Mouse Bone Marrow Dendritic Cells Research Articles

Discovery of Novel Small Molecule Dual Inhibitor Targeting Toll-Like Receptors 7 and 9.

The aberrant secretion of proinflammatory cytokines by immune cells is the principal cause of inflammatory diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Toll-like receptor 7 (TLR7) and TLR9, sequestered to the endosomal compartment of dendritic cells and macrophages, are closely associated with the initiation and progression of these diseases. Therefore, the development of drugs targeting dysregulated endosomal TLRs is imperative to mitigate systemic inflammation. Here, we applied the principles of computer-aided drug discovery to identify a novel low-molecular-weight compound, TLR inhibitory compound 10 (TIC10), and its potent derivative (TIC10g), which demonstrated dual inhibition of TLR7 and TLR9 signaling pathways. Compared to TIC10, TIC10g exhibited a more pronounced inhibition of the TLR7- and TLR9-mediated secretion of the proinflammatory cytokine tumor necrosis factor-α in a mouse macrophage cell line and mouse bone marrow dendritic cells in a concentration-dependent manner. While TIC10g slightly prevented TLR3 and TLR8 activation, it had no impact on cell surface TLRs (TLR1/2, TLR2/6, TLR4, or TLR5), indicating its selectivity for TLR7 and TLR9. Additionally, mechanistic studies suggested that TIC10g interfered with TLR9 activation by CpG DNA and suppressed downstream pathways by directly binding to TLR9. Western blot analysis revealed that TIC10g downregulated the phosphorylation of the p65 subunit of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPKs), including extracellular-signal-regulated kinase, p38-MAPK, and c-Jun N-terminal kinase. These findings indicate that the novel ligand, TIC10g, is a specific dual inhibitor of endosomal TLRs (TLR7 and TLR9), disrupting MAPK- and NF-κB-mediated proinflammatory gene expression.

Breast Cancer Vaccine Containing a Novel Toll-like Receptor 7 Agonist and an Aluminum Adjuvant Exerts Antitumor Effects

Mucin 1 (MUC1) has received increasing attention due to its high expression in breast cancer, in which MUC1 acts as a cancer antigen. Our group has been committed to the development of small-molecule TLR7 (Toll-like receptor 7) agonists, which have been widely investigated in the field of tumor immunotherapy. In the present study, we constructed a novel tumor vaccine (SZU251 + MUC1 + Al) containing MUC1 and two types of adjuvants: a TLR7 agonist (SZU251) and an aluminum adjuvant (Al). Immunostimulatory responses were first verified in vitro, where the vaccine promoted the release of cytokines and the expression of costimulatory molecules in mouse BMDCs (bone marrow dendritic cells) and spleen lymphocytes. Then, we demonstrated that SZU251 + MUC1 + Al was effective and safe against a tumor expressing the MUC1 antigen in both prophylactic and therapeutic schedules in vivo. The immune responses in vivo were attributed to the increase in specific humoral and cellular immunity, including antibody titers, CD4+, CD8+ and activated CD8+ T cells. Therefore, our vaccine candidate may have beneficial effects in the prevention and treatment of breast cancer patients.

Gold Nanoparticles Stimulate the Maturation and Activation of Mouse Bone Marrow Dendritic Cells

Nanomedicine is an interdisciplinary science that links nanotechnology, nanoscience and nanoengineering to the field of health and the use of nanoscale tools, in this case, nanoparticles have great therapeutic potential against target cells such as dendritic cells (DC). DCs are highly motile cells whose main function is antigen presentation; they are mediators between innate and adaptive immunity. In this investigation, DCs were differentiated and matured with granulocyte-macrophage colony stimulating factor (GM-CSF), 25 ug/mL and spherical gold nanoparticles (AuNPs) of approximately 30 nm at concentrations of 5 to 200 ug/mL. DCs were obtained from the bone marrow of adult mice; their characterization was performed by means of RT-PCR, scanning electron microscopy (SEM) and confocal to know their morphology and the location of the nanoparticles inside. The bone marrow DCs (bmDCs) possibly differentiated into cCD1 dendritic cells, their mature morphological status was determined by the presence of co-stimulatory factors (CD40, CD86 and CD11c), in addition to MHC II and CIITA when in contact with AuNPs, the results were confirmed by SEM observation due to their star-like shape. In terms of the location of the AuNPs they would be clustered and outside the nucleus. The discovery of nanoparticles that do not cause significant cytotoxicity, activate dendritic cells, and produce cytokines, causing phenotypic changes in them, enhancing, and modulating the adaptive immune response or generating a targeted immunotherapy in the presence of different pathogens in the organism.

