Induced CD4+CD25+T Lymocytes In RhG-CSF Mobilized Peripheral Blood By TGF-β₁

Objectives: 1) To sort CD4+CD25+ T lymphocytes and CD4+CD25– T lymphocytes from rhG-CSF ( human recombination granulocyte colony stimulating factor ) mobilized peripheral blood. 2) To induce CD4+CD25– T lymphocytes from rhG-CSF mobilized peripheral blood become CD4+CD25+ T by TGF-β1,and analyze the character of phenotype , we also value the the function of suppress through suppresive experiment.3) To improve the function of TGF-β1 in induce CD4+CD25– T lymphocytes turn to CD4+CD25+ T lymphocytes.Methods: Collected ten doners'blood of peripheral blood stem cell transplantation, the ten doners were given rhG-CSF10μg/(kg·d) five days as routine, at the five day,collected their blood through blood seperation instrument as mobilezed peripheral blood (G-PB),before they were given the injection of rhG-CSF,we also collect unmobilezed peripheral blood (PB).And then,we isolated CD4+CD25–T lymphocytes from G-PB and PB with a CD4+CD25+T lymphocytes selection kit by MACS(magnetic activated cell sorting), induced the cells with TGF-β1, used FCM ( flow cytometry),RT-PCR(reverse transcription-PCR),cell proliferation and suppression assay to test the expression of CD25+, Foxp3 and suppressive function,compaired the difference of the rate and the suppressive activity of CD4+CD25+T cells between 2 groups. Results: 1) Both G-PB and PB can get >90% high purity of CD4+CD25–T lymphocytes by MACS. 2) After treated with anti-CD3mAb and TGF-β1,there was a difference between CD4+CD25–T lymphocytes isolated from G-PB and PB,the expression of CD25+ were (77.9±2.3)% and (65.7±4.2)% respectively(P<0.05); 3) RT-PCR showed that G-PB derived CD4+CD25+T cells induced by TGF-β1 expressed Foxp3 higher than which derived from PB ; 4) The cells isolated from both G-PB and PB induced through TGF-β1 had suppressive properties,they all can suppress the proliferation of monocyte from healthy volunteer. The cpm of 3H-TdR incorporation assay were 11739±352 and 18732±437, positive and negative control were 38283±328,1300±94 (P<0.05).Conclusion: 1)We can sort CD4+CD25+ T lymphocytes and CD4+CD25– T lymphocytes from rhG-CSF mobilized peripheral blood by MACS. 2) TGF-β1 play a important role in induce CD4+CD25– T lymphocytes turn to CD4+CD25+T lymphocytes,which can induce CD4+CD25– T lymphocytes from G-PB and PB became CD4+CD25+T lymphocytes and the latter cells have suppressive function,RT-PCR shows they all express Foxp3. 3)rhG-CSF mobilized peripheral blood can be the important source of CD4+CD25+ regulatory T lymphocytes.