Combination of DC/AML fusion vaccine with SAR leads to induction of tumor-specific T-cell immunity ex vivo. (A) Fusion efficiency was established using immunohistochemical staining, AML cells stained with CD123 (left), DCs stained with CD86 (middle), and DC/AML fusion cells stained with both CD123 and CD86 (right). (B) T-cell activation was analyzed by flow cytometry at baseline (day 0), and after in vitro fusion veTcs vs uTcs (day 5). Cells were stained with the activation markers CD25 APC, CD69 PE, 41bb APC, and PD1 BV421. (C) 41bb expression by flow cytometry; in vitro coculture of veTcs with autologous tumor cells led to further antigen-specific activation in the presence of SAR in both CD4 and CD8 subsets vs the control engager. (D) The capacity of veTcs to target autologous leukemia cells in the presence of TCE in vitro was assessed by flow cytometry. Dying target cells are identified by cells that dually stain for tumor labeled with TFL4 (APC channel) and cleaved granzyme B substrate (FITC channel). Mean of 3 separate assays is shown, pairwise comparisons all not significant. (E) Detection of intracellular IFN-γ of CD4 and CD8 by flow cytometry after coculture of veTcs with autologous tumor cells and the addition of SAR (30 ng). Mean with standard error of the mean (SEM) for 3 separate assays is shown. (F) Representative flow plots of intracellular IFN-γ for CD8 and CD4 T cells. Statistical analysis was performed by 1-way analysis of variance using GraphPad. ∗P ≤ .05; ∗∗P ≤ .01. PD1, programmed cell death protein 1.