Derived Dendritic Cells Research Articles

Niflumic acid renders dendritic cells tolerogenic and up-regulates inhibitory molecules ILT3 and ILT4

Niflumic acid is a member of non-steroidal anti-inflammatory agents, from which aspirin was recently shown to inhibit maturation of human-monocyte derived dendritic cells (DCs). DCs are crucial regulators of the immune response, capable of inducing immunity as well as tolerance. In our in vitro study we showed a tolerogenic effect of NFA on phenotype and function of LPS-matured monocyte-derived DCs. Different drug concentrations dose-dependently down-regulated the expression of co-stimulatory molecules, particularly CD80 and lowered the expression of dendritic cell marker CD1a. Opposingly, the expressions of two inhibitory surface molecules, associated with tolerogenic DCs, immunoglobulin-like transcripts (ILT)3 and ILT4 were induced in treated DCs. The levels of TNFα production by NFA-treated DCs did not change significantly compared to controls, whereas the IL-12p70 and IL-10 production was completely abrogated at higher drug concentrations. However, at lower drug concentrations, the production of IL-12p70 was increased. There were no significant differences in the uptake of FITC labeled dextran by treated DCs compared to untreated cells. In allogeneic cultures with whole CD4 + T cells, dendritic cells differentiated in the presence of NFA appeared poor stimulators of CD4 + T-cell proliferation, even compared to immature DCs (iDCs). These results indicate the immunosuppressive properties of NFA, which may be therapeutically useful in controlling chronic immune and/or inflammatory diseases, by modulating DC characteristics towards tolerogenic DCs.

Protection against Influenza A Virus by Memory CD8 T Cells Requires Reactivation by Bone Marrow-Derived Dendritic Cells

Influenza A virus is the causative agent of an acute inflammatory disease of the airway. Although Abs can prevent infection, disease and death can be prevented by T cell-mediated immunity. Recently, we showed that protection against lethal influenza A (PR8/34) virus infection is mediated by central memory CD8 T cells (T(CM)). In this study, using relB(-/-) mice we began to investigate the role of bone marrow (BM)-derived dendritic cells (DCs) in the mechanism of protection. We found that in the absence of functional DCs, memory CD8 T cells specific for the nucleoprotein epitope (NP(366-374)) fail to protect even after adoptive transfer into naive recipients. Through an analysis of Ag uptake, activation of memory CD8 T cells, and display of peptide/MHC complex by DCs in draining LNs and spleen early after virus infection, we established that lack of protection is associated with defective Ag presentation by BM-derived DCs and defective homing of memory T cells in the lymph nodes draining the airway tract. Collectively, the data suggest that protection against the influenza A virus requires that memory CD8 T cells be reactivated by Ag presented by BM-derived DCs in the lymph nodes draining the site of infection. They also imply that protection depends both on the characteristics of systemic adaptive immunity and on the coordinated interplay between systemic and local immunity.

Staphylococcal enterotoxin B can activate NKT cells independent of Cd1d

Staphylococcal enterotoxin B (SEB) is a superantigen that can activate 5 to 30% of the T cell population and unlike conventional antigens, it directly binds to the MHC Class II molecules and activates T cells with variable TCR β chain such as Vβ8. Such activation triggers massive cytokine release that leads to rash, fever, and can often lead to multi‐organ failure, coma and death. The precise role of NKT cells in the cytokine production and toxicity induced by SEB is not clear. In this study, we investigated the role of NKT cells following SEB immunization. We found that SEB administration in vivo triggered Vascular Leak Syndrome (VLS) in the lungs of wild‐type (WT) mice while it was significantly decreased in Cd1d−/−mice lacking NKT cells. This was reversed when WT spleenocytes were injected into the Cd1d−/−mice. Cd1d−/−mice also exhibited decreased cytokine production in response to SEB, particularly IL‐4. When bone marrow derived dendritic cells (DCs) from WT and Cd1d−/−mice were tested for their ability to activate NKT cells, we noted that DCs from Cd1d−/−mice were able to activate NKT cells. Together, our studies demonstrate that NKT cells can be directly activated independent of CD1d. Thus, the plethora of cytokines produced following SEB can also be attributed to initial NKT cell activation (Supported in part by NIH grants: AI053703, ES09098, AI058300, DA016545, HL058641 and P01AT003961).

Matrilysin (MMP-7) Is a Novel Broadly Expressed Tumor Antigen Recognized by Antigen-Specific T Cells.

