1. Cytosine-Phosphorothionate-Guanine Oligodeoxynucleotides Exacerbates Hemophagocytosis by Inducing Tumor Necrosis Factor–Alpha Production in Mice after Bone Marrow Transplantation 
Jiajia Liu, Yong-Mei Guo, Nobuyuki Onai, Hideaki Ohyagi, Makoto Hirokawa, Naoto Takahashi, Hiroyuki Tagawa, Kumi Ubukawa, Isuzu Kobayashi, Hiroyuki Tezuka, Yoshihiro Minamiya, Toshiaki Ohteki, Kenichi Sawada 
Biology of Blood and Marrow Transplantation 
Volume 22, Issue 4, Pages (April 2016)
DOI: /j.bbmt
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

2. Figure 1
Three types of murine BMT models. (A) Chimerism after BMT. PB collected on day 7 was analyzed using flow cytometry. Images are representative of 2 independent experiments. (B) Survival curves for BMT mice. On day −1, C57BL/6 (B6) mice were lethally irradiated. On day 0, the mice were intravenously administered allogeneic BM cells (5 × 106) with splenocytes (1 × 107) from BALB/c mice (BALB[+S]-B6, indicated as GVHD; n = 25) or without splenocytes (BALB[−S]-B6, indicated as Allo-BMT; n = 8). As controls, lethally irradiated B6 mice were given syngeneic BM cells (5 × 106) from B6 mice (B6-B6, indicated as Syn-BMT; n = 11) or not (no BMT; n = 9). Data were pooled from 4 independent experiments. Body weights (C) and clinical GVHD scores (D) were determined at the indicated times (n = 6 to 12/group). Data are means ± SD pooled from 2 independent experiments. ∗∗P < .01. NS indicates no significance.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

3. Figure 2
Hemophagocytosis is a common feature in BMT mice. (A) On day 7 after BMT, RBC-lysed PB cells were stained with May-Grünwald-Giemsa (upper panel) and analyzed using fluorescent activated cell sorter (lower panel). Images are representative of at least 3 independent experiments. Naïve B6 mice were used as controls. Scale bars represent 20 μm. (B) At the indicated times (days 7, 14, 21, 28, 35, and 42) after BMT, the TER119+CD11c+ cell fractions among RBC-lysed PB cells were determined using fluorescent activated cell sorter (n = 4 to 6/group). Data shown are means ± SD pooled from 2 independent experiments. ∗P < .05 and ∗∗P < .01 versus syngeneic BMT (B6-B6). NS indicates no significance. (C) On day 11 after BMT, the TER119+CD11c+ cell fraction among spleen cells was analyzed using fluorescent activated cell sorter (n = 3 to 6/group). Data shown are mean ± SD pooled from 2 independent experiments. ∗P < .05 and ∗∗P < .01.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

4. Figure 3
CpG induces HPS in BMT mice. (A) On day 7 after Syn-BMT, the indicated doses of CpG were administered to the mice (n = 3/group). RBC-lysed PB cells were analyzed using fluorescent activated cell sorter 18 hours after CpG administration. (B) On day 7 after Syn-BMT, PBS or CpG (100 μg) was administered to the indicated mice (n = 3/group), and RBC-lysed PB cells were analyzed using fluorescent activated cell sorter at the indicated times thereafter. Data are shown as means ± SD. ∗P < .05 and ∗∗P < .01 versus control (PBS). (C) On day 7 after BMT, PBS or CpG (100 μg) was administered to the indicated mice (n = 5 to 8/group). RBC-lysed PB cells (upper panel), BM cells (BM, middle panel) and spleen cells (SP, lower panel) were analyzed using fluorescent activated cell sorter 18 hours, 96 hours, and 96 hours, respectively, after administration. (D) Spleen size (upper panel) and weight (lower panel) were evaluated 96 hours after CpG administration. (E-G) Blood cell counts (WBC, hemoglobin, and platelets) were evaluated 18 hours and 96 hours after CpG administration (D), while rectal temperature (F) and serum triglyceride (G) were determined after 18 hours and 96 hours, respectively. Data shown are the means ± SD pooled from at least 2 independent experiments. ∗P < .05 and ∗∗P < .01. NS indicates no significance; ND, not determined.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

5. Figure 4
CpG induces production of TNF-α, IL-6, and IFN-γ in all BMT mice. (A) Survival curves for BMT mice administered CpG. On day 7 after BMT, CpG at the indicated dose was administered to Syn-BMT, Allo-BMT, and GVHD mice. The total number of mice used for each experiment is shown in the inset. Data were pooled from 3 independent experiments. ∗∗P < .01 and ∗∗∗P < .001 versus GVHD mice without CpG (e). (B) Cytokine production. On day 7 after BMT, PBS, or CpG (100 μg) was administered to the indicated mice (n = 6/group). At the indicated times after CpG administration, serum TNF-α, IL-6, and IFN-γ were measured using ELISAs. Data shown are mean ± SD from 2 independent experiments.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

