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.

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.

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.

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.

Differential inhibitory action of the fungal toxin orellanine on alkaline phosphatase isoenzymes.

The inhibitory action of orellanine (3,3',4,4'-tetrahydroxy-2,2'-dipyridyl-1,1'-dioxide), a fungal toxin of Cortinarius orellanus Fr. and C. orellanoides R. Hry., on alkaline phosphatase isoenzymes was studied. Orellanine specifically inhibited alkaline phosphatase activity in LLC-PK1 renal epithelial cell cultures and in the colon carcinoma cell line Caco-2 without affecting gamma-glutamyl transpeptidase activity. Kinetic studies revealed that orellanine acts on renal alkaline phosphatase as a noncompetitive inhibitor, whereas the intestinal and placental isoforms are inhibited competitively.

Interaction between hormone-dependent and hormone-independent human breast cancer cells.

We developed two different models based on in vitro co-culture of hormone-dependent and hormone-independent cell lines to simulate the cell population heterogeneity of human breast cancer tumours. Oestrogen-dependent (MCF-7, ZR 75.1) and oestrogen-independent cell lines (MDAMB-231 BT-20) were grown under serum-free conditions. Co-culture of hormone-dependent and hormone-independent cell lines resulted in an increased cell yield compared to single cell cultures carried out at the same seeding ratios. Such an increase was not affected by addition of oestradiol and single growth factors (EGF, bFGF and IGF-I). These results allow us to conclude that in a heterogeneous cell population like human breast tumours, interaction between hormone-dependent and hormone-independent cell lines may result in a complex regulation of cell growth.

A novel and sensitive method for the detection of secreted cell products using time-resolved fluorescence.

A new test has been developed for the quantitative detection of products secreted from isolated cells, based on the use of lanthanide- rather than enzyme-linked streptavidin. Used as a label, europium (Eu3+) can be measured with high sensitivity by time-resolved fluorescence. The main advantages of this assay are both an increased sensitivity and measuring range of cell released substances, when compared to the standard "wet" ELISA. Thus, the immunoglobulin secretion rate of 10(5) splenocytes could be easily measured by time-resolved fluoroimmunoassay (TR-FIA), while it remained below the detection limit of the 'wet' ELISA. In contrast to the classical ELISPOT test, this assay does not detect single antibody secreting cells (ASC), but would be useful for precise quantification of secreted cell products, such as immunoglobulins, cytokines, growth factors.

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.

Influence of culture conditions on the estrogenic cell growth stimulation of human breast cancer cells.

17 beta-Estradiol is a potent mitogen for hormone-dependent cell lines (MCF-7, T47D and ZR 75.1). However, the degree of hormone sensitivity is very much influenced by culture conditions. In order to understand which factors modulate estrogenic effects on cell growth, four different culture conditions were used: (a) medium with dextran-coated charcoal-treated fetal calf serum (DCC-FCS); (b) medium with dextran-coated charcoal-treated growth factor-inactivated serum (DCC-FCSd); (c) serum-free medium, after a 24-h incubation with serum to allow cell attachment; and (d) serum-free medium on collagen IV-treated plates. In all cell lines the highest cell growth stimulation was achieved when estradiol was added in the presence of 5% DCC-FCS, whereas reducing or removing serum from the culture medium resulted in a decrease in cell proliferation stimulation. We postulate that serum contains some still unknown components able to modulate the degree of estrogenic action in endocrine-dependent breast cancer cell lines.

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.

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.

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.

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.

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.

Immune response in the lungs following oral immunization with bacterial lysates of respiratory pathogens.

We have investigated the local immune response of the BALB/c mouse respiratory tract after oral immunization with a bacterial lysate of seven common respiratory pathogens. After two immunization on five consecutive days, we examined the immunoglobulin (immunoglobulin G [IgG], IgM, and IgA) secretion rates of cells isolated from the lungs and compared them with those of spleen cells of orally immunized and nonimmunized animals by using a new test system based on time-resolved fluorescence. The procedure followed the principle of the classical ELISPOT test with nitrocellulose-bottomed microtiter plates, but europium (Eu3+)-linked streptavidin rather than enzyme-conjugated streptavidin was used, with the advantage of quantifying secreted immunoglobulins instead of detecting single antibody-secreting cells. Lymphocytes isolated from the lungs of treated animals revealed significant increases in total and antigen-specific IgA synthesis compared with the rates of the controls, whereas IgG and IgM production rates showed no remarkable differences. In addition, the sera of treated mice revealed higher antigen-specific IgA titers but not increased IgM and IgG levels. We conclude that priming the gut-associated lymphoid tissue with bacterial antigens of pneumotropic microorganisms can elicit an enhanced IgA response in a distant mucosal effector site, such as the respiratory tract, according to the concept of a common mucosa-associated immune system.

[Enhanced antibody production by lung lymphocytes after oral immunization with Bordetella pertussis surface antigens]. / Gesteigerte Antikörperproduktion von Lungenlymphozyten nach oraler Immunisierung mit Bordetella-pertussis-Oberflächenantigenen.

The purpose of our study was to evaluate the effect of oral vaccination with Bordetella pertussis surface antigens on the immune response at the site of antigen application. We orally immunized female BALB/c mice on five consecutive days and repeated this procedure after a free interval of 10 days. Lymphocytes of the lung (LL), Peyer's patches (PPL) and lamina propria of the gut (LPL) were isolated and the immunoglobulin secretion rate was measured with time-resolved immunofluorescence. Oral immunization was found to enhance the IgA secretion rate by 69.9% in LL compared to unimmunized animals. The IgG synthesis in LL was increased by 28.1% and the IgM synthesis by 14.1%. In addition, an improvement of 47.8% was observed for the IgG secretion in LPL and PPL. Thus, our results demonstrate a strong local immune response after oral immunization with Bordetella pertussis.

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.

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.