- Email: [email protected]
Workshop N: Dendritic cells and co-stimulation
lInstitut fur Genetik der Universitat K61n, K6ln und 2MPI fur Immunbiologie, Freiburg, Germany
N. 1 Generation of CD 11 cCre-transgenic mice for conditional mutagenesis in dendritic cells C. BURKHARDT!, I. LIEBERAMl, 1. BROCKER2, and I. FORSTER! The bacteriophage PI-derived Cre/loxP site-specific recombination system has been adapted recently for the conditional inactivation of genes in mice. This is achieved by targeted insertion of two loxP recognition sequences into the mouse genome, and deletion of the intervening DNA segment in the presence of the recombinase Cre. To study the functional importance of certain genes and their products in dendritic cells, we are generating transgenic mice in which the recombinase Cre is specifically expressed in dendritic cells (DC). For this purpose, we have chosen the CDllc-promoter which has previously been shown to be highly DC-specific (1. Brocker et aI., J.Exp. Med. 185, 541, 1997). Furthermore, the DC-specific expression pattern of the ~2-integrin CDllc has been confirmed by RNAse protection analysis of purified bone marrow derived DC versus macrophages. To this end, two independent transgenic founder lines have been obtained with either one or multiple copies of the CDllcCre transgene. These mice are presently being analysed for their Cre-expression pattern as well as Cre-mediated deletion of loxP-fianked target genes in vivo.
CellGenix GmbH, Freiburg, and IDepartment of Medicine I, Hematology-Oncology, University Medical Center Freiburg, Germany
N. 2 Generation of dendritic cells from monocytes under serum-free conditions A. GARBE, G. SCHULZ, and A. LINDEMANN! After uptake of antigen Langerhans cells (LC) migrate to the regional lymph nodes and differentiate to potent antigen presenting cells (APC) able to prime naive T cells and to initiate an antigen-specific immune response. We and others have shown before that human dendritic cells (DC) of the LC-phenotype can be generated from monocytes with interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF) in vitro. Tumor necrosis factor alpha (TNF-a) or lipopolysaccaride (LPS) upregulate the accessory molecules B7-1 (CD80) and B7-2 (CD86) on the cell surface and further increase the expression of MHC class-II, thus inducing highly efficient APC. Since these cultures contained fetal bovine serum, DCs may also present serum derived antigens which may impair specificity of T cell induction. We show now that DCs can be generated under serum-free conditions in the presence of transforming growth factor beta (TGF-~I). IL-4 and GM-CSF induced CDla+ DCs from monocytes within 6 days, but numbers and purity of CDla+ cells were increased when TGF-~l was added. However, TNF-a induced upregulation of CD80, CD86 and MHC class II was partially inhibited in TGF-~1 supplemented cultures. Therefore, in order to generate DCs under serum-free condi-
582 . 29th Annual Meeting 1998 tions for in vivo vaccination a sequential culture protocol is proposed. During the first culture period IL-4, GM-CSF and TGF-~l are used to differentiate LC-type cells that most effectively take up and process antigen. After antigen uptake, TNF-a is added after depletion of TGF-~l to induce potent CD80+ and CD86+ APCs.
!Institute of Immunology, University of Witten/Herdecke; 2Department of Dermatology, University of Munster; 3Department of Dermatology, University of Wurzburg, Germany
N.3 Dendritic cells strongly induce the migration and antigen-specific proliferation of Tcells co-cultured within a 3-D collagen matrix M. GUNZER\ D. KARAKUyu-MEYER 1, B. NIGGEMANN!, S. GRABBE 2, P. FRIEDL3, E. KAMPGEN3, and K. S. ZANKER! Dendritic cells (DC) are professional antigen presenting cells, which are specifically effective in the priming of naive T cells. In liquid cultures as well as fixed tissue sections of lymphatic areas it has been shown, that DC form tight clusters with T cells, but to date little is known about the underlying dynamics of these physical cell interactions. To address this question, T cells of DO 11.10 T cell receptor transgenic mice, which are specific for a class II MHC restricted peptide of chicken ovalbumin (OVA), together with DC, pulsed with the relevant OVA-peptide were embedded within a 3-D collagen matrix. The behavior of several hundred individual DC and T cells over a period of 4-5 consecutive days was recorded by time-lapse videomicroscopy. The migration of both cell types relative to each other, DC-T cell interactions, cell divisions as well as changes in cell numbers were analysed by direct visual analysis of the videorecordings. Computer assisted cell tracking gave access to the number of migrating cells in a population as well as the velocities of individual cells. During the course of co-culture the mere presence of DC strongly upregulated the velocities of individual T cells as well as the percentage of locomoting T cells in the population, which was only little less pronounced in non-specific controls (non-pulsed DC plus non-transgenic T cells). In contrast, T cells, incubated in the 3-D collagen gels in the absence of DC, displayed a steady decrease in both migratory parameters. Only in the specific combinations, however, we frequently observed T cell divisions. As a result, the number of transgenic T lymphocytes in coculture with OVA pulsed DC increased 5-fold. Actively migrating T cells only stopped for approx. 30 min to complete cytokinesis, before the daughter cells commenced movement into different directions. Larger clusters of DC and trapped T cells, as characteristic for liquid cultures, were completely absent within the collagen gels. In conclusion, 3-D collagen gel support physiologic processes such as cell migration and cell division. In our model pulsed as well as non-pulsed DC strongly stimulated T cell migration, however, only specifically labelled DC were able to induce T cell proliferation.
Department of Hematopathology, University of Kiel, Germany
N. 4 Monoclonal antibody Ki-M4 - specific for follicular dendritic cells - does not recognize the long isoforrn ,of C021
H. J. HEIDEBRECHT, J. PETERS, H. H. WACKER, and R. PARWARESCH
a.
Recently Liu et al. Exp. Med., 185, 165, 1997) published, that the monoclonal antibody (mAb) Ki-M4 - established in our lab 15 years ago (PARWARESCH et al., Blood 62, 585, 1983) recognizes the long isoform of CD21, a glycoprotein of about 145 kDa. This antigen should be a
29th Annual Meeting 1998 . 583
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specific molecule of follicular dendritic cells (FDC). Already 1 years ago three different groups (MOORE et al., Immunology 84, 9194, 1987; WEIS et al., l Exp. Med 167, 1047, 1988 and FUJISAKU et al., lBiol. Chern. 2649, 2118, 1989) published the eDNA sequence of the long isoform of CD21 as a molecule which is expressed in human B lymphocyte celline Raji and in human mature B lymphocytes. In the last 15 years we performed series of experiments to determine the nature of the Ki-M4 antigen. In immunohistochemical stainings Ki-M4 selectively detects FDC's and sinus lining cells in human lymphnodes. Human mature B lymphocytes and Raji cells could never be stained with Ki-M4. Furtheron we were not able to detect the CD21 antigen with mAb Ki-M4 in Western Blot or immunoprecipitation experiments. In some of our immunoprecipitation experiments Ki-M4 detected a protein of about 50 kDa. Because of our datas we are sure that mAb Ki-M4b does not recognize the long isoform of CD21. The function of this FDC-specific molecule is hitherto unknown.
IDepartment of Immunology, Georg-August University, Gottingen, and ZDepartment of Endocrinology, University Clinic, Essen, Germany
N.5 Monocyte-derived dendritic cells (MoDe): significant increase of allostimulating by inhibition of PDE4 with 3-isobuty-l-methylxanthine D. HEISE!, K. ZIETZ!, l H. PETERS!, and R. GIESELERZ We previously showed that a transient incubation of monocytes during their differentiation into MoDC with dibutyric cyclic adenosine monophosphate (db-cAMP) upregulates their stimulatory activity [1]. We have now sought to define whether cAMP is the definite second messenger to increase accessory activity. To this end, monocytes were initially incubated with db-cAMP, washed and supplied with GM-CSF, IL-4 and IFN-y to differentiate into MoDC [2]. Part of the onsets were then cultured in the permanent presence of phosphodiesterase type 4 (PDE 4) inhibitor 3-isobutyl-l-methylxanthine. On day 6, differentiated MoDC (n = 5, each) were analysed phenotypically for HLA-A/B/C, HLA-DR, CD40, CD50, CD54, CD80, CD83, and CD86 and transferred to mixed leukocyte cultures (MLC). Functional assays were run for five days, added [3H]thymidine, and incubated for 18 hrs. When compared to cultures without inhibitor, the MLC tests revealed an uP. to threefold increase in the presence of both db-cAMP and PDR4 inhibitor (i.e. 27 500 dpm:83 ,600 dpm). On the other hand, we interestingly did not detect any significant phenotypic differences between MoDC generated without dc-cAMP and inhibitor, in the presence of both, or with cAMP alone. The question therefore arose as to which effect secondary to the elevation' of intracellular cAMP could possibly explain the observed increase in allostimulation. We now show that cAMP might selectively upregulate the influx of CaZ+. Others have earlier shown that ~f\ elevation of intracellular CaZ+ alone by the addition of Calcium ionophore may cause the differentiation of highly stimulatory DC from blood monocytes [3]. We here provide first evidepJe that the effects of both cAMP and Caz+ are directly linked with each other, and that they ar~ two key elements of a chain of events in response to an extracellular first messenger. Studies 011 ~he nature of this first mediator are currently underway. [1] NAJAR HM, RUHL S, BRU-CAPDEVltLE AC, PETERS JH (1990). J Leukoc Bioi 47: 429-439. [2] GIESELER R, HEISE D, SORURI'A, Sd-rWARTZ P, PETERS, JH. Develop Immunol: in press. [3] CZERNIECKI BJ, CARTER C, RIVOLTINI L et al. (1997). J Immunol159: 2823-2837. Supported by Astra, Wedel, Germany
584 . 29th Annual Meeting 1998 Department of Dermatology, University of Wiirzburg, and lDepartment of Immunobiology, University of Bonn, Germany
N. 6 Stabilization of MHC class I molecules on human monocyte derived dendritic cells P. KElKAVOUSSI, C. CARSTENS l, C. SCHEICHER, W. FRIES, E.B. BROCKER, N. KOCH l, and E. KAMPGEN Tissue-derived "immature" dendritic cells (DC) such as epidermal Langerhans cells are specialized to carry antigen in an immunocompetent form into draining lymphnodes to initiate T cell responses. As a remarkable feature and prerequite to their function once formed MHC class II /peptide complexes are stably expressed on mature DC (Kdmpgen et al. PNAS 88:3014, 1991). Recently, Cella et al. (Nature 388:782, 1997) reported that LPS or TNF-a induced maturation results in stabilization of MHC class II but not MHC class I molecules on human monocyte derived DC (moDC). We have reevaluated this important issue and analyzed biosynthesis and decay of MHC class I molecules in moDC over prolongued culture times. MoDC were generated under FCS free culture conditions from peripheral blood monocytes via a 7 day treatment with GM-CSF and IL-4 and matured to day 10 by addition of TNF-a or monocyte conditioned medium (MCM,containinglb-l~, 11-6, TNF-a and PGE2). Cells were metabolically labeled either at d7 or dID and chased up to 72h. Immunoprecipitation with mAbs revealed a high catabolism of MHC class I between d7 and dID in all DC populations regardless of culture conditions. A continous high turnover of MHC class I between diD and d14 was observed in GMCSF/IL-4 "immature" moDC, whereas TNF-a treated moDC displayed an increased half-life of class I molecules to about 48h. A remarkable stability of class I molecules between diD and d14 was observed, even in the absence of any cytokines, if moDCs had been matured from d7 to dID with MCM. Our data suggest, that maturation of DC downregulates both, the biosynthesis of MHC class II and class I molecules, however in a sequential course of events. In addition, loading of DC with class I-restricted peptides for immunotherapy should prove best using MCM matured DC at the end of the maturation process.
Department of Dermatology, and llnstitute of Pathology, Julius-Maximilians University, Wiirzburg, Germany
N. 7 Human monocytes activate different expression patterns of f>roteins of the Jak/Stat pathway during differentiation into dendritic cells or macrophage W. FRIES, M. NEUMANN!, P. KEIKAVOUSSI, A. KOLB-MAURER, E.-B. BROCKER, and E. KAMPGEN Monocytic precursor cells (mPC) can be differentiated in vitro into dendritic cells (DC) or macrophages (MF) by different cytokine conditions. High concentrations of GM-CSF and IL-4 for 7d lead to "immature" antigen-processing DC, which further mature to antigen-presenting DC by a 3d incubation with monocyte conditioned medium (MCM) or an equivalent cytokine cocktail. Alternatively, adherent CDl4+/CD68+ M can be generated by incubation with M-CSF and low-dose GM-CSF. To study signal transduction events that might govern cell fate determination cell extracts from different time points were analysed by means of SDS-PAGE and immunoblotting with specific antibodies. Gel shift and super-shift assays were used for functional studies. The mPC (dO) could be shown to contain distinct levels of Statl, 3, and nuclear (active) StatS, but no Jakl, 2, 3 and Stat6. Incubation of mPC with GM-CSF and IL4led to a general upregulation of all analyzed signal transduction proteins, the strongest for StatS. Jak3 and Stat6 were detectable at d7, but exclusively in the cytosol. Treatment of "immature" DC with MCM (d8-dl0)
29th Annual Meeting 1998 . 585 resulted in a strong upregulation of Jak3, mainly located in the cytosolic fraction, and a weaker upregulation of Jak1 and 2, which appear to be membrane-bound. Compared to immature d7 DC, macrophages as products of an alternative monocytic differentiation pathway show higher levels of nuclear StatS and 6 and detectable Jakl. Following treatment of macrophages with MCM, StatS and 6 are strongly downregulated, while the Jak3 level increases. No change was observed for Jakl expression. In summary, induction of DC development from monocytic precursor cells (dO-d7) as well as DC functional maturation (d8-dIO) are paralleled by distinct expression patterns of Jak/Stat proteins, which are clearly different from the macrophage pathway. Ongoing studies aim at further dissecting the functional role of these signal transduction pathways.
Departments of Dermatology and INeurology, University of Wiirzburg, Germany
N. 8 Expression of IL-18 and Caspase-l (ICE) mRNA is not correlated in dendritic cells C. SCHEICHER, M. KUPPNER\ P. KEIKAVOUSSI, F. WEILBACH 1, G. TERBECK, E.B. BROCKER, K. TOYKA 1, and E. KAMPGEN Based on RT-PCR-data we recently reported that dendritic cells (DC) from mouse bone marrow, spleen, or skin (LC) as well as human DC derived from monocytes (moDC) express mRNA for Interferon Gamma Inducing Factor (IL-18). This cytokine has been shown to promote the development of a Thl immune response in synergism with IL-12. We established the model that immature DC such as epidermal Langerhans cells (LC) or GM-CSF/IL-4 induced human moDC constitutively express IL-18, but completely downregulate expression of IL-18 mRNA upon induction of DC maturation. Using intracellular staining and western blotting we have now analyzed the presence of IL-18 protein in human and mouse DC from different sources, but were unable to detect IL-18 protein. Since Interleukin-l converting enzyme (ICE = Caspasel) has been shown to cleave pro-IL-18 and generate mature secretable IL-18, we further looked for Caspase-l mRNA expression in our DC populations. So far we could detect Caspase-l mRNA in all DC populations examined without correlation to the regulated expression of IL-18 mRNA. We therefore conclude that Caspase-l may not be sufficient to give rise to mature IL-18 in DC.
Research Center of Infectious Biology and IDepartment of Dermatology, University of Wiirzburg, Germany.
N. 9 Interleukin-2 prevents epidermal Langerhans cells from infection with
Leishmania major
A. SCHARNER,
c. SCHEICHER\ E. KAMPGEN\ and H. MOLL
In the course of cutaneous infection with Leishmania major (L. major) epidermal Langerhans cells (LC) have been shown to be critically involved in the induction of L. major specific immunity. Thereby LC take up parasites at the site of dermal infection and migrate to the draining lymph node to activate specific T cells. Since mobilization of LC usually results from inflammatory stimuli we were interested to analyze the effect of cytokines on the uptake of L. major by LC. After depletion of Thy-l + dendritic epidermal T cells freshly isolated epidermal cells CEC) from BALB/c mice were incubated with L. major in the presence of various concentrations of IL-2, IL-4 and IL-IO. The infection rate of LC was determined by staining with acridinorange/ ethidium bromide and light microscopy or, alternatively, by doublefluorescence FACscan analy-
586 . 29th Annual Meeting 1998 sis using fluorochrome-labeled parasites and monoclonal antibodies to MHC class II on LC. Of the cytokines tested, only IL-2 significantly downregulated the infection rate of LC in a concentration, time and temperature dependent manner. Addition of IL-10 inhibited this IL-2-effect, whereas IL-4 had no influence. Interestingly, the same IL-2 effect was observed in KO-mice, that do not express the IL-2 receptor alpha chain. However, FACscan analysis revealed the presence of the IL-2R-~-chain on freshly isolated LC, which is lost after 10 hours of culture. This is the first report on functional effects of IL-2 on epidermal LC, resulting in a marked decline in susceptibility to infection with L. major. Surprisingly, IL-2 effects were observed in the absence of the IL-2R-a-chain, currently thought to be required for IL-2 signalling in the mouse. Thus the question remains, how IL-2 signalling is brought about in LC.
lInstitute of Microbiology, University of Lausanne, Lausanne, 2Institute of Biochemistryand Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, 3Basel Institute of Immunology, Basel, Switzerland
N. lOT cell priming by dendritic cells lowers the antigen threshold for Tcelli Bcell collaboration
F. LUTHI l , I. MAILLARD\ S. VACHERON2, T. BROCKER3, H. DIGGELMANN 1, and H. ACHA-ORBEA2 B lymphocytes, contrary to dendritic cells, have limited capabilities to induce a primary immune response. They only receive T cell help when they present antigen or superantigen to primed T cells, and they efficiently activate primed but not virgin T cells. To better understand why activated B cells expressing co-stimulation molecules are poor antigen presenters in primary immune responses in vivo we took advantage of the mouse mammary tumor virus (MMTV) infection model. MMTV(SIM) superantigens are presented by both MHC class II I-E and I-A but the latter present much less efficiently. Even at highest virus concentrations, no detectable superantigen response is found in mice lacking IE expression. With transgenic or chimeric mice expressing I-E molecules exclusively on dendritic cells we show that priming of superantigen-reactive T cells by I-E expressing dendritic cells is required to allow specific T cell/B cell collaboration involving MMTV-infected B cells in the context of MHC class II I-A. These results suggest that priming by dendritic cells enables T cells to interact efficiently with B cells and that priming on dendritic cells lowers the threshold of antigen/MHC complexes required for T cell/B cell collaboration. These data suggest that the signal delivered through the T cell receptor required to prime T cells is far greater than that required for B cells to elicit help from primed T cells.
Research Center Borstel, Borstel, Germany
N. 11 CD34-positive peripheral blood stem cells have essential accessory functions for the stimulation of human T lymphocytes with lipopolysaccharide (LPS), but not with recall-antigens T. MATTERN, G. GIRROLEIT, H.-D. FLAD, and A. J. ULMER Recently, we have described that LPS is able to induce proliferation of human peripheral blood T lymphocytes and the secretion of IFN-y by these cells (j. Immunol. 153: 2996, 1994,]. Immunol. 160: 3412, 1998). We found that the activation of T lymphocytes by LPS was dependent on a direct cell-to-cell contact to monocytes and on B7-CD28 interactions, but MHC unrestricted.
29th Annual Meeting 1998 . 587 Furthermore, the presence of IL-12 was obligatory. Our data suggest the requirements for a further very rare blood cell population with accessory properties for LPS-induced T cell stimulation to occur. This cell type was now identified as CD34-positive stem cells. Depletion of CD34-positive stem cells from mononuclear cells (MNC) abrogated the response of T cells to LPS, but not to recall-antigens like tetanus toxoid (TT) or PPD. Addition of purified CD34-positive cells restored the LPS-induced T cell proliferation, but had no influences on antigen-induced proliferation. In summary, our data demonstrate that CD34-positive peripheral blood stem cells function as potent accessory cells during activation of T lymphocytes with LPS. The mechanisms by which CD34-positive cells provide their accessory activity are presently under investigation. [Supported in part by DFG (SFB 367, project C5)].
IInstitute of Pathology, Department of Molecular Pathology, and 2University Hospital Wurzburg, Department of Dermatology, Julius-Maximilians University, Wurzburg, Germany
N. 12 Differential activation of specific transcription factors is a hallmark of maturation of human dendritic cells M. NEUMANN!, W. FRIES 2, E. SERFLING 1, E.-B. BROCKER2, and E. KAMPGEN2 Dendritic cells are potent antigen presenting cells which undergo specific developmental stages. During this maturation they loose their antigen-processing and gain an antigen-presenting capacity. Specific changes in gene expression are a prerequisite for this maturation process. We used an in vitro maturation system for the analysis of the differential activation of Rel/NF-KB and octamer transcription factors during the development of human dendritic cells derived from peripheral blood monocytes (PBMC). The concentration of active, nuclear p65/RelA and p52/NF-KB2 remained almost constant, whereas levels of p50/NF-KBl increased slightly. In contrast, a dramatic increase of nuclear RelB and c-Rel was notable. As far as the octamer factors were concerned, Oct-2 decreased drastically, wheras Oct-l remained unchanged. A comparison with PBMC-derived macrophages revealed marked differences between both cell types. For example, only a slight increase of c-Rel was notable and Oct-2 levels did not decrease. Gelshift-assays showed an overall increase in KB-specific DNA binding in the course of dendritic cell maturation as well as the appearance of new binding complexes. Such assays performed with an octamer binding site displayed a slight increase in Oct-l- and a strong drop in Oct-2-specific binding activity. Taken together, our data indicate a possible involvement of the differential activation of Rel/NF-KB as well as octamer transcription factors in the changes of gene expression accompanying dendritic cell maturation.
Clinical Research Unit, Department of Dermatology, University of Mainz, Mainz, Germany
N. 13 Maturation of epidermal Langerhans cells: Increased expression of ~- and y-actin isoforms as a Iiasis of specialized cell functions X. Ross, R. Ross, and A. B. RESKE-KUNZ Epidermal Langerhans cells (LC) represent immature dendritic cells. Following activation by antigen contact, they migrate to draining lymph nodes and mature into potent immunostimulatory cells for naive T cells. These processes are accompanied by morphological alterations. Applying a differential screening procedure we isolated differentially expressed cDNAs involved in the maturation events including cDNAs of the cytoskeletal actin isoforms ~- and
588 . 29th Annual Meeting 1998 y-actin. Stronger signals with hybridization probes derived from cultured LC compared with probes derived from freshly isolated LC indicate upregulation of actin expression. Upregulated expression of actin was confirmed by RT-PCR, Western blot, immunofluorescence analysis and flow cytometry. In vivo, FITC-modified dendritic cells, present in draining lymph nodes of mice following application of the contact sensitizer FITC, exhibited high actin levels as shown by cytofluorometrical measurements. Staining with fluorescence-labelled phalloidin that selectively binds to polymerized F-actin, indicates an increase in F-actin levels in cultured LC. Thus our data show that maturation of LC, which involves formation of dendritic structures and movement of formerly immobile cells, is accompanied by augmented expression of actin and formation of additional actin filaments. Furthermore, actin mRNA, often used as reference to assess mRNA amounts for Northern blotting or competitive RT-PCR because of its high and ubiquitous expression, is an inappropriate standard for the analysis of LC and DC.
Department of Dermatology, University of Wiirzburg, and lInstitute of Immunology, University of Witten/Herdecke, Germany
N. 14 Specific interactions of human T-cells with autologous dendritic cells are dynamic, short-lived and repetitive A. SCHAFER, E. KAMPGEN, M. GUNZER 1, P. KEIKAVOUSSI, K. S. ZANKER!, E.-B. BROCKER, and P. FRIEDL Antigen-specific T cell activation and proliferation are thought to result from long-lived adhesive interactions between T lymphocytes and dendritic cells (DC), leading to the formation of stable multicellular aggregates (clusters). Using autologous monocyte-derived DCs and peripheral CD4+ T cells in the process of oxidative mitogenesis, the biophysics, interaction kinetics and frequencies and the resulting T cell proliferation were investigated in liquid culture as compared to coculture in a three-dimensional (3-D) collagen matrix model. Using highly sensitive videomicroscopy in combination with computer-assisted cell tracking, oxidative mitogenesis was characterized by vigorous T cell motility and migration, followed by highly dynamic and short-lived T cell-DC interactions, lasting from minutes to few hours. In liquid culture, an initial phase up to 40 h of similarly transient T cell-DC encounters was followed by a period of more stable interactions and cluster formation. In contrast, in 3-D collagen lattices, T cell-DC interactions usually lasting few minutes, lacked cluster formation (>72 h) despite efficient 3H thymidine uptake and prolonged survival of both, T cell and DC. In conclusion, while unspecific T cell-DC interactions were expected to be short-lived, we now propose that also specific T cell activation and proliferation may result from highly dynamic and rather short-lived T cellDC interactions interconnected with frequent periods of active T cell-migration.
Institute for Immunology, Medical Faculty, University of Dresden, Dresden, and Institute of Immunology, University of Munich, Munich, Germany
N. 15 M-DC8+ leukocytes - function and phenotype of novel human dendritic cell population isolated by a one-step immunomagnetic separation K. SCHAKEL, E. MAYER, C. FEDERLE, M. SCHMITZ, G. RIETHMULLER, and E. P. RIEBER Dendritic cells (DC) have a unique capacity to present primary antigens to T cells and play therefore a pivotal role in the induction of immune responses. This makes DC attractive for the development of immunmodulatory strategies. In particular, the use of DC has been proposed
29th Annual Meeting 1998 . 589 for therapeutic priming of cytolytic T cells against defined tumor peptides or viral antigens. Similarly, DC may serve as tools for reprogramming T cells in order to combat autoimmune or atopic diseases. Direct isolation of human DC for experimental purposes however, is hampered by their low frequency and by the lack of selective markers allowing large scale purification from blood. Here we describe the novel monoclonal antibody (mAb) M-DC8, which was generated by immunizing mice with fresWy prepared human mononuclear blood cells higWy enriched for dendritic cells. The mAb M-DC8 specifically reacted with 0,5-1 % of blood leukocytes showing the characteristic function and morphology of DC. Using a one step immunomagnetic procedure M-DC8+ cells could be directly isolated from fresh mononuclear cells. M-DC8+ cells were negative for T cell, B cell, NK cell and monocyte markers (CD2, CD19, CD56 and CD14, respectively), they expressed HLA class II molecules, CD33 and costimulatory molecules CD86 and CD40 at moderate levels, whereas expression of CD80 only appeared after in vitro culture. This pattern of surface antigens is shared with a population of blood cells recognized as immature DC. In addition, M-DC8+ cells characteristically express Fc(RIII (CD16) and efficiently ingested latex beads and antibody coated sheep red blood cells. M-DC8+ cells displayed an outstanding capacity to present antigen as shown in various in vitro assays. In particular, they were excellent stimulators of autologous mixed leukocyte reactions, they activated T cells against primary antigens such as keyhole limpet hemocyanin. Furthermore, they induced differentiation of purified allogeneic CD8+ T cells into alloantigen-specific cytotoxic effector cells and activated cytotoxic T cells against tumor peptides. The mAb M-DC8 might thus be a valuable tool to determine circulating DC for diagnostic purposes and to isolate these cells for in vitro and in vivo studies of antigen specific T cell priming.
