Fig. 8: Recall immunity and long-term leukemia control.
From: Demethylating therapy increases anti-CD123 CAR T cell cytotoxicity against acute myeloid leukemia
a Schematic diagram depicting the isolation of residual anti-CD123 CAR T cells from the BM or mice 28 days post inoculation for further in vitro and in vivo analysis. b Kaplan–Meier analysis of percentage survival for each treatment group PBS (n = 6; CTLA-4negative anti-CD123 CAR Tc n = 9; CTLA-4positive anti-CD123 CAR Tc n = 9). Secondary recipient mice were engrafted with 0.5 ×106 MOLM-13Luc cells on day 0. On day 7, mice were randomly distributed and received either 100 μL PBS, 2.5 ×106 CTLA-4positive or CTLA-4negative anti-CD123 CAR Tc isolated from primary engrafted mice. Attrition of mice: as described above. c Specific cytotoxicity of MOLM-13 cells following co-culture with NTD or anti-CD123 CTLA-4negative (n = 3) or CTLA-4positive CAR Tc (n = 3). d Specific cytotoxicity of anti-CD123 CTLA-4positive (n = 3) and CTLA-4negative CAR Tc (n = 3) against 3 independent primary patient AML PB PBMCs with varying CD123+ expression following a 16 h co-incubation. For c and d, data represent three independent experiments with a fixed number of target cells/well for all E:T ratios. In both cases, counting beads were used to quantify the absolute number of residual live target cells. Residual live target cells were CellTrace violet+ 7-AAD−. e Scatter plot showing the percentage quantification of TNFα from live CTLA-4negative or CTLA-4positive CD3+ anti-CD123 CAR Tc following a 24 h co-culture with media only (n = 4) or MOLM-13 cells (n = 4). f Representative flow cytometry plot from each condition from e is shown. g Representative histograms of 3 independent experiments depicting the proliferation of CTLA-4positive (left panel) and CTLA-4negative (right panel) CD123 CAR T cells or NTD T cells as examined by CellTrace violet dye dilution. Cells were co-cultured for 96 h with MOLM-13 cells at an E:T ratio of 1:1. Each dotted line represents one cell division. All graphed data are represented as mean ± SEM. p-values were calculated using b two-sided Mantel–Cox test (log-rank) or two-sided unpaired student’s t-test (Mann–Whitney) (e).
