Intestinal CD103(+)CD11b(-) dendritic cells restrain colitis via IFN-γ-induced anti-inflammatory response in epithelial cells.

A crosstalk between commensals, gut immune cells, and colonic epithelia is required for a proper function of intestinal mucosal barrier. Here we investigated the importance of two distinct intestinal dendritic cell (DC) subsets in controlling intestinal inflammation. We show that Clec9A-diphtheria toxin receptor (DTR) mice after depletion of CD103(+)CD11b(-) DCs developed severe, low-dose dextran sodium sulfate (DSS)-induced colitis, whereas the lack of CD103(+)CD11b(+) DCs in Clec4a4-DTR mice did not exacerbate intestinal inflammation. The CD103(+)CD11b(-) DC subset has gained a functional specialization that able them to repress inflammation via several epithelial interferon-γ (IFN-γ)-induced proteins. Among others, we identified that epithelial IDO1 and interleukin-18-binding protein (IL-18bp) were strongly modulated by CD103(+)CD11b(-) DCs. Through its preferential property to express IL-12 and IL-15, this particular DC subset can induce lymphocytes in colonic lamina propria and in epithelia to secrete IFN-γ that then can trigger a reversible early anti-inflammatory response in intestinal epithelial cells.

A high-throughput alphavirus-based expression cloning system for mammalian cells.

We have developed a widely applicable functional genomics strategy based on alphavirus expression vectors. The technology allows for rapid identification of genes encoding a functional activity such as binding of a defined ligand. Complementary DNA (cDNA) libraries were expressed in mammalian cells following infection with recombinant Sindbis virus (SIN replicon particles), a member of the Alphavirus genus. Virus-infected cells that specifically bound a ligand of choice were isolated using fluorescence-activated cell sorting (FACS). Replication-competent, infective SIN replicon particles harboring the corresponding cDNA were amplified in a next step. Within one round of selection, viral clones encoding proteins recognized by monoclonal antibodies or Fc-fusion molecules could be isolated and sequenced. Moreover, using the same viral libraries, a plaque-lift assay was established that allowed the identification of secreted, intracellular, and membrane proteins.

In vivo-matured Langerhans cells continue to take up and process native proteins unlike in vitro-matured counterparts.

We have been able to identify the cell subset derived from Langerhans cells in the total dendritic cell population of the peripheral lymph node and hence to follow their trafficking under normal physiological conditions as well as upon skin irritation. As expected, the rapid mobilization of Langerhans cells triggered by inflammatory signals into the draining lymph node correlated with an up-regulation of costimulatory molecules and with an enhanced immunostimulatory capacity. Surprisingly, however, these cells, instead of shutting down, maintain the capacity to capture and process protein Ags during the couple of days they stay alive in stark contrast to in vitro-matured dendritic cells.

Protective T-cell-based immunity induced in neonatal mice by a single replicative cycle of herpes simplex virus.

Newborns are very susceptible to infections because their immune systems are not fully developed and react to antigen exposure preferentially with unresponsiveness. UV-inactivated herpes simplex virus type 1 (HSV-1) represents such an antigen and does not induce an immune response in neonates. In contrast, protective T cells were primed in newborn mice by a single replicative cycle of DISC HSV-1 given once within 24 h of birth. Each of the HSV-1-primed CD4(+) or CD8(+) T cells induced in wild-type or interferon-deficient mice conferred resistance to naive animals exposed to a lethal virus challenge. Inactivated HSV-1, injected at variable doses up to 10(4) times that of DISC HSV-1, was ineffective in inducing any detectable immune responses in neonates. Thus, the capacity of HSV-1 to replicate once, but not the number of virus particles per se, was decisive in inducing protective T-cell-associated immunity in newborn mice.

Anatomical origin of dendritic cells determines their life span in peripheral lymph nodes.

Dendritic cells (DCs) exhibit considerable heterogeneity in their anatomical location, surface phenotype, and functional properties. In this study, we demonstrate that peripheral lymph nodes contain at least four major, functionally separable, and independently derived, DC subsets, which can be clearly demarcated by their CD11c, CD40, and CD8 expression pattern. Surprisingly, all DCs derived directly from the bone marrow, the myeloid- and the lymphoid-related subsets, turned over fast with t(1/2) of a couple of days. In contrast, DCs exported from the skin, both dermal and epidermal, accumulated 3- to 4-fold slower, turnover that is dramatically increased by cutaneous inflammation.

The antigen dose determines T helper subset development by regulation of CD40 ligand.