Identification and targeting of antigens involved in malignant transformation, survival advantage of tumor cells or their metastatic spread might represent a promising approach in the development of immunotherapeutic strategies to treat malignant diseases. Using oligonucleotide microarray analysis we found that matrix metalloproteinase- (MMP-)7 is extensively upregulated in a broad variety of malignant tissues. MMPs are a family of endopeptidases that degrade extracellular matrix (ECM). MMP-7 can promote cancer invasion by proteolytic cleavage of ECM and basement membrane proteins. In addition, MMP-7 has been shown to accelerate the proliferation of umbilical vein endothelial cells in vitro and promote angiogenesis. Thus, targeting MMP-7 may affect both tumor cells and tumor vessels. In order to analyze the feasibility to induce MMP-7-specific CTLs, monocyte derived dendritic cells (DCs) were generated from a healthy donor, electroporated with full length MMP-7-in-vitro-transcript (IVT) that coded for the entire MMP-7-protein and used as antigen presenting cells (APCs) for the in vitro induction of antigen-specific CTLs. These in vitro generated CTLs demonstrated an antigen-specific and HLA class I-restricted cytolytic activity against tumor cells endogenously expressing MMP-7. The specificity and HLA-restriction was confirmed in cold target inhibition assays and by using blocking antibodies. In the next set of experiments, we wanted identify HLA class I-presented MMP-7-peptides that are recognized by our CTL lines. To accomplish this, peptides bound to HLA class I molecules were isolated from surgically removed RCC specimens that overexpressed MMP-7. By applying mass spectrometry-based peptide sequencing of the HLA ligands, a HLA-A3-binding peptide derived from the novel antigen MMP-7 was identified. To analyze the immunogenicity of the new MMP-7 derived peptide with respect to its ability to elicit primary T cell responses, we induced MMP-7-specific CTLs in vitro. DCs derived from a HLA-A3+ healthy donor were pulsed with the HLA-A3-binding antigenic peptide and utilized as APCs. The induced CTL lines demonstrated antigen-specific killing: T cells only recognized autologous DCs loaded with the cognate MMP-7-A3 peptide or HLA matched tumor cells expressing MMP-7, whereas they did not lyse target cells pulsed with an irrelevant HLA-A3-binding peptide. The objective of our study is the development of a DC-based adoptive immunotherapy for patients with malignant diseases. Therefore, we sought to analyze, whether our previous findings could be applied in patients with malignant diseases in an autologous setting. To achieve this, we generated DCs from an HLA-A3+ ALL patient in complete remission after chemotherapy and used them as APCs for CTL induction after electroporation with MMP-7-IVT. The autologous ALL blasts, frozen at the time of diagnosis, were used as targets in Cr-release assays. These cells were efficiently lysed by the CTLs specific for MMP-7, confirming the expression analysis by RT-PCR. Importantly, the normal autologous purified DCs, CD3+ T and CD19+ B cells were not recognized. In conclusion, our study describes the identification of a novel broadly expressed T-cell epitope derived from the MMP-7-protein that represents an interesting candidate to be applied in immunotherapies of human malignancies capable of targeting neo-vascularization and malignant cells in developing tumors.

Novel C5a Agonist-Based Dendritic Cell Vaccine in a Murine Model of Melanoma

A novel method to improve targeting and presentation of poorly immunogenic tumor-related antigens was investigated. This was performed with a molecular adjuvant constructed by covalently linking a response selective peptide agonist of C5a (YSFKDMP(MeL)aR) to known melanoma tumor-related antigens. C57Bl/6J mice were injected subcutaneously with bone marrow derived dendritic cells (DCs) pulsed with a melanoma epitope (TRP2-P2/Agonist), melanoma epitope tyrosinase (TYR/Agonist), a nonfunctional reverse conformation C5a agonist bound to TYR(reverse peptide) or DMSO-PBS vehicle. Mice were injected with the pulsed DCs and cytokines IL-2 and GM-CSF three times prior to subcutaneous challenge with B16-F10 melanoma cells. All groups subsequently received DC vaccine boosters twice per week. Tumor growth was reduced and survival enhanced in mice immunized with the combination of TRP2-P2/Agonist and TYR/Agonist compared to mice receiving reverse peptide or vehicle.

Human Allogeneic and Murine Xenogeneic Dendritic Cells Are Cytotoxic to Human Tumor Cells via Two Distinct Pathways

Human monocyte-derived dendritic cells (DCs), stimulated with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 1 week, major histocompatibility complex killed human tumor cells in 24-hour cytotoxicity assays. These immature DCs were >90% CD11c, major histocompatibility complex class II(+), but <1% were CD83(+) cells. Within 24 hours, these DCs ingested tumor membranes. The DC cells also lysed Jurkat lymphoma cells, but not Jurkat cells genetically knocked out of the Fas-associated death domain (FADD) or caspase-8. DC2.4, a cloned murine DC line, also displayed cytotoxicity toward U-251 cells, although these murine DCs were less potent than human DC. DC2.4 did not kill Jurkat cells, rat T9 glioma cells, or human Caco-2 colon cancer cells, suggesting that a unique receptor or ligand interaction exists between the DC and U-251 cells. This interaction was destroyed by the paraformaldehyde fixation of the tumor cells. Supernatants from the cultures of DC2.4 and tumor cells were analyzed by the Griess reaction for signs of nitric oxide (NO) production. Augmented NO production occurred in DC2.4/U-251 and DC2.4/Jurkat cultures but was not seen in the human DC/U-251 cultures. These studies suggest that DCs possess different mechanisms of tumoricidal activity.

Enhanced serum antigen-specific IgG 1 and proinflammatory cytokine production in nicotinic acetylcholine receptor α7 subunit gene knockout mice

Human and murine immune cells such as mononuclear leukocytes consisting of mainly T and B cells, bone marrow derived dendritic cells (DCs) and macrophages all express various nicotinic acetylcholine (ACh) receptor (nAChR) subunits. Activated T cells and DCs have the ability to synthesize ACh by choline acetyltransferase, suggesting the role of non-neuronal cholinergic system expressed in immune cells in the regulation of immune cell function. Stimulation of human leukemic T and B cell lines with nicotine causes a transient Ca 2+-signaling that is antagonized by α-bungarotoxin, suggesting the involvement of α7 subunit. Furthermore, α7 nAChRs have been shown to negatively regulate synthesis and release of tumor necrosis factor (TNF)-α in macrophages. These findings suggest that immune cell function is regulated by its own non-neuronal cholinergic system, at least in part, via α7 nAChR-mediated pathways. In the present study, we tested the role of α7 nAChRs in the regulation of immune function by measuring total serum and antigen-specific IgG 1 and IgM, and production of TNF-α, gamma interferon (IFN-γ) and interleukin (IL)-6 in activated spleen cells of nAChR α7 subunit gene knockout (α7 KO) and wild-type C57BL/6J mice immunized with ovalbumin (OVA). We found that serum levels of total and anti-OVA-specific IgG 1 were significantly elevated in α7 KO mice, though there were no significant differences in serum levels of total and anti-OVA-specific IgM between the two genotypes. Production of TNF-α, IFN-γ and IL-6 in spleen cells was significantly facilitated in α7 KO mice. Expression of AChE mRNA was not different between the two genotypes. These results suggest that α7 nAChRs are involved in the regulation of cytokine production, through which modulates TNF-α, IFN-γ and IL-6 productions, leading to modification of antibody production, but are not involved in expression of cholinergic components in immune cells.

CXCR4 engagement promotes dendritic cell survival and maturation

It has been reported that human monocyte derived-dendritic cells (DCs) express CXCR4, responsible for chemotaxis to CXCL12. However, it remains unknown whether CXCR4 is involved in other functions of DCs. Initially, we found that CXCR4 was expressed on bone marrow-derived DCs (BMDCs). The addition of specific CXCR4 antagonist, 4-F-Benzoyl-TN14003, to the culture of mouse BMDCs decreased their number, especially the mature subset of them. The similar effect was found on the number of Langerhans cells (LCs) but not keratinocytes among epidermal cell suspensions. Since LCs are incapable of proliferating in vitro, these results indicate that CXCR4 engagement is important for not only maturation but also survival of DCs. Consistently, the dinitrobenzene sulfonic acid-induced, antigen-specific in vitro proliferation of previously sensitized lymph node cells was enhanced by CXCL12, and suppressed by CXCR4 antagonist. These findings suggest that CXCL12–CXCR4 engagement enhances DC maturation and survival to initiate acquired immune response.

Signaling through FcγRIII is required for optimal T helper type (Th)2 responses and Th2-mediated airway inflammation

Although inhibitory Fcγ receptors have been demonstrated to promote mucosal tolerance, the role of activating Fcγ receptors in modulating T helper type (Th)2-dependent inflammatory responses characteristic of asthma and allergies remains unclear. Here, we demonstrate that signaling via activating Fcγ receptors in conjunction with Toll-like receptor 4 stimulation modulated cytokine production from bone marrow–derived dendritic cells (DCs) and augmented their ability to promote Th2 responses. Ligation of the low affinity receptor FcγRIII was specifically required for the enhanced Th2 responses, as FcγRIII−/− DCs failed to augment Th2-mediated airway inflammation in vivo or induce Th2 differentiation in vitro. Further, FcγRIII−/− mice had impaired Th2 cytokine production and exhibited reduced airway inflammation, whereas no defect was found in FcγRI−/− mice. The augmentation of Th2 immunity was regulated by interleukin 10 production from the DCs but was distinct and independent of the well-established role of FcγRIII in augmenting antigen presentation. Thus, our studies reveal a novel and specific role for FcγRIII signaling in the regulation of Th cell responses and suggest that in addition to immunoglobulin (Ig)E, antigen-specific IgG also contributes to the pathogenesis of Th2-mediated diseases such as asthma and allergies.

2,3,7,8-Tetrachlorodibenzo- p-dioxin modulates functional differentiation of mouse bone marrow-derived dendritic cells : Downregulation of RelB by 2,3,7,8-tetrachlorodibenzo- p-dioxin

We have previously shown that benzo( a)pyrene inhibits the growth and functional differentiation of mouse bone marrow (BM)-derived dendritic cells (DCs) [Hwang, J.A., Lee, J.A., Cheong, S.W., Youn, H.J., Park, J.H., 2007. Benzo( a)pyrene inhibits growth and functional differentiation of mouse bone marrow-derived dendritic cells. Downregulation of RelB and eIF3 p170 by benzo( a)pyrene. Toxicol. Lett. 169, 82–90]. Since the toxic effects of benzo( a)pyrene are aryl hydrocarbon receptor (AhR)-dependent, we examined the effects of the very potent AhR agonist 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on the growth and functional differentiation of mouse BM-derived DCs. Ten nanomolars of TCDD had significant effects on functional differentiation of mouse DCs derived from BM cultured in the presence of GM-CSF and IL-4. The yields of DCs, flow-cytometrically analyzed for co-expression of CD11c/MHCII or CD11c/CD86, were reduced for TCDD-treated cultures, but TCDD itself had no effect on the growth of BM. DCs from TCDD-treated cultures expressed higher levels of MHCII and CD86, whereas expression of CD11c was reduced, compared with vehicle-treated cultures. Production of IL-10, but not IL-12, by the DCs from TCDD-treated cultures was decreased. Allogeneic T-cell stimulating ability of TCDD-treated DCs was increased compared to control DCs. The effects of TCDD were dependent on aryl hydrocarbon receptor (AhR), because α-naphthoflavone, an AhR antagonist, suppressed the effects of TCDD on IL-10 production and T-cell stimulating ability. RT-PCR revealed the downregulation of RelB, a transcription factor necessary for DCs differentiation and function. Taken together, although benzo( a)pyrene and TCDD exert their effects via binding to AhR, their effects on the growth and functional differentiation of bone marrow-derived DCs are different.

Lead effects on development and function of bone marrow-derived dendritic cells promote Th2 immune responses

Although lead (Pb) has significant effects on the development and function of macrophages, B cells, and T cells and has been suggested to promote allergic asthma in mice and humans, Pb modulation of bone marrow (BM)-derived dendritic cells (DCs) and the resultant DC effects on Th1 and Th2 development have not been examined. Accordingly, we cultured BM cells with murine granulocyte macrophage-colony stimulating factor (mGM-CSF) ± PbCl 2. At day 10, culture supernatant (SN) and non-adherent cells were harvested for analysis. Additionally, day 10 non-adherent BM-DCs were harvested and recultured with mGM-CSF + LPS ± Pb for 2 days. The day 10 Pb exposure significantly inhibited BM-DC generation, based on CD11c expression. Although fewer DCs were generated with Pb, the existing Pb-exposed DCs had significantly greater MHC-II expression than did the non-Pb-exposed DCs. However, these differences diminished upon LPS stimulation. After LPS stimulation, CD80, CD86, CD40, CD54, and MHC-II were all up-regulated on both Pb-DCs and DCs, but Pb-DCs expressed significantly less CD80 than did DCs. The CD86:CD80 ratio suggests a Pb-DC potential for Th2 cell development. After LPS stimulation, IL-6, IL-10, IL-12 (p70), and TNF-α levels significantly increased with both Pb-DCs and DCs, but Pb-DCs produced significantly less cytokines than did DCs, except for IL-10, which further supports Pb-DC preferential skewing toward type-2 immunity. In vitro studies confirm that Pb-DCs have the ability to polarize antigen-specific T cells to Th2 cells. Pb-DCs also enhanced allogeneic and autologous T cell proliferation in vitro, and in vivo studies suggested that Pb-DCs inhibited Th1 effects on humoral and cell-mediated immunity. The Pb effect was mainly on DCs, rather than on T cells, and Pb's modification of DC function appears to be the main cause of Pb's promotion of type-2-related immunity, which may relate to Pb's enhanced activation of the Erk/MAP kinase pathway.

Methamphetamine Inhibits β-Chemokines and Co-Stimulatory Molecule Expression by Dendritic Cells

The US is currently experiencing a serious epidemic of methamphetamine (Meth) use entangled with HIV-1 infection. Blood monocyte derived dendritic cells (DC ) are the first line of defense against HIV-1 infection and are the initial target of HIV-1 in injection drug users. Chemokines are known to be HIV-1 suppressing molecules and are positively associated with non- progression of HIV disease. Co-stimulatory molecules are necessary for DC maturation, effective antigen presentation, cell migration, and T cell proliferation. Although previous studies suggest that Meth deregulates various immune responses, the role of Meth on gene expression and production of β-chemokines and co-stimulatory molecules by DC has not been studied. We hypothesize that Meth induced immune defects may be mediated by dysregulation of β-chemokines (MIP-1α/CCL3, MIP-1β/CCL4 and RANTES/CCL5), co-stimulatory and maturation molecules (CD83 and CCR7) by DC. Our results show that Meth significantly downregulates the gene expression and production of β-chemokines and co stimulatory molecule by DC from normal subjects. In HIV-1 infected subjects, RANTES variant In1.1c that has been associated with accelerated HIV-1 disease progression was significantly higher compared to normal controls. Further, Meth significantly inhibited total RANTES gene expression with a reciprocal upregulation of RANTES variant In1.1c in a dose dependent manner by both immature DC (IDC) and mature DC (MDC) from normal subjects. These studies report for the first time that Meth deregulates β-chemokines and co-stimulatory molecule expression by DC. The results emanating from these studies may help to support the therapeutic application of chemokines to restore anti-HIV-1 immune responses to prevent or control HIV-1 infection in meth using populations.

Immunostimulatory activity of the heat shock protein DnaK from Francisella tularensis (45.18)

Abstract Francisella tularensis is the causative agent of tularemia, a severe debilitating disease of humans and other mammals. Since this microorganism is also considered as a ‘category A pathogen’ and a potential biowarfare agent, there is a need for an effective vaccine. Several antigens of F. tularensis including the heat shock protein DnaK have been proposed for use in a potential subunit vaccine. In this study we investigated the immunostimulatory properties of F. tularensis DnaK on murine bone marrow derived dendritic cells (DC). Recombinant DnaK was produced using a bacterial expression system and purified using a three-step purification procedure, i.e., affinity, ion exchange and size exclusion chromatography. DnaK induced DC to produce the proinflammatory cytokines IL-6, TNF-α and IL-12p40 in a dose dependant manner. DnaK did not induce IL-23p19 production. IL-10 production was low compared to IL-12p40. DnaK also induced DC maturation as demonstrated by an up-regulation of CD40 and CD86 expression. The responses were not mediated by endotoxin contamination, since the immunostimulatory activity of DnaK was resistant to polymixin B treatment, but sensitive to boiling or proteinase K digestion. An understanding of the interactions between virulence antigens of F. tularensis and immune cells is crucial for the development of a safe and efficacious vaccine against F. tularensis.

