing (PCPS) of cancer-driving antigens could generate attractive neoepitopes to be targeted by T cell receptor (TCR)-based adoptive T cell therapy. Based on a spliced peptide prediction algorithm, TCRs were generated against putative KRASG12V- and RAC2P29L-derived neo-splicetopes with high HLA-A*02:01 binding affinity. TCRs generated in mice with a diverse human TCR repertoire specifically recognized the respective target peptides with high efficacy. However, we failed to detect any neo-splicetope-specific T cell response when testing the in vivo neo-splicetope generation and obtained no experimental evidence that the putative KRASG12V- and RAC2P29L-derived neo-splicetopes were naturally processed and presented. Furthermore, only the putative RAC2P29L-derived neo-splicetopes was generated by in vitro PCPS. The experiments pose severe questions on the notion that available algorithms or the in vitro PCPS reaction reliably simulate in vivo splicing and argue against the general applicability of an algorithm-driven 'reverse immunology' pipeline for the identification of cancer-specific neo-splicetopes.

Link between organ-specific antigen processing by 20S proteasomes and CD8(+) T cell-mediated autoimmunity.

Adoptive transfer of cross-reactive HSP60-specific CD8(+) T cells into immunodeficient mice causes autoimmune intestinal pathology restricted to the small intestine. We wondered whether local immunopathology induced by CD8(+) T cells can be explained by tissue-specific differences in proteasome-mediated processing of major histocompatibility complex class I T cell epitopes. Our experiments demonstrate that 20S proteasomes of different organs display a characteristic composition of alpha and beta chain subunits and produce distinct peptide fragments with respect to both quality and quantity. Digests of HSP60 polypeptides by 20S proteasomes show most efficient generation of the pathology related CD8(+) T cell epitope in the small intestine. Further, we demonstrate that the organ-specific potential to produce defined T cell epitopes reflects quantities that are relevant for cytotoxic T lymphocyte recognition. We propose tissue-specific antigen processing by 20S proteasomes as a potential mechanism to control organ-specific immune responses.

Humoral anti-proteasomal autoimmunity in dilated cardiomyopathy.

Virus-induced chronic inflammation, autoimmune processes and impaired protein quality control may be involved in the pathogenesis of dilated cardiomyopathy (DCM). The ubiquitin-proteasome system is important in the modulation of inflammatory processes and the immune response. Proteasomes were identified as targets of a humoral autoimmune response in systemic inflammatory diseases, which provoked us to investigate anti-proteasomal immunity in DCM in detail: a total of 90 DCM patients with impaired left-ventricular function (LVEF < or = 45%) were enrolled in this study. Autoimmune response to cardiac proteasomes was found to be enhanced in DCM patients, revealing the detection of predominantly alpha subunits of the 20S proteasome complex. Proteasome antibody (ProtAb) levels were found to be particularly enhanced at stages of advanced heart failure: moderately decreased LVEF and considerably increased NT-pro BNP levels were observed in DCM patients who tested positive for ProtAb (P < 0.05). A linear regression model suggested a link between the detection of cardiotropic viruses in endomyocardial biopsies and anti-proteasomal immunity (P < 0.01). Likewise, ProtAb levels were enhanced in a murine model of chronic enterovirus myocarditis. Our data also point to a potential interaction of ProtAb with the cell surface: ProtAb exerted negative inotropic effects in field-stimulated cardiomyocytes. In conclusion, humoral autoreactive anti-proteasome immune responses appear to be enhanced in DCM. Viral infection of the myocardium may be linked to the induction of anti-proteasomal immunity in DCM.

Lack of evidence for a pathogenic role of proteasome-directed autoimmunity in dilated cardiomyopathy.

The proteasome has been identified as a target of the humoral autoimmune response in different inflammatory disease entities including dilated cardiomyopathy (DCM). However, the role of proteasome autoantibodies (ProtAb) remains to be studied. Here, we have isolated human ProtAb by affinity-purification from the IgG fractions obtained from DCM patients, which predominantly detected the outer ring subunits alpha3 of the 20S proteasome. In an attempt to study the cellular effects potentially exerted by these ProtAb, simultaneous calcium and cell contractility measurements were performed in rat cardiomyocytes revealing no short-term effects upon human ProtAb exposure. Immunofluorescence staining and FACS analysis pointed towards a failure of human ProtAb to bind to the intact cell membrane, whereas human ProtAb detected 20S proteasomes in the cytoplasm and nucleus. The lack of the cell surface interaction of human ProtAb was in agreement with the failure of these autoantibodies to interfere with the cellular viability. Further, we investigated whether the removal of ProtAb by immunoadsorption (IA) resulted in functional improvement in DCM patients. IA was performed in 90 DCM patients (left ventricular ejection fraction < or =45%, ProtAb detection at baseline in 30% of these DCM patients). Improvement of LVEF was not associated with the initial detection and removal of ProtAb in DCM patients. ProtAb were reconstituted to baseline levels as soon as after 3 months post-IA/IgG treatment despite the overall improvement of LVEF in this study group. In conclusion, our data argue against a direct impact of ProtAb in the pathogenesis of DCM.

Differential interferon responses enhance viral epitope generation by myocardial immunoproteasomes in murine enterovirus myocarditis.

