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. Author manuscript; available in PMC: 2018 Aug 5.
Published in final edited form as: Nat Med. 2018 Feb 5;24(3):352–359. doi: 10.1038/nm.4478

Fig. 1. Generation of the anti-CD19 chimeric antigen receptor (CAR) constructs to induce JAK-STAT pathway activation.

Fig. 1

(a) A schematic diagram of the anti-CD19 CAR constructs used in this study. An FMC63-derived single-chain variable fragment (scFv) is linked to CD28 and CD3z (28-z); 4-1BB and CD3z (BB-z); and CD28, the cytoplasmic domain of the IL-2 receptor β chain with or without an internal deletion and CD3z with the YXXQ motif (28-IL2RB-z (YXXQ) and 28-ΔIL2RB-z (YXXQ)). All CAR-genes were linked to truncated nerve growth factor receptor (ΔNGFR) via a P2A sequence. (b, c) The indicated CAR-transduced T cells were rested in cytokine-free media overnight and stimulated with the CD19+ acute lymphoblastic leukemia cell line NALM-6 or the CD19− cell line K562 at an effector to target (E:T) ratio of 1:1. T cells mock-transduced and treated with IL-2 (300 IU/ml) or IL-21 (50 ng/ml) were used as a control. The phosphorylation of STAT3 and STAT5 within the CD8+ ΔNGFR+ T cell population was analyzed by flow cytometry at variable time points. The data shown are representative FACS plots (b) and the mean fluorescence intensity of the phosphorylated STAT3 and STAT5 (c, n=4 different donor samples; repeated measures one-way ANOVA with Tukey’s multiple comparisons test for each time point; F=63.57 (pSTAT3 at 60 min), 39.49 (pSTAT3 at 120 min), 71.41 (pSTAT3 at 240 min), 103.8 (pSTAT5 at 60 min), 201 (pSTAT5 at 120 min), 113.7 (pSTAT5 at 240 min); degree of freedom=15). (d) CAR constructs with either STAT3 or STAT5 binding domain alone. For the 28-ΔIL2RB (FLSL)-z (YXXQ) CAR, tyrosine within the YLSL motif of the IL-2 receptor β was replaced by phenylalanine to abrogate STAT5 association and the YXXQ motif was introduced by site-directed mutagenesis to the C-terminus end of CD3z (e) T cells transduced with the indicated CAR constructs were analyzed for phosphorylation of STAT3 and STAT5 120 minutes after stimulation with NALM-6 (e, n=4 different donor samples; repeated measures one-way ANOVA with Tukey’s multiple comparisons test; F=75.44 for pSTAT3, F=99.46 pSTAT5; degree of freedom=11). In c and e, horizontal lines denote mean values. ns, not significant.