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.     

Yersinia enterocolitica YopP inhibits MAP kinase-mediated antigen uptake in dendritic cells

Yersinia enterocolitica (Ye) targets mouse dendritic cells (DCs) and inhibits their ability to trigger T cell activation. Here we have investigated whether Ye might interfere with antigen presentation in DCs. Infection of DCs with the Ye wild-type strain reduced OVA uptake by DCs as demonstrated by flow cytometry and confocal laser scan microscopy. In contrast, DCs infected with Yersinia outer protein P (YopP)-deficient mutant strain rapidly internalized OVA. Furthermore, transfection of DCs with YopP, but not with a cysteine protease deficient YopP-C172A mutant, reduced uptake of OVA. This finding suggests that YopP, a virulence factor of Ye, inhibits OVA uptake by DCs. By the use of MAPK inhibitors we provide evidence that YopP mediates reduction of OVA uptake by its ability to block MAPK signalling pathways in host cells. Using transferrin (Tf) as specific marker for clathrin-mediated endocytosis (CME) and lucifer yellow (LY) as specific marker for macropinocytosis (MP) we could show that YopP inhibits CME, whereas other Yops inhibit MP. In keeping with these data, activation and proliferation of OVA-specific T cells was reduced when DCs were treated with MAPK inhibitors. Together, our data demonstrate that (i) MAPK play an important role in antigen uptake by CME in DCs, and (ii) that YopP inhibits this pathway of antigen uptake in DCs, which might contribute to evasion of adaptive immunity.     

Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport

The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major virulence factor of C. neoformans whereby polysaccharide packaged in lipid vesicles crosses the cell wall and the capsule network to reach the extracellular environment.     

Glucuronoxylomannan in the Cryptococcus species capsule as a target for Chimeric Antigen Receptor T-cell therapy

Background aimsThe genus Cryptococcus comprises two major fungal species that cause clinical infections in humans: Cryptococcus gattii and Cryptococcus neoformans. To establish invasive human disease, inhaled cryptococci must penetrate the lung tissue and reproduce. Each year, about 1 million cases of Cryptococcus infection are reported worldwide, and the infection's mortality rate ranges from 20% to 70%. Many HIV⁺/AIDS patients are affected by Cryptococcus infections, with 220,000 cases of cryptococcal meningitis reported worldwide in this population every year (C. neoformans infection statistics, via the Centers for Disease Control and Prevention, https://www.cdc.gov/fungal/diseases/cryptococcosis-neoformans/statistics.html). To escape from host immune cell attack, Cryptococcus covers itself in a sugar-based capsule composed primarily of glucuronoxylomannan (GXM). To evade phagocytosis, yeast cells increase to a >45-µm perimeter and become titan, or giant, cells. Cryptococci virulence is directly proportional to the percentage of titan/giant cells present during Cryptococcus infection. To combat cryptococcosis, the authors propose the redirection of CD8⁺ T cells to target the GXM in the capsule via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR).ResultsGXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk in the extracellular domain, a CD28 transmembrane domain and CD28 and CD3-? signaling domains. After lentiviral transduction of human T cells with the GXMR-CAR construct, flow cytometry demonstrated that 82.4% of the cells expressed GXMR-CAR on their surface. To determine whether the GXMR-CAR⁺ T cells exhibited GXM-specific recognition, these cells were incubated with GXM for 24 h and examined with the use of brightfield microscopy. Large clusters of proliferating GXMR-CAR⁺ T cells were observed in GXM-treated cells, whereas no clusters were observed in control cells. Moreover, the interaction of GXM with GXMR-CAR⁺ T cells was detected via flow cytometry by using a GXM-specific antibody, and the recognition of GXM by GXMR-CAR T cells triggered the secretion of granzyme and interferon gamma (IFN-γ). The ability of GXMR-CAR T cells to bind to the yeast form of C. neoformans was detected by fluorescent microscopy, but no binding was detected in mock-transduced control T cells (NoDNA T cells). Moreover, lung tissue sections were stained with Gomori Methenamine Silver and evaluated by NanoZoomer (Hamamatsu), revealing a significantly lower number of titan cells, with perimeters ranging from 50 to 130 µm and giant cells >130 µm in the CAR T-cell treated group when compared with other groups. Therefore, the authors validated the study's hypothesis by the redirection of GXMR-CAR⁺ T cells to target GXM, which induces the secretion of cytotoxic granules and IFN-γ that will aid in the control of cryptococcosisConclusionsThus, these findings reveal that GXMR-CAR⁺ T cells can target C. neoformans. Future studies will be focused on determining the therapeutic efficacy of GXMR-CAR⁺ T cells in an animal model of cryptococcosis.     

