Potential Roles of CCR5+ CCR6+ Dendritic Cells Induced by Nasal Ovalbumin plus Flt3 Ligand Expressing Adenovirus for Mucosal IgA Responses

We assessed the role of CCR5⁺/CCR6⁺/CD11b⁺/CD11c⁺ dendritic cells (DCs) for induction of ovalbumin (OVA)-specific antibody (Ab) responses following mucosal immunization. Mice given nasal OVA plus an adenovirus expressing Flt3 ligand (Ad-FL) showed early expansion of CCR5⁺/CCR6⁺/CD11b⁺/CD11c⁺ DCs in nasopharyngeal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLNs). Subsequently, this DC subset became resident in submandibular glands (SMGs) and nasal passages (NPs) in response to high levels of CCR-ligands produced in these tissues. CD11b⁺/CD11c⁺ DCs were markedly decreased in both CCR5^−/− and CCR6^−/− mice. Chimera mice reconstituted with bone marrow cells from CD11c-diphtheria toxin receptor (CD11c-DTR) and CCR5^−/− or CD11c-DTR and CCR6^−/− mice given nasal OVA plus Ad-FL had elevated plasma IgG, but reduced IgA as well as low anti-OVA secretory IgA (SIgA )Ab responses in saliva and nasal washes. These results suggest that CCR5⁺CCR6⁺ DCs play an important role in the induction of Ag-specific SIgA Ab responses.     

Residual Endotoxin Contaminations in Recombinant Proteins Are Sufficient to Activate Human CD1c+ Dendritic Cells

Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c⁺ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002–2 ng/ml). We show that CD1c⁺ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c⁺ DCs was closely correlated with high CD14 expression levels observed in CD1c⁺ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.     

Impaired pro-inflammatory cytokine production and increased Th2-biasing ability of dendritic cells exposed to Taenia excreted/secreted antigens: A critical role for carbohydrates but not for STAT6 signaling

In cysticercosis, a parasitic disease caused by cestodes, the details of early interactions between parasite antigens and innate cells from the host are not well understood. In this study, the role of cestode-conditioned dendritic cells (DCs) in priming Th1 versus Th2 responses to bystander antigen was examined by using CD11c⁺ DCs as antigen-presenting cells and naive CD4⁺ DO11.10 lymphocytes specific to ovalbumin (OVA) as responding cells. No conventional maturation was induced in DCs exposed to Taenia crassiceps excreted/secreted antigens (TcES). The ability of TcES to affect Toll-like receptor (TLR)-mediated maturation and the pro-inflammatory response was analyzed by co-pulsing DCs with TcES and TLR ligands. DCs exposed to TcES blocked TLR4, TLR9 and Toxoplasma soluble antigen-induced phenotypic maturation. TcES-exposed DCs also blocked secretion of pro-inflammatory cytokines and alloreactive T cell proliferation, while preserving IL-10 production. DCs pulsed with TcES + OVA suppressed IFN-γ, whereas they induced greater IL-4 production by CD4⁺ DO11.10 cells. TcES with chemically-altered glycans failed to modulate TLR-mediated activation of DCs and their Th1-inhibitng ability, which was STAT6-independent. Our results reflect the capacity of TcES glyco-antigens to modulate Th1-type and inflammatory responses mediated through DC activation.     

Isoflavones,Genistein and Daidzein,Regulate Mucosal Immune Response by Suppressing Dendritic Cell Function

Lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, has been shown to have a strong adjuvant effect towards inhaled antigens contributing to airway inflammation. Isoflavones are anti-inflammatory molecules present in abundant quantities in soybeans. We investigated the effect of isoflavones on human dendritic cell (DC) activation via LPS stimulation and subsequent DC-mediated effector cell function both in vitro and in a mouse model of upper airway inflammation. Human monocyte-derived DCs (MDDC) were matured with LPS (or TNF-α) +/− isoflavones (genistein or daidzein). The surface expression levels of DC activation markers were analyzed by flow cytometry. Mature DCs +/− isoflavones were washed and cultured with freshly-isolated allogenic naïve CD4⁺ T cells for 5 days or with autologous natural killer (NK) cells for 2 hours. The percentages of proliferating IFN-γ⁺ CD4⁺ T cells and cytokine levels in culture supernatants were assessed. NK cell degranulation and DC cytotoxicity were measured by flow cytometry. Isoflavones significantly suppressed the activation-induced expression of DC maturation markers (CD83, CD80, CD86) and MHC class I but not MHC class II molecules in vitro. Isoflavone treatment inhibited the ability of LPS-DCs to induce IFN-γ in CD4⁺ T cells. NK cell degranulation and the percentage of dead DCs were significantly increased in isoflavone-treated DC-NK co-culture experiments. Dietary isoflavones suppressed the mucosal immune response to intra-nasal sensitization of mice to ovalbumin. Similar results were obtained when isoflavones were co-administered during sensitization. These results demonstrate that soybean isoflavones suppress immune sensitization by suppressing DC-maturation and its subsequent DC-mediated effector cell functions.     

Antigen-Specific IgG ameliorates allergic airway inflammation via Fcγ receptor IIB on dendritic cells

Background

There have been few reports on the role of Fc receptors (FcRs) and immunoglobulin G (IgG) in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs) in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa.

Methods

In FcγRIIB deficient (KO) and C57BL/6 (WT) mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA). Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c⁺ MHC class II⁺ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c⁺ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs) in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs) differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL).

Results

In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c⁺ MHC class II⁺ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c⁺ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously.

Conclusion

Antigen-specific IgG ameliorates allergic airway inflammation via FcγRIIB on DCs.     

The Polyunsaturated Fatty Acids Arachidonic Acid and Docosahexaenoic Acid Induce Mouse Dendritic Cells Maturation but Reduce T-Cell Responses In Vitro

Long-chain polyunsaturated fatty acids (PUFAs) might regulate T-cell activation and lineage commitment. Here, we measured the effects of omega-3 (n-3), n-6 and n-9 fatty acids on the interaction between dendritic cells (DCs) and naïve T cells. Spleen DCs from BALB/c mice were cultured in vitro with ovalbumin (OVA) with 50 μM fatty acids; α-linolenic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid or oleic acid and thereafter OVA-specific DO11.10 T cells were added to the cultures. Fatty acids were taken up by the DCs, as shown by gas chromatography analysis. After culture with arachidonic acid or DHA CD11c⁺ CD11b⁺ and CD11c⁺ CD11b^neg DCs expressed more CD40, CD80, CD83, CD86 and PDL-1, while IA^d remained unchanged. However, fewer T cells co-cultured with these DCs proliferated (CellTrace Violet^low) and expressed CD69 or CD25, while more were necrotic (7AAD⁺). We noted an increased proportion of T cells with a regulatory T cell (Treg) phenotype, i.e., when gating on CD4⁺ FoxP3⁺ CTLA-4⁺, CD4⁺ FoxP3⁺ Helios⁺ or CD4⁺ FoxP3⁺ PD-1⁺, in co-cultures with arachidonic acid- or DHA-primed DCs relative to control cultures. The proportion of putative Tregs was inversely correlated to T-cell proliferation, indicating a suppressive function of these cells. With arachidonic acid DCs produced higher levels of prostaglandin E₂ while T cells produced lower amounts of IL-10 and IFNγ. In conclusion arachidonic acid and DHA induced up-regulation of activation markers on DCs. However arachidonic acid- and DHA-primed DCs reduced T-cell proliferation and increased the proportion of T cells expressing FoxP3, indicating that these fatty acids can promote induction of regulatory T cells.     

