Interestingly, only CY but not other drugs, in combination with DN Treg-cell transfer, helped the survival of BM cell
in the recipients (Fig. 1). It still remains elusive why, other than rapamycin, FK506 or CyA, only CY treatment could help the induction-mixed chimerism even though they all preferentially target-activated cells. CY, predominantly toxic to proliferating cells, has SB431542 been shown to have a great advantage in prolonging heart graft survival but not in achieving tolerance in fully MHC-mismatched transplantation. Unfortunately, prolonged treatment with this drug elicits severe side effects in patients. A comprehensive approach is to reduce the use of immunosuppressive drugs by combining them with another treatment. Indeed, using CY one or two times along with donor-specific transfusion Selleck BKM120 (DST) helps BM transplantation and promotes mixed chimerism [[42-44]]. However, fetal GVHD developed in these mice. Although the pathophysiology detail of GVHD remains elusive, donor CD4+ and CD8+ T cells have been held critically responsible. In our protocol, donor CD4+ and CD8+ T-cells transplantation developed GVHD and mortality (Fig. 2). In contrast, donor DN-T
cell transfer groups survived more than 100 days with no pathological evidence of GVHD (Fig. 2). Moreover, previous studies indicated that DN Treg cells could suppress T cell-mediated GVHD [[27, 45]]. More importantly, the benefits of DN Treg cells in GVHD are supported in a clinical study. All patients who demonstrated more than 1% DN Treg cells did not develop GVHD after
hematopoietic stem cell transplantation [], which hints on the role of DN Treg cells in suppressing GVHD. Hence, the results that DN Treg cells can suppress GVHD give a VAV2 strong rationale for its clinical application in BM transplantation. General immunosuppression can control T cells but hamper antitumor and infection in patients. Reducing the clonal size of donor-reactive T cells has been recognized as a prerequisite for inducing tolerance in transplantation [[47, 48]]. Clonal deletion of donor reactive T cells permits donor graft survival while keep antitumor and antiinfection immunity in recipients. It has been shown that the DST combined with anti-CD154 blocking antibody can induce clonal T-cell exhaustion [[49, 50]]. Previous studies have shown that clonal deletion of developing T cells was correlated with the induction of mixed chimerism [[43, 44, 51]]. It was reported a high frequency of DN-T cells bearing autoreactive TCR that caused deletion of CD4+ or CD8+ T cells []. In this study, after adoptive transfer of donor DN Treg cells, the recipient T-cell proliferation was significantly inhibited (Fig. 3C). The percentages of several major TCR subtypes in recipients were reduced in CD4+ and CD8+ T cells (Fig. 3A and B), implying that these TCRs could be the major responsive subtypes in rejecting allografts.