| Hepatocellular carcinoma(HCC)is one of the most common malignant carcinomas in China.Hepatitis B virus(HBV)infection is the major cause of HCC in China.Currently,antiviral therapies have been proved to be the effective strategy to inhibit the replication of HBV virus for most of the HBV infection.However,they fail to eradicate HBV infected liver cells.The effect of screening for HCC in high-risk populations is still contraversial.Cellular immunity,demonstrated as the presence of antigen specific CD8+T cells is critical to eliminate the tumor cells in anti-tumor immunity.Glypican-3(GPC3),as one of the tumor-associated antigens,expressed in placenta and fetal liver,overexpressed in more than 70%HCC tissues,but not in normal liver tissues.Clinical studies demonstrated that HCC patients with higher anti-GPC3 specific CTL frequencies had a significantly longer overall survival.In this study,we aimed to induce anti-GPC3 specific cellular immunity to eliminate GPC3-expressing liver cells for preventing/delaying the development of primary liver cancer.Previous studies showed that TLR7/8 agonist(CL097)conjugated vaccines could enhance the antigen cross-presentation and induce antigen-specific immnune responses.In this study,we found that CL097 conjugated vaccines could also upregulate the expression of relative proteins in MHC-I pathway,such as TAP1/TAP2,Tapasin,and MHC-I alpha chain proteins with the comparison to antigen only treated dendritic cells with the help of HPLC/MS.To enhance the GPC3 immunogenicity,the mannosylated DOTAP liposomes were used as the antigen delivery system to target the monocyte-derived dendritic cells and CL097 was used as adjuvants.We synthesized liposome encapsulated GPC3 and CL097 vaccines(LP/Man-GPC3-CL097).The targeted monocytes matured to dendritic cells and upregulated the expression of MHC-I,CD83 and CCR7.To clarify the effect of CL097 conjugated liposomes encapsulated antigens on the intracellular antigen processing pathway,we treated DCs with different forms of antigens for 3 hours in 37℃.With the help of Immunofluorescence technique,we found that liposomes encapsulated antigens and CL097 were taken up by DCs more effectively,processed and presented through early endosome,Endoplasmic reticulum(ER)and Golgi apparatus in a MHC-I pathway,escaping from lysosomes,which is benifit for antigen cross-presentation on the cell surfaces.LP/Man-GPC3-CL097 enhanced the antigen uptake by CD11c+ cells and antigen delivery to draining lymph nodes and liver tissues in vivo.In LP/Man-GPC3-CL097 immunized wild type C57BL/6J mice,GPC3-specific IFN-y producing CD8+ T cells were elicited and liver infiltrated T lymphocytes secreted effective cytokines such as IFN-y and Granzyme B,and LP/Man was used as control.Meanwhile,liver infiltrated T lymphocytes could kill the GPC3-expressing Hepal-6 cell lines at the E/T ratio of 40:1.LP/Man-GPC3-CL097 immunization protects mice from GPC3+ tumor cell challenge in a CD8-dependent manner.To investigate the effect of LP/Man-GPC3-CL097 on HBV-related liver cancer development,HBV-transgenic mice received 25μg/g body weight diethylnitrosamine(DEN)via intraperitoneal injection at 2 weeks of age.On 8,10,12 and 14 weeks of age,mice were immunized with LP/Man-GPC3-CL097 respectively.LP/Man was used as control.By 22 weeks of age,tumor numbers(P<0.01)and tumor sizes(tumor≤1mm,P<0.05)decreased in the immunized mice.Meanwhile,the percentage of liver infiltrated IFN-y producing CD8+ T cells and secreted IFN-y and Ganzyme B were much higher than the control group.Conclusion:Liposome encapsulated tumor associated antigen GPC3 and adjuvants CL097 could accelerate dendritic cells maturation,enhance the uptake by dendritic cells,and upregulate the expression of relative proteins in MHC-I pathway.Immunization with LP/Man-GPC3-CL097 could induce the production of anti-GPC3 specific CD8+T cells and inhibit/delay the primary liver cancer development.Our study provide a possible strategy for HCC treatment in the future. |
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