Figure 2.
Galectin-9 impairs CAR19 T cells. (A) Cartoon depicting the in vitro experimental design of cocultures of CAR19 T cells with B-ALL cells. (B) Expression levels of TIM-3 in CAR19 T cells (left) and galectin-9 in CAR19 T-cell–resistant B-ALL cells (right), determined by FACS after 24 hours of CAR T-cell:B-ALL coculture. (C-D) Apoptosis assay (Annexin-V/7-AAD) and T-cell proliferation measured by an eFluor 670 retention assay, after incubation of CAR19 T cells with or without rhGalectin-9 (1 μg/mL) for 48 hours. Left panels show representative FACS profiles. Right panels show average data. (E) CAR19 T-cell–mediated cytotoxicity against SEM and NALM6 B-ALL cells at different E:T ratios in the presence/absence of rhGalectin-9 (1 μg/mL) for 48 hours. (F) Western blot detection of galectin-9 in the indicated nontransduced and galectin-9–overexpressing B-ALL cells (SEM OE Gal9 and NALM6 OE Gal9). GAPDH was used as a housekeeping control. (G) CAR19 T-cell–mediated cytotoxicity against nontransduced and galectin-9–overexpressing SEM and NALM6 B-ALL cells at different E:T ratios for 48 hours. All data are shown as mean ± standard error of the mean. Three independent experiments were performed with T cells from 3 different donors. ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001. E:T, effector:target; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; h, hours; IL-7, interleukin-7; IL-15, interleukin-15; MFI, mean fluorescence intensity; OE, overexpressing; PBMC, peripheral blood mononuclear cell; rhGalectin-9, recombinant human galectin-9; UT, untransduced; WB, western blot; WT, wild type.

Galectin-9 impairs CAR19 T cells. (A) Cartoon depicting the in vitro experimental design of cocultures of CAR19 T cells with B-ALL cells. (B) Expression levels of TIM-3 in CAR19 T cells (left) and galectin-9 in CAR19 T-cell–resistant B-ALL cells (right), determined by FACS after 24 hours of CAR T-cell:B-ALL coculture. (C-D) Apoptosis assay (Annexin-V/7-AAD) and T-cell proliferation measured by an eFluor 670 retention assay, after incubation of CAR19 T cells with or without rhGalectin-9 (1 μg/mL) for 48 hours. Left panels show representative FACS profiles. Right panels show average data. (E) CAR19 T-cell–mediated cytotoxicity against SEM and NALM6 B-ALL cells at different E:T ratios in the presence/absence of rhGalectin-9 (1 μg/mL) for 48 hours. (F) Western blot detection of galectin-9 in the indicated nontransduced and galectin-9–overexpressing B-ALL cells (SEM OE Gal9 and NALM6 OE Gal9). GAPDH was used as a housekeeping control. (G) CAR19 T-cell–mediated cytotoxicity against nontransduced and galectin-9–overexpressing SEM and NALM6 B-ALL cells at different E:T ratios for 48 hours. All data are shown as mean ± standard error of the mean. Three independent experiments were performed with T cells from 3 different donors. ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001. E:T, effector:target; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; h, hours; IL-7, interleukin-7; IL-15, interleukin-15; MFI, mean fluorescence intensity; OE, overexpressing; PBMC, peripheral blood mononuclear cell; rhGalectin-9, recombinant human galectin-9; UT, untransduced; WB, western blot; WT, wild type.

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