IDepartment of Dermatology, University of Freiburg, Germany, 2Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, USA, 3Department of Dermatology, University of Munster, Germany
N. 16 Dendritic cell migration in vivo: Quantification, kinetics, organ specificity and activating capacity for T lymphocyte mediated immunity ].C. SIMONI,]. M. WEISS!, V. DELATIREt, B. MAlt, K. MANKE2, S. GRABBE3, and M. B. LAPPIN! Dendritic cells (DC) are likely to have an increasingly important role in vaccination therapy, therefore, this study sought to determine the migratory capacity and immunogenic function of murine bone-marrow (BM)-derived DC following intradermal (i.d.) and intravenous (i.v.) injection in vivo. DC were enriched from BM cultures using Metrizamide. Following centrifugation, the low-buoyant density cells, referred to throughout as DC, were CDllchigh, lab high, B7-1 high and B7_2 high and potently activated alloreactive T cells in MLR. In contrast, the high-density cells expressed low levels of the above markers, comprised mostly granulocytes based on GR1 expression, and were poor stimulator cells in MLR. Following i.d. injection of fluorescently labeled cells into syngeneic recipient mice, DC but not granulocytes migrated to the T-dependent areas of draining lymph nodes (LN). DC numbers in LN were quantified by flow-cytometric analysis, on days 1,2,3,5 and 7 following DC transfer. Peak numbers of around 90 DC per draining LN were found at the 48 hr time point. There was very little migration of DC to non-draining LN, thymus or spleen at any of the timepoints studied. In contrast, following i.v. injection, DC accumulated mainly in the spleen, liver and lungs of recipient mice but were largely absent from peripheral LN and thymus. The ability of DC to induce T cell-mediated immune responses was examined using Trinitrobenzenesulphate (TNBS)-derivatized DC (TNBS-DC) to transfer contact hypersensitivity responses (CHS) to naive syngeneic recipients. Following i.d. injection, as few as 10 5 TNBS-DC, but not TNBS-granulocytes, transferred CHS responses. However, the same number of TNBS-DC failed to transfer CHS following i.v. injec-
590 . 29th Annual Meeting 1998 tion. In summary, this study provides new and quantitative data on the organ specific migration of murine BM-derived DC following i.d. and i.v. injection. The demonstration that the route of DC administration determines the potency of CHS transfer, strongly suggests that the route of immunization should be considered in the design of vaccine protocols using DC.
IDepartment of Dermatology, University of Freiburg, 2Department of Biochemistry University of Muenster, 3Department of Tumoroncology, University of Homburg, Germany.
N. 17 Interaction with the extracellular matrix (ECM) component hyaluronan (HA) induces maturation of human dendritic cells (DC) c. C. TERMEER!, J. HENNIES I, J. M. WEISS!, U. VOITH!, R. SCHMITTS3, E. SCHOPF!, P. PREHM 2, and J. C. SIMONI The glycosaminoglycan HA is a major component of the cutaneous ECM. Physiologically, HA exists as a high molecular weight polymer (HMW-HA), but is cleaved into lower molecular weight fragments (sHA) at sites of inflammation. Since DC migrating into and through inflamed tissues will contact HA, we wished to investigate the effects of HMW-HA and sHA-fragments both derived from LPS-free HEALON®, on human DC, generated from peripheral blood progenitors by culture in GM-CSF and IL-4. FACS-analysis revealed that only sHA (2-20 glucurionic acid-molecules) but not HMW-HA induced phenotypic changes in DC similar to those seen after LPS-stimulation or culture in monocyte conditioned medium, i.e. an upregulation of HLA-DR, B7-1, B7-2, CD83, ICAM-l, CD14 and CD44 invariant forms and a down-regulation of CDla and CDllS. Likewise, sHA-fragments increased the production of the proinflammatory cytokines IL-l~, TNF-a and IL-12 by DC as determined by ELISA. These phenotypical changes were paralleled by functional data, since sHA increased dramatically the capacity of DC to stimulate the proliferation of resting alloreactive CD3+ T-cells. Furthermore, we found DC's to rapidly internalize sHA-fragments but not HMW-HA. Importantly, this internalization was not mediated by known cellular HA-receptors (HAR), since blocking studies with mAb's against the maior HA-receptor CD44, RHAMM or ICAM-l had no effect. In addition, DC's generated from the bone marrow of HAR knockout mice, showed similar activation upon sHAstimulation as DC from wildtype mice. Further DC, in contrast to fibroblasts, keratinocytes or melanoma cells, DC did not express significant HA-synthetase mRNA as shown by rtPCR and did not secrete HA as determined by RIA. Our findings indicate that HA-fragments encountered by DC at sites of inflammation contribute to DC activation and maturation, thus enhancing their capacity to induce T cell-mediated immune responses.
Department of IDermatology, Freiburg University,2lnstitute of Genetics, Forschungszentrum Karlsruhe, Germany
N. 18 CD44 variant isoforms playa functional role for dendritic cell T cell interactions J.M. WEISS I, c.c. TERMEER!, M. LAPPIN', J. SLEEMAN2, E. SCHOPF!, and J.c. SIMON! Recently we demonstrated that following antigen contact, epidermal Langerhans cells (LC) and dendritic cells (DC) upregulate pan CD44 epitopes and epitopes encoded by variant exons CD44v4 and CD44v6. Functionally, antibodies against CD44 epitopes arrested LC in the epidermis, prevented the binding of activated LC and DC to the T cell zones of lymph nodes (LN),
29th Annual Meeting 1998 . 591 and inhibited their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Since CD44 has recently been demonstrated to costimulate T cell proliferation by a CD28 independent mechanism we extended our studies to investigate the function of CD44 isoforms in DC interaction with T cells. In primary allogeneic MLR responses with bone marrow derived murine DC as stimulators we found that mAbs against pan CD44 epitopes, and epitopes encoded by CD44v4 and v6 significantly inhibited DC-induced T cell proliferation by 75, 70 and 25%, respectively. Preincubation of either DC or T cells with these anti CD44 mAbs revealed a selective and specific effect on DC but not on T cells. These findings suggest that pan CD44 epitopes and CD44v encoded isoforms contribute to the costimulatory activity of DC. In summary we show that the modulated expression of pan CD44 and CD44 variant exon encoded isoforms is not only functional in DC LN-adhesion and migration but also critical for their capacity to stimulate alloreactive T cells.
IMax-Delbriick-Centre of Molecular Medicine, MDC, Berlin, Germany, 2Bender & Co., 3Institute of Molecular Pathology, IMP, Vienna, Austria
N. 19 Gene delivery into antigen presenting dendritic cells by receptor mediated transfection S. S. DIEBOLD 1, E. WAGNER2, M. COTIEN3, and M. ZENKE 1 Dendritic cells (DC) are professional antigen presenting cells that represent a particularly attractive cell type for use in immunotherapy of diseases, such as cancer. In peripheral organs like skin DC get exposed to a variety of pathogens such as viruses and bacteria which they capture through specific cell surface receptors. The present study capitalizes on using such surface receptors for gene delivery into DC by receptor-mediated endocytosis. We demonstrate that polyethylenimine/DNA (PEl/DNA) complexes can be effectively transduced into primary human DC as adenovirus or mannose PEl/DNA transfection complexes (Ad/PEl/DNA and ManPEI/DNA, respectively). Ad/PEl/DNA complexes have plasmid DNA bound to the adenovirus particles by PEl and deliver DNA into cells via the adenovirus infection route. Such transfection complexes yiel'd high transduction levels and sustained expression of luciferase and green fluorescent protein (GFP) reporter genes and are almost as effective as recombinant adenovirus vectors. ManPEI/DNA complexes rely on uptake by receptor mediated endocytosis via mannose receptor that is highly expressed on DC. Additionally, incorporation of adenovirus particles in ManPEI/DNA transfection complexes further enhances transduction efficiencies and transgene expression. Different plasmid DNAs can be transduced simultaneously. We also demonstrate that Ad/PEI transfected DC are competent in stimulating T cell proliferation in allogeneic and autologous mixed lymphocyte reactions. Thus, Ad/PEl and ManPEI transfection represent particularly versatile transduction systems for DC, with ManPEI being exclusively built up of synthetic compounds.
N. 1 Generation of CD 11 cCre-transgenic mice for conditional mutagenesis in dendritic cells C. BURKHARDT!, I. LIEBERAMl, 1. BROCKER2, and I. FORSTER! The bacteriophage PI-derived Cre/loxP site-specific recombination system has been adapted recently for the conditional inactivation of genes in mice. This is achieved by targeted insertion of two loxP recognition sequences into the mouse genome, and deletion of the intervening DNA segment in the presence of the recombinase Cre. To study the functional importance of certain genes and their products in dendritic cells, we are generating transgenic mice in which the recombinase Cre is specifically expressed in dendritic cells (DC). For this purpose, we have chosen the CDllc-promoter which has previously been shown to be highly DC-specific (1. Brocker et aI., J.Exp. Med. 185, 541, 1997). Furthermore, the DC-specific expression pattern of the ~2-integrin CDllc has been confirmed by RNAse protection analysis of purified bone marrow derived DC versus macrophages. To this end, two independent transgenic founder lines have been obtained with either one or multiple copies of the CDllcCre transgene. These mice are presently being analysed for their Cre-expression pattern as well as Cre-mediated deletion of loxP-fianked target genes in vivo.
CellGenix GmbH, Freiburg, and IDepartment of Medicine I, Hematology-Oncology, University Medical Center Freiburg, Germany
N. 2 Generation of dendritic cells from monocytes under serum-free conditions A. GARBE, G. SCHULZ, and A. LINDEMANN! After uptake of antigen Langerhans cells (LC) migrate to the regional lymph nodes and differentiate to potent antigen presenting cells (APC) able to prime naive T cells and to initiate an antigen-specific immune response. We and others have shown before that human dendritic cells (DC) of the LC-phenotype can be generated from monocytes with interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF) in vitro. Tumor necrosis factor alpha (TNF-a) or lipopolysaccaride (LPS) upregulate the accessory molecules B7-1 (CD80) and B7-2 (CD86) on the cell surface and further increase the expression of MHC class-II, thus inducing highly efficient APC. Since these cultures contained fetal bovine serum, DCs may also present serum derived antigens which may impair specificity of T cell induction. We show now that DCs can be generated under serum-free conditions in the presence of transforming growth factor beta (TGF-~I). IL-4 and GM-CSF induced CDla+ DCs from monocytes within 6 days, but numbers and purity of CDla+ cells were increased when TGF-~l was added. However, TNF-a induced upregulation of CD80, CD86 and MHC class II was partially inhibited in TGF-~1 supplemented cultures. Therefore, in order to generate DCs under serum-free condi-
582 . 29th Annual Meeting 1998 tions for in vivo vaccination a sequential culture protocol is proposed. During the first culture period IL-4, GM-CSF and TGF-~l are used to differentiate LC-type cells that most effectively take up and process antigen. After antigen uptake, TNF-a is added after depletion of TGF-~l to induce potent CD80+ and CD86+ APCs.
!Institute of Immunology, University of Witten/Herdecke; 2Department of Dermatology, University of Munster; 3Department of Dermatology, University of Wurzburg, Germany
N.3 Dendritic cells strongly induce the migration and antigen-specific proliferation of Tcells co-cultured within a 3-D collagen matrix M. GUNZER\ D. KARAKUyu-MEYER 1, B. NIGGEMANN!, S. GRABBE 2, P. FRIEDL3, E. KAMPGEN3, and K. S. ZANKER! Dendritic cells (DC) are professional antigen presenting cells, which are specifically effective in the priming of naive T cells. In liquid cultures as well as fixed tissue sections of lymphatic areas it has been shown, that DC form tight clusters with T cells, but to date little is known about the underlying dynamics of these physical cell interactions. To address this question, T cells of DO 11.10 T cell receptor transgenic mice, which are specific for a class II MHC restricted peptide of chicken ovalbumin (OVA), together with DC, pulsed with the relevant OVA-peptide were embedded within a 3-D collagen matrix. The behavior of several hundred individual DC and T cells over a period of 4-5 consecutive days was recorded by time-lapse videomicroscopy. The migration of both cell types relative to each other, DC-T cell interactions, cell divisions as well as changes in cell numbers were analysed by direct visual analysis of the videorecordings. Computer assisted cell tracking gave access to the number of migrating cells in a population as well as the velocities of individual cells. During the course of co-culture the mere presence of DC strongly upregulated the velocities of individual T cells as well as the percentage of locomoting T cells in the population, which was only little less pronounced in non-specific controls (non-pulsed DC plus non-transgenic T cells). In contrast, T cells, incubated in the 3-D collagen gels in the absence of DC, displayed a steady decrease in both migratory parameters. Only in the specific combinations, however, we frequently observed T cell divisions. As a result, the number of transgenic T lymphocytes in coculture with OVA pulsed DC increased 5-fold. Actively migrating T cells only stopped for approx. 30 min to complete cytokinesis, before the daughter cells commenced movement into different directions. Larger clusters of DC and trapped T cells, as characteristic for liquid cultures, were completely absent within the collagen gels. In conclusion, 3-D collagen gel support physiologic processes such as cell migration and cell division. In our model pulsed as well as non-pulsed DC strongly stimulated T cell migration, however, only specifically labelled DC were able to induce T cell proliferation.
Department of Hematopathology, University of Kiel, Germany
N. 4 Monoclonal antibody Ki-M4 - specific for follicular dendritic cells - does not recognize the long isoforrn ,of C021
H. J. HEIDEBRECHT, J. PETERS, H. H. WACKER, and R. PARWARESCH
a.
Recently Liu et al. Exp. Med., 185, 165, 1997) published, that the monoclonal antibody (mAb) Ki-M4 - established in our lab 15 years ago (PARWARESCH et al., Blood 62, 585, 1983) recognizes the long isoform of CD21, a glycoprotein of about 145 kDa. This antigen should be a
29th Annual Meeting 1998 . 583
°
specific molecule of follicular dendritic cells (FDC). Already 1 years ago three different groups (MOORE et al., Immunology 84, 9194, 1987; WEIS et al., l Exp. Med 167, 1047, 1988 and FUJISAKU et al., lBiol. Chern. 2649, 2118, 1989) published the eDNA sequence of the long isoform of CD21 as a molecule which is expressed in human B lymphocyte celline Raji and in human mature B lymphocytes. In the last 15 years we performed series of experiments to determine the nature of the Ki-M4 antigen. In immunohistochemical stainings Ki-M4 selectively detects FDC's and sinus lining cells in human lymphnodes. Human mature B lymphocytes and Raji cells could never be stained with Ki-M4. Furtheron we were not able to detect the CD21 antigen with mAb Ki-M4 in Western Blot or immunoprecipitation experiments. In some of our immunoprecipitation experiments Ki-M4 detected a protein of about 50 kDa. Because of our datas we are sure that mAb Ki-M4b does not recognize the long isoform of CD21. The function of this FDC-specific molecule is hitherto unknown.
