1. The Fifth Epidermal Growth Factor–like Region of Thrombomodulin Alleviates Murine Graft-versus-Host Disease in a G-Protein Coupled Receptor 15 Dependent Manner 
Bin Pan, Xiangmin Wang, Shinsuke Kojima, Chie Nishioka, Akihito Yokoyama, Goichi Honda, Kailin Xu, Takayuki Ikezoe 
Biology of Blood and Marrow Transplantation 
Volume 23, Issue 5, Pages (May 2017)
DOI: /j.bbmt
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

2. Figure 1
TME5 alleviated systemic aGVHD in mice. In the C57BL/6 → BALB/c allo-HSCT model, lethally irradiated recipients were transplanted with WT TCD-BM (5 × 106/mouse) and WT spleno-T cells (5 × 105/mouse) from donors. Day of transplantation was set as day 0. Recipients were intraperitoneally injected with PBS, rTM (1000 µg/kg), TME5 (125 µg/kg), or TME45 (194 µg/kg) on every other day for up to 4 weeks (15 in each group). Survival (A) and GVHD scores (B) were monitored every 2 days. (C) Blood samples were collected at indicated time points and were subjected to cytometric bead array (CBA) analysis for concentration of cytokines (5 in each group). (D) Livers and colons were obtained on day 21 and subjected to histologic analysis (4 in each group). One representative hematoxylin and eosin staining photo of each group was shown. (E) Lymphocytes were isolated from the liver and colon on day 21 and were delivered to flow cytometric analysis (4 in each group). Figure shows percentages of Th 1, Th 17, and Treg populations gated on CD4+ T cells. CD4+IFN-γ+IL-17– represents Th1; CD4+IFN-γ–IL-17+ represents Th17; CD4+FoxP3+ represents Tregs. Statistical comparison of survival was performed with log-rank test. Data are shown as mean ± SD and are compared using unpaired t test or 1-way ANOVA test. *P < .05.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

3. Figure 2
TME5 suppressed allo-reaction in vitro. To induce MLR, WT spleno-T cells were stimulated with irradiated splenocytes (7.5 Gy) from BALB/c in the presence of PBS or TME5. Unstimulated T cells were used as control. On day 3 proliferation of MLRs were detected with (A) BrdU incorporation assay (6 in each group). On day 5, lymphocytes and supernatants were collected from PBS- and TME5 (100 ng/mL)-treated MLRs. (B) Lymphocytes were subjected to flow cytometric analysis on Th 1, Th 17, and Tregs (3 in each group). Figure shows percentages of Th 1, Th,17, and Treg populations gated on CD4+ T cells. (C) Supernatants were measure for concentration of cytokines, by using CBA (6 in each group). Data are shown as mean ± SD and are compared using 1-way ANOVA test. *P < .05; n.s., no significance.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

4. Figure 3
TME5 alleviated aGVHD in a GPR15-dependent manner. (A) For pull-down assay with rTM and TME5, rTM and TME5 (with V5 tag) were preincubated with mouse IgG (control) or anti-TM (epitope: TME5) followed by incubation with membrane proteins. After incubation, rTM- or TME5-containing complexes were precipitated by anti-TM (epitope: D1) plus agarose A/G beads or anti-V5 plus agarose A/G beads, respectively. The precipitated proteins were delivered to Western blot analysis with antibodies to GPR15, TM and V5. (B-E) In C57BL/6 → BALB/c allo-HSCT model, lethally irradiated recipients were transplanted with WT TCD-BM (5 × 106/mouse) from donors. Donor-derived WT or GPR15-knockout (GPR15KO) spleno-T cells (5 × 105/mouse) were co-transferred to recipients. Recipients were intraperitoneally injected with PBS or TME5 (125 µg/kg) on every other day for up to 4 weeks (15 in each group). Survival (B) and GVHD scores (C) were monitored every 2 days. (D) Livers and colons were obtained on day 21 and subjected to histologic analysis (4 in each group). One representative hematoxylin and eosin staining photo was shown. (E) Lymphocytes were isolated from liver and colon on day 21 and were delivered to flow cytometry analysis (4 in each group). Figure shows percentages of Th 1, Th 17, and Treg populations gated on CD4+ T cells. Comparison of survival was performed with log-rank test. Data are shown as mean ± SD and are compared using 1-way ANOVA test. Figure represents 1 of 3 independent experiments.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