Dendritic cell-derived TNF-α is responsible for development of IL-10-producing CD4 T cells

Immature dendritic cells (DCs) appear to be involved in peripheral immune tolerance via induction of IL-10-producing CD4⁺ T cells. We examined the role of TNF-α in generation of the IL-10-producing CD4⁺ T cells by immature DCs. Immature bone marrow-derived DCs from wild type (WT) or TNF-α^−/− mice were cocultured with CD4⁺ T cells from OVA specific TCR transgenic mice (OT-II) in the presence of OVA_323-339 peptide. The WT DCs efficiently induced the antigen-specific IL-10-producing CD4⁺ T cells, while the ability of the TNF-α^−/− DCs to induce these CD4⁺ T cells was considerably depressed. Addition of exogenous TNF-α recovered the impaired ability of the TNF-α^−/− DCs to induce IL-10-producing T cells. However, no difference in this ability was observed between TNF-α^−/− and WT DCs after their maturation by LPS. Thus, TNF-α appears to be critical for the generation of IL-10-producing CD4⁺ T cells during the antigen presentation by immature DCs.     

The Gene Expression Profile of CD11c+CD8α? Dendritic Cells in the Pre-Diabetic Pancreas of the NOD Mouse

Two major dendritic cell (DC) subsets have been described in the pancreas of mice: The CD11c⁺CD8α⁻ DCs (strong CD4+ T cell proliferation inducers) and the CD8α⁺CD103⁺ DCs (T cell apoptosis inducers). Here we analyzed the larger subset of CD11c⁺CD8α⁻ DCs isolated from the pancreas of pre-diabetic NOD mice for genome-wide gene expression (validated by Q-PCR) to elucidate abnormalities in underlying gene expression networks. CD11c⁺CD8α⁻ DCs were isolated from 5 week old NOD and control C57BL/6 pancreas. The steady state pancreatic NOD CD11c⁺CD8α⁻ DCs showed a reduced expression of several gene networks important for the prime functions of these cells, i.e. for cell renewal, immune tolerance induction, migration and for the provision of growth factors including those for beta cell regeneration. A functional in vivo BrdU incorporation test showed the reduced proliferation of steady state pancreatic DC. The reduced expression of tolerance induction genes (CD200R, CCR5 and CD24) was supported on the protein level by flow cytometry. Also previously published functional tests on maturation, immune stimulation and migration confirm the molecular deficits of NOD steady state DC. Despite these deficiencies NOD pancreas CD11c⁺CD8α⁻ DCs showed a hyperreactivity to LPS, which resulted in an enhanced pro-inflammatory state characterized by a gene profile of an enhanced expression of a number of classical inflammatory cytokines. The enhanced up-regulation of inflammatory genes was supported by the in vitro cytokine production profile of the DCs. In conclusion, our data show that NOD pancreatic CD11c⁺CD8α⁻ DCs show various deficiencies in steady state, while hyperreactive when encountering a danger signal such as LPS.     

Paclitaxel enhances early dendritic cell maturation and function through TLR4 signaling in mice

Subclinical doses of Paclitaxel (PTX) given 1 day prior to a HER-2/neu (neu)-targeted, granulocyte-macrophage colony stimulating factor (GM-CSF)-secreting whole-cell vaccine enhances neu-specific T cell responses and slows neu⁺ tumor growth in tolerized HER-2/neu (neu-N) mice. We demonstrate that co-administration of PTX and Cyclophosphamide (CY) synergizes to slow tumor growth, and that in vitro, DC precursors exposed to PTX before LPS maturation results in greater co-stimulatory molecule expression, IL-12 production, and the ability to induce CD8⁺ T cells with enhanced lytic activity against neu⁺ tumors. PTX treatment also enhances maturation marker expression on CD11c⁺ DCs isolated from vaccine-draining lymph nodes. Ex vivo, these DCs activate CD8⁺ T cells with greater lytic capability than DC’s from vaccine alone-treated neu-N mice. Finally, PTX treatment results in enhanced antigen-specific, IFN-γ-secreting CD8⁺ T cells in vivo. Thus, administration of PTX with a tumor vaccine improves T cell priming through enhanced maturation of DC.     

Bordetella Adenylate Cyclase Toxin Differentially Modulates Toll-Like Receptor-Stimulated Activation,Migration and T Cell Stimulatory Capacity of Dendritic Cells

Adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent Bordetella pertussis. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC) enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR)-activated murine and human dendritic cells (DCs). cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 in vitro. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4⁺ and CD8⁺ T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4⁺ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4⁺CD25⁺Foxp3⁺ T regulatory cells in vitro. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8⁺ T cell proliferation and limited the induction of IFN-γ producing CD8⁺ T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during B. pertussis infection.     

