1. Absence of donor Th17 leads to augmented Th1 differentiation and exacerbated acute graft-versus-host disease
by Tangsheng Yi, Dongchang Zhao, Chia-Lei Lin, Chunyan Zhang, Ying Chen, Ivan Todorov, Thomas LeBon, Fouad Kandeel, Stephen Forman, and Defu Zeng
Blood
Volume 112(5):
September 1, 2008
©2008 by American Society of Hematology

2. IL-17−/− donor cells induced exacerbated acute GVHD
IL-17−/− donor cells induced exacerbated acute GVHD. (A) Sublethally irradiated BALB/c recipients were transplanted with graded numbers (1.25∼2.5 × 106) spleen cells and 2.5 × 106 TCD-BM cells from wild-type (WT) or IL-17−/− C57BL/6 donors.
IL-17−/− donor cells induced exacerbated acute GVHD. (A) Sublethally irradiated BALB/c recipients were transplanted with graded numbers (1.25∼2.5 × 106) spleen cells and 2.5 × 106 TCD-BM cells from wild-type (WT) or IL-17−/− C57BL/6 donors. The recipients were checked for clinical signs of GVHD and body weight every 5 days and monitored for survival daily. Body weight change and survival curves are shown. There were 12 mice in each group, and 3 replicated experiments were combined, except the group given TCD-BM alone had 4 recipients. (B,C) Histopathology of liver, gut, and lung of recipients given TCD-BM alone or TCD-BM and spleen cells (2.5 × 106) from WT or IL-17−/− donor 13 days after HCT. One representative histopathology and mean (± SE) of histologic score of 6 examined recipients in each group are shown. (D,E) Sublethally irradiated BALB/c recipients were transplanted with sorted T cells (0.5 × 106) from WT or IL-17−/− donors. Clinical score and survival curves are shown. There were 8 recipients in each group; 2 replicated experiments were combined. (F) Liver mononuclear cells of the recipients were stained for H-2b (donor marker), CD4, CD8, and intracellular IL-17 on day 10 after HCT. Gated H-2b+ cells are shown in IL-17 versus CD4 or CD8. The percentage of IL-17 CD4+ or CD8+ T cells is shown beside the gating boxes. Mean (± SE) of the IL-17+ CD4+ or CD8+ T cells in the liver of recipients given WT donor T cells are 0.69 (± 0.02) or 0.77 (± 0.08). IL-17+ T cells in the spleen of the recipients given IL-17−/− donor T cells were not detectable (< 0.05%).
Tangsheng Yi et al. Blood 2008;112:
©2008 by American Society of Hematology

3. Augmented Th1 differentiation in recipients given IL-17−/− donor cells.
Augmented Th1 differentiation in recipients given IL-17−/− donor cells. (A,B) Serum levels of IFN-γ and TNF-α of recipients given WT or IL-17−/− donor spleen cells 5 days after HCT. (C) Mesenteric lymph nodes (MLN) and spleen cells of the recipients given WT or IL-17−/− donor cells were stained for H-2b, CD4, CD8, and intracellular IFN-γ, 13 days after HCT. Gated H-2b+ cells are shown in IFN-γ versus CD4 or CD8. The percentage of IFN-γ+ cells is shown beside the gating boxes. One representative of 4 recipients in each group is shown. (D,E) Means (± SE) of the percentages of IFN-γ+ CD4+ or CD8+ donor T cells in MLN and spleen.
Tangsheng Yi et al. Blood 2008;112:
©2008 by American Society of Hematology

4. Increased infiltration of Th1 cells in liver of recipients given IL-17−/− donor cells.
Increased infiltration of Th1 cells in liver of recipients given IL-17−/− donor cells. (A) Liver mononuclear cells from recipients given WT or IL-17−/− donor cells were stained for H-2b, CD4, CD8, and intracellular IFN-γ, 13 days after HCT. Gated H-2b+ cells are shown in IFN-γ versus CD4 or CD8. The percentage of IFN-γ+CD4+ or CD8+ T cells is shown beside the gating boxes. One representative of 4 recipients in each group is shown. (B) Means (± SE) of the percentages of donor IFN-γ+ CD4+ or CD8+ T cells of the 4 recipients and of the yield of IFN-γ+ CD4+ or CD8+ T cells of the 4 recipients. (C) MLN cells of the recipients were stained for H-2b, CD4, CD8, and CCR5. Histogram of CCR5 (solid line) versus isotype control (shaded area) of H-2b+ CD4+ or CD8+ cells are shown. One representative of 4 recipients is shown. Means (± SE) of the percentage of CCR5+ cells among CD4+ or CD8+ donor T cells are 43.1% (± 3.9%) or 10.8% (± 2.0%) in recipients given WT donor cells and 67.6% (± 2.8%) or 23.3% (± 1.6%) in recipients given IL-17−/− donor cells. (D) Means (± SE) of chemokine expression levels by liver of the recipients given WT or IL-17−/− donor cells 5 days after HCT. Relative gene expression levels were normalized within each sample to the house keeping gene GAPDH and were presented relative to the expression in liver tissue of syngeneic HCT recipients. There were 4 mice in each group.
Tangsheng Yi et al. Blood 2008;112:
©2008 by American Society of Hematology