Murine models of coxsackievirus B3 (CVB3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection, with host-specific outcomes ranging from complete recovery in resistant mice to chronic disease in susceptible hosts. To identify susceptibility factors that modulate the course of viral myocarditis, we show that type-I interferon (IFN) responses are considerably impaired in acute CVB3-induced myocarditis in susceptible mice, which have been linked to immunoproteasome (IP) formation. Here we report that in concurrence with distinctive type-I IFN kinetics, myocardial IP formation peaked early after infection in resistant mice and was postponed with maximum IP expression concomitant to massive inflammation and predominant type-II IFN responses in susceptible mice. IP activity is linked to a strong enhancement of antigenic viral peptide presentation. To investigate the impact of myocardial IPs in CVB3-induced myocarditis, we identified two novel CVB3 T cell epitopes, virus capsid protein 2 [285-293] and polymerase 3D [2170-2177]. Analysis of myocardial IPs in CVB3-induced myocarditis revealed that myocardial IP expression resulted in efficient epitope generation. As opposed to the susceptible host, myocardial IP expression at early stages of disease corresponded to enhanced CVB3 epitope generation in the hearts of resistant mice. We propose that this process may precondition the infected heart for adaptive immune responses. In conclusion, type-I IFN-induced myocardial IP activity at early stages coincides with less severe disease manifestation in CVB3-induced myocarditis.

Generation of in silico predicted coxsackievirus B3-derived MHC class I epitopes by proteasomes.

Proteasomes are known to be the main suppliers of MHC class I (MHC-I) ligands. In an attempt to identify coxsackievirus B3 (CVB3)-MHC-I epitopes, a combined approach of in silico MHC-I/transporters associated with antigen processing (TAP)-binding and proteasomal cleavage prediction was applied. Accordingly, 13 potential epitopes originating from the structural and non-structural protein region of CVB3 were selected for further in vitro processing analysis by proteasomes. Mass spectrometry demonstrated the generation of seven of the 13 predicted MHC-I ligands or respective ligand precursors by proteasomes. Detailed processing analysis of three adjacent MHC-I ligands with partially overlapping sequences, i.e. VP2(273-281), VP2(284-292) and VP2(285-293), revealed the preferential generation predominantly of the VP2(285-293) epitope by immunoproteasomes due to altered cleavage site preferences. The VP2(285-293) peptide was identified to be a high affinity binder, rendering VP2(285-293) a likely candidate for CD8 T cell immunity in CVB3 infection. In conclusion, the concerted usage of different in silico prediction methods and in vitro epitope processing/presentation studies was supportive in the identification of CVB3 MHC-I epitopes.

BSc2118 is a novel proteasome inhibitor with activity against multiple myeloma.

OBJECTIVES: The ubiquitin-proteasome system emerged as a new therapeutic target in cancer treatment. The purpose of this study was to elucidate the effects of the novel proteasome inhibitor BSc2118 on t(4;14) positive and negative multiple myeloma (MM) cells and normal peripheral blood mononuclear cells (PBMNC). METHODS: Human MM cell lines OPM-2, RPMI-8226, and U266 and primary MM cells from bone marrow aspirates were exposed to BSc2118. Cytotoxicity levels were evaluated using the MTT-test. BSc2118-induced apoptosis was analyzed by annexin-V assay. Further methods used included proteasomal activity determination, cell cycle analysis, western blot, and transcription factor assays. RESULTS: In OPM-2, RPMI-8226, U266 cell lines and primary MM cells, BSc2118 caused dose-dependent growth inhibitory effects. After 48 h, dose-dependent apoptosis occurred both in cell lines and primary myeloma cells irrespective of t(4;14). A significant G2-M cell cycle arrest occurred after 24 h. Furthermore, we observed a marked inhibition of intracellular proteasome activity, an increase in intracellular p21 levels, and an inhibition of NF-kappaB activation. The toxicity against PBMNC remained low, suggesting a broad therapeutic range of this agent. CONCLUSION: Taken together, BSc2118 shows significant antimyeloma activity and may be considered as a promising agent in cancer drug development.

Virus-induced type I IFN stimulates generation of immunoproteasomes at the site of infection.

IFN-gamma is known as the initial and primary inducer of immunoproteasomes during viral infections. We now report that type I IFN induced the transcription and translation of immunoproteasome subunits, their incorporation into the proteasome complex, and the generation of an immunoproteasome-dependent CD8 T cell epitope in vitro and provide in vivo evidence that this mechanism occurs prior to IFN-gamma responses at the site of viral infection. Type I IFN-mediated generation of immunoproteasomes was initiated by either poly(I:C) or HCV RNA in human hepatoma cells and was inhibited by neutralization of type I IFN. In serial liver biopsies of chimpanzees with acute HCV infection, increases in immunoproteasome subunit mRNA preceded intrahepatic IFN-gamma responses by several weeks, instead coinciding with intrahepatic type I IFN responses. Thus, viral RNA-induced innate immune responses regulate the antigen-processing machinery, which occurs prior to the detection of IFN-gamma at the site of infection. This mechanism may contribute to the high effectiveness (95%) of type I IFN-based therapies if administered early during HCV infection.