Expression of Th17/Tregs Axis Associated Regulatory Factors in Patients with Acute Graft-Versus-Host Disease
Authors: Xuan Li , Wu Xiuli , Gao Li , Wu Meiqing , Jin Zhenyi , Wang Xinyu , Xu Yan , Liu Hui , Fan Zhiping , Li Yangqiu , Liu Qifa 
Published: October 2019

Abstract
Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the most valid methods for the treatment of malignant hematological diseases. Graft-versus-host disease (GVHD)，which remains the main complication after allo-HSCT，leads to the high transplant-related mortality. GVHD occurs as a result of T cell activation. Acute GVHD (aGVHD) is suggested to be predominantly related to T helper 1 (Th1) responses. Recent investigations have discovered the possible role of Th17 cells and regulatory T cells (Tregs) in the development of aGVHD. Th17 cells are found to have a direct role in the development of aGVHD, and adoptive transfer of in vitro-differentiated Th17 cells is capable of inducing lethal aGVHD. Clinical and experimental studies have suggested that Tregs could prevent and treat aGVHD, while preserve graft-versus-tumor activity. However, the controls of Th17/Tregs axis on the occurrence and development of aGVHD are not fully understood. The aim of this study is to investigate the expression levels and clinical significance of Th17/Tregs axis associated regulatory factors in patients with aGVHD.
Methods The expression levels of Th17/Tregs axis associated regulatory factors (IL-17A, IL-23R, RORc, STAT-1, STAT-3, Foxp3, CD25, CTLA-4, GITR and TLR8) were analyzed in peripheral blood from 20 patients with aGVHD at the following two time points (at the onset of aGVHD and two weeks after the treatment), using real-time reverse transcription polymerase chain reaction with SYBR Green I staining. Fifteen patients responded effectively to the treatment within two weeks, and 5 did not respond within two weeks. Twenty healthy donors were selected for the control. The β2-microglobulin gene was used as an endogenous reference, and the relative mRNA expression level of each gene was evaluated by the 2-ΔCt×100% method.
Results The expression levels of RORc, STAT-1 and STAT-3 genes in the untreated patients with aGVHD were significantly higher than that in the healthy donors (P<0.001, P<0.001 and P<0.001), while the expression levels of IL-17A and IL-23R genes were similar between the untreated patients with aGVHD and the healthy donors (P=0.147, P=0.190). The expression levels of GITR and TLR8 genes in the untreated patients with aGVHD were also significantly higher than that in the healthy donors (P=0.003, P<0.001). The expression levels of Foxp3, CD25 and CTLA-4 genes were similar between the untreated aGVHD patients and the healthy donors (P=0.298, P=0.934 and P=908). The expression level of TLR8 gene in the patients with aGVHD was significantly decreased after effective treatment (P=0.035).The expression levels of IL-17A, IL-23R, RORc, STAT-1, STAT-3, Foxp3, CD25, CTLA-4 and GITR genes in untreated patients with aGVHD had no significant change after effective treatment (P=0.778, P=0.875, P=0.730, P=0.177, P=0.925, P=0.397, P=0.778, P=0.470 and P=0.638). The expression level of Foxp3 gene was significantly decreased after ineffective treatment (P=0.043). The expression levels of IL-17A, IL-23R, RORc, STAT-1, STAT-3, CD25, CTLA-4, GITR and TLR8 genes were similar in patients at aGVHD onset and after ineffective treatment (P=0.686, P=0.500, P=0.686, P=0.345, P=0.225, P=0.345, P=0.345, P=0.893 and P=0.345).
Conclusions Th17/Tregs axis associated regulatory factors might influence in the occurrence and development of aGVHD．

Disclosures
Xuan: It was supported by National Natural Science Foundation of China (81270647, 81300445, 81200388); National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding. Wu:It was supported by National Natural Science Foundation of China (81270647, 81300445, 81200388); National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding; It was supported by the Technology Plan of Guangdong Province of China (2012B031800403); the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding. Liu:It was supported by National Natural Science Foundation of China (81270647, 81300445, 81200388); National High Technology Research and Development Program of China (863 Program) (2011AA020105); National Public Health Grand Research Foundation (201202017): Research Funding; It was supported by Natural Science Foundation of Guangdong Province (S2012010009299); the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1);: Research Funding; It was supported by the Technology Plan of Guangdong Province of China (2012B031800403); the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding.