Double negative Treg cells promote nonmyeloablative bone marrow chimerism by inducing T‐cell clonal deletion and suppressing NK cell function

Abstract

The establishment of immune tolerance and prevention of chronic rejection remain major goals in clinical transplantation. In bone marrow (BM) transplantation, T cells and NK cells play important roles for graft rejection. In addition, graft-versus-host-disease (GVHD) remains a major obstacle for BM transplantation. In this study, we aimed to establish mixed chimerism in an irradiation-free condition. Our data indicate that adoptive transfer of donor-derived T-cell receptor (TCR) αβ IntroductionInjection of donor bone marrow (BM) was first reported to induce skin allograft tolerance by establishing chimerism in neonatal hosts [1]. Thereafter, induction of mixed chimerism by BM transplantation has been considered promising among the numerous methods developed for tolerance induction in transplantation.Correspondence: Dr. Zhu-Xu Zhang e-mail: zhuxu.zhang@lhsc.on.caMixed chimerism refers to a state in which allogeneic hematopoietic cells coexist with recipient cells, resulting in a state of tolerance toward both the donor and the host, thus avoiding chronic rejection and side effects of any drug treatments in transplantation [2]. Although mixed chimerism has produced clinical benefits in transplantation [3,4], sustained chimerism in patients and large animal models has not yet been achieved. In addition, GVHD is still a major obstacle in BM transplantation. Obviously, this approach needs further improvement to be practical in the clinic. Results Adoptive transfer of DN Treg cells facilitates allogeneic-mixed chimerism in BM transplantationTo develop a suitable clinical method, we tried to establish mixed chimerism with an irradiation-free protocol by transferring DN Treg cells and using clinically available immune suppressive drugs. Cyclophosphamide (CY), cyclosporine A (CyA), FK506, and rapamycin (RAPA) were tested in this study. Recipient BALB/c mice were treated with the immunosuppressive agents before and after BM transplantation. CY: 200 mg/kg on day 0 and 100 mg/kg on day 3; CyA: 15 mg/kg from day 0 to 9; FK506: 16 mg/kg from day 0 to 9; RAPA: 2 mg/kg from day 0 to 9; phosphate-buffered saline (PBS): 0.3 mL/mouse from day 0 to 9. DN Treg cells were purified from C57BL/6 mice and were activated by platecoated anti-CD3 in presence of IL-2. The purity was confirmed by anti-CD3, CD4, CD8, TCRγδ, and NK1.1 (Fig. 1A). Next, we studied whether mixed chimerism would lead to graft tolerance. Skin grafts from BM donor strain C57BL/6 mice were transplanted onto chimeric BALB/c mice and control mice 30 days after BM transplantation. The control mice were treated with BM and CY only. Donor skin grafts survived longer than 100 days in chimeric mice but were rejected shortly in control CY-treated mice (mean ± SD = 12 ± 3 days, Fig. 1D). Skin grafts from third-party control C3H (H-2k) mice were used to determined if chimeric mice corroborate donor-specific tolerance. Skin grafts from C3H mice were rejected shortly in chimeric mice (Fig. 1D, mean ± SD = 11 ± 2 days), suggesting that antigen-specific tolerance was established ...

The impact of donor characteristics on the immune cell composition of mixture allografts of granulocyte–colony‐stimulating factor–mobilized marrow harvests and peripheral blood harvests

“…Regarding CD3+CD4–CD8– regulatory T cells, we found that a successful expansion of CD3+CD4–CD8– Tregs at 60 days after allo‐HSCT might help avoid severe manifestations of acute GVHD after unmanipulated HBMT . In a mouse model, CD3+CD4–CD8– T cells were identified as potential regulatory cells that could alleviate GVHD and promote nonmyeloablative marrow chimerism . Therefore, the higher dose of CD3+CD4–CD8– T cells in allografts of younger donors may partly account for the lower incidence of GVHD as we previously described .…”

Immunoregulatory CD4-CD8- T cells as a potential therapeutic tool for transplantation, autoimmunity, and cancer

“…In stark contrast to CD4 + and CD8 + T cells, the co-injection of non-transgenic DN T cells with T cell-depleted BM into a sublethally irradiated host ensured prolonged recipient survival in the absence of clinical signs of GVHD (He et al, 2007; Su et al, 2012; Figure 2B ), indicating that DN T cells are not alloreactive nor pathogenic in this context. Moreover, this led to stable mixed chimerism and, as opposed to BM transplantation alone, promoted donor allotolerance (He et al, 2007; Su et al, 2012; Figure 2B ). Together, these findings demonstrate that, as opposed to CD4 + or CD8 + T cells, the transfer of allogeneic DN T cells does not cause GVHD.…”

“…The tolerance of donor spleen cells may be explained, at least in part, by the accumulation of 2C TCR DN T cells, which can effectively inhibit pathogenic CD8 + T cell responses, in recipient mice (Young et al, 2003a). Subsequently, the role of non-transgenic DN T cells in both parent to F1 and fully MHC-mismatched bone marrow (BM) transplantation following either a myeloablative (He et al, 2007) or non-myeloablative regimen (Su et al, 2012) was investigated ( Figure 2B ). In stark contrast to CD4 + and CD8 + T cells, the co-injection of non-transgenic DN T cells with T cell-depleted BM into a sublethally irradiated host ensured prolonged recipient survival in the absence of clinical signs of GVHD (He et al, 2007; Su et al, 2012; Figure 2B ), indicating that DN T cells are not alloreactive nor pathogenic in this context.…”

“…Subsequently, the role of non-transgenic DN T cells in both parent to F1 and fully MHC-mismatched bone marrow (BM) transplantation following either a myeloablative (He et al, 2007) or non-myeloablative regimen (Su et al, 2012) was investigated ( Figure 2B ). In stark contrast to CD4 + and CD8 + T cells, the co-injection of non-transgenic DN T cells with T cell-depleted BM into a sublethally irradiated host ensured prolonged recipient survival in the absence of clinical signs of GVHD (He et al, 2007; Su et al, 2012; Figure 2B ), indicating that DN T cells are not alloreactive nor pathogenic in this context. Moreover, this led to stable mixed chimerism and, as opposed to BM transplantation alone, promoted donor allotolerance (He et al, 2007; Su et al, 2012; Figure 2B ).…”

“…Indeed, TCR transgenic and non-transgenic DN T cells can also inhibit NK cells (He et al, 2007; Su et al, 2012), B cells (Zhang et al, 2006; Hillhouse et al, 2010; Ford McIntyre et al, 2011), and dendritic cells (Gao et al, 2011). The combination of their distinct phenotypic characteristics and their unique antigen-specific immunoregulatory properties toward multiple cellular targets has prompted investigators to examine the role of DN T cells in various disease models.…”