Kathrin Textoris-Taube

Immunoproteasome LMP2 60HH variant alters MBP epitope generation and reduces the risk to develop multiple sclerosis in Italian female population

Background Albeit several studies pointed out the pivotal role that CD4+T cells have in Multiple Sclerosis, the CD8+ T cells involvement in the pathology is still in its early phases of investigation. Proteasome degradation is the key step in the production of MHC class I-restricted epitopes and therefore its activity could be an important element in the activation and regulation of autoreactive CD8+ T cells in Multiple Sclerosis. Methodology/Principal Findings Immunoproteasomes and PA28-αβ regulator are present in MS affected brain area and accumulated in plaques. They are expressed in cell types supposed to be involved in MS development such as neurons, endothelial cells, oligodendrocytes, macrophages/macroglia and lymphocytes. Furthermore, in a genetic study on 1262 Italian MS cases and 845 controls we observed that HLA-A*02+ female subjects carrying the immunoproteasome LMP2 codon 60HH variant have a reduced risk to develop MS. Accordingly, immunoproteasomes carrying the LMP2 60H allele produce in vitro a lower amount of the HLA-A*0201 restricted immunodominant epitope MBP111–119. Conclusion/Significance The immunoproteasome LMP2 60HH variant reduces the risk to develop MS amongst Italian HLA-A*02+ females. We propose that such an effect is mediated by the altered proteasome-dependent production of a specific MBP epitope presented on the MHC class I. Our observations thereby support the hypothesis of an involvement of immunoproteasome in the MS pathogenesis.

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.

Immunoproteasomes Are Essential for Clearance of Listeria monocytogenes in Nonlymphoid Tissues but Not for Induction of Bacteria-Specific CD8+ T Cells

Microbial infections induce the replacement of constitutive proteasomes by immunoproteasomes (I-proteasomes). I-proteasomes support efficient generation of MHC class I epitopes and influence immunodominance hierarchies of CD8+ T cells. Recently, the function of I-proteasomes in antimicrobial responses was challenged by showing that the lack of I-proteasomes has no effect on induction and function of lymphocytic choriomeningitis virus-specific CD8+ T cells. Here, we show that infection with Listeria monocytogenes rapidly induces I-proteasomes in nonlymphoid tissues, which leads to enhanced generation of protection relevant CD8+ T cell epitopes. I-proteasome-deficient mice (β5i−/− mice) exhibited normal frequencies of L. monocytogenes-specific CD8+ T cells. However, clearance of L. monocytogenes in liver but not spleen was significantly impaired in I-proteasome-deficient mice. In summary, our studies demonstrate that induction of I-proteasomes is required for CD8+ T cell-mediated elimination of L. monocytogenes from nonlymphoid but not lymphoid tissues.

The 20S Proteasome Splicing Activity Discovered by SpliceMet

The identification of proteasome-generated spliced peptides (PSP) revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL) thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS). For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected.

PA28 and the proteasome immunosubunits play a central and independent role in the production of MHC class I-binding peptides in vivo

Proteasomes play a fundamental role in the processing of intracellular antigens into peptides that bind to MHC class I molecules for the presentation of CD8+ T cells. Three IFN‐γ‐inducible catalytic proteasome (immuno)subunits as well as the IFN‐γ‐inducible proteasome activator PA28 dramatically accelerate the generation of a subset of MHC class I‐presented antigenic peptides. To determine whether these IFN‐γ‐inducible proteasome components play a compounded role in antigen processing, we generated mice lacking both PA28 and immunosubunits β5i/LMP7 and β2i/MECL‐1. Analyses of MHC class I cell‐surface levels ex vivo demonstrated that PA28 deficiency reduced the production of MHC class I‐binding peptides both in cells with and without immunosubunits, in the latter cells further decreasing an already diminished production of MHC ligands in the absence of immunoproteasomes. In contrast, the immunosubunits but not PA28 appeared to be of critical importance for the induction of CD8+ T‐cell responses to multiple dominant Influenza and Listeria‐derived epitopes. Taken together, our data demonstrate that PA28 and the proteasome immunosubunits use fundamentally different mechanisms to enhance the supply of MHC class I‐binding peptides; however, only the immunosubunit‐imposed effects on proteolytic epitope processing appear to have substantial influence on the specificity of pathogen‐specific CD8+ T‐cell responses.

