Effect Of ShPD-1 On Function Of Dendritic Cells Derived From Patients With Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC) is one of most severe malignancy that enormously threaten human health and life, the death rate occupy the third position of the malignancy death[1]. In our country, the hepatocellular carcinoma fatality rate is only in behind of gastric carcinoma in country side while it got the second position behind pulmonary carcinoma in city. The incidence of HCC is on the increase and it is becoming more and more significant both clinically and epidemiologically. It is responsible for more than 500000 deaths with over 600000 new cases yearly worldwide[2]. HCC has severe malignancy, most frequent cause of mortality and worse prognostic. Meanwhile in our country, when most of the patients made a definite diagnose of HCC belong to advanced stage. However, only 10% to 20% of patients undergo surgery because of poor liver function, metastases, or both, and of those having surgery, the 5-year survival rate is only 25% to 50%[3]. There is no effective systemic therapeutic modalities have been established for patients with HCC. Therefore, the new effective way to inhibit the tumor cell growth will be the key in our present study. With the depth of tumor immunology research, it proved that there has existed a specific immune response for tumor in our body, which is pointed a new direction for cancer therapy, that immune therapy is an important means of cancer therapy, in which DC-based tumor vaccine immune therapy to become a focus for researchers in recent years. Now part of DC tumor vaccine has entered the phaseⅠ,Ⅱclinic trial[4]. Dendritic cells (DC) play a crucial role in the initiation of an immune response. They have the ability take up antigen efficiently. After processing, they present the antigen on their surface in association with MHC molecules, and stimulated naive T cells to proliferation and differentiation via their antigen-specific receptores[5]. Because of these immunostimulating properties, the DC may be effective for the prevention of carcinogenesis, as well as for the treatment of cancers. It has indicated that tumor vaccine using DCs as the vehicle to deliver cancer antigens for an effective induction of T-cell antitumor immunity and immunologic memory in vitro and animal trials. Most clinic trials indicate that DC immunotherapy has a favorable perspective[6].In our antitumor immunity mechanisms, the cytoimmunity mediated of T cell is main way. Two signals are required to activate a na?ve T cell. The first signal is initiated by T-cell receptor (TCR) recognition of antigen in the form of peptides presented by the MHC-Ⅱ. The second signal is mediated by engagement of the co-stimulatory molecule CD28 on na?ve T cells with its ligands,B7 costimulatory molecules which are expressed on mature DCs. Only the double signals costimulation can activate T cell utility[7].If second signal deficiency, it will induce T cells in the condition of clonal anerge or non-response[8].Accordingly, the costimulatory signal of B7/CD28 is significant effect for activation of T cells. In recent years,PD-1/PD-L1 as a member of B7/CD28 superfamily, the effect is appreciated by more and more people[9]. programmed death ligand-1( PD-L1) as a new member of B7 family is constitutively expressed at low levels on both hematopoietic cells, including resting T, B, myeloid, and dendritic cells[10].programmed death-1 (PD-1) expressed on activated T, B, and myeloid cells [11].Ligation of PD-L1 and PD-1 was shown to induce a co-inhibitory signal in activated T cells and to promote T-cell apoptosis, anergy, and exhaustion[12]. Foundation study indicate that the cytokines VEGF,IL-10,TGF-βwhich is exist in tumor microenvironment ,can induce expressed PD-L1 on dendritic cells up-regulation. It can make the dendritic cells derived from tumor patients in a condition of immature and functional disturbance, a low ability to activate T cell and not generate effective antitumor immunity. So it makes the tumor cells immune escape[13]. Recent studies indicates that PD-L1 is one of the important molecules in tumor immune escape, so blocking PD-L1/PD-1 inhibitory signals is a significance strategy in tumor immunity.Objective: To detect the effect of shPD-1 blocked PD-L1/PD-1 inhibitory signals on dendritic cells derived from patients with HCC in stimulate autoallergic T cells proliferation, secret cytokines and stimulate CTL killing activity. To investigate reinforce the immunostimulation ability of DC by blocking PD-L1/PD-1 inhibitory signals,Method: Peripheral blood mononuclear cells ( PBMCs) were isolated from peripheral blood of patients with solid tumor by Ficoll-hypaque gradient centrifugation. and then monocytes(Mo) were purified through adhensiveness. DCs were obtained after incubation of monocytes with the media containing granulocyte/macrophage colony stimulating factor (GM-CSF) ,interleukin-4 ( IL-4) and tumor necrosis factor-α(TNF-α).The phenotypes of DCs were detected by flow cytometry, mixed lymphocyte reaction was analyzed by MTT assay and IL -12p70 in the cell culture supernatant were detected by ELISA. The cytotoxicity of CTLs to cancer cells was assayed by MTT. Result: After blocking PD-L1 on DCs, DCs showed a stronger ability to stimulate T cells proliferation and secret IL -12p70. CTLs stimulated with DCs showed the higher killing effect on target cells(P<0.05). Conclusion: Blocking PD-L1 on DCs can significantly enhance their ability to activate T cells, and to promote antitumor immunity.