The Role Of STAT1 In Immune Tolerance Induction Of T Cells Form Allo-HSCT Donor Pretreated By G-CSF

Objective: Hematologists, in their clinical practice, has noticed that despite the presence of a higher number of T cells in peripheral blood hematopoietic stem cell (PBHSC) grafts than that in bone marrow grafts, the incidence and severity of acute graft-versus host disease (aGVHD) is surprisingly lower after PBHSC transplantation than that after bone marrow transplantation. This phenomenon supports a critical role for G-CSF as a novel mediator of T cell tolerance. It is also well known that Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, one of all-important signal pathways in immunity, plays an important role in the process of T cell tolerance induction. At present, STAT1 as a key molecule in the development of GVHD and dendritic cell (DC) maturation has been proved by many experiments. However, the expression levels of the JAK-STAT members involved in the processes of G-CSF induced T cell tolerance remain unknown. Therefore, we measured the expression level of STAT1 mRNA by Real-Time Quantitative PCR in our study and preliminarily analyzed its role in the process of G-CSF induced T cell tolerance.Methods: 1 Sample Collection: Each 10ml of the periphery blood specimen was collected from each of the 23 peripheral blood hematopoietic stem cell donors before and after the treatment of recombined human granulocyte colony stimulating factor (rhG-CSF) as mobilization agent from May of 2006 to July of 2007.2 Groupings:The samples were divided into two paired groups: group A (the periphery blood specimen from the 23 donors before treated with G-CSF as mobilization agent) and group B (the periphery blood specimen from the 23 donors after treated with G-CSF as mobilization agent).3 Collecting CD4+ T lymphocytes by using MACS: First the mononuclear cells were separated from the collected periphery blood samples, then, the CD4+ T cells were obtained by using MACS separating technique. The purity and the number and of the sorted CD4+ T cells was measured and counted with FCM.4 The ratio of Th1 versus Th2 cells was measured by FCM.5 RNA extraction and cDNA synthesis: total RNA was extracted from the CD4+ cells obtained form donors by using Trizol kit, and cDNA was synthesized by reverse transcription reaction.6 The expression level of G-CSFR,T-bet, GATA-3 and STAT1 was measured by Real-Time Quantitative PCR(RQ-PCR). 7 Cultures of the CD4+ T cells were cultured with G-CSF at different concentration. 24 or 48 hours later, the proliferation of CD4+ cell were detected using MTT method.8 Total RNA was extracted from the CD4+ cells after cultured by using Trizol kit, and cDNA was synthesized by reverse transcription reaction.9 The expression level of G-CSFR,T-bet, GATA-3 and STAT1 in the cultured cells was measured by Real-Time Quantitative PCR(RQ-PCR). Results:1 The ratio of Th1 to Th2 decreased after the treatment with G-CSF.2 A statistically significant increase was confirmed in the number of CD4+T cells and in the ratio of CD4+T cells versus all of the mononuclear cells from donors after who were treated with G-CSF.3 No significant difference in the expression level of G-CSFR,T-bet,GATA-3,STAT1 in the CD4+ T cells between before and after G-CSF treatment was detected.4 Proliferative index (PI) were increased dependent on the stimulate time when purified CD4+ T cells were cultured in the presence of G-CSF. PI gets the peak after 24-48 hours. PI decreased markedly after 72 hours. 5 After stimulated by G-CSF, there are significant differences in the expression level of G-CSFR,T-bet,GATA-3,STAT1 in the CD4+ T cells.Conclusions:1 G-CSF decreased the ratio of Th1 to Th2, which may be one of the mechanisms of T cell tolerance induced by G-CSF2 .G-CSF stimulated the proliferation of CD4+T cells in the donors after 3-day treatment.3 G-CSF stimulated the proliferation of CD4+T cells in vitro.PI gets the peak after 24-48 hours and decreased markedly after 72 hours.4 The distinction expression level of G-CSFR between vivo and vitro may caused by the difference environment and concentration of G-CSF between vivo and vitro.5 The distinction expression level of GATA-3 and T-bet between vivo and vitro may caused by the difference environment and concentration of G-CSF between vivo and vitro.6 The expression level of STAT1, an important signal molecule for Th1 cell immunological reaction, was not decreased in vivo but do decrease in vitro, This result may be explained by the crosstalk between STAT1 and other members in the complicated signal transduction pathways in vivo, and by its potential regulation effect on the genesis and function maintenance of Treg cells. Further studies are necessary to explore the precise role of STAT1 in T cell tolerance inducted by G-CSF.