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The prime function of proteasomes is the control of protein homeostasis in cells (i.e., the removal of proteins that are not properly folded, damaged by stress conditions like reactive oxygen species formation, or degraded on the basis of regular protein turnover). During viral infection, the standard proteasome is replaced by the so-called immunoproteasome (IP) in an IFN-γ–dependent manner. It has been proposed that the IP is required to protect cell viability under conditions of IFN-induced oxidative stress. In this study, we investigated the requirement for IP to cope with the enhanced need for protein degradation during lymphocytic choriomeningitis virus (LCMV) infection in mice lacking the IP subunit LMP7. We found that IP are upregulated in the liver but not in the spleen during LCMV infection, although the total proteasome content was not altered. The expression of standard proteasome subunits is not induced in LMP7-deficient mice, indicating that enhanced proteasomal activity is not required during viral infection. Furthermore, ubiquitin accumulation, apoptosis induction, and viral titers were similar in LCMV-infected mice lacking LMP7 compared with wild-type mice. Taken together, these data indicate that the IP is not required to regulate protein homeostasis during LCMV infection.

BackgroundAllergic asthma is a chronic disease and medical treatment often fails to fully control the disease in the long term, leading to a great need for new therapeutic approaches. Immunoproteasome inhibition impairs T helper cell function and is effective in many (auto-) inflammatory settings but its effect on allergic airway inflammation is unknown.MethodsImmunoproteasome expression was analyzed in in vitro polarized T helper cell subsets. To study Th2 cells in vivo acute allergic airway inflammation was induced in GATIR (GATA-3-vYFP reporter) mice using ovalbumin and house dust mite extract. Mice were treated with the immunoproteasome inhibitor ONX 0914 or vehicle during the challenge phase and the induction of airway inflammation was analyzed.ResultsIn vitro polarized T helper cell subsets (Th1, Th2, Th17, and Treg) express high levels of immunoproteasome subunits. GATIR mice proved to be a useful tool for identification of Th2 cells. Immunoproteasome inhibition reduced the Th2 response in both airway inflammation models. Furthermore, T cell activation and antigen-specific cytokine secretion was impaired and a reduced infiltration of eosinophils and professional antigen-presenting cells into the lung and the bronchoalveolar space was observed in the ovalbumin model.ConclusionThese results show the importance of the immunoproteasome in Th2 cells and airway inflammation. Our data provides first insight into the potential of using immunoproteasome inhibition to target the aberrant Th2 response, e.g. in allergic airway inflammation.

Polymyositis (PM) is a chronic autoimmune inflammatory myopathy resulting in muscle weakness. The limited approved therapies and their poor efficacy contribute to its comorbidity. We investigated the therapeutic use of ONX 0914 and KZR-616, selective inhibitors of the immunoproteasome, in C protein-induced myositis (CIM), a mouse model of PM that closely resembles the human disease. Diseased mice (day 13 post-immunization) were treated with 10 mg/kg ONX 0914 or KZR-616 or vehicle on alternate days until day 28. Endpoints included muscle strength assessed by a grip strength meter, serum creatine kinase activity, histology, and immunohistochemistry analysis. Treatment with ONX 0914 or KZR-616 prevented the loss of grip strength in mice after CIM induction, while vehicle-treated animals displayed progressive muscle weakness. Immunoproteasome inhibition lowered PM-associated leukocyte infiltration of the muscle and prevented increased serum creatine kinase levels. LMP7-deficient mice were resistant to CIM induction as they depicted no alteration in the grip strength, creatine kinase (CK) levels, nor showed muscular alterations. In conclusion, selective inhibition of the immunoproteasome displays therapeutic efficacy in a pre-clinical mouse model of PM with suppression of muscle inflammation and preservation of muscle strength. Positive results from this study support the rationale for using KZR-616 in clinical studies. published

The IFN stimulated gene 15 (ISG15) encodes a 15-kDa ubiquitin-like protein, that is induced by type I IFNs and is conjugated to the bulk of newly synthesized polypeptides at the ribosome. ISG15 functions as an antiviral molecule possibly by being covalently conjugated to viral proteins and disturbing virus particle assembly. Here, we have investigated the effect of ISGylation on degradation and antigen presentation of viral and cellular proteins. ISGylation did not induce proteasomal degradation of bulk ISG15 target proteins neither after overexpressing ISG15 nor after induction by IFN-β. The MHC class I cell surface expression of splenocytes derived from ISG15-deficient mice or mice lacking the catalytic activity of the major de-ISGylating enzyme USP18 was unaltered as compared to WT mice. Fusion of ubiquitin or FAT10 to the long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus accelerated the proteasomal degradation of NP while fusion to ISG15 did not detectably speed up NP degradation. Nevertheless, MHC-I restricted presentation of two epitopes of NP were markedly enhanced when it was fused to ISG15 similarly to fusion with ubiquitin or FAT10. Thus, we provide evidence that ISG15 can enhance the presentation of antigens on MHC-I most likely by promoting co-translational antigen processing. published

MECL-1 (β2i), LMP2 (β1i), and LMP7 (β5i) are the proteolytically active subunits of the immunoproteasome (IP), a special type of proteasome mainly expressed in hematopoietic cells. Targeting the IP in autoimmune diseases proved to be therapeutically effective in preclinical mouse models. In endotoxin-stimulated human PBMCs, IP inhibition reduces the secretion of several proinflammatory cytokines, with the suppression of IL-23 being the most prominent. In this study, we investigated why the production of IL-23, a key mediator of inflammation in autoimmunity, is blocked when the IP is inhibited in LPS-stimulated human PBMCs. CD14+ monocytes could be identified as the main producers of IL-23 in LPS-stimulated PBMCs. We found that IP inhibition with the irreversible LMP7/LMP2 inhibitor ONX 0914 induced apoptosis in CD14+ monocytes, whereas CD4+, CD3+, CD19+, and CD56+ cells remained unaffected. A high expression of IPs renders monocytes susceptible to IP inhibition, leading to an accumulation of polyubiquitylated proteins and the induction of the unfolded protein response. Similar to IP inhibition, inducers of the unfolded protein response selectively kill CD14+ monocytes in human PBMCs. The blockage of the translation in CD14+ monocytes protects these cells from ONX 0914–induced cell death, indicating that the IP is required to maintain protein turnover in monocytes. Taken together, our data reveal why IP inhibition is particularly effective in the suppression of IL-23–driven autoimmunity.