Downregulation of Key Early Events in the Mobilization of Antigen-bearing Dendritic Cells by Leukocyte Immunoglobulin-like Receptor B4 in a Mouse Model of Allergic Pulmonary Inflammation

Leukocyte Immunoglobulin-like Receptor B4 (LILRB4) null mice have an exacerbated T helper cell type 2 (Th2) immune response and pulmonary inflammation compared with Lilrb4^+/+ animals when sensitized intranasally with ovalbumin (OVA) and low-dose lipopolysaccharide (LPS) followed by challenge with OVA. Moreover, OVA-challenged Lilrb4 ^−/− mice exhibit greater migration of antigen (Ag)-bearing dendritic cells (DCs) to lymph nodes and accumulation of interleukin 4- and interleukin 5-producing lymph node lymphocytes. The main objective of this study was to determine how the absence of LILRB4 leads to a greater number of DCs in the lymph nodes of Ag-challenged mice and increased lung Th2 inflammation. Mice were sensitized intranasally with PBS alone or containing OVA and LPS; additional cohorts were subsequently challenged with OVA. Expression of chemokine (C-C motif) ligand 21 (CCL21) in the lung was assessed immunohistologically. OVA ingestion and expression of LILRB4 and chemokine (C-C motif) receptor 7 (CCR7) were quantified by flow cytometry. Inhalation of OVA and LPS induced upregulation of LILRB4 selectively on lung Ag-bearing DCs. After sensitization and challenge, the lung lymphatic vessels of Lilrb4 ^−/− mice expressed more CCL21, a chemokine that directs the migration of DCs from peripheral tissue to draining lymph nodes, compared with Lilrb4^+/+ mice. In addition, lung DCs of challenged Lilrb4 ^−/− mice expressed more CCR7, the CCL21 receptor. The lungs of challenged Lilrb4 ^−/− mice also contained significantly greater numbers of CD4+ cells expressing interleukin-4 or interleukin-5, consistent with the greater number of Ag-bearing DCs and Th2 cells in lymph nodes and the attendant exacerbated Th2 lung pathology. Our data establish a new mechanism by which LILRB4 can downregulate the development of pathologic allergic inflammation: reduced upregulation of key molecules needed for DC migration leading to decreases in Th2 cells in lymph nodes and their target tissue.     

Canine Recombinant Adenovirus Vector Induces an Immunogenicity-Related Gene Expression Profile in Skin-Migrated CD11b+ -Type DCs

Gene expression profiling of the blood cell response induced early after vaccination has previously been demonstrated to predict the immunogenicity of vaccines. In this study, we evaluated whether the analysis of the gene expression profile of skin-migrated dendritic cells (DCs) could be informative for the in vitro prediction of immunogenicity of vaccine, using canine adenovirus serotype 2 (CAV2) as vaccine vector. CAV2 has been shown to induce immunity to transgenes in several species including sheep and is an interesting alternative to human adenovirus-based vectors, based on the safety records of the parental strain in dogs and the lack of pre-existing immunity in non-host species. Skin-migrated DCs were collected from pseudo-afferent lymph in sheep. Both the CD11b⁺ -type and CD103⁺ -type skin-migrated DCs were transduced by CAV2. An analysis of the global gene response to CAV2 in the two skin DC subsets showed that the gene response in CD11b⁺ -type DCs was far higher and broader than in the CD103⁺ -type DCs. A newly released integrative analytic tool from Ingenuity systems revealed that the CAV2-modulated genes in the CD11b⁺ -type DCs clustered in several activated immunogenicity-related functions, such as immune response, immune cell trafficking and inflammation. Thus gene profiling in skin-migrated DC in vitro indicates that the CD11b⁺ DC type is more responsive to CAV2 than the CD103⁺ DC type, and provides valuable information to help in evaluating and possibly improving viral vector vaccine effectiveness.     

