Michael Basler

A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis

The immunoproteasome, a distinct class of proteasome found predominantly in monocytes and lymphocytes, is known to shape the antigenic repertoire presented on class I major histocompatibility complexes (MHC-I). However, a specific role for the immunoproteasome in regulating other facets of immune responses has not been established. We describe here the characterization of PR-957, a selective inhibitor of low–molecular mass polypeptide-7 (LMP7, encoded by Psmb8), the chymotrypsin-like subunit of the immunoproteasome. PR-957 blocked presentation of LMP7-specific, MHC-I–restricted antigens in vitro and in vivo. Selective inhibition of LMP7 by PR-957 blocked production of interleukin-23 (IL-23) by activated monocytes and interferon-γ and IL-2 by T cells. In mouse models of rheumatoid arthritis, PR-957 treatment reversed signs of disease and resulted in reductions in cellular infiltration, cytokine production and autoantibody levels. These studies reveal a unique role for LMP7 in controlling pathogenic immune responses and provide a therapeutic rationale for targeting LMP7 in autoimmune disorders.

Immuno- and Constitutive Proteasome Crystal Structures Reveal Differences in Substrate and Inhibitor Specificity

Constitutive proteasomes and immunoproteasomes shape the peptide repertoire presented by major histocompatibility complex class I (MHC-I) molecules by harboring different sets of catalytically active subunits. Here, we present the crystal structures of constitutive proteasomes and immunoproteasomes from mouse in the presence and absence of the epoxyketone inhibitor PR-957 (ONX 0914) at 2.9 Å resolution. Based on our X-ray data, we propose a unique catalytic feature for the immunoproteasome subunit β5i/LMP7. Comparison of ligand-free and ligand-bound proteasomes reveals conformational changes in the S1 pocket of β5c/X but not β5i, thereby explaining the selectivity of PR-957 for β5i. Time-resolved structures of yeast proteasome:PR-957 complexes indicate that ligand docking to the active site occurs only via the reactive head group and the P1 side chain. Together, our results support structure-guided design of inhibitory lead structures selective for immunoproteasomes that are linked to cytokine production and diseases like cancer and autoimmune disorders.

Proteasomes in immune cells: more than peptide producers?

When cells are stimulated with pro-inflammatory cytokines, most of their constitutively expressed proteasomes are replaced with immunoproteasomes, which increase the production of peptides for presentation on MHC class I molecules. In addition, cortical thymic epithelial cells selectively express a type of proteasome known as the thymoproteasome that is required for the positive selection of thymocytes. Here, we discuss how these specialized types of proteasome shape the T cell receptor repertoire of cytotoxic T lymphocytes and propose that immunoproteasomes have functions, in addition to antigen processing, that influence cytokine production and T cell differentiation, survival and function. We also discuss how inhibitors of immunoproteasomes can suppress undesired T cell responses in autoimmune diseases.

Prevention of Experimental Colitis by a Selective Inhibitor of the Immunoproteasome

The proteasome, a multicatalytic protease, is responsible for the degradation of intracellular proteins. Stimulation of cells with inflammatory cytokines, such as IFN-γ, leads to the replacement of the constitutive catalytic proteasome subunits by the inducible subunits low molecular mass polypeptide (LMP)2 (β1i), multicatalytic endopeptidase complex-like-1 (β2i), and LMP7 (β5i), which are required for the production of certain MHC class I-restricted T cell epitopes. In this study, we investigated the effect of immunoproteasomes on the development of dextran sulfate sodium-induced colitis. Colitis induction in LMP2-, LMP7-, and multicatalytic endopeptidase complex-like-1–deficient mice caused reduced weight loss compared with wild-type mice. Although colon lengths were shortened in wild-type mice, no reduction was observed in immunoproteasome-deficient mice. In accordance with this, proinflammatory cytokines, such as TNF-α and IL-1β, were not upregulated in these mice. Blockage of LMP7 by a novel LMP7-selective inhibitor (PR-957) strongly reduced pathological symptoms of dextran sulfate sodium-induced colitis. Production of numerous cytokines in PR-957–treated mice was suppressed, resulting in reduced inflammation and tissue destruction. Taken together, these results demonstrate that an immunoproteasome-specific inhibitor can be used to attenuate autoimmune diseases like colitis.

