Figure 3.
Loss of GABARAP impairs ICD-induced phagocytosis and antitumor T-cell activation. (A) For phagocytosis assay, MM cells and DCs were prestained with different dyes (either far-red or carboxyfluorescein diacetate succinimidyl ester [CFSE]). Dye-stained AMO1, H929, and 5TGM1 cells either WT or GABARAPKO were left untreated or treated with BTZ (5, 2.5, and 7.5 nM, respectively) for 16 hours. Then, they were cocultured with dye-stained DCs. Analysis was performed after 4 hours. Shown in the graph is the fold increase in the percentage of double-positive DCs in treated cells compared with untreated cells, as assessed by flow cytometry. (B) Phagocytosis assay of BTZ-treated (5 nM; 16 hours) or -untreated stained AMO1 WT, GABARAPKO, and GABARAPKO with the addition of exogenous recombinant CRT (rCRT) cocultured with stained DCs for 4 hours. Fold increase in the percentage of double-positive DCs in treated cells compared with untreated cells is shown. Representative overlay histograms confirm the exposure of surface CRT in the different conditions (right), as assessed by flow cytometry. (C) Phagocytosis assay of BTZ-treated (7.5 nM; 16 hours) or -untreated stained KMS11 WT or GABARAPOE cocultured with stained DCs for 4 hours. Fold increase in the percentage of double-positive DCs in treated cells compared with untreated cells is shown. (D) BTZ-treated (16 hours) or -untreated U266 either WT or GABARAPKO cells were cocultured with HLA-matched DCs and T cells from the same healthy donors. After 5 days, T cells were negatively selected from all 4 coculture conditions (α, WT untreated; β, WT treated with BTZ, δ, GABARAPKO untreated; and γ, GABARAPKO treated with BTZ) and then cultured for 24 hours with new U266 cells at 1:5 target:effector (T:E) ratio, followed by 7-AAD staining and quantification of MM cell lysis by flow cytometry. Shown in the graph is the fold change increase of MM cell lysis induced by the T cells retrieved from the treated conditions versus the untreated ones. For panels A-D, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 (unpaired Student t test). GFP, green fluorescent protein.

Loss of GABARAP impairs ICD-induced phagocytosis and antitumor T-cell activation. (A) For phagocytosis assay, MM cells and DCs were prestained with different dyes (either far-red or carboxyfluorescein diacetate succinimidyl ester [CFSE]). Dye-stained AMO1, H929, and 5TGM1 cells either WT or GABARAPKO were left untreated or treated with BTZ (5, 2.5, and 7.5 nM, respectively) for 16 hours. Then, they were cocultured with dye-stained DCs. Analysis was performed after 4 hours. Shown in the graph is the fold increase in the percentage of double-positive DCs in treated cells compared with untreated cells, as assessed by flow cytometry. (B) Phagocytosis assay of BTZ-treated (5 nM; 16 hours) or -untreated stained AMO1 WT, GABARAPKO, and GABARAPKO with the addition of exogenous recombinant CRT (rCRT) cocultured with stained DCs for 4 hours. Fold increase in the percentage of double-positive DCs in treated cells compared with untreated cells is shown. Representative overlay histograms confirm the exposure of surface CRT in the different conditions (right), as assessed by flow cytometry. (C) Phagocytosis assay of BTZ-treated (7.5 nM; 16 hours) or -untreated stained KMS11 WT or GABARAPOE cocultured with stained DCs for 4 hours. Fold increase in the percentage of double-positive DCs in treated cells compared with untreated cells is shown. (D) BTZ-treated (16 hours) or -untreated U266 either WT or GABARAPKO cells were cocultured with HLA-matched DCs and T cells from the same healthy donors. After 5 days, T cells were negatively selected from all 4 coculture conditions (α, WT untreated; β, WT treated with BTZ, δ, GABARAPKO untreated; and γ, GABARAPKO treated with BTZ) and then cultured for 24 hours with new U266 cells at 1:5 target:effector (T:E) ratio, followed by 7-AAD staining and quantification of MM cell lysis by flow cytometry. Shown in the graph is the fold change increase of MM cell lysis induced by the T cells retrieved from the treated conditions versus the untreated ones. For panels A-D, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 (unpaired Student t test). GFP, green fluorescent protein.

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