FAT10 is the only ubiquitin-like modifier which directly targets its substrate proteins for rapid degradation by the proteasome. While the conjugation and proteasomal targeting of FAT10 are fairly well understood, the biological functions of FAT10 have remained largely elusive. Here we have investigated the role of FAT10 in cytokine responses in mice upon viral infection. We used lymphocytic choriomeningitis virus (LCMV) infection of mice to induce the IFN-γ and TNF-α-dependent expression of FAT10. We found that TCR-stimulated splenocytes derived from LCMV-infected FAT10-/- mice secreted less IFN-γ and expressed less mRNA for IL-12 p40 but secreted more IFN-α and IFN-β compared to FAT10+/- mice. The reduction in IFN-γ secretion could be assigned to CD8+ T cells. Nevertheless, LCMV viral clearance was similar in FAT10-/- as compared to FAT10+/- mice. Since FAT10 has previously been reported to promote influenza A virus (IAV) replication in vitro we have studied the effect of FAT10 deficiency during IAV infection in mice. Unexpectedly, IAV titers and disease symptoms were not changed in FAT10-/- mice even though the Fat10 mRNA was rapidly induced in the lung upon IAV infection. In conclusion, we find that FAT10 fine-tunes the balance of interferons during viral infection by lowering the production of type I and enhancing type II interferons.

The proteasome is a multicatalytic protease in the cytosol and nucleus of all eukaryotic cells that controls numerous cellular processes through regulated protein degradation. Proteasome inhibitors have significantly improved the survival of multiple myeloma patients. However, clinically approved proteasome inhibitors have failed to show efficacy against solid tumors, neither alone nor in combination with other therapies. Targeting the immunoproteasome with selective inhibitors has been therapeutically effective in preclinical models for several autoimmune diseases and colon cancer. Moreover, immunoproteasome inhibitors prevented the chronic rejection of allogeneic organ transplants. In recent years, it has become apparent that inhibition of one single active center of the proteasome is insufficient to achieve therapeutic benefits. In this review we summarize the latest insights how targeting multiple catalytically active proteasome subunits can interfere with disease progression in autoimmunity, growth of solid tumors, and allograft rejection. published

The TNF and IFN-γ-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) is most prominently expressed in immunological tissues but information regarding basal expression and inducibility of FAT10 in the different types of immune cells is still lacking. Hence, we investigated FAT10 mRNA expression in the major human and murine immune cell subsets, and FAT10 protein expression in human leukocytes. We isolated the different human leukocytes from peripheral blood and the murine immune cell subsets from spleen. The purified leukocytes were left untreated or stimulated with TNF and INF-γ or LPS to induce FAT10 followed by quantitative real-time PCR or western blot analysis. Basal expression of FAT10 mRNA and protein was generally low but strongly up-regulated by IFN-γ and TNF in all immune cell subsets. LPS treatment induced FAT10 expression marginally in human CD8+ T cells and murine granulocytes, but it increased Fat10 expression significantly in murine regulatory T cells. Yet, in human CD8+ T cells, natural killer cells, natural killer T cells, and dendritic cells, the FAT10 mRNA was expressed without induction. Similarly, murine macrophages, monocytes, and regulatory T cells expressed Fat10 in the absence of stimulation. In summary, our findings suggest particular functions of FAT10 in these cell types. Furthermore, we observed not only a cell type-specific but also a species-specific basal FAT10 expression profile. Our data will serve as a guideline for future investigations to further elucidate FAT10’s role in the immune system. published

Background and Purpose Multicatalytic endopeptidase complex-like-1 (β2i), low molecular mass polypeptide (LMP) 2 (β1i) and LMP7 (β5i) are the proteolytically active subunits of the immunoproteasome, a special type of proteasome mainly expressed in haematopoietic cells. Targeting LMP7 has been shown to be therapeutically effective in preclinical models of autoimmune diseases. In this study, we investigated the selectivity and biological activity of LU-005i, a recently described inhibitor of the immunoproteasome. Experimental Approach The specificity of LU-005i and other immunoproteasome-selective inhibitors was characterized using fluorogenic peptide substrates. The effect of proteasome inhibition on cytokine release was investigated in endotoxin-stimulated mouse splenocytes or human peripheral blood mononuclear cells (PBMCs). The effect of proteasome inhibition on inflammatory bowel disease in the dextran sulfate sodium (DSS)-induced colitis model was assessed by measuring weight loss and colon length. Key Results LU-005i is the first human and mouse immunoproteasome-selective inhibitor that targets all three proteolytically active immunoproteasome subunits. LU-005i inhibited cytokine secretion from endotoxin-stimulated mouse splenocytes or human PBMCs. Furthermore, differentiation of naive T helper cells to T helper 17 cells was impaired in the presence of LU-005i. Additionally, LU-005i ameliorated DSS-induced colitis. Conclusion and Implications This study with a novel pan-immunoproteasome inhibitor substantiates that the immunoproteasome is a promising drug target for the treatment of inflammatory diseases and that exclusive inhibition of LMP7 is not necessary for therapeutic effectiveness. Our results will promote the design of new generations of immunoproteasome inhibitors with optimal therapeutic efficacy for clinical use in the treatment of autoimmunity and cancer.

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible variant of the proteasome which has been implicated in regulating inflammatory responses and antigen presentation. In the thymus, medullary thymic epithelial cells (mTECs) and cortical thymic epithelial cells (cTECs) do express different proteasome subunits exerting chymotrypsin-like activities suggesting distinct functions in thymic T cell selection. Employing the lymphocytic choriomeningitis virus (LCMV) infection model, we could show that the immunoproteasome subunit LMP7 was absolutely required for the generation of LCMV GP118-125-specific T cells although the class I mediated presentation of GP118-125 was not dependent on LMP7. Using bone marrow chimeras and adoptive transfer of LMP7-deficient CD8+ T cells into RAG1-deficient mice we show that LMP7-deficient mice lacked GP118-125-specific T cell precursors and that LMP7 was required in radioresistant cells – most likely thymic epithelial cells - to enable their selection. Since LMP7 is strongly expressed in negatively selecting mTECs but barely in positively selecting cTECs our data suggest that LMP7 was required to avoid excessive negative selection of GP118-125-specific T cell precursors. Taken together, this study demonstrates that the immunoproteasome is a crucial factor for filling up holes within the cytotoxic T cell repertoire. This article is protected by copyright. All rights reserved

The immunoproteasome, a distinct class of proteasomes, which is inducible under inflammatory conditions and constitutively expressed in monocytes and lymphocytes, is known to shape the antigenic repertoire presented on major histocompatibility complex (MHC) class I molecules. Moreover, inhibition of the immunoproteasome subunit LMP7 ameliorates clinical symptoms of autoimmune diseases in vivo and was shown to suppress the development of T helper cell (Th) 1 and Th17 cells and to promote regulatory T-cell (Treg) generation independently of its function in antigen processing. Since Th1 and Th17 cells are detrimental and Treg cells are critical for transplant acceptance, we investigated the influence of the LMP7-selective inhibitor ONX 0914 in a mixed lymphocyte reaction (MLR) in vitro as well as on allograft rejection in a MHC-disparate (C57BL/6 to BALB/c) and a multiple minor histocompatibility antigen (miHA)-disparate (B10.Br to C3H) model of skin transplantation in vivo. Although we observed reduced allo-specific IL-17 production of T cells in vitro, we found that selective inhibition of LMP7 had neither an influence on allograft survival in an MHC-mismatch model nor in a multiple minor mismatch skin transplantation model. We conclude that inhibition of the immunoproteasome is not effective in prolonging skin allograft survival in skin allotransplantation. published

