Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity

There is currently a need for vaccines that stimulate cell-mediated immunity-particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)-against viral and tumor antigens. The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells (DCs). DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells. Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research. We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals. Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells. Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses.     

Maturation of circulating dendritic cells and imbalance of T-cell subsets in patients with squamous cell carcinoma of the head and neck

Interactions between dendritic cells (DCs) and T cells play a pivotal role in the regulation and maintenance of immune responses. In cancer patients, various immunological abnormalities have been observed in these immune cells. Here, we investigated proportions and the phenotype of DCs and the cytokine profile of T-cell subsets in the peripheral blood of patients with squamous cell carcinoma of the head and neck (SCCHN), using multicolor flow cytometry. The percentage of myeloid (CD11c+), but not plasmacytoid (CD123+) DCs, was significantly lower (P<0.05) and expression of HLA-DR was significantly decreased in total and myeloid DCs of cancer patients compared to healthy donors. Simultaneous analyses of T-cell subsets in the patients’ circulation showed significantly increased proportions of CD4+ T cells expressing Th1 and Th2 cytokines after ex vivo stimulation without any skewing in the Th1/Th2 ratio. The relative level of HLA-DR expression on myeloid or total DCs positively correlated with the Th1/Th2 ratio (P<0.01), and the proportion of total circulating DCs was inversely correlated with that of regulatory CD4+CD25+ T cells (P<0.01). These results suggest that the decreased proportion of circulating DCs and decreased HLA-DR expression in DCs may have a major impact on systemic immune responses in patients with SCCHN.     

Scavenger receptor class B is required for hepatitis C virus uptake and cross-presentation by human dendritic cells

Class B scavenger receptors (SR-Bs) bind lipoproteins and play an important role in lipid metabolism. Most recently, SR-B type I (SR-BI) and its splicing variant SR-BII have been found to mediate bacterial adhesion and cytosolic bacterial invasion in mammalian cells. In this study, we demonstrate that SR-BI is a key host factor required for hepatitis C virus (HCV) uptake and cross-presentation by human dendritic cells (DCs). Whereas monocytes and T and B cells were characterized by very low or undetectable SR-BI expression levels, human DCs demonstrated a high level of cell surface expression of SR-BI similar to that of primary human hepatocytes. Antibodies targeting the extracellular loop of SR-BI efficiently inhibited HCV-like particle binding, uptake, and cross-presentation by human DCs. Moreover, human high-density lipoprotein specifically modulated HCV-like particle binding to DCs, indicating an interplay of HCV with the lipid transfer function of SR-BI in DCs. Finally, we demonstrate that anti-SR-BI antibodies inhibit the uptake of cell culture-derived HCV (HCVcc) in DCs. In conclusion, these findings identify a novel function of SR-BI for viral antigen uptake and recognition and may have an important impact on the design of HCV vaccines and immunotherapeutic approaches aiming at the induction of efficient antiviral immune responses.     

Hepatitis C virus protects human B lymphocytes from Fas-mediated apoptosis via E2-CD81 engagement

HCV infection is often associated with B-cell regulatory control disturbance and delayed appearance of neutralizing antibodies. CD81 is a cellular receptor for HCV and can bind to HCV envelope protein 2 (E2). CD81 also participates to form a B cell costimulatory complex. To investigate whether HCV influences B cell activation and immune function through E2 -CD81 engagement, here, human Burkitt's lymphoma cell line Raji cells and primary human B lymphocytes (PHB) were treated with HCV E2 protein and cell culture produced HCV particles (HCVcc), and then the related cell phenotypes were assayed. The results showed that both E2 and HCVcc triggered phosphorylation of IκBα, enhanced the expression of anti-apoptosis Bcl-2 family proteins, and protected Raji cells and PHB cells from Fas-mediated death. In addition, both E2 protein and HCVcc increased the expression of costimulatory molecules CD80, CD86 and CD81 itself, and decreased the expression of complement receptor CD21. The effects were dependent on E2-CD81 interaction on the cell surface, since CD81-silenced Raji cells did not respond to both treatments; and an E2 mutant that lose the CD81 binding activity, could not trigger the responses of both Raji cells and PHB cells. The effects were not associated with HCV replication in cells, for HCV pseudoparticle (HCVpp) and HCVcc failed to infect Raji cells. Hence, E2-CD81 engagement may contribute to HCV-associated B cell lymphoproliferative disorders and insufficient neutralizing antibody production.     

