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    • Immunological monitoring of immunotherapy trials Chris Schmidt, Nathan Martinez, Michelle Neller Queensland Institute of Medical Research, Brisbane Cancer immunotherapy trials conducted over the last few years have concentrated on the analysis of immunological markers of response to antigenically well defined vaccines. Improvements in the quantity or quality of T cell responses against the vaccine, in a patient population, are proposed to allow a rational basis for improved clinical outcomes. However, using current methodologies, only weak immune correlates of clinical response have been found. Immunotherapies using either allogeneic or autologous tumour cells as the source of antigens have been more clinically effective than defined antigen vaccines, but measuring anti-tumour immune responses elicited by them remains a challenge. A comprehensive examination of the anti-tumour (rather than anti-vaccine) immune responses of patients can be undertaken if autologous tumour cell lines are generated. This is feasible for ~70% of melanoma patients. Autologous tumour cell lines also allow the antigens targeted by patients’ T cells to be determined1. Knowledge of the hierarchy of melanoma epitopes targeted by an individual patient’s T cells provides the basis for a detailed characterisation of the effector and memory response. Our preliminary data support the prevailing notion that CD8+ Th1 effector T cells are a critical component of clinically effective anti-tumour immune responses. However, vaccinating for CD8+ T cell responses in humans is in its infancy. As we learn to induce CD8+ T cells, we need to know the desired goal: the optimal balance between terminally differentiated effectors and latent memory cells for the most effective clinical response must be determined. We propose that autologous tumour cell lines provide the key to a comprehensive analysis of anti-tumour immune responses. 1 Lennerz,V. et al. The response of autologous T cells to a human melanoma is dominated by mutated neoantigens. Proc. Natl. Acad. Sci. U. S. A. 102, 16013-16018 (2005). 1
    • The Danish experience of DC based immunotherapy of patients with disseminated breast cancer and renal cell carcinoma. – Results on clinical outcome, biomarkers and immune parameters. Inge Marie Svane MD Center for Cancer Immune Therapy (CCIT), Department of Hematology and Department of Oncology, University Hospital Herlev, Copenhagen, Denmark p53 targeted DC therapy has been tested in patients with verified progressive breast cancer. Almost 1/3 of the patients attained stable disease. This indication of an effect was supported by 1) a positive correlation between p53 expression of tumor and observed SD, 2) therapy induced p53 specific T cells in 4/7 patients with SD but only in 2/9 patients with PD, and 3) significant response associated changes in serum YKL-40 and IL-6 levels. We performed a comprehensive analysis of the effector stage of the p53 specific CD8+ T cells by the use of Dextramer Technology and multicolour FACS. Furthermore, we prospectively examined for more general treatment associated quantitative and qualitative changes in T cell subpopulations including Tregs. We found that the frequency of CD4+ CD25high regulatory T cells was almost doubled after only four weeks of weekly vaccination and low-dose IL-2. Patients with progressive cytokine-refractory metastatic RCC were vaccinated with DCs loaded with either a cocktail of survivin and telomerase peptides or tumour lysate depending on their HLA haplotype. Disease stabilization was observed in 13/27 patients and an antigen specific immune response was demonstrated in 5/6 patients tested. Significant response associated changes in IL-6 and YKL-40 were observed during treatment. The impact of administration of dendritic cell (DC) vaccination in combination with low-dose IL-2 on the frequency of CD4+CD25highFoxp3+ regulatory T cells were analysed. We found that the frequency of Treg cells in peripheral blood increased more than 7-fold at the 4th vaccine compared to pre-treatment and decreased again at the 6th vaccine but remained significantly higher than the pre-treatment levels. 2
    • DECAVAREC : An interleukin-12 secreting denditic cell-based cancer vaccine for treatment of metastatic renal carcinoma Thomas Felzmann, Alexander M. Dohnal TRIMED Biotech, Vienna, Austria DECAVAREC is an observer blind, multi-centre, randomised Phase II study to investigate the safety and efficacy for the treatment of metastatic renal carcinoma (mRCC) with Trivax, a dendritic cell-based interleukin-12 secreting autologous cancer vaccine. Main inclusion criterion is male and female patients between 18-80 years with written informed consent suffering from mRCC fter tumour nephrectomy or nephron- sparing surgery and on therapy with Sunitinib. Main exclusion criteria is no tumour resection or other conditiond requiring alternative cancer treatment such as chemotherapy, immune suppressive medication or cytokine therapy. Primary efficacy criterion is tumour progression scale according to the RECIST criteria; co-primary criteria are progression free survival and overall survival. Secondary efficacy criteria are performance status; DTH-skin test for in vivo assessment of anti-tumour immunity; and immunological in vitro assay to determine anti-tumour immunity. After stage I, a decision on whether to stop with continue with stage II will be made based on the primary efficacy criterion. For stage I a total of 70 patients is planned on an ad hoc basis randomised into treatment and control groups. Currently, nine urologic treatment centres in Austria and one in the Czech Republic have committed to participate in our study and negotiations are going on with several other CEE hospitals. All legal requirements are fulfilled including positive votes from the ethics committees and a formal by the Austrian drug agency in accordance with new EU and EMEA uidelines and directives. The first patients are currently accrued into the study. In anticipation of the considerably larger number of patients required for stage II we are still looking for additional partners who wish to participate in DECAVAREC. 3
    • Towards the next generation of dendritic cell vaccines. Carl G. Figdor Department of Tumor Immunology and Medical Oncology, Nijmegen Centre for Molecular Life Sciences (NCMLS), Postbox 9101, 6500 HB Nijmegen (c.figdor@ncmls.ru.nl), The Netherlands. We exploit dendritic cells (DCs) to vaccinate melanoma patients. We recently demonstrated a statistical significant correlation between favorable clinical outcome and the presence of vaccine-related tumor antigen specific T cells in delayed type hypersensitivity (DTH) skin biopsies. While we find immunological responses in 30-50% of the patients, favorable clinical outcome is only observed in a minority of the treated patients. Therefore, it is obvious that current DC-based protocols need to be improved to increase clinical efficacy. For this reason, we study in small proof of principle trials the fate, interactions and effectiveness of the injected DCs. We recently compared DC loaded with tumor antigen specific MHC class I binding peptides alone, in combination with MHC class II binding peptides or with defined tumor antigen mRNA (gp100 and tyrosinase). The results show that the presence of supplementary tumor antigen-specific MHC class II epitopes result in an T helper response that might be beneficial for the clinical outcome in these patients. Furthermore, comparing different routes of administration we observed that intranodal injection is not always successful (MRI) and that only a small proportion of the intradermally administered DCs reach the lymph nodes (scintigraphy). Our preliminary data clearly indicate that the cells that reach the lymph nodes are fully mature DCs that are able to induce an immune response in vivo. Currently we study whether pre-conditioning of the vaccination site results in enhanced migration. Updated results and strategies to improve DC vaccination will be presented. This work was supported by grants 1999-1950, 2000-2301, 2003-2893, 2003-2917 and 2004-3127 from the Dutch Cancer Society, and EU projects DC-THERA, and CANCER IMMUNOTHERAPY, the TIL-foundation and the NOTK. 4