Antigen processing for direct presentation on MHC class I molecules is a multistep process requiring the concerted activity of several cellular complexes. The essential steps at the beginning of this pathway, namely protein synthesis at the ribosome and degradation via the proteasome, have been known for years. Nevertheless, there is a considerable lack of factors identified to function between protein synthesis and degradation during antigen processing. Here, we analyzed the impact of the chaperone BAG6 on MHC class I cell surface expression and presentation of virus-derived peptides. Although an essential role of BAG6 in antigen processing has been proposed previously, we found BAG6 to be dispensable in this pathway. Still, interaction of BAG6 and the model antigen tyrosinase was enhanced during proteasome inhibition pointing towards a role of BAG6 in antigen degradation. Redundant chaperone pathways potentially mask the contribution of BAG6 to antigen processing and presentation. published

BACKGROUND PLGA microsphere-based vaccination has been proven to be effective in immunotherapy of syngeneic model tumors in mice. The critical step for the translation to humans is the identification of immunogenic tumor antigens and potent vaccine formulations to overcome immune tolerance. METHODS HLA-A*0201 transgenic mice were immunized with eight different human prostate cancer peptide antigens co-encapsulated with TLR ligands into PLGA microspheres and analyzed for antigen-specific and functional cytotoxic T lymphocyte responses. RESULTS Only vaccination with STEAP1262-270 peptide encapsulated in PLGA MS could effectively crossprime CTLs in vivo. These CTLs recognized STEAP1262–270/HLA-A*0201 complexes on human dendritic cells and prostate cancer cell lines and specifically lysed target cells in vivo. Vaccination with PLGA microspheres was much more potent than with incomplete Freund's adjuvant. CONCLUSIONS Our data suggests that there exist great differences in the immunogenicity of human PCa peptide antigens despite comparable MHC class I binding characteristics. Immunogenic STEAP1262–270 peptide encapsulated into PLGA microspheres however was able to induce vigorous and functional antigen-specific CTLs and therefore is a promising novel approach for immunotherapy against advanced stage prostate cancer. Prostate © 2015 Wiley Periodicals, Inc.

Immunoproteasome (IP) inhibition holds potential as a novel treatment option for various immune-mediated pathologies. The IP inhibitor ONX 0914 reduced T cell cytokine secretion and Th17 polarization and showed pre-clinical efficacy in a range of autoimmune disorders, transplant-allograft rejection, virus-mediated tissue damage, and colon cancer progression. However, the molecular basis of these effects has remained largely elusive. Here, we have analyzed the effects of ONX 0914 in primary human and mouse lymphocytes. ONX 0914-treatment impaired primary T cell activation in vitro and in vivo. IP inhibition reduced ERK-phosphorylation sustainment, while leaving NF-κB and other signaling pathways unaffected. Naïve T and B cells expressed nearly exclusively immuno- or mixed proteasomes but no standard proteasomes and IP inhibition but not IP-deficiency induced mild proteostasis stress, reduced DUSP5 expression and enhanced DUSP6 protein levels due to impaired degradation. However, accumulation of DUSP6 did not cause the reduced ERK-phosphorylation in a non-redundant manner. We show that broad-spectrum proteasome inhibition and immunoproteasome inhibition have distinct effects on T cell activation at the molecular level. Notably, ONX 0914-treated T cells recovered from proteostasis stress without apoptosis induction, apparently via Nrf1-mediated up-regulation of standard proteasomes. In contrast, B cells were more susceptible to apoptosis after ONX 0914-treatment. Our data thus provide mechanistic insights how IP inhibition functionally impedes T and B cells likely accounting for its therapeutic benefits. published

In addition to antigen processing, immunoproteasomes were recently shown to exert functions influencing cytokine production by monocytes and T cells, T-helper cell differentiation, and T-cell survival. Moreover, selective inhibition of the immunoproteasome subunit LMP7 ameliorated symptoms of autoimmune diseases including CD4(+) T-cell mediated EAE. In this study, we show that LMP7 also plays a crucial role in the pathogenesis of lymphocytic choriomeningitis virus (LCMV)-induced meningitis mediated by CTLs. Mice lacking functional LMP7 display delayed and reduced clinical signs of disease accompanied by a strongly decreased inflammatory infiltration into the brain. Interestingly, we found that selective inhibition and genetic deficiency of LMP7 affect the pathogenesis of LCMV-induced meningitis in a distinct manner. Our findings support the important role of LMP7 in inflammatory disorders and suggest immunoproteasome inhibition as a novel strategy against inflammation-induced neuropathology in the CNS.

HLA-F adjacent transcript 10 (FAT10) is a cytokine-inducible ubiquitin-like modifier that is highly expressed in the thymus and directly targets FAT10-conjugated proteins for degradation by the proteasome. High expression of FAT10 in the mouse thymus could be assigned to strongly autoimmune regulator–expressing, mature medullary thymic epithelial cells, which play a pivotal role in negative selection of T cells. Also in the human thymus, FAT10 is localized in the medulla but not the cortex. TCR Vβ-segment screening revealed a changed T cell repertoire in FAT10-deficient mice. Analysis of five MHC class I– and II–restricted TCR-transgenic mice demonstrated an altered thymic negative selection in FAT10-deficient mice. Furthermore, the repertoire of peptides eluted from MHC class I molecules was influenced by FAT10 expression. Hence, we identified FAT10 as a novel modifier of thymic Ag presentation and epitope-dependent elimination of self-reactive T cells, which may explain why the fat10 gene could recently be linked to enhanced susceptibility to virus-triggered autoimmune diabetes.

The 20S immunoproteasome (IP) is an interferon(IFN)-γ − and tumor necrosis factor (TNF) −inducible variant of the 20S constitutive proteasome (CP) in which all its peptidolytically active subunits β1, β2, and β5 are replaced by their cytokine inducible homologues β1i (LMP2), β2i (MECL-1), and β5i (LMP7). These subunit replacements alter the cleavage specificity of the proteasome and the spectrum of proteasome-generated peptide ligands of MHC class I molecules. In addition to antigen processing, the IP has recently been shown to serve unique functions in the generation of pro-inflammatory T helper cell subtypes and cytokines as well as in the pathogenesis of autoimmune diseases, but the mechanistic involvement of the IP in these processes has remained elusive. In this study we investigated whether the IP differs from the CP in the interaction with two IFN-γ/TNF inducible factors: the 11S proteasome regulator PA28αβ and the ubiquitin-like modifier FAT10 (ubiquitin D). Using thermophoresis, we determined the affinity of PA28αβ for the CP and IP to be 12.2 nM +/− 2.8 nM and 15.3 nM +/− 2.7 nM, respectively, which is virtually identical. Also the activation of the peptidolytic activities of the IP and CP by PA28αβ did not differ. For FAT10 we determined the degradation kinetics in cycloheximide chase experiments in cells expressing almost exclusively IP or CP as well as in IFN-γ stimulated and unstimulated cells and found no differences between the degradation rates. Taken together, we conclude that neither differences in the binding strength to, nor activation by PA28αβ, nor a difference in the rate of FAT10-mediated degradation can account for distinct functional capabilities of the IP as compared to the CP.