Although the amount of antigen and the strength of T cell stimulation have been suggested to regulate Th1 vs. Th2 polarization, it remains unclear how the antigen dose and the strength of signal is detected by the T cell and translated into differential cytokine production. Using co-cultures of dendritic cells (DC) and ovalbumin (OVA)-specific CD4+ T cells obtained from RAG-2)(-/-) DO11.10 mice, we show here that high-dose antigen induced Th1 development by up-regulation of CD40 ligand (CD40L), whereas low-dose antigen stimulation failed to induce CD40L and promoted Th2 development. CD40-CD40L interaction was essential for IL-12 production by DC. In the absence, de novo IL-4 production by T cells and autocrine Th2 development was induced. Furthermore, our results demonstrate that LFA-1/ ICAM interaction promotes Th1 differentiation by lowering the antigen dose required for CD40L up-regulation. Thus, we propose that (1) peptide-MHC density and (2) accessory molecules such as LFA-1 determine T helper polarization by regulation of CD40L.

CTL priming by CD8(+) and CD8(-) dendritic cells in vivo.

Two distinct developmental pathways are driving the formation of myeloid- and lymphoid-related dendritic cells (DC) which differ in anatomical localization and phenotype. In terms of function, it has been hypothesized that only the myeloid-related CD8(-) DC are able to initiate immune responses, whereas the lymphoid-related CD8(+) DC have been suggested to induce tolerance. Here we show that both subsets activate CD8(+) T cells in vitro and induce protective anti-viral CTL responses in vivo. Thus, vaccine strategies using peptide-pulsed DC do not have to take into account DC subsets for priming.

Three chemokines with potential functions in T lymphocyte-independent and -dependent B lymphocyte stimulation.

Three clustered mouse chemokine genes, ABCD-1, -2 and -3, are all expressed highly in dendritic cells and, at various levels, in activated B cells. T cell-independently activated B cells express ABCD-1 and -2, but not -3. T cell-dependently activated B cells express all three. ABCD-1 attracts activated CD8+ cytotoxic T cells and CD4+ helper T cells of type 1 and 2. ABCD-2 preferentially attracts type 2 helper T cells, while ABCD-3 does not attract T cells at all. Both ABCD-1 and ABCD-2 bind to the same receptor (CCR4). In addition, ABCD-1 binds to a second, unknown, receptor on a separate T cell population. The three chemokines might guide T cell-independent as well as -dependent responses with two types of CD4+ T cells.

CD8(+) T cells mediate CD40-independent maturation of dendritic cells in vivo.

Induction of cytotoxic T lymphocyte (CTL) responses against minor histocompatibility antigens is dependent upon the presence of T cell help and requires the interaction of CD40 on dendritic cells (DCs) with CD40 ligand on activated T helper cells (Th). This study demonstrates that CD40 is neither involved in Th-dependent nor Th-independent antiviral CTL responses. Moreover, the data show that DC maturation occurs in vivo after viral infection in the absence of CD40 and Th. This maturation did not require viral infection of DCs but was mediated by peptide-specific CD8(+) T cells. Surprisingly, naive CD8(+) T cells were able to trigger DC maturation within 24 h after activation in vivo and in vitro. Moreover, peptide-activated CD8(+) T cells were able to induce maturation in trans, as DCs that failed to present the relevant antigen in vivo also underwent maturation. Upon isolation, the in vivo-stimulated DCs were able to convert a classically Th-dependent CTL response (anti-HY) into a Th-independent response in vitro. Thus, antiviral CD8(+) T cells are sufficient for the maturation of DCs in the absence of CD40.

OX40-deficient mice are defective in Th cell proliferation but are competent in generating B cell and CTL Responses after virus infection.

OX40, a member of the TNF receptor superfamily, is expressed on activated T cells and implicated in stimulation of T cells and T-dependent humoral responses. We generated OX40-/- mice and found that the formation of extrafollicular plasma cells, germinal centers, and antibody responses was independent of OX40. After infection with LCMV and influenza virus, OX40-/- mice retain primary and memory cytotoxic T cell responses with normal expansion and decline of specific CTL. In contrast, CD4+ T cell proliferation and the number of IFN-gamma-producing CD4+ T cells were reduced in OX40-/- mice. Moreover, the number of CD4+ T cells infiltrating the lungs of influenza virus-infected OX40-/- mice was reduced. These results define a unique role of OX40 in the generation of optimal CD4+ T cell responses in vivo.