    • Human Hemato-Lymphoid System Mice as Preclinical in vivo Models Markus G. Manz Institute for Research in Biomedicine, 6500 Bellinzona, Switzerland Because practical and ethical restrictions limit in vivo studies of the human hemato- lymphoid system, substitute human to small animal xeno-transplantation models have been employed. Previous models, however, sustained only limited development and maintenance of human lymphoid cells. We recently showed that intra-hepatic injection of CD34+ human cord blood cells into 2x2 Gy irradiated newborn Rag2-/-γc-/- mice leads to de novo development of B, T, and dendritic cells, formation of structured primary and secondary lymphoid organs, and production of some limited functional immune responses. Beyond, some human CD34+ hematopoietic progenitor cells were maintained in bone marrow of primary recipients and engrafted in secondary recipient animals, albeit with reduced constitution efficacy. Rag2-/-γc-/- Human-Hemato-Lymphoid-System mice were infected with human specific lymphotropic viruses, EBV and HIV. EBV infected animals produced some T cell responses, however, animals also developed EBV driven B cell proliferation, similar as observed in immunodeficient patients. Upon HIV-infection high plasma viremia was observed for up to 190 days, the longest time followed. A marked relative CD4 T cell depletion in peripheral blood occurred in CXCR4-tropic strain-infected mice compared to CCR5-tropic strain-infected animals. Only rare specific Ig responses were observed. Thus, this straightforward to generate and cost-effective in vivo model in many aspects resembles human adaptive immune system development and function, as well as human lymphotropic viral induced disease. However, human hematopoietic stem cell and engraftment maintenance is not sustained long-term, human myeloid cell development is minor, and human adaptive immune responses are limited and non-consistent. Therefore, as is, the model is not suitable for e.g. large-scale, pre-clinical predictive vaccine candidate testing. In order to reach human stem cell and engraftment maintenance for prolonged times, to enhance myeloid differentiation, and to improve adaptive immunity, we are currently replacing weak or non mouse to human cross-reactive cytokines as well as mouse with human MHC. 5
    • Brain tumour stem cells could be favourable targets for immunotherapy Tony Avril 1, Guillaume Gapihan1, Stephan Saikali2, Elodie Vauléon1, Abderrahmane Hamlat3, Sylma Diabira3 and Véronique Quillien1, 4. 1- CRLCC, Centre Eugene Marquis, Rennes (France); 2- Department of Pathology, CHU Pontchaillou, Rennes; 3- Department of Neurosurgery, CHU Pontchaillou, Rennes; 4- UMR 6061 – CNRS, University of Rennes1; Rennes. Despite a combination of surgery, radiotherapy and chemotherapy, glioblastoma multiforme (GBM) has a very poor prognosis due to the inevitable recurrence of the tumour within the central nervous system. Brain Tumour Stem Cells (BTSCs) are thought to play a key role in this resistance to conventional treatment, and are therefore regarded as interesting targets. In particular, immunotherapy could be an attractive strategy to kill BTSCs remaining after surgical treatment. Primary culture of GBM cells in a special media without serum allows the formation of neurospheres and hence provides an easy method of isolating and expanding BTSCs. In our laboratory, BTSCs are expanded in a serum-free medium previously described by Reynolds & Weiss (1992), in the presence of EGF and bFGF, after mechanical dissociation of GBM samples. The emergence of the first neurospheres is observed 4 to 21 days after starting the culture; neurosphere cultures are then passaged every 21 days after trypsin dissociation. Up to the present, 4 neurosphere cultures have been amplified from 21 GBM samples, using up to 5, 7, 7 and 10 passaging cycles for each respective culture. Phenotype analyses show that cells derived from all the neurosphere cultures strongly express nestin and A2B5 (markers of neural stem cells and progenitor cells), while 3 out of 4 express CD133 and only one of these with high expression. In terms of immunological characterisation, cells derived from all these neurosphere cultures express HLA class-I molecules. In addition, EGFRvIII, a tumour antigen previously described in GBM, is found on the cell surface in one case. These preliminary results show that BTSC are potentially interesting targets for immunotherapy. We are now performing proteomic profiling on these cells with a chip- based nano-LC-MS/MS technology in order to find new potential antigens. 6
    • Oxidation by hypochlorous acid breaks tumor tolerance and stimulates T cells which recognize autologous primary tumor. Cheryl L-L. Chiang1, Jonathan A. Ledermann2 ,Egla Aitkens3 , Elizabeth Benjamin4, David R. Katz1 and Benjamin M. Chain1,5 1 Division of Infection and Immunity, UCL, London UK 2 Department of Oncology, UCL, London UK 3 Department of Oncology, UCL Hospitals, London UK 4 Department of Histopathology. Rockefeller Building, UCL. UK Ovarian cancer commonly relapses after remission and activating tumour-specific T cells with dendritic cells (DCs) loaded with tumour cells is a promising approach to target residual microscopic tumours. Hypochlorous acid, a product of neutrophil myeloperoxidase, is a powerful enhancer of antigen processing and presentation. In this study we examine whether ovarian epithelial cells (SK-OV-3) exposed to hypochlorous acid can be used to break tolerance in ovarian cancer patients by stimulating T cell responses which recognize common tumor antigens as well as autologous tumor. SK- OV-3 cells expressing HER-2/neu and MUC1 ovarian antigens were killed by hypochlorous acid (HOCl) oxidation. Treatment with 60μM HOCl for 1 h induced necrosis in all the cells. Oxidised, but not live SK-OV-3 were engulfed by monocyte- derived DCs. Autologous T cells primed with DCs of HLA-A2+ healthy volunteers loaded with oxidised SK-OV-3 (HLA-A2-) recognised oxidised SK-OV-3 and HLA-A2- restricted epitopes of MUC1 and HER-2/neu. Responses were absent with heat- or hydrochloric acid (HCl)-killed SK-OV-3, thus HOCl oxidation and not cell death/necrosis enhanced the immunogenicity of SK-OV-3. Oxidised SK-OV-3 primed T cells did not respond to oxidised melanoma, or vice versa. In a second series of experiments, cells of ovarian cancer patients in remission, whose tumours overexpressed MUC1 and/or HER-2/neu, were tested using the same model. DCs of HLA-A2+ and A2- patients pulsed with oxidised SK-OV-3 stimulated T cells specific for oxidised SK-OV-3, and HER-2/neu and MUC1 epitopes. Oxidised SK-OV-3 loaded DCs further matured with CD40 agonistic antibody or monophosphoryl lipid A, primed stronger CD4+ and CD8+ responses than immature DCs. The T cells stimulated with DC and oxidised SK- OV-3 cells also recognised autologous tumour cells isolated from ascites, demonstrating that the SK-OV-3 cells shared significant antigenic reprtoire with primary tumour. In conclusion, immunization with mature DCs loaded with a generic oxidized tumor cell line breaks tumor tolerance and stimulates a polyclonal anti-tumor response which recognizes autologous tumor. These findings suggest a new immunotherapeutic strategy to extend remission in ovarian cancer. 7
    • Sequentially matured human DC with CD4+ T cells as secondary signals favour Th1, CTL and long term memory T cells responses Thomas Simon*, Séverine Tanguy-Royer*, Pierre-Joseph Royer, Nicolas Boisgerault, Jean-François Fonteneau and Marc Grégoire *both authors contributed equally Corresponding author: Dr. Marc Gregoire, INSERM U892, Institut de Biologie, 44093 Nantes Cedex 01, France. Phone: +33-2-40-08-41-50; Fax: +33-2-40-08-40-82. E-mail: marc.gregoire@nantes.inserm.fr In periphery, dendritic cells (DC) exposed to maturation stimuli migrate to lymph nodes where they generate T cell specific immune responses. In lymph node, CD4+ helper T cells are necessary to licence and condition DC, which then become empowered to initiate CD8+ cytotoxic T cell (CTL) responses. In this work, we studied DC phenotype and functions with regards to the kinetic of exposure to these peripheral and lymph node maturation stimuli. We developed an in vitro DC activation model mimicking this sequential exposure. We used as first signals TNF-α and polyI:C (inflammatory and pathogen signals) and as second signals CD40-L plus IFN-γ or activated CD4+ T cells. Our results show that the sequential maturation, above all with activated CD4+ T cells, increased dramatically DC maturation according to DC phenotype and cytokine secretion. Interestingly, Th1 polarization and CTL activation was widely favoured. Furthermore, this sequential maturation allows the generation T cell with a long-term memory phenotype. Thus sequential delivery of maturation stimuli should be considered in the future to improve DC vaccination efficiency. 8
    • The maturation cocktail for DC generation influences induction of MUC1 specific immune responses Joris Vanderlocht, Mariska B. Huls, Birgit L.M.G. Senden-Gijsbers, Wilfred. T.V. Germeraad and Gerard M.J. Bos, Department of Internal Medicine, Sub-Division of Haematology, University Hospital Maastricht, Maastricht, the Netherlands In a recent EU-funded project for the development of a Dendritic cell vaccine against Mucin1 (MUC1) positive tumours, DCs were supplied by IDM, Paris. These DCs were elutriated from a leukapheresis product that was differentiated for 7 days with IL-13 and GM-CSF. The resulting immature dendritic cells were further matured by the TLR2 and 4 ligand Ribomunyl and IFN-a RI-cocktail). However, the most often used DC currently evaluated in clinical trials uses IL-4, GM-CSF in combination with TNF- and PGE2 (TP-cocktail; which sometimes also includes IL-1n and IL-6). In the current project we compared the 2 maturation cocktails in greater detail starting with IDM’s immature DC. DC generated with the different maturation cocktails differ in their morphology and with the RI cocktail a more uniform upregulation of co-stimulatory molecules was obtained. TP-DC had a better migration capacity which can be explained by CCR7 and MMP-9 upregulation. The most striking difference between the generated DC involves the cytokine profile. DC generated with the RI-cocktail showed an enhanced production of more than 30 cytokines including IL-12, IL-6, IL-1l , TNF-d , IL-18, IL-23, but also IL-10. The DC generated with the DP cocktail produced only 4 factors in higher quantities. These differences could be linked to a difference in the capacity to polarize naïve T cells towards a Th1 profile. In co-culture experiments with naïve T cells, RI-DC induced a higher percentage of phenotypic markers indicating Th1 polarisation on CD4+ T cells. In addition RI-DC were superior in the induction of CTLs reactive for MUC1. However when analyzing the functionality of such sorted antigen-specific T cells, we did not see a difference in the intrinsic capacity to kill target tumour cells. To investigate whether the dendritic cells differ in their induction of suppressive T cells we are currently using unsorted CTLs. We conclude that RI-DC have the capacity to induce a broader immune response that is needed for cancer kill compared to the standard TP-cocktail, but it might be that these DC have to be applied intranodally or directly into the tumor to compensate for a possible minimal migration capacity (at least observed in vitro). 9