Whole protein and defined CD8(+) and CD4(+) peptides linked to penetratin targets both MHC class I and II antigen presentation pathways

Cytoplasmic delivery and cross-presentation of proteins and peptides is necessary for processing and presentation of antigens for the generation of cytotoxic T cells. We previously described the use of the 16 amino acid peptide penetratin from the Drosophila Antennapedia homeodomain (penetratin, Antp) to transport cytotoxic T lymphocyte epitopes derived from ovalbumin (OVA) or the Mucin-1 tumor-associated antigen into cells. We have now shown that penetratin covalently conjugated to OVA protein and linked in tandem to CD4(+) and/or CD8(+) T-cell epitopes from OVA-stimulated T cells in vitro (B3Z T-cell hybridoma and OT-I and OT-II T cells). The induction of these responses was directly mediated by the penetratin peptide as linking a nonspecific 16-mer peptide to OVA or mixing did not induce CD8(+) or CD4(+) T-cell responses in vitro. Furthermore, interferon (IFN)-γ-secreting CD4(+) and CD8(+) T cells were induced which suppressed B16.OVA tumor growth in C57BL/6 mice. Tumor protection was mediated by a CD8(+) T-cell-dependent mechanism and did not require CD4(+) help to protect mice 7 days after a boost immunization. Alternatively, 40 days after a boost immunization, the presence of CD4(+) help enhanced antigen-specific IFN-γ-secreting CD8(+) T cells and tumor protection in mice challenged with B16.OVA. Long-term CD8 responses were equally enhanced by antigen-specific and universal CD4 help. In addition, immunization with AntpOVA significantly delayed growth of B16.OVA tumors in mice in a tumor therapy model.     

Dendritic cells loaded with exogenous antigen by electroporation can enhance MHC class I–mediated antitumor immunity

To develop an efficient antitumor immunotherapy, we have examined if dendritic cells (DCs) loaded with soluble antigens by electroporation present more antigens via the MHC (major histocompatibility complex) class I pathway, which mediate a cytotoxic T-cell response. DCs loaded with ovalbumin (OVA) by electroporation presented more MHC class I–restricted determinants compared with DCs pulsed with OVA. When electroporated DCs were pulsed with OVA for additional times, both MHC class I– and II–restricted presentation of OVA were increased compared with each single procedure, including electroporation or simple pulse. Immunization with DCs loaded with OVA by electroporation induced higher cytotoxicity of splenocytes to E.G7 cells, a clone of EL4 cells transfected with an OVA cDNA, than immunization with DCs pulsed with OVA. In the animal study, immunization with DCs loaded with OVA or tumor cell lysates by electroporation induced an effective antitumor immunity against tumor of E.G7 cells or Lewis lung carcinoma cells, respectively. In addition, immunization with DCs loaded with antigen by combination of electroporation and pulse, completely protected mice from tumor formation, and prolonged survival, in both tumor models. These results demonstrated that electroporation would be a useful way to enhance MHC class I–mediated antitumor immunity without functional deterioration, and that the combination of electroporation and pulse could be a simple and efficient antigen-loading method and consequently lead to induction of strong antitumor immunity.Abbreviations DCs dendritic cells - MHC major histocompatibility complex - OVA ovalbumin - TAA tumor-associated antigen - CTL cytotoxic T lymphocyte - LDH lactate dehydrogenase     

Quantum dots for tracking dendritic cells and priming an immune response in vitro and in vivo

