Study On CD4~+CD25~+ Regulatory T Cells In Disease And Expanding CD4~+CD25~+ Regulatory T Cells In Vitro

There were T cells which produced specific reaction to autoantigen in normal adults, but one couldn't get immune disease because the control of various kinds of tolerance mechanism. The immunologists had different ideas to the exist of regulatory T cells for the past many years because of the scared mark of cells and molecular basis of depressant effect. After the detection of CD4+CD25+ regulatory T cells(Tregs) by Sakaguchi in 1995,they are in the focus of intense research in immunology.Naturally occurring CD4+CD25+regulatory T cells are generated in thymus,and they represent 5%-10% of total CD4+T lymphocytes in periheral blood and spleen of normal adults .Tregs display immunoanergic and immunosuppressive activity. Activated Tregs can inhibit the proliferation and cytokines secretion of CD4+ and CD8+ T cells,and also inhibit NK,B cells and DC.Tregs is a kind of multi-cytokine secreting(interleukin 4,interleukin 10 and TGF-beta),affecting T cell suppressing cell. Tregs inhibit the proliferation of effector cells by cell-cell contact and/or secreting soluble cytokine such as TGF-βand IL-10. Its function in autoimmune disease occurring and developing was firstly studied. And gradually its effects in organ transplantion,tumor immunity,chronic inflammatory reaction and persistent infection were also explored. But there are several limitations to use them: one is the rare populations.So it is a quention which immunologists and clinicians search for constantly that how to culture and expand Tregs in vitro and then utilize in vivo.Our projects are focusing on study the change and clinical significance of CD4+CD25+ T cells in normal adults and patients with morbid state,then in vitro isolation , purification and expansion of CD4+CD25+T cells and in vitro function assays, related mechanism studies,explore the express of CD4+CD25+ T cells in neonatal cord blood and in vitro isolation , purification and expansion of CD4+CD25+T cells in neonatal cord blood. Our study will contribute the experiment substructure to the clinical application of CD4+CD25+T cells in autoimmune disease,tumor and graft rejection.②The expression of Foxp3 between normal controls and patients:The percentage of Foxp3+ in CD4+CD25+, and CD4+CD25high T cells in patients with AA was lower than that in normal controls (P<0.05),and the percentage is also low in patients with autoallergic disease compare with normal controls(P<0.05). The percentage of Foxp3+ is higher than that in normal controls(P<0.01).At first,the percentage and absolute numbers of CD4+ T cells,CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells in peripheral blood monouclear cell(PBMC) were examined with flow cytometry in normal controls,25 patients with AA, 37 patients with autoallergic disease,60 patients with cancer. The expression of Foxp3 in CD4+CD25+, CD4+CD25low and CD4+CD25high T cells was analyzed by flow cytometry.The levels of Foxp3mRNA in PBMC were determined by RT-PCR.①The comparesion of the percentage CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells between normal controls and patients:Compare with normal controls, the percentage of CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells are decreased in patients with AA(P<0.05), especially in patients with SAA(P<0.001), and they are also decreased in patients with autoallergic disease (P<0.05). The percentage of CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells are incresed in patients with cancers(Ⅰ+Ⅱ),but the difference is ont significance(P > 0.05),and they are obviously increased in patients with cancers(Ⅲ+Ⅳ) (P<0.01).③The expression of Foxp3 mRNA between normal controls and patients:it is lower in patients with AA and autoallergic disease than that in normal controls,and it is higher in patients with cancer. These findings suggeste that the expression of Tregs and Foxp3 is down regulated in patiens with AA and autoallergic disease,lead to Tregs'losing the ability of immunesuppression.It maybe provide a new idea of immunotherapy in patients with AA and immuneallergic disease through increasing the expression of Foxp3. It means that tumor cells could induce the proliferation,amplification and activation of CD4+CD25+ Treg by expressing tumor antigens or secreting immune inhibition factors,so it can impair effects of effector cells on tumor,and improve the tumor survival and development.It maybe provide an original way to immunotherapy in patients with tumor by removing Tregs of patients with tumor or block up the related cytokine.And then,we make a study of isolation, purification and expansion of CD4+CD25+T cells in vitro.①We use the micro-magnetic beads to isolate the CD4+CD25+T cells. After the columns, the purity of the cells are usually about 80%-89%.It confirm the method of isolation is feasible.②The freshly purified CD4+CD25+T cells are anergic to the stimulators but they suppress the proliferation of CD4+ T cells and is dose-dependent.The inhibition ratio is 74% at a 1:2 ratio(CD4+:CD4+CD25+ T cells) by MTT.③The amplificated CD4+CD25+ T cells also keep their immunity characteristics,and the immunosuppressive action is enhanced.We adopted 2 kinds of concentration of IL-2(200u/mL and 500u/mL),and all plused anti-CD3/anti-CD28 beads. The data showed that it can be expanded by both of protocols but, in terms of yield, plate-bound IL-2 500u/mL is better than another one. Expansion gets its peak on day 9, then goes down.④The amplificated CD4+CD25- T cells can express the marker of CD25 but have no immunosuppression ability.Many studies have made remarkable progress along with understand to Tregs by immunologists,but we have ont know the molecule mechanism of Tregs about its proceed,growth and mature.In order to explore the feature of Tregs ,we quantify the proportion of CD4+CD25highFoxp3+ regulatory T cells in neonatal cord blood and adult peripheral blood with flow cytometry , and explore the clinical significance of Treg in neonatal cord blood,and isolate,dentify,expand Tregs. It showde that the percentage and absolute numbers of CD4+ T cells were both increased in neonatal cord blood (P<0.01),the percentage of CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells were decreased in neonatal cord blood(P<0.05),but their absolute numbers were increased(P<0.01) compared with adult PBMC. The percentage of Foxp3 in CD4+CD25+ and CD4+CD25high T cells in neonatal cord blood were both lower than that in adult peripheral blood(P<0.01), the expression levels of Foxp3 mRNA were also lower than that in adult peripheral blood(P<0.05). It can't distinguish CD4+CD25+ T and CD4+CD25-T cells in adult peripheral blood with flow cytometry ,but they can be distinguished well in neonatal cord blood.We observe the high express of Foxp3 in CD4+CD25highT cells.It confirm that CD4+CD25highT cells play a important role which educe the ability of immunosuppression in regulatory cells. We examine the express of Foxp3 in neonatal cord blood no matter with flow cytometry or RT-PCR,the expression levels of Foxp3 are both lower than that in adult PBMC.Tregs of neonatal cord blood maybe incipient T cells and seldomly be stimulated by antigen,so it can't be activated.Tregs can obtain suppression function by reexcitation of antigen in periphery even though there are more Tregs in thymus. We isolate CD4+CD25+T cells from neonatal cord blood with micro-magnetic beads, the purity of the cells are usually about 83%-85%. The freshly purified and amplificated CD4+CD25+T cells are anergic to the stimulators but they suppress the proliferation of CD4+ T cells and is dose-dependent. Expansion gets its peak on day 12.Our study proved the prospect of Tregs'clinical application.With the progress of medical science,we have used neonatal cord blood to treat many kinds of disease.It maybe provide theory basis of Tregs'clinical application and transplantation immunity. It's significance that induce CD4+CD25highT cells in neonatal cord blood to mature and then treat various kinds of autollergic diease,allergic disorder,supress allograft rejection.