Activated murine B lymphocytes and dendritic cells produce a novel CC chemokine which acts selectively on activated T cells.

Genes were isolated using the suppression subtractive hybridization method by stimulation of pro/pre B cells with anti-CD40 and interleukin (IL)-4 to mature S mu-Sepsilon-switched cells. One of the strongly upregulated genes encodes a novel murine CC chemokine we have named ABCD-1. The ABCD-1 gene has three exons separated by 1. 2- and 2.7-kb introns. It gives rise to a 2.2-kb transcript containing an open reading frame of 276 nucleotides. Two polyadenylation sites are used, giving rise to cDNAs with either 1550 or 1850 bp of 3' untranslated regions. The open reading frame encodes a 24 amino acid-long leader peptide and a 68 amino acid-long mature protein with a predicted molecular mass of 7.8 kD. ABCD-1 mRNA is found in highest quantities in activated splenic B lymphocytes and dendritic cells. Little chemokine mRNA is present in lung, in unstimulated splenic cells, in thymocytes, and in lymph node cells. No ABCD-1 mRNA is detected in bone marrow, liver, kidney, or brain, in peritoneal exudate cells as well as in the majority of all unstimulated B lineage cells tested. It is also undetectable in Concanavalin A-activated/IL-2-restimulated splenic T cells, and in bone marrow-derived IL-2-induced natural killer cells and IL-3-activated macrophages. Recombinant ABCD-1 revealed a concentration-dependent and specific migration of activated splenic T lymphoblasts in chemotaxis assays. FACS(R) analyses of migrated cells showed no preferential difference in migration of CD4(+) versus CD8(+) T cell blasts. Murine as well as human T cells responded to ABCD-1. Freshly isolated cells from bone marrow, thymus, spleen, and lymph node, IL-2-activated NK cells, and LPS-stimulated splenic cells, all did not show any chemotactic response. Thus, ABCD-1 is the first chemokine produced in large amounts by activated B cells and acting selectively on activated T lymphocytes. Therefore, ABCD-1 is expected to play an important role in the collaboration of dendritic cells and B lymphocytes with T cells in immune responses.

Flow-cytometric evaluation of oxidative burst in phagocytic cells of children with cystic fibrosis.

OBJECTIVES: The objective of this study was to assess the dye 2', 7'-dichlorofluorescein (DCF) assay in screening for alterations in polymorphonuclear cell (PMN) and monocyte (MC) oxidative burst of cystic fibrosis (CF) patients. STUDY DESIGN: 56 CF patients aged between 2 and 20 years were investigated. Purified cells were stimulated with phorbolmyristate acetate (PMA) and zymosan (ZX). A range for DCF fluorescence for PMA- and ZX-stimulated and non-stimulated cells was established based on data from 60 healthy controls. RESULTS: PMNs showed both enhancement and impairment. A deficient oxidative burst was detected in a total of 14 CF patients caused by abnormally high mean fluorescence intensity (MFI) of resting cells. Enhanced oxidative burst was seen in 6 CF patients. CF patients responded differently to PMA or ZX stimulation. Pseudomonas aeruginosa colonization significantly enhanced (p<0.005) the MFI of resting PMNs. MCs of CF patients showed a significantly (p<0.05) enhanced oxidative burst after stimulation with PMA compared to healthy controls, but no differences could be observed after stimulation with ZX. Serum concentrations of interleukin-6 were elevated in all CF patients, in particular in those with activation of both PMNs and MCs. CONCLUSION: The DCF assay shows for the first time the heterogeneity of the oxidative burst reaction in CF patients. In our opinion, the DCF assay is a reliable method for detecting pathological oxidative burst in CF patients.

Oral immunization with poly-(D,L-lactide-co-glycolide) and poly-(L-lactic acid) microspheres containing pneumotropic bacterial antigens.

Encouraged by recent findings showing the usefulness of nonreplicating antigen delivery systems in the induction ofmucosal immune responses, we investigated microspheres as a means to deliver LW 50020, an immunomodulator consisting of lysates of seven common respiratory pathogens. BALB/c mice were orally immunized with LW 50020 encapsulated into poly-(D,L-lactide-co-glycolide) (PLG) and poly-(L-lactic acid) (PLA) microspheres prepared by either a solvent-evaporation or a solvent-extraction double-emulsion technique. Particle uptake into intestinal Peyer's patches, induction of antibodies in sera and secretion of immunoglobulins by isolated Peyer's patches, lungs and spleen lymphocytes were investigated. Our results revealed size and surface characteristic-dependent uptake of microspheres into Peyer's patches. Microsphere translocation into Peyer's patches was efficient for 0.8-microm microspheres, but poor for 2.0-microm and surface-modified microspheres. We showed an enhanced immune response in the lungs and sera following oral immunization with 0.8-microm PLG solvent-evaporation microspheres. The immunomodulation was statistically significant as compared to free LW 50020. In contrast, oral immunization with other preparations caused reduced or absent modulation of the immune response compared to 0.8-microm microspheres and free antigen. These findings indicate that microspheres displaying small particle sizes, rapid antigen release and high antigen content provide optimal tools to deliver orally applied antigens.