IDepartment of Immunology, Georg-August University, Gottingen, and ZDepartment of Endocrinology, University Clinic, Essen, Germany
N.5 Monocyte-derived dendritic cells (MoDe): significant increase of allostimulating by inhibition of PDE4 with 3-isobuty-l-methylxanthine D. HEISE!, K. ZIETZ!, l H. PETERS!, and R. GIESELERZ We previously showed that a transient incubation of monocytes during their differentiation into MoDC with dibutyric cyclic adenosine monophosphate (db-cAMP) upregulates their stimulatory activity [1]. We have now sought to define whether cAMP is the definite second messenger to increase accessory activity. To this end, monocytes were initially incubated with db-cAMP, washed and supplied with GM-CSF, IL-4 and IFN-y to differentiate into MoDC [2]. Part of the onsets were then cultured in the permanent presence of phosphodiesterase type 4 (PDE 4) inhibitor 3-isobutyl-l-methylxanthine. On day 6, differentiated MoDC (n = 5, each) were analysed phenotypically for HLA-A/B/C, HLA-DR, CD40, CD50, CD54, CD80, CD83, and CD86 and transferred to mixed leukocyte cultures (MLC). Functional assays were run for five days, added [3H]thymidine, and incubated for 18 hrs. When compared to cultures without inhibitor, the MLC tests revealed an uP. to threefold increase in the presence of both db-cAMP and PDR4 inhibitor (i.e. 27 500 dpm:83 ,600 dpm). On the other hand, we interestingly did not detect any significant phenotypic differences between MoDC generated without dc-cAMP and inhibitor, in the presence of both, or with cAMP alone. The question therefore arose as to which effect secondary to the elevation' of intracellular cAMP could possibly explain the observed increase in allostimulation. We now show that cAMP might selectively upregulate the influx of CaZ+. Others have earlier shown that ~f\ elevation of intracellular CaZ+ alone by the addition of Calcium ionophore may cause the differentiation of highly stimulatory DC from blood monocytes [3]. We here provide first evidepJe that the effects of both cAMP and Caz+ are directly linked with each other, and that they ar~ two key elements of a chain of events in response to an extracellular first messenger. Studies 011 ~he nature of this first mediator are currently underway. [1] NAJAR HM, RUHL S, BRU-CAPDEVltLE AC, PETERS JH (1990). J Leukoc Bioi 47: 429-439. [2] GIESELER R, HEISE D, SORURI'A, Sd-rWARTZ P, PETERS, JH. Develop Immunol: in press. [3] CZERNIECKI BJ, CARTER C, RIVOLTINI L et al. (1997). J Immunol159: 2823-2837. Supported by Astra, Wedel, Germany
584 . 29th Annual Meeting 1998 Department of Dermatology, University of Wiirzburg, and lDepartment of Immunobiology, University of Bonn, Germany
N. 6 Stabilization of MHC class I molecules on human monocyte derived dendritic cells P. KElKAVOUSSI, C. CARSTENS l, C. SCHEICHER, W. FRIES, E.B. BROCKER, N. KOCH l, and E. KAMPGEN Tissue-derived "immature" dendritic cells (DC) such as epidermal Langerhans cells are specialized to carry antigen in an immunocompetent form into draining lymphnodes to initiate T cell responses. As a remarkable feature and prerequite to their function once formed MHC class II /peptide complexes are stably expressed on mature DC (Kdmpgen et al. PNAS 88:3014, 1991). Recently, Cella et al. (Nature 388:782, 1997) reported that LPS or TNF-a induced maturation results in stabilization of MHC class II but not MHC class I molecules on human monocyte derived DC (moDC). We have reevaluated this important issue and analyzed biosynthesis and decay of MHC class I molecules in moDC over prolongued culture times. MoDC were generated under FCS free culture conditions from peripheral blood monocytes via a 7 day treatment with GM-CSF and IL-4 and matured to day 10 by addition of TNF-a or monocyte conditioned medium (MCM,containinglb-l~, 11-6, TNF-a and PGE2). Cells were metabolically labeled either at d7 or dID and chased up to 72h. Immunoprecipitation with mAbs revealed a high catabolism of MHC class I between d7 and dID in all DC populations regardless of culture conditions. A continous high turnover of MHC class I between diD and d14 was observed in GMCSF/IL-4 "immature" moDC, whereas TNF-a treated moDC displayed an increased half-life of class I molecules to about 48h. A remarkable stability of class I molecules between diD and d14 was observed, even in the absence of any cytokines, if moDCs had been matured from d7 to dID with MCM. Our data suggest, that maturation of DC downregulates both, the biosynthesis of MHC class II and class I molecules, however in a sequential course of events. In addition, loading of DC with class I-restricted peptides for immunotherapy should prove best using MCM matured DC at the end of the maturation process.
Department of Dermatology, and llnstitute of Pathology, Julius-Maximilians University, Wiirzburg, Germany
N. 7 Human monocytes activate different expression patterns of f>roteins of the Jak/Stat pathway during differentiation into dendritic cells or macrophage W. FRIES, M. NEUMANN!, P. KEIKAVOUSSI, A. KOLB-MAURER, E.-B. BROCKER, and E. KAMPGEN Monocytic precursor cells (mPC) can be differentiated in vitro into dendritic cells (DC) or macrophages (MF) by different cytokine conditions. High concentrations of GM-CSF and IL-4 for 7d lead to "immature" antigen-processing DC, which further mature to antigen-presenting DC by a 3d incubation with monocyte conditioned medium (MCM) or an equivalent cytokine cocktail. Alternatively, adherent CDl4+/CD68+ M can be generated by incubation with M-CSF and low-dose GM-CSF. To study signal transduction events that might govern cell fate determination cell extracts from different time points were analysed by means of SDS-PAGE and immunoblotting with specific antibodies. Gel shift and super-shift assays were used for functional studies. The mPC (dO) could be shown to contain distinct levels of Statl, 3, and nuclear (active) StatS, but no Jakl, 2, 3 and Stat6. Incubation of mPC with GM-CSF and IL4led to a general upregulation of all analyzed signal transduction proteins, the strongest for StatS. Jak3 and Stat6 were detectable at d7, but exclusively in the cytosol. Treatment of "immature" DC with MCM (d8-dl0)
29th Annual Meeting 1998 . 585 resulted in a strong upregulation of Jak3, mainly located in the cytosolic fraction, and a weaker upregulation of Jak1 and 2, which appear to be membrane-bound. Compared to immature d7 DC, macrophages as products of an alternative monocytic differentiation pathway show higher levels of nuclear StatS and 6 and detectable Jakl. Following treatment of macrophages with MCM, StatS and 6 are strongly downregulated, while the Jak3 level increases. No change was observed for Jakl expression. In summary, induction of DC development from monocytic precursor cells (dO-d7) as well as DC functional maturation (d8-dIO) are paralleled by distinct expression patterns of Jak/Stat proteins, which are clearly different from the macrophage pathway. Ongoing studies aim at further dissecting the functional role of these signal transduction pathways.
Departments of Dermatology and INeurology, University of Wiirzburg, Germany
N. 8 Expression of IL-18 and Caspase-l (ICE) mRNA is not correlated in dendritic cells C. SCHEICHER, M. KUPPNER\ P. KEIKAVOUSSI, F. WEILBACH 1, G. TERBECK, E.B. BROCKER, K. TOYKA 1, and E. KAMPGEN Based on RT-PCR-data we recently reported that dendritic cells (DC) from mouse bone marrow, spleen, or skin (LC) as well as human DC derived from monocytes (moDC) express mRNA for Interferon Gamma Inducing Factor (IL-18). This cytokine has been shown to promote the development of a Thl immune response in synergism with IL-12. We established the model that immature DC such as epidermal Langerhans cells (LC) or GM-CSF/IL-4 induced human moDC constitutively express IL-18, but completely downregulate expression of IL-18 mRNA upon induction of DC maturation. Using intracellular staining and western blotting we have now analyzed the presence of IL-18 protein in human and mouse DC from different sources, but were unable to detect IL-18 protein. Since Interleukin-l converting enzyme (ICE = Caspasel) has been shown to cleave pro-IL-18 and generate mature secretable IL-18, we further looked for Caspase-l mRNA expression in our DC populations. So far we could detect Caspase-l mRNA in all DC populations examined without correlation to the regulated expression of IL-18 mRNA. We therefore conclude that Caspase-l may not be sufficient to give rise to mature IL-18 in DC.
Research Center of Infectious Biology and IDepartment of Dermatology, University of Wiirzburg, Germany.
N. 9 Interleukin-2 prevents epidermal Langerhans cells from infection with
Leishmania major
A. SCHARNER,
c. SCHEICHER\ E. KAMPGEN\ and H. MOLL
In the course of cutaneous infection with Leishmania major (L. major) epidermal Langerhans cells (LC) have been shown to be critically involved in the induction of L. major specific immunity. Thereby LC take up parasites at the site of dermal infection and migrate to the draining lymph node to activate specific T cells. Since mobilization of LC usually results from inflammatory stimuli we were interested to analyze the effect of cytokines on the uptake of L. major by LC. After depletion of Thy-l + dendritic epidermal T cells freshly isolated epidermal cells CEC) from BALB/c mice were incubated with L. major in the presence of various concentrations of IL-2, IL-4 and IL-IO. The infection rate of LC was determined by staining with acridinorange/ ethidium bromide and light microscopy or, alternatively, by doublefluorescence FACscan analy-
586 . 29th Annual Meeting 1998 sis using fluorochrome-labeled parasites and monoclonal antibodies to MHC class II on LC. Of the cytokines tested, only IL-2 significantly downregulated the infection rate of LC in a concentration, time and temperature dependent manner. Addition of IL-10 inhibited this IL-2-effect, whereas IL-4 had no influence. Interestingly, the same IL-2 effect was observed in KO-mice, that do not express the IL-2 receptor alpha chain. However, FACscan analysis revealed the presence of the IL-2R-~-chain on freshly isolated LC, which is lost after 10 hours of culture. This is the first report on functional effects of IL-2 on epidermal LC, resulting in a marked decline in susceptibility to infection with L. major. Surprisingly, IL-2 effects were observed in the absence of the IL-2R-a-chain, currently thought to be required for IL-2 signalling in the mouse. Thus the question remains, how IL-2 signalling is brought about in LC.
lInstitute of Microbiology, University of Lausanne, Lausanne, 2Institute of Biochemistryand Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, 3Basel Institute of Immunology, Basel, Switzerland
N. lOT cell priming by dendritic cells lowers the antigen threshold for Tcelli Bcell collaboration
F. LUTHI l , I. MAILLARD\ S. VACHERON2, T. BROCKER3, H. DIGGELMANN 1, and H. ACHA-ORBEA2 B lymphocytes, contrary to dendritic cells, have limited capabilities to induce a primary immune response. They only receive T cell help when they present antigen or superantigen to primed T cells, and they efficiently activate primed but not virgin T cells. To better understand why activated B cells expressing co-stimulation molecules are poor antigen presenters in primary immune responses in vivo we took advantage of the mouse mammary tumor virus (MMTV) infection model. MMTV(SIM) superantigens are presented by both MHC class II I-E and I-A but the latter present much less efficiently. Even at highest virus concentrations, no detectable superantigen response is found in mice lacking IE expression. With transgenic or chimeric mice expressing I-E molecules exclusively on dendritic cells we show that priming of superantigen-reactive T cells by I-E expressing dendritic cells is required to allow specific T cell/B cell collaboration involving MMTV-infected B cells in the context of MHC class II I-A. These results suggest that priming by dendritic cells enables T cells to interact efficiently with B cells and that priming on dendritic cells lowers the threshold of antigen/MHC complexes required for T cell/B cell collaboration. These data suggest that the signal delivered through the T cell receptor required to prime T cells is far greater than that required for B cells to elicit help from primed T cells.
