Expression of Regulatory γδ T Cells in Patients with Acute Graft-Versus-Host Disease
Authors: Xuan Li , Gao Li , Wu Xiuli , Fan Zhiping , Huang Fen , Yi Zhangshan , Li Yangqiu , Liu Qifa 
Published: November 2019

Abstract
Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the most effective therapy for hematologic malignancies. Acute graft-versus-host disease (aGVHD) is a major complication after allo-HSCT and leads to high transplant-related mortality. Regulatory γδ T cells (γδ Tregs), as a novel subset of γδ T cells with Foxp3 expression and immunosuppressive effect, are found to effectively alleviate aGVHD in mice model. However, the effect of γδ Tregs 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 γδ Tregs in patients with aGVHD.
Methods The immunophenotyping of γδ Tregs was analyzed in peripheral blood from 15 patients with aGVHD at the following two time points (at the onset of aGVHD and two weeks after the treatment), using flow cytometry. Twelve patients responded effectively to the treatment within two weeks, and 3 did not respond within two weeks. The expression levels of immunoregulatory-associated molecules (Foxp3, CD25, CTLA-4, GITR, TLR8, RORc, STAT-1 and STAT-3) were analyzed in peripheral blood, using real-time RT-PCR with SYBR GreenⅠstaining. Liquichip technology was used to analyze the concentration of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10, IL-17 and tumour necrosis factor-α (TNF-α) in cultural supernatant.
Results The proportions of γδ T, Vδ1 T and Vδ2 T cells did not differ significantly between the untreated patients and those with effective treatment (P=0.308, P=0.117 and P=0.099). However, the proportions of Foxp3+γδ T, Foxp3+Vδ1 T and Foxp3+Vδ2 T cells were significantly increased after effective treatment (P=0.023, P=0.008 and P=0.028). The mRNA expression levels of Foxp3, RORc and TLR8 genes were significantly increased after effective treatment (P=0.060, P=0.001 and P=0.041). The expression levels of CD25, CTLA-4, GITR, STAT-1 and STAT-3 genes were similar between the untreated patients and those with effective treatment (P=0.95, P=0.421, P=0.605, P=0.186 and P=0.809). In addition, the concentration of IL-10 was decreased after effective treatment (P=0.005), and the concentration of IFN-γ, IL-4, IL-17 and TNF-α was similar between the two groups (P=0.662, P=0.314, P=0.152 and P=0.254). At the same time, we found that the proportion of Foxp3+γδ T cells and the expression level of Foxp3 gene both had a decreased tendency after ineffective treatment. The concentration of IL-10 had a increased tendency after ineffective treatment.
Conclusions γδ Tregs might influence in the occurrence and development of aGVHD.

Disclosures
Liu: Science and technology planning project of Guangdong Province (2014B020226004);: Research Funding; the project of health collaborative innovation of Guangzhou city (201400000003-1, 201400000003-4, 201508020254).: Research Funding; National Natural Science Foundation of China (81270647, 81300445, 81470349, 81400141, U1401221, 81401315).: Research Funding; Natural Science Foundation of Guangdong Province (2014A030310171, 2016A030310390) ;: Research Funding.