Figure 8. Downregulation of TET2 expression by JQ1 treatment contributes to reinvigoration of CAR T cells from patients with CLL.

(A) TET2 mRNA reduction in CD19 CAR T cells transduced and expanded in the presence of 150 nM (–)-JQ1 or (+)-JQ1 (n = 8; paired t test). (B) Depiction of the organization of the human (h)TET2 catalytic domain and structures of FLAG-tagged TET2-CS and TET2-HxD with highlighted targets for mutagenesis in red (top left panel). Schematic of the sequential oxidations of 5-mC to 5-hmC and to 5-fC and to 5-caC catalyzed by TET2 is shown (top right panel). Immunoblot of TET2 protein levels in HEK293T cells is depicted. HSP90 was used as a loading control (bottom left panel). Dot blots for 5-mC, 5-hmC, and 5-caC in genomic DNA isolated from the above HEK293T cells transfected with an empty vector, TET2-CS, and TET2-HxD are shown (bottom right panel, blots are representative of 3 independent experiments). (C) OCR, (D) SRC, and (E) MRC of expanded CLL patient CAR T cells transduced with vector alone or TET2-CS and subsequently treated with (–)-JQ1 or (+)-JQ1 for 4 days (n = 3–4; paired t test). (F) Levels of PD-1 expression on CLL patient CD8⁺ CAR⁺ T cells transduced with TET2-CS or TET2-HxD (representative histograms, left panel; graphical data summary, right panel; n = 7, paired t test). (G) Frequency of CD8⁺ PD-1⁺ CLL patient T cells transduced with vector alone or lentiviral vectors encoding TET2-CS and TET2-HxD followed by treatment with (–)-JQ1 or (+)-JQ1 (n = 6–12, 2-tailed t test). Data are shown as the mean ± SEM. (H) Levels of TNF-α and IL-2 elaborated by CD8⁺CAR⁺ T cells transduced with vector alone or TET2-CS and subsequently treated with (–)-JQ1 or (+)-JQ1 and stimulated with irradiated K562_CD19 or K562_CD19/PD-L1 cells (n = 4, paired t test). *P ≤ 0.05, **P ≤ 0.01, NS, not significant.