Bacterial ghosts (BGs)—Advanced antigen and drug delivery system

Bacterial ghosts (BGs) are empty bacterial envelopes of Gram-negative bacteria produced by controlled expression of cloned gene E, forming a lysis tunnel structure within the envelope of the living bacteria. BGs are devoid of cytoplasmic content and possess all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat. BGs are ideally suited as an advanced drug delivery system (ADDS) for toxic substances in tumor therapy. The inner space of BGs can be loaded with either single components or combinations of peptides, drugs or DNA which provides an opportunity to design new types of (polyvalent) drug delivery vehicles. Uptake of BGs loaded with Doxorubicin (Dox) by CaCo2 cells led to effective Dox release from endo-lysosomal compartments and accumulation in the nucleus. Viability and proliferative capacity of the cells were significantly decreased (2–3 orders of magnitude) after internalization of Dox loaded BGs as compared to cells incubated with free Dox. The same effect was observed with leukemia cells. Melanoma cells also revealed a high capability to internalize BGs. These results indicate that BGs are able to target a range of types of cancer. BGs have also been investigated as DNA delivery vectors. Studies show DNA loaded BGs are efficiently phagocytosed and internalized by both professional APCs and tumor cells with up to 82% of cells expressing the plasmid-encoded reporter gene. Our studies with BGs as an ADDS system contribute (i) to optimize drug delivery for the treatment of cancer; (ii) define specific conditions for selection and preparation of BG formulations; (iii) and provide a background for the clinical application of BGs in cancer therapy.     

Primary Alloproliferative TH1 Response Induced by Immature Plasmacytoid Dendritic Cells in Collaboration with Myeloid DCs

The role played by dendritic cell (DC) subsets in the immune response to alloantigens is not well defined. In vitro experiments have extensively shown that freshly isolated myeloid (M)DCs induce a strong T lymphocyte proliferation whereas plasmacytoid (P)DCs do not, unless activated by CD40 ligation. The aim of these studies was to explore whether the interplay among PDCs, MDCs and T cells modulates alloresponse. Freshly isolated MDCs and PDCs were merged in different proportions and used as antigen presenting cells (APCs) in mixed lymphocyte cultures (MLC). As described, isolated PDCs only induced a mild alloresponse, while MDCs were potent inducers of alloproliferation. Unexpectedly, when PDCs were merged with even low numbers of MDCs (down to 100 cells) and used as APCs, a potent Th1 cell proliferation was detected. Survival and maturation of PDCs was increased in these MLC conditions, which could partially explain the magnitude of the T-cell response. Interestingly, the proportion of IFNgamma-producing cells generated in such cultures was higher compared to MDC-stimulated cultures. These data suggest that the interaction between both DC subsets is determinant to generate a potent Th1 response, at least in an allogeneic situation, and may be relevant to the outcome of allogeneic stem cell transplantation.     

Blocking of integrins inhibits HIV‐1 infection of human cervical mucosa immune cells with free and complement‐opsonized virions

The initial interaction between HIV‐1 and the host occurs at the mucosa during sexual intercourse. In cervical mucosa, HIV‐1 exists both as free and opsonized virions and this might influence initial infection. We used cervical explants to study HIV‐1 transmission, the effects of opsonization on infectivity, and how infection can be prevented. Complement opsonization enhanced HIV‐1 infection of dendritic cells (DCs) compared with that by free HIV‐1, but this increased infection was not observed with CD4⁺ T cells. Blockage of the α4‐, β7‐, and β1‐integrins significantly inhibited HIV‐1 infection of both DCs and CD4⁺ T cells. We found a greater impairment of HIV‐1 infection in DCs for complement‐opsonized virions compared with that of free virions when αM/β2‐ and α4‐integrins were blocked. Blocking the C‐type lectin receptor macrophage mannose receptor (MMR) inhibited infection of emigrating DCs but had no effect on CD4⁺ T‐cell infection. We show that blocking of integrins decreases the HIV‐1 infection of both mucosal DCs and CD4⁺ T cells emigrating from the cervical tissues. These findings may provide the basis of novel microbicidal strategies that may help limit or prevent initial infection of the cervical mucosa, thereby reducing or averting systemic HIV‐1 infection.     

