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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 1

Binding of soluble and MHC class II–bound IDH1R132H 15-mer and 20-mer peptides by anti-IDH1R132H antibody (H09) in peptide-based ELISA.

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Binding of soluble and MHC class II–bound IDH1R132H 15-mer and 20-mer pe...
(A) IDH1R132H IHC using H09 on glioma tissue p001 and p018. A high magnification image of the boxed area is shown at right. Scale bar: 40 μm (left and center); 8 μm (right). (B) IDH1 15-mer and 20-mer peptide library encompassing amino acid 132. (C) IDH1 15-mer and 20-mer peptide-based H09 ELISA. MOG, negative control; DMSO, vehicle control. One-way ANOVA, Tukey corrected, n = 3. (D and E) MHC class II–bound IDH1R132H p122–136 peptide-based ELISA plates. H09 (1°) was preincubated with specific (red, IDH1R132H–HLA-DR1) and control (black, CLIP–HLA-DR1) tetramer (4-mer) and subsequently subjected to p122–136 IDH1R132H-based ELISA. 2°, secondary antibody. pIDH1R132H, p122–136 IDH1R132H. One-way ANOVA, Tukey corrected, n = 3 (E). ***P < 0.001.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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