Activation of the pro-inflammatory transcription factor NF-κB requires signal-induced proteasomal degradation of the inhibitor of NF-κB (IκB) in order to allow nuclear translocation. Most cell types are capable of expressing two types of 20S proteasome core particles, the constitutive proteasome and immunoproteasome. Inducible under inflammatory conditions, the immunoproteasome is mainly characterized through an altered cleavage specificity compared to the constitutive proteasome. However, the question whether immunoproteasome subunits affect NF-κB signal transduction differently from constitutive subunits is still up for debate. To study the effect of immunoproteasomes on LPS- or TNF-α-induced NF-κB activation, we used IFN-γ stimulated peritoneal macrophages and mouse embryonic fibroblasts derived from mice deficient for the immunoproteasome subunits low molecular mass polypeptide (LMP) 2, or LMP7 and multicatalytic endopeptidase complex-like 1 (MECL-1). Along the canonical signaling pathway of NF-κB activation no differences in the extent and kinetic of IκB degradation were observed. Neither the nuclear translocation and DNA binding of NF-κB nor the production of the NF-κB dependent cytokines TNF-α, IL-6, and IL-10 differed between immunoproteasome deficient and proficient cells. Hence, we conclude that immunoproteasome subunits have no specialized function for canonical NF-κB activation. published

Animal tumor models and human cancer studies have provided convergent evidence that chronic psychological stress plays a decisive role in modulating anti-tumor T cell immunity. However, whether chronic stress also affects anti-cancer vaccine strategies that rely on the induction of functional tumor-specific TCD8+ cells has not been investigated yet. In this study we provide direct evidence that chronic stress suppresses the therapeutic efficacy of a biodegradable poly(d,l-lactide-co-glycolide) microsphere (PLGA-MS) based cancer vaccine in a murine melanoma model. Exposure of mice to social disruption stress (SDR), a well-established model mimicking psychological chronic stress in humans, significantly impaired tumor protection in response to cancer vaccination under both prophylactic and therapeutic conditions. Vaccine failure in stressed mice correlated with significantly reduced generation of interferon-γ (IFN-γ)-producing TCD8+ effectors and CTL-mediated killing. Phenotypic analysis of dendritic cells (DCs) revealed that both migratory and lymphoid-resident DCs failed to undergo full maturation upon antigen uptake. Notably, decreased DC maturation was associated with a significant impairment of peripheral DCs to migrate to draining LNs and to prime subsequent TCD8+ responses in vivo. In conclusion, chronic stress represents an important factor mediating immunosuppression in cancer-vaccinated hosts by impairing DC functions and subsequent TCD8+ priming. Potentially, the mechanistic insights gained in this study open new avenues in utilizing the full potential of anti-cancer vaccination strategies. published

Multiple sclerosis (MS) is a chronic demyelinating immune mediated disease of the central nervous system. The immunoproteasome is a distinct class of proteasomes found predominantly in monocytes and lymphocytes. Recently, we demonstrated a novel function of immunoproteasomes in cytokine production and T cell differentiation. In this study, we investigated the therapeutic efficacy of an inhibitor of the immunoproteasome (ONX 0914) in two different mouse models of MS. ONX 0914 attenuated disease progression after active and passive induction of experimental autoimmune encephalomyelitis (EAE), both in MOG₃₅-₅₅ and PLP₁₃₉₋₁₅₁-induced EAE. Isolation of lymphocytes from the brain or spinal cord revealed a strong reduction of cytokine-producing CD4(+) cells in ONX 0914 treated mice. Additionally, ONX 0914 treatment prevented disease exacerbation in a relapsing-remitting model. An analysis of draining lymph nodes after induction of EAE revealed that the differentiation to Th17 or Th1 cells was strongly impaired in ONX 0914 treated mice. These results implicate the immunoproteasome in the development of EAE and suggest that immunoproteasome inhibitors are promising drugs for the treatment of MS.

New treatment options and drug targets for colorectal carcinoma are a pressing medical need. Inflammation and pro-inflammatory cytokines produced by Th1 and Th17 cells like IL-6, TNF, IL-17 and IL-23 promote the development and growth of colorectal cancer (CRC). The immunoproteasome is a proteasome subtype highly expressed in immune cells but also in the intestine. Since the immunoproteasome promotes Th1 and Th17 differentiation and pro-inflammatory cytokine production, we investigated here whether deficiency or inhibition of the immunoproteasome subunit LMP7 would interfere with CRC development and exacerbation in preventive and therapeutic mouse models. Treatment with the LMP7 inhibitor ONX 0914 blocked tumor initiation and progression in either chemically-induced (AOM/DSS) or transgenic mouse models (ApcMin/+) of colon carcinogenesis. ONX 0914 treatment strongly reduced tumor numbers and CRC-associated loss of body weight while the survival rates were significantly enhanced. Moreover, genetic LMP7 deficiency markedly reduced the tumor burden in AOM/DSS induced wild type and ApcMin/+ mice. In conclusion, we show that the immunoproteasome is involved in CRC development and progression and we identify LMP7 as a new potential drug target for the treatment of CRC. published

During the last years it has emerged that the ubiquitin-like modifier FAT10 is directly involved in cancer development. FAT10 expression is highly up-regulated by proinflammatory cytokines IFN-γ and TNF-α in all cell types and tissues and it was also found to be upregulated in many cancer types such as glioma, colorectal, liver or gastric cancer. While proinflammatory cytokines within the tumor microenvironment probably contribute to FAT10 overexpression, an increasing body of evidence argues that pro-malignant capacities of FAT10 itself largely underlie its broad and intense overexpression in tumor tissues. FAT10 thereby regulates pathways involved in cancer development such as the NF-κB- or Wnt-signaling. Moreover, FAT10 directly interacts with and influences downstream targets such as p53 or β-catenin, leading to enhanced survival, proliferation, invasion and metastasis formation of cancer cells but also of non-malignant cells. In this review we will provide an overview of the regulation of FAT10 expression as well as its function in carcinogenesis. published

In experimental tumor immunotherapy, incomplete Freund's adjuvant (IFA) has been considered as the "gold standard" for T-cell vaccination in mice and humans in spite of its considerable adverse effects. Recently, we succeeded in eliciting strong CTL responses in mice after vaccination with biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres (MS). In our study, we compared the immune response to IFA and PLGA-MS containing ovalbumin (OVA) and CpG-oligodeoxynucleotide (MS-OVA/CpG) or we used a mixture of MS-OVA/CpG and MS-polyI:C. A single vaccination with MS-OVA/CpG elicited long-lasting titers of IgG1 and IgG2a, but only low IgE titers, and also the T-cell response was biased toward Th(1) differentiation. Antigen presentation to CD4(+) and CD8(+) cells and activation of a cytotoxic T-cell response in mice vaccinated with PLGA-MS and IFA lasted for over 3 weeks. Preconditioning of the injection site with TNF-α and heterologous prime-boost regimen further enhanced the cytotoxic response. PLGA-MS were as efficient or superior to IFA in eradication of preexisting tumors and suppression of lung metastases. Taken together, PLGA-MS are well-defined, biodegradable and clinically compatible antigen carrier systems that compare favorably with IFA in their efficacy of tumor immunotherapy in mouse models and hence deserve to be tested for their effectiveness against human malignant diseases.

