Fig. 8: Sensitivity of unstimulated vs. stimulated PBLCs to CDT, MAF, and TMZ.

a CDT treatment induced significantly more cell death in stimulated than unstimulated PBLCs after 24 h. b CDTm, which is mutated in the enzymatic active CDT B-unit, induced neither cell death induction in unstimulated nor stimulated PBLCs. (a, b, n = 3–5, mean value, SD, t-test **p < 0.01, ***p < 0.001). c Representative pictures of γH2AX (green) staining in unstimulated and stimulated CD3 (red) T cells within PBLC. CDT induced massive DNA damage in stimulated compared with unstimulated cells (quantification of γH2AX intensity is shown in Fig. S11a). d Mafosfamide treatment induced after 24 h significantly more cell death (apoptosis and clearly necrosis) in unstimulated than stimulated PBLCs (n = 3, mean value, SD, t-test, *p < 0.05, **p < 0.01, ***p < 0.001). e Without MGMT inhibition, TMZ did not induce significant cell death ( < 10 %) in PBLCs. Apoptosis and necrosis were measured after 72 h. f MGMT depletion by pre-treatment with O⁶BG increased significantly TMZ-induced cell death in stimulated cells, already at very low doses of 6.25 and 25 µM TMZ (n = 4, mean value, SD, t-test, *p < 0.05, **p < 0.01). g The sensitizing effect of CD3/CD28 stimulation towards TMZ was also obvious in magnetic bead-isolated and MGMT depleted Treg, Th, and CTL (cell death was measured 72 h after TMZ treatment, n = 5, mean value, SD, t-test, **p < 0.01, ***p < 0.001). h Apoptosis was measured in unstimulated and stimulated Th by SubG1 quantification