Robust Immune Response Induced by Schistosoma mansoni TSP-2 Antigen Coupled to Bacterial Outer Membrane Vesicles.

PurposeThe use of adjuvants can significantly strengthen a vaccine’s efficacy. We sought to explore the immunization efficacy of bacterial outer membrane vesicles (OMVs) displaying the Schistosoma mansoni antigen, SmTSP-2, through a biotin-rhizavidin coupling approach. The rationale is to exploit the nanoparticulate structure and the adjuvant properties of OMVs to induce a robust antigen-specific immune response, in light of developing new vaccines against S. mansoni.Materials and MethodsOMVs were obtained from Neisseria lactamica and conjugated with biotin. The recombinant SmTSP-2 in fusion with the biotin-binding protein rhizavidin (rRzvSmTSP-2) was produced in E. coli and coupled to biotinylated OMVs to generate an OMV complex displaying SmTSP-2 on the membrane surface (OMV:rSmTSP-2). Transmission electron microscopy (TEM) and dynamic light scattering analysis were used to determine particle charge and size. The immunogenicity of the vaccine complex was evaluated in C57BL/6 mice.ResultsThe rRzvSmTSP-2 protein was successfully coupled to biotinylated OMVs and purified by size-exclusion chromatography. The OMV:rSmTSP-2 nanoparticles showed an average size of 200 nm, with zeta potential around – 28 mV. Mouse Bone Marrow Dendritic Cells were activated by the nanoparticles as determined by increased expression of the co-stimulatory molecules CD40 and CD86, and the proinflammatory cytokines (TNF-α, IL-6 and IL-12) or IL-10. Splenocytes of mice immunized with OMV:rSmTSP-2 nanoparticles reacted to an in vitro challenge with SmTSP-2 with an increased production of IL-6, IL-10 and IL-17 and displayed a higher number of CD4+ and CD8+ T lymphocytes expressing IFN-γ, IL-4 and IL-2, compared to mice immunized with the antigen alone. Immunization of mice with OMV:rSmTSP-2 induced a 100-fold increase in specific anti-SmTSP-2 IgG antibody titers, as compared to the group receiving the recombinant rSmTSP-2 protein alone or even co-administered with unconjugated OMV.ConclusionOur results demonstrate that the SmTSP-2 antigen coupled with OMVs is highly immunogenic in mice, supporting the potential effectiveness of this platform for improved antigen delivery in novel vaccine strategies.

Gynura procumbens ethanol extract and its fractions inhibit Th1, Th2 and Th17 but induce Treg cells differentiation during atherosclerosis development

Introduction: Gynura procumbens (Lorr.) Merr. (GP) displays cardio-protective effect, which may hinder atherogenesis induced by oxidized low-density lipoprotein (oxLDL) and leukocytes. The current study was undertaken to elucidate the chemical constituents of GP ethanol extract and its aqueous, chloroform, ethyl acetate, and hexane fractions, and their effects on CD4+ T cell differentiation during atherogenesis. Methods: Initially, the bioactive constituents in GP ethanol extract and its fractions were analyzed using gas chromatography-mass spectrometry (GC-MS). Generated mouse bone marrow dendritic cells (BMDC) were loaded with oxLDL and GP ethanol extract and its fractions for 24 hours and co-cultured with mouse CD4+ T cells for 72 hours. For the determination of T-bet, GATA-3, RORγt, Foxp3, DLL-3, and Jagged-1 mRNA gene expression, the floating cells (CD4+ T cells) and adherence cells (BMDC) were isolated from their total RNAs and reverse transcribed into cDNA.Results: GC-MS analysis showed that GP ethanol extract and its fractions contained various volatile compounds. GP ethanol extract and its fractions also increased the DLL-3 gene but suppressed Jagged-1 gene expression in oxLDL-treated BMDC. Furthermore, GP ethanol extract and its fractions suppressed T-bet, GATA-3, and RORγt gene expression but increased the expression of the Foxp3 gene in differentiated CD4 + T cells. Conclusion: GP ethanol extract and its fractions are composed of various bioactive chemical components that can induce anti-atherogenic effects by inhibiting pro-atherogenic cells such as Th1, Th2, and Th17 cells while increasing anti-atherogenic cells, Treg cells.

