Anti-CD20 cytotoxicity after single-agent or combination pretreatment. (A) Relative viability of L1236 cells after exposure to ATO at the indicated concentrations for 48 hours, normalized to the untreated control (UT). (B) As in panel A, but after ATRA. (C) Relative viability of L1236 cells pretreated with 10 µM ATO, 40 µM ATRA, or dimethyl sulfoxide (UT), subsequently exposed to CD20 antibodies rituximab (20 µg/mL) or tositumomab (10 µg/mL) or no antibody for 3 days. Percentages reflect normalization to the no-treatment setting. (D) CD20 expression after single-agent and combination treatments (as indicated) in L1236 cells, at the transcript level (top; relative values by RQ-PCR, normalized to the untreated control [UT]), and at the protein level (bottom; by immunoblot analysis, with α-tubulin as a loading control). ATRA, 20 μM; ATO, 5 μM; compound 40, 10 μM. (E) CD20 surface expression by immunofluorescence image-based flow cytometry (bottom) and matching brightfield pictures (top) in untreated (UT) SU-DHL4 B-NHL cells and L1236 cells exposed to ATO/compound 40 as in panel D (ATO/40), or untreated (UT); shown are 2 representative examples each. Of note, the L1236 signals are displayed with a fourfold amplification compared with the SU-DHL4 signals. (F) CD20 surface expression by conventional flow cytometry in L1236 cells as in panel E. (G) Relative viability of L1236 cells exposed to the CD20 antibodies rituximab or tositumomab (as in panel C) after the indicated pretreatments (as in panel D, ie, ATO at only 5 µM). (H) Rituximab-mediated ADCC values in L1236 (left) or L428 cHL cells (right) preexposed to the indicated drugs or combinations (as in panel G), 6 hours after the addition of the NFAT-luciferase–engineered effector T cells, relative to no preexposure (UT). Data are presented as mean ± SD; *P < .05.