    • T-cell responses in stage IV melanoma patients vaccinated with dendritic cells electroporated with defined RNA Voland S, Schmitt-Haendle M, Haendle I, Erdmann M, Schliep S, Schaft N, Dörrie J, Kämpgen E, Schuler-Thurner B, Schuler G Department of Dermatology, Hartmannstrasse 14, 91052 Erlangen, Germany Objective: In this study we investigated the immunogenicity of a tumor vaccine consisting of autologous monocyte-derived dendritic cells (DC), which are electroporated with defined RNA encoding for the tumor associated antigens (TAA) MageA3, MelanA and Survivin, and in half of the patients in addition with a RNA encoding for E/L- Selectin which is designed to redirect the injected mature DC from blood to lymph nodes via high endothelial venules. Patients and study design: Stage IV melanoma patients with progressive disease after at least one systemic therapy are included in this study. DC are electroporated with the three TAA, and in half of the patients in addition with E/L-Selectin. Patients are apheresed at the beginning of the study and after 4 vaccinations to provide cells for DC generation and immunomonitoring. The vaccine was given at day 1, 14, 42 and day 70 as intravenous infusion. Immunomonitoring: T-cell responses were monitored on day 1 and on day 70 using freshly isolated PBMC and overlapping peptides covering the whole sequence of the three TAA. PBMC were stimulated with pools of peptides together with IL-2 and IL-7 for 7 days. Peptide specific responses were detected using an Interferon gamma elispot assay. Results: Up to now 8 patients reached the second leukapharesis after 4 vaccinations. In two patients a strong induction or expansion of T-cell responses could be detected. These patients had received a vaccine with ELS. Conclusion: In contrast to previous reports the intravenous route appears to be effective. It is possible that this is a unique property of the DC-RNA vaccine used here. A more detailed study of the immune responses and trafficking will show whether the introduction of the E/L-Selectin RNA induces as expected a more diverse homing pattern of T cells by redirecting the intravenously injected DC from blood to lymph nodes. 10
    • High immunogenic potential of p53, survivin and hTERT mRNA-electroporated dendritic cells in primary breast cancer Özcan Met1,2*, Eva Balslev3, Inge Marie Svane1,2; 1 Center for Cancer Immune Therapy (CCIT), Department of Hematology, 2Department of Oncology, and 3Department of Pathology, University Hospital Herlev, Copenhagen, Denmark Dendritic cells (DCs) loaded with tumor associated antigens (TAAs) are frequently employed in immunotherapy of cancer. Messenger RNA-based gene transfer is an attractive approach for antigen formulation and delivery into DCs and studies on the induction of anti-tumor immune response by DCs transfected with TAA-encoding mRNA has demonstrated the power and feasibility of this approach. Several TAAs have been identified in breast cancer, some of which appear to play a critical role in tumorigenesis. Universal TAAs such p53, survivin and hTERT are widely expressed in different tumor types and thus are of particular interest as targets in immunotherapy. In this study, we describe the frequencies of TAA-specific T cells in breast cancer patients after short-term in vitro stimulation with autologous DCs transfected with mRNA encoding p53, survivin or hTERT. ELISPOT analysis showed T-cell reactivity against TAA mRNA-transfected DC-targets in more than 40% of tested patients. The observed T-cell responses were highly associated with breast cancer as TAA-specific T-cell responses were only detected in one out of 10 healthy donors at the most. Furthermore, the T-cell response induced by p53 mRNA-transfected DCs was predominant in patients with high expression of p53 in tumor, indicating a correlation between p53 expression and p53-specific T-cell reactivity. Thus, p53, survivin and hTERT mRNA-transfected DCs are capable of inducing specific T-cell responses ex vivo in patients with primary breast cancer, thereby constituting a useful strategy for immunotherapy of breast cancer. 11
    • pST1 derived mRNA leads to more and longer protein expression after electroporation: implications of vector choice on activation of monocyte derived DC and antigen presentation. An M.T. Van Nuffel, Carlo Heirman, Kris Thielemans and Aude Bonehill Vrije Universiteit Brussel, Medical School, Laboratory of Molecular and Cellular Therapy, Brussels, Belgium E-mail: An.Van.Nuffel@vub.ac.be mRNA electroporation is a very efficient technique to modify human dendritic cells (DC) with the added advantage of being safe and easily applicable for clinical use. In this regard, the mRNA quality and stability are of primordial importance to yield reproducible results. Most commonly, the pGEM vector containing a T7 promotor and a 64 adenine poly-A tail is used for in vitro transcription of capped mRNA. Recently the pST1 plasmid, containing two human β-globin 3’ untranslated regions (UTR) and blunt ending on a 120 adenines poly-A tail, has been described to yield more stable mRNA with a higher translation efficiency, particularly after electroporation in immature DC. Therefore, the aim of this study was to compare mRNA obtained from these two vectors, encoding either a functional CD40L protein or the tumorspecific antigen MelanA. Electroporating DC with pGEM derived CD40L mRNA yields mature, CTL and Th1 activating DCs. However, the CD40L protein appears only transiently on the surface of DC. Therefore in particular, the mRNA stability and translation efficiency is of major importance. After electroporation of immature DC significantly higher and longer CD40L expression was observed with pST1 derived CD40L mRNA. This resulted in a slightly more mature phenotype and in substantially higher cytokine/chemokine secretion. When analyzing their ability to activate naive MelanA specific CD8+ T cells, we found that DC electroporated with pST1 derived CD40L mRNA and loaded with MelanA peptide induced more MelanA specific tetramer positive T cells, more TNF- α/IFN-γ secreting T cells and more cytolytic T cells. Furthermore, we observed that CD40L modified DC were able to induce maturation of their surrounding, CD40L negative DC. In a next set of experiments, we investigated the antigen presentation capacity of DC electroporated with pST1 derived MelanA encoding mRNA. Preliminary data suggest that in a coculture with specific TILs electroporation of pST1 derived MelanA mRNA in DC leads to a longer-lasting antigen presentation. In conclusion we show the pivotal role of the choice of plasmid used to generate mRNA. 12
    • Role of microRNA-155 in mature dendritic cells Isabelle Dunand-Sauthier1, Leonardo Capponi1, Antoine Geinoz1, Emmanuèle Barras1, Hans Acha-Orbea2 and Walter Reith1 1 Department of Pathology and Immunology, University of Geneva Medical School, Geneva, Switzerland 2 Department of Biochemistry, Faculty of Biology and Medicine, University of Lausanne, Epalinges, Switzerland MicroRNAs are evolutionarily-conserved small single-stranded non-coding RNAs that bind to the 3' untranslated regions (3'UTR) of target mRNAs and regulate their translation and/or degradation. They have been implicated in numerous biological processes, including cell proliferation, apoptosis, morphogenesis, differentiation, diseases and cancer. To study the potential role of microRNAs in dendritic cells (DC), we have used two different microarray platforms to characterize changes in the pattern of microRNA expression that occur during lipopolysaccharide (LPS)-induced maturation of human monocyte-derived DC (MO-DC). These experiments demonstrated that the expression of a specific microRNA (miR-155) was up-regulated strongly and consistently in LPS- treated MO-DC. By means of miR-155-specific qRT-PCR experiments, we next found that miR-155 expression was induced strongly (10-20 fold) and rapidly (within les than 1-2 hours) in MO-DC stimulated with LPS, as well as in response to various other maturation stimuli, including several different Toll-Like Receptor (TLR) ligands and pro- inflammatory cytokines. Furthermore, maturation-induced miR-155 expression was found to be conserved in mice, as it was also increased dramatically during the maturation of mouse splenic DC (up to 40 fold), mouse bone-marrow-derived DC (up to 5 fold) and a transformed mouse DC cell line (up to 150 fold). A strong increase in miR-155 expression is thus a general and evolutionarily-conserved feature of DC maturation. To identify target mRNAs regulated by miR-155, we have used a combination of computational approaches and expression analysis. Finally, the function of miR-155 in DC is being studied by examining the consequences of either expressing it ectopically or inhibiting its expression in the mouse DC cell line. Modulation of dendritic cell functions by miR-146a and miR-155 13
