AML cells pretreated with GSK621 induce a vaccination effect in vivo. (A) Schema of the vaccination assay performed with C1498 cells or MLL-AF9 cells in immunocompetent C57BL/6 mice. (B) Representative interferon gamma enzyme-linked immunospot (ELISpot) assay using single-cell suspensions from the spleens of 1 animal of each arm of the experiment activated by culture with the parental AML cells. Splenocytes were cocultured with a syngeneic C57BL/6 melanoma cell line (B16F10) or the ovalbumin antigen alone (OVA) as negative controls, or anti-CD3 as a nonspecific positive control. Each column represents ELISpots from splenocytes of 1 spleen seeded in triplicates. (C) Kaplan-Meier curves showing the overall survival in each group (5 mice per group) vaccinated with MLL-AF9 cells pretreated as indicated and fixed before injection, then challenged with live MLL-AF9 cells. Log-rank test. (D) Tumor growth on the challenge site of individual animals in panel C. Of note, none of the mice vaccinated with GSK621-treated cells developed tumors (red line on x-axis). (E) Kaplan-Meier curves showing the overall survival in each group (5 mice per group) vaccinated with MLL-AF9 cells pretreated with GSK621 before injection, then challenged with live MLL-AF9 cells. One group was depleted of CD4/CD8 T cells with intraperitoneal injections of neutralizing antibodies starting 2 days before the challenge (dashed line). The mice of the other group received a control IgG (solid line). Log-rank test. (F) Tumor growth on the challenge site of individual animals in panel E.