5. Neutralizing IFN-γ in recipients given IL-17−/− donor cells ameliorated acute GVHD. (A,B) BALB/c recipients given spleen cells (2.5 × 106) and TCD-BM cells (2.5 × 106) from IL-17−/− donors were injected intraperitooneally with anti–IFN-γ mAb or control rat ...
Neutralizing IFN-γ in recipients given IL-17−/− donor cells ameliorated acute GVHD. (A,B) BALB/c recipients given spleen cells (2.5 × 106) and TCD-BM cells (2.5 × 106) from IL-17−/− donors were injected intraperitooneally with anti–IFN-γ mAb or control rat IgG (0.5 mg/mouse) on days 0, 5, 10, and 15 after HCT. Body weight change and survival curves are shown. There were 8 mice in each group; 2 replicated experiments were combined. (C) Means (± SE) of serum levels of IFN-γ and TNF-α of the above recipients 5 days after HCT. (D) Means (± SE) of donor T-cell yield in liver of recipients treated with anti–IFN-γ or rat IgG 5 days after HCT. There were 4 recipients in each group. (E) MLN cells of the recipients treated with anti–IFN-γ or rat IgG were stained for H-2b (donor marker), CD4, CD8, and CCR5 or isotype control 5 days after HCT. Gated on H-2b+CD4+or CD8+ T cells, a histogram of anti-CCR5 (solid line) and isotype control (shaded area) is shown. One representative of 4 recipients in each group is shown. Means (± SE) of the CCR5+CD4+ T cells in recipients treated with anti–IFN-γ or rat IgG are 15.4% (± 1.5%) or 32.3% (± 1.4%). Means (± SE) of the CCR5+CD8+ T cells in recipients treated with anti–IFN-γ or rat IgG are 28.4% (± 1.1%) or 65.3% (± 5.2%). (F) Means (± SE) of chemokine expression levels in liver of the recipients treated with anti–IFN-γ or rat IgG 5 days after HCT. Relative gene expression levels were normalized within each sample to the house keeping gene GAPDH and were presented relative to the expression in synergic HCT recipients. There were 4 mice in each group.
Tangsheng Yi et al. Blood 2008;112:
©2008 by American Society of Hematology

6. Adding back of IL-17 to recipients given IL-17−/− donor cells ameliorated acute GVHD. (A,B) Recipients given spleen (2.5 × 106) and TCD-BM cells (2.5 × 106) from IL-17−/− donor cells were injected intraperitoneally with recombinant IL-17 (0.3 μg/mouse) on d...
Adding back of IL-17 to recipients given IL-17−/− donor cells ameliorated acute GVHD. (A,B) Recipients given spleen (2.5 × 106) and TCD-BM cells (2.5 × 106) from IL-17−/− donor cells were injected intraperitoneally with recombinant IL-17 (0.3 μg/mouse) on days 0, 3, and 7 after HCT. Body weight change and survival curves are shown. There were 12 recipients in each group; 3 replicated experiments were combined. (C,D) Histopathology of liver, gut, and lung of recipients treated with PBS or IL days after HCT. Arrow points to the necrosis area in liver and infiltrated epithelial and lamina propria areas in gut of recipients given PBS treatment only. One representative of histopathology and mean (± SE) of histology score of 6 examined recipients in each group is shown. (E) Mean (± SE) of the percentage of donor-type IFN-γ+CD4+ or CD8+ T cells in MLN, spleen, and liver of recipients treated with IL-17 or PBS 13 days after HCT. There were 4 recipients in each group.
Tangsheng Yi et al. Blood 2008;112:
©2008 by American Society of Hematology

7. IL-17 down-regulation of Th1 differentiation required host DCs
IL-17 down-regulation of Th1 differentiation required host DCs. (A-C) Sorted CD4+ T cells (2 × 105) from WT or IL-17−/− donors were stimulated with plate-bound anti-CD3 and anti-CD28 as well as IL-12 (10 ng/mL) in culture for 72 hours.
IL-17 down-regulation of Th1 differentiation required host DCs. (A-C) Sorted CD4+ T cells (2 × 105) from WT or IL-17−/− donors were stimulated with plate-bound anti-CD3 and anti-CD28 as well as IL-12 (10 ng/mL) in culture for 72 hours. The percentage of IFN-γ+ or IL-17+ cells among cultured donor CD4+ T cells were determined by intracellular staining, and the IFN-γ concentration in culture supernatant was measured by ELISA. One representative of 4 replicated intracellular staining experiments, the mean (± SE) percentage of IFN-γ+ cells among donor CD4+ T cells, and the mean (± SE) of the IFN-γ concentration in the culture supernatants are shown. (D-F) Sorted donor CD4+ T cells (2 × 105) from WT or IL-17−/− donors were cocultured with enriched DCs from irradiated host BALB/c mice for 96 hours. The percentage of IFN-γ+ or IL-17+ cells among cultured donor CD4+ T cells were determined by intracellular staining, and the IFN-γ concentration in culture supernatant was measured by ELISA. One representative of 4 replicated intracellular staining experiment, the mean (± SE) of the percentage of IFN-γ+ cells among donor CD4+ T cells, and the mean (± SE) of the IFN-γ concentration in the culture supernatants are shown. (G-I) Sorted naive CD44loCD4+ T cells from WT or IL-17−/− donors were cocultured with enriched DCs from irradiated host BALB/c mice for 5 days. The percentage of IFN-γ+ or IL-17+ cells among cultured donor CD4+ T cells were determined by intracellular staining, and the IFN-γ concentration in culture supernatant was measured by ELISA. Dead cells were excluded by fixable aqua dead cell stain kit. One representative of 3 replicated experiments, the mean (± SE) percentage of IFN-γ+ cells among donor CD4+ T cells, and the mean (± SE) of the IFN-γ concentration in the culture supernatants of triplicates are shown. (J) IFN-γ concentration in the culture supernatants of naive donor WT and IL-17−/− CD4+ T cells and host DCs with or without the presence of neutralizing anti–IL-12 Antibody. (K) IFN-γ concentration in the culture supernatants of IL-17−/− donor CD4+ T cells and host DCs with or without addition of IL-17 or IL-17 plus IL-12.
Tangsheng Yi et al. Blood 2008;112:
©2008 by American Society of Hematology