    • Bence Rethi, Katalin Kis-Tóth, Zoltán Veréb, Peter Gogolak, Enikő Sonkoly, Andor Pivarcsi and Éva Rajnavölgyi MicroRNAs have been implicated in the regulation of lymphocyte differentiation and antigen presenting cell functions. In macrophages, TLR pathways and inflammatory cytokines regulate the expression of several miRNAs, including miR-125b, miR-146a and miR-155. Target analysis suggested that these microRNAs may act as regulators of macrophage inflammatory responses, illustrated by miR146a, that is induced by LPS and acts as a negative regulator on the expression of the TLR4 signalling components IRAK1 and TRAF6, possibly decreasing sensitivity for further LPS-mediated activation. Individual microRNAs are characterized by hundreds of putative target sites on several different mRNA molecules, possibly reflecting multiple actions of the same microRNA molecules on cell functions. To assess the potential roles of miR-146a and miR-155 in human dendritic cells (DCs) we analyzed the expression pattern of these microRNAs during in vitro differentiation and activation of DCs and studied whether experimental manipulation of miR-146a and miR-155 levels modulate DC functions. We found that miR-146a is upregulated during monocyte-derived DC differentiation and the expression pattern of both miR-146a and miR-155 was influenced by the type of DC activation stimuli. Migratory response of DCs to MIP-3β was compromised of miR-146a transfected DCs and the production of TNF, IL-12, IL-6 and IL-10 cytokines by activated DCs was downmodulated by both miR-146a and miR-155. These results indicate a potential negative regulation of DC effector functions by miR146a and miR155, however their roles at physiological expression levels and the mechanisms of their actions are yet to be further clarified. Role of cytokine environment and cytokine receptor expression in human monocytes in the generation of functionally distinct dendritic cells 14
    • Lucia Conti, Marco Cardone, Barbara Varano, Patrizia Puddu, Filippo Belardelli and Sandra Gessani Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy. Myeloid dendritic cells (DCs) and macrophages evolve from a common precursor. However, factors controlling monocyte differentiation toward DC or macrophage are poorly defined. We report that surface density of GM-CSF receptor (GM-CSFR) α subunit in human peripheral blood monocytes varies among donors. Although no correlation was found between the extent of GM-CSFR and monocyte differentiation into DC driven by GM-CSF and IL-4, GM-CSFR expression strongly influenced the generation of CD1a+ dendritic-like cells in the absence of IL-4. CD1a+ cells generated in the presence of GM-CSF express CD40, CD80, MHC I and II, DC-SIGN, MR, CCR5, and partially retain CD14 expression. Interestingly, they spontaneously induce the expansion of CD4+ and CD8+ allogeneic T lymphocytes producing IFN-γ, and migrate toward CCL4 and CCL19. Upon stimulation with TLR ligands, they acquire the phenotypic features of mature DCs. In contrast, the allostimulatory capacity is not further increased upon LPS activation. However, by blocking LPS-induced IL-10, a higher T cell proliferative response and IL-12 production was observed. Interestingly, IL-23 secretion was not affected by endogenous IL-10. These results highlight the importance of GM- CSFR expression in monocytes for cytokine-induced DC generation and point to GM- CSF as a direct player in the generation of functionally distinct DCs. 15
    • Divergent Effects of Hypoxia on Dendritic Cell Functions Alessandra Mancino1, Tiziana Schioppa3, Paola Larghi7, Fabio Pasqualini7, Manuela Nebuloni4, I-Hsuan Chen6 , Silvano Sozzani5, Jonathan M. Austyn6, Alberto Mantovani1,2, Antonio Sica7 1 Istituto Clinico Humanitas, IRCCS, 20089 Rozzano, Milan, Italy; 2University of Milan, 20133 Milan, Italy; 3Cancer Research UK, Translational Oncology, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ; 4Institute of Pathology, Department of Clinical Sciences L. Sacco, University of Milan, 20157 Milan, Italy; 5Department of Biomedical Sciences and Biotechnology, Section of General Pathology and Immunology University of Brescia, Italy; 6Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, UK; 7Fondazione Humanitas per la Ricerca, 20089 Rozzano, Milan, Italy. Dendritic cells (DC) are professional antigen-presenting cells (APC) patrolling tissues to sense danger signals and to activate specific immune responses. In addition they play a role in inflammation and tissue repair. Here we show that oxygen availability is necessary to promote full monocyte-derived DC differentiation and maturation. Low oxygen tension (hypoxia) inhibits expression of several differentiation and maturation markers (CD1a, CD40, CD80, CD83, CD86 and MHC class II molecules) in response to lipopolysaccharide (LPS), as well as their stimulatory capacity of T cell functions. These events were paralleled by impaired upregulation of the chemokine receptor CCR7, a necessary event for the homing of mature DC to lymph nodes. In contrast, hypoxia strongly up-regulates production of pro-inflammatory cytokines, TNFα and IL-1β in particular, and the inflammatory chemokine receptor CCR5. Subcutaneous injection of hypoxic DC in the footpad of mice resulted in enhanced leukocyte recruitment at the site of injection and defective DC homing to draining lymph nodes. Thus hypoxia uncouples the promotion of inflammatory and tissue repair from sentinel functions in DC, a safeguard mechanism against self-reactivity of damaged tissues. 16
    • Low doses of denileukin difitox (ONTAK® ) induce survival rather than depletion of CD4+CD25+ regulatory T cells in vitro Gabi Theiner, A. Baur, MB. Lutz, G. Schuler Denileukin diftitox (ONTAK®, DAB(389)IL-2) is a cytotoxic fusion protein, composed of interleukin 2 (IL-2) and two diphtheria toxin fragments, designed to specifically delete cells overexpressing the IL-2 receptor (IL-2R). Ex vivo studies have shown that ONTAK® interacts with the high-affinity IL-2R and undergoes internalization. Subsequent cleavage and release of the diphtheria toxin into the cytosol induces rapid cell death. Originally, the drug has been approved for treatment of cutaneous T-cell lymphoma. Recently tumor patients receiving antigen-specific therapeutic tumor vaccination were pretreated with ONTAK® aiming to deplete CD4+CD25+ regulatory T cells (Treg), which are thought to suppress anti-tumor immunity. With respect to Treg depletion the reported effects of ONTAK® in vivo were controversial. Thus we wished to carefully evaluate the impact of this drug on resting and activated conventional T cells (Tcon) and Treg in vitro. Indeed ONTAK® killed only Tcon that had been preactivated. ONTAK ® clearly and dose-dependently induced apoptosis in Treg that where activated simultaneously, even in low doses, while resting and preactivated Treg showed no apoptosis. Surprisingly, low doses of ONTAK® improved survival of resting and preactivated Treg. Possibly, ONTAK® was not internalized and remained on the cell surface delivering an IL-2R signal. In fact, we found a strong increase of STAT-5 phosphorylation upon ONTAK ® treatment indicating intracellular IL-2R signalling. Additionally this effect correlated with Caspase-3 activation, but seemed Bcl-2 independent. Our findings indicate that low concentrations of ONTAK® induce the survival of resting Treg rather than cause their depletion possibly by delivering an IL-2 signal. Our data may explain the discrepant in vivo effects regarding depletion of Treg in patients by ONTAK® which has to be taken into account for the design of future clinical trials. Antigen cross presentation by dendritic cells: control of phagosome maturation and acidification 17
