Fig. 9: AZA pre-treatment of the leukemia cells promotes intracellular retention of CTLA-4 while enhancing Lck and ZAP70 signaling in the anti-CD123 CAR T cells.

a Scatter plot showing the extracellular expression levels of CTLA-4 in CD4⁺ anti-CD123 CAR or NTD Tc following a 96 h co-culture in the presence of media only (n = 4), naive MOLM-13 cells (n = 4), or MOLM-13 cells pre-treated with 1 μM AZA (n = 4). Data were pooled from four independent experiments. b Representative flow cytometry plot depicting intracellular levels of CTLA-4 in CD4⁺ anti-CD123 CAR or NTD Tc. c Scatter plot showing the intracellular expression levels of CTLA-4 in CD4⁺ anti-CD123 CAR or NTD Tc following a 96 h co-culture in the presence of media only (n = 4), naive MOLM-13 cells (n = 4), or MOLM-13 cells pre-treated with 1 μM AZA (n = 4). Since raw intracellular expression levels could include surface levels of CTLA-4, extracellular and intracellular staining from the same sample were set up side by side and analyzed. The scatter plot depicts the difference between intracellular levels with extracellular levels to give the “true” intracellular expression of CTLA-4 in the cells. Data were pooled from four independent experiments. d Representative flow cytometry plot depicting the phosphorylated level of Lck (pLck) in CD4⁺ anti-CD123 CAR T cells that have been exposed for 96 h to media only, untreated MOLM-13 cells, or MOLM-13 cells pre-treated with 1 μM AZA. e Representative flow cytometry plot depicting the phosphorylated level of Zap70 (pZap70) in CD4⁺ anti-CD123 CAR T cells that have been exposed for 96 h to media only, untreated MOLM-13 cells, or MOLM-13 cells pre-treated with 1μM AZA. f Scatter plot depicting the fold change in percentage expression of pLck in CD4⁺ anti-CD123 CAR Tc (media only n = 4; untreated MOLM-13 n = 4; AZA-treated MOLM-13 n = 4). Data were pooled from four independent experiments. g Scatter plot depicting the fold change in percentage expression of pZap70 in CD4⁺ anti-CD123 CAR Tc (media only n = 4; untreated MOLM-13 n = 4; AZA-treated MOLM-13 n = 4). Data were pooled from four independent experiments and represented as mean ± SEM. p-values were calculated using two-sided one-way ANOVA (Kruskal–Wallis with Dunn’s multiple comparisons).