1. Volume 18, Issue 3, Pages 367-379 (March 2003)
Regulatory Dendritic Cells Protect Mice from Murine Acute Graft-versus-Host Disease and Leukemia Relapse 
Katsuaki Sato, Naohide Yamashita, Naomi Yamashita, Masanori Baba, Takami Matsuyama 
Immunity 
Volume 18, Issue 3, Pages (March 2003)
DOI: /S (03)
Copyright © 2003 Cell Press Terms and Conditions

2. Figure 1 rDCs Regulate the Function of T Cells
The panels that are listed in the legend but do not appear here are posted as Supplemental Data. (A) Cells were stained with the stated mAbs, and cell surface expression was analyzed by flow cytometry. Data are represented by a dot plot.
(B) CD4+ T cells (H-2b) were cultured with mDCs or rDCs (H-2d) at various T cell:DC ratios, and the proliferative response was measured.
(C and D) I-Kb+CD4+ T cells or I-Kd+CD4+ T cells obtained from the transplanted recipients (H-2d) (C) or (H-2q) (D) were cultured with mDCs (H-2d) (C) or (H-2q) (D) in the presence or absence of mDCs or rDCs obtained from the indicated strains at various T cell:DC ratios, and the proliferative response was measured. Primed CD4+ T cells (H-2b) plus mDCs (H-2d) (C) or primed CD4+ T cells (H-2d) plus mDCs (H-2q) (D) versus any other group, P < 0.01.
(E and F) I-Kb+CD8+ T cells or I-Kd+CD8+ T cells obtained from the transplanted recipients (H-2d) (E) or (H-2b) (F) were cultured with medium alone (none) or the indicated types of DCs at a T cell/DC ratio of 10:1 and were subjected to CTL assay against the targeted cells obtained from various strains. CD8+ T cells alone versus any other group, P < 0.01.
(G and H) CD4+ T cells (H-2b) (G) or (H-2d) (H) were cultured with or without the indicated types of DCs (H-2d) (G) or (H-2q) (H) at a T cell/DC ratio of 10:1 in a first coculture. In another experiment, CD4+ T cells obtained from a first coculture were then cultured with medium alone (none) or mDCs obtained from various strains at a T cell/DC ratio of 10:1 in the presence or absence of IL-2 in a second coculture, and the proliferative response was measured. Primed CD4+ T cells (H-2b) plus mDCs (G) or primed CD4+ T cells (H-2d) plus mDCs (H) versus any other group, P < Five replicate experiments with similar results were pooled.
Immunity  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

3. Figure 2
rDCs Protected the Recipients of Allogeneic BMS from the Lethality Caused by Acute GVHD
(A and B) Recipients (H-2d) of BM cells or BMS (H-2b) were injected with or without the indicated types of DCs 2 days after transplantation. (C) Recipients (H-2b) of BMS (H-2q) were injected with or without the indicated types of DCs 2 days after transplantation. (D) Recipients (H-2d) of BMS (H-2b) were injected with or without various doses of rDCs (H-2d) on the indicated days after transplantation. (E and F) Recipients (H-2d) of BMS (H-2b) injected with rDCs (H-2d) were treated with the indicated Abs (E) or injected with the indicated types of CD4+CD25+ T cells (H-2b) (F) after transplantation as described in Experimental Procedures. Untreated recipients of allogeneic BMS versus any other group, P < Two replicate experiments with similar results were pooled.
Immunity  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

4. Figure 3
rDCs Impair Allogeneic Ag-Specific Responses of CD4+ T Cells and CD8+ T Cells in Transplanted Mice
Recipients (H-2d) of BMS (H-2b) were injected with or without mDCs or rDCs (H-2d), and spleen mononuclear cells and sera were obtained from each group 5 days after transplantation. (A) Cells were stained with the stated mAbs, and cell surface expression was analyzed by flow cytometry. Data are expressed as percent positive cells. (B) I-Kb+CD4+ T cells obtained from spleen mononuclear cells were cultured with medium alone (none) or mDCs (H-2d or H-2q) in the presence or absence of IL-2 at a T cell/DC ratio of 10:1, and the proliferative response was measured. (C) I-Kb+CD8+ T cells obtained from spleen mononuclear cells were subjected to CTL assay against P815 cells or EL4 cells. (D) Concentrations of IFN-γ, TNF-α, and IL-12p40 in sera were evaluated by ELISA. Untreated recipients of allogeneic BMS versus any other group, P < (E) Recipients (H-2d) of BMS (H-2b) were injected with CFSE-labeled rDCs (H-2d) 2 days after transplantation, and CFSE-labeled rDCs in spleen mononuclear cells were determined at the indicated days after injection by flow cytometry. Five replicate experiments with similar results were pooled.
Immunity  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

