APG101 treatment did not affect donor engraftment, T-cell phenotype, function, or homing in allogeneic BM-transplanted animals. (A-E) Lethally irradiated B6D2F1 recipient mice (H-2^bxd, CD45.2⁺) were reconstituted with BM from B6 mice (H-2^b, CD45.2⁺) plus B6.SJL-derived spleen cells (H-2^b, CD45.1⁺) and were treated with PBS or APG101 starting 1 day before transplantation. Fourteen days after transplantation mice were killed and spleen cells were analyzed for total cell count and the numbers of CD45.1⁺ T, NK, NK-T cells, and T_reg (A) and T-cell phenotype (B) by flow cytometry. To measure the proliferative capacity and cytotoxicity of allogeneic T cells, CD45.1⁺ T cells were purified from spleens of PBS and APG101-treated mice 14 days after transplantation. CFSE-labeled T cells were stimulated in vitro with anti-CD3 and CD28 antibodies, irradiated DBA/2 (H-2^d) spleen cells (alloantigen) or C3H (H-2^k) spleen cells (third party). Proliferation of CD3/CD28-activated CD4⁺ and CD8⁺ CD45.1⁺ T cells was measured after 4 days, whereas allogeneic and third-party proliferation was determined after 6 days (C). Cytotoxic specificity of splenic CD45.1⁺ T cells was analyzed on P815 and EL4 cells in chromium release assays. Addition of CMA (10nM) or APG101 (100 ng/mL) during the assay indicates the cytotoxic effector mechanisms used (D). Different days after transplantation, spleen, and liver of transplanted mice were analyzed for the percentage of infiltrating CD45.1⁺ T cells by flow cytometry (E). Data represent the mean value ± SD of 6 mice analyzed in panel A, of triplicates from CD45.1⁺ T cells, which were re-isolated and pooled from spleens of 30 mice treated in panel C, of triplicates from spleen cells, which were pooled from 30 mice treated and adjusted for an effector-target ration of CD45.1⁺ T cells:target cells as described in M+M in panel D, and the mean values ± SD of 3 mice analyzed in panel E. (B) is representative for 1 mouse of 6 analyzed. All data are representative for 1 experiment of at least 2 experiments performed. No statistical significant differences between APG101 or PBS-treated animals were detected in all experiments performed.