The immunoproteasome in antigen processing and other immunological functions

Treatment of cells with interferon-γ leads to the replacement of the constitutive catalytic proteasome subunits β1, β2, and β5 by the inducible subunits LMP2 (β1i), MECL-1 (β2i), and LMP7 (β5i), 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 β5i 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.

Immunoproteasomes Down-Regulate Presentation of a Subdominant T Cell Epitope from Lymphocytic Choriomeningitis Virus

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.

Immunodominance of an Antiviral Cytotoxic T Cell Response Is Shaped by the Kinetics of Viral Protein Expression

Lymphocytic choriomeningitis virus (LCMV) infection induces a protective CTL response consisting of gp- and nucleoprotein (NP)-specific CTL. We find that a small load of LCMV led to immunodominance of NP-CTL, whereas a large viral load resulted in dominance of gp-CTL. This is the first study describing that immunodominance is not fixed after infection with a given pathogen, but varies with the viral load instead. We assumed higher Ag sensitivity for NP-CTL, which would explain their preferential priming at low viral load, as well as their overstimulation resulting in selective exhaustion at high viral load. The higher Ag sensitivity of NP-CTL was due to faster kinetics of NP-epitope presentation. Thus, we uncover a novel factor that impinges upon immunodominance and is related to the kinetics of virus protein expression. We propose that CTL against early viral proteins swiftly interfere with virus replication, resulting in efficient protection. If these “early” CTL fail in immediate virus control, they are activated in the face of higher viral load compared with “late” CTL and are therefore prone to be exhausted. Thus, the observed absence of early CTL in persistent infections might not be the cause, but rather the consequence of viral persistence.

An altered T cell repertoire in MECL-1-deficient mice

Immunoproteasome subunits low-molecular mass polypeptide (LMP)2 and LMP7 affect Ag presentation by MHC class I molecules. In the present study, we investigated the function of the third immunosubunit LMP10/multicatalytic endopeptidase complex-like (MECL)-1 (β2i) in MECL-1 gene-targeted mice. The number of CD8+ splenocytes in MECL-1−/− mice was 20% lower than in wild-type mice. Infection with lymphocytic choriomeningitis virus (LCMV) elicited a markedly reduced cytotoxic T cell (CTL) response to the LCMV epitopes GP276–286/Db and NP205–212/Kb in MECL-1−/− mice. The weak CTL response to GP276–286/Db was not due to an impaired generation of this epitope but was attributed to a decreased precursor frequency of GP276–286/Db-specific T cells. The expansion of TCR-Vβ10+ T cells, which contain GP276–286/Db-specific cells, was reduced in LCMV-infected MECL-1−/− mice. Taken together, our data reveal an in vivo function of MECL-1 in codetermining the T cell repertoire for an antiviral CTL response.

Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis

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.

Cross-Presentation of the Long-Lived Lymphocytic Choriomeningitis Virus Nucleoprotein Does Not Require Neosynthesis and Is Enhanced via Heat Shock Proteins

Many viral proteins that contain MHC class I-restricted peptides are long-lived, and it is elusive how they can give rise to class I epitopes. Recently, we showed that direct presentation of an epitope of the long-lived lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) required neosynthesis in accordance with the defective ribosomal products hypothesis. In this study, we report that LCMV-NP can be cross-primed in mice using either LCMV-NP-transfected human HEK293 or BALB/c-derived B8 cells as Ag donor cells. In addition, we establish that contrary to direct presentation, cross-presentation required accumulation of the mature LCMV-NP and could not be sustained by the newly synthesized LCMV-NP protein, intermediate proteasomal degradation products, or the minimal NP396 epitope. Nevertheless, NP cross-presentation was enhanced by heat shock and was blunted by inhibitors of heat shock protein 90 and gp96. We propose that cross-presentation has evolved to sustain the presentation of stable viral proteins when their neosynthesis has ceased in infected donor cells.