Differential Proteomic Study Of Human CD4～+ CD25～+T Cells And CD4～+ CD25～+ T Cells

Several mechanisms contribute to immune tolerance, including the thymic deletion of autoreactive T cells and the induction of anergy in the periphery. In addition to these passive mechanisms, evidence has accumulated for the active suppression of autoreactivity by a population of regulatory T cells.A large quantity of literature identifies naturally occurring CD4+CD25+ T cells as key regulatory T cells involved in the maintenance of self-tolerance also the control graft rejection. The enhancement of regulatory-cell function might prove useful for the treatment of immune-mediated diseases,whereas the downregulation of these cells might be beneficial for the enhancement of the immunogenicity of vaccines that are specific for tumour antigens.Although the role of CD4+CD25+ regulatory T cells(Treg )in maintenance of immune tolerance has been proved by sufficient studies.The molecular basis for the suppressive activity of CD4+CD25+ Treg remains a matter of debate.Technologies of differential display proteomics provide a platform for systematic comparative research at the protein level.The strategy combining two-dimensional electrophoresis (2D-GE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry ( MALDI-TOF-MS) is most common in practice. It has proven to be very valuable in the screen and identification of function-associated molecules,biomarkers and drug targets.To explore the molecular basis for the suppressive activity of human CD4+CD25+ T cells, We applied differential proteomic technology to analyze differential proteins of human CD4+CD25+ and CD4+CD25- T cells.We isolated CD4+CD25+ and CD4+CD25" T cells and performed function assays.By magnetic activation cell sorting(MACS),we obtained higher purity of human splenic CD4+CD25+ and CD4+CD25" T cells.The proliferation assays of isolated CD4+CD25+ and CD4+CD25- T cells showed that CD4+CD25+ T cells were anergic or hyporesponsive relative to CD4+CD25- T cells.Indirect and direct suppression assays evidenced the suppressive properties of CD4+CD25+ T cells.Then we separated the whole cell proteins of CD4+CD25+ and CD4+CD25- T cells bytwo dimensional gel electrophoresis and obtained well displayed gel maps.The digitized 2-D maps were were quantitatively analyzed using 2-D analysis software packages. By analyzing the differential expression of proteomes between the two cell subsets, a proteome differential expression map was created. The proteome patterns of two cell subsets are similar on the whole. Most of protein spots concentrated in a gel area covering p/ from 4 to 8 and Mr from 14 to65 kDa. Average 704 protein spots were detected in CD4+CD25T cells by gel image analysis, and 685 spots in CD4+CD25+ T cells. Based on querying the quantitatively and qualitatively variable proteins between two cell subsets using ImageMaster 2D Database software and statistical data analysis, There were 25 protein spots whose expression levels showed significant variations(fold of difference > 4), 17 of them had higher levels in CD4+ CD25- compared to CD4+CD25+ T cells, and 8 spots vice versa.By peptide mass fingerprinting strategy,we identified 13 proteins derived from gels of CD4+ CD25- T cells and 2 proteins from those of CD4+CD25+ T cells among 25 differential spots. The proteins we found expressed at higher levels in CD4+CD25' T cells included enzymatic and structural proteins, signal transduction proteins (LCKBP1 and lasp-1 ) ,HSP70 family(Bip protein) and apoptosis associated protein(B chain of Caspase-7) ,etc. However,two proteins we found expressed at higher levels in CD4+CD25 +T cells were both proteins related to nucleic acid metabolism.These results will facilitate further functional studies.