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Fig. 2 | Molecular Cancer

Fig. 2

From: Antibody-mediated delivery of a viral MHC-I epitope into the cytosol of target tumor cells repurposes virus-specific CD8+ T cells for cancer immunotherapy

Fig. 2

TEDbody-mediated CMV-pMHCI presentation proceeds via the conventional MHC-I antigen-processing pathway. A A representative flow cytometric histogram of CMV-pMHCI display on the surface of MDA-MB-231 cells, detected by the CMV-pMHCI-specific C1–17 Ab (red) in comparison with the control involving only the secondary Ab (blue). The cells were treated with the indicated peptide, TEDbody, or control Ab (4 μM) at 4 °C for 3 h or at 37 °C for 18 h prior to flow cytometric analysis. B Percentage rates of tumor cell lysis by ex vivo-expanded CMVp-CTLs after the cancer cells were treated with the indicated peptide, TEDbody, or control Ab (20, 100, or 500 nM) for 12 h at 37 °C, prior to coculture with CMVp-CTLs for 18 h at an E:T ratio of 5:1. C IFN-γ secretion caused by the activation of CMVp-CTLs in response to CMV-pMHCI presentation on MDA-MB-231 cells after the cells were treated with the indicated peptide, TEDbody, or control Ab (0.2 μM) for 12 h at 37 °C in the absence or presence of MG132 (20 μM), ERAP1-IN-1 (20 μM), or brefeldin A (200 nM). The bar graphs show the mean ± SEM (n ≥ 3). D A representative flow cytometric histogram of CMV-pMHCI display on the surface of wild-type and TAP1 knockout MDA-MB-231 cells (red), compared to the control involving only the secondary Ab (blue). The cells were treated with the indicated TEDbody or control Ab (4 μM) at 37 °C for 18 h prior to flow cytometric analysis with the C1–17 Ab. E Representative confocal fluorescence microscopy images of MDA-MB-231 cells treated with the indicated TEDbody or control Ab (4 μM) at 37 °C for 18 h and monitoring of colocalization of CMV-pMHCI (red) with early endosome marker EEA1 (green) or a Golgi marker called 58 K Golgi (green). Nuclei were stained with Hoechst 33342 (blue). Scale bar: 20 μm. The images are representative of three independent experiments. F Percentage rates of tumor cell lysis by ex vivo-expanded CMVp-CTLs, after the cancer cells were treated with the indicated peptide, TEDbody, or control Ab (1 μM) at 37 °C for 12 h, prior to coculture with CMVp-CTLs for 18 h at the indicated E:T ratio. G and H Real-time kinetics of TEDbody-induced cell lysis of MDA-MB-231-EGFP cells by ex vivo-expanded PKH26-labeled CMVp-CTLs (G) and representative time-lapse fluorescence microscopy images (H). MDA-MB-231-EGFP cells treated with the indicated peptide, TEDbody, or control Ab (1 μM) for 12 h and then cocultivated with PKH26-labeled CMVp-CTLs at an E:T ratio of 3:1 inside the Lionheart FX automated microscopy system for the indicated periods. Lysis of MDA-MB-231-EGFP cells (green) by PKH26-labeled CMVp-CTLs (red) was registered based on a loss of the EGFP signal. In (G), the cell index refers to green fluorescence intensity from the total cancer cell area after normalization to that from the total cancer cell area at time point 0. In (H), scale bar: 20 μm. In (B), (C), (F), and (G), error bars present the mean ± SEM (n = 3). In (B), (C), (F), and (G), *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the vehicle-treated control (B,C) or inCT-treated control (F,G); ns: not significant

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