Therapy of established tumour with a hybrid cellular vaccine generated by using granulocyte–macrophage colony-stimulating factor genetically modified dendritic cells

Dendritic cells (DCs) are the most powerful of all antigen-presenting cells and play a critical role in the induction of primary immune responses. DC-based vaccination represents a potentially powerful strategy for cancer immunotherapy. In this study, a new approach for a DC-based melanoma vaccine was described. Splenic DCs from C57BL/6 mice were fused with B16 melanoma cells, and the resultant B16/DC hybrid cells expressed major histocompatibility complex (MHC) molecules - B7 as well as the B16 tumour marker M562 - which were enriched by Ia-mediated positive selection with a MiniMACS column. The fusion rates were 12.7-26.8%. To generate hybrid tumour vaccines with potentially greater potent therapeutic efficacy, we genetically engineered DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) prior to cell fusion. Recombinant adenovirus vector was used to mediate gene transfer into DCs with high efficiency and DCs expressed GM-CSF at 96-138 ng/105 cells/ml 24 hr after GM-CSF gene transfer. GM-CSF gene-modified DCs (DC.GM) exhibited higher expression of B7 and co-stimulatory capacity in mixed lymphocyte reaction (MLR). Fusion of DC.GM with B16 cells generated B16/DC.GM hybrid cells secreting GM-CSF at 59-63 ng/105 cells/ml. Immunization of C57BL/6 mice with the B16/DC hybrid vaccine elicited a specific cytotoxic T-lymphocyte (CTL) response and protected the immunized mice from B16 tumour challenge, reduced pulmonary metastases and extended the survival of B16 tumour-bearing mice. The B16/DC.GM hybrid vaccine was able to induce a CTL response and protective immunity more potently and tended to be therapeutically more efficacious than the B16/DC vaccine. In vivo depletion of T-cell subsets demonstrated that both CD8+ and CD4+ T cells were essential for the therapeutic effects of B16/DC and B16/DC.GM hybrid vaccines. Additionally, other non-specific effector cells may also contribute to tumour rejection induced by the B16/DC.GM hybrid vaccine. These data indicate that a DC-based hybrid tumour vaccine may be an attractive strategy for cancer immunotherapy, and that GM-CSF gene-modified DCs may lead to the generation of hybrid vaccines with potentially increased therapeutic efficacy.     

Ubiquitin-fusion degradation pathway plays an indispensable role in naked DNA vaccination with a chimeric gene encoding a syngeneic cytotoxic T lymphocyte epitope of melanocyte and green fluorescent protein

Antitumour immunity against murine melanoma B16 was achieved by genetic immunization with a naked chimeric DNA encoding a fusion protein linking green fluorescent protein (GFP) to the N-terminus of a major CD8(+) cytotoxic T lymphocyte (CTL) epitope of tyrosinase-related protein 2 (TRP-2(181-188)) of murine melanoma, designated as pGFP-TRP-2. Tumour growth was profoundly suppressed in C57BL/6 mice immunized with pGFP-TRP-2, while mice vaccinated with pTRP-2 showed rapid tumour growth and died within 40 days after tumour challenge. Splenocytes of mice immunized with pGFP-TRP-2 showed high CTL activity specific for TRP-2(181-188). GFP-TRP-2 expressed in COS-7 cells was rapidly degradated in vitro and the degradation was almost completely prevented by adding a proteasome inhibitor, MG-132, in the culture. Furthermore, the antimelanoma immunity induced by genetic immunization with pGFP-TRP-2 was completely cancelled in mice deficient in proteasome activator PA28alpha/beta. Taken together, GFP-TRP-2 processed by cytosolic proteasome played a central role in breaking peripheral tolerance to a melanoma/melanocyte antigen, TRP-2(181-188), by activating CD8(+) CTL specific for TRP-2(181-188). TRP-2(181-188) fused to GFP may be readily cut off from GFP by the ubiquitin-fusion degradation (UFD) pathway and efficiently presented to major histocompatibility complex class I molecules, resulting in effective induction of CD8(+) T cells specific for the CTL epitope. Furthermore, CD4(+) T cells specific for GFP were shown to play a crucial role in the antimelanoma immunity, probably potentiating activity of TRP-2-specific CTL and/or the "ubiquitin-proteasome pathway". It is noteworthy to document that genetic immunization with pGFP plus pTRP-2(181-188) failed to exert the antitumour immunity.     