Restoration of innate immune activation accelerates Th1‐cell priming and protection following pulmonary mycobacterial infection

The immune mechanisms underlying delayed induction of Th1‐type immunity in the lungs following pulmonary mycobacterial infection remain poorly understood. We have herein investigated the underlying immune mechanisms for such delayed responses and whether a selected innate immune‐modulating strategy can accelerate Th1‐type responses. We have found that, in the early stage of pulmonary infection with attenuated Mycobacterium tuberculosis (M.tb H37Ra), the levels of infection in the lung continue to increase logarithmically until days 14 and 21 postinfection in C57BL/6 mice. The activation of innate immune responses, particularly DCs, in the lung is delayed. This results in a delay in the subsequent downstream immune responses including the migration of antigen‐bearing DCs to the draining lymph node (dLN), the Th1‐cell priming in dLN, and the recruitment of Th1 cells to the lung. However, single lung mucosal exposure to the TLR agonist FimH postinfection is able to accelerate protective Th1‐type immunity via facilitating DC migration to the lung and draining lymph nodes, enhancing DC antigen presentation and Th1‐cell priming. These findings hold implications for the development of immunotherapeutic and vaccination strategies and suggest that enhancement of early innate immune activation is a viable option for improving Th1‐type immunity against pulmonary mycobacterial diseases.     

Topical rather than intradermal application of the TLR7 ligand imiquimod leads to human dermal dendritic cell maturation and CD8+ T‐cell cross‐priming

Toll‐like receptor (TLR) ligands are attractive candidate adjuvants for therapeutic cancer vaccines, since TLR signaling stimulates and tunes both humoral and cellular immune responses induced by dendritic cells (DCs). Given that human skin contains a dense network of DCs, which are easily accessible via (intra‐)dermal delivery of vaccines, skin is actively explored as an antitumor vaccination site. Here we used a human skin explant model to explore the potential of TLR ligands as adjuvants for DC activation in their complex microenvironment. We show that topical application of Aldara skin cream, 5% of which comprises the TLR7 agonist imiquimod, significantly enhanced DC migration as compared with that resulting from intradermal injection of the TLR7/8 ligand R848 or the soluble form of imiquimod. Moreover, Aldara‐treated DCs showed highest levels of the costimulatory molecules CD86, CD83, CD40, and CD70. Topical Aldara induced the highest production of pro‐inflammatory cytokines in skin biopsies. When combined with intradermal peptide vaccination, Aldara‐stimulated DCs showed enhanced cross‐presentation of the melanoma antigen MART‐1, which resulted in increased priming and activation of MART‐1‐specific CD8⁺ T cells. These results point to advantageous effects of combining the topical application of Aldara with antitumor peptide vaccination.     

Regulation of the tolerogenic function of steady‐state DCs

Dendritic cells (DCs) are master regulators of T‐cell responses. After sensing pathogen‐derived molecular patterns (PAMPs), or signals of inflammation and cellular stress, DCs differentiate into potent activators of naïve CD4⁺ and CD8⁺ T cells through a process that is termed DC maturation. By contrast, DCs induce and maintain peripheral T‐cell tolerance in the steady state, that is in the absence of overt infection or inflammation. However, the immunological steady state is not devoid of DC‐activating stimuli, such as commensal microorganisms, subclinical infections, or basal levels of proinflammatory mediators. In the presence of these activating stimuli, DC maturation must be calibrated to ensure self‐tolerance yet allow for adequate T‐cell responses to infections. Here, we review the factors that are known to control DC maturation in the steady state and discuss their effect on the tolerogenic function of steady‐state DCs.     

