Enhancement Of Human Regulatory γδ T Cells In Vitro Induced By Granulocyte Colony-Stimulating Factor
Authors: Wu Xiuli , Xuan Li , Wang Xu , Yu Sijian , Yi Zhengshan , Dai Min , Zhang Yu , Zheng Zhongxin , Liu Qifa , Li Yangqiu 
Published: October 2019

Abstract

Background and Objective
Graft versus host disease (GVHD) is the main complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Regulatory γδ T cells (γδ Tregs), which express Foxp3 and primarily belong to CD27+CD25high phenotype, are a novel subset of cells with immunosuppressive function and have the potential application prospect in GVHD therapy. Peripheral blood mononuclear cells (PBMCs) could be induced to generate γδ Tregs in vitro by stimulating with anti-TCR γδ and transforming growth factor-β (TGF-β). Our previous studies demonstrated that granulocyte colony-stimulating factor (G-CSF) had immunomodulatory effect on T cells; G-CSF mobilization influenced the distribution and clonality of TRGV and TRDV repertoire (T cell receptors of γδ T cells), and significant positive correlation was observed between the invariable clonality of TRDV1 gene repertoire after G-CSF mobilization and low incidence of GVHD in recipients. To further characterize the immunoregulatory functions of G-CSF, this study explored the possibility of γδ Tregs induced by G-CSF in vitro.

Methods
PBMCs of healthy donors were cultured in vitro by stimulating with anti-TCR γδ and different cytokines for 9-12 days. The culture system was grouped based on the difference of added cytokines, including: (1) TGF-β (2) G-CSF (3) TGF-β+G-CSF (4) blank group. The induced cells were used as effector cells in the carboxylfluorescein diacetate succinimidyl ester (CFSE) assays and cell immunophenotyping was analyzed by flow cytometry. Autologous CD4+T cells were purified by microbeads, labeled with CFSE, used as responder cells, and finally co-cultured with effector cells. After 5 days incubation, cells were harvested and analyzed for flow cytometry by gating on the CFSE-labeled cells. The expression levels of Foxp3 and CD25 genes were detected by real-time polymerase chain reaction.

Results
After 9 days’ culture in vitro, the proportion of Foxp3+ γδ T cells was 7.87% in the blank group, which was significantly lower than that in the TGF-β, G-CSF and TGF-β + G-CSF group (62.3%, 52.9% and 63.5%) (P<0.001). The proportions of CD25+γδ T and CD27+γδ T cells were 3.9% and 3.5% in the blank group, which were also significantly lower than that in the TGF-β, G-CSF and TGF-β + G-CSF group (CD25+γδ T cells: 43.5%, 43.4% and 44.4%; CD27+γδ T cells: 41.5%, 41.9% and 44.0%, respectively) (P<0.001, P<0.001). The effector cells induced by anti-TCR γδ with TGF-β, G-CSF and TGF-β + G-CSF all manifested a more significant suppressive effect on responder cells than cells induced only by anti-TCR γδ. However, there was no significant difference in the effect of cell proliferation suppression in the TGF-β, G-CSF and TGF-β + G-CSF groups. The expression level of Foxp3 gene was 0.384% in the blank group, which was significantly lower than that in the TGF-β, G-CSF and TGF-β + G-CSF group (0.623%, 0.639% and 0.843%, respectively) (P<0.001). The expression level of Foxp3 in TGF-β+ G-CSF group was significantly higher than that in the TGF-β and G-CSF group (P=0.001, P=0.007). The expression level of CD25 gene was similar among the four groups (P=0.457).

Conclusions
The generation of γδ Tregs could be enhance in vitro induced by G-CSF. This may be one of the reasons that G-CSF play an immunoregulatory role on decreased GVHD onset during allo-HSCT.

Disclosures:
Wu: National Natural Science Foundation of China (No. 81200388); the Technology Plan of Guangdong Province of China (No. 2012B031800403); the project of the Zhujiang Science & Technology Star of Guangzhou city (No. 2013027).: Research Funding.