A structural model of 20S immunoproteasomes: effect of LMP2 codon 60 polymorphism on expression, activity, intracellular localisation and insight into the regulatory mechanisms

Abstract The immunoproteasome subunit low molecular weight protein 2 (LMP2) codon 60 polymorphism has been associated with autoimmune diseases. It has also been demonstrated to influence susceptibility to TNF-α-induced apoptosis in blood cells and proteasome activity in aged human brain. In the present study, an in silico model of immunoproteasome was used to examine the effect of the R60H polymorphism in the LMP2 subunit. The investigation of immunoproteasome expression, activity and intracellular localisation in an in vitro cellular model, namely lymphoblastoid cell lines, showed no major variations in functionality and amount, while a significant difference in antibody affinity was apparent. These data were integrated with previous results obtained in different tissues and combined with a structural model of the LMP2 polymorphism. Accordingly, we identified three prospective mechanisms that could explain the biological data for the polymorphism, such as modulation of the binding affinity of a putative non-catalytic modifier site on the external surface of the immunoproteasome core, or the modification of any channel between α and β rings.

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.

Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes.

Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes.

Antitopes Define Preferential Proteasomal Cleavage Site Usage

Protein degradation by proteasomes is a major source of peptides presented by major histocompatibility v complex class I proteins. Importantly, interferon γ-induced immunoproteasomes in many cases strongly enhance the generation of antigenic peptides both in vitro and in vivo. Whether this is due to enhanced substrate turnover or to a change in proteasomal cleavage specificity is, however, largely unresolved. To overcome the problems of peptide quantification inherent to mass spectrometry, we introduced the “antitope” as substrate-specific internal standard. The antitope is a non-functional peptide that is generated by proteasomal cleavage within the epitope, resulting in partial overlaps with the functional epitope. Using antitopes as internal standards we demonstrate that the observed enhanced immunoproteasome-dependent presentation of the bacterial listeriolysin O T-cell epitope LLO(296–304) is indeed due to altered cleavage preferences. This method is also applicable to other major histocompatibility class I epitopes as is shown for two potential epitopes derived from Coxsackievirus.

The N-terminal Flanking Region of the TRP2360–368 Melanoma Antigen Determines Proteasome Activator PA28 Requirement for Epitope Liberation

Proteasomes are known to produce major histocompatibility complex (MHC) class I ligands from endogenous antigens. The interferon-γ-inducible proteasome activator PA28 plays an important role in the generation of MHC ligands by proteasomes. Generation of the HLA-A*0201 restricted melanoma antigen TRP2360–368 by the proteasome has been shown to be dependent on the function of PA28 in vitro and in vivo (Sun, Y., Sijts, A. J., Song, M., Janek, K., Nussbaum, A. K., Kral, S., Schirle, M., Stevanovic, S., Paschen, A., Schild, H., Kloetzel, P. M., and Schadendorf, D. (2002) Cancer Res. 62, 2875–2882). Here we analyzed the role of the epitope sequence environment in determining this PA28 dependence. Experiments using the melanoma TRP2288–296 epitope and the murine cytomegalovirus-derived pp89 epitope precursor peptide for epitope replacement revealed that the TRP2360–368 flanking sequences can transfer PA28 dependence onto otherwise PA28 independent epitopes. Moreover, the N-terminal flanking sequence is sufficient to establish PA28 dependence of an epitope by allowing PA28-induced coordinated dual cleavages. These results show that N-terminal flanking sequences strongly influence epitope generation efficiency and that PA28 function is particularly relevant for the generation of normally poorly excised peptide products.