GoKawiila,b, CAR-T cells were repetitively incubated with PLC/PRF/5 cells with or without TGFβ (10 ng/ml). One of two independent experiments is shown. (a) CD45− tumour cells were quantified by flow cytometry 3-4 days after each killing round. (b) CAR-T expansion was determined as fold increase of CAR+ cells over initial seeding. c,d, CAR-T cells were cultured for the indicated time on plates coated with recombinant human GPC3 (10 μg/ml), with or without TGFβ (10 ng/ml). (c) CAR-T expansion was determined as in b. (d) Supernatant IFNγ levels were evaluated at day 7 (Donor #1) or day 5 (Donor #2). e, TGFβ levels were evaluated in the lysate of wild-type Huh-7 cells and TGFβ-overexpressing Huh-7 (Huh7-oeTGFβ) cells by ELISA. n = 3 independent assays. f,g, Huh7-oeTGFβ tumour-bearing mice received 7 × 106 untransduced T or CAR-T cells. n = 10 mice. h, CAR+ cells in tumours were quantified by flow cytometry 7 days postinfusion. n = 5 mice. i,j, PD1 and LAG3 expression was evaluated by flow cytometry in the tumours of mice treated with unarmoured or dnTGFβRII-armoured CAR-T cells 14 days postinfusion. Unarmoured: n = 5 mice; armoured: n = 4 mice (one was excluded for not receiving the intended CAR-T dose). A representative dot plot is shown. k, Serum IFNγ levels were evaluated by Meso Scale Discovery assays 7 days postinfusion (n = 5). l, Serum AFP levels were evaluated by ELISA before, 7 days, and 14 days postinfusion. n = 5 mice (an infusion error existed for one mouse on day 14). Data are mean ± s.d. for d, and mean ± s.e.m. for f,h-l. Two-sided unpaired Student’s t-test for d; one-sided ratio paired t-test for e; two-way ANOVA with Šídák’s multiple comparisons test for f; Kaplan–Meier method with log-rank test for g; one-way ANOVA with Tukey’s honest significant difference test for k,l; two-sided Mann-Whitney U test for h-j. AFP, alpha-fetoprotein; ELISA, enzyme-linked immunosorbent assay; LAG3, lymphocyte-activation gene 3; PD1, programmed cell death protein 1.
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