Research Center Borstel, Borstel, Germany
N. 11 CD34-positive peripheral blood stem cells have essential accessory functions for the stimulation of human T lymphocytes with lipopolysaccharide (LPS), but not with recall-antigens T. MATTERN, G. GIRROLEIT, H.-D. FLAD, and A. J. ULMER Recently, we have described that LPS is able to induce proliferation of human peripheral blood T lymphocytes and the secretion of IFN-y by these cells (j. Immunol. 153: 2996, 1994,]. Immunol. 160: 3412, 1998). We found that the activation of T lymphocytes by LPS was dependent on a direct cell-to-cell contact to monocytes and on B7-CD28 interactions, but MHC unrestricted.
29th Annual Meeting 1998 . 587 Furthermore, the presence of IL-12 was obligatory. Our data suggest the requirements for a further very rare blood cell population with accessory properties for LPS-induced T cell stimulation to occur. This cell type was now identified as CD34-positive stem cells. Depletion of CD34-positive stem cells from mononuclear cells (MNC) abrogated the response of T cells to LPS, but not to recall-antigens like tetanus toxoid (TT) or PPD. Addition of purified CD34-positive cells restored the LPS-induced T cell proliferation, but had no influences on antigen-induced proliferation. In summary, our data demonstrate that CD34-positive peripheral blood stem cells function as potent accessory cells during activation of T lymphocytes with LPS. The mechanisms by which CD34-positive cells provide their accessory activity are presently under investigation. [Supported in part by DFG (SFB 367, project C5)].
IInstitute of Pathology, Department of Molecular Pathology, and 2University Hospital Wurzburg, Department of Dermatology, Julius-Maximilians University, Wurzburg, Germany
N. 12 Differential activation of specific transcription factors is a hallmark of maturation of human dendritic cells M. NEUMANN!, W. FRIES 2, E. SERFLING 1, E.-B. BROCKER2, and E. KAMPGEN2 Dendritic cells are potent antigen presenting cells which undergo specific developmental stages. During this maturation they loose their antigen-processing and gain an antigen-presenting capacity. Specific changes in gene expression are a prerequisite for this maturation process. We used an in vitro maturation system for the analysis of the differential activation of Rel/NF-KB and octamer transcription factors during the development of human dendritic cells derived from peripheral blood monocytes (PBMC). The concentration of active, nuclear p65/RelA and p52/NF-KB2 remained almost constant, whereas levels of p50/NF-KBl increased slightly. In contrast, a dramatic increase of nuclear RelB and c-Rel was notable. As far as the octamer factors were concerned, Oct-2 decreased drastically, wheras Oct-l remained unchanged. A comparison with PBMC-derived macrophages revealed marked differences between both cell types. For example, only a slight increase of c-Rel was notable and Oct-2 levels did not decrease. Gelshift-assays showed an overall increase in KB-specific DNA binding in the course of dendritic cell maturation as well as the appearance of new binding complexes. Such assays performed with an octamer binding site displayed a slight increase in Oct-l- and a strong drop in Oct-2-specific binding activity. Taken together, our data indicate a possible involvement of the differential activation of Rel/NF-KB as well as octamer transcription factors in the changes of gene expression accompanying dendritic cell maturation.
Clinical Research Unit, Department of Dermatology, University of Mainz, Mainz, Germany
N. 13 Maturation of epidermal Langerhans cells: Increased expression of ~- and y-actin isoforms as a Iiasis of specialized cell functions X. Ross, R. Ross, and A. B. RESKE-KUNZ Epidermal Langerhans cells (LC) represent immature dendritic cells. Following activation by antigen contact, they migrate to draining lymph nodes and mature into potent immunostimulatory cells for naive T cells. These processes are accompanied by morphological alterations. Applying a differential screening procedure we isolated differentially expressed cDNAs involved in the maturation events including cDNAs of the cytoskeletal actin isoforms ~- and
588 . 29th Annual Meeting 1998 y-actin. Stronger signals with hybridization probes derived from cultured LC compared with probes derived from freshly isolated LC indicate upregulation of actin expression. Upregulated expression of actin was confirmed by RT-PCR, Western blot, immunofluorescence analysis and flow cytometry. In vivo, FITC-modified dendritic cells, present in draining lymph nodes of mice following application of the contact sensitizer FITC, exhibited high actin levels as shown by cytofluorometrical measurements. Staining with fluorescence-labelled phalloidin that selectively binds to polymerized F-actin, indicates an increase in F-actin levels in cultured LC. Thus our data show that maturation of LC, which involves formation of dendritic structures and movement of formerly immobile cells, is accompanied by augmented expression of actin and formation of additional actin filaments. Furthermore, actin mRNA, often used as reference to assess mRNA amounts for Northern blotting or competitive RT-PCR because of its high and ubiquitous expression, is an inappropriate standard for the analysis of LC and DC.
Department of Dermatology, University of Wiirzburg, and lInstitute of Immunology, University of Witten/Herdecke, Germany
N. 14 Specific interactions of human T-cells with autologous dendritic cells are dynamic, short-lived and repetitive A. SCHAFER, E. KAMPGEN, M. GUNZER 1, P. KEIKAVOUSSI, K. S. ZANKER!, E.-B. BROCKER, and P. FRIEDL Antigen-specific T cell activation and proliferation are thought to result from long-lived adhesive interactions between T lymphocytes and dendritic cells (DC), leading to the formation of stable multicellular aggregates (clusters). Using autologous monocyte-derived DCs and peripheral CD4+ T cells in the process of oxidative mitogenesis, the biophysics, interaction kinetics and frequencies and the resulting T cell proliferation were investigated in liquid culture as compared to coculture in a three-dimensional (3-D) collagen matrix model. Using highly sensitive videomicroscopy in combination with computer-assisted cell tracking, oxidative mitogenesis was characterized by vigorous T cell motility and migration, followed by highly dynamic and short-lived T cell-DC interactions, lasting from minutes to few hours. In liquid culture, an initial phase up to 40 h of similarly transient T cell-DC encounters was followed by a period of more stable interactions and cluster formation. In contrast, in 3-D collagen lattices, T cell-DC interactions usually lasting few minutes, lacked cluster formation (>72 h) despite efficient 3H thymidine uptake and prolonged survival of both, T cell and DC. In conclusion, while unspecific T cell-DC interactions were expected to be short-lived, we now propose that also specific T cell activation and proliferation may result from highly dynamic and rather short-lived T cellDC interactions interconnected with frequent periods of active T cell-migration.
Institute for Immunology, Medical Faculty, University of Dresden, Dresden, and Institute of Immunology, University of Munich, Munich, Germany
N. 15 M-DC8+ leukocytes - function and phenotype of novel human dendritic cell population isolated by a one-step immunomagnetic separation K. SCHAKEL, E. MAYER, C. FEDERLE, M. SCHMITZ, G. RIETHMULLER, and E. P. RIEBER Dendritic cells (DC) have a unique capacity to present primary antigens to T cells and play therefore a pivotal role in the induction of immune responses. This makes DC attractive for the development of immunmodulatory strategies. In particular, the use of DC has been proposed
29th Annual Meeting 1998 . 589 for therapeutic priming of cytolytic T cells against defined tumor peptides or viral antigens. Similarly, DC may serve as tools for reprogramming T cells in order to combat autoimmune or atopic diseases. Direct isolation of human DC for experimental purposes however, is hampered by their low frequency and by the lack of selective markers allowing large scale purification from blood. Here we describe the novel monoclonal antibody (mAb) M-DC8, which was generated by immunizing mice with fresWy prepared human mononuclear blood cells higWy enriched for dendritic cells. The mAb M-DC8 specifically reacted with 0,5-1 % of blood leukocytes showing the characteristic function and morphology of DC. Using a one step immunomagnetic procedure M-DC8+ cells could be directly isolated from fresh mononuclear cells. M-DC8+ cells were negative for T cell, B cell, NK cell and monocyte markers (CD2, CD19, CD56 and CD14, respectively), they expressed HLA class II molecules, CD33 and costimulatory molecules CD86 and CD40 at moderate levels, whereas expression of CD80 only appeared after in vitro culture. This pattern of surface antigens is shared with a population of blood cells recognized as immature DC. In addition, M-DC8+ cells characteristically express Fc(RIII (CD16) and efficiently ingested latex beads and antibody coated sheep red blood cells. M-DC8+ cells displayed an outstanding capacity to present antigen as shown in various in vitro assays. In particular, they were excellent stimulators of autologous mixed leukocyte reactions, they activated T cells against primary antigens such as keyhole limpet hemocyanin. Furthermore, they induced differentiation of purified allogeneic CD8+ T cells into alloantigen-specific cytotoxic effector cells and activated cytotoxic T cells against tumor peptides. The mAb M-DC8 might thus be a valuable tool to determine circulating DC for diagnostic purposes and to isolate these cells for in vitro and in vivo studies of antigen specific T cell priming.