Maturation of Peyer's patch dendritic cells in vitro upon stimulation via cytokines or CD40 triggering.

In mouse Peyer's patches (PP), dendritic cells (DC) are localized in T cell areas as NLDC145+ CD11c+ cells, and in the dome and corona region of the follicle as NLDC145- CD11c+ cells, respectively, suggesting the presence of two different DC populations with distinct roles in antigen uptake, processing, and presentation. However, it is not clear how this relates to DC maturation. In this report, we demonstrate that freshly-isolated CD11c+ DC have the properties of immature DC since they endocytose soluble antigens, phagocytose particulate material such as latex beads, synthetize major histocompatibility complex (MHC) class II and invariant chain, but, at the same time, display low stimulatory activity for resting T cells, as shown in mixed-lymphocyte reaction and oxidative mitogenesis assays. When cultured for 24 h in the presence of the cytokines granulocyte-macrophage colony-stimulating factor and tumor necrosis factor or anti-CD40, the cells undergo dramatic phenotypic and functional changes characteristic of DC maturation. After 24 h stimulation in vitro, CD11c+ cells lose the ability to take up proteins such as ovalbumin, and in parallel with this decline, the biosynthesis of MHC class II and invariant chain is dramatically down-regulated or eliminated. On the other hand cells treated in vitro exhibit on the cell surface higher levels of MHC class II, of co-stimulatory molecules (CD80, CD86), of adhesion molecules (CD44, intercellular adhesion molecule-1), and acquire expression of the interdigitating DC surface marker NLDC145. Concomitantly, the ability to stimulate naive T cells drastically increased after in vitro treatment with both stimuli. Taken together, our results indicate that the majority of DC in the PP are immature in terms of their antigen-uptake capacity. These sentinel antigen presenting cells are strategically positioned at the dome region of PP, where antigens are transcytosed via the M cells from the gut lumen. A second population of mature interdigitating NLDC145+ CD11c+ DC stimulates naive unprimed T cells in interfollicular areas by up-regulation of surface ligands and accessory signals.

Phenotypic and functional characterization of CD11c+ dendritic cell population in mouse Peyer's patches.

The antigen-presenting cell system in the gastrointestinal tract, one of three main sites (skin and lung being the others) of primary antigen contact, is poorly understood. Our study focused on dendritic cells (DC) as possible candidates for antigen uptake, processing and presentation in mucosal inductive sites, such as Peyer's patches (PP). To investigate the morphology, immunophenotype and stimulatory activity of intestinal DC, a procedure was developed to obtain a cell population by using collagenase digestion of PP, density centrifugation and cell sorting on the basis of CD11c expression. The resultant low-density cell fraction consisted of a nonadherent cell population expressing different intensities of CD11c that could at least be characterized by typical DC morphology (e.g. abundant cytoplasma with veil-like cytoplasmatic dendrites, irregularly shaped nuclei, multivesicular and multilamellar bodies), constitutive levels of surface MHC class II, the presence of macrophage-specific markers, such as F4/80, Mac-I and Fc receptors, respectively, on subpopulations of CD11c+ sorted cells and expression of adhesion and co-stimulatory receptors like ICAM-1 and CD44. The capability of this low-density CD11c+ fraction to stimulate T cell responses was demonstrated in primary allogeneic mixed-lymphocyte reactions (MLR). Herein, we show that the freshly isolated CD11c+ cells showed weak accessory function, but develop this capacity following short-term culture in vitro in the presence of granulocyte/macrophage colony-stimulating factor. Although the nature and functional capacity of the isolated CD11c+ needs further clarification, these preliminary results describing phenotype and accessory function provide some evidence that these cells isolated from the PP may be immature forms of DC and play a crucial role as antigen-presenting cells with important implications for understanding the complex network regulating intestinal antigen uptake, processing and presentation.