Mature dendritic cells infected with canarypox virus elicit strong anti-human immunodeficiency virus CD8+ and CD4+ T-cell responses from chronically infected individuals

Recombinant canarypox virus vectors containing human immunodeficiency virus type 1 (HIV-1) sequences are promising vaccine candidates, as they replicate poorly in human cells. However, when delivered intramuscularly the vaccines have induced inconsistent and in some cases transient antigen-specific cytotoxic T-cell (CTL) responses in seronegative volunteers. An attractive way to enhance these responses would be to target canarypox virus to professional antigen-presenting cells such as dendritic cells (DCs). We studied (i) the interaction between canarypox virus and DCs and (ii) the T-cell responses induced by DCs infected with canarypox virus vectors containing HIV-1 genes. Mature and not immature DCs resisted the cytopathic effects of canarypox virus and elicited strong effector CD8+ T-cell responses from chronically infected HIV+ individuals, e.g., cytolysis, and secretion of gamma interferon (IFN-gamma) and beta-chemokines. Furthermore, canarypox virus-infected DCs were >30-fold more efficient than monocytes and induced responses that were comparable to those induced by vaccinia virus vectors or peptides. Addition of exogenous cytokines was not necessary to elicit CD8+ effector cells, although the presence of CD4+ T cells was required for their expansion and maintenance. Most strikingly, canarypox virus-infected DCs were directly able to stimulate HIV-specific, IFN-gamma-secreting CD4 helper responses from bulk as well as purified CD4+ T cells. Therefore, these results suggest that targeting canarypox virus vectors to mature DCs could potentially elicit both anti-HIV CD8+ and CD4+ helper responses in vivo.     

Simultaneous ex vivo quantification of antigen-specific CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cell responses using in vitro transcribed RNA

Assessment of antigen-specific T-cell responses has been greatly facilitated by development of ELISPOT and intracellular cytokine flow cytometry (CFC) assays. The use of autologous antigen presenting cells transfected with in vitro transcribed RNA as stimulators allows in principle quantification of antigen-specific T-cells independent of the knowledge of the epitopes. We describe here a cytokine secretion assay that enables simultaneous assessment of both antigen-specific CD4+ as well as CD8+ T-cells directly from clinical samples without the need for generation of dendritic cells. To this aim, bulk PBMCs were electroporated with RNA encoding the antigen fused to trafficking signal sequences derived from a MHC class I molecule and used as stimulators. With human cytomegalovirus (HCMV) phosphoprotein 65 (pp65) as antigen we show that for measuring ex vivo T-cell responses in ELISPOT and CFC such stimulators are superior or at least equivalent to a pool of overlapping peptides representing the entire pp65 sequence as well as to untagged pp65 encoding RNA. This approach avoids the time consuming generation of dendritic cells as immune stimulators and, in particular when used in the context of the CFC, is robust, broadly applicable and fast.     

Aggregation of Human Recombinant Monoclonal Antibodies Influences the Capacity of Dendritic Cells to Stimulate Adaptive T-Cell Responses In Vitro