An optimized method for the induction and purification of mouse bone marrow dendritic cells

Dendritic cells (DCs) play an essential role in the initiation of adaptive immune responses, but they are rare in all organs. The traditional methods used to increase the yield and purity of DCs are the early removal of granulocyte culture medium and the isolation of high-purity DCs by magnetic-activated cell sorting (MACS). This study provides a more rapid and economical optimization method to obtain more high-purity DCs. (i) We harvested 18% more bone marrow (BM) cells by using forceps to crack the epiphysis instead of cutting it with scissors during BM cell extraction. (ii) When the cells in the culture medium that is discarded on day 3 in the traditional method were centrifuged and then added back to the petri dish, the DC yield on day 5 increased by 61%. (iii) On the third day, the addition of fresh medium and the retention of the original medium rather than discarding it increased the number of DCs harvested on the fifth day by 137%. (i-iii) The improved method cost an average of 74% less than the conventional method and yielded the same number and function of cells. (iv) The initial number of BM cells was increased by 15% in 4-week-old mice compared with 8-week-old mice. (v) The Percoll density centrifugation (PDS) method was used to purify DCs on day 6 after induction, and the purity of the DCs was greater than 90%, which showed no significant difference from the MACS method. However, the yield of the PDS method increased by 21%. In addition, the PDS method has a lower cost, with an average purification cost of 4 CNY ($0.58) compared with 648 CNY ($93.25) for MACS, reducing the cost by 99%. Therefore, high-purity and high-yield DCs can be rapidly obtained through a five-step improvement in the process of BM cell extraction, induction and purification.

Synthetic MUC1 breast cancer vaccine containing a Toll-like receptor 7 agonist exerts antitumor effects.

Adjuvant immunotherapy has recently emerged as a potential treatment strategy for breast cancer. The tumor-associated protein mucin 1 (MUC1) has received increasing attention due to its high expression in numerous types of common tumors, in which MUC1 acts as a cancer antigen. However, the simple mixed composition of an adjuvant and a peptide is not a sufficient rationale for a MUC1 peptide-based vaccine. The present study developed a novel Toll-like receptor 7 (TLR7) agonist-conjugated MUC1 peptide vaccine (T7-MUC1), which elicited an effective immune response and a robust antitumor effect in a mouse breast cancer model. In vitro, T7-MUC1 significantly increased the release of cytokines in mouse bone marrow dendritic cells and spleen lymphocytes, and induced the dendritic cell-cytokine-induced killer response against tumor cells with high MUC1 expression. In vivo, it was observed that the 4T1 tumor weights in mice immunized with the T7-MUC1 conjugate were reduced by ≥70% compared with those in the control group. Furthermore, the therapeutic responses in vivo were attributed to the increase in specific humoral and cellular immunity, including high antibody titers, antibody-dependent cell-mediated cytotoxicity and cytotoxic T-lymphocyte activity. The percentages of CD3+/CD8+ T-cells were significantly higher in the T7-MUC1 treatment group compared with those in the control group. Therefore, the results of the present study suggested that the T7-MUC1 vaccine inhibited tumor growth in mice and thus may have potential as a therapeutic candidate in clinical trials for breast cancer immunotherapy.

Co-delivery of CpG and antigen using hyaluronic acid bioconjugates-decorated nanoparticles to promote maturation and activation of dendritic cells