Dendritic cells (DCs) play a key role in initiating adaptive immune response by presenting antigen to T cells in lymphoid organs. Here, we investigate the potential of quantum dots (QDs) as fluorescent nanoparticles for in vitro and in vivo imaging of DCs, and as a particle-based antigen-delivery system to enhance DC-mediated immune responses. We used confocal, two-photon, and electron microscopies to visualize QD uptake into DCs and compared CD69 expression, T cell proliferation, and IFN-gamma production by DO11.10 and OT-II T cells in vivo in response to free antigen or antigen-conjugated to QDs. CD11c(+) DCs avidly and preferentially endocytosed QDs, initially into small vesicles near the plasma membrane by an actin-dependent mechanism. Within 10 min DCs contained vesicles of varying size, motion, and brightness distributed throughout the cytoplasm. At later times, endocytosed QDs were compartmentalized inside lysosomes. LPS-induced maturation of DCs reduced the rate of endocytosis and the proportion of cells taking up QDs. Following subcutaneous injection of QDs in an adjuvant depot, DCs that had endocytosed QDs were visualized up to 400 microm deep within draining lymph nodes. When antigen-conjugated QDs were used, T cells formed stable clusters in contact with DCs. Antigen-conjugated QDs induced CD69 expression, T cell proliferation, and IFN-gamma production in vivo with greater efficiency than equivalent amounts of free antigen. These results establish QDs as a versatile platform for immunoimaging of dendritic cells and as an efficient nanoparticle-based antigen delivery system for priming an immune response.     

Cryptococcus neoformans capsular polysaccharide component galactoxylomannan induces apoptosis of human T-cells through activation of caspase-8

The major virulence factor of Cryptococcus neoformans is its polysaccharide capsule composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM) and mannoproteins. A variety of immunomodulating activities have been described for GXM and mannoproteins but little is known about possible interactions of GalXM with the immune system. In the present article, we investigate the effect of purified soluble GalXM on human T lymphocytes. The results indicate that, GalXM (i) can affect selected immune responses; (ii) causes significant impairment of T cell proliferation and increases interferon-gamma and interleukin-10 production; and (iii) induces apoptosis of T lymphocytes through activation of caspase-8 that terminates with fragmentation of DNA. These results are the first to suggest a role for GalXM in C. neoformans virulence by demonstrating that it can target human T cells, and that it may impair the development of an effective specific T cell response.     

Parallel beta-helix proteins required for accurate capsule polysaccharide synthesis and virulence in the yeast Cryptococcus neoformans

The principal capsular polysaccharide of the opportunistic fungal pathogen Cryptococcus neoformans consists of an alpha-1,3-linked mannose backbone decorated with a repeating pattern of glucuronyl and xylosyl side groups. This structure is critical for virulence, yet little is known about how the polymer, called glucuronoxylomannan (GXM), is faithfully synthesized and assembled. We have generated deletions in two genes encoding predicted parallel beta-helix repeat proteins, which we have designated PBX1 and PBX2. Deletion of either gene results in a dry-colony morphology, clumpy cells, and decreased capsule integrity. Two-dimensional nuclear magnetic resonance spectroscopy of purified GXM from the mutants indicated that both the wild-type GXM structure and novel, aberrant linkages were present. Carbohydrate composition and linkage analysis determined that these aberrant structures are correlated with the incorporation of terminal glucose residues that are not found in wild-type capsule polysaccharide. We conclude that Pbx1 and Pbx2 are required for the fidelity of GXM synthesis and may be involved in editing incorrectly added glucose residues. PBX1 and PBX2 knockout mutants showed severely attenuated virulence in a murine inhalation model of cryptococcosis. Unlike acapsular strains, these mutant strains induced delayed symptoms of cryptococcosis, though the infected animals eventually contained the infection and recovered.     

Glucuronoxylomannan of Cryptococcus neoformans exacerbates in vitro yeast cell growth by interleukin 10-dependent inhibition of CD4+ T lymphocyte responses

Glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, is the most important virulence factor of this fungus. We analyzed the molecular events related to protective immune responses against a non-encapsulated strain of C. neoformans, mediated by murine splenic CD4(+) T lymphocytes in vitro, and the impact of GXM addition upon these events. Both the lymphoproliferation of CD4(+) T cells and the control of fungus growth were dependent on B7 co-stimulation. Addition of GXM did not modify CD4(+) T cell proliferation, but exacerbated infection in cultures obtained from normal and infected hosts. GXM enhanced the secretion of IL-10 and IL-4, while it reduced the production of pro-inflammatory cytokines TNF-alpha and IFN-gamma. The blockade of IL-10 activity with neutralizing antibodies increased TNF-alpha production and reduced yeast cell growth. The findings suggest that GXM exacerbates infection by down-regulating cell-mediated protective immune response and that IL-10 is implicated in yeast evasion.     

