Fig. 1
Dasatinib reduced the differentiation and exhaustion of both CD28 and 4-1BB/CART cells during ex vivo expansion, thus enhanced their therapeutic efficacy in mice models. a The differentiation and exhaustion in CD28/CART cells were evaluated by expression of CD45RO, CD62L, PD1, TIM3 and LAG3 on 5–7 days after CAR-carrying virus transduction. b–d 5–7 days after transduction, CD28/CART cells were cultured with dasatinib 10 nM, 30 nM,100 nM and equivalent volume of DMSO to dasatinib in control for 72 h. The quantification of b CD62L, c PD1, TIM3 and LAG3, d CD25 and CD69, respectively, showing the effects of dasatinib on CD28/CART cells differentiation, exhaustion and activation. e Schematic diagram of experimental setup depicting that NSG mice were injected with 1 × 106 Nalm6-luc cells followed by administration of 1 × 106 DMSO and 30 nM dasatinib pretreated CD28/CART cells, respectively, 5 days later, and tumor burden was determined by bioluminescent imaging system every week. f The dynamics of tumor burden in two groups of Nalm6-bearing mice was assessed by bioluminescent imaging (n = 5 per group). g The mean average radiance on representative d33 (n = 5 per group). h Dasatinib treated CD28/CART group showed significant prolonged survival compared with control (p = 0.0018, log-rank Mantel-Cox test). i 4-1BB CART cells were treated with dasatinib 30 nM and equivalent volume of DMSO to dasatinib in control for nine consecutive days, and the expression of CD45RO and CD62L was evaluated on day 3, 6 and 9, respectively. j the number of CART cells was calculated by cell counts on day 3, 6 and 9, respectively. k The quantification of CD62L on day 9 showing the inhibitory effect of dasatinib on 4-1BB CART cell differentiation with the prolonged ex vivo culture. l Real-time PCR was performed to identify differentiation-related transcription factor TCF1. m The quantification depicting the expression of inhibitory receptors on day 9. n The quantification showing the effect of dasatinib on activation-related markers (CD25 and CD69) in 4-1BB CART cells on day 9. o NSG mice were injected with 1 × 106 luciferase expressing Nalm6 on day0, and 5 days later, mice received 1 × 106 DMSO and dasatinib 30 nM pretreated 4-1BB/CART cells, respectively. Tumor burden was determined by bioluminescent imaging every week. p The mean average radiance on representative d26 (n = 5 per group). q 7 days after CART cells infusion, mice blood was collected, and the percentage of CART cells was determined by flow cytometry. r Dasatinib-treated CART group showed prolonged survival compared with control (p = 0.0021, log-rank Mantel-Cox test). All data of in vitro experiments was given as n = 3 replicates and representative of three donors; *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant; error bars represent the mean ± SEM as determined by a two-tailed unpaired t test