Search

Establishing mixed chimerism is a promising approach for inducing donor-specific transplant tolerance. The establishment and maintenance of mixed chimerism may enable long-term engraftment of organ transplants while minimizing the use of immunosuppressants. Several protocols for inducing mixed chimerism have been reported; however, the exact mechanism underlying the development of immune tolerance remains to be elucidated. Therefore, understanding the kinetics of engraftment during early post-transplant period may provide insight into establishing long-term mixed chimerism and permanent transplant tolerance. In this study, we intentionally induced allogeneic mixed chimerism using a nonmyeloablative regimen by host natural killer (NK) cell depletion and T cell-depleted bone marrow (BM) grafts in a major histocompatibility complex (MHC)-mismatched murine model and analyzed the kinetics of donor (C57BL/6) and recipient (BALB/c) engraftment in the weeks following transplantation. Donor BM cells were well engrafted and stabilized without graft-versus-host disease (GVHD) as early as one week post-bone marrow transplantation (BMT). Donor-derived thymic T cells were reconstituted four weeks after BMT; however, the emergence of newly developed T cells was more obvious at the periphery as early as two weeks after BMT. Also, the emergence and changes in ratio of recipient- and donor-derived NKT cells and antigen presenting cells (APCs) including dendritic cells (DCs) and B cells were noted after BMT. Here, we report a longitudinal analysis of the development of donor- and recipient-originated hematopoietic cells in various lymphatic tissues of intentionally induced mixed chimerism mouse model during early post-transplant period. Through the understanding of immune reconstitution at early time points after nonmyeloablative BMT, we suggest guidelines on intentionally inducing durable mixed chimerism.

Therapeutic effects of combined cell therapy with mesenchymal stem cells (MSCs) and regulatory T cells (Treg cells) have recently been studied in acute graft-versus-host-disease (aGVHD) models. However, the underlying, seemingly synergistic mechanism behind combined cell therapy has not been determined. We investigated the origin of Foxp3+ Treg cells and interleukin 17 (IL-17+) cells in recipients following allogeneic bone marrow transplantation (allo-BMT) to identify the immunological effects of combined cell therapy. Treg cells were generated from eGFP-expressing C57BL/6 mice (Tregegfp cells) to distinguish the transferred Treg cells; recipients were then examined at different time points after BMT. Systemic infusion of MSCs and Treg cells improved survival and GVHD scores, effectively downregulating pro-inflammatory Th×and Th17 cells. These therapeutic effects of combined cell therapy resulted in an increased Foxp3+ Treg cell population. Compared to single cell therapy, adoptively transferred Tregegfp cells only showed prolonged survival in the combined cell therapy group on day 21 after allogeneic BMT. In addition, Foxp3+ Treg cells, generated endogenously from recipients, significantly increased. Significantly higher levels of Tregegfp cells were also detected in aGVHD target organs in the combined cell therapy group compared to the Treg cells group. Thus, our data indicate that MSCs may induce the long-term survival of transferred Treg cells, particularly in aGVHD target organs, and may increase the repopulation of endogenous Treg cells in recipients after BMT. Together, these results support the potential of combined cell therapy using MSCs and Treg cells for preventing aGVHD.

Mesenchymal stromal cells (MSCs) are seen as an ideal source of cells to induce graft acceptance; however, some reports have shown that MSCs can be immunogenic rather than immunosuppressive. We speculate that the immunomodulatory effects of regulatory T cells (Tregs) can aid the maintenance of immunoregulatory functions of MSCs, and that a combinatorial approach to cell therapy can have synergistic immunomodulatory effects on allograft rejection. After preconditioning with Fludarabine, followed by total body irradiation and anti-asialo-GM-1(ASGM-1), tail skin grafts from C57BL/6 (H-2k(b)) mice were grafted onto the lateral thoracic wall of BALB/c (H-2k(d)) mice. Group A mice (control group, n = 9) did not receive any further treatment after preconditioning, whereas groups B and C (n = 9) received cell therapy with MSCs or Tregs, respectively, on days -1, +6 and +13 relative to the skin transplantation. Group D (n = 10) received cell therapy with MSCs and Tregs on days -1, +6 and +13. Cell suspensions were obtained from the spleens of five randomly chosen mice from each group on day +7, and the immunomodulatory effects of the cell therapy were evaluated by flow cytometry and real-time PCR. Our results show that allograft survival was significantly longer in group D compared to the control group (group A). Flow cytometric analysis and real-time PCR for splenocytes revealed that the Th2 subpopulation in group D increased significantly compared to the group B. Also, the expression of Foxp3 and STAT 5 increased significantly in group D compared to the conventional cell therapy groups (B and C). Taken together, these data suggest that a combined cell therapy approach with MSCs and Tregs has a synergistic effect on immunoregulatory function in vivo, and might provide a novel strategy for improving survival in allograft transplantation.