Cryptococcus neoformans cryoultramicrotomy and vesicle fractionation reveals an intimate association between membrane lipids and glucuronoxylomannan

Cryptococcus neoformans is an encapsulated pathogenic fungus. The cryptococcal capsule is composed of polysaccharides and is necessary for virulence. It has been previously reported that glucuronoxylomannan (GXM), the major capsular component, is synthesized in cytoplasmic compartments and transported to the extracellular space in vesicles, but knowledge on the organelles involved in polysaccharide synthesis and traffic is extremely limited. In this paper we report the GXM distribution in C. neoformans cells sectioned by cryoultramicrotomy and visualized by transmission electron microscopy (TEM) and polysaccharide immunogold staining. Cryosections of fungal cells showed high preservation of intracellular organelles and cell wall structure. Incubation of cryosections with an antibody to GXM revealed that cytoplasmic structures associated to vesicular compartments and reticular membranes are in close proximity to the polysaccharide. GXM was generally found in association with the membrane of intracellular compartments and within different layers of the cell wall. Analysis of extracellular fractions from cryptococcal supernatants by transmission electron microscopy in combination with serologic, chromatographic and spectroscopic methods revealed fractions containing GXM and lipids. These results indicate an intimate association of GXM and lipids in both intracellular and extracellular spaces consistent with polysaccharide synthesis and transport in membrane-associated structures.     

An IFN-gamma-IL-18 signaling loop accelerates memory CD8+ T cell proliferation

Rapid proliferation is one of the important features of memory CD8(+) T cells, ensuring rapid clearance of reinfection. Although several cytokines such as IL-15 and IL-7 regulate relatively slow homeostatic proliferation of memory T cells during the maintenance phase, it is unknown how memory T cells can proliferate more quickly than na?ve T cells upon antigen stimulation. To examine antigen-specific CD8(+) T cell proliferation in recall responses in vivo, we targeted a model antigen, ovalbumin(OVA), to DEC-205(+) dendritic cells (DCs) with a CD40 maturation stimulus. This led to the induction of functional memory CD8(+) T cells, which showed rapid proliferation and multiple cytokine production (IFN-gamma, IL-2, TNF-alpha) during the secondary challenge to DC-targeted antigen. Upon antigen-presentation, IL-18, an IFN-gamma-inducing factor, accumulated at the DC:T cell synapse. Surprisingly, IFN-gamma receptors were required to augment IL-18 production from DCs. Mice genetically deficient for IL-18 or IFN-gamma-receptor 1 also showed delayed expansion of memory CD8(+) T cells in vivo. These results indicate that a positive regulatory loop involving IFN-gamma and IL-18 signaling contributes to the accelerated memory CD8(+) T cell proliferation during a recall response to antigen presented by DCs.     

Interactions of Haemophilus ducreyi and purified cytolethal distending toxin with human monocyte-derived dendritic cells, macrophages and CD4+ T cells

To evaluate the early stages of the host response to chancroid bacterium Haemophilus ducreyi, we investigated the in vitro responses of monocyte-derived dendritic cells (DCs) and macrophages (MQs) to this pathogen and Haemophilus influenzae. The phagocytic activities and pro-inflammatory cytokine secretion profiles of the antigen-presenting cells (APCs) were analyzed after exposure to gentamycin-killed bacteria, H. ducreyi lipooligosaccharide (LOS), and purified cytolethal distending toxin (HdCDT). T-cell proliferation and cytokine release were examined after co-culturing isolated autologous CD4+ T cells with antigen-pulsed APCs. Both the DCs and MQs phagocytosed H. ducreyi and H. influenzae, as estimated by flow cytometry. All of the strains induced APC secretion of TNF-alpha, IL-6, IL-8, and IL-12, as measured by ELISA. Other human cells, particularly endothelial cells and fibroblasts, also produced cytokines when stimulated with these bacteria. Purified LOS at concentration 1 microg/ml induced two to threefold lower levels of cytokines than the whole bacteria, which indicates that other components are involved in immune activation. HdCDT inhibited partially the production of the aforementioned cytokines. High levels of IFN-gamma, but not of IL-4 and IL-13, were secreted by T cells after activation by either DCs or MQs that were pre-exposed to bacteria, indicating the Th1 nature of the immune response. The levels of T-cell proliferation induced by H. ducreyi were lower than those induced by H. influenzae. HdCDT-treated APCs did not display cytokine responses or T-cell proliferation. These results indicate that HdCDT intoxication, which results in progressive apoptosis of APCs, may hamper early stage immune responses.     

Oral tolerance induction with antigen conjugated to cholera toxin B subunit generates both Foxp3+CD25+ and Foxp3-CD25- CD4+ regulatory T cells

Oral administration of Ag coupled to cholera toxin B subunit (CTB) efficiently induces peripheral immunological tolerance. We investigated the extent to which this oral tolerance is mediated by CD25+CD4+ regulatory T cells (T(reg)). We found that total T(reg), KJ1-26+ T(reg) and CTLA-4+ T(reg) were all increased in Peyer's patches, mesenteric lymph nodes, and, to a lesser extent, in spleen of mice after intragastric administration of OVA/CTB conjugate, which also increased TGF-beta in serum. This could be abolished by co-administering cholera toxin or by treatment with anti-TGF-beta mAb. CD25+ T(reg), but also CD25-CD4+ T cells from OVA/CTB-treated BALB/c or DO11.10 mice efficiently suppressed effector T cell proliferation and IL-2 production in vitro. Following adoptive transfer, both T cell populations also suppressed OVA-specific T cell and delayed-type hypersensitivity responses in vivo. Foxp3 was strongly expressed by CD25+ T(reg) from OVA/CTB-treated mice, and treatment also markedly expanded CD25+Foxp3+ T(reg). Furthermore, in Rag1(-/-) mice that had adoptively received highly purified Foxp3-CD25-CD4+ OT-II T cells OVA/CTB feeding efficiently induced CD25+ T(reg) cells, which expressed Foxp3 more strongly than naturally developing T(reg) and also had stronger ability to suppress effector OT-II T cell proliferation. A remaining CD25- T cell population, which also became suppressive in response to OVA/CTB treatment, did not express Foxp3. Our results demonstrate that oral tolerance induced by CTB-conjugated Ag is associated with increase in TGF-beta and in both the frequency and suppressive capacity of Foxp3+ and CTLA-4+ CD25+ T(reg) together with the generation of both Foxp3+ and Foxp3-CD25- CD4+ T(reg).     

The Receptor for Advanced Glycation End Products (RAGE) Affects T Cell Differentiation in OVA Induced Asthma

The receptor for glycation end products (RAGE) has been previously implicated in shaping the adaptive immune response. RAGE is expressed in T cells after activation and constitutively in T cells from patients with diabetes. The effects of RAGE on adaptive immune responses are not clear: Previous reports show that RAGE blockade affects Th1 responses. To clarify the role of RAGE in adaptive immune responses and the mechanisms of its effects, we examined whether RAGE plays a role in T cell activation in a Th2 response involving ovalbumin (OVA)-induced asthma in mice. WT and RAGE deficient wild-type and OT-II mice, expressing a T cell receptor specific for OVA, were immunized intranasally with OVA. Lung cellular infiltration and T cell responses were analyzed by immunostaining, FACS, and multiplex bead analyses for cytokines. RAGE deficient mice showed reduced cellular infiltration in the bronchial alveolar lavage fluid and impaired T cell activation in the mediastinal lymph nodes when compared with WT mice. In addition, RAGE deficiency resulted in reduced OT-II T cell infiltration of the lung and impaired IFNγ and IL-5 production when compared with WT mice and reduced infiltration when transferred into WT hosts. When cultured under conditions favoring the differentiation of T cells subsets, RAGE deficient T cells showed reduced production of IFNγ but increased production of IL-17. Our data show a stimulatory role for RAGE in T activation in OVA-induced asthma. This role is largely mediated by the effects of RAGE on T cell proliferation and differentiation. These findings suggest that RAGE may play a regulatory role in T cell responses following immune activation.     

