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. 2022 Aug 8;119(33):e2112006119. doi: 10.1073/pnas.2112006119

Fig. 3.

Fig. 3.

IL13 WT, E12Y, C4, and D7 CARs mediate comparable IL13Rα2-dependent in vitro effector function. IL13Rα2-dependent degranulation (A) and IFN-γ production (B) for IL13 WT, E12Y, C4, and D7 CAR and untransduced (mock) T cells. T cell lines were cocultured at a 1:1 E:T ratio with IL13Rα2+ GBM lines U251T and PBT030-2 or HT1080 engineered to express IL13Rα2 (HT1080-IL13Rα2); after 5 h, the degranulation marker CD107a and intracellular IFN-γ were measured by flow cytometry, and the percentage of positive cells was graphed (mean ± SD; n = 3 wells). (C) Secreted IFN-γ production by indicated T cell lines in response to increasing concentrations of immobilized rhIL13Rα2-Fc (mean ± SD; n = 3 wells). P < 0.0001 for all IL13-CAR T cell variants and mock T cells. (D) Tumor killing by IL13-CAR and mock T cell lines cultured at a 1:10 E:T ratio for 2 days. Flow cytometry was used to determine the remaining viable tumor count, and tumor killing was determined as the percentage of normalized to mock (mean ± SD; n = 3 wells). (E) CAR T cell killing in experiments using four separate donors at a 1:50 E:T ratio against PBT030-2, U251T, and HT1080-IL13Rα2, showing similar levels of tumor killing for WT, E12Y, C4, and D7. Plots represent remaining tumor cells at day 7 (the dotted line represents seeded tumor cells). (F) Live CAR T cells in experiments presented following the 7-day coculture presented in E. No live CAR T cells were observed in tumor-only and mock CAR T cell experiments; levels of CAR T cell persistence were as follows: WT > D7 > E12Y > C4. A–F are representative of at least three independent experiments. (G) Expression of IL13Rα2 in PBT-neg, PBT-low, and PBT-high cell lines by flow cytometry. (H) Tumor killing of PBT-neg, PBT-low, and PBT-high at E:T ratios of 1:4 (Left) and 1:10 (Right). Noted statistic comparisons for E12Y across groups are representative of trends for all CAR variants. IFN-γ (I) and IL2 (J) secreted from CAR T cell cocultures with IL13Rα2-engineered cell lines at an E:T ratio of 1:1 were quantified by Luminex assay. Noted statistic comparisons compare each CAR variant individually to mock. All data shown from A–D are means ± SD and H–J are means ± SEM (****P < 0.0001, ***P < 0.005, **P < 0.01, *P < 0.05).