Figure 3.

GM-CSF CRISPR-knockout CAR-T cells exhibit reduced expression of GM-CSF, similar levels of key cytokines and chemokines, and enhanced antitumor activity. (A) CRISPR Cas9 GM-CSF^k/o CART19 cells exhibit reduced GM-CSF production compared with wild-type CART19 cells, but other cytokine production and degranulation are not inhibited by the GM-CSF gene disruption. CART19 cells and GM-CSF^k/o CART19 cells stimulated with NALM6; n = 3 experiments, 2 replicates per experiment. Data are mean ± SEM. ***P < .001, Student t test. n.s., P > .05, Student t test. (B) GM-CSF k/o CAR-T cells have reduced serum human GM-CSF in vivo compared with CAR-T treatment, as assayed by multiplex; 5 or 6 mice per group (4 to 6 at time of bleed, 8 days post–CAR-T cell injection). Data are mean ± SEM. ***P < .001, ****P < .0001, Student t test. (C) GM-CSF^k/o CART19 cells in vivo enhance overall survival compared with wild-type CART19 cells in a high tumor burden relapse xenograft model of ALL utilizing a NALM6 cell line; 5 or 6 mice per group. **P < .01, log-rank test. Human (D) and mouse (E) multiplex of serum cytokines and chemokines. A statistically significant reduction of human GM-CSF in GM-CSF^k/o CART19 cells compared to wild-type CAR-T cells was observed, but no other statistically significant differences between GM-CSF^k/o CART19 cells and wild-type CART19 cells were seen, further implicating that critical T-cell cytokines and chemokines are not adversely depleted by reducing GM-CSF expression; 5 or 6 mice per group (4 to 6 at time of bleed). ****P < .0001, Student t test.