5. Figure 4
Characterization of Donor-Derived CD4+CD25+ T Cells in Transplanted Mice
The panels that are listed in the legend but do not appear here are posted as Supplemental Data. Allogeneic transplantation was performed as described in Figure 3, and I-Kb+CD4+ T cells were obtained from spleen mononuclear cells in each group.
(A and B) I-Kb+CD4+ T cells were assayed for phenotype (A) and cytokine profile (B) by flow cytometry. Data are expressed as percent positive cells. CD4+ T cells obtained from normal mice versus any other group, P < 0.01.
(C) CD4+CD25+ T cells isolated from normal mice (H-2b) or I-Kb+CD4+CD25+ T cells isolated from I-Kb+CD4+ T cells in each group of recipients were assayed for phenotype by flow cytometry. Data are represented by a dot plot.
(D) Unprimed CD4+CD25+ T cells (H-2b) or I-Kb+CD4+ CD25+ T cells isolated from rDC-treated recipients on the indicated days after transplantation were assayed for CD152 expression by flow cytometry. Data are expressed as percent positive cells. Unprimed CD4+CD25+ T cells versus any other group, P < 0.01.
(E) CD4+ T cells (H-2b), unprimed CD4+CD25+ T cells (H-2b), and/or I-Kb+CD4+CD25+CD152+ T cells were cultured with or without mDCs or rDCs (H-2d or H-2q) at a T cell/DC ratio of 10:1, and the proliferative response was measured.
(F) CD4+ T cells (H-2b) were cultured with mDCs (H-2d) at a T cell/DC ratio of 10:1 in the presence or absence of the different numbers of unprimed CD4+CD25+ T cells or I-Kb+CD4+CD25+CD152+ T cells isolated from rDC-treated recipients on day 5 after transplantation, and the proliferative response was measured.
(G) CD4+ T cells (H-2b) were cultured with mDCs (H-2d) in the presence or absence of unprimed CD4+CD25+ T cells or I-Kb+CD4+CD25+ T cells isolated from rDC-treated recipients on the indicated days after transplantation at a T cell/CD25+T cell/DC ratio of 10:1:1, and the proliferative response was measured.
(H) CD4+ T cells (H-2b) were cultured with or without mDCs (H-2d) in the presence or absence of I-Kb+CD4+CD25+ CD154+ T cells, I-Kb+CD4+CD25+CD152+ T cells, or IL-2 at a T cell/CD25+T cell/DC ratio of 10:10:1. For Transwell experiments, I-Kb+CD4+CD25+CD152+ T cells plus mDCs (H-2d) were either added directly to the coculture of CD4+ T cells (H-2b) with mDCs (H-2d) or were separated in 24-well plates. Following depletion of mDCs, T cells were transferred to 96-well plates to measure the proliferative response. CD4+ T cells plus mDCs (E, G, H) or CD4+ T cells, unprimed CD4+CD25+ T cells plus mDCs (G) versus any other group, P < Five replicate experiments with similar results were pooled.
Immunity  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

6. Figure 5
Involvement of cAMP and p27kip1 in an Induction of Allogeneic Ag-Specific Tolerant CD4+ T Cells by rDCs
(A, C, and D) CD4+ T cells (H-2b) were obtained from the coculture of CD4+ T cells (H-2b) with the indicated types of DCs (H-2d) or I-Kb+CD4+ CD25+CD152+ Tr cells as described in Figure 4 at a T cell/DC ratio of 10:1 or at a T cell/CD25+ T cell ratio of 1:1. (B and E) Allogeneic transplantation was performed as described in Figure 3, and I-Kb+CD4+ T cells obtained from spleen mononuclear cells in each group were then cultured with mDCs (H-2d or H-2q) at a T cell/DC ratio of 10:1. Following these procedures, CD4+ T cells were isolated from the coculture. (A and B) The concentration of intracellular cAMP was measured. (C, D, and E) The expression of p27kip1 was analyzed by immunoblot. Five replicate experiments with similar results were pooled.
Immunity  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

7. Figure 6
GVL Effect in rDC-Treated Leukemia-Bearing Recipients of Allogeneic BMS
Leukemia (H-2d)-bearing recipients (H-2d) received BM (H-2b), BMS (H-2b), and/or rDCs (H-2d) as described in Experimental Procedures. (A and B) Recipients were monitored for survival (A) and body weight (B). (C) Liver and spleen were obtained from representative recipients at the time of death or 60 days after transplantation to determine their weight. Recipients that received TBI plus the inoculation with P815 cells versus any other group, P < Two replicate experiments with similar results were pooled.
Immunity  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions