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Ephrin receptor A2, the epithelial receptor for Epstein-Barr virus entry, is not available for efficient infection in human gastric organoids

Fig 3

Despite comparable EPHA2 expression levels in organoids vs. epithelial cell lines there is no efficient EBV infection in human gastric organoids.

(A) Scheme depicting B cell-mediated transfer infection of organoid-derived monolayers. (B) EBV transfer-infected organoid-derived monolayers were checked at 6 dpi by fluorescence microscopy. Scale: 200 μm. Representative image of at least three independent experiments. (C) At 6 dpi immunofluorescence was performed on EBV transfer-infected organoid-derived monolayers for epithelial marker Occludin, GFP-expressing EBV and lymphocyte marker CD45. DNA was counterstained with Hoechst. (I) depicts close-up of infected primary epithelial/organoid cell (GFP+, Occludin+ and CD45-). (II) depicts close-up of infected remaining B cells (GFP+, CD45+). Scale: 200 μm. Representative images of three independent experiments. (D) Flow cytometry gating strategy for evaluation of EBV infection efficiency. Left plot depicts FSC/SSC with gated cell population in P1. Middle plot depicts FSC/PI with gated viable cells in R1. Right plot depicts CD45-APC/EBV-GFP displaying localization of different cell populations. Q1: CD45+/GFP- = uninfected B cells, Q2: CD45+/GFP+ = infected B cells, Q3: CD45-/GFP- = uninfected epithelial cells and Q4: CD45-/GFP+ = infected epithelial cells. (E) At 4–6 dpi, EBV transfer-infected organoid-derived monolayers from different donors were analyzed for EBV infection rate by flow cytometry. Results are shown as means of three independent experiments with SD. #1 and 42 refers to patient IDs.

Fig 3