Figure 4.

Pre-treatment of leukemia cells with AZA potentiates CAR T cells in co-cultures. (A and B) Nalm-6 cells were primed with AZA (0.4 μM, 24 hrs) or remained untreated. Equal numbers of viable cells were then co-cultured with CAR T cells for another 24 hrs at (E) T = 1:1, 1:10, 1:20. Proportions of CD3⁺ CD10⁺ T cell/leukemia-cell conjugates were determined as number of events in Q2/Q1 + Q2. (C) single CD10⁺ target cell numbers were determined in Q1. (D) Proportions of apoptotic 7AAD⁺ target cells (P4) were analyzed in CD3⁺ gate. (E) CFSE-labeled CAR T cells cultured alone (in the presence of IL-2 only) or co-cultured with Nalm-6 cells for 48 hrs at (E) T=1:1 exhibit dye dilution during 48 hr culture with each peak representing one division. It is relevant that all CFSE⁺ T cells analyzed prior to culture were located in P6 (data not shown). Rapidly dividing CFSE^dim T cells that underwent 4 divisions were determined in P10 gate. Each condition was done in duplicate, with 1 representative histogram for each condition shown. (F) CD3⁺ T cell numbers per culture. (G) PD-1 expression in CAR T cells before and after 24-hour co-culture with Nalm-6 cells analyzed in CD3⁺ T cell gates. One representative flow cytometry analysis is shown for each condition. (H) hPD-L1/hPD-L2 expression in Nalm-6 cells.