    • Sebastian Amigorena INSERM U365, ImmunitŽ et Cancer, Pavillon Pasteur, Institut Curie, 26 rue d’Ulm, F-75245 PARIS Cedex 05. Dendritic cells initiate most CD8+ T cell immune responses. To do so, dendritic cells take up antigens in the periphery and generate proteolytic peptides which are on MHC class I molecules. These peptide-MHC complexes are exposed on the surface of dendritic cells, where they engage cognate T cell receptors upon establishment of tight physical contacts between the dendritic cells and the T lymphocytes. In the last few years, we have analyzed the intracellular event that lead to MHC class I loading with peptides from phagocytosed antigens. We showed that dendritic cells have developed number of specialization of their phagocytic pathway that allow them to be the most efficient antigen cross presenting cells. These specializations include the ability of DCs to export internalized proteins to the cytosol, the capacity of dendritic cell phagosomes to recruit ER resident proteins and a remarkable lack of acidification that protects phagocytosed antigens from complete degradation. More recently, we have analyzed the mechanisms of control of the phagosomal pH in dendritic cells. The NADPH oxidase NOX2 is recruited to the DC’s early phagosomes and mediates sustained production of low levels of reactive oxygen species, causing active and maintained alkalinization of the phagosomal lumen. DCs lacking NOX2 show enhanced phagosome acidification and increased antigen degradation, resulting in impaired cross presentation. Therefore, NOX2 plays a critical role in conferring DCs the ability to function as specialized phagocytes adapted to process antigen rather than just kill the pathogen. we show that Rab27a, a small GTPase present on lysosome-related organelles, controls the recruitment of the NADPH oxidase NOX2 to phagosomes. In Rab27a-defective dendritic cells, phagosome acidification is increased and cross presentation is less efficient than in wild type cells. Although a particular subpopulation of DCs (that bear the marker CD8 ) cross present phagocytosed antigens more efficiently than other DC subtypes, the intracellular mechanisms responsible for this specialization are unknown. We now show that CD8+ DCs display a more alkaline phagosomal pH and lower degradation rates of internalized proteins than other spleen DCs. Limited acidification in CD8+ DCs is due to the efficient recruitment of the NADPH oxidase NOX2 to phagosomes in this particular DCsubpopulation. In CD8- DCs, in spite of high total superoxide production, NOX2 assembles at the plasma membrane, and not on phagosomes. In Rac2-defective CD8+ DCs, NOX2 was redistributed from phagosomes to the plasma membrane, resulting in a pattern of sub cellular distribution very similar to that of CD8- DCs. We conclude that Rac2-dependent phagosomal NOX2 assembly in CD8+, but not in CD8- DCs, limits acidification and degradation, thus promoting antigen cross presentation. Immunotherapy of established lesions caused by high risk HPV 16 18
    • CJM Melief1. MJP Welters1, MJG Löwik2, APG Vloon1, JW Drijfhout1, ARPM Valentijn3, AR Wafelman3, GJ Fleuren4, R Offringa1, SH van der Burg5 and GG Kenter2 Depts. of 1Immunohematology and Blood Transfusion, 2Gynaecology, 3Pharmacy, 4 Pathology and 5Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands. A therapeutic vaccine was designed based on long overlapping peptides covering the complete amino acid sequence of the HPV16 E6 and E7 oncogenic proteins, thereby harboring all potential T helper and CTL epitopes. Previously, we demonstrated that HPV16 specific T-cell immunity induced by this vaccine,delivered in Montanide ISA 51 adjuvant was able to terminate persistent infections and eradicate established HPV16+ tumors inrabbits. Currently, 20 patients with histologically proven HPV16+ vulvar intraepithelial neoplasia (VIN) grade III were vaccinated 4 times with a 3-week interval by s.c. injection of the long peptides emulsified in Montanide ISA 51. Immunological monitoring was performed at the systemic level by the analysis of blood samples, drawn before each vaccination and after the last vaccination, and at the local level by the analysis of HPV16-specific T-cells in tissue biopsies of the VIN lesion (before and after vaccination) as well as a biopsy from the last vaccination site. In all 20 patients, already after 2 vaccinations strong and broad vaccine-induced systemic proliferative responses, accompanied with the production of IFNg and IL-5 were detected. This type of response is similar to the memory T-cell responses observed in healthy individuals with HPV16-specific immunity. Importantly, circulating HPV16 E6 and E7 specific T-cells produced IFNg upon stimulation with naturally processed and presented antigen. Notably, vaccination resulted in the induction of both CD4+ and CD8+ HPV16-specific T-cells. Multiple epitopes were recognized in each patient. Analysis of the local immune response demonstrated the presence of HPV16-specific Th1/Th2 cells infiltrating both the vaccination site and the VIN lesion after vaccination in 6 out of 9 patients analyzed.A complete clinical response was seen in 5 out of 20 patients, as determined by complete clearance of lesions by macroscopy and microscopy. In 4 of these patients HPV 16 was also cleared as determined by PCR.The majority of the remaining 15 patients experienced a partial remission of lesions and substantial improvement of disease-associated complaints. In conclusion, our peptide-based vaccine elicits a strong and broad HPV16-specific T-cell response that displays the capacity to migrate into the persistently HPV16-infected lesion of patients with high grade VIN and causes complete regressions in a substantial proportion of patients. 19
    • DC cancer vaccines: development of new strategies for antigen loading Katrin Birkholz1, Jan Dörrie1, Niels Schaft1, Michael Schwenkert2, Christian Kellner2, Georg Fey2, Gerold Schuler1 1) Department of Dermatology, University Hospital Erlangen, Erlangen, Germany 2) Chair of Genetics, University of Erlangen-Nuremberg, Erlangen, Germany Although DC-based vaccination has shown encouraging responses in melanoma and renal cell carcinoma, further improvement of the efficiency of these vaccines is needed. To find the most efficient DC antigen-loading technique, different methods, such as direct peptide loading, electroporation of tumor-antigen RNA, or the use of antibody- antigen constructs need to be thoroughly analyzed and compared. Therefore, the cancer- testis antigen MAGE-A3 was chosen as a tumor model. As a functional read-out system for the efficiency of antigen-presentation, we transfected CD4+ T cells with RNA encoding TCR recognizing MAGE-A3 epitopes. After co-incubation with the DC, cytokine release was quantified. As one possible loading strategy, we electroporated the DC with MAGE-A3-DCLAMP RNA. The DCLAMP sequence targets the antigen to lysosomes, which leads to MHC class II presentation. Indeed, electroporation of mature (m)DC with MAGE-A3-DCLAMP RNA resulted in HLA-DP4 class II-restricted presentation of the MAGE-A3 peptide KKLLTQHFVQENYLEY (KKL, aa 243-258) and induction of cytokine release (i.e. INFγ and IL-2) by autologous MAGE-A3/DP4-specific CD4+ T cells. For a different loading strategy, we targeted DEC-205, an endocytosis receptor expressed on the surface of DC. We designed antibody-antigen constructs, consisting of a single-chain variable fragment (scFv) directed against DEC-205, genetically linked to different parts of the MAGE-A3 antigen (i.e. the KKL, and EVDPIGHLY (aa 168-176) peptides). The fusion proteins were expressed in 293T cells and displayed binding to immature (i)DC and mDC. We could show in preliminary experiments that loading of DC with the anti-DEC-205scFv-MAGE-A3-KKL construct led to antigen presentation, whereas incubation of DC with the heat-inactivated construct or a control construct did not. Taken together, we have established tools to thoroughly compare different antigen-loading strategies of DC, which hopefully will contribute to the future generation of better DC-based anti-cancer vaccines. 20
    • Many antigen presenting cells, or much antigen per presenting cell; studies on priming and expansion of melanA-specific T cells N. Schaft*, J. Dörrie*, Verena Wellner, Chris Wohn, Tanja Schunder, Ina Müller, G. Schuler *These authors contributed equally RNA-group, Dept. Dermatology, University Hospital Erlangen, Erlangen, Germany RNA transfection is an increasingly important method to load dendritic cells (DC) with antigen for therapeutic tumor vaccination. The best established method is electroporation, leading to a satisfying and homogenous expression of the introduced antigen. Lipofection with Transmessenger, a reagent designed for RNA transfection, represents an alternative method. We compared both methods with respect to transfection efficiency and found that Transmessenger resulted in very heterogenous expression: only <20% of the DC expressed the introduced antigen, however some of them in up to 20 fold higher amounts compared to electroporated DC. Next, we addressed how this difference in antigen amount would contribute to the quantity and quality of antigen-specific CD8+ T cells that were primed and expanded with these DC in in vitro stimulation assays. We examined the percentage of MHC tetramer-binding T cells, their phenotype, and their functional avidity using the model-antigen melanA. We found that few DC expressing large amounts of the antigen expanded specific T cells substantially better than 10 times more DC expressing intermediate amounts of the antigen. The stronger the T-cell expansion was, the higher was the number of lytic effectors. To examine functional avidity of the T cells, we established an ELISPOT-based protocol: equal numbers of MHC tetramer- binding T cells were used on target cells that had been loaded with titrated concentration of the corresponding melanA-derived peptide. We observed no substantial difference in avidity between T cells that had been stimulated with the differently loaded DC. These data indicate that higher amounts of antigen on the DC are advantageous for efficient T cell stimulation. However, the resulting T cells need to be further analysed, especially in restimulation experiments. 21