Vault nanocapsules as adjuvants favor cell-mediated over antibody-mediated immune responses following immunization of mice

Background

Modifications of adjuvants that induce cell-mediated over antibody-mediated immunity is desired for development of vaccines. Nanocapsules have been found to be viable adjuvants and are amenable to engineering for desired immune responses. We previously showed that natural nanocapsules called vaults can be genetically engineered to elicit Th1 immunity and protection from a mucosal bacterial infection. The purpose of our study was to characterize immunity produced in response to OVA within vault nanoparticles and compare it to another nanocarrier.

Methodology and Principal Findings

We characterized immunity resulting from immunization with the model antigen, ovalbumin (OVA) encased in vault nanocapsules and liposomes. We measured OVA responsive CD8⁺ and CD4⁺ memory T cell responses, cytokine production and antibody titers in vitro and in vivo. We found that immunization with OVA contain in vaults induced a greater number of anti-OVA CD8⁺ memory T cells and production of IFNγ plus CD4⁺ memory T cells. Also, modification of the vault body could change the immune response compared to OVA encased in liposomes.

Conclusions/Significance

These experiments show that vault nanocapsules induced strong anti-OVA CD8⁺ and CD4⁺ T cell memory responses and modest antibody production, which markedly differed from the immune response induced by liposomes. We also found that the vault nanocapsule could be modified to change antibody isotypes in vivo. Thus it is possible to create a vault nanocapsule vaccine that can result in the unique combination of immunogen-responsive CD8⁺ and CD4⁺ T cell immunity coupled with an IgG1 response for future development of vault nanocapsule-based vaccines against antigens for human pathogens and cancer.     

Inhibition of Breast Cancer Resistance Protein (ABCG2) in Human Myeloid Dendritic Cells Induces Potent Tolerogenic Functions during LPS Stimulation

Breast cancer resistance protein (ABCG2), a member of the ATP-binding cassette transporters has been identified as a major determinant of multidrug resistance (MDR) in cancer cells, but ABC transporter inhibition has limited therapeutic value in vivo. In this research, we demonstrated that inhibition of efflux transporters ABCG2 induced the generation of tolerogenic DCs from human peripheral blood myeloid DCs (mDCs). ABCG2 expression was present in mDCs and was further increased by LPS stimulation. Treatment of CD1c⁺ mDCs with an ABCG2 inhibitor, Ko143, during LPS stimulation caused increased production of IL-10 and decreased production of pro-inflammatory cytokines and decreased expression of CD83 and CD86. Moreover, inhibition of ABCG2 in monocyte-derived DCs (MDDCs) abrogated the up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in these cells in response to LPS. Furthermore, CD1c⁺ mDCs stimulated with LPS plus Ko143 inhibited the proliferation of allogeneic and superantigen-specific syngenic CD4⁺ T cells and promoted expansion of CD25⁺FOXP3⁺ regulatory T (Treg) cells in an IL-10-dependent fashion. These tolerogenic effects of ABCG2 inhibition could be abolished by ERK inhibition. Thus, we demonstrated that inhibition of ABCG2 in LPS-stimulated mDCs can potently induce tolerogenic potentials in these cells, providing crucial new information that could lead to development of better strategies to combat MDR cancer.     

Changes in lamina propria dendritic cells on the oral administration of exogenous protein antigens during weaning

Two critical periods of maximum exposure to antigens occur in young mammals, immediately after birth and at weaning, as a result of colonization by commensal bacteria and the ingestion of new diets. At weaning, active immune responses of antibody production against dietary proteins are known to occur, but simultaneously, oral tolerance is acquired for harmless food proteins. However, regulated mechanisms of the immune system at weaning remain to be elucidated although its immune responses may be somewhat similar to those in adulthood. Considering that tolerogenic antigen-presenting cells (APCs) are likely to be a key factor in the acquisition of oral tolerance, in the present study, we examined the changes of dendritic cells (DCs) in the lamina propria (LP) on exposure to food proteins at weaning. C57BL/6 female mice were weaned at the age of 3 weeks and orally administered 10 mg of ovalbumin (OVA) for ten consecutive days after weaning. The administration led to a decrease in the plasma level of immunoglobulin specific for OVA, suggesting the acquisition of oral tolerance. The uptake of fluorescence-labeled OVA was significantly observed for CD11c⁺LPDCs. When we analyzed the changes of two types of LPDCs, PDCA-1⁺ MHC II⁺ DCs and CD103⁺ MHC II⁺ DCs, ten consecutive gavages of OVA marginally, but not significantly, augmented only the frequency of PDCA-1⁺ MHC II⁺ DCs. Considering that the change of APCs likely appears immediately on the response to antigen intake, we found the statistically significant increase in the frequency of PDCA-1⁺ DCs, but not in that of CD103⁺ DCs, even after two treatments, indicating PDCA-1⁺ DCs to be recruited in the LP within 2 days of exposure to food proteins. These results suggest that the behavior of tolerogenic PDCA-1⁺ DCs may change at weaning with the removal of the immunoprotective components of maternal milk.     

