G-CSF Affects the Distribution and Clonality of TRGV and TRDV Repertoire of T Cells and the Expression Pattern of CD3 Genes
Authors: Xuan Li , Wu Xiuli , Liu Qifa , Zhang Yu , Fan Zhiping , Li Yangqiu , Ling Yiwen 
Published: October 2019

Abstract
Abstract 1946
The immune modulatory effect of granulocyte colony-stimulating factor (G-CSF) on T cells resulted in an unexpected low incidence of graft-versus-host disease (GVHD) in allogeneic peripheral blood stem cell transplantation (allo-PBSCT). The antigen recognition of T cells was based on a heterodimer composed either of the αβ chain or of the γδ chain of the TCR, which was associated with four transmembrane proteins, named CD3γ, CD3ε, CD3δ and CD3ζ, respectively. Recent data indicated that γδ+ T cells might participate in mediating graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation. However, whether G-CSF could influence the T cell receptors (TCR) of γδ+ T cells (TRGV and TRDV repertoire) and CD3 genes remains unclear. To further characterize this feature, we compared the distribution and clonality of TRGV and TRDV repertoire of T cells and the expression pattern of CD3 genes before and after G-CSF mobilization, and investigated the correlation between the changes of TCR repertoire and GVHD in patients undergoing G-CSF mobilized allo-PBSCT.
The complementarity-determining region 3 (CDR3) sizes of three TRGV and eight TRDV subfamily genes were analyzed in peripheral blood mononuclear cells (PBMCs) from 20 donors before and after G-CSF mobilization, using RT-PCR and genescan technique. To determine the expression levels of TRGV subfamily genes and CD3 genes, we performed quantitative analysis of TRGVI∼III subfamilies and CD3γ, δ, ε and ζ chain genes by real-time PCR.
All of three TRGV subfamily expression levels were decreased significantly after G-CSF mobilization. The pattern of TRGV subfamily expression levels was TRGVII>TRGV I>TRGV III before mobilization, and changed to TRGV I>TRGV II>TRGV III after G-CSF mobilization. Most TRGV and TRDV subfamilies revealed polyclonality from pre-G-CSF-mobilized and G-CSF-mobilized samples. Oligoclonality was detected in TRGV and TRDV subfamilies in 3 donors before mobilization and in another 4 donors after G-CSF mobilization. The expression patterns and clonality of TRGVII, TRDV3 and TRDV1 were found more frequently to change after G-CSF mobilization. Significant positive correlation was observed between the invariable clonality of TRDV1 gene repertoire after G-CSF mobilization and low incidence of GVHD in recipients (P=0.015, OR=0.047). In addition, the expression patterns of CD3 genes before and after G-CSF mobilization were CD3ζ> CD3ε> CD3δ > CD3γ. The expression levels of CD3 ζ and ε genes after G-CSF mobilization (2.194%, 0.867%) were significantly lower than that before mobilization (7.615%, 1.839%) (P=0.031, P=0.009); while the expression levels of CD3 δ and γ genes before mobilization (1.097% and 0.271%) were not significantly different from that after G-CSF mobilization (0.333% and 0.077%) (P=0.093, P=0.070). Significant positive correlation was observed between the relative expression levels of CD3 ζ chain genes before and after G-CSF mobilization (rs=0.576,P=0.016), but no significant correlation of gene expression levels were found in CD3γ, δ and ε genes before and after G-CSF mobilization. The expression level of CD3ζ genes before and after G-CSF mobilization and CD3δ gene after mobilization all respectively showed a negative correlation to the age of donors (rs= −0.549, P=0.023; rs= −0.679, P=0.003; rs= −0.570, P=0.017).
In conclusion, G-CSF mobilization not only influences the distribution and clonality of TRGV and TRDV repertoire, but also influences the expression levels of CD3 ε and ζ genes. This alteration of TRGV and TRDV repertoire might play a role in mediating GVHD in G-CSF mobilized allo-PBSCT.

Disclosures:
Liu: National Natural Science Foundation of China (No.30971300), Guangdong Natural Science Foundation (No.10451051501005778), Science and Technology Planning Project of Guangdong Province of China (No.2009A030200007) and China Postdoctoral Science Foundation (: Research Funding.