    • Querying a DC "omics" database according to a pathway based logic. Duccio Cavalieri Department of Pharmacology, University of Florence. duccio.cavalieri@unifi.it-www.ducciocavalieri.org In this talk I will present a vision on the use of pathway based approaches to query databases of immunologically related data with focus on dendritic cells. The exposure of the dendritic cell to different stimuli leads to changes in gene expression depending on the extent at which the interacting molecules or organisms react with TLR or lectin receptors. Regulation of gene expression and protein activity is central to the function of molecular and cellular systems. High throughput methods provide various partial views of the participating genes, RNAs and proteins, and their interactions. Genes never act alone in a biological system, but participate in a cascade of networks. Pathways represent the biologist’s way of describing the nature of biological interactions and control networks. Integration of “omics” data in the context of the biological and cellular pathways is particularly relevant for the detection of subtle but co-ordinated changes (Cavalieri D and De Filippo C., Bioinformatic Methods for Integrating Whole-Genome Expression Results into Cellular Networks. may 2005 Drug Discovery Today). Our contribution to the DC community within and outside the DC-Thera network has recently led to the development of a method called EuGene (Cavalieri D, Castagnini C, Toti S, Maciag K, Kelder T, Gambineri L, Angioli S, Dolara P. Eu.Gene Analyzer a tool for integrating gene expression data with pathway databases. Bioinformatics. 2007 Jun 28; Bioinformatics 2007 Free download at http//www.ducciocavalieri.org/) that implements both statistical algorithms to determine which pathways are most affected by transcriptional changes and connection to visualization tool to map expression data from multiple whole-genome expression experiments on metabolic pathways. Our research aims at establishing the methodological bases and developing the computational infrastructures to investigate the networks of molecular interactions characterizing specific DC differentiation programs. The methodological aim of the project is to develop a repository of pathways and genomics datasets regarding DC’s and algorithms for the integrative analysis of high-throughput molecular data in the light of the structural and functional organization of the genome. These computational methods will contribute fulfilling gaps in the bioinformatics analysis of genomic data where the integration of different types of information still represent a major, and partially unresolved, computational issue. The analytical results will support the reconstruction of regulatory networks and the molecular signatures of DC maturation. Strategically, the project has the objective to create an interdisciplinary research group integrating biological, bioinformatics and statistical expertise which will guarantee the development of a systemic approach to decipher the complex biological system behind DC maturation and the immune response. 22
    • Pathway Based analysis allows comparison between different datasets and microarray platforms Luca Beltrame Department of Pharmacology University of Florence-Center of Excellence Cisi Background-The wide use of whole genome microarray based techniques for the investigation of the cell transcriptome has stirred the application of various statistical approaches for classifying samples and genes. However, comparisons made on the level of gene lists obtained by different statistical methods or from different datasets hardly converge. Such discrepancies are often reduced when analyzing apparently affected biologically related groups of genes, e.g. metabolic or signalling pathways using group testing procedures, e.g. over-representation analysis, functional class scoring (FCS), or global tests. Results-We have used EuGene, a new pathway based analysis method, to run GSEA and Fisher exact test analysis on datasets from different laboratories and using different platforms for Renal Carcinoma. The datasets used different patients, different microarray platforms, different statistical analyses to generate prognostic markers. The overlap of the gene lists of genes classified as differentially expressed in the datasets was less than 50%. We then analyzed the experiments at the pathway level. We used EuGene to generate E- scores from GSEA. We then used a descriptive cluster analysis approach to identify groups of pathways with similar expression levels, highlighting the cellular modules associated to the tumor development. Discussion-Cluster analysis with the appropriate distance measure allowed to reconstruct the cellular modules involved in the cancer process and identified common pathways associated with the pathology. Group analysis allowed to compare datasets irrespective of the platform used and of the author, revealing a fundamental approach to compare publicly available microarray datasets. 23
    • Proteomic analysis of human dendritic cells Sonja Buschow et al. A comprehensive protein inventory of clinical grade immature and cytokine cocktail matured (Il-6, IL-1β, TNFα, PGE2; 48 hours) monocyte derived human dendritic cells (DC) from a healthy donor has been established by using high accuracy, high sensitivity protein identification technology. We have identified 2794 proteins in DCs by liquid chromatography tandem mass spectrometry, comprising of 2623 proteins detected in immature DCs, 1896 proteins in mature DCs, and 1725 proteins in both cell types. Differences were quantified by the exponentially modified Protein Abundance Index (emPAI) method, a label free spectral counting procedure for determining relative protein amounts of LC-MS/MS data. Protein expression profiles were compared with gene expression profiles generated by microarray technology. In total, we could map 96 percent of the detected proteins to expressed genes for the immature DCs and 90 percent for the mature DCs. Therefore, we conclude that our data contains sufficient quantitative information to define subsets of up- and down-regulated proteins for functional analysis. We find 202 proteins upregulated by more than 2 fold in mature DCs and 463 proteins upregulated in immature DCs. Functional annotation by gene ontology classification and pathway analysis by several bioinformatics approaches is currently ongoing. Preliminary results obtained from querying DAVID (http://david.abcc.ncifcrf.gov/) and Ontologizer (http://www.charite.de/ ch/medgen/ontologizer/) show statistically significant enrichment for immunological- relevant signaling pathways and immunology-related GO terms of the upregulated proteins in mature DCs. 24
    • Translational profiling of activated monocyte-derived DCs Maurizio Ceppi, Giovanna Clavarino and Philippe Pierre* Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Méditerranée. Campus de Luminy, Case 906 13288 Marseille, Cedex 09, France As a first step, the rate of protein synthesis in Lipopolysaccharide (LPS)-stimulated human DCs has been quantified with the help of puromycin incorporation experiment (Schmidt etal, submited). An important increase in protein synthesis was observed as soon as 1h after LPS-stimulation. Following this activation, a continous decrease of protein synthesis was observed from 8h to 24h of maturation, reaching a level inferior to immature DC translation activity. We have developped a protocol for the isolation of polysomal-bound mRNA molecules out of human DCs at various stages of maturation. To obtain a global view of translationally engaged mRNA molecules, total and polysomal-bound mRNA populations have been compared in immature (0h) and activated DCs (4h and 16h post-LPS stimulation) with the help of Affymetrix Microarrays. A preliminary analysis indicates that 10 to 20% of the genes are translationally regulated in DCs at 16h post-LPS, whereas 80 to 90% of the genes are only regulated at the transcriptional level. No major difference could be observed at 4h post-LPS, indicating that mRNA translation regulation takes place in a “late stage” of DC-activation. Four mRNA isolation experiments, each including six Affymetrix microarrays analysis, have been successfully performed. Thus, translation-related gene expression data out of four different blood donors are currently available and we are now validating the molecules, which are translationally regulated during maturation 25
