The Expression of Th17-Associated Cytokines in Human Acute Graft-versus-Host Disease Yuejun Liu, Yifeng Cai, Lan Dai, Guanghua Chen, Xiao Ma, Ying Wang,

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The Expression of Th17-Associated Cytokines in Human Acute Graft-versus-Host Disease Yuejun Liu, Yifeng Cai, Lan Dai, Guanghua Chen, Xiao Ma, Ying Wang, Ting Xu, Song Jin, Xiaojin Wu, Huiying Qiu, Xiaowen Tang, Caixia Li, Aining Sun, Depei Wu, Haiyan Liu Biology of Blood and Marrow Transplantation Volume 19, Issue 10, Pages 1421-1429 (October 2013) DOI: 10.1016/j.bbmt.2013.06.013 Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 CD4+FoxP3+Treg cells decreased and Th17 cells increased in PBMCs of aGVHD patients. Peripheral blood of transplant patients was analyzed for CD4+FoxP3+Treg and Th17 cell percentages. (A) Representative plot of flow cytometric detection of CD4+FoxP3+Treg and Th17 cells. (B) (C) The percentages of Tregs and Th17 cells were evaluated in patients without aGVHD (grade 0), mild aGVHD (grade 1 to 2), severe aGVHD (grade 3 to 4), and healthy donors (HD). *P < .05, †P < .01, ‡P < .001. Biology of Blood and Marrow Transplantation 2013 19, 1421-1429DOI: (10.1016/j.bbmt.2013.06.013) Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 The expression of FoxP3 was down-regulated and RORγt expression was up-regulated in aGVHD patients. (A) Shows FoxP3 expression from PBMCs of patients without aGVHD (grade 0), patients with mild aGVHD (grade 1 to 2), patients with severe aGVHD (grade 3 to 4), and healthy donors (HD). (B) Shows RORγt expression from PBMCs of patients without aGVHD (grade 0), patients with mild aGVHD (grade 1 to 2), patients with severe aGVHD (grade 3 to 4), and healthy donors (HD). (C) RORγt expression negatively correlated with FoxP3 expression (r2 = .1237, P < .01). *P < .05, †P < .01, ‡P < .001. Biology of Blood and Marrow Transplantation 2013 19, 1421-1429DOI: (10.1016/j.bbmt.2013.06.013) Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Th17-associated cytokine expressions in GVHD patients. (A) IL-17, (B) IL-23, (C) IL-22, (D) TGF-β, (E) IL-6, (F) IL-21, (G) IL-1β, and (H) IL-23R expressions were analyzed from PBMCs of patients without aGVHD (grade 0), patients with mild aGVHD (grade 1 to 2), patients with severe aGVHD (grade 3 to 4), and healthy donors (HD). (A) IL-17 and (C) IL-22 expressions in CD4+ T cells were examined. (A) IL-17, (B) IL-23, and (C) IL-22 serum levels were also measured and compared among aGVHD (grade 0), aGVHD (grade 1 to 2), and aGVHD (grade 3 to 4) patients. *P < .05, †P < .01, ‡P < .001. Biology of Blood and Marrow Transplantation 2013 19, 1421-1429DOI: (10.1016/j.bbmt.2013.06.013) Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Correlation between Th17-related cytokine and FoxP3 and IL-17 expressions. Correlations among TGF-β (A), IL-6 (B) expressions, and FoxP3 expression were analyzed. Correlations among IL-17 expression and TGF-β (C), IL-6 (D), IL-1β (E), IL-21 (F), IL-22 (G), IL-23 (H), serum levels of IL-23 (I), and IL-23R (J) expressions are shown. Biology of Blood and Marrow Transplantation 2013 19, 1421-1429DOI: (10.1016/j.bbmt.2013.06.013) Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Dynamic changes of Th1, Th17, and FoxP3+ Treg Cells and IL-17, IL-23, and IL-22 expression during the onset and resolution of aGVHD (A) UPN 3, (B) UPN 5, (C) UPN 13, and (D) UPN 16 were measured for their Treg, Th1, and Th17 cell percentages before, during, or after the onset of aGVHD. Serum levels of IL-17, IL-23, and IL-22, as well as the transcripts of IL-17 and IL-22 RORγt in CD4+ T cells, were also examined. (E) Serum levels of IL-17, IL-23, and IL-22 in aGVHD patients were measured before, during, and after the onset of GVHD. Each line represents an individual patient. Biology of Blood and Marrow Transplantation 2013 19, 1421-1429DOI: (10.1016/j.bbmt.2013.06.013) Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions