Triple combination inhibits c-MYC in PBMCs containing leukemic cells ex vivo from ATL patients. (A) Expression of CD25 on CD4⁺T cells of PBMCs isolated from acute ATL (n = 4), smoldering/chronic ATL patients (n = 4), and healthy donors (n = 5). The expression was measured on day 0 (before) and day 6 (after) ex vivo culture. rIL-2 cytokine (100 U/ml) was added into the culture of PBMCs from acute ATL patients and healthy donors. (B) The bar graphs represent average percentages of CD4⁺CD25⁺ on T cells of healthy donors (n = 5) and acute and chronic ATL patients (n = 8) after 6-day ex vivo culture. Error bars represent mean ± SEM. Student t test was used to determine statistical differences. ****P < .0001. (C) Western blot analysis of c-MYC after 6-day ex vivo culture treated with 1 μM of I-BET762, copanlisib, and bardoxolone methyl or combination for 2 hours (n = 8). (D) Quantitative real-time PCR analysis of TSCD22D3 (GILZ) and TNFRSF8 (CD30) transcripts in PBMC cultures of patients treated with DMSO or 1 μM of the triple combination for 4 hours (n = 5). (E) Cell viability of the PBMCs from an acute ATL patient and (F) CD4⁺ T cells isolated from a healthy donor, cultured with I-BET762 (250 nM), copanlisib (125 nM), and bardoxolone methyl (62.5 nM), n = 3. One-way ANOVA or Student t test was used to determine statistical differences. *P < .05, **P < .01, ****P < .0001.