As shown in Fig. 3A, the CD4+ TCR clonal deletion were found, particularly on Vβ2, 7, 8.1/2, and 8.3 after DN Treg-cell transfer (Fig. 3A). Similarly, CD8+ TCR Vβ2, 7, 8.1/2, and 8.3 were significantly reduced after DN Treg-cell treatment (Fig. 3B). Taken together, these data indicate that adoptive transfer of DN Treg cells induces recipient T-cell selective clonal deletion
in both CD4+ and CD8+ T cells. To further study if clonal deletions in CD4+ and CD8+ T cells hamper their antidonor responses, total lymphocytes (5 × 104/well) from treated BALB/c mice were used for T-cell proliferation assay and donor-type C57BL/6 spleen cells (5 × 105/well) were used as stimulators. As shown in Fig. 3C, T-cell proliferation in DN Treg cells-treated Fulvestrant research buy mice was significantly reduced compared with PBS-treated mice (mean ± SD =4,836 ± 2,686 cpm versus 23,907 ± 7,077 cpm, p < 0.01). Whereas, T-cell proliferation to the third-party control C3H spleen cells remained at a similar level as PBS-treated
group (Fig. 3C), indicating that DN Treg-cell transfer induced C57BL/6 antigen-specific www.selleckchem.com/products/azd5363.html TCR Vβ deletion and alloimmunity to other antigens still remains. Next, we further studied the mechanism of DN Treg cell-mediated T-cell deletion. DN Treg cells were purified from FasL null (gld), Fas null (lpr), and perforin null (perforin−/−) mice and were used for adoptive transfer before BM transplantation. As showed in Fig. 3C, T-cell proliferation was reduced in mice treated with perforin null DN Treg cells but not in those treated with other DN Treg cells, indicating a perforin-dependent mechanism for
DN Treg cell-mediated T-cell clonal deletion. Besides T cells, NK cells play an important role in BM graft rejection [[20-23, 31]]. We therefore examined the Selleck Ponatinib effect of adoptive transfer of DN Treg cells on recipient NK-cell function. To focus on NK cells and eliminate T cell-mediated rejection, CD4+ T cells and CD8+ T cells in all BALB/c mice were depleted by i.p. injection of CD4 depletion antibody (GK1.5) and CD8 depletion antibody (YTS169.4) on day −4 and −1. Efficiency of depletion (>98%) were confirmed in blood by flow cytometry before DN Treg-cell transfer (Fig. 4A). In a control group, NK cells were depleted by anti-Asialo GM1 on day −4 and −1 before BM transplantation and the depletion was confirmed by anti-CD3 and anti-CD49b staining (Fig. 4B). Recipient BALB/c mice received DN Treg cells on day 0, and immunosuppressive treatment on day 0 and 3 as described in Fig. 1. On day 6, BALB/c BM cells (recipient strain, 107, labeled with CFSE and Far-red) together with C57BL/6 BM cells (donor strain, 107, labeled with CFSE alone) were i.v. injected to BALB/c mice and spleen cells were analyzed 2 days after. As shown in Fig. 4C, most of the donor C57BL/6-derived cells were rejected in PBS-treated mice (killing rate mean ± SD = 95.
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