5. Figure 4
GPR15 mediated immuno-suppressive function of TME5 in allo-reaction in vitro. (A) WT and (B) GPR15KO T cells were stimulated with anti-mouse CD3ε, anti-mouse CD28 and rmIL-2 for 72 hours. Unstimulated T cells were used as control. Proteins from WT T cells were analyzed by Western blot. GPR15KO T cells were delivered to flow cytometric analysis for expression of green fluorescent protein (GFP). (C and D) 2 × 106/mL WT or GPR15KO T cells were stimulated with irradiated allo-geneic splenocytes in the presence of PBS or TME5 (100 ng/mL). (C) On day 3, proliferation of MLRs were detected with BrdU incorporation assay (6 in each group). (D) On day 5, lymphocytes were subjected to flow cytometric analysis, and counts of Th 1, Th 17, nTregs, and iTregs were calculated from total cell numbers and percentages of each populations (3 in each group). (E and F) GPR15KO T cells were transfected with Gpr15-expression vector or a control vector followed by stimulation with irradiated allo-geneic splenocytes for 3 days in the presence of PBS or TME5 (100 ng/mL). (E) Proliferation of MLRs were detected with BrdU incorporation assay (6 in each group). (F) qPCR were applied to analyze mRNA levels of cytokines in lymphocytes. Data represent fold changes (6 in each group). Data are shown as mean ± SD and are compared using 1-way ANOVA test. *P < .05.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

6. Figure 5
TME5 suppressed production of IL-6 in T cells. (A and B) GPR15KO T cells were transfected with Gpr15-expression vector or a control vector. (A) Transfected T cells as well as WT T cells were stimulated with anti-mouse CD3ε, anti-mouse CD28 and rmIL-2 for 12 hours in the presence of PBS or gradient doses of TME5 (10 ng/mL, 100 ng/mL, and 200 ng/mL). Cells were subjected to qPCR analysis for expression level of IL-6 mRNA. Unstimulated T cells were used as control (6 in each group). Data are shown as mean ± SD and are compared using 1-way ANOVA test. *P < .05. (B) Transfected T cells as well as WT T cells were stimulated with anti-mouse CD3ε and anti-mouse CD28 for 12 hours in the presence of PBS or TME5 (100 ng/mL). After washing with PBS, cells were restimulated with anti-mouse CD3, anti-mouse CD28, and rmIL-2 (10 ng/mL) for 1 hour in the presence of PBS or TME5 (100 ng/mL). Cytoplasmic and nuclear proteins were analyzed by performing Western blot with indicated antibodies. Figure showed here represents 1 of 3 independent experiments.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

7. Figure 6
TME5 reduced activation of dendritic cells. (A) BMDCs from WT and GPR15KO mice were primed with rmTNF-α (20 ng/mL) for 24 hours in the presence of PBS or TME5 (100 ng/mL). Levels of TNF-α and IL-6 mRNAs were measured by qPCR. Data represent fold changes (6 in each group). (B) BMDCs were primed with rmTNF-α (20 ng/mL) for 30 minutes in the presence of PBS or TME5 (100 ng/mL). Cytoplasmic and nuclear proteins were analyzed by performing Western blot with indicated antibodies. (C) BMDCs were primed with rmTNF-α (20 ng/mL) for 24 hours in the presence of PBS or TME5 (100 ng/mL). After washing with PBS, primed BMDCs were co-cultured with 1 × 106 BALB/c-derived T cells for 5 days. T cells were collected and delivered to flow cytometric analysis, and counts of Th 1, Th 17, nTregs, and iTregs were calculated from total cell numbers and percentages of each populations (3 in each group). Figure shows populations gated on CD4+ T cells. Data are shown as mean ± SD and are compared using unpaired t test or 1-way ANOVA test. *P < .05. Figure represents 1 of 3 independent experiments.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

8. Figure 7
TME5-treated BMDCs induced less severe aGVHD. In BALB/c → C57BL/6 allo-HSCT model, lethally irradiated recipients (WT or GPR15KO) were transplanted with donor-derived TCD-BM (5 × 106/mouse) and spleno-T cells (5 × 106/mouse). Recipient-derived WT or GPR15KO BMDCs were primed with rmTNF-α for 24 hours in the presence of PBS or TME5 (100 ng/mL) and were co-transferred to WT or GPR15KO recipients, respectively (10 in each group). Survival (A) and GVHD scores (B) were monitored every 2 days. (C) Blood samples were collected at indicated time points and were subjected to CBA analysis for concentration of cytokines (4 in each group). (D) Livers and colons were collected on day 14 (4 in each group). Total RNA were extracted from tissue lysates followed by qPCR analysis for mRNA levels of cytokines. Statistical comparison of survival was performed with log-rank test. Data are shown as mean ± SD and are compared using 1-way ANOVA test.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

9. Figure 8
Proposed models of TME5-mediated anti-inflammatory function. GRK indicates G-protein coupled receptor kinases; p50 and p65, p50 and p65 subunits of NF-κB; ERK, extracellular signal-regulated kinase.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions