Combining Exosomes Derived from Immature DCs with Donor Antigen-Specific Treg Cells Induces Tolerance in a Rat Liver Allograft Model.

Allograft tolerance is the ultimate goal in the field of transplantation immunology. Immature dendritic cells (imDCs) play an important role in establishing tolerance but have limitations, including potential for maturation, short lifespan in vivo and short storage times in vitro. However, exosomes (generally 30-100 nm) from imDCs (imDex) retain many source cell properties and may overcome these limitations. In previous reports, imDex prolonged the survival time of heart or intestine allografts. However, tolerance or long-term survival was not achieved unless immune suppressants were used. Regulatory T cells (Tregs) can protect allografts from immune rejection, and our previous study showed that the effects of imDex were significantly associated with Tregs. Therefore, we incorporated Tregs into the treatment protocol to further reduce or avoid suppressant use. We defined the optimal exosome dose as approximately 20 µg (per treatment before, during and after transplantation) in rat liver transplantation and the antigen-specific role of Tregs in protecting liver allografts. In the co-treatment group, recipients achieved long-term survival, and tolerance was induced. Moreover, imDex amplified Tregs, which required recipient DCs and were enhanced by IL-2. Fortunately, the expanded Tregs retained their regulatory ability and donor-specificity. Thus, imDex and donor-specific Tregs can collaboratively induce graft tolerance.

Co-transfection of dendritic cells with AFP and IL-2 genes enhances the induction of tumor antigen-specific antitumor immunity.

Dendritic cells (DCs) are highly efficient, specialized antigen-presenting cells and DCs transfected with tumor-related antigens are regarded as promising vaccines in cancer immunotherapy. The aim of the present study was to investigate whether DCs co-transfected with the α-fetoprotein (AFP) and human interleukin-2 (IL-2) genes were able to induce stronger therapeutic antitumor immunity in transfected DCs. In this study, DCs from hepatocellular carcinoma (HCC) patients were co-transfected with the IL-2 gene and/or the AFP gene. The reverse transcription-PCR (RT-PCR) data revealed that the DCs transfected with the adenovirus AdAFP/IL-2 expressed AFP and IL-2. The DCs co-transfected with IL-2 and AFP (AFP/IL-2-DCs) enhanced the cytotoxicities of cytotoxic T lymphocytes (CTLs) and increased the production of IL-2 and interferon-γ significantly compared with their AFP-DC, green fluorescent protein (GFP)-DC, DC or phosphate-buffered saline (PBS) counterparts. In vivo data suggested that immunization with AFP-DCs enhances antigen-specific antitumor efficacy more potently than immunization with IL-2-DCs or AFP-DCs. These findings provide a potential strategy to improve the efficacy of DC-based tumor vaccines.

Tolerance induction by exosomes from immature dendritic cells and rapamycin in a mouse cardiac allograft model.

BACKGROUND: Dendritic cells (DCs) release bioactive exosomes that play an important role in immune regulation. Because they express low levels of class I major histocompatibility complex (MHC) and co-stimulatory molecules, exosomes derived from donor immature DCs (imDex) prolong allograft survival by inhibiting T-cell activation. However, this effect is limited and does not induce immunological tolerance when imDex are administered alone. Thus, we tested the effect of combined treatment with donor imDex and low-dose rapamycin on inducing tolerance in a mouse cardiac transplantation model. METHODS: ImDex were obtained from the culture supernatant of immature DCs derived from donor mouse (C57BL/6) bone marrow and were injected with suboptimal doses of rapamycin into recipient mouse (BALB/c) before and after transplantation. The capacity of this treatment to induce immune tolerance was analyzed in vitro and in vivo using the mouse cardiac transplantation model. RESULTS: Donor imDex expressed moderate levels of MHC class II and low levels of MHC class I and co-stimulatory molecules, but neither imDex nor subtherapeutic rapamycin dose alone induced cardiac allograft tolerance. Combined treatment with imDex and rapamycin, however, led to donor specific cardiac allograft tolerance. This effect was accompanied by decreased anti-donor antigen cellular response and an increased percentage of spleen CD4(+)CD25(+) T cells in recipients. Furthermore, this donor specific tolerance could be further transferred to naïve allograft recipients through injection of splenocytes, but not serum, from tolerant recipients. CONCLUSION: Combined with immunosuppressive treatment, donor imDex can prolong cardiac allograft survival and induce donor specific allograft tolerance.

Phosphatidylinositol 3'-kinase signaling pathway is essential for Rac1-induced hypoxia-inducible factor-1(alpha) and vascular endothelial growth factor expression.

We have previously demonstrated the roles of RhoA, Rac1, and Cdc42 in hypoxia-driven angiogenesis. However, the role of oncogenes in hypoxia signaling is poorly understood. Given the importance of Rho proteins in the hypoxic response, we hypothesized that Rho family members could act as mediators of hypoxic signal transduction. We investigated the cross-talk between hypoxia and oncogene-driven signal transduction pathways and explored the role of Rac1 on hypoxia-induced hypoxia-inducible factor (HIF)-1α and VEGF expression. Since the phosphatidylinositol 3'-kinase (PI3K) pathway is involved in signal transduction of many oncogenes, we explored the role of PI3K on Rac1-mediated expression of HIF-1α and VEGF in hypoxia. We showed that LY-294002, a PI3K inhibitor, suppressed HIF-1α and VEGF induction under hypoxic conditions by up to 50%. Activation of Rac1 resulted in an upregulation of hypoxia-induced HIF-1α expression, which was blocked by LY-294002. These data suggested that Rac1 is an intermediate in the PI3K-mediated induction of HIF-1α. Interestingly, there was a significant downregulation of the tumor suppressor genes p53 and von Hippel-Lindau tumor suppressor (VHL) in cells expressing a constitutively active form of Rac1. Rac1-mediated inhibition of p53 and VHL could therefore be implicated in the upregulation of HIF-1α expression.

Dendritic cells fused with allogeneic hepatocellular carcinoma cell line compared with fused autologous tumor cells as hepatocellular carcinoma vaccines.

PURPOSE: To investigate the specific antitumor responses against autologous hepatocellular carcinoma (HCC) cells of dendritic cells (DCs) fused with allogeneic HCC cell line, and evaluated the feasibility of BEL7402 as an alternative strategy to deliver shared HCC antigens to DCs. METHODS: Previous studies demonstrated fusions of patient-derived DCs and autologous tumor cells could induce T-cell responses against autologous tumors. These fusion cells require patient-derived tumor cells, which are not, however, always available. Here, we report the fusing of autologous DCs with allogeneic HCC cell line to induced cytotoxic T-lymphocyte (CTL) response against autologous HCC cells compare with autologous tumor cells. RESULTS: These DC/ BEL7402 fusion cells co-expressed tumor-associated antigens and DC-derived costimulatory and major histocompatibility complex molecules. Both CD4+ and CD8 T+ cells were activated by the fusion cells as demonstrated by the proliferation of T-cells, the production of cytokines and the simultaneous induction of specific CTL responses. Significantly, CTL induced by dendritic cell/allogeneic BEL7402 fusion cells were able to kill autologous HCC cells by human leukocyte antigen-A2 restricted mechanisms. The results did not show significant difference between DC fusion with autologous hepatocellular carcinoma cells and DC fusion with allogeneic hepatocellular carcinoma cell line. CONCLUSIONS: The fusion of allogeneic HCC cell line and autologous DCs may have applications in antitumor immunotherapy through cross-priming against shared tumor antigens and may provide a platform for adoptive immunotherapy.

Improvement of dendritic-based vaccine efficacy against hepatitis B virus-related hepatocellular carcinoma by two tumor-associated antigen gene-infected dendritic cells.

Recently, studies on dendritic cell (DC) vaccine have focused on the development of more effective DC vaccine regimen, such as the application of multiple tumor-associated antigen-targeted DC vaccine. This approach could be used to enhance efficacy of DC-based vaccine against tumors and infectious diseases. In this study, we analyzed whether DC from patients with hepatocellular carcinoma can be infected with the alpha-fetoprotein (AFP) gene and/or HBsAg gene (hepatocellular carcinoma-related antigen). Further, it was examined whether vaccination using these genetically engineered DC can induce stronger therapeutic antitumor immunity. Results revealed that DC infected with AdAFP (adenovirus AFP)/HBsAg can express AFP and HBsAg by reverse transcription-polymerase chain reaction and Western blot techniques. Compared with those before transfection, the expressions of membrane molecules increased dramatically. Specific T cells generated by DCs infected with AdAFP/HBsAg specifically recognized human leukocyte antigen-matched HepG2.2.15 cell lines. Moreover, the cytotoxic activity of cytotoxic T lymphocytes against HepG2.2.15 with DCs expressing AFP was significantly augmented by coinfection with the HBsAg gene. Administration with such vaccine also significantly increased the production of interleukin-12p70 and interferon-gamma. Most importantly, in vivo results suggested that inhibitors of tumor growth were most significant in severe combined immunodeficiency mice model, which was treated with induced cytotoxic T lymphocyte by the AFP/HBsAg-DC vaccine. These results indicate that a vaccination therapy using DCs coinfected with the two tumor-associated antigen genes is an effective strategy for immunotherapy in the activation of DCs, CD4(+) T cells, and CD8(+) T cells, and may be useful in the clinical application of cancer vaccine therapy.

