Cilengitide administration allows for CD8-mediated cytotoxicity. Allo-HCT was performed as described. (A) CD4⁺/CD8⁺ Tc’s were isolated from BALB/c recipients on day 14 after allo-HCT and cilengitide or PBS treatment and used as effector cells against L1210 cells in the indicated ratios of target:effector. Values are the percentage of annexin V⁺/PI⁺ cells ± SD from 1 representative experiment. Experiments were repeated 3 times in triplicates with similar results. (B-C) Splenocytes were isolated on day 14 after allo-HCT from BALB/c recipients and stained for CD8 and CD107a. (B) Quantification of CD107a⁺ population as percentage ± SD of all CD8⁺ Tc’s. One representative of 2 independent experiments is shown, each performed with at least 4 animals per group. (C) Representative flow cytometry data for the indicated groups described in (B) are shown. (D) Expansion and rejection of A20^luc cells in BALB/c recipients receiving BM alone, with Tc’s and PBS or cilengitide. Days 0, 6, and 13 as representative time points are shown (left panel). Photons emitted from the A20^luc cells in vivo are shown over time for the indicated groups (n = 3 per group). The experiment was performed 3 times with at least 3 animals per group and showed similar results (right panel). (E) Mice from the indicated groups were euthanized on day 13 after allo-HCT. LN (axillary, mesenteric, inguinal) and BM from the femur was isolated and analyzed for the presence of B220^high A20^luc cells by flow cytometry. The percentage (mean ± SD) of B220^high A20^luc cells in the BM (left) and LN (right panel) is shown. The experiment was performed once with at least 3 mice in each group. (F) Survival of BALB/c recipients after allo-HCT receiving additional A20^luc and PBS or cilengitide. Survival is improved in the BM/A20^luc+Tc+cilengitide-treated group (⁠⁠) as compared with BM/A20^luc+Tc+PBS (⁠⁠; P = .005), BM/A20^luc+cilengitide (⁠⁠; P = .025), and BM/A20^luc alone (⁠⁠; P = .003). The experiment was performed twice, and the number of mice per group is indicated in the survival graph.