Figure 7. Downregulation of BATF expression by JQ1 treatment contributes to the maintenance of the T_SCM and T_CM phenotypes.

(A and B) Validation of BATF knockdown with qPCR (A, n = 4) and immunoblotting (B, representative blots of 3 experiments and quantified protein levels). Error bars indicate the SD. *P < 0.05, **P < 0.01 in a 1-sample t test compared to control siRNA (Ctsh), which was assigned a value of 1. (C) CD3⁺ T cells were stimulated with aAPC/mOKT3 and transduced with control or siBATF and truncated nerve growth factor receptor (ΔNGFR). The frequency of CD45RA^+/–CD62L⁺CCR7⁺ cells within the ΔNGFR⁺CD8⁺ T cell population 14 days after initial stimulation is shown (n = 8, paired ANOVA). (D) The IL-2, IFN-γ, and TNF-α secretion levels were assessed by intracellular flow cytometry in the ΔNGFR⁺CD8⁺ T cell population upon restimulation with aAPC/mOKT3. Frequencies of IL-2–secreting cells and cells producing all 3 cytokines are shown (n = 6, paired ANOVA). (E and F) CD3⁺ T cells were stimulated with aAPC/mOKT3 and transduced with the control ΔNGFR or BATF-IRES-ΔNGFR. The CD8⁺NGFR⁺ cells were isolated on day 7 and immunoblotted for BATF (E, representative blots of 4 experiments). Frequencies of CD45RA⁺CD62L⁺CCR7⁺ cells (left y axis) and CD45RA^–CD62L⁺CCR7⁺ cells (right y axis) within the ΔNGFR⁺CD8⁺ T cell population were assessed 14 days following stimulation (F, n = 9, paired ANOVA). (G) IL-2 production of NGFR⁺CD8⁺ T cells upon restimulation with aAPC/mOKT3 was assessed by intracellular flow cytometry (n = 4, paired ANOVA). (H–J) Naive T cells were pretreated with IL-7, transduced with lentiviral shRNAs against BATF, and stimulated with aAPC/mOKT3 or transplanted into irradiated NSG mice. Frequency of CD45RA^+/–CD62L⁺CCR7⁺ cells within the ZsGreen⁺CD8⁺ T cell population was evaluated 10 days after stimulation with aAPC/mOKT3 (I, n = 5, paired ANOVA) or 11 days following T cell transplantation into NSG mice (J, n = 6, paired ANOVA). NS, not significant.