Crosstalk between dendritic cell subsets and implications for dendritic cell-based anticancer immunotherapy

Dendritic cells (DCs) are a family of professional antigen-presenting cells that have an indispensable role in the initiation of innate and adaptive immune responses against pathogens and tumor cells. The DC family is very heterogeneous. Two main types of naturally occurring DCs circulate in peripheral blood, each with its unique phenotypic and functional characteristics: myeloid DCs and plasmacytoid. There is an ample number of studies that have focused on the bi-directional crosstalk between DCs and natural killer cells or T cells. However, the crosstalk among the different DC subsets, in the context of infectious diseases and cancer, has until now not received much attention. Here, we review all available literature that has dealt with the crosstalk between plasmacytoid and myeloid DCs and the potential mode of action. Emphasis will be given to the therapeutic potential of the combination of DC subsets for DC-based immunotherapy.     

The effects of cichorium intybus extract on the maturation and activity of dendritic cells

Background

Cichorium intybus is a medicinal plant commonly used in traditional medicine for its benefits in immune-madiated disorders. There are several evidences showing that C. intybus can modulate immune responses. In the present study we have investigated the effects of the ethanolic root extract of this plant on the immune system by targeting dendritic cells (DCs). For this purpose, phenotypic and functional maturity of murine DCs after treatment with the extract was analyzed by flow cytometry and mixed lymphocyte reaction (MLR) assay.

Results

C. intybus did not change the expression of CD40, CD86 and MHC-II molecules as important co-stimulatory markers on DCs compared to the control, indicating that it could not promote DCs phenotypic maturation. Treatment of DCs with lower concentrations of the extract resulted in an increased production of IL-12 by these cells with no change in IL-10 release. The capacity of treated DCs to stimulate allogenic T cells proliferation and cytokines secretion was examined in the co-cuture of these cells with T cells in MLR. C. intybus at higher concentrations inhibited proliferation of allogenic T cells and in lower concentrations changed the level of cytokines such that IL-4 decreased and IFN-γ increased.

Conclusions

These results indicated that C. intybus extract at higher concentrations can inhibit T cell stimulating activity of DCs, whereas at lower concentrations can modulate cytokine secretion toward a Th1 pattern. These data may in part explain the traditional use of this plant in treatment of immune-mediated disorders.     

Tumor-derived microparticles in tumor immunology and immunotherapy

Microvesicles or microparticles, a type of cytoplasm membrane-derived extracellular vesicles, can be released by cancer cells or normal cell types. Alteration of F-actin cytoskeleton by various signals may lead to the cytoplasm membrane encapsulating cellular contents to form microparticles, which contain various messenger molecules, including enzymes, RNAs and even DNA fragments, and are released to extracellular space. The release of microparticles by tumor cells (T-MPs) is a very common event in tumor microenvironments. As a result, T-MPs not only influence tumor cell biology but also profoundly forge tumor immunology. Moreover, T-MPs can act as a natural vehicle that delivers therapeutic drugs to tumor cells and immune cells, thus, remodeling tumor microenvironments and resetting antitumor immune responses, thus, conferring T-MPs a potential role in tumor immunotherapies and tumor vaccines. In this review, we focus on the double-edged sword role of T-MPs in tumor immunology, specifically in TAMs and DCs, and emphasize the application of drug-packaging T-MPs in cancer patients. We aim to provide a new angle to understand immuno-oncology and new strategies for cancer immunotherapy.     

Easy performance of 6-color confocal immunofluorescence with 4-laser line microscopes

Confocal laser scanning microscopy is an advanced technique for imaging tissue samples in vitro and in vivo at high optical resolution. The development of new fluorochrome variants do not only make it possible to perform multicolor flow cytometry of single cells, but in combination with high resolution laser scanning systems also to investigate the distribution of cells in lymphoid tissues by confocal immunofluorescence analyses, thus allowing the distinction of various cell populations directly in the tissue. Here, we provide a protocol for the visualization of at least six differently fluorochrome-labeled antibodies at the same time using a conventional confocal laser scanning microscope with four laser lines (405 nm, 488 nm, 555 nm, and 639 nm laser wavelength) in both murine and human tissue samples. We further demonstrate that compensation correction algorithms are not necessary to reduce spillover of fluorochromes into other channels when the used fluorochromes are combined according to their specific emission bands and the varying Stokes shift for co-excited fluorochromes with the same laser line.