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. Author manuscript; available in PMC: 2020 Jun 1.
Published in final edited form as: Clin Cancer Res. 2019 Sep 4;25(23):7126–7138. doi: 10.1158/1078-0432.CCR-19-0421

Figure 1. GD2.CAR constructs containing CD28 costimulatory endodomain enable superior ex vivo expansion of CAR-NKTs.

Figure 1.

(A) Following a 10-day primary stimulation with α-GalCer-pulsed autologous PBMCs, NKTs were re-stimulated and transduced 2 days later with the indicated CAR constructs or no construct (NT: non-transduced). Representative flow cytometry analysis of indicated GD2.CAR construct expression 3 days after transduction by gating on 1A7+ (14G2a anti-idiotype)/ Vα24-Jα18+ NKT cells (n=13). (B) Quantification of total NKT cell fold expansion, mean ± SD following 10 days of secondary expansion (n=13). NKT cell number was determined using the Cellometer Auto Cell Viability Counter with AOPI staining. *p < 0.05, ****p < 0.0001, NS: not significant, one-way ANOVA. (C) Quantification of CAR+ NKT cell expansion calculated following 10 days of secondary expansion. *p < 0.05, *** p < 0.001, Student’s paired t-test. (D) NKTs transduced with the indicated GD2.CAR constructs were stained with annexin-V and 7-AAD 5 days after transduction. Shown are representative results from one of four donors. (E) Quantification of annexin-V +/7-AAD- NKTs ± SD in both CAR+ gated and CAR- gated populations for indicated GD2.CAR NKT groups (n=4). *p < 0.05, **p < 0.01, NS: not significant, one-way ANOVA. (F) TRAIL expression in CAR+ gated populations for the indicated GD2.CAR NKTs 7 days post-transduction. Shown are TRAIL median fluorescence intensity (MFI) results from a representative donor and mean ± SD of TRAIL expression MFI from six donors. ***p < 0.001, NS: not significant, one-way ANOVA. (G) Levels of activated caspase 8 were evaluated in the indicated CAR+ and CAR- NKT populations using FAM-LETD-FMK labeling 7 days post-transduction. Shown are representative results from one of three donors.