Objective To study the maturation and activation effects of hyaluronic acid (HA) modified polymer nanoparticles co-delivering adjuvants and antigens on mouse bone marrow dendritic cells (BMDCs). Methods HA-modified polylactic acid-glycolic acid copolymer (PLGA) and cationic lipid DOTAP were used as nanocarriers (DOTAP-PLGA) to co-deliver adjuvant CpG with model antigen ovalbumin (OVA). In the drug-loaded nanocarriers, CpG was covalently bound to the surface of HA, and OVA was physically blended into DOTAP-PLGA nanocarriers. The nanoparticles were characterized by transmission electron microscopy and dynamic light scattering. The in vitro release of CpG and OVA in the nanoparticles was investigated. The uptake and distribution of nanoparticles in mouse BMDCs were studied by flow cytometry and laser scanning confocal microscopy. The maturation and cytokine expression of mouse BMDCs were evaluated by flow cytometry and enzyme-linked immunosorbent assay, respectively. Results The CpG-HA-OVA-PLGA nanoparticles loading CpG and OVA were prepared. The average particle size was (305.1±2.2) nm and the polydispersity index was 0.203. A core-shell structure of the nanoparticles modified by HA was clearly observed by transmission electron microscopy. Cellular experiment results showed that CpG-HA-OVA-PLGA nanoparticles could be efficiently uptaken by mouse BMDCs, and promote lysosomal release of CpG and cytoplasmic delivery of antigen OVA. Compared with free OVA group and free OVA+CpG group, the CpG-HA-OVA-PLGA nanoparticles significantly up-regulated the expression of co-stimulatory molecules CD86 and CD40 (all P<0.01), major histocompatibility complex I (MHC-I) (P<0.01), and cytokine tumor necrosis factor-α (TNF-α) (P<0.01). Conclusions HA-modified CpG and OVA nanoparticle co-delivery vectors can effectively promote the maturation and activation of dendritic cells, which provides a basis for the development of novel vaccine vectors for the co-delivery of antigens and adjuvants. Key words: Nanoparticles; Co-delivery; CpG; Antigen; Dendritic cells

Immune response elicited by graphene oxide-based nanovaccine

Objective To study the antigen-specific immune response induced by the graphene oxide (GO) in mice. Methods OVA-loaded GO nano-immunocomplexes (GO-OVA) were prepared by co-incubation of nano GO with model antigen ovalbumin (OVA). Nano GO was characterized by atomic force microscopy and laser particle size analyzer. The cytotoxicity of GO to mouse bone marrow dendritic cells (BMDCs) was detected by cell counting kit (CCK-8). The GO-OVA uptake of BMDCs were observed by fluorescent staining. C57BL/6 mice were divided into OVA group, aluminum adjuvant OVA (Al-OVA) group and GO-OVA group (6 mice in each group) by body weight for in vivo immunization. The levels of OVA-specific antibody IgG (total IgG, IgG1, and IgG2a) in serum of mice were detected by enzyme-linked immunosorbent assay (ELISA). The T lymphocyte subsets in spleen and inguinal lymph nodes of mice were detected by flow cytometry. Results The average particle size of the prepared nano GO was (294.34±4.68) nm, and the polydispersity coefficient was 0.208. Nano GO has less toxicity to mouse BMDCs. The results of in vitro experiments indicated that GO-OVA nanovaccine can be efficiently internalized by mouse BMDCs. The antigen-specific IgG antibodies induced by the GO-OVA was similar to that of aluminum adjuvant and the difference was not statistically significant (P>0.05), and the Th1-type response was predominant. The proportions of CD4+ and CD8+ T lymphocytes in the spleen and inguinal lymph nodes in GO-OVA group were significantly higher than those in OVA and Al-OVA groups, and the differences were statistically significant (all P<0.05). Conclusions GO-OVA nano-immunocomplexes can induce both humoral and cellular immune responses in mice, which provides basis for the development of novel vaccine vectors and adjuvants. Key words: Graphene oxide; Nanovaccine; Humoral immune response; Cellular immune response

Mouse β-defensin-14 for inducing the maturation of dendritic cells

Backgroundβ-defensins are an excellent antimicrobial peptide against microbial infection in which dendritic cells (DCs) play a crucial role by improving the innate and adaptive immune defense. However, it is unclear whether BDs affect DC maturation. This work aimed to study the effects of mouse β-defensin-14 (MBD-14) on DC maturation. MethodsVia in vitro using mouse bone marrow DCs, the maturation of DCs was evaluated by cell morphological staining, flow cytometry, endocytosis assay, and allogeneic mixed lymphocyte reaction, respectively. And it was also assessed by in vivo establishing a mouse air-pouch model for flow cytometric determination, cytokine analysis, and histological staining.Additionally, CLI-095, an inhibitor of Toll-like receptor-4 (TLR-4), was used to determine whether TLR-4 is possibly involved in DC maturation. ResultsIt was found MBD-14 promoted DCs to form more filopodia and lamellipodia, increased the expression of DC maturation markers (CD40 and MHC-II), decreased their endocytic capacity, and enhanced T-cell proliferation. The analyses of the air-pouch exudates were consistent with the in vitro results of MBD-14 activating DCs. And when CLI-095 was applied, DC maturation was inhibited partly. ConclusionsThis work demonstrates that MBD-14 can promote the maturation of DCs in which TLR-4 is possibly involved.

Enhanced immunization via dissolving microneedle array-based delivery system incorporating subunit vaccine and saponin adjuvant.

