Immunobiologic Properties Of Dendritic Cell Subsets From The Donors' Peripheral Blood Mobilized With G-CSF

To research the immunobiologic properties of different DC subsets derived from the PBMCs which mobilized with G-CSF,this study was designed to include the following two aspects.Part 1 Immunobiologic Properties of Monocytes Derived Dendritic Cells from the Donors' Peripheral Blood Mobilized with G-CSFMethodsDC derived from the adherent PBMCs were propagated in vitro under the condition of GM-CSF and IL-4 for 7 days then divided into groups and induced with IL-10,ATG or TNFa for another 2 days.To collect the supernatant and cells.DC were identified with flow cytometry,ELISA,antigen phagocytosis,CFSE attenuated test to examine the expressing of HLA-DR,CD 1a,CD11c,CD40 and CD80,the secretion of IL- 12(P70),the function of antigen phagocytosis and the activity of allogeneic stimulus response.ResultsImmature DC can be generated and the yield of CD1a⁺ DC derived from adherent monocytes was 1.62±0.57×10⁴ per 2×10⁶ PBMCs after induced with GM-CSF and IL-4 for 9 days.After induced with IL-10,ATG and/or TNFa:1.The expressing of HLA-DR,CD11c,CD40,CD80 and the secretion of IL-12 increased obviously,and the activity of allogeneic stimulus response enhanced after induced with TNFa.2.The expressing of CD1a on DC in group IL-10/TNFa increased and other expressing or the secretion of IL-12,the activity of allogeneic stimulus response all decreased compared with group TNFa.3.The expressing of HLA-DR,CD11c,CD40,CD80,the secretion of IL-12,and the activity of allogeneic stimulus response decreased in group ATG/TNFa compared with TNFa.4.In group ATG/TNFa the expressing of CD1a decreased,CD11c and CD80 increased obviously,and the activity of allogeneic stimulus response enhanced compared with group IL-10/TNFa.5.The immature DC could be induced to mature under the condition of TNFa and the phagotrophic function decreased obviously.6.The expressing of HLA-DR or CD11c on iDC derived from the donors which mobilized with G-CSF was lower than the healthy blood donors as control,and the expressing of CD11c,CD40,CDS0,the serection of IL-12 and the activity of allogeneic stimulus response were lower after induced with TNFa too.Conclusions1.It could be generated immature DC from the donors' PBMCs whith mobilized G-CSF. The immature DC could be induced to mature with TNFa,and IL-10 or ATG could assign their tolerogenic properties.2.The phenotypes and function of those DC from the donors which mobilized with G-CSF lower partly than the healthy blood donors'.3.Although the yield of CD1a⁺DC was inferior to the blood donors',but we obtained a large number of PBMCs from a single donor which mobilized with G-CSF.Therefor the PBMCs which mobilized with G-CSF probably become a important resource of different DC.Part 2 Immunobiologic Properties of Different Dendritic Cell Subsets from the Donors' Peripheral Blood Mobilized with G-CSFMethodsMyeloid dendritic cells(MDC) or plasmacytoid dendritic cells(PDC) were separated by immunomagnetic beads and then induced with TNFa or CpG₂₀₀₆OND to mature MDC(mMDC) or mature PDC(mPDC).The methods of investigation as follows: 1.Phenotype of DC subsets were identified by flow cytometry;2.The ailogeneic T-cell-stimulating capacity were determined by MLR;3.The lebels of supernatant INF-Y and IL-10 were detected by ELISA after MLR,the secretion of INF-γand IL-4 in CD4+ cells were identified by flow cytometry;4.The proportion of CD4⁺CD25^highT lymphocytes or expression of Foxp3mRNA were detected by flow cytometry or RT-PCR after MLR.Results1.The purity of separated MDC was about ninety-six percent.MDC express high constitutive levels of HLA-DR,CD11c and CD33,moderate levels of CD4,considerable low levels of CD40,CD45RA,CD80,and the percentage of CD40,CD80 was up-regulation after induced with TNFa.The purity of separated PDC was about ninety-five percent.PDC expressed high constitutive levels of HLA-DR,CD4 and CD45RA,considerable low levels ofCD11c,CD33,CD40,CD80,and the percentage of CD40,CD80 was up-regulation after induced with CpG2006 ODN.2.MDC especially mMDC had strong potential to stimulate allogeneic T lymphocytes proliferation,while PDC and mPDC had a weak stimulate potential.3.The secretion of INF-γamong T lymphocytes after stimulated by mMDC were higher than MDC or PDC.The secretion of IL-10 increased obviously after stimulated by PDC specially mPDC.4.CD4⁺ cells expressed more IFN-γthan the control in group MDC and mMDC,which the mMDC was the most,while the PDC had no effect.All the groups of DC had no influence on the expressing of IL-4 inside of CD4⁺ cells.5.MDC and mMDC had no influence on inducing of CD4⁺CD25^high regulatory T lymphocytes in MLR,but PDC had the effect,the effect of mPDC was more obviously.6.The expressing of Foxp3 mRNA had no significant difference in all groups. Conclusions1.Although the expressing of HLA-DR was high,the two directly isolated DC subsets from PBMCs were in the state of immature and could be induced to mature in the suitable condition.No matter how MDC were in the state of mature or immature,they had strong potential to stimulate allogeneic T lymphocytes proliferation and promoted T lymphocytes to product IFN-γ,and the raise of secretion probably promoted the polarization of Th1.2.Unlike mDC,PDC were less efficient capture,process and present antigens to the MHC molecules and so had low efficiency of presenting antigens and weak T-cell-stimulating capacity.Although PDC promoted the production of IFN-γamong T lymphocytes,it seemed that were not produced by Th1.PDC could not promote Th2 cells to product IL-4,which the polarization of Th2 probably reflected the production of IL-10.3.PDC could induce the generation of CD4⁺CD25^high regulatory T cells.