IDepartment of Dermatology, University of Freiburg, Germany, 2Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, USA, 3Department of Dermatology, University of Munster, Germany
N. 16 Dendritic cell migration in vivo: Quantification, kinetics, organ specificity and activating capacity for T lymphocyte mediated immunity ].C. SIMONI,]. M. WEISS!, V. DELATIREt, B. MAlt, K. MANKE2, S. GRABBE3, and M. B. LAPPIN! Dendritic cells (DC) are likely to have an increasingly important role in vaccination therapy, therefore, this study sought to determine the migratory capacity and immunogenic function of murine bone-marrow (BM)-derived DC following intradermal (i.d.) and intravenous (i.v.) injection in vivo. DC were enriched from BM cultures using Metrizamide. Following centrifugation, the low-buoyant density cells, referred to throughout as DC, were CDllchigh, lab high, B7-1 high and B7_2 high and potently activated alloreactive T cells in MLR. In contrast, the high-density cells expressed low levels of the above markers, comprised mostly granulocytes based on GR1 expression, and were poor stimulator cells in MLR. Following i.d. injection of fluorescently labeled cells into syngeneic recipient mice, DC but not granulocytes migrated to the T-dependent areas of draining lymph nodes (LN). DC numbers in LN were quantified by flow-cytometric analysis, on days 1,2,3,5 and 7 following DC transfer. Peak numbers of around 90 DC per draining LN were found at the 48 hr time point. There was very little migration of DC to non-draining LN, thymus or spleen at any of the timepoints studied. In contrast, following i.v. injection, DC accumulated mainly in the spleen, liver and lungs of recipient mice but were largely absent from peripheral LN and thymus. The ability of DC to induce T cell-mediated immune responses was examined using Trinitrobenzenesulphate (TNBS)-derivatized DC (TNBS-DC) to transfer contact hypersensitivity responses (CHS) to naive syngeneic recipients. Following i.d. injection, as few as 10 5 TNBS-DC, but not TNBS-granulocytes, transferred CHS responses. However, the same number of TNBS-DC failed to transfer CHS following i.v. injec-
590 . 29th Annual Meeting 1998 tion. In summary, this study provides new and quantitative data on the organ specific migration of murine BM-derived DC following i.d. and i.v. injection. The demonstration that the route of DC administration determines the potency of CHS transfer, strongly suggests that the route of immunization should be considered in the design of vaccine protocols using DC.
IDepartment of Dermatology, University of Freiburg, 2Department of Biochemistry University of Muenster, 3Department of Tumoroncology, University of Homburg, Germany.
N. 17 Interaction with the extracellular matrix (ECM) component hyaluronan (HA) induces maturation of human dendritic cells (DC) c. C. TERMEER!, J. HENNIES I, J. M. WEISS!, U. VOITH!, R. SCHMITTS3, E. SCHOPF!, P. PREHM 2, and J. C. SIMONI The glycosaminoglycan HA is a major component of the cutaneous ECM. Physiologically, HA exists as a high molecular weight polymer (HMW-HA), but is cleaved into lower molecular weight fragments (sHA) at sites of inflammation. Since DC migrating into and through inflamed tissues will contact HA, we wished to investigate the effects of HMW-HA and sHA-fragments both derived from LPS-free HEALON®, on human DC, generated from peripheral blood progenitors by culture in GM-CSF and IL-4. FACS-analysis revealed that only sHA (2-20 glucurionic acid-molecules) but not HMW-HA induced phenotypic changes in DC similar to those seen after LPS-stimulation or culture in monocyte conditioned medium, i.e. an upregulation of HLA-DR, B7-1, B7-2, CD83, ICAM-l, CD14 and CD44 invariant forms and a down-regulation of CDla and CDllS. Likewise, sHA-fragments increased the production of the proinflammatory cytokines IL-l~, TNF-a and IL-12 by DC as determined by ELISA. These phenotypical changes were paralleled by functional data, since sHA increased dramatically the capacity of DC to stimulate the proliferation of resting alloreactive CD3+ T-cells. Furthermore, we found DC's to rapidly internalize sHA-fragments but not HMW-HA. Importantly, this internalization was not mediated by known cellular HA-receptors (HAR), since blocking studies with mAb's against the maior HA-receptor CD44, RHAMM or ICAM-l had no effect. In addition, DC's generated from the bone marrow of HAR knockout mice, showed similar activation upon sHAstimulation as DC from wildtype mice. Further DC, in contrast to fibroblasts, keratinocytes or melanoma cells, DC did not express significant HA-synthetase mRNA as shown by rtPCR and did not secrete HA as determined by RIA. Our findings indicate that HA-fragments encountered by DC at sites of inflammation contribute to DC activation and maturation, thus enhancing their capacity to induce T cell-mediated immune responses.
Department of IDermatology, Freiburg University,2lnstitute of Genetics, Forschungszentrum Karlsruhe, Germany
N. 18 CD44 variant isoforms playa functional role for dendritic cell T cell interactions J.M. WEISS I, c.c. TERMEER!, M. LAPPIN', J. SLEEMAN2, E. SCHOPF!, and J.c. SIMON! Recently we demonstrated that following antigen contact, epidermal Langerhans cells (LC) and dendritic cells (DC) upregulate pan CD44 epitopes and epitopes encoded by variant exons CD44v4 and CD44v6. Functionally, antibodies against CD44 epitopes arrested LC in the epidermis, prevented the binding of activated LC and DC to the T cell zones of lymph nodes (LN),
29th Annual Meeting 1998 . 591 and inhibited their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Since CD44 has recently been demonstrated to costimulate T cell proliferation by a CD28 independent mechanism we extended our studies to investigate the function of CD44 isoforms in DC interaction with T cells. In primary allogeneic MLR responses with bone marrow derived murine DC as stimulators we found that mAbs against pan CD44 epitopes, and epitopes encoded by CD44v4 and v6 significantly inhibited DC-induced T cell proliferation by 75, 70 and 25%, respectively. Preincubation of either DC or T cells with these anti CD44 mAbs revealed a selective and specific effect on DC but not on T cells. These findings suggest that pan CD44 epitopes and CD44v encoded isoforms contribute to the costimulatory activity of DC. In summary we show that the modulated expression of pan CD44 and CD44 variant exon encoded isoforms is not only functional in DC LN-adhesion and migration but also critical for their capacity to stimulate alloreactive T cells.
IMax-Delbriick-Centre of Molecular Medicine, MDC, Berlin, Germany, 2Bender & Co., 3Institute of Molecular Pathology, IMP, Vienna, Austria
N. 19 Gene delivery into antigen presenting dendritic cells by receptor mediated transfection S. S. DIEBOLD 1, E. WAGNER2, M. COTIEN3, and M. ZENKE 1 Dendritic cells (DC) are professional antigen presenting cells that represent a particularly attractive cell type for use in immunotherapy of diseases, such as cancer. In peripheral organs like skin DC get exposed to a variety of pathogens such as viruses and bacteria which they capture through specific cell surface receptors. The present study capitalizes on using such surface receptors for gene delivery into DC by receptor-mediated endocytosis. We demonstrate that polyethylenimine/DNA (PEl/DNA) complexes can be effectively transduced into primary human DC as adenovirus or mannose PEl/DNA transfection complexes (Ad/PEl/DNA and ManPEI/DNA, respectively). Ad/PEl/DNA complexes have plasmid DNA bound to the adenovirus particles by PEl and deliver DNA into cells via the adenovirus infection route. Such transfection complexes yiel'd high transduction levels and sustained expression of luciferase and green fluorescent protein (GFP) reporter genes and are almost as effective as recombinant adenovirus vectors. ManPEI/DNA complexes rely on uptake by receptor mediated endocytosis via mannose receptor that is highly expressed on DC. Additionally, incorporation of adenovirus particles in ManPEI/DNA transfection complexes further enhances transduction efficiencies and transgene expression. Different plasmid DNAs can be transduced simultaneously. We also demonstrate that Ad/PEI transfected DC are competent in stimulating T cell proliferation in allogeneic and autologous mixed lymphocyte reactions. Thus, Ad/PEl and ManPEI transfection represent particularly versatile transduction systems for DC, with ManPEI being exclusively built up of synthetic compounds.