Subvisible proteinaceous particles which are present in all therapeutic protein formulations are in the focus of intense discussions between health authorities, academics and biopharmaceutical companies in the context of concerns that such particles could promote unwanted immunogenicity via anti-drug antibody formation. In order to provide further understanding of the subject, this study closely examines the specific biological effects proteinaceous particles may exert on dendritic cells (DCs) as the most efficient antigen-presenting cell population crucial for the initiation of the adaptive immune response. Two different model IgG antibodies were subjected to three different types of exaggerated physical stress to generate subvisible particles in far greater concentrations than the ones typical for the currently marketed biotherapeutical antibodies. The aggregated samples were used in in vitro biological assays in order to interrogate the early DC-driven events that initiate CD4 T-cell dependent humoral adaptive immune responses – peptide presentation capacity and co-stimulatory activity of DCs. Most importantly, antigen presentation was addressed with a unique approach called MHC-associated Peptide Proteomics (MAPPs), which allows for identifying the sequences of HLA-DR associated peptides directly from human dendritic cells.The experiments demonstrated that highly aggregated solutions of two model mAbs generated under controlled conditions can induce activation of human monocyte-derived DCs as indicated by upregulation of typical maturation markers including co-stimulatory molecules necessary for CD4 T-cell activation. Additional data suggest that highly aggregated proteins could induce in vitro T-cell responses. Intriguingly, strong aggregation-mediated changes in the pattern and quantity of antigen-derived HLA-DR associated peptides presented on DCs were observed, indicating a change in protein processing and presentation. Increasing the amounts of subvisible proteinaceous particles correlated very well with the pronounced increase in the peptide number and clusters presented in the context of class II HLA-DR molecules, suggesting a major involvement of a mass-action mechanism of altering the presentation.     

The Attenuated Brucella abortus Strain 19 Invades,Persists in,and Activates Human Dendritic Cells,and Induces the Secretion of IL-12p70 but Not IL-23

Background

Bacterial vectors have been proposed as novel vaccine strategies to induce strong cellular immunity. Attenuated strains of Brucella abortus comprise promising vector candidates since they have the potential to induce strong CD4⁺ and CD8⁺ T-cell mediated immune responses in the absence of excessive inflammation as observed with other Gram-negative bacteria. However, some Brucella strains interfere with the maturation of dendritic cells (DCs), which is essential for antigen-specific T-cell priming. In the present study, we investigated the interaction of human monocyte-derived DCs with the smooth attenuated B. abortus strain (S) 19, which has previously been employed successfully to vaccinate cattle.

Methodology/Principal findings

We first looked into the potential of S19 to hamper the cytokine-induced maturation of DCs; however, infected cells expressed CD25, CD40, CD80, and CD86 to a comparable extent as uninfected, cytokine-matured DCs. Furthermore, S19 activated DCs in the absence of exogeneous stimuli, enhanced the expression of HLA-ABC and HLA-DR, and was able to persist intracellularly without causing cytotoxicity. Thus, DCs provide a cellular niche for persisting brucellae in vivo as a permanent source of antigen. S19-infected DCs produced IL-12/23p40, IL-12p70, and IL-10, but not IL-23. While heat-killed bacteria also activated DCs, soluble mediators were not involved in S19-induced activation of human DCs. HEK 293 transfectants revealed cellular activation by S19 primarily through engagement of Toll-like receptor (TLR)2.

Conclusions/Significance

Thus, as an immunological prerequisite for vaccine efficacy, B. abortus S19 potently infects and potently activates (most likely via TLR2) human DCs to produce Th1-promoting cytokines.     

Hizikia fusiforme extract enhances dendritic cell maturation in vitro and in vivo

ABSTRACT

Dendritic cells (DCs) are play critical roles in the priming and regulation of immune responses. DCs rapidly process and convey these antigens to prime antigen-specific T cells. Therefore, regulation of DCs functions is important for immunity and immunotherapies. Immune adjuvants for DCs activation are needed to improve the efficacy of vaccines against tumors and many infectious diseases. Therefore, we demonstrate that H. fusiformis extract can regulate DCs maturation and activation. H. fusiformis extract induced costimulatory molecules (CD 80 and CD86), antigen-presenting molecules (major histocompatibility complex (MHC) I and II), CCR7 expression, and interleukin (IL)-12 production in DCs. These effects are associated with upregulation of mitogen-activated protein kinase (MAPK) signaling pathway. In addition, H. fusiformis extract induces costimulatory molecules on splenic DCs and activated CD8⁺ T cells in vivo. Taken together, these findings suggest that H. fusiformis extract may be a potential efficient immune therapeutic compound in DCs-mediated immunotherapies.     

Adaptive immune responses during Shigella dysenteriae type 1 infection: an in vitro stimulation with 57 kDa major antigenic OMP in the presence of anti-CD3 antibody

An effort was made to understand the role of the 57 kDa major antigenic fraction of Shigella outer membrane protein (OMP) in the presence of T-cell antigen receptor in activation of adaptive immune responses of the cell mediated immune (CMI) restored patients. The expression of HLA-DR/CD4 out of CD3⁺ T-cells was significantly dominant over the HLA-DR/CD8 and comparable to unstimulated cells of infected or healthy controls. CD4⁺ T-cell activation together with HLA-DR is associated with the expression of CD25⁺ (IL2Rα) for IL-2 growth factors with decreased IL-4 levels, required for maintaining the homeostasis of CD4⁺ T cell. Furthermore, the positive expression of the CD45 antigen is possibly required for acquiring the memory for CD4⁺ cells signals and facilitates the interaction with CD54 antigen. As a result, antigen-specific secondary signal is generated for B-cell activation to produce IgG2a and IgG2b. This suggests that antibody mediated-adaptive immune responses are generated due to anti-CD3 induced helper T-cell activity. The above mentioned findings reflect that the antigen alone might not exacerbate the selective T-cell responses. But these antigens in the presence of anti-CD3 antibody might help to elicit adaptive immune response via T-cell receptor (TCR) activation.     

Immunomodulatory drugs improve the immune environment for dendritic cell-based immunotherapy in multiple myeloma patients after autologous stem cell transplantation

Multiple myeloma (MM) is characterized by a malignant proliferation of plasma cells in the bone marrow with associated organ damage. Although the prognosis of MM has improved recently, the disease remains incurable for the large majority of patients. The eradication of residual disease in the bone marrow is a main target on the road toward cure. Immune cells play a role in the control of cancer and can be tools to attack residual MM cells. However, the myeloma-associated immune deficiency is a major hurdle to immunotherapy. We evaluated ex vivo the effects of low doses of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide on several immune cell types from MM patients after autologous stem cell transplantation and with low tumor burden. We observed that these drugs increased CD4⁺ and CD8⁺ T-cell proliferation and cytokine production, enhanced the lytic capacity of cytotoxic T lymphocytes and reduced the suppressive effects of regulatory T cells on CD8⁺ T-cell responses. In addition, we found that functional dendritic cells (DCs) can be generated from mononuclear cells from MM patients. The presence of IMiDs improved the quality of antigen-specific T cells induced or expanded by these DCs as evidenced by a higher degree of T-cell polyfunctionality. Our results provide a rationale for the design of early phase clinical studies to assess the efficacy of DC-based immunotherapy in combination with posttransplant maintenance treatment with IMiDs in MM.     

Differential expression of cell surface markers by ovine respiratory tract dendritic cells.

Dendritic cells (DCs) are key antigen-presenting cells central to the induction of primary immune responses. Despite the prevalence of respiratory disease in sheep and the increasing use of the ovine lung as a model for human disease, ovine respiratory tract DCs (RTDCs) have not yet been characterized. Using single and double immunocytochemical staining, expression of a number of potential DC markers (MHC class II, CD1b, SIRPalpha, and CD205) by ovine RTDC populations has been determined. MHC class II staining revealed widespread populations of DCs either adjacent to respiratory airway epithelium or within the lung parenchyma. CD1b was expressed by a small subpopulation of both airway and parenchymal RTDCs. Expression of SIRPalpha was limited to a small subpopulation of airway RTDCs but was absent from the lung parenchyma. CD205 was widely expressed by airway RTDCs but expressed only by a small subpopulation of parenchymal RTDCs. In addition, the majority (87%) of parenchymal CD205+ cells exhibited a non-DC-like morphology and did not express MHC class II, suggesting that these single CD205+ cells were not DCs. Phenotypic differences between airway and parenchymal RTDCs may be related to functional differences between the two populations.     

Dendritic cells,engineered to secrete a T-cell receptor mimic peptide,induce antigen-specific immunosuppression in vivo

A T-cell receptor mimic peptide (TCRpep) consisting of an 8-amino-acid peptide, homologous to the transmembrane region of the T-cell receptor (TCR) alpha chain, blocks T-cell activation after systemic application. When dendritic cells (DCs) were transduced to secrete the TCRpep and injected into mice, evidence of immunosuppression was observed. In a CD8-driven allergy model, the injection of DCs transduced with the TCRpep reduced inflammation markedly and in a CD4+ T cell-dependent model of multiple sclerosis (experimental autoimmune encephalitis, EAE), injection of TCRpep-secreting DCs abrogated EAE symptoms and prolonged survival. These effects were antigen specific, because transduced DCs that did not express the respective antigen failed to convey protection in the allergy model as well as in the EAE model. Thus these data show that DCs expressing the TCRpep are able to suppress T-cell activation and might be a useful tool for inducing antigen-specific immune suppression in vivo.     

Conserved hierarchy of helper T cell responses in a chimpanzee during primary and secondary hepatitis C virus infections

Control of hepatitis C virus (HCV) infection could be influenced by the timing and magnitude of CD4+ T cell responses against individual epitopes. We characterized CD4+ T cells targeting seven Pan troglodytes (Patr) class II-restricted epitopes during primary and secondary HCV infections of a chimpanzee. All Patr-DR-restricted HCV epitopes bound multiple human HLA-DR molecules, indicating the potential for overlap in epitopes targeted by both species. Some human MHC class II molecules efficiently stimulated IL-2 production by chimpanzee virus-specific T cell clones. Moreover, one conserved epitope designated NS3(1248) (GYKVLVLNPSV) overlapped a helper epitope that is presented by multiple HLA-DR molecules in humans who spontaneously resolved HCV infection. Resolution of primary infection in the chimpanzee was associated with an initial wave of CD4+ T cells targeting a limited set of dominant epitopes including NS3(1248.) A second wave of low-frequency CD4+ T cells targeting other subdominant epitopes appeared in blood several weeks later after virus replication was mostly contained. During a second infection 7 years later, CD4+ T cells against all epitopes appeared in blood sooner and at higher frequencies but the pattern of dominance was conserved. In summary, primary HCV infection in this individual was characterized by T cell populations targeting two groups of MHC class II-restricted epitopes that differed in frequency and kinetics of appearance in blood. The hierarchial nature of the CD4+ T cell response, if broadly applicable to other HCV-infected chimpanzees and humans, could be a factor governing the outcome of HCV infection.     

Cancer Immunotherapy Employing an Innovative Strategy to Enhance CD4+ T Cell Help in the Tumor Microenvironment

Chemotherapy and/or radiation therapy are widely used as cancer treatments, but the antitumor effects they produce can be enhanced when combined with immunotherapies. Chemotherapy kills tumor cells, but it also releases tumor antigen and allows the cross-presentation of the tumor antigen to trigger antigen-specific cell-mediated immune responses. Promoting CD4+ T helper cell immune responses can be used to enhance the cross-presentation of the tumor antigen following chemotherapy. The pan HLA-DR binding epitope (PADRE peptide) is capable of generating antigen-specific CD4+ T cells that bind various MHC class II molecules with high affinity and has been widely used in conjunction with vaccines to improve their potency by enhancing CD4+ T cell responses. Here, we investigated whether intratumoral injection of PADRE and the adjuvant CpG into HPV16 E7-expressing TC-1 tumors following cisplatin chemotherapy could lead to potent antitumor effects and antigen-specific cell-mediated immune responses. We observed that treatment with all three agents produced the most potent antitumor effects compared to pairwise combinations. Moreover, treatment with cisplatin, CpG and PADRE was able to control tumors at a distant site, indicating that our approach is able to induce cross-presentation of the tumor antigen. Treatment with cisplatin, CpG and PADRE also enhanced the generation of PADRE-specific CD4+ T cells and E7-specific CD8+ T cells and decreased the number of MDSCs in tumor loci. The treatment regimen presented here represents a universal approach to cancer control.     

Immune evasion by Yersinia enterocolitica: differential targeting of dendritic cell subpopulations in vivo

CD4(+) T cells are essential for the control of Yersinia enterocolitica (Ye) infection in mice. Ye can inhibit dendritic cell (DC) antigen uptake and degradation, maturation and subsequently T-cell activation in vitro. Here we investigated the effects of Ye infection on splenic DCs and T-cell proliferation in an experimental mouse infection model. We found that OVA-specific CD4(+) T cells had a reduced potential to proliferate when stimulated with OVA after infection with Ye compared to control mice. Additionally, proliferation of OVA-specific CD4(+) T cells was markedly reduced when cultured with splenic CD8α(+) DCs from Ye infected mice in the presence of OVA. In contrast, T-cell proliferation was not impaired in cultures with CD4(+) or CD4(-)CD8α(-) DCs isolated from Ye infected mice. However, OVA uptake and degradation as well as cytokine production were impaired in CD8α(+) DCs, but not in CD4(+) and CD4(-)CD8α(-) DCs after Ye infection. Pathogenicity factors (Yops) from Ye were most frequently injected into CD8α(+) DCs, resulting in less MHC class II and CD86 expression than on non-injected CD8α(+) DCs. Three days post infection with Ye the number of splenic CD8α(+) and CD4(+) DCs was reduced by 50% and 90%, respectively. The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation. Together, we show that Ye infection negatively regulates the stimulatory capacity of some but not all splenic DC subpopulations in vivo. This leads to differential antigen uptake and degradation, cytokine production, cell loss, and cell death rates in various DC subpopulations. The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4(+) and CD8α(+) DCs. These events may contribute to reduced T-cell proliferation and immune evasion of Ye.     

IL-10 alters DC function via modulation of cell surface molecules resulting in impaired T-cell responses

IL-10 is a potent inhibitor of T-cell activation and has tolerizing effects on these cells. These effects are primarily mediated via modulation of antigen presenting cell function. Here, it is demonstrated that IL-10 completely inhibits LPS-induced DC maturation, resulting in altered DC-T-cell interactions and reduced T-cell responses. IL-10 inhibited LPS-induced upregulation of costimulatory molecules, MHC Class II, and the secretion of IL-12, TNF-alpha, IL-6, and IL-1beta by DCs, although it upregulated the SLAM (CD150) expression at both the mRNA and protein levels. IL-10 pre-treated DC did not respond to subsequent LPS activation and its stimulatory ability for allogeneic and antigen-specific T-cells was severely impaired. Importantly, T-cells derived from co-cultures with Ag-pulsed, IL-10-treated DC were impaired in their responses to subsequent Ag-specific restimulation. Transwell and DC-derived plasma membrane experiments indicated that the capacity of IL-10-treated DC to induce T-cell unresponsiveness results from alterations in the cell surface molecules rather than modulation of cytokine secretion.     

Dendritic cells in the human decidua

Dendritic cells (DCs) in the pregnant human uterine mucosa have been poorly characterized, although they are likely to regulate immune responses to both placental trophoblast cells and uterine infections. In this study an HLA-DR+, CD11c+ lin- (CD3-, CD19-, CD56-, CD14-) population has been identified by three-color flow cytometry. The cell isolates were prepared either by collagenase digestion or mechanically from first-trimester decidual tissue. The decidual DCs comprised approximately 1.7% of CD45+ cells in the isolates and had the phenotype of immature myeloid DCs. No CD1a+ Langerhans cells or CD123+ plasmacytoid DCs were detected. The decidual DCs were DC-SIGN-, DEC-205+, CD40+. Two subsets could be distinguished on the basis of relative expression of HLA-DR, which also differed in expression of DC-activation markers. The DCs were identified in situ by immunohistology by DEC-205 staining. Cells with dendritic processes were found scattered through both the decidua basalis (in which trophoblast cells are infiltrating) and the decidua parietalis. They were also visible in endothelial-lined spaces. This is the first study to identify and describe the phenotype and distribution of human decidual DCs.