In vitro activation and differentiation of naïve CD4 and CD8 T cells into HCV Core- and NS3-specific armed effector cells: A new role for CD4 T cells

Viral clearance in hepatitis C virus (HCV) infection has been correlated with strong, multi-specific and sustained T cell responses. The number of functionally active effector T cells determines the outcome of infection. Only a small number of antigen-specific naïve T cells are originally present. Upon infection, they undergo activation, clonal expansion and differentiation to become effector cells. In this study, we determined the ability of dendritic cells (DCs) to prime T cells in vitro to become effector cells upon stimulation with various TLR ligands or IFNα. T cell priming and activation was determined by proliferation and production of effector molecules, IFN-γ and Granzyme B (GrB). HCV Core-specific T cells showed significant increase in proliferation, and the number of HCV Core-specific CD4⁺ and CD8⁺ T cells producing IFN-γ and GrB was higher than control or NS3-specific T cells. These in vitro-primed CD4⁺ and CD8⁺ T cells exhibit the phenotype of just-activated and/or armed effector lymphocytes confirming the transition of naïve T cells to effector cells. This is the first study demonstrating the activation of GrB⁺CD4⁺ T cells against antigen(s) derived from HCV. Our study suggests a novel role of CD4⁺ T cells in immunity against HCV.     

The Role of the E3 Ligase Cbl-B in Murine Dendritic Cells

Dendritic cells (DCs) are potent antigen-presenting cells with a promising potential in cancer immunotherapy. Cbl proteins are E3 ubiquitin ligases and have been implicated in regulating the functional activity of various immune cells. As an example, c-Cbl negatively affects DC activation. We here describe that another member of the Cbl-protein family (i.e. Cbl-b) is highly expressed in murine bone-marrow-derived DCs (BMDCs). Differentiation of cblb−/− bone marrow mononuclear cells into classical BMDCs is unaltered, except enhanced induction of DEC-205 (CD205) expression. When tested in mixed-lymphocyte reaction (MLR), cblb−/− BMDCs exhibit increased allo-stimulatory capacity in vitro. BMDCs were next in vitro stimulated by various toll like receptor (TLR)-agonists (LPS, Poly(I:C), CpG) and exposed to FITC-labeled dextran. Upon TLR-stimulation, cblb−/− BMDCs produce higher levels of proinflammatory cytokines (IL-1α, IL-6 and TNF-α) and exhibit a slightly higher level of FITC-dextran uptake. To further characterize the functional significance of cblb−/− BMDCs we tested them in antigen-specific T cell responses against ovalbumin (OVA) protein and peptides, activating either CD8⁺ OT-I or CD4⁺ OT-II transgenic T cells. However, cblb−/− BMDCs are equally effective in inducing antigen-specific T cell responses when compared to wildtype BMDCs both in vitro and in vivo. The migratory capacity into lymph nodes during inflammation was similarly not affected by the absence of Cbl-b. In line with these observations, cblb−/− peptide-pulsed BMDCs are equally effective vaccines against OVA-expressing B16 tumors in vivo when compared to wildtype BMDCs. We conclude that in contrast to c-Cbl, Cbl-b plays only a limited role in the induction of Ag-specific T cell responses by murine BMDCs in vitro and in vivo.     

Dendritic Cells from Oral Cavity Induce Foxp3+ Regulatory T Cells upon Antigen Stimulation

Evidence is accumulating that dendritic cells (DCs) from the intestines have the capacity to induce Foxp3⁺CD4⁺ regulatory T cells (T-regs) and regulate immunity versus tolerance in the intestines. However, the contribution of DCs to controlling immunity versus tolerance in the oral cavity has not been addressed. Here, we report that DCs from the oral cavity induce Foxp3⁺ T-regs as well as DCs from intestine. We found that oral-cavity-draining cervical lymph nodes contained higher frequencies of Foxp3⁺ T-regs and ROR-γt⁺ CD4⁺T cells than other lymph nodes. The high frequency of Foxp3⁺ T-regs in the oral-cavity-draining cervical lymph nodes was not dependent on the Toll like receptor (TLR) adaptor molecules, Myd88 and TICAM-1 (TRIF). In contrast, the high frequency of ROR-γt⁺ CD4⁺T cells relies on Myd88 and TICAM-1. In vitro data showed that CD11c⁺ DCs from oral-cavity-draining cervical lymph nodes have the capacity to induce Foxp3⁺ T-regs in the presence of antigen. These data suggest that, as well as in the intestinal environment, antigen-presenting DCs may play a vital role in maintaining tolerance by inducing Foxp3⁺ T-regs in the oral cavity.     

CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

Myeloid dendritic cells (DCs) can capture HIV-1 via the receptor CD169/Siglec-1 that binds to the ganglioside, GM3, in the virus particle membrane. In turn, HIV-1 particles captured by CD169, an I-type lectin, whose expression on DCs is enhanced upon maturation with LPS, are protected from degradation in CD169⁺ virus-containing compartments (VCCs) and disseminated to CD4⁺ T cells, a mechanism of DC-mediated HIV-1 trans-infection. In this study, we describe the mechanism of VCC formation and its role in immune evasion mechanisms of HIV-1. We find HIV-1-induced formation of VCCs is restricted to myeloid cells, and that the cytoplasmic tail of CD169 is dispensable for HIV-1 trafficking and retention within VCCs and subsequent trans-infection to CD4⁺ T cells. Interestingly, introduction of a di-aromatic endocytic motif in the cytoplasmic tail of CD169 that results in endocytosis of HIV-1 particles, suppressed CD169-mediated HIV-1 trans-infection. Furthermore, super-resolution microscopy revealed close association of CD169 and HIV-1 particles in surface-accessible but deep plasma membrane invaginations. Intriguingly, HIV-1 particles in deep VCCs were inefficiently accessed by anti-gp120 broadly neutralizing antibodies, VRC01 and NIH45-46 G54W, and thus were less susceptible to neutralization. Our study suggests that HIV-1 capture by CD169 can provide virus evasion from both innate (phagocytosis) and adaptive immune responses.     

A novel in vivo inducible dendritic cell ablation model in mice

Dendritic cells (DCs) are involved in T cell activation via their uptake and presentation of antigens. In vivo function of DCs was analyzed using transgenic mouse models that express diphtheria toxin receptor (DTR) or the diphtheria toxin-A subunit (DTA) under the control of the CD11c/Itgax promoter. However, CD11c⁺ cells are heterogeneous populations that contain several DC subsets. Thus, the in vivo function of each subset of DCs remains to be elucidated. Here, we describe a new inducible DC ablation model, in which DTR expression is induced under the CD11c/Itgax promoter after Cre-mediated excision of a stop cassette (CD11c-iDTR). Crossing of CD11c-iDTR mice with CAG-Cre transgenic mice, expressing Cre recombinase under control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter, led to the generation of mice, in which DTR was selectively expressed in CD11c⁺ cells (iDTRΔ mice). We successfully deleted CD11c⁺ cells in bone marrow-derived DCs in vitro and splenic CD11c⁺ cells in vivo after DT treatment in iDTRΔ mice. This mouse strain will be a useful tool for generating mice lacking a specific subset of DCs using a transgenic mouse strain, in which the Cre gene is expressed by a DC subset-specific promoter.