    • Different forms of Saccharomyces cerevisiae elicit specific responses in dendritic cells Lisa Rizzetto1, Giorgio Napolitani2, Mirela Kuka3, Ugo D'Oro3, Duccio Cavalieri1 1 Dept. of Preclinic and Clinic Farmacology, University of Florence, Italy 2 Institute for Research in Biomedicine, Bellinzona, Switzerland 3 Novartis Vaccines, Siena, Italy How our immune system discriminates between pathogenic and non pathogenic microorganisms is still an open question. We propose to use different vegetative and non vegetative forms of the non pathogenic Saccharomyces cerevisae yeast to investigate the rules of the recognition game. Phagocytosis-mediated antigen presentation together with TLR-dependent gene expression of inflammatory cytokines and co-stimulatory molecules, instruct development of antigen-specific acquired immunity. Monocyte-derived dendritic cell (MoDC) recognize S. cerevisiae yeast trough different TLRs such as TLR4 and TLR2. TLR-2 is essential in the recognition of fungal-derived components in collaboration with dectin-1, a lectin family receptor for the fungal cell wall component ß-glucan. After recognition of cell wall components, MoDC phagocytise yeast cells and nucleic acids, in particular ssRNA, are recognized by intracellular TLRs such as TLR7, TLR8 and TLR9 eliciting an additional robust pro-inflammatory response with cytokine production. In order to deeper insight the mechanisms of DC-S. cerevisiae interaction, we evaluated the role of RNA and different cell wall in different conditions. The different composition of cell wall in different growth conditions may influence the immune recognition of this yeast. We evaluated the ability of vegetative and senescent cells of S. cerevisiae to induce MoDC cytokine responses. By measuring cytokine production we observed that cells in different senescence states elicit peculiar immunological response. To reconstruct the signalling networks responsible for differences in the response we have performed transcriptional analysis of hMoDC and used Eu.Gene (Cavalieri et. al, 2007) to reconstruct pathways and regulatory programs. 26
    • Global transcriptomic analysis identifies defects in key functional clusters of genes in dendritic cells derived from leukaemic MUTZ-3 precursors. Jane Rasaiyaah, Paul Kellam and Benjamin Chain Division of Infection and Immunity, UCL, London Dendritic cells (DC) derived from acute myeloid leukaemia (AML) cells have received considerable interest for their potential to stimulate immunotherapeutic responses. MUTZ-3, an immortalized AML derived cell line, has provided a useful model for this approach. In this study we use a global transcriptomic approach to compare MUTZ-3 derived DC (M3DC) with conventional monocyte derived DC (MDDC). An immunoselected starting population of CD14+ cells were used to generate a homogeneous non-dividing population of M3DC with phenotypic characteristics of classic MDDC. Whole genome transcriptome analysis of M3DC and MDDC, coupled with functional ontology based data analysis, revealed three clusters of significantly down-regulated genes, with immunologically related functions in pathogen recognition, DC maturation and cytokine/chemokine signaling. Further analysis of a proportion of these genes confirmed down-regulation of protein expression. The molecular differences in protein and message expression were consistent with a profoundly impaired response of M3DC to microbial stimulation. This defect in response to pathogen associated molecular patterns (PAMPS) could be restored in part by priming M3DC with IFNγ. The leukaemic phenotype therefore reflects not only deregulated proliferative capacity, but altered patterns of gene expression leading to substantial perturbation of normal antigen presenting cell function. 27
    • A module network approach to the unraveling of the molecular pathways underlying the physiological diversity of the in vivo malaria parasite Plasmodium falciparum N. Pochet, J. P. Daily, D. Scanfeld, K. Le Roch, D. Plouffe, M. Kamal, O. Sarr, S. Mboup, O. Ndir, D. Wypij, K. Levasseur, E. Thomas, P. Tamayo, C. Dong, Y. Zhou, E. S. Lander, D. Ndiaye, D. Wirth, E. A. Winzeler, J. P. Mesirov, A. Regev In malaria little is understood about the specific mechanisms underlying disease pathogenesis, since neither in vitro cultures nor animal models accurately reflect the human-pathogen interaction. Indeed, despite the extensive transcriptional profiling of different strains of parasites in vitro, we obtained little novel insight into the in vivo biology and pathogenesis. In our recent work, we have examined for the first time the transcriptional profiles of Plasmodium parasites isolated directly from the blood of 43 patients with mild disease in Senegal. We clustered the samples’ expression profiles, using a Non-Negative Matrix Factorization (NMF) algorithm and discovered that expression profiles cluster into three distinct groups. Cluster 2 samples were similar to early ring stage profiles of the parasite (3D7 strain) grown in vitro. However, additionally we found two novel genome-wide transcriptional states that have not been previously observed in vitro. These new transcriptional states were not related to parasite life cycle stage, data of collection/hybridization or parasite genotype and hence represent parasite biology not previously identified. With a novel comparative genomics approach, we characterized Cluster 2 state as a normal fermentative (glycolytic) growth, Cluster 1 state as a putative starvation response, accompanied by oxidative phosphorylation, and Cluster 3 state as a stress response, associated with elevated inflammation in the patients. The two novel states (particularly the ‘stress response’ state) are associated with patient characteristics suggestive of more severe disease. Host data associated with these novel states suggest that differences in parasite biology may contribute to disease outcomes. This is important as it suggests a novel pathogenesis model and may provide previously unidentified parasite biology that could be targeted. In addition, the identification of severe disease signatures would provide prognostic information and finally, parasite response to drugs when it exists in alternative states would provide insight into drug effect. Reproducing these novel states may require testing multiple in vitro conditions. In the future, we intend to build on these results: (i) to replicate and elaborate on the in vivo findings in a distinct clinical cohort and (ii) to establish a signature-based readout for high-throughput screening of Plasmodium steady state RNA to develop an in vitro system that recapitulates these novel physiological states. These would allow us to establish an in vitro screening approach which is faithful to the in vivo physiological states of the parasite, towards more effective approaches to understanding pathogenesis, drug discovery and treatment in malaria. 28
    • Comparison of the transcriptomes of CD34-derived and human skin Langerhans cells Henri de la Salle(1), Sylvie Tourne(1), Doulaye Dembelé (2), Huguette Bausinger (1), Dominique Fricker (1), Susanne Koch (3), Sylvia Schnautz (3), Philippe Kastner (2), Daniel Hanau (1), Thomas Bieber (3) (1) EFS-Alsace, Strasbourg, France; (2) IGBMC Strasbourg, France; (3) University of Bonn, Germany The characterization of human skin Langerhans cells is difficult due to limiting numbers of cells which are available in each experiment. This limit can be bypassed by the use of Langerhans cells differentiated from CD34+ precursors. Nevertheless, how these in vitro differentiated cells represent an accurate model of skin cells is not known. To investigate this question we have initiated a program of comparison of the transcriptomes of CD34+ progenitors-derived and skin Langerhans cells. CD34+ cord blood cells were differentiated into Langerhans cells and immunopurified using anti-CD207 antibodies, giving 95% of Langerin positive cells. Human skin Langerhans cells were also immunopurified using anti-CD1a antibodies from epidermal sheet of plastic surgery skin samples, giving 90-95% Langerin-positive cell populations. Keratinocytes were also saved. The transcriptomes of the three cell types were compared to those of monocytes- derived dendritic cells and peripheral blood dendritic cells available in databanks and analyzed by a clustering algorithm, which generates sets of genes preferentially expressed in different cell types. By this way, genes preferentially expressed in only one or both types of Langerhans cells were identified. These genes belong to the different categories of receptors, transporters, signal transducer, nuclear factors, cellular transport, secreted proteins including extracellular matrix proteins and, of course, un-characterized genes. This analysis provides markers that might be followed in order to optimize the differentiation of precursors into closer models of skin Langerhans cells, revealed genes whose characterization makes sense in order to better understand the function of Langerhans cells and, points to the necessity to develop the analysis of the pathways particular Langerhans cells. 29
