INTRODUCTION

METHODS
Mice
Bone marrow transplantation and acute GVHD induction
Treg cell generation
Treg cell therapy
Histopathological analysis of acute GVHD
Flow cytometry
ELISA assay for cytokine levels
Statistical analysis

RESULTS
Phenotypes of ex vivo expanded Treg cells according to Treg source
![]() | Figure 1.Immunophenotypes of ex vivo expanded retinoic acid-induced regulatory T (Treg) cells according to Treg source. Retinoic acid-induced CD4+CD25+ Treg cells showed > 96% purity using flow cytometry. Treg cells induced from C57BL/6 (donor, H-2b), BALB/c (host, H-2d), or DBA1J (third-party, H-2q) splenic CD4+ T cells were characterized by positive expression of intracellular Foxp3, (A) cytotoxic T lymphocyte antigen-4 (CLTA-4), and surface expression of the indicated markers (B) inducible costimulator (ICOS), (C) intercellular adhesion molecule 1 (ICAM-1), (D) CD103, (E) programmed death-1 (PD-1) in the gated CD4 T-cell populations. The reported percentages are those of double-positive cells. Results are representative of two independent experiments. |
Regardless of Treg source, CD4+CD25+CD62L+ Treg cells are protective for lethal acute GVHD
![]() | Figure 2.Improved survival in groups of mice treated with three types of Treg cell after bone marrow transplantation (BMT). Recipient mice (BALB/c, H-2d) were divided into five groups and irradiated with 800 cGy followed by intravenous injection with 5 × 106 T-cell depleted bone marrow (TCDBM) cells as a control. Four of the groups were also infused with 5 × 105 CD4+CD25– T cells from donor spleen cells of C57BL/6 background, to induce graft-versus-host disease (GVHD). Three of these four groups were then designated as treatment groups: 5 × 105 donor-, host-, or third-party-derived Treg cells were infused into mice after BMT. Survival rates in an acute GVHD mouse model with adoptive transfer of three types of Treg cells are compared to those in a GVHD control group following allogeneic BMT. |
![]() | Figure 3.Improved clinical graft-versus-host disease (GVHD) scores in groups of mice treated with three types of Treg cell therapy after bone marrow transplantation. The degree of clinical acute GVHD (A) was assessed weekly using a scoring system that summed changes in five clinical parameters: weight loss, posture, activity, fur texture, and skin integrity. All animals were monitored for mean serial weight measurements (B) and clinical signs. Most effects were observed to be accentuated in the presence of donor-derived Treg cell therapy. TCD-BM, cell-depleted bone marrow cells; Treg, regulatory T. |
Regardless of Treg source, CD4+CD25+CD62L+ Treg cell therapy reduces the histopathological manifestations of acute GVHD
![]() | Figure 4.Improvement of histopathological scores in target organs of groups of mice that received adoptive transfer of reglatory T (Treg) cells after bone marrow transplantation (BMT). Histological scores were assessed weekly in target organs of acute graft-versus-host disease (GVHD): skin, small intestine, and liver. (A) Histological GVHD scores were evaluated in a mouse model of GVHD, as well as in mice infused with donor-derived Treg cells, host-derived Treg cells, or third-party Treg cells, using samples from skin (×200), small intestine (×200), and liver tissues (×200) 15 days after BMT (with individual criteria for each specific tissue; H&E). (B) Regardless of the source of Treg cells, groups of mice treated with infusion of Treg cells demonstrated significant reductions in total GVHD histological scores compared with the GVHD control group. |
Third-party-derived CD4+CD25+CD62L+ Treg cells did not modulate the balance of Th1 and Th2 cells in lethal acute GVHD induced by CD4+CD25– T cells
![]() | Figure 5.Only donor-derived regulatory T (Treg) cells modulate the balance of T-helper 1 (Th1) and Th2 cells in lethal acute graft-versus-host disease (GVHD). Treatment of mice with donor-derived Treg cells resulted in a significant reduction in CD4+ interferon-γ (IFN-γ)+ Th1 cells, but an enhancement of CD4+ interleukin (IL)-10+ and CD4+IL-4+ Th2 cells, when compared with host-derived or third-party-derived Treg cells 15 days after bone marrow transplantation. Donor-derived Treg cells led to a greater increase in (A) IL-10 and (B) IL-4 levels and a greater decrease in IFN-γ levels versus treatment with host- or third-party-derived Treg cells in a murine model of acute GVHD. (C) Data are presented as the ratio of Th1/Th2 among CD4+ T cells, which was calculated as the ratio of CD4+IL-4+ Th2 cells divided by the percentage of CD4+IFN-γ+ Th1 cells. The purity of all cell subsets was > 95% as determined by flow cytometry analysis. Data are shown as mean ± SEM. TCD-BM, cell-depleted bone marrow cells. a p < 0.05. |
Third-party-derived CD4+CD25+CD62L+ Treg cell therapy provides an immunomodulatory effect associated with reciprocal regulation of 17+ Th17/Treg cells
![]() | Figure 6.All three groups of Treg cell therapy provide an immunomodulatory effect associated with reciprocal regulation of 17+ T-helper (Th17)/regulatory T (Treg) cells. Regardless of cell source, Treg cell therapy resulted in a significant reduction in CD4+ interleukin (IL)-17+ Th17 cells, but an enhancement in CD4+Foxp3+ Treg cells compared with graft-versus-host disease (GVHD) control at 15 days after bone marrow transplantation. (A) Treg cell therapy groups led to an increase in Foxp3 levels and a greater decrease in IL-17 levels and (B) IL-6 levels as compared with the GVHD control in a murine model of acute GVHD. However, statistical analysis revealed no significant differences. (C) Data are presented as the ratio of Treg/Th17 among CD4+ T cells, which was calculated as the ratio of CD4+Foxp3+ Treg cells divided by the percentage of CD4+IL-17+Th17 cells. The purity of all cell subsets was > 95% as determined by flow cytometry analysis. Data are shown as mean ± SEM. IFN-γ, interferon-γ; TCD-BM, cell-depleted bone marrow cells; NS, not significant. a p < 0.05. |

DISCUSSION
