Figure 2.

DRrejTs exhibit cytotoxicity against EBV-associated lymphoma superior to LMP1-CARTs and LMP2-rejTs

(A) In vitro⁵¹Cr release assay of iPSC-derived DRrejTs and PBMC-derived LMP1-CARTs (effectors) against HLA-A∗2402⁺ ENKL and HLA-A∗2402⁻ LCLs (targets). Error bars represent ± SD. Data represent three independent triplicate experiments. (B) NOG mice were intraperitoneally inoculated with HLA-matched ENKL cells expressing firefly luciferase and treated either with PBMC-derived LMP1-CARTs (n = 7) or iPSC-derived DRrejTs (n = 8). No treatment indicates that mice were injected with ENKL cells but not treated with T cells (n = 5). LMP1-CARTs or DRrejTs were injected intraperitoneally once a week (3 doses). Images of all mice from each group of two independent experiments are shown. (C) Kaplan-Meier curve representing percentage survival of experimental groups: no treatment, LMP1-CARTs, or DRrejTs. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by log-rank test. (D) In vitro⁵¹Cr release assay of iPSC-derived DRrejTs and LMP1-CAR-transduced HPV-rejTs (LMP1-CAR/HPV-rejTs) (effectors) against HLA-A∗2402⁺ LCLs and LMP1⁻ tumors (targets). Error bars represent ± SD. Data represent three independent triplicate experiments. (E) iPSC-derived DRrejTs and control rejTs were cocultured with HLA-A∗2402⁺ GFP/FFluc ENKL (ratio 5:1). Cultures were collected, stained with anti-CD3 antibody, and analyzed by flow cytometry on days 0, 5, and 7. The plots represent three independent experiments. Cells were counted using counting beads on flow cytometry. Graph summarizes the results of total number of NK-YS cells. Error bars represent ± SEM. ∗∗∗∗p < 0.0001 by two-way ANOVA. (F) In vitro⁵¹Cr release assay of iPSC-derived DRrejTs and LMP2-rejTs (effectors) against HLA-A∗2402⁺ LCL and HLA-A∗2402⁻ LCL (targets). Error bars represent ± SD. Data represent three independent triplicate experiments. (G) iPSC-derived DRrejTs and control rejTs were cocultured with HLA-A∗2402⁻ LCL (ratio 5:1). Cultures were collected, stained with anti-CD3 and anti-CD19 antibodies, and analyzed by flow cytometry on days 0, 5, and 7. The plots represent three independent experiments. Cells were counted using counting beads on flow cytometry. Graph summarizes the results of total cell number of LCLs. Error bars represent ± SEM. ∗∗∗∗p < 0.0001 by two-way ANOVA. E:T ratio, effector-to-target ratio. (H) In vitro⁵¹Cr release assay of DRrejT (effector) against HLA-A∗2402⁻ ENKL and EBV⁻ tumor (targets). Error bars represent ± SD. (I) In vitro⁵¹Cr release assay of DRrejTs (effectors) against HLA-A∗2402⁺ ENKL primary cells and EBV⁻ tumors (targets). Error bars represent ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by unpaired Student’s t test.