Fig 2: IL-33 gene restores MHC-I expression and signaling in MHC-I-loss metastatic carcinoma cells, as well as immune recognition of tumours.(a,b) MHC-I surface expression correlates with increased IL-33 expression. Mean Fluorescence Intensity (MFI) of MHC-I for stably transfected clones: metastatic A9, A9+IL-33, primary TC1; P < 0.05 when compared TC1 cells with A9 cells (Student’s t-test). (c) Functional B3Z-assay: the expression of IL-33 enhanced the ability of the tumour cells to present the MHC-I OVA-peptide complexes on their surface to be specifically recognized by B3Z T-cells. Values were normalized to vector alone control; *P < 0.05 compared with IL-33 expressing A9 cells (Student’s t-test).

Fig 3: IL-33 expression is reduced in human metastatic prostate cancer.IL-33 gene is down-regulated in human metastatic prostate cancer compared to human benign and primary prostate tumors. The data was obtained from the Gene Expression Atlas created by the European Biostatistics Institute (Gene Expression Atlas; https://www.ebi.ac.uk/gxa/home; access date: 30/01/2013). A high-throughput immunoblot approach was utilized to characterize proteomic alterations in human prostate cancer progression, focusing on the transition from clinically localized prostate cancer to metastatic disease. They examined protein expression levels and gene transcript levels and found that the proteins that were in agreement with gene expression could be used as a predictor of clinical outcome.

Fig 4: Genetic complementation of immune evasive tumours shows clear phenotypic shift towards immune recognition.(a) To overcome the issue of multi-focality, FACS analysis was used to count tumour-infiltrating lymphocytes (TILs). The expression of IL-33 by the tumour skews the TILs towards a cytotoxic T cell response, as a statistically significant increase in the number of CD8-positive cells can be shown in genetically modified (A9+IL-33) tumours versus in metastatic (A9), (Student’s t-test), **P < 0.005. (b–q) Immunohistochemical staining was used to support the FACS analysis. CD8 is primarily a marker for cytotoxic T cells, but also found on natural killer cells and dendritic cells; MHC-I is found on all nucleated cells; CD68 is a marker of monocytes and macrophages; Ly6G is a marker for monocytes, granulocytes and neutrophils; FoxP3 is a marker of regulatory T cells. Greater numbers of CD8-positive cytotoxic T cells can be seen within the genetically modified (A9+IL-33) tumours (b,d) versus unmodified metastatic (A9) tumours (c). IL-33 expression increases MHC-1 expression on tumour cell surface; A9+IL-33 (g) versus unmodified metastatic A9 (f), thereby increasing tumour antigen presentation. Fewer regulatory T cells are present in IL-33 expressing tumours, as indicated by lower FoxP3 staining; metastatic A9 (o) versus A9+IL-33 (p). Increased macrophage and neutrophil response is seen in IL-33 expressing tumours; unmodified Metastatic A9 (i,l) versus A9+IL-33 (j,m) respectively. (e,h,k,n,q) Negative controls (rat IgG targeting Keyhole Limpet Hemocyanin (KLH)) were included to show that non-specific staining was minimized. 10 μm thick sections were stained with appropriate antibodies and imaged at 5x (b) or 20x (c–q) magnification. Size bar = 400 μm in (b); Size bar = 100 μm in (c–q).

Fig 5: Eosinophil recruitment into tumour tissue is induced by IL-33 expression (C57BL/6 mouse).(a,b) Eosinophils are located on the border between metastatic (A9) tumour and normal tissue. (c) IL-33-induced changes allow eosinophilic infiltration into tumour tissue, which expresses IL-33. 10 μm thick tumour sections were stained with Giemsa stain and imaged at 5x (Size bar = 400 μm (a)) and 10x magnification (Size bar = 200 μm (b,c)).