The analysis of cell types involved in cross-priming of particulate Ag is essential to understand and improve immunotherapies using microparticles. In this study, we show that murine splenic dendritic cells (DCs) as well as macrophages (MΦs) are able to efficiently endocytose poly(D,L-lactate-co-glycolate) acid (PLGA) microspheres (MS) and to cross-present encapsulated Ags in the context of MHC class I molecules in vitro. A comparison of purified CD8+ and CD8− DCs indicated that both DC subtypes are able to present OVA-derived epitopes on MHC class I and II in vitro. To determine the contribution of DCs and MΦs to cross-priming of PLGA MS in vivo, DCs were depleted in transgenic CD11c-DTR mice, and MΦs were depleted by clodronate liposomes in wild-type mice before immunizing mice with OVA-encapsulated MS. Our results show that the depletion of DCs or MΦs alone only led to minor differences in the OVA-specific immune responses. However, simultaneous depletion of DCs and MΦs caused a strong reduction of primed effector cells, indicating a redundancy of both cell populations for the priming of PLGA MS-encapsulated Ag. Finally, we analyzed PLGA MS trafficking to draining lymph nodes after s.c. injection. It was evident that fluorescent particles accumulated within draining lymph nodes over time. Further analysis of PLGA MS-positive lymphatic cells revealed that mainly CD8− DCs and MΦs contained MS. Moreover, immune responses in BATF3 knockout mice lacking CD8+ DCs were normal. The results presented in this work strongly suggest that in vivo cross-priming of PLGA MS-encapsulated Ag is performed by CD8− DCs and MΦs.

Therapeutic DNA vaccination is an attractive adjuvant option to conventional methods in the fight against cancer, like surgery radiotherapy and chemotherapy. Despite strong antitumor effects that were observed in small animals with different antigens, DNA-based vaccines remain weakly immunogenic in large animals and primates compared to protein-based vaccines. Here, we sought to enhance the immunogenicity of a therapeutic nontransforming cervical cancer DNA vaccine (HPV-16 E7SH) by introduction of a highly optimized CpG cassette into the plasmid backbone as well as by an optimized DNA delivery using an advanced electroporation (EP) technology. By integrating the means for agent administration and EP into a single device, this technology enables a simple, one-step procedure that facilitates reproducibility. We found that highly optimized CpG motifs alone triggers an enhanced IFN-γ and granzyme B response in Elispot assays as well as stronger tumor regression. Furthermore, these effects could be dramatically enhanced when the CpG cassette containing plasmid was administered via the newly developed EP technology. These data suggest that an optimized application of CpG-enriched DNA vaccines may be an attractive strategy for the treatment of cancer. Collectively, these results provide a basis for the transfer of preclinical therapeutic DNA-based immunization studies into successful clinical cancer trials.

The mRNA of the ubiquitin-like modifier FAT10 has been reported to be overexpressed in 90% of hepatocellular carcinoma (HCC) and in over 80% of colon, ovary and uterus carcinomas. Elevated FAT10 expression in malignancies was attributed to transcriptional upregulation upon the loss of p53. Moreover, FAT10 induced chromosome instability in long-term in vitro culture, which led to the hypothesis that FAT10 might be involved in carcinogenesis. In this study we show that interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha synergistically upregulated FAT10 expression in liver and colon cancer cells 10- to 100-fold. Real-time RT-PCR revealed that FAT10 mRNA was significantly overexpressed in 37 of 51 (72%) of human HCC samples and in 8 of 15 (53%) of human colon carcinomas. The FAT10 cDNA sequences in HCC samples were not mutated and intact FAT10 protein was detectable. FAT10 expression in both cancer tissues correlated with expression of the IFN-gamma- and TNF-alpha-dependent proteasome subunit LMP2 strongly suggesting that proinflammatory cytokines caused the joint overexpression of FAT10 and LMP2. NIH3T3 transformation assays revealed that FAT10 had no transforming capability. Taken together, FAT10 qualifies as a marker for an interferon response in HCC and colon carcinoma but is not significantly overexpressed in cancers lacking a proinflammatory environment.

The modification of proteins by ubiquitin has a major role in cells of the immune system and is counteracted by various deubiquitinating enzymes (DUBs) with poorly defined functions. Here we identified the ubiquitin-specific protease USP8 as a regulatory component of the T cell antigen receptor (TCR) signalosome that interacted with the adaptor Gads and the regulatory molecule 14-3-3β. Caspase-dependent processing of USP8 occurred after stimulation of the TCR. T cell-specific deletion of USP8 in mice revealed that USP8 was essential for thymocyte maturation and upregulation of the gene encoding the cytokine receptor IL-7Rα mediated by the transcription factor Foxo1. Mice with T cell-specific USP8 deficiency developed colitis that was promoted by disturbed T cell homeostasis, a predominance of CD8(+) γδ T cells in the intestine and impaired regulatory T cell function. Collectively, our data reveal an unexpected role for USP8 as an immunomodulatory DUB in T cells.

The proteasome is the main protein-degrading machine within the cell, producing ligands for MHC class I molecules. It is a cylindrical multicatalytic protease complex, and the catalytic activity is mediated by the three subunits β1, β2, and β5 which possess caspase-, trypsin-, and chymotrypsin-like activities, respectively. By stimulation with interferon (IFN)-γ the replacement of these subunits by β1i, β2i, and β5i is induced leading to formation of immunoproteasomes with altered proteolytic and antigen processing properties. The genes coding for these immunosubunits are restricted to jawed vertebrates but have so far not been found in the genomes of birds, e.g., chicken, turkey, quail, black grouse and zebra finch. However, the chicken genome sequences are not completely assigned; therefore, we investigated the presence of immunoproteasome on protein level. 20S proteasome was purified from the chicken brain, blood, spleen, and bursa of Fabricius, followed by separation via two-dimensional (2D) gel electrophoresis. We analyzed the protein spots derived from the spleen and brain by mass spectrometry and could identify all 14 proteasomal subunits, but there were no differences detectable in the spot patterns. Moreover, we stimulated the chicken spleen cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin aiming at the induction of immunoproteasome, but in spite of the induction of proliferation and IFN-γ, no evidence for immunoproteasome formation in chicken could be obtained. This result was substantiated by the finding that 20S proteasomes isolated from immune and non-immune tissues showed very similar peptidolytic activities. Taken together, our results indicate that chicken lack immunoproteasomes also on protein level.