6. Figure 5
Blockade of TNF-α completely prevents hemophagocytosis in all CpG-injected mice. (A) On day 7 after BMT, PBS, or CpG (100 μg) was administered to the indicated mice (n = 3 to 6/group). PBS or etanercept (400 μg) was then administered. RBC-lysed PB cells (upper panel), BM cells (middle panel), and spleen cells (SP, lower panel) were then analyzed using fluorescent activated cell sorter 18 hours, 96 hours, and 96 hours, respectively, after reagent administration. Data shown are means ± SD pooled from 2 independent experiments. ∗P < .05 and ∗∗P < .01. NS indicates no significance; ND, not determined. (B) Effect of TNF inhibition on survival among BMT mice administered CpG. On day 7 after BMT, CpG (100 μg) was intravenously administered to GVHD mice (n = 8-10/group). Four hours before CpG administration, PBS, anti-IFN-γ mAb (R4-6A2, 400 μg), isotype IgG (400 μg) or TNF inhibitor (etanercept, 400 μg) was subcutaneously administered to the mice. Just after CpG administration, PBS, anti-IFN-γ mAb (400 μg), isotype IgG (400 μg), or etanercept (400 μg) was administered again. Data were pooled from 2 independent experiments. (C-E) On day 7 after BMT in GVHD mice, PBS or CpG (50 μg) was administered (n = 3 to 6/group) to the indicated mice, after which PBS, isotype antibody (400 μg), R4-6A2 (400 μg), or etanercept (Eta, 400 μg) was administered. Serum concentrations of IFN-γ (C) and TNF-α (D) were respectively measured 8 hours and 1 hour after CpG-injection. (E) RBC-lysed PB cells were analyzed using fluorescent activated cell sorter 18 hours after CpG administration, and the TER119+CD11c+ cell fractions are shown. Data are means ± SD pooled from 2 independent experiments. ∗P < .05 and ∗∗P < .01. NS indicates no significance.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

7. Figure 6
TNF-α derives from recipient mice whereas hemophagocytes are from the donor. (A) On day 7 after Syn-BMT in B6 and TNF-α−/− mice, CpG (100 μg) or PBS was administered (n = 5/group) followed by etanercept (400 μg) or PBS, and serum TNF-α was measured using ELISA. (B) BM cells from B6, TNF-α−/−, or BALB/c mice plus splenocytes (BALB[+S]) were transplanted into lethally irradiated B6 or TNF-α−/− mice, as indicated. Lethally irradiated B6 (no BMT) cells were used as control. On day 7 after BMT, CpG (100 μg) was administered (n = 3/group), and 1 hour later serum TNF-α was measured using an ELISA. Results are representative of 2 independent experiments. (C) Lethally irradiated B6 mice were given BM cells from SJL (CD45.1) mice (SJL-B6) or BM cells plus splenocytes from BALB/c mice (H-2kd) (BALB[+S]-B6). On day 11 after BMT, RBC-lysed PB cells, spleen (SP) cells, and BM cells were analyzed using fluorescent activated cell sorter. Images are representative of 2 independent experiments.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

8. Figure 7
CpG induces hemophagocytosis in TNF-α−/− mice. (A) On day 7 after B6 or TNF-α−/− Syn-BMT, CpG (50 μg), or PBS were administered (n = 5 or 6/group) followed by etanercept (400 μg) or PBS. RBC-lysed PB cells were analyzed using fluorescent activated cell sorter 18 hours later and the Ter119+CD11c+ cell fractions are shown. (B) On day 7 after BMT, Syn-BMT and GVHD mice (n = 5 or 6/group) were administered PBS or etanercept (400 μg). RBC-lysed PB cells were analyzed using fluorescent activated cell sorter 18 hours later, and the Ter119+CD11c+ cell fractions are shown. The values for TNF-α, IL-6, and IFN-γ are from the data shown in Figure 4B at 0 hour. (C) On day 7 after BMT, CpG (50 μg) was administered to GVHD mice (n = 5 or 6/group) followed by PBS or etanercept (400 μg). At the indicated times thereafter (0 hour, 1 hour, 8 hours, and 24 hours), serum IFN-γ was measured using an ELISA. A representative of 2 independent experiments is shown as means ± SD. ∗P < .05 and ∗∗P < .01. NS indicates no significance.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions