ATRA increases FRβ expression and m909 CAR T-cell recognition of AML cell lines. (A) AML cell lines were treated with (dotted black line) and without (solid black line) 10 nM ATRA for 5 days (d1-d5). Surface FRβ expression was determined each day by flow cytometry with m909-IgG (black) or human IgG isotype control (gray). (B) FRβ mRNA expression was determined before (untreated) and after 3 days and 5 days of 10 nM ATRA treatment. Relative mRNA is shown compared to untreated HL60. Bars represent mean ± SEM of n = 5 replicate wells. P values were calculated for each cell line compared to untreated controls. (C) m909 CAR T cells secrete higher IFN-γ (IFNg) in response to THP1 cells pretreated for 5 days with 10 nM ATRA (gray bars) compared to untreated cells (black bars) in overnight cocultures. (D) THP1-fLuc cells were pretreated with (THP1-ATRA) or without (THP1) 10 nM ATRA for 5 days before coculture with m909 CAR or GFP control T cells at an E:T ratio of 1:1. Percent lysis was determined by residual luciferase activity after overnight coculture. (E) m909 CAR T cells secrete higher IFN-γ after 3 days of coculture in the presence of 10 nM ATRA (gray bars) and AML target cell lines compared to cultures without ATRA (black bars). No significant differences in IFN-γ secretion were observed for m909 T cells activated in the presence of C30-FRβ with or without ATRA. In panels C-E, graphs represent mean ± SEM from n = 3 independent experiments using 3 distinct T-cell donors. P values were calculated for each T-cell subset to compare between untreated and ATRA-treated cell lines. *P < .05; **P < .01; ***P < .001. ns, P > .05.