Humoral and cellular immune responses in the murine respiratory tract following oral immunization with cholera toxin or Escherichia coli heat-labile enterotoxin.

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are the strongest mucosal immunogens identified to date and are also good adjuvants when given orally together in combination with unrelated antigens. We used these potent immunogens to monitor local and systemic immune responses following oral immunization of BALB/c mice, and compared their action on the following: (a) immunoglobulin production rates (IgG, IgM and IgA) in mucosal inductive (Peyer's patches-PPs), effector (intestinal lamina propria-LP, respiratory tract) and systemic (spleen) sites; (b) analysis of systemic antigen-specific antibodies (IgG subclasses, IgA and IgE); (c) time monitoring of fecal anti-CT and anti-LT antibodies, and (d) in vivo relevance of interleukin-6 (IL-6) to mucosal responses. Both mucosal immunogens elicited specific antibody responses (IgA, IgG) not only in the gastrointestinal tract (PP's and intestinal LP), but also in the respiratory tract and spleens of orally immunized mice. These mucosal responses were accompained by elevated secretion of IL-6 in all investigated tissues, indicating involvement of this cytokine in B-cell maturation processes. Furthermore, oral immunization with CT and LT induced elevated serum titers of IgG1 followed by IgG2a, IgG2b, IgG3 and IgA, while high antigen-specific IgA and IgG1 responses were found in fecal extracts. These findings illustrate the action of orally administered CT and LT, respectively, on several humoral and cellular immune responses not only at the gastrointestinal tract, the application site, but also in distant mucosal effector sites such as the respiratory tract. These data suggest the potential use of these mucosal adjuvants in oral immunization strategies to improve the local immune response in remote mucosal tissues, in accordance with the concept of a common mucosa-associated immune system.

Preparation and characterization of poly-(D,L-lactide-co-glycolide) and poly-(L-lactic acid) microspheres with entrapped pneumotropic bacterial antigens.

Poly-(lactide-co-glycolide) microspheres with entrapped antigen have shown considerable promise as controlled release vaccines. To enhance the immunomodulatory effect of LW 50020, a bacterial lysate of seven common respiratory pathogens used perorally as an immunomodulator, we prepared poly-(D,L-lactide-co-glycolide) (PLG) and poly-(L-lactic acid) (PLA) microspheres with entrapped immunomodulator by solvent evaporation or solvent extraction double emulsion techniques. Physical properties, such as particle size, LW 50020 entrapment rate, antigen release patterns and morphological characteristics were investigated. All preparations displayed a high degree of antigen loading up to 95%, whereas size, surface morphology and antigen release patterns were significantly influenced by the method of preparation and the polymer components used. Solvent evaporation microspheres are porous particles from 0.8 micron to 2.0 microns in diameter, that show a rapid antigen release for PLG, and a moderate antigen release for PLA microspheres within 33 days. Solvent extraction microspheres have proven to be particles from 1.1 microns to 5.0 microns in diameter showing a smooth surface and a medium antigen release rate over 33 days. SDS-PAGE and immunoblotting of extracted antigen confirmed that the molecular weight and antigenicity of the immunomodulator remained unaltered by the entrapment procedure.

Features of oral immunization.

In this review, we focus on some key areas concerning the unique properties of the mucosal immune system. They are: (1) the fact that the common mucosal immune system consists of different compartments; (2) the advantages of oral vaccination, which can be exploited to antigen-specific sIgA-mediated local immune responses as well as systemic immunity; (3) efficacious oral immunization against respiratory infections; (4) oral tolerance with respect to activation of T cells which, after declining, can be repeatedly reinduced without changes in profile or magnitude, and (5) the use of transgenic plants as a new vaccine source for a new vaccination strategy, i.e. employing edible dietary vaccines.

Chronic granulomatous disease assessed by single-cell granulocyte oxidative burst activity.

Here we report on a case of chronic granulomatous disease (CGD) in a 3-year-old boy who suffered from severe repeated bacterial infections including multiple liver abscesses. The case is of interest because (1) the disease is very rare (it is the first case of CGD diagnosed at the Clinic for Pediatric Medicine, University of Innsbruck), (2) the diagnosis, based on clinical parameters and the nitrobluetetrazolium test was completed and validated by single-cell measurements of respiratory-burst activity of the patient's granulocytes in a fluorescence-activated cell sorter (FACS), and (3) the applied FACS method, adapted in our laboratory, presents one of the most sensitive and reliable methods to evaluate this aspect of disturbed granulocyte function.