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. 2019 Aug 7;7:209. doi: 10.1186/s40425-019-0685-y

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© The Author(s). 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Fig. 4 — PD-1 knockdown impaired CAR-T cells in vitro proliferative potential. a 0.4 × 10⁴ purified CAR-T cells were co-cultured with Raji-luc cells at the E:T ratio of 01:1 for 72 h. Lysis of Raji-luc cells at different time were measured by luminescence, and S4-CART19 presented restrained lysis ability than SCR-CART19. b The absolute numbers of T cells were recorded daily to evaluate the in vitro proliferation potential which was driven by target cells stimulation, and the proliferation was significantly impaired by PD-1 knockdown. c The ZsGreen positive rates in different T cell populations were continuously recorded which were divided by the mean positive rates on the fifth day in each group to get the relative value of the positive rate. d The absolute numbers of CAR-T cell were continuously recorded, and the PD-1 silenced CAR-T cells presented impaired proliferative potential. e The daily doubling time of CAR-T cells were calculated, and the proliferation of S3-CART19 and S4-CART19 slowed down significantly with the prolongation of cultivation, compared with SCR-CART19. f Compared with SCR-CART19, the doubling time of S4-CART19 and S3-CART19 increased significantly from day 8 to day 9 and from day 10 to day 11, respectively. g 0.5 × 10⁴ purified CAR-T cells were co-cultured with A549–19 cells at the E:T ratio of 01:1 for 72 h. The absolute numbers of T cells were recorded daily, and SCR-CART19 proliferated more significantly than S3-CART19 and S4-CART19. h 0.5 × 10⁵ Raji-luc cells were co-cultured with 0.5 × 10⁴ purified CAR-T cells for 72 h, and SCR-CART19 presented higher lysis ability than S3-CART19 and S4-CART19. i 0.5 × 10⁵ A549–19luc cells were co-cultured with 0.25 × 10⁴ purified CAR-T cells for 72 h, and SCR-CART19 presented higher lysis ability than S3-CART19 and S4-CART19.CAR-T 0.01 < *P < 0.05; **P < 0.01. Statistical significance was determined using the ANOVA method for multiple comparisons. Data represent the mean ± SEM of triplicates and are representative of at least 3 independent experiments or are presented individually