Potent influence of bovine serum proteins in experimental dendritic cell-based vaccination protocols

Typically autologous dendritic cells (DCs) intended for vaccination are generated from bone marrow derived stem cells or blood monocytes, loaded with antigen and introduced into the organism. However, addition of serum to DC culture medium is often necessary. Thus, serum proteins will be taken up and presented by the DCs together with other antigens. If heterologous serum is used, some of the serum proteins might be antigenic and thus induce a strong immune response when introduced in the recipient. We used the murine model of malignant melanoma, B16, to investigate the consequences of addition of fetal calf serum (FCS) to the medium for culturing murine DCs. The results showed that vaccination of mice with DCs cultured in vitro in the presence of FCS but in the absence of extraneous tumour antigens, protected the mice from challenge with B16 tumour cells similarly cultured in FCS. This protection could not be elicited by vaccination with FCS alone. Interestingly, the protective effect of DC vaccination was abolished when the challenging B16 tumour cells were free of serum proteins. Thus, these results show that DCs grown in the presence of FCS are able to induce immunity, which may be mistaken to be tumour immunity.     

Augmented induction of CD8+ cytotoxic T-cell response and antitumour resistance by T helper type 1-inducing peptide

The effector CD8(+) T cells recognize major histocompatibility complex (MHC) class I binding altered self-peptides expressed in tumour cells. Although the requirement for CD4(+) T helper type 1 (Th1) cells in regulating CD8(+) T cells has been documented, their target epitopes and functional impact in antitumour responses remain unclear. We examined whether a potent immunogenic peptide of Mycobacterium tuberculosis eliciting Th1 immunity contributes to the generation of CD8(+) T cells and to protective antitumour immune responses to unrelated tumour-specific antigens. Peptide-25, a major Th epitope of Ag85B from M. tuberculosis preferentially induced CD4(+) Th1 cells in C57BL/6 mice and had an augmenting effect on Th1 generation for coimmunized unrelated antigenic peptides. Coimmunization of mice with Peptide-25 and ovalbumin (OVA) or Peptide-25 and B16 melanoma peptide [tyrosinase-related protein-2 (TRP-2)] for MHC class I led to a profound increase in CD8(+) T cells specific for OVA and TRP-2 peptides, respectively. This heightened response depended on Peptide-25-specific CD4(+) T cells and interferon-gamma-producing T cells. In tumour protection assays, immunization with Peptide-25 and OVA resulted in the enhancement of CD8(+) cytotoxic cell generation specific for OVA and the growth inhibition of EL-4 thymoma expressing OVA peptide leading to the tumour rejection. These phenomena were not achieved by immunization with OVA alone. Peptide-25-reactive Th1 cells counteractivated dendritic cells in the presence of Peptide-25 leading them to activate and present OVA peptide to CD8(+) cytotoxic T cells.     

Immunization with tumor-associated epitopes fused to an endoplasmic reticulum translocation signal sequence affords protection against tumors with down-regulated expression of MHC and peptide transporters

Treatment of human cancers with an inherent antigen-processing defect due to a loss of peptide transporters (TAP-1 and TAP-2) and/or MHC class I antigen expression remains a considerable challenge. There is now an increasing realization that tumor cells with down-regulated expression of TAP and/or MHC class I antigens display strong resistance to cytotoxic T lymphocyte (CTL)-mediated immune control, and often fail to respond to the conventional immunotherapeutic protocols based on active immunization with tumor-associated epitopes (TAE) or adoptive transfer of tumor-specific T cells. In the present study, we describe a novel approach based on immunization with either genetically modified tumor cells or naked DNA vectors encoding TAE fused to an endoplasmic reticulum (ER) signal sequence (ER-TAE) which affords protection against challenge by melanoma cells with down-regulated expression of TAP-1/2 and MHC class I antigens. In contrast, animals immunized with a vaccine based on TAE alone showed no protection against tumor challenge. Although MHC-peptide tetramer analysis showed a similar frequency of antigen-specific CTL in both ER-TAE- and TAE-immunized mice, functional analysis revealed that CTL activated following immunization with ER-TAE displayed significantly higher avidity for TAE when compared to animals immunized with the TAE alone. These observations provide a new strategy in anti-cancer vaccine design that allows activation of a highly effective and well-defined CTL response against tumors with down-regulated expression of TAP and MHC class I antigens.     

Dendritic cell vaccine therapy by immunization with fusion cells of interleukin-2 gene-transduced, spleen-derived dendritic cells and tumour cells

We examined the preventive and therapeutic effects of fusion cells prepared from spleen-derived dendritic cells (DCs) transduced with the interleukin-2 (IL-2) gene and QRsP fibrosarcoma cells in a mouse lung metastasis model. The IL-2 or LacZ gene was introduced into spleen-derived DCs using an adenoviral vector. Irradiated QRsP tumour cells were fused with IL-2 gene-transduced DCs (fusion/IL-2) or LacZ gene-transduced DCs (fusion/LacZ) by polyethylene glycol. These fusion cells expressed major histocompatibility complexes (MHC) class I and II, CD86, CD11c and CD8alpha. Splenocytes from mice vaccinated with fusion cells showed increased production of interferon-gamma (IFN-gamma) and cytotoxic T-lymphocyte (CTL) activity as compared with those vaccinated with DCs or tumour cells alone, and CTL levels were higher in fusion/IL-2-vaccinated mice than in fusion/LacZ-vaccinated mice. In our experiments on the protective and therapeutic effects on lung metastasis, mice vaccinated with fusion/IL-2 fusion/LacZ or fusion showed a significant reduction in pulmonary metastasis compared with those given DCs, tumour or phosphate-buffered saline. The introduction of the IL-2 gene into fusion cells produced more potent preventive and therapeutic effects. These results suggest that immunization with fusion cells prepared from spleen-derived DCs and tumour cells is capable of inducing preventive and therapeutic anti-tumour immunity against lung metastasis, and modification by the IL-2 gene may increase anti-tumour efficacy.     

Significant anti-tumour activity of adoptively transferred T cells elicited by intratumoral dendritic cell vaccine injection through enhancing the ratio of CD8+ T cell/regulatory T cells in tumour

We have shown that immunization with dendritic cells (DCs) pulsed with hepatitis B virus core antigen virus‐like particles (HBc‐VLP) packaging with cytosine–guanine dinucleotide (CpG) (HBc‐VLP/CpG) alone were able to delay melanoma growth but not able to eradicate the established tumour in mice. We tested whether, by modulating the vaccination approaches and injection times, the anti‐tumour activity could be enhanced. We used a B16‐HBc melanoma murine model not only to compare the efficacy of DC vaccine immunized via footpads, intravenously or via intratumoral injections in treating melanoma and priming tumour‐specific immune responses, but also to observe how DC vaccination could improve the efficacy of adoptively transferred T cells to induce an enhanced anti‐tumour immune response. Our results indicate that, although all vaccination approaches were able to protect mice from developing melanoma, only three intratumoral injections of DCs could induce a significant anti‐tumour response. Furthermore, the combination of intratumoral DC vaccination and adoptive T cell transfer led to a more robust anti‐tumour response than the use of each treatment individually by increasing CD8⁺ T cells or the ratio of CD8⁺ T cell/regulatory T cells in the tumour site. Moreover, the combination vaccination induced tumour‐specific immune responses that led to tumour regression and protected surviving mice from tumour rechallenge, which is attributed to an increase in CD127‐expressing and interferon‐γ‐producing CD8⁺ T cells. Taken together, these results indicate that repeated intratumoral DC vaccination not only induces expansion of antigen‐specific T cells against tumour‐associated antigens in tumour sites, but also leads to elimination of pre‐established tumours, supporting this combined approach as a potent strategy for DC‐based cancer immunotherapy.     

Targeting MOG expression to dendritic cells delays onset of experimental autoimmune disease

Haematopoietic stem cell (HSC) transfer coupled with gene therapy is a powerful approach to treating fatal diseases such as X-linked severe combined immunodeficiency. This ability to isolate and genetically manipulate HSCs also offers a strategy for inducing immune tolerance through ectopic expression of autoantigens. We have previously shown that retroviral transduction of bone marrow (BM) with vectors encoding the autoantigen, myelin oligodendrocyte glycoprotein (MOG), can prevent the induction of experimental autoimmune encephalomyelitis (EAE). However, ubiquitous cellular expression of autoantigen driven by retroviral promoters may not be the best approach for clinical translation and a targeted expression approach may be more acceptable. As BM-derived dendritic cells (DCs) play a major role in tolerance induction, we asked whether targeted expression of MOG, a target autoantigen in EAE, to DCs can promote tolerance induction and influence the development of EAE. Self-inactivating retroviral vectors incorporating the mouse CD11c promoter were generated and used to transduce mouse BM cells. Transplantation of gene-modified cells into irradiated recipients resulted in the generation of chimeric mice with transgene expression limited to DCs. Notably, chimeric mice transplanted with MOG-expressing BM cells manifest a significant delay in the development of EAE suggesting that targeted antigen expression to tolerogenic cell types may be a feasible approach to inducing antigen-specific tolerance.     

慢病毒载体及其应用进展

慢病毒载体具有可感染分裂细胞及非分裂细胞,转移基因片段容量较大,目的基因表达时间较长,不易引发宿主免疫反应等诸多优点,因此,慢病毒载体已成为基因治疗中载体研究的热点.我们以HIV-1来源的慢病毒载体为代表综述了慢病毒载体的结构特点,发展,及其应用研究进展.     

Transduction of dendritic cells by antigen-encoding lentiviral vectors permits antigen processing and MHC class I-dependent presentation

BACKGROUND: Because antigen-presenting dendritic cells (DCs) play a major role in the polarization of T cells, including T(H)2 cells involved in allergy, strategies to modify DCs genetically are required. OBJECTIVE: The purpose of this investigation was to transduce murine bone marrow-derived DCs with lentiviral vectors encoding antigen to demonstrate antigen processing and MHC class I-dependent presentation. METHODS: Bone marrow leukocytes were incubated with antigen-encoding lentiviral constructs and cultured with GM-CSF, IL-4, and Flt-3 ligand. The capacity of the resulting DCs to express, process, and present antigen was tested in vitro. RESULTS: An average of 40% of DCs expressed antigen after 1 week of culture when antigen encoded by the lentiviral vector construct was green fluorescent protein. To demonstrate that transduced antigen can be presented by DCs on MHC class I, we chose the lymphocytic choriomeningitis virus glycoprotein (gp) as a model antigen, inasmuch as it is recognized by CD8 T cells from transgenic mice expressing an MHC class I-restricted T-cell receptor specific for the epitope of positions 33 through 41 of gp. DCs transduced with lentiviral construct encoding gp and matured with LPS activated transgenic T cells in an antigen-specific fashion. Using transporter associated with antigen presentation (TAP)-deficient mice, we show that presentation of the gp33-41 epitope is TAP-dependent, confirming processing of gp by the endogenous pathway. CONCLUSIONS: These results demonstrate that CD8 T cells can recognize MHC class I epitopes processed from antigen in DCs transduced with lentiviral vectors. Lentiviral transduction of DCs and antigen presentation to CD8 T cells could be exploited for immunotherapy, because allergen-specific CD8 T cells have been shown to be suppressive in IgE-dependent allergy models.     