The cytotoxic T cell response to pathogens is usually directed against a few immunodominant epitopes, while other potential epitopes are either subdominant or not used at all. In C57BL/6 mice, the acute cytotoxic T cell response against lymphocytic choriomeningitis virus is directed against immunodominant epitopes derived from the glycoprotein (gp33–41) and the nucleoprotein (NP396–404), while the gp276–286 epitope remains subdominant. Despite extensive investigations, the reason for this hierarchy between epitopes is not clear. In this study, we show that the treatment of cells with IFN-γ enhanced the presentation of gp33–41, whereas presentation of the gp276–286 epitope from the same glycoprotein was markedly reduced. Because proteasomes are crucially involved in epitope generation and because IFN-γ treatment in vitro and lymphocytic choriomeningitis virus infection in vivo lead to a gradual replacement of constitutive proteasomes by immunoproteasomes, we investigated the role of proteasome composition on epitope hierarchy. Overexpression of the active site subunits of immunoproteasomes LMP2, LMP7, and MECL-1 as well as overexpression of LMP2 alone suppressed the presentation of the gp276–286 epitope. The ability to generate gp276–286-specific CTLs was enhanced in LMP2- and LMP7-deficient mice, and macrophages from these mice showed an elevated presentation of this epitope. In vitro digests demonstrated that fragmentation by immunoproteasomes, but not constitutive proteasomes led to a preferential destruction of the gp276 epitope. Taken together, we show that LMP2 and LMP7 can at least in part determine subdominance and shape the epitope hierarchy of CTL responses in vivo.

PA28alpha/beta is a regulatory complex of the 20S proteasome which consists of two IFN-gamma inducible subunits. Both subunits, alpha and beta, contribute equally to the formation of hexa- or heptameric rings which can associate with the 20S proteasome. Previously, we have shown that overexpression of the PA28alpha subunit enhanced the MHC class I-restricted presentation of two viral epitopes and that purified PA28alpha/beta accelerated T cell epitope generation by the 20S proteasome in vitro, indicating a role for PA28alpha/beta in antigen presentation. This conclusion was recently confirmed in PA28beta gene targeted mice which were severely deficient in MHC class I-restricted antigen presentation. These mice displayed a defect in the assembly of immunoproteasomes, suggesting that a lack of the proteasome subunits LMP2, LMP7, and MECL-1 may account for the deficiency in antigen presentation. In this study we investigated whether the effect of PA28alpha/beta on antigen presentation is dependent on a change of proteasome subunit composition. We have analyzed the assembly and subunit composition of proteasomes in fibroblast transfectants overexpressing both, alpha and beta subunits of PA28. In these transfectants we found a marked enhancement in the presentation of the immunodominant H-2Ld-restricted pp89 epitope of murine cytomegalovirus, although the 20S proteasome composition was the same as in recipient cells. We, therefore, conclude that PA28alpha/beta can enhance antigen processing independently of changes in 20S proteasome subunit composition or assembly.

The complete inhibition of proteasome activities interferes with the production of most MHC class I peptide ligands as well as with cellular proliferation and survival. In this study we have investigated how partial and selective inhibition of the chymotrypsin-like activity of the proteasome by the proteasome inhibitors lactacystin or epoxomicin would affect Ag presentation. At 0.5–1 μM lactacystin, the presentation of the lymphocytic choriomeningitis virus-derived epitopes NP118 and GP33 and the mouse CMV epitope pp89–168 were reduced and were further diminished in a dose-dependent manner with increasing concentrations. Presentation of the lymphocytic choriomeningitis virus-derived epitope GP276, in contrast, was markedly enhanced at low, but abrogated at higher, concentrations of either lactacystin or epoxomicin. The inhibitor-mediated effects were thus epitope specific and did not correlate with the degradation rates of the involved viral proteins. Although neither apoptosis induction nor interference with cellular proliferation was observed at 0.5–1 μM lactacystin in vivo, this concentration was sufficient to alter the fragmentation of polypeptides by the 20S proteasome in vitro. Our results indicate that partial and selective inhibition of proteasome activity in vivo is a valid approach to modulate Ag presentation, with potential applications for the treatment of autoimmune diseases and the prevention of transplant rejection.

A means of regulating the fate of intracellular proteins is their covalent conjugation to ubiquitin-like proteins. A recently discovered ubiquitin-like protein is called “diubiquitin” because it consists of two ubiquitin-like domains in head-to-tail arrangement. Human diubiquitin is encoded at the telomeric end of the MHC class I locus and was previously found to be expressed in dendritic cells and mature B cells. We have extended the expression analysis of diubiquitin by reverse transcriptase-PCR and Northern blotting in primary endothelial cells and human cancer cell lines derived from nine different tissues. Diubiquitin expression was found to be generally and synergistically inducible with the cytokines IFN- and TNFbut not with IFN- . Diubiquitin mRNA expression was induced within 2 h after cytokine stimulation and was independent of protein neosynthesis but dependent on proteasome activity. The mouse homologue of diubiquitin which is also encoded in the MHC class I locus was likewise induced with IFN- and TNF- . A general and synergistic induction with IFN- and TNF- suggests that diubiquitin may exert its functions in antigen presentation or other cellular processes controlled by these two cytokines.

Dendritic cells (DC) are highly specialized professional antigen presenting cells which are pivotal for the initiation and control of the cytotoxic T cell response. Upon stimulation by cytokines, bacteria, or CD40L DC undergo a maturation process from an antigen-receptive state to a state of optimal stimulation of T cells. We investigated the composition of proteasomes of DC derived from human peripheral blood monocytes before and after stimulation by CD40L, LPS, or proinflammatory cytokines (TNF-alpha + IL-6 + IL-1beta). Immunoprecipitation of proteasomes and analysis on two-dimensional gels revealed that during maturation the inducible proteasome subunits LMP2, LMP7, and MECL-1 are up-regulated and that the neosynthesis of proteasomes is switched exclusively to the production of immunoproteasomes containing these subunits. The proteasome regulator PA28 is markedly up-regulated in mature DC and in addition a so - far unidentified 21-kDa protein co-precipitates with the proteasome in LPS - stimulated DC. These changes in proteasome composition may be functionally linked to special properties of DC like MHC class I up-regulation or cross-priming. Our findings imply that the spectrum of class I-bound peptides may change after DC maturation which could be relevant for the design of DC - based vaccines.

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.

The presentation of viral antigens on MHC class I molecules requires their intracellular fragmentation into peptides of appropriate length and anchor residue positions. Evidence has accumulated that the proteasome is the endoprotease in charge of the generation of MHC class I ligands in the cytoplasm. The generation of T cell epitopes derived from the leader peptides of endoplasmic reticulum (ER) targeted proteins, however. has been reported to be independent of the proteasome. Here we show that the H-2Db restricted antigen presentation of the immunodominant T cell epitope derived from the ER leader of the glycoprotein of lymphocytic choriomeningitis virus (LCMV) is completely abolished by administration of the proteasome inhibitor lactacystin. Thus our data support the role of the proteasome in class I restricted antigen processing and extend it to an ER leader derived epitope from a viral glycoprotein.