Comparative analysis of DC fused with allogeneic hepatocellular carcinoma cell line HepG2 and autologous tumor cells as potential cancer vaccines against hepatocellular carcinoma.

Fusions of patient-derived dendritic cells (DCs) and autologous tumor cells induce T-cell responses against autologous tumors in animal models and human clinical trials. These fusion cells require patient-derived tumor cells, which are not, however, always available. Here we fused autologous DCs from patients with hepatocellular carcinoma (HCC) to an allogeneic HCC cell line (HepG2). These fusion cells co-expressed tumor-associated antigens (TAAs) and DC-derived costimulatory and MHC molecules. Both CD4(+) and CD8(+) T cells were activated by the fusion cells. Cytotoxic T lymphocytes (CTLs) induced by the fusion cells were able to kill autologous HCC by HLA-A2- and/or HLA-A24-restricted mechanisms. CTL activity against shared TAAs indicates that the presence of alloantigens does not prevent the development of CTLs with activity against autologous HCC cells. These fusion cells may have applications in anti-tumor immunotherapy through cross-priming against shared tumor antigens and may provide a platform for adoptive immunotherapy.

Quantitation assay for absorption and first-pass metabolism of emodin in isolated rat small intestine using liquid chromatography-tandem mass spectrometry.

Emodin has numerous biochemical and pharmacological activities, though information about its intestinal absorption and first-pass metabolism is scarce. The purpose of this study was to evaluate intestinal absorption and metabolism of luminally administered emodin in an isolated rat small intestine using the method of LC/MS/MS. About 22.55% of the administered emodin appeared at the vascular side, chiefly as free emodin (12.01%), but some emodin glucuronide (8.69%) and sulfate (1.84%) were also detected. Free glucuronide (5.23%) and sulfate (1.08%) moieties were found in the luminal perfusate. This model serves as a valuable tool for understanding intestinal handling of emodin, and our results confirm absorption and first-pass metabolism of emodin in the rat small intestine. Phase II metabolic enzymes such as glucuronyl transferase or sulfate transferase may also play an important role in the first-pass metabolism of emodin in the small intestine, which may ultimately reduce the bioavailability (and thus the efficacy) of orally administered emodin.

alpha-fetoprotein and interleukin-18 gene-modified dendritic cells effectively stimulate specific type-1 CD4- and CD8-mediated T-Cell response from hepatocellular carcinoma patients in Vitro.

The T-helper 1 (Th1) immune reaction is most important in dendritic cell (DC)-based immunotherapy. Interleukin (IL)-18, a Th1-biasing cytokine, plays a pivotal role in inducing cytotoxic T lymphocyte (CTL) responses. In this study, we analyzed whether dendritic cells (DCs) from patients with hepatocellular carcinoma (HCC) can be transduced with the IL-18 gene and/or alpha-fetoprotein (AFP) gene, and we examined whether vaccinations using these genetically engineered DC can induce stronger therapeutic antitumor immunity. The results showed that DC transfected with AdIL-18/AFP can expressed IL-18 and AFP by reverse transcriptase-polymerase chain reaction and enzyme-linked immunoassay. Compared with those before transfection, the expressions of membrane molecules were increased dramatically. Specific T cells generated by DC transfected with AdIL-18/AFP recognized HLA-matched HepG2 cell lines specifically. Most importantly, The cytotoxic activity of CTLs against HepG2 with DC expressing AFP(AFP-DC) was significantly augmented by co-transduction with the IL-18 gene. Administration with such vaccine also significantly increased the production of interleukin-12p70 and interferon-gamma. These results indicate that a vaccination therapy using DC co-transduced with the TAA gene and IL-18 genes is effective strategy for immunotherapy in terms of the activation of DCs, CD4+ T, cells and CD8+ T cells, and may be useful in the clinical application of a cancer vaccine therapy.

[Immune response of HBsAg gene-modified dendritic cell-based vaccine in HepG2. 2. 15 hepatocellular carcinoma cells].

