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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 2. Co-expression of IL-15 enhances survival and reduces exhaustion-associated phenotype in GD2.CAR NKTs.

Figure 2.

(A) Expression of phosphorylated STAT5 (pSTAT5) was evaluated in NKTs expressing the GD2.28z or GD2.28z.15 CAR 7 days post-transduction following 12 hours of culture in serum- and cytokine-starved conditions. Shown is a representative histogram from one of four donors (left) and mean ± SD of MFI for all donors (n=4). **p < 0.01, one-way ANOVA. (B) Expression of intracellular Bcl-2 was evaluated in NKTs expressing the GD2.28z or GD2.28z.15 CAR 7 days post-transduction following 12 hours of culture in serum- and cytokine-starved conditions. Shown is a representative histogram from one of four donors (left) and Bcl-2 expression MFI for each individual donor expressing the GD2.28z versus the GD2.28z.15 CAR (n=4). *p < 0.05, Student’s paired t-test. (C) Expression of intracellular Bcl-xL was evaluated in NKTs expressing the GD2.28z or GD2.28z.15 CAR 7 days post-transduction following 12 hours of culture in serum- and cytokine-starved conditions. Shown is a representative histogram from one of four donors (left) and Bcl-xL expression MFI for each individual donor expressing the GD2.28z versus the GD2.28z.15 CAR (n=4). *p < 0.05, Student’s paired t-test. (D) Following 21-day in vitro expansion, NKTs from 14 individual donors transduced with the indicated constructs or non-transduced (NT) NKTs were evaluated for CD4 expression and (E) 11 of them were also examined for CD62L expression by flow cytometry. NS: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, Student’s paired t-test. (F) NKTs transduced with the GD2.28z or GD2.28z.15 CAR or NT were evaluated for expression of the indicated exhaustion markers 7 days after transduction (n=8). *p < 0.05, **p < 0.01, ****p < 0.0001, NS: not significant, one-way ANOVA. (G) SPICE analysis of exhaustion marker expression from three of the donors shown in F. Pie charts reflect proportions of indicated NKT groups expressing indicated numbers (0-3) of exhaustion markers. Colored arcs indicate the specific combinations of exhaustion markers expressed.