Proteasomes generate peptides bound by major histocompatibility complex (MHC) class I molecules. Avoiding proteasome inhibitors, which in most cases do not distinguish between individual active sites within the cell, we used a molecular genetic approach that allowed for the first time the in vivo analysis of defined proteasomal active sites with regard to their significance for antigen processing. Functional elimination of the δ/low molecular weight protein (LMP) 2 sites by substitution with a mutated inactive LMP2 T1A subunit results in reduced cell surface expression of the MHC class I H-2Ld and H-2Dd molecules. Surface levels of H-2Ld and H-2Dd molecules were restored by external loading with peptides. However, as a result of the active site mutation, MHC class I presentation of a 9-mer peptide derived from a protein of murine cytomegalovirus was enhanced about three- to fivefold. Our experiments provide evidence that the δ/LMP2 active site elimination limits the processing and presentation of several peptides, but may be, nonetheless, beneficial for the generation and presentation of others.

The primary structures of the interferon-gamma-inducible mouse 20S proteasome subunit MECL-1 and its alternate homolog MC14 were determined. Northern analysis of mouse tissues revealed that MECL-1 mRNA predominantly occurred in thymus, lymph nodes, and spleen, whereas small amounts were detected in non-lymphoid tissues such as kidney, muscle, and testis. Unexpectedly, probing RNA blots with MC14 showed that tissues with high MECL-1 expression contained little MC14 and vice versa. A very similar reciprocal tissue expression was subsequently found for the homologous subunit pairs LMP2 and delta as well as LMP7 and MB1. The subunit protein composition of 20S proteasomes purified from liver, thymus, and lung reflected RNA expression. The impact of a regulated reciprocal tissue expression is discussed with respect to thymic selection and the induction of tolerance in potentially autoreactive T cells.

Major Histocompatibility Class I (MHC-I) molecules are present at the cell surface either as fully conformed trimolecular complexes composed of heavy chain, beta-2-microglobulin ( 2m) and antigenic peptide or as various open forms, devoid of the peptide and/or 2m. While the role of fully conformed MHC-I is well studied, the physiological role of open conformers is neglected. We have shown that fully conformed MHC-I and open MHC-I conformers segregate at the PM and during endosomal trafficking resulting in the exclusion of open MHC-I from the early endosomal/juxtanuclear recycling route. As a result, open MHC-I conformers are internalized with a higher rate than fully conformed counterparts. Although the majority of internalized open MHC-I is directed into the acidic late endosomal (LE) compartments, only a fraction of them is degraded. Namely, a significant fraction of open MHC-I is present in a subset of LEs with the capacity of recycling and/or exocytosis. Therefore, it should be examined whether exogenous peptide loading may occur during traveling of MHC-I proteins through LE compartments, especially in a subset of less acidic LEs that detach from the core of perinuclear acidic LEs and migrate toward the cell periphery. Given that the acidic LE environment is not favorable for Peptide loading, an endosomal compartment with the recycling capacity and less acidic environment that allows stabilization of newly formed trimolecular complexes is proper site for exogenous peptide loading. We propose that a LE compartment which collect and retain open MHC-I conformers should be taken into consideration as a site of exogenous peptide loading.

Background Posttraumatic stress disorder (PTSD) is associated with an enhanced risk for cardiovascular and other inflammatory diseases. Chronic low-level inflammation has been suggested as a potential mechanism linking these conditions. Methods We investigated plasma cytokine levels as well as spontaneous and lipopolysaccharide (LPS)-stimulated cytokine production by peripheral blood mononuclear cells (PBMCs) in a group of 35 severely traumatized PTSD patients compared to 25 healthy controls. Results Spontaneous production of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α by isolated PBMCs was significantly higher in the PTSD compared to the control group and even correlated with PTSD symptom severity within the PTSD group. In contrast, circulating plasma levels of pro- and anti-inflammatory cytokines such as IL-6, IL-8, IL-10, TNF-α, or monocyte chemotactic protein (MCP)-1 were not significantly altered in PTSD patients compared to healthy controls. Conclusions Our findings indicate that PBMCs of PTSD patients are already pre-activated in vivo, providing further evidence for low-grade inflammation in PTSD. This might possibly represent one psychobiological pathway from PTSD to poor physical health.

The heterodimeric cytokine IL-23 is important for the maintenance of Th17 cells, which are pivotal mediators of autoimmune diseases like rheumatoid arthritis, colitis, and multiple sclerosis. Prostaglandin E2 (PGE2) is a soluble regulator of inflammation that has both pro- and anti-inflammatory properties. PGE2 has been shown to elevate the IL-23 production by dendritic cells (DC). Monocytes are also producers of IL-23 but the effect of PGE2 on IL-23 production by human monocytes has hardly been investigated. We show here that PGE2 blocks the production of IL-23 by LPS-stimulated monocytes in an IL-10 and IL-1β independent manner. This effect was due to the down-regulation of the p40 subunit of IL-23 on mRNA and protein level. The p40 subunit is shared by IL-12 and, consistently, PGE2 also lowered the IL-12 production by monocytes. These effects of PGE2 were cAMP-dependent since the cAMP enhancer forskolin strongly reduced IL-23 and IL-12 production by monocytes. Taken together, PGE2 acts in an anti-inflammatory manner by lowering IL-23 production by monocytes while it has the opposite effect in DC. Our data may help to reconcile controversial point of views on the pro- and anti-inflammatory nature of PGE2 by making a strong case for a cell type-dependent function.

Proteasomes have recently been shown to be central to the production of antigenic peptides that can bind to major histocompatibility complex class I molecules. Here, Marcus Groettrup and colleagues discuss the structural architecture of the proteasome and its proteolytic mechanisms with respect to the generation of antigenic peptides. They suggest that exchanges of proteolytic proteasome subunits and proteasome activators cooperate to optimize the production of antigens for cytotoxic T cells.

The 20S proteasome is the enzyme complex responsible for the processing of antigens bound by major histocompatibility complex class I molecules. The role of the interferon-gamma (IFN-gamma)-inducible proteasome subunits LMP2 and LMP7 in this process is, however, still controversial. We have studied the effects of IFN-gamma-independent LMP incorporation on the quality of peptides processed from the murine cytomegalovirus IE pp89 25-mer polypeptide substrate through dual cleavages by 20S proteasomes. The incorporation of a single LMP subunit or both LMP2 and LMP7 induces changes in 20S proteasome subunit stoichiometry, alters its cleavage site preference and in consequence, the quality of the generated peptides. When the several hydrolytic activities are tested with short fluorogenic peptide substrates, the Vmax, S0.5 (Km), or both values of 20S proteasomes are altered, depending on the combination of LMP. There exists, however, no obvious correlation between the observed changes in hydrolytic activities against short fluorogenic peptides and the changes in dual cleavage site usage within the 25-mer polypeptide substrate. As judged from the calculated Hill coefficients, the presence of both LMP subunits induces a drastic increase in positive cooperativity between the proteasome subunits.