PurposeTo enhance the immunogenicity of the model subunit vaccine, ovalbumin (OVA) was combined with platycodin (PD), a saponin adjuvant. To reduce the toxicity of PD, OVA, and adjuvant were loaded together into liposomes before being incorporated into a dissolving microneedle array.MethodsOVA- and PD-loaded liposomes (OVA-PD-Lipos) were prepared using the film dispersion method. Their uptake behavior, toxicity to mouse bone marrow dendritic cells (BMDCs), and hemolytic activity to rabbit red blood cells (RBCs) were evaluated. The OVA-PD-Lipos were incorporated into a dissolving microneedle array. The chemical stability of OVA and the physical stability of OVA-PD-Lipos in microneedle arrays were investigated. The immune response of Institute of Cancer Research mice and potential skin irritation reaction of rabbits to OVA-PD-Lipos-MNs were evaluated.ResultsThe uptake of OVA by mouse BMDCs was greatly enhanced when OVA was prepared as OVA-PD-Lipos, and in this form, the toxicity of PD was dramatically reduced. OVA was chemically stable as OVA-PD-Lipos, when OVA-PD-Lipos was incorporated into a dissolving microneedle array. Institute of Cancer Research mice treated with OVA-PD-Lipos-MNs showed a significantly enhanced immune response. PD combined with OVA elicited a balanced Th1 and Th2 humoral immune response in mice, with minimal irritation in rabbit skin.ConclusionThe dissolving microneedle array-based system is a promising delivery vehicle for subunit vaccine and its adjuvant.

Targeted antigen delivery to dendritic cell via functionalized alginate nanoparticles for cancer immunotherapy

The purpose of the present study was to identify an “easy-to-adopt” strategy to enhance immune responses using functionalized alginate (ALG) nanoparticles (MAN-ALG/ALG=OVA NPs), which were prepared by CaCl2 cross-linking of two different types of ALG. The mannose (MAN) modified ALG (MAN-ALG) was used for dendritic cell targeting. The other component, composed of ovalbumin (OVA), a model antigen, is conjugated to ALG (ALG=OVA) via pH sensitive Schiff base bond. Grafting of alginate was demonstrated by FT-IR and 1H NMR, while the morphological structure, particle size, Zeta potential of MAN-ALG/ALG=OVA NPs were measured using TEM and DLS. The OVA releasing behavior of MAN-ALG/ALG=OVA NPs was determined as a function of pH. Antigen uptake was examined by flow cytometry and confocal laser scanning microscopy in vitro using mouse bone marrow dendritic cells (BMDCs). The results showed that MAN-ALG/ALG=OVA NPs facilitated antigen uptake of BMDCs and cytosolic release of the antigen. Significant up-regulation of cytokine secretion and expression levels of the surface co-stimulatory molecules were also observed in MAN-ALG/ALG=OVA NPs-treated BMDCs, compared to free OVA. In vivo bio-distribution study using Cy7 (a near-infrared fluorescence dye) labeled MAN-ALG/ALG=OVA NPs showed efficient in vivo trafficking of the nanoparticles from the injection site to the draining lymph nodes. Moreover, MAN-ALG/ALG=OVA NPs were found to enhance cross-presentation of OVA to B3Z T cell hybridoma in vitro. Subcutaneous administration of MAN-ALG/ALG=OVA NPs also induced major cytotoxic T lymphocytes (CTL) response and inhibition of E.G7 tumor growth in C57BL/6 mice. In summary, we report here that the MAN-ALG/ALG=OVA NPs have the potential as a potent nanovaccine for cancer immunotherapy.

Heat shock proteins 70 and 90 from Clonorchis sinensis induce Th1 response and stimulate antibody production