Sodium chloride modified silica nanoparticles as a non-viral vector with a high efficiency of DNA transfer into cells

Development of reliable vectors is a major challenge in gene therapy. Previous gene transfer methods using non-viral vectors, such as liposomes or nanoparticles, have resulted in relatively low levels (35 to approximately 50%) of gene expression. We have developed a silicon nanoparticle (SNAP) system, a novel non-viral vector, for DNA transfer into cells. SNAP was synthesized chemically and modified with sodium chloride or sodium iodide. Electronmicroscopy of SNAP and fluorescence microscopy of fluorescence-labeled SNAP revealed that they were generated uniformly, had diameters of 10-100 nm, and showed a better efficiency (about 70%) of DNA transfection into cells as well as protection of DNA against degradation. The microscopy also demonstrated the adhesion of SNAP with HT1080 cell surface and entry of SNAP into the cells without cytotoxicity. Intravenous and/or intra-abdominal administration of the SNAP to mice revealed the accumulation of SNAP in the cells of the brain, liver, spleen, lung, kidney, intestine, prostate and the testis without any pathological cell changes or mortality, suggesting that they passed through the blood-brain, blood-prostate, and blood-testis barriers. These findings indicate that the SNAP generated has good biological characteristics as a potential promising vector for gene transfer, gene therapy and drug delivery.     

HIV-2 derived lentiviral vectors: gene transfer in Parkinson's and Fabry disease models in vitro

Lentiviral vectors are prime candidate vectors for gene transfer into dividing and non-dividing cells, including neuronal cells and stem cells. For safety, HIV-2 lentiviral vectors may be better suited for gene transfer in humans than HIV-1 lentiviral vectors. HIV-2 vectors cross-packaged in HIV-1 cores may be even safer. Demonstration of the efficacy of these vectors in disease models will validate their usefulness. Parkinson's disease and Fabry disease provide excellent models for validation. Parkinson's disease is a focal degeneration of dopaminergic neurons in the brain with progressive loss of ability to produce the neurotransmitter dopamine. Current treatment entails administration of increasing doses of L-dopa, with attendant toxicity. We explore here the hypothesis that gene transfer of aromatic acid decarboxylase (AADC), a key enzyme in the pathway, will make neuronal cells more efficiently convert L-dopa into dopamine. Fabry disease on the other hand is a monogenic inherited disease, characterized by alpha-galactosidase A (AGA) deficiency, resulting in glycolipid accumulation in several cell types, including fibroblasts. Animal models for preclinical investigations of both of these diseases are available. We have designed monocistronic HIV-1 and HIV-2 vectors with the AADC transgene and monocistronic and bicistronic HIV-2 vectors with the AGA and puromycin resistance transgenes. They were packaged with either HIV-2 cores or HIV-1 cores (hybrid vectors). Gene transfer of AADC gene in neuronal cells imparted the ability on the transduced cells to efficiently convert L-dopa into dopamine. Similarly, the AGA vectors induced Fabry fibroblasts to produce high levels of AGA enzyme and caused rapid clearance of the glycolipids from the cells. Both monocistronic and bicistronic vectors were effective. Thus, the insertion of a second gene downstream in the bicistronic vector was not deleterious. In addition, both the self-packaged vectors and the cross-packaged hybrid vectors were effective in gene transfer.     

Adenovirus transduction and culture conditions affect the immunogenicity of murine dendritic cells

Adenovirus vectors encoding carcinoembryonic antigen (Ad-CEA) or costimulatory molecules CD80, intercellular adhesion molecule-1 (ICAM-1) and leucocyte function-associated antigen-3 (LFA-3) (Ad-STIM) were used to transduce murine bone marrow-derived dendritic cells (BMDC). BMDC were characterized for expression of activation markers and for their ability to elicit protective immunity against MC38-CEA tumours in wildtype and CEA-transgenic (CEA-tg) mice. To determine optimal culture conditions, studies were conducted using BMDC cultured in heterologous bovine serum or autologous mouse serum. Transduction of cells grown in presence of heterologous serum increased the expression of costimulatory molecules, major histocompatibility complex class II, of IL-6 and IL-12. Upon vaccination, tumour protection was not specific and was observed also with untransduced cells. Transduced BMDC cultured in the presence of autologous serum showed low expression of the activation markers, did not express IL-6 and had reduced ability to stimulate T-cell proliferation. Nonetheless, CEA-specific CD8+ T-cell response was enhanced upon coinfection of Ad-STIM and Ad-CEA in both mouse strains, although this immune response was not sufficient to protect CEA-tg mice from tumour challenge. These studies support the use of BMDC transduced with Ad vectors encoding tumour antigens for cancer immunotherapy and demonstrate that culture conditions greatly affect the immunological properties of these cells.     

