The Mechanism Study Of G-CSF-induced T-lymphocyte Polarization Toward A Type-2 Phenotype

Objectve:G-CSF are now be widely used in stem cells mobilization in peripheral blood stem cells(PBSC).Despite a 10-fold increase in the number of CD3 +cells in the apheresis product used for the transplantation of stem cells, patients undergoing peripheral blood stem cells transplantation(PBSCT)do not delnonstrate an apparent increased incidence or severity of acute graft-versus-host disease(GVHD).The Recently,new properties of G-CSF as a modulator of the immunological response were discovered.G-CSF may affect T-cell homeostasis by multiple mechanisms,inducing polarization of cytokine secretion on of T-cell proliferation,and enhancement of T-cell apoptosis.CD4+ lymphocytes can be functionally subdivided into TH1 and TH2 cells based on their pattern of cytokine expression.TH1 cells can secrete tumor necrosis factor(TNF)-αand IFN-γ, whereas TH2 cells are capable of producing IL-2,IL-4,and IL-10.Recent results have suggested that acute GVHD is mediated by TH1 lymphocytes and that TH2 lymphocytes may exert a protective effect but may have a role in mediating chronic GHVD.Therefore,in this study we investigated the effects of G-CSF on TH1 and TH2 cell development in mitogen-stimulated human CD4+ cells and studied the mechanisms of G-CSF-mediated changes Within the lymphocyte subpopulations.Methods:In this study,peripheral blood for in vitro studies was obtained from normal volunteers.For studies involving in vivo G-CSF stimulation of normal donors,samples were obtained from normal stem cell donors before and after 5 days of G-CSF treatment(10μg/kg subcutaneously daily)before apheresis donation.Peripheral blood mononuclear cells(PBMCs)from normal unstimulated donors were separated using density gradient centrifugation with lymphocyte separation media.For lymphocyte subset separation,cells were applied to either a CD4+ affinity column.Cultures of normal unstimulated donor cells were cultured with G-CSF at different concentration.24 or 48 hours later,the proliferation of CD4+ cell were detected using MTT method;The antigen response capability were measured by mixed lymphocyte reaction(MLR).Cells were then surface stained with anti-CD4 and intracellularly stained with either anti-IFN-γor anti-IL-4.Samples were analyzed using an flow cytometer to get the TH1/TH2 ratio.Total RNA was extracted,and the real-time RT-PCR were preformed to detected the gene expression.Some gene include the G-CSFR,IL-4,IFN-γand polarization related gene(CD28,CTLA-4,ICOS,GATA3,T-bet,FOXP3) were detected.Result:When PBMC preparations,purified CD4+cells(above 95%pure by flow cytometry)were cultured in the presence of G-CSF.After the G-CSF stimulation,the proliferative index(PI)were increased dependent on the stimulate time.After 4-8 hours,the PI began to increase,then,get the peak after 24-48 hours.After 72 hours,the PI decreased markedly.The data of LTT shown the G-CSF can restrain the CD4+ cell reactivity to PHA stimulation.The antigen response capability were both decreased and had no different with the G-CSF stimulation in vitro or in vivo.The ratios of TH1/TH2 were descended after the stimulation.Some gene's expression level were increased after the G-CSF stimulation,for example IL-4,CTLA-4,GATA3;while,the other gene's expression were decreased,for example IFN-γand T-bet gene.There were significant relativity between IFN-γand T-bet gene,and also between IL-4 and GATA-3 gene.Conclusion:Investigations performed by our group in healthy subjects treated with G-CSF to mobilize PBMC have already demonstrated a directly effect.G-CSF can induce suppression of lymphocyte proliferation after mitogenic challenge.Our results demonstrated that G-CSF treatment modulates the ratios of TH1/TH2 of CD4+T-cell.The ratio's reduction may be caused by the GATA3 gene's increase and the T-bet gene's decrease. Objectives:Along with the