Decitabine Can Increase the Induction of Regulatory γδ T Cells with Enhanced Immunosuppression on Graft-Versus-Host Disease From Adult Human Peripheral Blood Mononuclear Cells
Authors: Hu Yongxian , Gu Yanjun , Sheng Lixia , Fu Huarui , Wu Kangni , Zhang Lifei , Liu Lizhen , Fu Shan , Xu Yulin , Xiao Haowen , Yu Xiaohong , Huang He 
Published: October 2019

Abstract
Abstract 1901
Regulatory γδ T cells (γδ Tregs) is a novel subset of cells with immunosuppressive function while methods for γδ Treg induction is rarely introduced and its role in graft-versus-host disease (GVHD) prevention remains unkown. Decitabine, a kind of hypomethylating agents, can act synergistically with TGF-β1 to convert a variety of αβ T cells to regulatory αβ T cells with suppressive function but its role in induction and function of γδ Tregs has not been reported. We show here the role of decitabine for the induction of γδ Tregs. Moreover, we provide functional analysis and underlying mechanisms of decitabine-induced γδ Tregs relative to γδ Tregs without decitabine induction as well as in vivo evidences of their preventions on GVHD. Human peripheral blood mononuclear cells (PBMCs) were cultured with IL-2, IL-15, TGF-β1 and zoledronic acid (ZOL). On day 2, 0.5μmol/ml decitabine was added to aliquots of PBMCs. On days 4, 7, and 10, half of the supernatant volume was replaced with media containing cytokines. On day 11, frequencies of γδ Tregs were detected by flow cytometry (FACS). We found the frequency of γδ Tregs was 36.2% in TGF-β1/IL-15/ZOL stimulated group (referred to as common γδ Tregs below) and 59.9% in IL-2/TGF-β1/IL-15/ZOL/decitabine stimulated group (referred to as decitabine-induced γδ Tregs) (p<0.05). In order to compare immunosuppressive function of the two populations, γδ T cells containing γδ Tregs were isolated by magnetic cell sorting system (MACS) and tested for their ability to suppress proliferation of alloreactive PBMCs using CFSE-based assay. After 5 days of in vitro culture, CFSE-labeled PBMCs proliferation was significantly reduced in the presence of enriched γδ Tregs even at 8:1(PBMCs: γδ Tregs) ratio. The inhibition rate was significantly different (decitabine-induced γδ Tregs VS common γδ Tregs at ratio 1:1 is 81.3% VS 68.2%, p<0.05). To clarify the underlying mechanisms we performed ELISA to measure levels of inhibitory cytokines IL-10, IL-4 and TGF-β1 in supernatant of CFSE-based assay. We noted an elevated IL-10 secretion in the decitabine-induced γδ Tregs group compared with common γδ Tregs group (92.7±11pg/ml VS 10.3±2pg/ml at ratio 1:1, p<0.01). We confirmed the result by intracellular IL-10 detection using FACS. Previous reports showed high levels of inducible T-cell costimulator (ICOS) were correlated with IL-10 synthesis. So γδ Tregs were monitored for ICOS expression by FACS. The result revealed that ICOS expression was up-regulated in decitabine-induced γδ Tregs in contrast to common γδ Tregs (MFI: 268 VS 54). Stability of Foxp3 is a critical factor in the immunosuppressive ability of Tregs. Thus we evaluated the frequency of γδ Tregs after 5 days in CFSE-based assay. We observed loss of Foxp3 expression in decitabine-induced γδ Tregs was negligible (<3%) while 15.5% common γδ Tregs lost foxp3 expression. To confirm the results in vitro we tested the functional ability to prevent GVHD in vivo. GVHD was induced in NOD/SCID mice following busulfan and anti-CD122 condition and 1×107 PBMCs transfusion. Animals were co-injected with either decitabine-induced γδ Tregs or common γδ Tregs at a ratio of 1:1. Survival time and GVHD manifestations of the transplanted mice were evaluated. As a result, transplantation of human PBMCs alone induced lethal GVHD with average survival time 25± 8 days while the survival time was 43± 5 days and 58±7 days in mice co-injected with common γδ Tregs and decitabine-induced γδ Tregs, respectively (p<0.05). Clinical manifestations such as hunched back, diarrhea, and body weight loss were statistically different among 3 groups. To investigate the infiltration of human lymphocytes into nonlymphoid tissues in GVHD mice, we performed immunohistochemical analysis of the liver and intestines using anti-human CD45. Remarkably abundant invasion of human CD45+ cells was observed around the veins in the liver and intestines transplanted with PBMCs alone while less invasion in mice co-injected with common γδ Tregs and the lest invasion in mice co-injected with decitabine-induced γδ Tregs. Altogether, our findings reveal that decitabine and the cytokines can efficiently syngenerize to induce γδ Tregs with enhanced immunosuppression on GVHD which are via higher levels of IL-10 production due to ICOS up-regulation as well as stability of Foxp3 expression. Thus γδ Tregs may be potentially exploited therapeutically in a variety of transplantation settings.

Disclosures:
No relevant conflicts of interest to declare.