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Proximity ligation assay evaluates IDH1R132H presentation in gliomas
Lukas Bunse, … , Wolfgang Wick, Michael Platten
Lukas Bunse, … , Wolfgang Wick, Michael Platten
Published January 2, 2015
Citation Information: J Clin Invest. 2015;125(2):593-606. https://doi.org/10.1172/JCI77780.
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Technical Advance Oncology

Proximity ligation assay evaluates IDH1R132H presentation in gliomas

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Abstract

For a targeted cancer vaccine to be effective, the antigen of interest needs to be naturally processed and presented on MHC by the target cell or an antigen-presenting cell (APC) in the tumor stroma. The presence of these characteristics is often assumed based on animal models, evaluation of antigen-overexpressing APCs in vitro, or assays of material-consuming immune precipitation from fresh solid tissue. Here, we evaluated the use of an alternative approach that uses the proximity ligation assay (PLA) to identify the presentation of an MHC class II–restricted antigen in paraffin-embedded tissue sections from patients with brain tumors. This approach required a specific antibody directed against the epitope that was presented. We used an antibody that specifically binds an epitope of mutated isocitrate dehydrogenase type 1 (IDH1R132H), which is frequently expressed in gliomas and other types of tumors. In situ PLA showed that the IDH1R132H epitope colocalizes with MHC class II in IDH1R132H-mutated glioma tissue. Moreover, PLA demonstrated colocalization between the class II epitope-containing melanoma antigen New York esophageal 1 and MHC class II. Collectively, our data suggest that PLA may be a useful tool to acquire information on whether an antigen is presented in situ, and this technique has potential to guide clinical studies that use antigen-specific cancer immunotherapy.

Authors

Lukas Bunse, Theresa Schumacher, Felix Sahm, Stefan Pusch, Iris Oezen, Katharina Rauschenbach, Marina Gonzalez, Gergely Solecki, Matthias Osswald, David Capper, Benedikt Wiestler, Frank Winkler, Christel Herold-Mende, Andreas von Deimling, Wolfgang Wick, Michael Platten

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Figure 3

Specific colocalization of IDH1R132H peptide and MHC class II in IDH1 mutant-overexpressing glioma cell line LN229 in vitro.

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Specific colocalization of IDH1R132H peptide and MHC class II in IDH1 mu...
(A) IDH1R132H–HLA-DR PLA on IDH1D252G R132H-overexpressing LN229 cells (LN229 DG RH) and IDH1D252G-overexpressing LN229 cells (LN229 DG), respectively. Data are representative of 3 experiments. Scale bar: 20 μm. (B) IDH1R132H–HLA-DR PLA on IDH1D252G R132H-overexpressing LN229 cells after treatment with HLA-DRA–specific siRNA (siHLA-DR) or siRNA control pool (siCONTROL). Data are representative of 2 experiments. Scale bar: 15 μm. (C) Quantification of HLA-DRA knockdown in IDH1D252G R132H-overexpressing LN229 cells by flow cytometry. Numbers in the histogram indicate percentages of HLA-DR–positive cells after treatment with siCONTROL or si-HLA-DRA. Data are representative of 2 experiments. Gray, isotype control; red, siCONTROL; and blue, siHLA-DR. (D) IDH1R132H–HLA-DR PLA with IDH1R132H (H09) costaining (green) in IDH1D252G R132H-overexpressing LN229 cells. A high-magnification image of the boxed area is shown below. Data are representative of 3 experiments. Scale bar: 20 μm (top); 10 μm (bottom). (E) Correlation of IDH1R132H expression (MFI) and PLA signal in IDH1R132H-overexpressing LN229 cells; n = 152, Spearman’s rho, r = 0.6352, P < 0.0001. (A, B, and D) Red, PLA signal; blue, DAPI.
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