| Objective CD4~+CD25~+regulatory T cell(Treg) has been identified recently as a new type of immuno-suppression cells. CD4~+CD25~+regulatory T cells, are characterized immuno-anergy and immuno-suppression. Their ability is to actively inhibit CD4~+CD25T cells, CD8~+ T cells, dendritic cells (DCs), natural killer (NK) cells, natural killer T (NKT) cells, in a cell-to-cell contact manner, and prevent activated CD8~+ T and NK from proliferating, and down-regulate costimulators expressed on DC. In short, CD4~+CD25~+regulatory T cell play an important function in maintaining immune homeostasis , suppressing all kind of nature and adoptive immune response. The prevalence of Treg in the peripheral blood of many cancer patients is increased when compared with normal individuals. In humans, initial studies have targeted the entire CD4~+CD25~+ circulating T cell population representing, as in mice, 5-10% of total CD4~+ T cells. CD4~+CD25~+regulatory T cell secrete IL-10, TGF-β, IFN-γand IL-4,but not IL-2. In addition to CD4 and CD25 markers, Treg also constitutively express CD152 (CTLA-4), CD45RB,CD44,ICAM-1,LFA-1,Foxp3 and GITR ,as well as chemokine receptor. Dendritic cells (DCs) are the most potent professional antigen presenting cells (APCs) in vivo. The mechanism of DC anti-tumor is DC can present tumor antigen peptides to T cells through the high expression MHC-I , II molecule, which can active T cells when associated with the high costimulation molecule expressed on DC surface such as B7-1(CD80),B7-2(CD86) and CD40. In vitro, CD4~+CD25~+regulatory T cell can suppress the function of DC, by the costimulation signals CTLA-4-B7. The above materials suggest CD4~+CD25~+regulatory T cell increase in the cancer patiets and suppress DC through CTLA-4 way. In order to demonstrate the deduction drawned above, the experiment was designed: put the Treg and DCs derived from CML patients and normal donors together to coculture, and the end, the suppression function of Treg on DC, was observed.Methods CD4~+CD25~+regulatory T cell were separated from peripheral blood of CML patients and normal donors by magnetic cell sorting(MACS) system. CTLA-4 expression on Treg were analyzed by flow cytometry. Bone marrow mononuclear cells (BMMNCs) derived from CML patients and healthy person were separately cultured to DC. We put them into four groups: A: CML-Treg coculturing with CML-DCs; B: CML-Treg coculturing with normal-DCs; C:normal-Treg coculturing with normal-DCs as control; D: normal-Treg coculturing with CML-DCs as control. Then adding anti-CTLA-4 to the coculturing system, the costimulator phenotypes (CD80, CD86) of DC were analyzed by flow cytometry, at the same time, the secretion of IL-10 and TGF-βwere analyzed by ELISA.Results The purity of sorted CD4~+CD25~+regulatory T cell was 85%—94%, with the survival rate of 92%～95%. CTLA-4 expression rate on Treg from CML patient and normal donor peripheral blood were 52.4%, 33.2% respectively. The costimulator phenotypes of DC had significant difference between before and after coculture with Treg(P<0.05). The secretion of IL-10 and TGF-βhad the high level on CML-Treg coculturing with CML-DCs group among the four groups, and the results were 629.22±124.04 pg/ml and 581.47±109.21 pg/ml (p<0.05).Conclusion We could successfully obtain Treg from peripheral blood and DC from bone marrow mononuclear cells after culturing. After they cocultured , the costimulators CD80 and CD86 expressed on DC were down-regulated, CD4~+CD25~+regulatory T cells secrete their associated cytokines. We observed the suppression function of Treg on DC, but the exact mechanism needs further research. The study provide a new way to increase function of DC and apply the DC vaccine.
|
PDF Full Text Request