1. Blocking Activator Protein 1 Activity in Donor Cells Reduces Severity of Acute Graft- Versus-Host Disease through Reciprocal Regulation of IL-17–Producing T Cells/Regulatory T Cells 
Min-Jung Park, Su-Jin Moon, Sung-Hee Lee, Eun-Kyung Kim, Eun-Ji Yang, Jun-Ki Min, Sung-Hwan Park, Ho-Youn Kim, Chul-Woo Yang, Mi-La Cho 
Biology of Blood and Marrow Transplantation 
Volume 20, Issue 8, Pages (August 2014)
DOI: /j.bbmt
Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

2. Figure 1
SR11302 inhibits alloreactive T cell proliferation in vitro through downregulation of AP-1/STAT3 signaling. (A) The cellular viability to SR11302 treatment in alloresponse condition in vitro was determined by MTT assay. A total of 2 × 105 RBC-lysed BALB/c splenic T cells (responders) were incubated with 2 × 105 irradiated RBC-lysed BALB/c splenic APC (syngeneic stimulator) or C57BL/6 splenic APC (allogeneic stimulator) for 4 days. (B) In a mixed lymphocyte reaction assay, a total of 2 × 105 RBC-lysed BALB/c splenic T cells (responders) were incubated with 2 × 105 irradiated RBC-lysed BALB/c splenic APC (syngeneic stimulator) or C57BL/6 (B6) splenic APC (allogeneic stimulator) for 4 days. Responder cells were cultured in the absence or presence of SR11302 (2.5 μM and 10 μM). SR11302 was added on day 0, and T cell proliferation was measured by [3H]-thymidine incorporation in each group. (C) The supernatants were collected, and ELISA was performed to determine the IFN-γ, IL-17, and IL-10 levels. Data represent means ± SD of triplicates (∗P < .05 versus alloresponse). (D) A total of 105 RBC-lysed BALB/c splenic T cells (responders) were incubated with 105 irradiated RBC-lysed BALB/c splenic APC (syngeneic stimulator) or B6 splenic APC (allogeneic stimulators). SR11302 (2.5 μM) was added on day 0 and cells were harvested on day 2. Then, western blotting analysis of AP-1 (D) and STAT3 phosphorylation associated protein expression (E) was performed in each sample. (1: syngeneic stimulator, 2: allogeneic stimulator in the absence of SR11302, 3: allogeneic stimulator in the presence of SR11302).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

3. Figure 2
Altered T cell differentiation after SR11302 treatment in donor allogeneic T cells is associated with reduced alloreactivity. (A) A total of 105 RBC-lysed BALB/c splenic T cells (responders) were incubated with 105 irradiated RBC-lysed BALB/c splenic APC (syngeneic stimulator) or C57BL/6 splenic APC (allogeneic stimulator) for 4 days. Responder cells were cultured in the absence or presence of SR11302 (2.5 μM and 10 μM). SR11302 was added on day 0 and cells were harvested on day 4. Then, intracellular staining for IFN-γ, IL-17, and Foxp3 in isolated CD4+ T cells was performed and analyzed by flow cytometric analysis. Right panel shows the absolute number of CD4+IFN-γ+, CD4+IL-17+, CD4+CD25+Foxp3+ T cells. (B) Splenic T cells were incubated for 72 hours with plate-bound anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) antibody in the absence or presence of 10 μM SR CD4+CD25+Foxp3+ Treg cell proportion was analyzed by flow cytometry. The data are expressed as the mean ± SD from 4 independent experiments. (C) The mean fluorescence intensity (MFI) of various Treg markers, including CTLA-4, ICOS, PD-1, and GITR, were analyzed by flow cytometry. The data are expressed as mean ± SD for 4 mice per group. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001, compared with the vehicle (control)-treated alloresponse.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

