Fig 1.
Cell immunogenicity monitoring using antigen-specific reporter T cells.
(A) Schematic representation. Reporter T cells (B10-TCR td 2D3 cell line) express WT1235 peptide (CMTWNQMNL)-specific TCR and a TCR signaling reporter construct. The reporter construct comprises 3x nuclear factor of activated T cells (NFAT)-response elements, IL-2 minimal promoter, and GFP. When the target cells and reporter T cells are cocultured, the interaction between WT1-specific TCR and WT1235 peptide/MHC-I complex activates TCR signaling. Upstreaming signaling events result in NFAT nuclear translocation and subsequent response-element binding. The response element, in turn, drives the expression of GFP. (B) Specificity of reporter T cells. T2-A24 cells were pulsed with 10 μM of the indicated HLA-A24-restricted peptides. WT1M236Y is a single amino acid substitution (M→Y) at position 2 of the native 9-mer WT1235, improving peptide/HLA-A*24 MHC-I complex stability. Cellular immunogenicity was evaluated by monitoring the activated reporter T cells with GFP expression (GFP positive %) using flow cytometry (S1 Fig). Data are represented as the mean ± SD of three independent experiments and Tukey’s multiple comparisons test was used. ****, p<0.0001. (C) Quantification of natural WT1235 epitopes on T2-A24 cells using reporter T cells.
Fig 2.
WT1 expression in mesothelioma cells.
(A) Immunofluorescence staining of WT1. Mesothelioma cells were stained with anti-WT1 antibody (WT1) (red), F-actin was stained with Alexa Fluor 488 Phalloidin (green), and cell nuclei were stained with DAPI (blue). Scale bar = 10 μm. (B) Western blot of WT1 and GAPDH in mesothelioma cells. (C) Densitometry values of expression relative to GAPDH are indicated. Data are represented as the mean ± SD of three independent experiments and Tukey’s multiple comparisons tests. *, p<0.05; **, p<0.01.
Fig 3.
HLA-ABC and HLA-A*24 expression and the cellular immunogenicity of mesothelioma cells.
(A) Representative flow cytometry histograms of mesothelioma cells stained for the entire HLA-ABC (β2-microglobulin) and HLA-A*24 molecules. Positive cells (red) overlap with the isotype control (blue). (B) The bar graph represents the expression of total HLA-ABC and HLA-A*24 on the cell surface. ΔMFI (mean fluorescence intensity (MFI) test-MFI isotype control) is shown. (C) The immunogenicity of mesothelioma cells was assessed using an in vitro immunogenicity assay. Data are represented as the mean ± SD of three independent experiments and Tukey’s multiple comparisons test was used. **, p<0.01; ***, p<0.001; ****, p<0.0001.
Fig 4.
The cellular immunogenicity of mesothelioma is augmented by the immunoproteasome-selective inhibitor ONX-0914.
(A) Structure of the 20S proteasome core and β-ring. The 20S proteasome core particle comprises four rings of seven subunits: two outer rings of α-subunits and two inner rings of β-subunits. The poly-ubiquitylated proteins are translocated into the 20S core where proteolysis occurs to produce short peptides. Aminopeptidases trim the short peptides to generate antigenic peptides of optimal length for binding to MHC-I molecules. (B) Three different β-subunits form a β-ring with different specificity and catalytic properties. Standard proteasome (β1c, β2c, and β5c) and immunoproteasome (β1i, β2i, and β5i) cleave substrates differently, producing different peptides (immunopeptidome). Proteasome inhibitors (MG132, epoxomicin, and bortezomib) mainly inhibit β5c and β5i, but also β1c and β2c. ONX-0914, an immunoproteasome selective inhibitor, mainly inhibits β5i but also β1i. (C) MESO-4 was treated with the indicated proteasome inhibitors for 3 hours, then co-cultured with reporter T cells for 20 hours, followed by a cellular immunogenicity assay. Data are represented as the mean ± SD of three independent experiments and Dunnett’s multiple comparisons test was used. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Representative data results from two independent experiments are shown.
Fig 5.
Expression of 20S proteasome β-subunits treated with ONX-0914, a selective inhibitor of the immunoproteasome.
(A) Representative western blots of protein extracts from MESO-4 treated with 20 nM ONX-0914 for 3 hours (indicated by +). Protein expressions of β1c, β2c, β5c, β1i, β2i, and β5i were analyzed by western blotting using the indicated antibodies. (B) Quantification of 20S proteasomal β-subunit expression was analyzed by western blotting and values were normalized to control (GAPDH). Data are represented as the mean ± SD of three independent experiments and an unpaired t-test was used. ns, not significant; **, p<0.01.
Fig 6.
MESO-4 pre-treated with ONX-0914 showed an increase in sensitivity to cytotoxic T cells.
ONX-0914-treated MESO-4 (1x105) and CD8-positive T cells, including WT1-specific cytotoxic T cells, were co-cultured at the ratios shown in the graph. Cytotoxicity was assessed by LDH assay. Data are represented as the mean ± SD of three independent experiments and Dunnett’s multiple comparisons test was used. *, p<0.05. Representative data results from two independent experiments are shown.
Fig 7.
IP causes internal destructive cleavage of the WT1235 epitope.
Precursor peptides containing the WT1235 epitope were digested with purified immunoproteasome (IP) (A) and standard proteasome (SP) (B) with and without ONX-0914 for 3 hours. Proteolysis products were evaluated by tandem mass spectrometry to measure production efficiency. Each fragmented peptide peak area was determined using extracted peak chromatograms. WT1235 epitope fragments are highlighted by black text. Analysis is representative of two independent experiments.