Effects of CK1ε and dual PI3K/CK1ε inhibition on normal and CLL human Tregs. (A) Protein expression of β-catenin was detected by intracellular flow cytometry in CLL patient T cells that were isolated by magnetic separation from previously frozen PBMCs and treated with PF-4800567, a CK1ε selective inhibitor, or PI3K inhibitors for 24 hours (n = 3). Graphs display mean + standard deviation (SD). (B) Normal human T cells were treated for 24 hours with the indicated inhibitors and then lysed in TRIzol Reagent. Axin2 messenger RNA levels were measured by qRT-PCR. Graph displays mean ΔΔCT. Error bars denote technical triplicates of a representative donor typical of 3 independent experiments. (C) A Treg suppression assay was performed as in Figure 3, following treatment with PI3K inhibitors (1 μM) with or without PF-4800567 (250 nM). (D-G) CLL patient T cells were isolated and cultured in Treg-polarizing conditions for 5 days in the presence of PF-4800567 (250 nM) and PI3K inhibitors (1 μM). Flow cytometry was performed to assess Treg percentage (D) and immunosuppressive markers (E-G) (n = 12). Graphs display mean + SD. *P < .05, **P < .005, ***P < .0005, ordinary 1-way ANOVA.