Fig. 1
From: Ex vivo Akt inhibition promotes the generation of potent CD19CAR T cells for adoptive immunotherapy
Akt inhibition does not compromise CD19CAR T cell proliferation and expansion. CD8+ T cells in PBMC from healthy donors were isolated by CD4+ T cell depletion following anti-CD4 microbeads labeling and magnetic selection with the EasySep system. The freshly isolated CD4 negative cells were activated with CD3/CD28 Dynabeads and transduced the next day with CD19R (EQ):CD28:ζ/EGFRt lentivirus at an MOI of 3, then expanded. The cultures were supplemented with 50 U/mL rhIL-2 and 1 μM Akt inhibitor every 48 h for 17–21 days before in vitro analysis. Transduced CD19CAR T cells without Akt inhibitor treatment were used as control. a Expanded CD8 + CD19CAR T cells were harvested and stained intracellularly with antibodies against phosphorylated Akt pS473 and total Akt. Fluorescence minus one (FMO) are depicted in red histograms. Histograms are of gated CD19CAR+ T cells. b Percentages of phosphorylated Akt positive cells from four different donors are presented. c Expanded CD8 + CD19 CAR T cells were labeled with 0.5 mM CFSE and then co-cultured with CD19+ LCLs as stimulators for 5 days in the presence of 50U/L rhIL-2. CFSE dilution of gated CD3+ and CAR+ double positive populations was determined using multicolor flow cytometry. Percentages of CFSE negative cells over non-stimulated cells are presented. d Percentages of CFSE negative CAR T cells derived from different T cell subsets are depicted. e CD8+ T cells were transduced with lentivirus encoding CD19CAR vector and expanded in a medium containing 50U/L rhIL2, in the presence and absence of 1 μM Akt inhibitor. Viable cell numbers were determined by Guava ViaCount at the indicated time points. f Data on day 17 from four different donors are presented. g Bulk T cells, purified TCM and purified naïve/memory T cells were transduced with lentivirus encoding second generation CD19CAR vector and expanded in a medium containing 50U/L rhIL2, in the presence and absence of 1 μM Akt inhibitor. Viable cell numbers were determined by Guava ViaCount at the indicated time points. Mock T cells from the same donor were used as controls