Bridging Small Molecules to Modified Bacterial Microparticles Using a Disulphide Linkage: MIS416 as a Cargo Delivery System

MIS416 is an intact minimal cell wall skeleton derived from Proprionibacterium acnes that is phagocytosed by antigen presenting cells, including dendritic cells (DCs). This property allows MIS416 to be exploited as a vehicle for the delivery of peptide antigens or other molecules (for example, nucleic acids) to DCs. We previously showed that covalent (non-cleavable) conjugation of OVA, a model antigen derived from ovalbumin, to MIS416 enhanced immune responses in DCs in vivo, compared to unconjugated MIS416 and OVA. Intracellular trafficking promotes the lysosomal degradation of MIS416, leading to the destruction of MIS416 plus the associated cargos conjugated to MIS416. However, lysosomal degradation of cargo may not be desired for some MIS416 conjugates. Here we have investigated whether a cleavable linkage could facilitate release of the cargo in the cytoplasm of DCs to avoid lysosomal degradation. DCs were treated in vitro with disulfide-containing conjugates, and as hypothesised faster release of SIINFEKL peptide in the cytoplasm of DCs was observed with the inclusion of a disulfide bond between MIS416 and cargo. The inclusion of a cleavable disulfide bond in the conjugates did not significantly alter the amount of SIINFEKL antigens presented on MHC I molecules on DCs as compared with conjugates without a disulfide bond. However, the conjugates containing disulfide-linkages performed either slightly better (p<0.05) than, or the same as conjugates without a disulfide bond with respect to in vitro OT-1 T-cell proliferation induced by the presentation of SIINFEKL antigens on DCs, or DC activation studies, respectively. However, disulfide-containing conjugates were less effective than conjugates without a disulfide bond in in vivo cytotoxicity assays. In conclusion, inclusion of a disulfide bond in MIS416-peptide conjugates was associated with efficient release of peptides in the cytoplasm of DCs, an important consideration for MIS416-mediated delivery of degradation-sensitive cargoes. However, treatment of DCs with disulfide-containing conjugates did not significantly alter the presentation of peptide antigens on MHC class I molecules to T-cells, or greatly enhance antigen-associated T-cell proliferation in vitro.     

Isolation and characterization of capsule structure mutant strains of Cryptococcus neoformans

The capsule of Cryptococcus neoformans is the most obvious virulence factor of this pathogenic yeast. The main capsule constituents are glucuronoxylomannans (GXM). Although several studies have focused on GXM composition and structure, very little is known about their genetics. To elucidate the relationship between the capsule structure and the pathophysiology of the cryptococcosis, genetic screening for mutant strains producing a structurally modified capsule was set up. Using monoclonal antibodies specific for different capsule sugar epitopes, we isolated strains with different mutated capsule structures (Cas mutants). According to their reactivities with various monoclonal antibodies, the mutants were classified into six groups (Cas1 to Cas6). One Cas2 mutant was used to clone the corresponding gene by complementation. This gene (USX1) encodes the previously identified UDP-xylose synthase. We demonstrated that it is necessary for both capsule xylosylation and C. neoformans virulence.     

Glucuronoxylomannan-mediated interaction of Cryptococcus neoformans with human alveolar cells results in fungal internalization and host cell damage

Infection by Cryptococcus neoformans begins with inhalation of infectious propagules. Fungi reach the lung tissue and interact with epithelial cells in a crucial but poorly understood process. In this study, the interaction of C. neoformans with the human alveolar epithelial cell lineage A549 was investigated, focusing on the relevance of the capsular polysaccharide in this process. The association of encapsulated strains with A549 cells was significantly inhibited by a monoclonal antibody to glucuronoxylomannan (GXM), a major component of the cryptococcal capsule. A purified preparation of GXM produced similar results, suggesting the occurrence of surface receptors for this polysaccharide on the surface of alveolar cells. A549 cells were in fact able to bind soluble GXM, as confirmed by indirect immunofluorescence analysis using the anti-polysaccharide antibody. C. neoformans is internalized after GXM-mediated interaction with A549 cells in a process that culminates with death of host cells. Our results suggest that C. neoformans can use GXM for attachment to alveolar epithelia, allowing the fungus to reach the intracellular environment and damage host cells through still uncharacterized mechanisms.     

Strategies for antigen choice and priming of dendritic cells influence the polarization and efficacy of antitumor T-cell responses in dendritic cell–based cancer vaccination

Dendritic cells (DCs) primed with tumor antigens (Ags) can stimulate tumor rejection. This study was aimed at evaluating the polarization of T-cell responses using various DC Ag-priming strategies for vaccination purposes. DCs cocultured with irradiated apoptotic tumor cells, DC-tumor fusions, and DCs pulsed with freeze-thaw tumor lysate Ags served as Ag-primed DCs, with EG7 tumor cells (class II negative) expressing OVA as the model Ag. DCs loaded with class I– and class II–restricted OVA synthetic peptides served as controls. Primed DCs were assessed by the in vitro activation of B3Z OVA-specific CD8 T cells and the proliferation of OVA-specific CD8 and CD4 T cells from OT-I and OT-II TCR transgenic mice, respectively. In vivo responses were measured by tumor regression following treatment with Ag-primed DCs and by CTL assays. Quantification of IL-2, IL-4, IL-5, IFN-, and TNF- by cytometric bead array (CBA) assay determined the polarization of TH1/TH2 responses, whereas H-2 K^b /SIINFEKL tetramers monitored the expansion of OVA-specific T cells. DC-EG7 hybrids stimulated both efficient class I and class II OVA responses, showing that DC-tumor hybrids are also capable of class II cross-presentation. The hybrids also induced the most potent CTLs, offered the highest protection against established EG7 tumors and also induced the highest stimulation of IFN- and TNF- production. DCs cocultured with irradiated EG7 were also effective at inducing OVA-specific responses, however with slightly reduced potency to those evoked by the hybrids. DCs loaded with lysates Ags were much less efficient at stimulating any of the OVA-specific T-cell responses, showed very little antitumor protection, and stimulated a weak TH1 response, overbalanced by an IL-5 TH2 response. The strategy of Ag-loading clearly influences the ability of DCs to polarize T cells for a TH1/TH2 response and thus determines the outcome of the elicited immune response, during various vaccination protocols.Abbreviations DC Dendritic cell - FSC Forward scatter - SSC Side scatter - TC Tumor cells This work was supported by Grant 9853 from the Leukaemia Research Fund, UK; a JRC studentship from GKT; and the Lewis Family Research Trust     

Polysaccharide diversity in VNI isolates of Cryptococcus neoformans from Roraima,Northern Brazil

Species of the Cryptococcus genus comprise environmental, encapsulated fungal pathogens that cause lethal meningitis in immunosuppressed individuals. In humans, fungal uptake of hypocapsular Cryptococcus by macrophages was associated with high fungal burden in the cerebrospinal fluid and long-term patient survival. On the basis of the key role of the cryptococcal capsule in disease, we analyzed the diversity of capsular structures in 23 isolates from pigeon excreta collected in the cities of Boa Vista, Bonfim and Pacaraima, in the state of Roraima (Northern Brazil). All isolates were identified as Cryptococcus neoformans (VNI genotype) by MALDI-TOF mass spectrometry. Through a combination of fluorescence microscopy, flow cytometry, ELISA and spectrophotometric methods, each isolate was characterized at the phenotypical level, which included measurements of growth rates at 30 and 37 °C, pigmentation, cell body size, capsular dimensions, serological reactivity, urease production and ability to produce extracellular glucuronoxylomannan (GXM), the main capsular component of C. neoformans. With the exception of melanization, a formidable diversity was observed considering all parameters tested in our study. Of note, hyper and hypo producers of GXM were identified, in addition to isolates with hyper and hypo profiles of reactivity with a polysaccharide-binding monoclonal antibody. Capsular dimensions were also highly variable in the collection of isolates. Extracellular GXM production correlated positively with capsular dimensions, urease activity and cell size. Unexpectedly, GXM concentrations did not correlate with serological reactivity with the cryptococcal capsule. These results reveal a high diversity in the ability of environmental C. neoformans to produce capsular components, which might impact the outcome of human cryptococcosis.