Effects On The Proliferation And Killing Of CD4~+ CD25~+ Regulatory T Cells By DC Stimulated By Toll-like Receptor 8 Agonist In Vitro

Objective This experiment studied the effect of Toll-like receptor 8(TLR-8)agonist on the proliferation and function of CD4+CD25+regulatory T cells in patients with acute myeloid leukemia.DC loaded with CD34+leukemia cells were stimulated with TLR-8 agonist(VTX-2337)and co-cultured with CD4+CD25+regulatory T cells,to observe their effects on the proliferation activity of CD4+CD25+regulatory T cells.CD4+CD25+regulatory T cells treated with TLR-8 agonist(VTX-2337)were co-cultured with CD34+leukemia cells and CTL,to observe their effects on CTL killing CD34+leukemia cell function.From the proliferation and function of CD4+CD25+regulatory T cells,a deeper exploration of new ways of leukemia cell immunotherapy in vitro provides a new method for killing leukemia cells in vivo.Method After fully communicating the research content and purpose of the study with the patient or the patient authorizer,and sign the informed consent.5ml anticoagulant blood in bone was collected from patients with primary acute myeloid leukemia(according to WHO classification,except acute promyelocytic type)during the hospitalization of the patient.and mononuclear cells were obtained after centrifugation,and then CD34+leukemia cells were sorted by MACS method,and further frozen for further research;when the patient reached the complete remission period,20ml autologous peripheral anticoagulant was collected again,and centrifuged,CD14+PBMC,CD4+CD25+Tregs were also obtained by MACS method.Granulocyte-macrophage colony stimulating factor(GM-CSF),interleukin(IL)-4 and tumor necrosis factor(TNF)-α were added to CD14+PBMC in vitro system for mature DC,and stored frozen for subsequent research.Will mature DC fuse with CD34+leukemia cells induced apoptotic by daunorubicin and bortezomib for 2 hours to prepare DC loaded CD34+leukemia cell for subsequent research.Autologous peripheral blood from patients with complete remission was collected and centrifuged to obtain mononuclear cells,CD34+leukemia cell-loaded DC and IL-2(20 IU/ml)were used to induce CTL,and then CTL were sorted by MACS method,reserved for subsequent research.TLR-8 agonist(VTX-2337)was co-cultured with DC loaded with CD34+leukemia cells and CD4+CD25+Tregs to observe the proliferation activity of CD4+CD25+Tregs.CD4+CD25+Tregs were treated with TLR-8 agonist(VTX-2337)and co-cultured with CTL and CD34+leukemia cells to observe the CTL killing rate.Result(1)The proliferation activity of CD4+CD25+Tregs in control group was 23.73%±3.36%,and the proliferation activity of CD4+CD25+Tregs in the TLR-8 agonist group was 2.95%±1.23%,P＜0.01,the difference was statistically significant.The proliferation activity of CD4+CD25+Tregs in the TLR-8 agonist group was significantly lower than the control group,suggesting that DC loaded with CD34+leukemia cells stimulated by TLR-8 significantly inhibited the proliferation of CD4+CD25+Tregs;(2)At effective target ratio 12.5:1,the killing rate of the control group was 13.81%±1.77%,the killing rate of the TLR-8 agonist group was 46.98%±7.98%,P<0.01,and the difference was statistically significant.At effective target ratio 25:1,the killing rate of the control group was 27.15%±5.99%,and the killing rate of the TLR-8 agonist group was 50.88%±8.83%,P＜0.01,and the difference was statistically significant.The killing rate of the TLR-8 agonist group increased significantly,suggesting that the TLR-8 agonist inhibited the immunosuppressive function of CD4+CD25+Tregs and thus enhanced the CTL killing function.Conclusion DC loaded CD34+leukemia cell stimulated by TLR-8 agonists can inhibit the proliferation activity of CD4+CD25+Tregs,and TLR-8 agonists can inhibit the immunosuppressive function of CD4+CD25+Tregs,and enhance the antitumor immune function of CTL.It is suggested that TLR-8 agonists can enhance the anti-tumor effect through the above two mechanisms...