BackgroundHeat shock proteins (HSPs) are found in all prokaryotes and most compartments of eukaryotic cells. Members of the HSP family mediate immune responses to tissue damage or cellular stress. However, little is known about the immune response induced by the oriental liver fluke, Clonorchis sinensis, even though this organism is carcinogenic to humans. We address this issue in the present study in mouse bone marrow dendritic cells (mBMDCs), using recombinant HSP70 and 90 from C. sinensis (rCsHSP70 and rCsHSP90).MethodsrCsHSP70 and rCsHSP90 were produced in an E. coli system. Purified recombinant proteins were treated in BMDCs isolated from C57BL/6 mice. T cells were isolated from Balb/c mice and co-cultured with activated mBMDCs. Expression of surface molecules was measured by flow cytometry and cytokine secretion was quantified using ELISA. C57BL/6 mice were divided into four groups, including peptide alone, peptide/Freund’s adjuvant, peptide/CsHSP70, peptide/CsHSP90, and were immunized intraperitoneally three times. Two weeks after final immunization, antibodies against peptide were measured using ELISA.ResultsBoth proteins induced a dose-dependent upregulation in major histocompatibility complex and co-stimulatory molecule expression and increased secretion of pro-inflammatory cytokines including interleukin (IL)-1β, -6, and -12p70 and tumor necrosis factor-α in mBMDCs. Furthermore, when allogenic T cells were incubated with mBMDCs activated by rCsHSP70 and rCsHSP90, the helper T cell (Th)1 cytokine interferon-γ was up-regulated whereas the level of the Th2 cytokine IL-4 was unchanged. These results indicate that rCsHSPs predominantly induce a Th1 response. Over and above these results, we also demonstrated that the production of peptide-specific antibodies can be activated after immunization via in vitro peptide binding with rCsHSP70 or rCsHSP90.ConclusionThis study showed for the first time that the HSP or HSP/peptide complexes of C. sinensis could be considered as a more effective vaccine against C. sinensis infection as results of the activator of host immune response as well as the adjuvant for antigenic peptide conjugate to induce peptide-specific antibody response in mice.

Silica nanoparticles activate purinergic signaling via P2X7 receptor in dendritic cells, leading to production of pro-inflammatory cytokines

We examined the mechanism of SNP-mediated stimulation of IL-1β and IL-18 production via P2R-mediated pathways in mouse bone marrow dendritic cells (mBMDCs). Examination of uptake of SNPs with diameters of 30, 70, and 300nm (SNP30, SNP70, and SNP300, respectively) by lipopolysaccharide-matured mBMDCs revealed that significant uptake of SNP30 occurred within as short a time as 1h. Production of IL-1β and IL-18 by cells exposed to SNPs increased dose-dependently, and was highest in cells exposed to SNP30. The SNP30-induced cytokine production was significantly inhibited by ATPase (apyrase) and by P2X7 receptor antagonist (A438079). ATP release was also highest in SNP30-exposed cells. Treatment of mBMDCs with exogenous ATP induced release of high levels of IL-1β and IL-18, and this release was also significantly inhibited by apyrase and A438079. The order of effectiveness of the three SNPs for inducing intracellular reactive oxygen species (ROS) production accorded well with those of cytokine production and ATP release. ROS production was inhibited by diphenyleneiodonium chloride (DPI). SNPs, especially SNP30, activate purinergic signaling in matured mBMDCs by inducing ATP release via P2X7 receptor. ATP induces ROS production via NADPH oxidase, and ROS activate inflammasomes, leading to caspase-1-dependent processing of pro-cytokines and release of IL-1β and IL-18.

Conjugation of toll-like receptor-7 agonist to gastric cancer antigen MG7-Ag exerts antitumor effects.

To investigate the effects of our tumor vaccines on reversing immune tolerance and generating therapeutic response. Vaccines were synthesized by solid phase using an Fmoc strategy, where a small molecule toll-like receptor-7 agonist (T7) was conjugated to a monoclonal gastric cancer 7 antigen mono-epitope (T7-MG1) or tri-epitope (T7-MG3). Cytokines were measured in both mouse bone marrow dendritic cells and mouse spleen lymphocytes after exposed to the vaccines. BALB/c mice were intraperitoneally immunized with the vaccines every 2 wk for a total of three times, and then subcutaneously challenged with Ehrlich ascites carcinoma (EAC) cells. Three weeks later, the mice were killed, and the tumors were surgically removed and weighed. Serum samples were collected from the mice, and antibody titers were determined by ELISA using an alkaline phosphate-conjugated detection antibody for total IgG. Antibody-dependent cell-mediated cytotoxicity was detected by the lactate dehydrogenase method using natural killer cells as effectors and antibody-labeled EAC cells as targets. Cytotoxic T lymphocyte activities were also detected by the lactate dehydrogenase method using lymphocytes as effectors and EAC cells as targets. Vaccines were successfully synthesized and validated by analytical high performance liquid chromatography and electrospray mass spectrometry, including T7, T7-MG1, and T7-MG3. Rapid inductions of tumor necrosis factor-α and interleukin-12 in bone marrow dendritic cells and interferon γ and interleukin-12 in lymphocytes occurred in vitro after T7, T7-MG1, and T7-MG3 treatment. Immunization with T7-MG3 reduced the EAC tumor burden in BALB/c mice to 62.64% ± 5.55% compared with PBS control (P < 0.01). Six or nine weeks after the first immunization, the monoclonal gastric cancer 7 antigen antibody increased significantly in the T7-MG3 group compared with the PBS control (P < 0.01). As for antibody-dependent cell-mediated cytotoxicity, antisera obtained by immunization with T7-MG3 were able to markedly enhance cell lysis compared to PBS control (31.58% ± 2.94% vs 18.02% ± 2.26%; P < 0.01). As for cytotoxic T lymphocytes, T7-MG3 exhibited obviously greater cytotoxicity compared with PBS control (40.92% ± 4.38% vs 16.29% ± 1.90%; P < 0.01). A successful method is confirmed for the design of gastric cancer vaccines by chemical conjugation of T7 and multi-repeat-epitope of monoclonal gastric cancer 7 antigen.

Maturation of mouse bone marrow dendritic cells (BMDCs) induced by Laminaria japonica polysaccharides (LJP)

The seaweed Laminaria japonica has been investigated in a laboratory research for its medical significance and LJP has been purified now. The objective of present study was to look at effect of LJP on structural, phenotypic and functional maturation of murine BMDCs. The structural maturation of BMDCs induced by LJP was evaluated by transmission electron microscopy (TEM); The phenotypic maturation of BMDCs was studied by flow cytometry(FCM) and functional maturation of BMDCs was analyzed by FITC-dextran, acid phosphatase (ACP) activity and enzyme linked immunosorbent assay (ELISA). We hereby proved that LJP markedly induced maturation of BMDCs with the data of decreased the number of lysosomes, upregulated expression of CD80, CD83, CD86, CD40 and MHC II key membrane molecules on BMDCs, downregulated phagocytosis, enriched production of IL-12 and TNF-α secreted by BMDCs. Therefore it should be concluded that LJP was with strong ability to induce maturation of BMDCs. Our data provided direct evidence to suggest that LJP could be considered as an immune stimulant in improving immune handicapped situation and as a useful adjuvant in vaccine designing.

Bioreducible alginate-poly(ethylenimine) nanogels as an antigen-delivery system robustly enhance vaccine-elicited humoral and cellular immune responses

Although polysaccharide nanogels have emerged as a novel antigen delivery system for vaccine development, whether modulating the redox sensitivity of nanogels could improve vaccine efficacy remains unclear. In the present study, we generated bioreducible cationic alginate-polyethylenimine (PEI) nanogels as a novel vaccine delivery system. Briefly, nanogels were prepared by the electrostatic interaction of negatively charged alginate sodium with branched PEI2k, followed by disulfide cross-linking to generate bioreducible nanogels (AP-SS). The AP-SS nanogels demonstrated great antigen-loading capacity and minimal cytotoxicity. The in vitro study showed that reducible AP-SS nanogels not only facilitated antigen uptake by mouse bone marrow dendritic cells (BMDCs), but also promoted intracellular antigen degradation and cytosolic release. Moreover, AP-SS nanogels significantly enhanced both MHC class I and II antigen presentation by BMDCs. Compared with the non-reducible nanogels, AP-SS nanogels more potently enhanced vaccine-induced antibody production and CD8+ T cell-mediated tumor cell lysis. Hence, the bioreducible alginate-PEI nanogels could serve as a potent adjuvant to improve vaccine-elicited humoral and cellular immune responses.

Toll-like receptor 3 agonist complexed with cationic liposome augments vaccine-elicited antitumor immunity by enhancing TLR3–IRF3 signaling and type I interferons in dendritic cells

Cancer vaccine-based immunotherapy is emerging as a novel therapeutic strategy for cancer treatment. However, its antitumor effect remains unsatisfied due to the poor immunogenicity of tumor antigens (Ags). Although polyriboinosinic: polyribocytidylic acid (PIC), a TLR3 agonist, has been reported as a promising adjuvant for cancer vaccines, its immunopotency may be limited by insufficient cellular penetration. In the present study, we incorporated PIC into DOTAP cationic liposome to generate PIC–DOTAP Liposome Complex (PDLC) nanoparticles. The results showed that PDLC was more potent than DOTAP or PIC to enhance vaccine-induced tumor-specific cytotoxic T lymphocyte (CTL) response and IFN-γ production. Moreover, two doses of PDLC vaccines remarkably suppressed tumor growth in mice, which involved the participance of CD8+ T cells and depended on the presence of Ag. The superior antitumor effect of PDLC vaccines could be attributable to enhanced maturation of mouse bone-marrow dendritic cells (BMDCs) and increased production of type I IFNs. More importantly, PDLC strengthened the TLR3 signaling in BMDCs by enhancing the interaction of PIC with TLR3 and augmenting downstream IRF-3 phosphorylation, as well as elevating IRF-3/IRF-7 mRNA transcription. Taking together, the complex of PIC and DOTAP liposomes enhanced PIC uptake and consequential TLR3 signaling in BMDCs, which in turn promoted DC maturation and type I IFN production, thereby augmenting the antitumor effect of cancer vaccines.

In vitroinduction of lymph node cell proliferation by mouse bone marrow dendritic cells following stimulation with differentEchinococcus multilocularisantigens

The immune response of mice experimentally infected with Echinococcus multilocularis metacestodes becomes impaired so as to allow parasite survival and proliferation. Our study tackled the question on how different classes of E. multilocularis antigens (crude vesicular fluid (VF); purified proteinic rec-14-3-3; purified carbohydrate Em2(G11)) are involved in the maturation process of bone-marrow-derived dendritic cells (BMDCs) and subsequent exposure to lymph node (LN) cells. In our experiments, we used BMDCs cultivated from either naïve (control) or alveolar echinococcosis (AE)-infected C57BL/6 mice. We then tested surface markers (CD80, CD86, MHC class II) and cytokine expression levels (interleukin (IL)-10, IL-12p40 and tumour necrosis factor (TNF)-α) of non-stimulated BMDCs versus BMDCs stimulated with different Em-antigens or lipopolysaccharide (LPS). While LPS and rec-14-3-3-antigen were able to induce CD80, CD86 and (to a lower extent) MHC class II surface expression, Em2(G11) and, strikingly, also VF-antigen failed to do so. Similarly, LPS and rec-14-3-3 yielded elevated IL-12, TNF-α and IL-10 expression levels, while Em2(G11) and VF-antigen didn't. When naïve BMDCs were loaded with VF-antigen, they induced a strong non-specific proliferation of uncommitted LN cells. For both, BMDCs or LN cells, isolated from AE-infected mice, proliferation was abrogated. The most striking difference, revealed by comparing naïve with AE-BMDCs, was the complete inability of LPS-stimulated AE-BMDCs to activate lymphocytes from any LN cell group. Overall, the presenting activity of BMDCs from AE-infected mice seemed to trigger unresponsiveness in T cells, especially in the case of VF-antigen stimulation, thus contributing to the suppression of clonal expansion during the chronic phase of AE infection.

Antitumor effects of murine bone marrow-derived dendritic cells infected with xenogeneic livin α recombinant adenoviral vectors against Lewis lung carcinoma

Transduction with recombinant, replication-defective adenoviral (rAd) vectors encoding a transgene is an efficient method for gene transfer into dendritic cells (DCs). Livin is a member of the inhibitor of apoptosis protein family. Lung cancer and many other tumors express livin at high levels; whereas, normal fully differentiated cells generally do not. Therefore, livin represents a tumor-specific target for cancer vaccine therapy. Self proteins like livin may not stimulate potent antitumor immune responses due to central immunologic tolerance. Small variations in protein sequence that may exist between homologous proteins of different species can break tolerance to the native antigen. To study immunogenicity of a xenogeneic livin protein, we constructed an recombinant adenoviral vectors containing the human livin α genes (rAd-hlivin α) and vaccinated C57BL/6 mice with mouse bone marrow dendritic cells (BMDCs) transfected with rAd-hlivin α gave rise to potent livin-specific cytotoxic T lymphocyte (CTL) capable of lysing Lewis lung carcinoma (LLC) cells. Moreover, vaccination of mice with rAd-hlivin α-transduced DCs (rAd-hlivin α DCs) induced a potent protective and therapeutic anti-tumor immunity to LLC in a subcutaneous model along with prolonged survival compared to mice vaccinated with control recombinant adenovirus-transduced DCs(rAd-c DCs) or DCs alone. Therefore, xenogeneic differences between human and murine sequences might be exploited to develop immunogenic tumor vaccines.