Fig. 6: Human reactivity to APOBEC3B-modified tumors.

a CD3+ T cells from healthy donor PBMCs were isolated and activated with CD3/CD28 beads. These T cells were cocultured with Mel888 cells previously transduced by lentivirus expressing GFP or APOBEC3B and pretreated for 12 h with human interferon gamma (hIFNγ). After 10 days of co-incubation, CD3+ T cells were isolated, stained with cell trace violet, and replated with hIFNγ pretreated Mel888 parental cells. After 3 days, supernatant was collected for hIFNγ ELISA, T cells underwent flow cytometric analysis for proliferation by cell trace violet dilution, and Mel888 cells were counted to assess target killing. Representative of three separate experiments. b hIFNγ ELISA, T-cell proliferation, and target killing from T cells cocultured with autologous Mel888 cells for both education and restimulation. Error bars indicate mean and SD. c Prior to coculture, CD14+ cells were isolated from healthy donor PBMCs and matured into monocyte-derived dendritic cells. CD3+ T cells were isolated from the same donor PBMCs and activated with CD3/CD28 beads. These T cells were cocultured with the mature dendritic cells and pulsed with pediatric glioma (SJPDGF1) or Mel888 lysate previously transduced by lentivirus expressing GFP or APOBEC3B. Lysates were added again on days 2 and 3 of coculture. Seven days later, CD3+ T cells were isolated, and cocultured with fresh monocyte-derived dendritic cells pulsed with parental SJPDGF1 lysate. Three days later, supernatant was collected for hIFNγ ELISA. d hIFNγ ELISA from vaccination using Mel888 or SJPDGF1 lysate for education and SJPDGF1 lysate for restimulation. All cocultures were performed at a 10:1 ratio of targets:effectors. Student’s t test was performed followed by Holm–Bonferroni correction for multiple comparisons. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.