Treatment of cells with interferon-g leads to the replacement of the constitutive catalytic proteasome subunits b1, b2, and b5 by the inducible subunits LMP2 (b1i), MECL-1 (b2i), and LMP7 (b5i), respectively, building the so-called immunoproteasome. The incorporation of these subunits is required for the production of numerous MHC class-I restricted T cell epitopes. Recently, new evidence for an involvement of the immunoproteasome in other facets of the immune response emerged. Investigations of autoimmune diseases in animal models and a genetic predisposition of b5i in human autoimmune disorders suggest a crucial function of the immunoproteasome in proinflammatory diseases. The recent elucidation of the high-resolution structure of the immunoproteasome will facilitate the design of immunoproteasome selective inhibitors for pharmacological intervention.

The induction of strong CD8 + T-cell responses against infectious diseases and cancer has remained a major challenge. Depending on the source of antigen and the infectious agent, priming of CD8 + T cells requires direct and/or cross-presentation of antigenic peptides on major histocompatibility complex (MHC) class I molecules by professional antigen-presenting cells (APCs). However, both pathways show distinct preferences concerning antigen stability. Whereas direct presentation was shown to efficiently present peptides derived from rapidly degraded proteins, cross-presentation is dependent on long-lived antigen species. In this report, we analyzed the role of antigen stability on DNA vaccination and recombinant vaccinia virus (VV) infection using altered versions of the same antigen. The long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) can be targeted for degradation by N-terminal fusion to ubiquitin or, as we show here, to the ubiquitin-like modifier FAT10. Direct presentation by cells either transfected with NP-encoding plasmids or infected with recombinant VV in vitro was enhanced in the presence of short-lived antigens. In vivo , however, the highest induction of NP-specific CD8 + T-cell responses was achieved in the presence of long-lived NP. Our experiments provide evidence that targeting antigens for proteasomal degradation does not improve the immunogenicity of DNA vaccines and recombinant VVs. Rather, it is the long-lived antigen that is superior for the efficient activation of MHC class I-restricted immune responses in vivo . Hence, our results suggest a dominant role for antigen cross-priming in DNA vaccination and recombinant VV infection.

Biodegradable poly(lactide-co-glycolide) (PLGA) microspheres (MS) deliver antigens and toll like receptor (TLR) ligands to antigen presenting cells (APC) in vitro and in vivo. PLGA-MS-microencapsulated model antigens are efficiently presented on MHC class I and II molecules of dendritic cells and stimulate strong cytotoxic and T helper cell responses enabling the eradication of pre-existing model tumors. The application of tumor lysates as a source of antigen for immunotherapy has so far not been very successful also due to a lack of suitable delivery systems. In this study we used PLGA-MS with co-encapsulated tumor lysates and CpG oligodeoxynucleotides (CpG-ODN) as well as microencapsulated polyI:C in order to elicit anti-tumor responses. Immunization of mice with such mixtures of MS yielded substantial cytotoxic T cell (CTL) responses and interfered with tumor growth in TRAMP mice, a pre-clinical transgenic mouse model of prostate carcinoma, which has previously resisted dendritic cell-based therapy. As an important step towards clinical application of PLGA-MS, we could show that γ-irradiation of PLGA-MS sterilized the MS, without reducing their efficacy in eliciting CTL and anti-tumor responses in subcutaneous tumor grafts. Since PLGA is approved for clinical application, sterilized PLGA-MS containing tumor lysates and TLR ligands hold promise as anti-tumor vaccines against prostate carcinoma in humans.

The proteasome is responsible for the generation of most epitopes presented on MHC class I molecules. Treatment of cells with IFN-γ leads to the replacement of the constitutive catalytic subunits β1, β2, and β5 by the inducible subunits low molecular mass polypeptide (LMP) 2 (β1i), multicatalytic endopeptidase complex-like-1 (β2i), and LMP7 (β5i), respectively. The incorporation of these subunits is required for the production of numerous MHC class I-restricted T cell epitopes. The structural features rather than the proteolytic activity of an immunoproteasome subunit are needed for the generation of some epitopes, but the underlying mechanisms have remained elusive. Experiments with LMP2-deficient splenocytes revealed that the generation of the male HY-derived CTL-epitope UTY246–254 was dependent on LMP2. Treatment of male splenocytes with an LMP2-selective inhibitor did not reduce UTY246–254 presentation, whereas silencing of β1 activity increased presentation of UTY246–254. In vitro degradation experiments showed that the caspase-like activity of β1 was responsible for the destruction of this CTL epitope, whereas it was preserved when LMP2 replaced β1. Moreover, inhibition of the β5 subunit rescued the presentation of the influenza matrix 58–66 epitope, thus suggesting that a similar mechanism can apply to the exchange of β5 by LMP7. Taken together, our data provide a rationale why the structural property of an immunoproteasome subunit rather than its activity is required for the generation of a CTL epitope.

Prostate cancer (PCa) is the most commonly diagnosed type of cancer in men in western industrialized countries. As a public health burden, the need for the invention of new cost-saving PCa immunotherapies is apparent. In this study, we present a DNA vaccine encoding for the prostate-specific antigen prostatic acid phosphatase (PAP) linked to the J-domain and the SV40 enhancer sequence. The PAP DNA vaccine induced a strong PAP-specific cellular immune response after electroporation (EP)-based delivery in C57BL/6 mice. Splenocytes from mice immunized with PAP recognized the naturally processed PAP epitopes, indicating that vaccination with the PAP-J gene broke its self-tolerance against PAP. Remarkably, DNA vaccination with PAP-J inhibited tumor growth in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse model that closely resembled human PCa. Therefore, this study highlights a novel cancer immunotherapy approach with the potential to control PCa in clinical settings.

The replacement of the catalytically active proteasome subunits β1, β2, and β5 by the immunoproteasome subunits low molecular mass polypeptide (LMP) 2 (β1i), multicatalytic endopeptidase complex-like–1 (MECL-1) (β2i), and LMP7 (β5i) is required for the production of numerous class I ligands. Hitherto, investigation of the immunoproteasome was confined to the analysis of mice deficient for one or two immunosubunits. In this study, we characterized LMP2−/−/MECL-1−/− double-deficient mice and used the well-defined LMP7-selective inhibitor ONX 0914 in these mice to generate mice lacking the activity of all immunoproteasome subunits. LMP2−/−/MECL-1−/− double-deficient mice had strongly reduced numbers of CD8+ T cells in the spleen. Nevertheless, infection with the lymphocytic choriomeningits virus induced a normal cytotoxic T cell response in these mice, although the T cell response to several class I epitopes was altered. Treatment of LMP2−/−/MECL-1−/− double-deficient mice with the LMP7-selective inhibitor ONX 0914 elicited a strong CTL response in lymphocytic choriomeningitis virus-infected mice. Thereby, the TCD8+ response to nucleoprotein 205–212, which is barely detectable in LMP2−/−/MECL-1−/− double-deficient mice, could be reverted to normal levels by LMP7 inhibition. Additional experiments could demonstrate that the increased CTL response to the nucleoprotein 205–212 in mice lacking functional immunoproteasome is due to an altered class I presentation of this epitope. Taken together, to our knowledge, this is the first study investigating viral infection in mice lacking activity of all three immunoproteasome subunits.