Pharmaceutical analysis of synthetic lipid A-based vaccine adjuvants in poly ( d, l-lactic- co-glycolic acid) nanoparticle formulations

The present study had two main objectives. First, was to compare the immune stimulatory effect of two synthetic lipid A analogues (7-acyl lipid A and pentaerythritol-based lipid A (PET lipid A)) on maturation/stimulation of bone marrow derived dendritic cells (DCs). Our second objective was to develop a liquid chromatography/mass spectrometry (LC-MS) method for the quantitative analysis of lipid A-based vaccine adjuvants. Treatment of immature DCs with 7-acyl lipid A and PET lipid A up regulated the surface expression of CD86 and CD40 molecules, and also induced similar profile of pro-inflammatory cytokine secretion. LC-MS analyses were performed using a Waters Micromass ZQ 4000 spectrometer, coupled to a Waters 2795 separations module with an autosampler. Calibration curves with R 2 > 0.999 were constructed over the concentration range of 1.25–20 μg/ml for the solution of 7-acyl lipid A and PET lipid A. The method was tested in a 3 day validation protocol. The accuracy of the assay at different concentrations tested ranged from 89 to 108% and from 92 to 107% for 7-acyl lipid A and PET lipid A, respectively. The limit of quantification for both 7-acyl lipid A and PET lipid A was 1.25 μg/ml (signal/noise (S/N)) ratio >15:1. The sensitivity of the method (the limit of detection) was 0.35 and 0.15 ng for 7-acyl lipid A and PET lipid A, respectively (S/N ratio between 4:1 or 3:1). As a preliminary application, this method has been successfully applied to the determination of 7-acyl lipid A and PET lipid A content in poly ( d, l-lactic- co-glycolic acid) nanoparticles (PLGA-NP).

Dendritic cells bearing HLA-G inhibit T-Cell activation in type 1 diabetes.

HLA-G is normally expressed on human trophoblast cells. It is a non-classical MHC molecule class I b. The role of HLA-G in diabetic type 1 is not known. We investigated the role of IFN-beta in induction HLA-G expression on the monocyte derived dendritic cells (DC) in diabetes type 1. Treatment of dendritic cell with IFN-beta in vitro from diabetic patients (n=20) and normal subjects (n=20) resulted to the production and expression of HLA-G on these cells from both groups. However, comparison of DC from the diabetic patients with DC from the controls revealed lower levels of HLA-G molecules in DC from diabetic patients. Using mixed lymphocyte reaction (MLR), it was found that DC expressing HLA-G mediated the inhibition of autologous T cell activation. It is concluded that IFN-beta can increase HLA-G in DC from diabetic patients; subsequently it may prevent the immune regularly pathway in the diabetic pathogenesis.

35: Histone deacetylase inhibitors induce indoleamine 2, 3-dioxygenase and modulate dendritic cell functions

Recent studies have demonstrated that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, reduces experimental acute graft-versus-host disease (GVHD). We investigated the cellular-molecular mechanisms of immuno-modulation by two HDAC inhibitors, SAHA and ITF 2357. Stimulation of bone marrow derived dendritic cells (DCs) with various TLR ligands (lipopolysaccharide (LPS), PGN, CpG) after pretreatment with either SAHA caused a significant reduction in the secretion of TNF-α, IL-12p70 and IL-6 compared to the untreated controls (P< 0.01).

Benzo( a)pyrene inhibits growth and functional differentiation of mouse bone marrow-derived dendritic cells : Downregulation of RelB and eIF3 p170 by benzo( a)pyrene

In this study, we have investigated effects of benzo( a)pyrene (BP) on growth and functional differentiation of mouse bone marrow (BM)-derived dendritic cells (DC). 1 μM BP dramatically inhibited growth of BM cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Although little alterations in surface expression of CD11c, major histocompatibility complex (MHC II), and CD86 molecules characteristic of mature DC were induced by BP, production of cytokines including IL-12, IL-10, and TNF-α, and allogeneic T cell stimulating ability were severely impaired. Some of the effects of BP were dependent on arylhydrocarbon receptor (AhR), because α-naphthoflavone, an AhR antagonist, suppressed the effects of BP on IL-12 production and T cell stimulating ability, but not on DC proliferation. Expression of RelB, a transcription factor necessary for DC differentiation and function, and eIF3 p170, a subunit of eukaryotic translation initiation factor (eIF)3, was reduced upon BP treatment.

Immune tolerance induced by adoptive transfer of dendritic cells in an insulin-dependent diabetes mellitus murine model

To investigate the effect and underlying mechanisms of immune-tolerance induced by the adoptive transfer of bone marrow (BM)-derived dendritic cells (DC) in insulin-dependent diabetes mellitus (IDDM) mice. The IDDM model was established by a low dose of streptozotocin (STZ) in Balb/c mice. Two DC subpopulations were generated from the BM cells with granulocyte-macrophage colony-stimulating factor with or without interleukin-4. The purity and the T cell stimulatory capability of DC were identified. These cells were used to modulate autoimmune response in pre-diabetic mice. Blood glucose was examined weekly; pancreas tissues were taken for histopathological analysis, and CD4(+) T cells were isolated to detect lymphocyte proliferation by MTT assay and the ratio of CD4(+)CD25(+) T cells by fluorescence-activated cell sorting (FACS). The cytokine secretion was determined by ELISA analysis. Two DC subsets were generated from BM, which have phenotypes of mature DC (mDC) and immature DC (iDC), respectively. The level of blood glucose decreased significantly by transferring iDC (P< 0.01) rather than mDC. Less lymphocyte infiltration was observed in the islets, and pancreatic structure was intact. In vitro, proliferation of lymphocytes decreased and the proportion of CD4(+)CD25(+) T cells increased remarkably, compared with the mDC-treated groups (P< 0.05), which were associated with increased level of the Th2 cytokine and reduced level of the Th1 cytokine after iDC transfer. Our data showed that iDC transfer was able to confer protection to mice from STZ-induced IDDM. The immune-tolerance to IDDM may be associated with promoting the production of CD4(+)CD25(+) T cells and inducing regulatory Th2 responses in vivo.

생약복합조성물(HemoHIM)의 수지상세포 활성화 효과

In our previous study, a novel herb mixture (HIM-Ⅰ) of Angelica gigas radix, Cnidium officinale rhizoma, and Paeonia japonica radix was developed to protect the intestinal and immune systems and to promote their recovery from radiation damage. A new herbal composition (HemoHIM) with the high immune modulating activity was developed from HIM-Ⅰ. In the present study, we examined the effects of HemoHIM on the maturation process of murine bone marrow (BM)-derived dendritic cells (DC). BM cells were cultured in the presence of IL-4 and GM-CSF and the generated immature DC were stimulated with HemoHIM for 24 hours. HemoHIM significantly enhanced the expression of co-stimulatory molecules, CD80 and CD86, especially. The activation capacity of HemoHIM-treated DC was significantly higher than that of immature DC, as analyzed by IL-2 and IFN-γ production and proliferation of the responding T cells in the co-culture with allogeneic T cells. The antigen-presenting capacity of HemoHIM-treated DC was also increased by the co-culture with OVA-specific T cells (HS-1), as analyzed by IL-2 and IFN-γ production and the proliferation. These results indicate that HemoHIM causes the maturation and activation of DC, which may be a part of mechanisms of immunomodulation by HemoHIM.

Enhanced antigen‐specific primary CD4+ and CD8+ responses by codelivery of ovalbumin and toll‐like receptor ligand monophosphoryl lipid A in poly(D,L‐lactic‐co‐glycolic acid) nanoparticles

The purpose of this research was to investigate the use of biodegradable poly(D,L-lactic-co-glycolic acid) nanoparticles (PLGA-NP) as a vaccine delivery system to codeliver antigen, ovalbumin (OVA) along with monophosphoryl lipid A (MPLA) as adjuvant for induction of potent CD4(+) and CD8(+) T cell responses. The primary CD4(+) T responses to OVA/MPLA NP were investigated using OVA-specific T cells from DO11.10 transgenic mice. Following adoptive transfer of these cells, mice were immunized s.c. by NP formulations. For assessing the CD8(+) responses, bone marrow derived dendritic cells (DCs) were pulsed with different OVA formulations, then, cocultured with CD8(+) T cells from OT-1 mice. T cell proliferation/activation and IFN-gamma secretion profile have been examined. Particulate delivery of OVA and MPLA to the DCs lead to markedly increase in in vitro CD8(+) T cell T cell proliferative responses (stimulation index >3000) and >13-folds increase in in vivo clonal expanded CD4(+) T cells. The expanded T cells were capable of cytokine secretion and expressed an activation and memory surface phenotype (CD62L(lo), CD11a(hi), and CD44(hi)). Codelivery of antigen and MPLA in PLGA-NP offers an effective method for induction of potent antigen specific CD4(+) and CD8(+) T cell responses.