The Effect of G-CSF Mobilization on the Expression of Immunoregulatory-Associated Molecules and G-CSFR Gene in the Peripheral Blood γδ+ T Cells
Authors: Xuan Li , Wu Xiuli , Dai Min , Jin Zhenyi , Wang Xinyu , Yu Sijian , Xu Yan , Liu Hui , Fan Zhiping , Li Yangqiu , Liu Qifa 
Published: October 2019

Abstract
Background Granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cell has been used more frequently than bone marrow as the source of stem cells in allogeneic hematopoietic stem cell transplantation. Although it contains more mature T cells, neither the incidence nor the severity of acute graft-versus-host disease (aGVHD) is higher compared with bone marrow transplantation. This might be due to the immunoregulatory effects of G-CSF on T cells, including that G-CSF directly modulated via its receptor on T cells or indirectly modulated T cell immune responses via effector cells and cytokines. Recent studies have shown that γδ+ T cells have immunoregulatory function, and might also participate in the pathogenesis of GVHD. However, these mechanisms are not fully understood, and whether G-CSF could influence the immunoregulatory-associated gene expression of γδ+ T cells remains unknown. The aim of this study is to investigate the effect of G-CSF mobilization on the expression of immunoregulatory-associated molecules and G-CSFR gene in the peripheral blood γδ+T cells.
Methods Peripheral blood γδ+ T cells were sorted by magnetic activated cell-sorting system. The expression levels of immunoregulatory- associated molecules (Foxp3, CD25, CTLA-4, GITR, TLR8, STAT-1, STAT-3 and RORc) and G-CSFR gene were analyzed in peripheral blood γδ+ T cells from 10 donors before and after G-CSF mobilization, using real-time reverse transcription polymerase chain reaction with SYBR Green I staining. 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 Foxp3, CD25, CTLA-4 and GITR genes in peripheral blood γδ+ T cells were similar before and after G-CSF mobilization (P=0.827, P=0.667, P=0.053 and P=0.129). The expression level of TLR8 gene in peripheral blood γδ+ T cells after G-CSF mobilization (0.731% ± 0.350%) was significantly higher than that before mobilization (0.188% ± 0.176%) (P=0.001). The expression level of STAT-1 gene in peripheral blood γδ+ T cells did not differ significantly before and after G-CSF mobilization (P=1.000), while the expression levels of STAT-3, RORc and G-CSFR genes in peripheral blood γδ+ T cells after mobilization (0.536% ± 0.234%, 0.683% ± 0.250%, 8.208% ± 6.175%) were significantly higher than that before mobilization (0.243% ± 0.134%, 0.356% ± 0.179%, 1.947% ± 1.442%) (P=0.003, P=0.003 and P=0.007).
Conclusions G-CSF mobilization might increase the expression levels of TLR8, STAT-3, RORc and G-CSFR genes in peripheral blood γδ+T cells.
The result suggested that G-CSF might play the role of immunoregulation by affecting γδ+ T cells.

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.