4. Figure 3
SR11302 suppresses the development of acute GVHD via blocking of c-Fos/c-Jun signaling. (A) C57BL/6 (B6) splenocytes (1 × 107 cells) were incubated with 10 μM of SR11302 or DMSO (control) for 2 hours at 37°C before adoptive transfer into lethally irradiated (800 cGy) BALB/c (recipient) mice. Recipients also received 5 × 106 bone marrow cells from B6 mice and were monitored for weight loss, survival, and clinical scores of aGVHD. Lethal aGVHD shown in BALB/c recipients was inhibited after administration of donor B6 splenocytes treated with SR Recipient mice receiving T cell–depleted bone marrow cells showed no signs of GVHD. Data combined from 3 independent experiments (n = 15 per group) are shown. (B) Histopathology of skin, small, and large intestine after BMT (n = 6 per group), which is representative of 2 independent experiments. Tissues isolated on day 14 after BMT were stained with H & E (original magnification, 200 ×) (left panel) and histological scores of each organ were higher in vehicle-treated animals, compared with those of SR11302–treated group (right pane). The data are expressed as the mean ± SD from 3 independent experiments for 5 mice per group. ∗P < .05, ∗∗∗P < .001, compared with the vehicle-treated GVHD group. (C) Fourteen days after BMT, splenocytes isolated from each group were stained with DAPI and anti-c-Fos or c-Jun antibodies, components of AP-1, and visualized by confocal microscopy. Representative examples of confocal staining for c-Fos and c-Jun in spleen from GVHD mice. (D) Representative examples of immunohistochemical staining for c-Fos and c-Jun, components of AP-1, in liver and small intestine tissue from GVHD mice. Positive immunoreactivity appears as a brown color and is counterstained with blue (original magnification, 400 ×).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

5. Figure 4
Analysis of CD4+ T helper cells in SR11302-treated GVHD mice. (A) C57BL/6 (B6) splenocytes (1 × 107 cells) were incubated with SR11302 (10 μM) or vehicle (DMSO) for 2 hours at 37°C before adoptive transfer into lethally irradiated (800 cGy) BALB/c mice. Recipient BALB/c mice also received 5 × 106 bone marrow cells from B6 mice. Intracellular cytokines were determined in the CD4+ T cells. Splenocytes of each group and were analyzed by flow cytometry on day 14 after BMT. Results are shown as mean ± SD (n = 5 per group). ∗P < .05, compared with the vehicle-treated GVHD group. (B) Fourteen days after BMT, the expression of Foxp3 and SOCS3 mRNA was determined by RT-PCR analysis from whole splenocytes isolated from each group. (C) Spleens isolated on day 14 after BMT in each group were analyzed by confocal microscopy for the expression of IFN-γ, IL-4, IL-17, and Foxp3 among CD4+ cells. The proportions of IFN-γ, IL-4, IL-17, or Foxp3 expressing CD4+ cells in spleen were represented by bar graph (right panel) (mean ± SD). The experiment was performed once with 6 mice per group. ∗P < .05, compared with the vehicle-treated GVHD group.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

6. Figure 5
SR11302 regulate signaling of STAT3 and STAT5. (A) C57BL/6 (B6) splenocytes (1 × 107 cells) were incubated with SR11302 (10 μM) or vehicle (DMSO) for 2 hours at 37°C before adoptive transfer into lethally irradiated (800 cGy) BALB/c mice. Recipient BALB/c mice also received 5 × 106 bone marrow cells from B6 mice. The signal transducers STAT3 and STAT5 in the spleens were analyzed by confocal microscopy on day 14 after BMT in each group. Data are representative of 3 independent experiments. (B) Fourteen days after BMT, isolated spleens from each group were analyzed by confocal microscopy for the expression of GITR and PD-1 among CD4+CD25+Foxp3+ regulatory T cells. The experiment was performed once with 6 mice per group. Bars represent the mean ± SD of 6 mice per group.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

7. Figure 6
SR11302 plays a critical role in Tregs-mediated inhibition of aGVHD severity. (A) Treg-depleted splenocytes (1 × 107 cells) from B6 mice were incubated with 10 μM of SR11302 or DMSO (control) for 2 hours at 37°C before adoptive transfer into lethally irradiated (800 cGy) BALB/c (recipient) mice. Recipients also received 5 × 106 bone marrow cells from B6 mice and were monitored for weight loss and survival. (B) Spleens were isolated on day 10 after BMT in each group. Then, intracellular staining for IFN-γ, IL-17, CD25, and Foxp3 in isolated CD4+ T cells was performed and analyzed by flow cytometric analysis.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions