1. Differential Effects of Gut-Homing Molecules CC Chemokine Receptor 9 and Integrin-β7 during Acute Graft-versus-Host Disease of the Liver 
Alina Schreder, Georgios Leandros Moschovakis, Stephan Halle, Jerome Schlue, Chun-Wei Lee, Angela Schippers, Sascha David, Günter Bernhardt, Arnold Ganser, Oliver Pabst, Reinhold Förster, Christian Koenecke 
Biology of Blood and Marrow Transplantation 
Volume 21, Issue 12, Pages (December 2015)
DOI: /j.bbmt
Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

2. Figure 1
β7 deficiency of donor T cells leads to reduced GVHD. Lethally irradiated BALB/c recipients were transplanted with 5 × 106 T cell–depleted BM and 1 × 106 C57BL/6 WT or integrin-β7 knockout T cells. (A) Survival of BMT recipients was monitored daily. (B) Mean percentage of starting body weight of BMT recipients was assessed twice per week. (C) Assessment of clinical GVHD scores of recipient mice was performed twice per week (n = 6 per group of WT and β7 knockout group, n = 2 for BM without T cells; 1 of 2 independent experiments is shown).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

3. Figure 2
CCR9 deficiency of donor T cells does not influence survival after BMT. (A) Lethally irradiated BALB/c recipients were transplanted with 5 × 106 T cell–depleted (TCD) BM and 1 × 106 C57BL/6 WT or CCR9 knockout (KO) T cells. Survival of BMT recipients was monitored daily. (B) Mean percentage and standard deviation (SD) of starting body weight of BMT recipients was assessed twice per week. (C) Assessment of clinical GVHD scores of recipient mice was done twice per week, and mean scores and SD are shown (n = 5 per group of WT and CCR9 KO group, n = 2 for BM without T cells; 1 of 2 independent experiments is shown). (D) Lethally irradiated BDF1 recipient mice were transplanted with 5 × 106 TCD BM and 1 × 106 C57BL/6 WT or CCR9 KO T cells. Survival of BMT recipients was monitored daily. (E) Mean percentage and SD of starting body weight of BMT recipients was assessed twice per week. (F) Assessment of clinical GVHD scores of recipient mice was done twice per week, and mean scores and SD are shown (n = 7 per group of WT and CCR9 KO group, n = 2 for BM without T cells; 1 of 2 independent experiments is shown). (G) Levels of inflammatory cytokines IFN-γ, MCP-1, and TNF-α were measured in the sera of C57BL/6 WT, β7 KO, or CCR9 KO T cell recipients (BALB/c) at days 7, 14, and 21 after BMT. Bars represent mean values, and error bars show the standard error of the mean. Data were pooled from 2 independent experiments (total n = 6 per group; n = 3 per group in each independent experiment; n = 1 for TCD BM, total n = 2).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

4. Figure 3
Reduced accumulation of β7 knockout (KO) donor T cells in target organs of acute GVHD. (A) Histopathology of colon, small intestine (SI), and liver of WT and integrin-β7 KO or (B) CCR9 KO recipients 21 days after BMT (total n = 5 to 6 per group; n = 2 to 3 per group in each independent experiment; n = 1 for T cell–depleted [TCD] BM, total n = 2). (C) Absolute numbers of donor CD4+ and CD8+ T cells in spleen (SPL), mesenteric lymph nodes (mLN), liver, and the gut of transplanted mice were analyzed on day 21 after BMT. After transplantation with WT or integrin-β7 KO T cells and TCD BM or (D) after transplantation with WT or CCR9 KO T cells and TCD BM. Data were combined from 2 independent experiments. Each dot represents the total cell count derived from 1 mouse (total n = 6 per group; n = 3 per group in each independent experiment). (E) Liver damage parameters glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were measured in the sera of integrin-β7 KO or (F) CCR9 KO recipient mice at day 21 after BMT. Bars indicate mean values. Error bars show standard error of the mean. Data were pooled from 2 independent experiments (total n = 6 per group; n = 3 per group in each independent experiment).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

5. Figure 4
Aberrant expression of MAdCAM-1 in the liver during experimental murine or human GVHD. Expression of MadCAM-1 was visualized using immunohistochemistry. Healthy mice were analyzed in comparison with GVHD animals at 21 days after transplantation. MAdCAM-1 expression can be found in Peyers's patches (PP) in untreated animals (A) but not in healthy liver (B). After induction of GVHD, MAdCAM-1 is expressed in the inflamed liver (C and D). One representative staining of 2 independent experiments is shown (E). Relative expression levels of MadCAM-1 mRNA in liver, small intestine, and colon at 3 weeks after transplantation was compared with T cell–depleted BM recipients using the ΔΔCt method. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) primers were used for the amplification of the housekeeping gene. HPRT was used for normalization. (n = 3). Representative data are shown from 1 of 2 experiments that gave similar results. Data are expressed as mean values ± standard error of the mean. (F) Expression of MAdCAM-1 in liver biopsies in patients suffering from acute, chronic, or post–donor lymphocyte infusion GVHD. MAdCAM-1 expression was visualized using immunohistochemistry and DAB signal amplification system. Two representative slides are shown in combination with isotype control and 1 patient without GVHD of the liver. In total, 15 GVHD patient samples and 1 control patient were analyzed.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

6. Figure 5
MadCAM-1 deficiency prolongs survival after allogeneic BMT and leads to reduced accumulation of donor CD4+ T cells in the intestine. (A) Lethally irradiated C57BL/6 or MAdCAM-1−/−recipients were transplanted with 5 × 106 T cell–depleted (TCD) BM supplemented with 3 × 106 T cells from BALB/c donors. Recipients of 5 × 106 TCD BM were used as control. Survival of BMT recipients was monitored daily. (B) Mean percentage and standard deviation (SD) of starting body weight of BMT recipients was assessed twice per week. (C) Assessment of clinical GVHD scores of recipient mice was done twice per week; mean scores and SD are shown. Data were combined from 2 independent experiments (total n = 10 or 11 per group; n = 5 to 6 per group in each independent experiment; n = 2 for TCD BM). (D) Histopathology of colon, small intestine (SI), and liver of WT and MAdCAM-1−/− mice 21 days after BMT (total n = 6 per group; n = 3 per group in each independent experiment; n = 1 for TCD BM, total n = 2). (E) Absolute number of donor CD4+ and CD8+ T cells in spleen (SPL), liver, mesenteric lymph nodes (mLN), and the gut of transplanted mice were analyzed at day 21 after transplantation of BALB/c donor T cells (+TCD BM) to WT or MAdCAM-1−/− recipient mice. Data were combined from 2 independent experiments. Each dot represents the total cell count derived from 1 mouse (total n = 6 per group; n = 3 per group in each independent experiment). (F) Expression of CD4, CD8, and integrin-β7 in the gut at day 21 post-BMT was visualized using immunohistochemistry. In total, 100 CD4 and 100 CD8 T cells were analyzed for the expression of integrin-β7 (n = 6 mice per group; n = 3 per group in each independent experiment). (G) Liver damage parameters glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were measured in the sera of MAdCAM-1−/− and WT recipient mice at day 21 after BMT. Bars indicate mean values. Error bars show standard error of the mean. Data were pooled from 2 independent experiments (total n = 6 per group; n = 3 per group in each independent experiment).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

7. Figure 6
Competitive homing of β7 knockout and WT T cells shows decreased infiltration of integrin-β7−/− lymphocytes to the intestine but not to the liver during active GVHD. TAMRA-labeled β7−/− lymphocytes and CFSE-labeled WT lymphocytes, and vice versa, were intravenously injected into BALB/c recipients at 3 weeks after allogeneic transplantation. Sixteen hours later, labeled lymphocytes were isolated from the intestine and the liver. Labeling effects were excluded by normalization of the ratio to 1:1 in each staining group (n = 3). Error bars show standard error of the mean, and bars show the mean of 2 independent experiments (total n = 6 per group).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions