Immune Responses of Human Immature Dendritic Cells Can Be Modulated by the Recombinant Aspergillus fumigatus Antigen Aspf1

Invasive aspergillosis is a significant cause of morbidity and mortality in patients after stem cell transplantation, in solid organ transplant recipients, and in patients with hematological malignancies. The interactions between human immature dendritic cells (iDCs) and Aspergillus fumigatus antigens are widely uncharacterized. We analyzed the immune response of iDCs to different recombinant A. fumigatus antigens (Aspf1 and Crf1). One of these antigens, the 18-kDa RNase Aspf1, triggered the increased level of expression of genes encoding proinflammatory cytokines and chemokines, and augmented the activation of NFκB and the apoptosis of iDCs. Furthermore, by fluorescence microscopy, we could demonstrate that in the first 3 h a major portion of Aspf1 accumulates on the cell surface. Finally, we could show an increased segregation of cytokines and chemokines after the stimulation of iDCs by an Aspf1 deletion mutant strain of A. fumigatus.Over the past decade, invasive aspergillosis (IA) has emerged as the most serious life-threatening infectious complication of intensive remission-induction chemotherapy and allogeneic hematopoietic stem cell transplantation. Aspergillus fumigatus is the species most commonly isolated from cases of IA. Despite improvements by the use of preventative strategies and the development of new antifungal drugs, IA has an incidence of 10 to 30% and is still associated with mortality rates as high as 90% in some surveys (4). In 2008, over 10,000 patients were hospitalized in the United States due to IA, resulting in 17.7 additional hospital days and $96,000 in additional costs per patient (32).In addition to neutrophils and macrophages, dendritic cells (DCs) play a major role in the defense against IA. DCs can transport antigens from the site of infection to the lymph nodes (18). Exposure to this pathogen leads to changes in the expression of their characteristic surface markers (CD40, CD80, CD83, and CD86) and induces the secretion of proinflammatory cytokines and chemokines (9, 10).There are few data regarding immunotherapy in patients with invasive fungal infections, and in particular, there are few data related to IA. This may be due, at least in part, to the complex antigenic properties of A. fumigatus, which have not yet been well characterized. However, several A. fumigatus proteins have been identified as immunogenic antigens. Among these are two proteins that have been used in this study: the 18-kDa immunoglobulin E (IgE)-binding protein Aspf1 and the glycosylphosphatidylinositol-anchored extracellular cell wall glucanase Crf1 (2). Aspf1 is a member of a family of conserved RNases that cleave defined phosphodiester bonds of the 28S rRNA of eukaryotic ribosomes (3, 16). In this study, we analyzed different aspects of the response of human monocyte-derived DCs to Crf1 and, especially, Aspf1.     

Generation of Immune Responses against Hepatitis C Virus by Dendritic Cells Containing NS5 Protein-Coated Microparticles

Dendritic cells (DCs) internalize and process antigens as well as activate cellular immune responses. The aim of this study was to determine the capacity of DCs that contain antigen-coated magnetic beads to induce immunity against the nonstructural hepatitis C virus (HCV) antigen 5 (NS5). Splenocytes derived from Fms-like tyrosine kinase receptor 3 (Flt3) ligand-pretreated BALB/c mice were incubated with magnetic beads coated with HCV NS5, lipopolysaccharide (LPS), and/or anti-CD40; purified; and used for immunization. Cellular immunity was measured using cytotoxic T-lymphocyte (CTL) and T-cell proliferation assays, intracellular cytokine staining, and a syngeneic tumor challenge using NS5-expressing SP2/0 myeloma cells in vivo. Splenocytes isolated from animals vaccinated with DCs containing beads coated with NS5, LPS, and anti-CD40 secreted elevated levels of interleukin-2 (IL-2) and gamma interferon in the presence of NS5. The numbers of CD4⁺, IL-2-producing cells were increased >5-fold in the group immunized with DCs containing beads coated with NS5, LPS, and anti-CD40, paralleled by an enhanced splenocyte proliferative response. Immunization promoted antigen-specific CTL activity threefold compared to the level for control mice and significantly reduced the growth of NS5-expressing tumor cells in vivo. Thus, strategies that employ NS5-coated beads induce cellular immune responses in mice, which correlate well with the natural immune responses that occur in individuals who resolve HCV.An estimated 170 million individuals (3% of the world''s population) are infected with hepatitis C virus (HCV), leading to cirrhosis, end-stage liver disease, and hepatocellular carcinoma. The current standard-of-care therapy for chronically infected HCV patients is the combined administration of pegylated alpha interferon (IFN-α) and ribavirin (28). A sustained response is seen in approximately 50 to 60% of individuals (33). Treatment is long term (6 to 12 months), costly, and associated with substantial toxicity (42). Clearly, more-effective regimens are needed (10).Clinical studies of the adaptive host immune response to acute HCV infection suggest a rationale for immunization approaches. Clearance of HCV is associated with early, multispecific, strong CD8⁺ T-cell immunity that is matched by vigorous and sustained CD4⁺ T-cell proliferation in response to multiple recombinant structural and nonstructural viral proteins (13, 18, 47). Activated T cells secrete proinflammatory cytokines (TH1 type), such as IFN-γ, coinciding with large reductions in viral load during acute infection (47). HCV infections that are successfully controlled result in durable memory populations (44). This observation is supported by a substantially lower rate of HCV persistence in reexposed humans with a history of acute resolving HCV (29). Rechallenge experiments with chimpanzees showed that antibody-mediated depletion of CD4⁺ T cells resulted in HCV persistence, which is in contrast to a marked reduction in duration and peak of viremia in nontreated animals (4, 20). The importance of CD4⁺ T cells is further emphasized by the loss of immune protection against reexposure to HCV and correlated with low CD4⁺ T-cell counts in intravenous drug users who had recovered from HCV but subsequently acquired human immunodeficiency virus infection (29). Whether recovery from acute HCV coincides ultimately with virus eradication is still a matter of debate (34). The disappearance of HCV-specific antibodies in some individuals 10 to 20 years after viral clearance indicates that a subgroup of patients achieves complete virus elimination (44).Persistent HCV infection can be attributed to a number of viral evasion strategies. First, the rapid spread of HCV in a host outpaces the immune response by several weeks (39, 46). Second, HCV is a strong inducer of type I IFN but appears to render hepatocytes resistant to antiviral activity (45, 46). Third, NK cell function can be inhibited by cross-linking tetraspanin CD81 on the cell surface with the major envelope protein of HCV (HCV E2), thus blocking NK cell activation, proliferation, and cytokine production (12). Finally, persistent HCV infections correlate with the permanent loss of HCV-specific T-cell proliferation during acute HCV infection, the functional exhaustion of an initially vigorous response, and the inability of effector T cells to migrate into the infected liver (10, 13, 18, 36, 39, 46). Insufficient CD4⁺ T-cell activity appears to be a key event leading or contributing to chronic HCV. Failure to sustain the CD4⁺ helper response renders virus-specific CD8⁺ T cells inadequate (e.g., loss of cytotoxicity and IFN-γ production), thus contributing to persistent viremia (39).Recapitulating successful immune responses and addressing HCV-specific defects in immunity are mandatory in efforts to improve current treatment options. The purported involvement of dendritic cells (DCs) in the impaired immune responses observed in patients with persistent HCV infection makes DCs a principal target for immunomodulatory approaches. Access to a sufficient quantity of mature DCs is a critical issue since only mature DCs are capable of targeting the antigen and inducing cellular immunity rather than tolerance. Previously, we outlined a novel method of generating large numbers of DCs in vivo (17). These, in turn, are enriched in vitro by phagocytosis of magnetic beads and separation in a magnetic field. Here we further demonstrate that, by immunizing mice with DCs that contain beads coated with the nonstructural HCV antigen NS5 and compounds known to induce DC maturation, significant levels of cellular immunity are elicited. The key elements of this approach reside in the combined enrichment, maturation, and antigen targeting of DCs in a single step and the generation of immune responses of the type known to promote viral clearance in humans.     

Coagulases as Determinants of Protective Immune Responses against Staphylococcus aureus

During infection, Staphylococcus aureus secretes two coagulases (Coa and von Willebrand factor binding protein [vWbp]), which, following an association with host prothrombin and fibrinogen, form fibrin clots and enable the establishment of staphylococcal disease. Within the genomes of different S. aureus isolates, coagulase gene sequences are variable, and this has been exploited for a classification of types. We show here that antibodies directed against the variable prothrombin binding portion of coagulases confer type-specific immunity through the neutralization of S. aureus clotting activity and protection from staphylococcal disease in mice. By combining variable portions of coagulases from North American isolates into hybrid Coa and vWbp proteins, a subunit vaccine that provided protection against challenge with different coagulase-type S. aureus strains in mice was derived.     

Dendritic Cell Immune Responses in HIV-1 Controllers

Purpose of Review

Robust HIV-1-specific CD8 T cell responses are currently regarded as the main correlate of immune defense in rare individuals who achieve natural, drug-free control of HIV-1; however, the mechanisms that support evolution of such powerful immune responses are not well understood. Dendritic cells (DCs) are specialized innate immune cells critical for immune recognition, immune regulation, and immune induction, but their possible contribution to HIV-1 immune defense in controllers remains ill-defined.

Recent Findings

Recent studies suggest that myeloid DCs from controllers have improved abilities to recognize HIV-1 through cytoplasmic immune sensors, resulting in more potent, cell-intrinsic type I interferon secretion in response to viral infection. This innate immune response may facilitate DC-mediated induction of highly potent antiviral HIV-1-specific T cells. Moreover, protective HLA class I isotypes restricting HIV-1-specific CD8 T cells may influence DC function through specific interactions with innate myelomonocytic MHC class I receptors from the leukocyte immunoglobulin-like receptor family.

Summary

Bi-directional interactions between dendritic cells and HIV-1-specific T cells may contribute to natural HIV-1 immune control, highlighting the importance of a fine-tuned interplay between innate and adaptive immune activities for effective antiviral immune defense.

     

Immune Responses in Patients with Metastatic Renal Cell Carcinoma Treated with Dendritic Cells Pulsed with Tumor Lysate

Patients with metastatic renal cell carcinoma (mRCC) have a limited life expectancy but still a subset of these patients develop immune and clinical responses after immunotherapy including dendritic cell (DC) vaccination. In a recently published phase I/II trials, fourteen HLA-A2 negative patients with progressive mRCC were vaccinated with autologous DC pulsed with allogeneic tumour lysate. Low-dose IL-2 administered subcutaneously was given concomitantly. In this study, we analysed lysate specific proliferation of PBMCs from these patients together with the TH1/TH2 balance of the responding T cells. Also, serum concentrations of IL-10, IL-12, IL-15, IL-17 and IL-18 from these patients and additional thirteen HLA-A2 positive mRCC patients treated with autologous DC pulsed with survivin and telomerase peptides were analysed during vaccination to identify systemic immune responses and potential response biomarkers. In HLA-A2 negative mRCC patients a spontaneous predominance of TH1 secreting tumour lysate specific T cells was observed prior to vaccination in patients attaining stable disease (SD) during treatment whereas patients with continued progressive disease (PD) had a mixed TH1/TH2 response. The TH1/TH2 balance was unchanged during vaccination also when tumour lysate specific T cell responses increased. An increase in IL-12, IL-17 and IL-18 serum concentrations was observed during vaccination but no difference between patients with SD and PD was observed. IL-10 or IL-15 was not measurable in serum.     

EBV LMP2A-specific T Cell Immune Responses Elicited by Dendritic Cells Loaded with LMP2A Protein

Type Ⅱ Epstein-Barr virus （EBV） associated malignancies such as nasopharyngeal carcinoma and non-Hodgkin＇s lymphomas consistently express latent membrane 2A （LMP2A） proteins, which have been suggested to be an ideal target for immunotherapy. In previous studies we have demonstrated that using LMP2A protein loaded dendritic cells, the most powerful antigen processing cells in the body can elicit specific and robust anti-tumor cellular immune response in vitro. In this paper, we further investigated the T cell profile of the anti-tumor immune response. We found that LMP2A specific CD4＋ and CD8＋ T cells could be stimulated by LMP2A protein loaded dendritic cells （DCs）. The Thl type immune response is dominant in the immune response mediated by LMP2A specific CD4^＋ T cells. The CD8^＋ cytotoxic T cells can lyse LMP2A bearing cells effectively and specifically. The CD8^＋ cytotoxic T cells can also secrete high level of intracellular IFN-γ, which indicates these cells are EBV-LMP2A specific cytotoxic T cells. Altogether, our studies proved that LMP2A protein loaded DCs can elicit anti-tumor cellular immune responses efficiently. This study provides a rationale for the DC-based immunotherapy against EBV-LMP2A expressing malignancies.     

Neutrophils Induce the Maturation of Immature Dendritic Cells: A Regulatory Role of Neutrophils in Adaptive Immune Responses

Th1-type immune cytokines are essential to establish adaptive immunity against various microbial pathogens, including Escherichia coli, which cause most urinary tract infections (UTIs). Dendritic cells (DCs) are vital to initiate Th1 immunity, while neutrophils, also referred to here as polymorphonuclear leukocytes (PMN) are reported to be involved in Th1 immunity initiation by secreting several chemokines and cytokines. We found that lipopolysaccharide (LPS)-triggered PMN (LPS-PMN) in vitro induced strong up-regulation of DCs surface markers CD40, CD80, MHC-II (Ia^b), and CD86 either by secreting soluble factors, such as TNF-α, or by PMN-DC cellular contact. LPS-PMN also stimulated DCs to produce IL-12 and TNF-α. Furthermore, purified DCs activated by LPS-PMN were able to present specific antigen to T cells and drive Th1 differentiation by producing large amount of IFN-γ but low amount of IL-4. Our results suggest a regulatory role of PMN for DCs function in adaptive immune responses, thereby providing a link between innate and adaptive immunity.     

Dendritic Cells and Monocytes as Accessory Cells in T-Cell Responses in Man

A method is described for simple and rapid preparation of human dendritic cells (DC) and monocytes (Mo) from peripheral blood. The phenotype of enriched DC and Mo was determined and compared by means of a panel of monoclonal antibodies (Mab's). The distribution and quantitative expression of HLA class II molecules encoded by the subloci DP, DQ, and DR were the same on the two cell types. During in vitro culture a rapid decrease of class II antigens on Mo was observed, whereas the expression of class II antigens on DC was relatively stable. The absence of monocyte markers on DC may indicate that this cell type does not belong to the monocyte/macrophage cell lineage. The phenotypic analysis shows that peripheral blood DC also lack differentiation antigens expressed by epidermal Langerhans cells (OKT6) and lymph node follicular dendritic reticulum cells (DRC-1). The relationship between peripheral blood DC and tissue-localized DC thus remains unsolved. With relatively high numbers of DC now available, production of DC lineage-specific Mab's may be approached.     

Dendritic Cells and Monocytes as Accessory Cells in T-Cell Responses in Man

The main antigen-presenting cells (APC) of human blood are reported to be the dendritic cells (DC), whereas monocytes (Mo) are only weakly or not at all capable of inducing T-cell immune responses to the soluble antigen purified protein derivative (PPD). In contrast, we found Mo to have a suppressive effect on the APC function of DC in vitro. Removal of Mo by adherence resulted in an increased APC activity, even though Mo produce more interleukin 1 (Il-1) than DC. Furthermore, addition of the prostaglandin synthesis inhibitor indomethacin gave rise to increased Il-1 production, HLA-class II expression, and a stronger antigen-specific T-cell response to PPD. Taken together, our studies indicate that the superior accessory cell function of DC compared with Mo in in vitro cultures may, at least partly, be attributed to the prostaglandin E2 production by Mo and more stable expression of HLA-class II molecules on DC.     

Impact of Malaria Preexposure on Antiparasite Cellular and Humoral Immune Responses after Controlled Human Malaria Infection

To understand the effect of previous malaria exposure on antiparasite immune responses is important for developing successful immunization strategies. Controlled human malaria infections (CHMIs) using cryopreserved Plasmodium falciparum sporozoites provide a unique opportunity to study differences in acquisition or recall of antimalaria immune responses in individuals from different transmission settings and genetic backgrounds. In this study, we compared antiparasite humoral and cellular immune responses in two cohorts of malaria-naive Dutch volunteers and Tanzanians from an area of low malarial endemicity, who were subjected to the identical CHMI protocol by intradermal injection of P. falciparum sporozoites. Samples from both trials were analyzed in parallel in a single center to ensure direct comparability of immunological outcomes. Within the Tanzanian cohort, we distinguished one group with moderate levels of preexisting antibodies to asexual P. falciparum lysate and another that, based on P. falciparum serology, resembled the malaria-naive Dutch cohort. Positive P. falciparum serology at baseline was associated with a lower parasite density at first detection by quantitative PCR (qPCR) after CHMI than that for Tanzanian volunteers with negative serology. Post-CHMI, both Tanzanian groups showed a stronger increase in anti-P. falciparum antibody titers than Dutch volunteers, indicating similar levels of B-cell memory independent of serology. In contrast to the Dutch, Tanzanians failed to increase P. falciparum-specific in vitro recall gamma interferon (IFN-γ) production after CHMI, and innate IFN-γ responses were lower in P. falciparum lysate-seropositive individuals than in seronegative individuals. In conclusion, positive P. falciparum lysate serology can be used to identify individuals with better parasite control but weaker IFN-γ responses in circulating lymphocytes, which may help to stratify volunteers in future CHMI trials in areas where malaria is endemic.     

Enhancing the Protective Immune Response against Botulism

The need for a vaccine against botulism has increased since the discontinuation of the pentavalent (ABCDE) botulinum toxoid vaccine by the Centers for Disease Control and Prevention. The botulinum toxins (BoNTs) are the primary virulence factors and vaccine components against botulism. BoNTs comprise three domains which are involved in catalysis (LC), translocation (HCT), and host receptor binding (HCR). Recombinant HCR subunits have been used to develop the next generation of BoNT vaccines. Using structural studies and the known entry properties of BoNT/A, an HCR subunit vaccine against BoNT/A that contained the point mutation W1266A within the ganglioside binding pocket was designed. HCR/A(W1266A) did not enter primary neurons, and the crystal structure of HCR/A(W1266A) was virtually identical to that of wild-type HCR/A. Using a mouse model, experiments were performed using a high-dose vaccine and a low-dose vaccine. At a high vaccine dose, HCR/A and HCR/A(W1266A) elicited a protective immune response to BoNT/A challenge. At the low-dose vaccination, HCR/A(W1266A) was a more protective vaccine than HCR/A. α-HCR IgG titers correlated with protection from BoNT challenge, although titers to block HCR/A entry were greater in serum in HCR/A-vaccinated mice than in HCR/A(W1266A)-vaccinated mice. This study shows that removal of receptor binding capacity enhances potency of the subunit HCR vaccine. Vaccines that lack receptor binding capacity have the added property of limited off-target toxicity.     

Dendritic Cells,Indoleamine 2,3 Dioxygenase and Acquired Immune Privilege

Dendritic cells (DCs) are specialized to stimulate T cell immunity. Paradoxically, some DCs suppress T cell responses and activate regulatory T cells. In this review, we focus on a potent counter-regulatory pathway mediated by plasmacytoid DCs (pDCs) expressing the immunosuppressive enzyme indoleamine 2,3 dioxygenase (IDO). IDO-expressing pDCs inhibit effector T cell responses, activate regulatory T cells, and attenuate pro-inflammatory responses in settings of chronic inflammation that manifest in clinical syndromes, such as infectious, allergic, and autoimmune diseases; cancer; and transplantation. Thus, IDO-expressing pDCs create immune privilege and provide novel opportunities to improve immunotherapy in multiple disease syndromes.     

Protective T Cell and Antibody Immune Responses against Hepatitis C Virus Achieved Using a Biopolyester-Bead-Based Vaccine Delivery System

Hepatitis C virus (HCV) infection is a major worldwide problem. Chronic hepatitis C is recognized as one of the major causes of cirrhosis, hepatocellular carcinoma, and liver failure. Although new, directly acting antiviral therapies are suggested to overcome the low efficacy and adverse effects observed for the current standard of treatment, an effective vaccine would be the only way to certainly eradicate HCV infection. Recently, polyhydroxybutyrate beads produced by engineered Escherichia coli showed efficacy as a vaccine delivery system. Here, an endotoxin-free E. coli strain (ClearColi) was engineered to produce polyhydroxybutyrate beads displaying the core antigen on their surface (Beads-Core) and their immunogenicity was evaluated in BALB/c mice. Immunization with Beads-Core induced gamma interferon (IFN-γ) secretion and a functional T cell immune response against the HCV Core protein. With the aim to target broad T and B cell determinants described for HCV, Beads-Core mixed with HCV E1, E2, and NS3 recombinant proteins was also evaluated in BALB/c mice. Remarkably, only three immunization with Beads-Core+CoE1E2NS3/Alum (a mixture of 0.1 μg Co.120, 16.7 μg E1.340, 16.7 μg E2.680, and 10 μg NS3 adjuvanted in aluminum hydroxide [Alum]) induced a potent antibody response against E1 and E2 and a broad IFN-γ secretion and T cell response against Core and all coadministered antigens. This immunological response mediated protective immunity to viremia as assessed in a viral surrogate challenge model. Overall, it was shown that engineered biopolyester beads displaying foreign antigens are immunogenic and might present a particulate delivery system suitable for vaccination against HCV.     

Vinpocetine Inhibited the CpG Oligodeoxynucleotide-induced Immune Response in Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (pDCs) exert dual roles in immune responses through inducing inflammation and maintaining immune tolerance. A switch of pDC phenotype from pro-inflammation to tolerance has therapeutic promise in the treatment of autoimmune diseases. Vinpocetine, a vasoactive vinca alkaloid extracted from the periwinkle plant, has recently emerged as an immunomodulatory agent. In this study, we evaluated the effect of vinpocetine on phenotype of pDCs isolated from C57BL/6 mice and explored its possible mechanism. Our data showed that vinpocetine significantly downregulated the expression of CD40, CD80, and CD86 on pDCs and increased the expression of translocator protein (TSPO), the specific receptor of vinpocetine, in pDCs. Vinpocetine significantly inhibited the Toll-like receptor 9 signaling pathway and reduced the secretion of related cytokines in pDCs through TSPO. Furthermore, viability of pDCs was significantly promoted by vinpocetine. These findings imply that vinpocetine serves as an immunomodulatory agent for pDCs and may be applied for the treatment of pDCs-related autoimmune diseases.     

Recombinant ESAT-6-Like Proteins Provoke Protective Immune Responses against Invasive Staphylococcus aureus Disease in a Murine Model

Staphylococcus aureus is a common pathogen found in the community and in hospitals. Most notably, methicillin-resistant S. aureus is resistant to many antibiotics, which is a growing public health concern. The emergence of drug-resistant strains has prompted the search for alternative treatments, such as immunotherapeutic approaches. To date, most clinical trials of vaccines or of passive immunization against S. aureus have ended in failure. In this study, we investigated two ESAT-6-like proteins secreted by S. aureus, S. aureus EsxA (SaEsxA) and SaEsxB, as possible targets for a vaccine. Mice vaccinated with these purified proteins elicited high titers of anti-SaEsxA and anti-SaEsxB antibodies, but these antibodies could not prevent S. aureus infection. On the other hand, recombinant SaEsxA (rSaEsxA) and rSaEsxB could induce Th1- and Th17-biased immune responses in mice. Mice immunized with rSaEsxA and rSaEsxB had significantly improved survival rates when challenged with S. aureus compared with the controls. These findings indicate that SaEsxA and SaEsxB are two promising Th1 and Th17 candidate antigens which could be developed into multivalent and serotype-independent vaccines against S. aureus infection.