development of cellular immunology and molecular biology,cell-mediated immunity have been developed in the clinical experiment,and have got determinate curative effect. Cytokine Induced Killer cells(CIK)as a new type of immune competent cell already used in many kinds of malignant tumor.Dendritic Cells(DC)as the most efficient antigen presenting cell,can handle and present Ag to mediate humane immune reaction.AS many tumor host can not induce antigenic specificity T cellullar immunologic response because of the lost of DC.How to induce functionality DC to use in the activity immunity has its important meanings.There have been many reports at follows,as a cytokine,FLT3-L can successfully induce mature DC include plasmacytoid lymphocyte DC and it can induce proliferation inhance specificity.Our research investigate different cytokines in the process of induce mature DC,comparing different co-cultures of DC-CIK and analysis its functions to tumor cells to support a reference for clinical use.Methods:The first stage of the experiment:The cells are collected from peripheral blood mononuclearcell(PBMNC).To use FLT3-L as the experiment group,GM-CSF as the control group.Cells were cultured according to the schedule,flow cytometry and immunofluorescence quantitative analysis were used to analysis immunophenotype of DC. The second stage of the experiment:Cells were collected from two resources,the first patient was a hemophagocytic syndrome who infected EBV,the second patient was a AML who was in paracmasia,EBV antigen peptide were added in the process of DC's cultivate and co-cultured CIK in the latter process of the patient with hemophagocytic syndrome.Comparing if it can cultivate single clone and inhance anti-tumour activity.Using two kinds of cytokine separately in the process of DC's cultivate and co-cultured with CIK in the latter process.Comparing differences between them with cell account,flow cytometry,genescan and cytotoxicity to find the best project.Results:1 Comparing the two kinds of cytokines in the process of DC's cultivate.Cells in both experiment group and control group are taper in the process of cultivate without significant differences. The total cellular score in both group is 2×107 before culture. The cellular score of FLT3-Lgroup in the third day is 1.10×107, GM-CSF group is 1.20×107;the cellular score of FLT3-Lgroup in the seventh day is 0.80×107,GM-CSF group is 0.80×107;the cellular score of FLT3-Lgroup in the tenth day is 0.60×107,GM-CSF group 0.53×107.The analysis of immunophenotype by flow cytometry:cells before culture:CD14+11.41%,CD83+0.28%,CD80+0.01%,CD86+1.32%. CD4+CD123+1.23%;cells after cultivated with FLT3-L:CD14+49.22%, CD83+6.96%,CD80+33.48%,CD86+52.05%,CD4+CD123+4.25%;cells after cultivated with GM-CSF:CD14+72.21%,CD83+2.90%,CD80+ 41.23%,CD86+68.31%,CD4+CD123+1.16%;mature DC increased after culture,the phentype of De in the two group have significant deviations.As the cellular percentage of mature DC is higher in GM-CSF group.2 Comparing the cultural method of DC-CIK in patient with hemophagocytic syndrome who infected EBVCell growth conditions and cell score:the number of DC has no significant change after 3 days culture.Barb shape of tuber can be seen on the cells in the inverted microscope at the second day of culture and they.showed half adherence.CIK proliferate after MabCD3 and MabCD28 added in the fourth day as clones can be seen. The speed of cell proliferation have no significant differences between the group of DC-CIK and CIK..The Cell population are 6.3 and 5.8 times more than before after 14 days' culture.Whe survival rate of cells in the culture is upon 95%.The analysis of immunophenotype by flow cytometry:DC before culture:HLA-DR+CD86+12.05%,CD83+2.32%,CD80+0.00%;DC after culture with GM-CSF:HLA-DR+CD86+91.17%,CD83+7.28%,CD80+36.20%。