The Role Of Zinc Finger Protein A20in The Dendritic Cells Derived From Acute Myeloid Leukemia

The active specific immunotherapy with dendritic cells (DCs) has made apreliminary and positive effect in the clinical treatment for cancer patients. Especiallyin the follow-up treatment of acute myeloid leukemia (AML), it could effectivelycontrol and elimination of minimal residual disease (MRD) and prolonged theremission for patients after chemotherapy or transplantation. However, the normalDCs had fewer sources, additional antigen load process and other shortage. So manyresearchers wanted to develop new therapeutic strategies, including leukemia tumorcells derived DCs (L-DCs). At present, the successful separations of L-DCs arechronic myeloid leukemia cells derived DCs (CML-DCs) and acute myeloid leukemiacell derived DCs (AML-DCs). CML-DCs had better mature and effective stimulateallogeneic T lymphocyte reaction, and had been used in clinic in some places. But,AML-DCs had many defects in phenotypic, function and differentiation, andimmature AML-DC was easy to induce immune tolerance, which was limited itsapplication in clinic. A20is an ubiquitin-modifying enzyme with a special zinc fingerstructure. It can negative regulation of DCs mature, the production of cytokines andimmune activity by limited the NF-κB signaling activity. So far, no one has evaluatedthe relationship between A20and AML-DCs. Therefore, we made research anddiscussion for the effect and mechanism of A20in AML-DCs in this subject, and wehope the results could provide helps for AML patients with individualizedimmunotherapy.Firstly, we isolated the peripheral blood mononuclear cells (PBMCs) from AMLpatients with initial treatment and healthy donors. Then, we improved theconventional DCs culture method to induce the mature AML-DCs. AML-DCs was then compared with normal DCs in morphology and phenotype. The results showedthat the AML-DCs had typical dendritic cell morphology under the microscope.Compared to normal DCs, AML-DCs surface molecules expressions were lower, suchas CD80, CD83and CD86. The fusion gene detection proved AML-DCs derived fromleukemia cells.Secondly, according to the zinc finger protein A20gene structure features, wedesigned three siRNA to down the expression of A20. The delivery of siRNA intoAML-DCs by lipofection is performed. We detect the the siRNA transfectionefficiency, A20expression and the changes of AML-DCs’ phenotypic by flowcytometric, real-time PCR. siRNA transfection efficiency was approximately55%.The flow cytometry and real time PCR results implied that A20expression was downregulated in AML-DCs with siRNA transfection, and the maturity of AML-DCs withsiRNA transfection has been improved.Thirdly, the peripheral blood T cells of the same patient in remission areseparation and purification by Nylon wool column. The T cells in vitro survival ismaintenance with IL-2. The results showed that purified T cells were high expressionof CD3and CD8. And those T cells could survive for about7days with a lowconcentration IL-2sustain. After we used the T cells co-cultured with A20-downregulated AML-DCs to induce the CTLs, the expression of cytokines (IFN-γ, IL-2,IL-4, TNF-α, Gramzyme B and Perforin) and extracellular factor (IL-12p70, IL-6)were improved. The flow cytometry and killing experiment results proved that theCTLs induced by AML-DCs with A20down-regulated had better specificity andkilling activity.Finally, we detected the signaling pathway of A20in AML-DCs. We choose thekey protein (RIP, TRAF6and NF-kappa B) of NF-κB signal pathway upstream anddownstream based on the new research literature of zinc finger protein A20signalingpathway. Then we use Western blot to analysis of the signal pathway of A20inAML-DCs by compared the different of siRNA group and control group. The resultsconfirmed that A20inhibited activation of the NF-κB pathway through inhibition of the RIP and NF-κB p65pathways in AML-DCs matured with TNF-α.In conclusion, our data suggested for the first time that the zinc finger proteinA20is the negative regulatory factors of the AML-DCs’ maturation and function. A20down regulated made AML-DCs’ surface molecules CD83, CD80and HLA-DRexpression improved. The AML-DCs with A20down-regulated could effectivelystimulate the activation of T cells, which indicated that the AML-DCs’ antigenpresenting and immune-stimulating ability has been enhanced. The CTLs induced byAML-DCs with A20down-regulated had high specificity, and killed the AML tumorcells effectively. This study provides us to establish a new immunotherapy foranti-AML tumor cells is enhanced DCs vaccine with the A20down-regulated byRNAi technology.