High transfection efficiency, gene expression, and viability of monocyte-derived human dendritic cells after nonviral gene transfer

Dendritic cells (DCs) are bone marrow-originated, professional antigen-capturing cells and APCs, which can function as vaccine carriers. Although efficient transfection of human DCs has been achieved with viral vectors, viral gene products may influence cellular functions. In contrast, nonviral methods have generally resulted in inefficient gene transfer, low levels of gene expression, and/or low cell viability. Monocyte-derived DCs are the most common source of DCs for in vitro studies and for in vivo applications. We hypothesized that reduction of the time to generate immature DCs (iDCs) might result in higher viability after transfection. Therefore, we established a protocol to generate human iDCs from CD14(+) monocytes within 3 days. These "fast" iDCs were phenotypically and functionally indistinguishable from conventional iDCs, showing high endocytic ability and low antigen-presenting capacity. Furthermore, the fast iDCs matured normally and had similar antigen-presenting capacity to conventional mature DCs. To optimize transfection of iDCs, we compared nonviral transfection of plasmid DNA and in vitro-transcribed (IVT) RNA with transfection reagents, electroporation, and nucleofection. Nucleofection of IVT RNA with the X1 program of an Amaxa Co. Nucleofector resulted in the most efficient transfection, with an average of 93% transfected iDCs, excellent long-term viability, and strong protein expression. Furthermore, the IVT RNA-transfected iDCs retained all phenotypic and functional characteristics of iDCs. This method is applicable to most purposes, including in vitro functional assays, in vivo DC immunotherapy, and DC-based vaccines.     

BCL-3基因沉默对BCL-3高表达的人大肠癌RKO细胞增殖和药物敏感性的影响

 目的：构建针对人BCL-3基因的RNA干扰（RNA interference,RNAi）慢病毒载体,观察其对大肠癌细胞株中BCL-3基因的沉默效应,以及基因沉默对细胞生物学行为及药物敏感性的影响。方法：运用RT-PCR及Western blotting方法检测5株大肠癌细胞株中BCL-3的表达状况;构建人BCL-3基因的RNAi慢病毒载体,并转染BCL-3高表达的大肠癌细胞株,运用Western  blotting方法鉴定其对BCL-3基因的沉默效果;BCL-3基因沉默后运用细胞增殖实验及软琼脂克隆形成实验检测其对细胞增殖能力的影响,运用MTT方法检测大肠癌细胞株对化疗药物敏感性的变化。结果：BCL-3高表达于大肠癌细胞株RKO中;成功构建了BCL-3　RNAi慢病毒载体,Western blotting实验显示其可抑制RKO细胞中BCL-3蛋白的表达;抑制BCL-3的表达后,增殖实验表明RKO细胞增殖能力下降,软琼脂克隆形成实验表明RKO细胞的克隆形成率下降;MTT实验表明BCL-3表达抑制后奥沙利铂对RKO细胞的半数抑制浓度（median inhibitory concentration, IC50）显著降低。结论: BCL-3表达抑制后,BCL-3高表达的大肠癌细胞株RKO的增殖能力下降,并且其对奥沙利铂的敏感性增强。     

Comparison between Sendai virus and adenovirus vectors to transduce HIV-1 genes into human dendritic cells

Immuno-genetherapy using dendritic cells (DCs) can be applied to human immunodeficiency virus type 1 (HIV-1) infection. Sendai virus (SeV) has unique features such as cytoplasmic replication and high protein expression as a vector for genetic manipulation. In this study, we compared the efficiency of inducing green fluorescent protein (GFP) and HIV-1 gene expression in human monocyte-derived DCs between SeV and adenovirus (AdV). Human monocyte-derived DCs infected with SeV showed the maximum gene expression 24 hr after infection at a multiplicity of infection (MOI) of 2. Although SeV vector showed higher cytopathic effect on DCs than AdV, SeV vector induced maximum gene expression earlier and at much lower MOI. In terms of cell surface phenotype, both SeV and AdV vectors induced DC maturation. DCs infected with SeV as well as AdV elicited HIV-1 specific T-cell responses detected by interferon gamma (IFN-gamma) enzyme-linked immunospot (Elispot). Our data suggest that SeV could be one of the reliable vectors for immuno-genetherapy for HIV-1 infected patients.     

Thyroid hormone (T3)-modification of polyethyleneglycol (PEG)-polyethyleneimine (PEI) graft copolymers for improved gene delivery to hepatocytes

Targeting of gene vectors to liver hepatocytes could offer the opportunity to cure various acquired and inherited diseases. Efficient gene delivery to the liver parenchyma has been obscured from efficient targeting of hepatocytes. Here we show that the thyroid hormone, triiodothyronine (T3), can be used to improve the gene transfer efficiency of nonviral gene vectors to hepatocytes in vitro and to the liver of mice in vivo. T3 conjugated to the distal ends of fluorescent labeled PEG-g-dextran resulted in T3-specific cellular endosomal uptake into the hepatocellular cell line HepG2. PEG-g-PEI graft copolymers with increasing molar PEG-ratios were synthesized, complexed with plasmid DNA, and transfected into HepG2 or HeLa cells. Gene transfer efficiency decreased as the number of PEG blocks increased. T3 conjugation to PEI and the distal ends of PEG blocks resulted in T3 specific gene transfer in HepG2 cells as evidenced by reduction of gene transfer efficiency after pre-incubation of cells with excess of T3. In vivo application of T3-PEG-g-PEI based gene vectors in mice after tail vein injection resulted in a significantly 7-fold increase of gene expression in the liver compared with PEG-g-PEI based gene vectors.     

Persistence of apoptosis-resistant T cell-activating dendritic cells promotes T helper type-2 response and IgE antibody production

Antigen-specific T cell-mediated apoptosis of dendritic cells (DCs) represents a unique down-regulatory mechanism that prevents the continuous activation of T cells by antigen-loaded DCs; this regulatory mechanism is impaired in allergy and as a consequence a large proportion of DCs tends to escape apoptosis following cognate interaction with CD4(+) T cells. However, the biological relevance of greater numbers of apoptosis-resistant DCs to the development of allergic IgE-mediated reactions remained to be determined. Here, we sought to investigate the in vitro and in vivo regulatory features of apoptosis-resistant DCs and to assess their role in host sensitization. Freshly isolated CD11c(+/hi)B220(-)DCs from ovalbumin (OVA)-sensitized, OVA-immunized and na?ve Balb/c mice were cultured with OVA-specific T cells and levels of T cell-mediated DCs apoptosis assessed by flow cytometry. Surviving apoptosis-resistant DCs were then recovered and subsequently co-cultured with OVA-specific CD62L(hi)CD44(low) na?ve T cells or passively transferred into naive syngenic recipients. In vitro profile of DC and T cell lymphokine production, chemokine receptors expression and in vivo, post-adoptive DC transfer T helper (T(H)) and IgE responses were assessed. Apoptosis-resistant DCs showed differential regulatory properties compared to their freshly isolated counterpart independent of the sensitization status of the donor. When co-cultured with na?ve OVA-specific T cells, apoptosis-resistant DCs from either sensitized or immunized mice induced T cells that produced increased levels of IL-4 and reduced levels of IFN-gamma and showed increased expression of T(H)-2 related CCR4 and CCR8 chemokine receptors. Finally, adoptive transfer of apoptosis-resistant DCs, induced higher levels of OVA-specific IgE responses in absence of antigen challenge in syngenic recipients compared to freshly isolated DCs from both sensitized and immunized mice. These data would suggest that sensitization-associated increased numbers of apoptosis-resistant T cell-activating DCs contribute to the generation/maintenance of IgE-mediated allergic reactions.