CIK before culture:CD3+74.88%,CD8+30.02%,CD3 CD8+28.08% CD3+CD56+1.27%;DC-CIK without EBV Ag:CD3+97.59%,CD8+55.49%, CD3+CD8+54.95%,CD3+CD56+8.50%;DC-CIK with EBV Ag:CD3+95.62%, CD8+53.55%,CD3+CD8+52.89%,CD3+CD56+8.44%。The analysis of genescan before and after culture:there has been a monoclone peak at TCRβ5.2 in the whole 24 TCRβ.Gaussian distribution can be seen in both DC-CIK without EBV Ag and DC-CIK with EBV Ag。The analysis of IFN-γsecretion:the ratio between effective cells and target cells are 5:1,10:1,20:1 separately.Toobtation the supernatant and dilute triple,using IFN-γELISA kit to determine the content of IFN-γ。Comparing CIK before culture,DC-CIK without EBV Ag,DC-CIK with EBV Ag,the ratio are 1:1.4:2.7;1:1.2:1.7; 1:1.1:1.7 separately,It indicated that according to different ratio, the group of DC-CIK with EBV Ag is 1.4~1.9times more than the group of DC-CIK without EBV Ag.and its cell cytotoxicity is powerful at the same time.3 Comparing the cultural method of DC-CIK in patient with AML who is in paracmasiaCell growth conditions and cell score:the number of DC has no significant change after 3 days culture.Barb shape of tuber can be seen on the cells in the inverted microscope at the second day of culture and they showed half adherence.CIK proliferate after MabCD3 and MabCD28 added in the fourth day as clones can be seen. The speed of cell proliferation have no significant differences between the group of DC-CIK and CIK..The cell population are 6.6 and 6.2 times more than before after 14 days' culture.The survival rate of cells in the culture is upon 95%.The analysis oF immunophenotype by flow cytometry:DC before culture:HLA-DR+CD86+6.90%,CD83+0.27%,CD80+0.13%,CD14+73.85%; DC after culture with FLT3-L::HLA-DR+CD86+76.79%,CD83+3.17%, CD80+11.87%,CD14+55.33%;DC after culture with GM-CSF: HLA-DR+CD86+43.87%,CD83+1.40%,CD80+6.74%,CD14+19.80%。The number of mature DC are increased in both group.Comparing the degree of maturity,FLT3-L is higher than GM-CSF。CIK before culture::CD3+78.57%,CD8+61.542%,CD4+22.85%,CD3+CD8+55.20%, CD3+CD56+5.03%;DC-CIK after culture with FLT3-L:CD3+91.43%, CD8+81.83%,CD4+12.59%,CD3+CD8+80.29%,CD3+CD56+47.31%;DC-CIK after culture with GM-CSF;CD3+91.97%,CD8+78.6%,CD4+12.56%, CD3+CD8+76.90%,CD3+CD56+57.72%。The immunophenotype of CD3+,CD8+,CD3+CD8+,CD3+CD56+are increased after culture,the average amplification are 12.09%,18.67%,23.40%,47.48%;the immunophenotype of CD4+are decreased after culture,the average decrease is 10.27%。Comparing the two group,FLT3-L had no significant amplification than GM-CSF,but the immunophenotype of CD3+CD56+is higher in GM-CSF about 10.41%。The analysis of genescan.before and after culture:Gaussian distribution can be seen in both group and there have no significant differences between them。The analysis of IFN-γsecretion:To obtation the supernatant and dilute five times,using IFN-γELISA kit to determine the content of IFN-γ。Comparing CTK before cultre,DC-CIK in FLT3-L group,DC-CIK in GM-CSF group,the ratio are 1:4:3,the average number according to 3 different holes separately,It indicated that the gruop of DC-CIK in FLT3-L is 33.3%more than the group of DC-CIK in GM-CSF.Conclusion:1 In the process of 14 day's co-culture with DC-CIK,we cultivated DC and CIK successfully and proved its feasibility in the condition of decurtate co-culture's time.2 It is:feasible for the patient with hemophagocytic syndrome who infected EBV to carry out DC-CIK adoptive immunotherapy,and it can be an adjunctive therapy in the clinical.The addition of EBV antigen in the process of DC's cultivate can stimulate T lymphocyte to produce single clone.3 It is feasible for the patient with AML who is in paracmasia to carry out DC-CIK adoptive immunotherapy,and it can be an adjunctive therapy in the clinical to decrease microresidual disease.4 Comparing the two kinds of cytokines,FLT3-L display a few superiority in the vitro cultivate process of DC,but not obviously.As its price is higher than GM-CSF and can not get easily, its use is restrict.