Cilengitide treatment improves survival after allo-HCT without affecting Tc proliferation. (A-C), (E-I) Allo-HCT was performed as described in the “Methods” section. (A) Distribution of donor-derived Tc^luc’s is displayed in representative BLI images on days 7 and 21 after allo-HCT with coadministration of cilengitide or PBS as control (left panel) and quantified over time measuring the emitted photons from expanding Tc^luc’s (n = 3 per group; right panel). Cilengitide administration had no significant impact on the expansion of Tc^luc’s in comparison with the PBS treatment. The experiment was performed 3 times with at least 3 animals per group and similar results. (B) Splenocytes were isolated 24 hours after allo-HCT from BALB/c recipients and analyzed for the percentage of proliferated CFSE⁺/H-2K^b+/CD3⁺ donor Tc’s. Mean value for the amount of expanded Tc’s ± SD (left panel) and a representative histogram depicting CFSE dilution for each group is shown (right panel). The experiment was performed twice with at least 3 animals per group. (C) Survival of animals receiving BM alone (⁠⁠), BM/Tc^luc+PBS (⁠⁠), or BM/Tc^luc+cilengitide (⁠⁠) (n = 12 in each group). Survival is improved in the cilengitide group as compared with the PBS-treated group (P < .0001). (D) CD4⁺/CD8⁺ Tc’s were exposed to cilengitide or camptothecin (10 µM) for 6 hours at the indicated concentrations, and viability was measured by flow cytometry for PI/annexin V. Annexin V and PI were used to differentiate between viable, necrotic, and apoptotic cells as follows: PI⁺/annexin V^– (necrotic cells), PI⁺/annexin V⁺ and PI^–/annexin V⁺ (apoptotic cells), and PI^–/annexin V^– (viable cells). In all, 100 000 Tc’s were analyzed for each sample, shown as absolute numbers ± SD. (E) Splenocytes from BALB/c recipients were isolated on day 14 after allo-HCT and stained for CD4, CD44, and CD62L. The quantification of T_N, T_EM, and T_CM among CD4⁺ Tc’s is shown as a percentage ± SD from 1 representative of 2 independent experiments with at least 4 animals per group and comparable results. (F) Splenocytes from BALB/c recipients were isolated on day 14 after allo-HCT and stained for CD4, IL-4, IFN-γ, and IL-17A. The quantification of IL-4, IFN-γ, and IL-17A among the CD4⁺ Tc’s population is shown as a percentage ± SD. The experiment was performed twice each time with 5 animals per group and comparable results. (G) Histopathology of the intestines and liver was quantified as indicated in the “Methods” section. The mean score ± SD for inflammation (upper panel) and apoptosis (lower panel) is shown for the liver and small and large intestines separately (n = 5 in each group) from 1 of 2 independent experiments, each performed with at least 3 animals per group. (H) Animals were euthanized on day 7 after allo-HCT, and serum was isolated from the indicated groups to measure TNF. The mean value ± SD is shown representing 3 individual animals per group. The experiment was performed once with 3 animals per group. (I) Mice were euthanized on day 30 after allo-HCT, and donor engraftment was analyzed by flow cytometry for the indicated immune cell types. The mean value ± SD is shown for 1 representative of 2 independent experiments (n = 6 per group).