OBJECTIVE: To study the cytotoxic T lymphocyte (CTL) response induced by dendritic cells (DC) transduced with recombinant adenovirus vector bearing hepatitis B virus surface antigen (HBsAg) gene in hepatocellular carcinoma HepG2. 2. 15 cells in vitro. METHODS: Full length HBsAg cDNAs were subcloned into pIND vector, followed by being cloned into pShuttle vector. The HBsAg gene fragments resulted from the pShuttle-S digested with PI-Sce and I-Ceu were linked to the linear adeno-X virus DNA. After packaged with HEK293 cells, the adenovirus expression vector was obtained. Then the recombinant adenovirus expression plasmid AdVHBsAg was transfected into human monocyte-derived dendritic cells, to construct AdVHBsAg hepatocarcinoma tumor vaccine. The effectiveness of transfection was detected by Western blot. Surface molecules of AdVHBsAg-DC were detected by FACS. Autologous T cell proliferation stimulated by AdVHBsAg-DC was detected by 3H-TdR assay. Cytotoxic CTL activity induced by AdVHBsAg-DC in vitro was detected by LDH assay. RESULTS: HBsAg gene in the inserted DNA of AdVHBsAg was confirmed by PCR, and predictive fragments proved by restriction enzyme digestion analysis were exhibited. Cell pathological changes appear after 10 days HEK293 cells transfected AdVHBsAg. Western blot analysis showed that HBV surface antigen gene was expressed in transfected DC, indicating that the transfection was effective. AdVHBsAg-DC was able to upregulate CD1a, CD11c, CD80, CD86 and HLA-DR. Autologus T cell proliferation induced by AdVHBsAg-DCs was significantly higher than that in DC control group and LacZ-DC group (P < 0.05). AdVHBsAg-DC activated CTL presented the specific killer ability to the hepatocellular carcinoma cells expressing HBsAg. CONCLUSION: DC transduced with recombinant adenovirus HBsAg can express HBV-related hepatocellular carcinoma antigen (HBsAg), and AdVHBsAg-DC can induce potent immune response against HBsAg-positive hepatocellular carcinoma cells in vitro.

Dendritic cell generated from CD34+ hematopoietic progenitors can be transfected with adenovirus containing gene of HBsAg and induce antigen-specific cytotoxic T cell responses.

Dendritic cells (DCs) are professional antigen presenting cells that are being considered as potential immunotherapeutic agents to promote host immune responses against tumor antigens. The use of such modified antigen-presenting cells for research or therapeutic have been limited by several factors, including maintaining DCs in a highly activated state, efficient transduction and expression, stable expression, identification of appropriate tumor-associated antigens, and absence of unintended functional changes or cytotoxicity. In this study, the feasibility of using CD34-DCs for tumor immunotherapy after transduction with a recombinant adenovirus containing HBsAg gene (AdVHBsAg), an HCC-associated antigen, was investigated. The gene transfer with recombinant adenovirus vectors (AdV) can obtained high levels of stable expression of HBsAg and its efficiency was increased in a multiplicity of infection (MOI)-dependent manner. Moreover, the AdVHBsAg infection had no appreciable effect on apoptosis of DCs compared with that of mock-infected DCs. The T cell lines, primed by the recombinant AdVHBsAg-infected DCs in vitro, recognized HBsAg-expressing tumor cell lines in a human leukocyte antigen (HLA) class I-restricted manner, and evoked a higher CTL response, which indicated that high potent and specific antitumor immune response could be induced by AdVHBsAg DC vaccine. It may be a promising the therapeutic modality for the treatment of HBsAg-expressing tumors, and will be a foundation for further study on DC vaccines and gene therapy for HCC.

[Cytotoxicity induced by HBsAg gene modified-dendritic cells against hepatocellular carcinoma cell HepG2.2.15].

BACKGROUND & OBJECTIVE: Up to now, there is no efficient immunotherapy for hepatocellular carcinoma (HCC). Dendritic cell (DC) vaccine could be a potential tool for HCC immunotherapy. This study was to evaluate the effect of dendritic cells (DCs) transfected with recombinant plasmid bearing hepatitis B virus surface antigen (HBsAg) gene, and the capability of generating specific cytotoxic T lymphocytes (CTL) response against HepG2.2.15 in vitro, which were induced by genetically modified DCs. METHODS: After cultured for 5 days, the DCs were transfected with pCR3.1-S by liposome. The HBsAg gene expression on pCR3.1-transfected DCs was identified by Western blot analysis, and immunofluorescence methods. The cytotoxicity against HepG2.2.15, which were induced by DCs, was tested by MTT assay. RESULTS: DCs up-regulated the expression of CD1a (55.0%), CD11c (98.6%), CD86 (86.1%), CD80 (66.1%), and HLA-DR (88.9%) after cultured for 5 days. Indirect immunofluorescence, and Western blot analysis showed that HBsAg gene was expressed on transfected DCs. The death rates of HepG2.2.15 cells induced by DCs transfected with pCR3.1-S were (52.3+/-2.8)% (E:T=5:1), (64.6+/-2.4)% (10:1), (78.8+/-2.6) (20:1), (82.1+/-2.4)% (40:1), while the pCR3.1- transfected and non-transfected DCs only induced relatively lower cytotoxicity (P< 0.05, n=4). CONCLUSION: DCs transfected with recombined plasmid expressed HBsAg efficiently, and the genetically modified DCs evoke a higher CTL response in vitro.