DZNep increases apoptosis in alloreactive CD8⁺ T cells in vivo. Donor C3H.SW mouse-derived TCD BM + CD44^loCD8⁺ T_N cells (Ly9.1) were transplanted into lethally irradiated B6 mice (Ly9.2). DZNep was administered subcutaneously to these recipients from day 3 to day 13, with vehicle treatment as control. (A-B) Graphs show (mean ± SD) the absolute number of donor-derived CD8⁺ T cells in spleen, lymph node, and liver on day 8 (n = 3 for each group) and day 14 (n = 5 for each group) after transplantation (A) and the percentage and absolute number of donor-derived CD25⁺CD8⁺ cells (B). (C) Dot plots and graphs show (mean ± SD) the fraction of annexin V–positive donor-derived CD8⁺ T cells isolated from B6 recipients on day 14 after transplantation (n = 5 for each group). (D) Donor CD8⁺ T cells were isolated from control recipients of T cells at day 14 after transplantation and cultured with IL-2 (5 ng/mL), with or without addition of DZNep (200nM) for 24 hours. Graphs (mean ± SD) show the ratio of dead cells versus live cells. (E) Donor T cells were isolated from the spleen at day 14 for CTL assay against MBL-2 cells. (F) In vivo CTL assay against infused host and donor-type B220 cells. B6 allogeneic target splenocytes were labeled with 3.0μM CFSE (CFSE^hi). Control targets were C3H.SW splenocytes labeled at a low CFSE concentration (0.3μM; CFSE^lo). On day 21, a 1:1 mixture of CFSE-labeled B6 and C3H.SW splenocytes (10⁷) were infused into B6 recipients of TCD BM or B6 recipients of TCD BM and CD8⁺ T cells, which had been treated with or without DZNep from day 3 to day 17. Spleens were harvested 18 hours later to assess killing of B6-derived CFSE^hi-target B cells. CD8⁺ T_N cells were cultured as controls. Representative results from 2 separate experiments are shown.