    • Epithelial cells as modulator of intestinal immuno-homeostasis. G. Matteoli1, Ilian Iliev1, E. Mazzini1, E. Mileti1 and M. Rescigno1 1 IFOM-IEO CAMPUS, Via Adamello 16, 20139, Milan, Italy The principal functions of the mucosal epithelial barrier are to digest and absorb food nutrients, as well as to protect the body from dangerous microorganisms. Our body is not fully equipped to transform all ingested food into its "recyclable" constituents, and we need the help of beneficial microorganisms for the digestion of complex macromolecules. These microbes, known collectively as the intestinal flora, have the difficult task of cohabiting and controlling each others' growth. Beneficial microorganisms also partially protect against pathogens by competing for metabolites, producing antimicrobial peptides, occupying epithelial/mucous niches, and preventing host–pathogen interactions; thus, it is our interest to tolerate them. At the same time, however, immunity to dangerous microorganisms has to be initiated. Alternatively, dendritic cells (DCs) in mucosal tissues are "educated" by epithelial cells (IECs) to suppress inflammation and promote immunological tolerance. Recent works suggest that differential epithelial cell (EC) responses to commensals and pathogens may control these two tolorogenic and immunogenic functions of mucosal DCs. Indeed, the IECs could "sense" the presence of noninvasive (commensal) versus invasive (pathogenic) microorganisms and transmit this information to antigen-presenting cells (APCs), such as DCs. Finally, APCs could be programmed to perform different tasks according to their tissue of origin, such that DCs or macrophages that are resident in the gut mucosa and regularly encounter commensal bacteria are tolerogenic, whereas "non- educated" DCs are recruited from nearby areas (dome of Peyer's patches) and from the blood, to initiate inflammation and the ensuing immune response to the invader. In agreement with this hypothesis, we described that different mediators released by IECs (TSLP, retinoic acid, TGFp , prostaglandins) are strikingly affecting the dendritic cells response. In fact, "IEC-conditioned" DCs are blocked irreversibly in their ability to release IL-12 and to activate Th1 T cells. They cannot produce inflammatory mediators even after encounter with pathogenic bacteria such as Salmonella. This interaction generates non-inflammatory and tolerogenic DCs that promote the development of Th2 responses, T reg cells, and B cell class switch recombination. Altogether, these responses allow us to tolerate commensal bacteria and food antigens and keep inflammation at bay. Deregulation of these mechanisms can lead to chronic inflammation and immune-related gut disorders like inflammatory bowel disease and cancer. Use of ontologies in pathways and high-throughput data analysis 30
    • Andrea Splendiani et al. The importance of the functional annotation of gene products for the interpretation of high-throughput data is clearly understood. In particular description of biological- pathways is a rich form of annotation capturing causal relations among processes, their temporal parts, the elements involved, and their functions. While a range of methods has been proposed for the analysis of ontological annotation of data, with the goal to score which “functions” best interpret an observed microarray experiment outcome, such methods fail to exploit the finer information represented in pathways. In this presentation we propose a way to exploit causal and compositional relations in pathways. In particular we focus on pathways represented in the Semantic Web context (which encompasses resources as Gene Ontology, Open Biomedical Ontologies and PathwayCommons), and we show, first, how these representations can transformed to suit specific analysis or visualization needs, and how this information can be used to enhance the interpretation of microarray data. In particular we will discuss how a set of pathways represented in the BioPAX format can be abstracted as an interaction network, and how pathway scoring methods can be seen as the computation of scoring function based on ontology patterns. We present the methodology the lead to this results, and we present a software platform that supports this operations. This software platform is released as opensource and freely available on the public repository bioinformatics.org. We show how this platform can be used by people without knowledge of the internals of ontology representation, and we show other features implemented to visualize and query ontologies. 31
    • Improvement of AMDA: the platform for automated data analysis of gene expression experiments Francesca Zollezzi et al. Microarrays have become common tools in many life-science laboratories. Despite their diffusion, it is still complex to analyze the huge amount of data generated by this powerful technology. Genopolis recently developed AMDA (Automated Microarray Data Analysis, BMC Bioinformatics. 2006 Jul 6;7:335), a tool that performs a fully automated microarray data analysis. The software is available as an R package and implements microarray data analysis as a series of steps including image file acquisition, quality controls on microarrays, normalizations, pre-processing, differential expressed genes selection, clustering, data representation by dimensionality reduction techniques and functional interpretation. Two new functions were integrated in AMDA. The first consists in a non specific filtering step which is based on the Interquartile Range (IQR). Data filtering is a preprocessing step to reduce the number of genes considered in further statistical analyses. Optimal data filtering is an essential step in microarray analysis to reduce the rate of false positive. This method evaluates the variability contained in signal level distributions for each array and filters out the probe sets that do not vary in the dataset. The second function extends AMDA analysis also to paired samples experimental designs. This kind of approach is characteristic in human studies and allows the comparison between correlated samples as for example blood samples from the same patient before and after a pharmacological treatment. Both these functions were written in R language and integrated in AMDA. Tests results obtained with the new AMDA version were compared to those obtained with the previous version. 32
    • A collaborative model for representing and managing microarray-related knowledge Marco Brandizi (marco@leafbioscience.com), Leaf Bioscience s.r.l. Standard formats and software tools have been developed which allow to represent and publicly share information about microarray experiments. Thanks to that, gene expression data may be shared, analysed and interpreted by the scientific community, in a collaborative manner. However, existing standards have limited support to the representation of the outcomes, experimental hypotheses or conclusions about an investigated biological question, which result from microarray data analysis. We argue that dealing with such kind of information would enhance the collaboration on analysing microarray data and, in general, it would improve the achievements of the scientific community working in this field. In order to make that possible, we propose a model which addresses this issue. We represent concepts like: sets of differentially expressed genes, results from clustering algorithms, claims about the role of genes in a biological pathway. Our model is based on the Semantic Web technologies, which are increasingly being used in Life Sciences. Ontologies, which are part of the Semantic Web, are also widely used in Life Sciences, since they allow to make order in a science with a variety of related phenomena, studied from multiple point of views and using different terminologies. The model we propose makes use of existing ontologies for the representation of microarray-related knowledge. We will introduce our model and its main features. Semantic wikis leverage on wiki tools for building a conceptually simple interface to build Semantic Web based contents. They allow to usefully combine knowledge represented in natural language, with constructs which make use of our OWL model. We show the potential of our application, or of a similar tool which could be built, to be an instrument for creating “collaborative gene expression atlas” about specific biological topics, studied by means of microarray experimentation. In doing that, we will present a project which may specifically interest the DCTHERA project. Our idea is to collect microarray data about dendritic cells and make them available by means of a standard microarray software product, such as BASE. Moreover, we propose to collect biological results that come from the analysis of such data, and use our model to publish a formalised version of such results, which could be integrated with the data repository. This kind of information would either be gathered from already performed analysis, or computed by means of existing software tools, in particular the ones produced by us and other participants to DCTHERA.We will also mention further possible developments, which include the integration with other related knowledge which is managed with the Semantic Web, such as the one proposed by the Health Care and Life Science Semantic Web Special Interest Group (HCLS-SIG). Availability: an instance of the demo hereby described is available at: http://artemisia.leafbioscience.com:8080/mannMakna. Details about the project are available at: http://brandizi.webhop.net/mysite/phdthesis. 33
    • The DC-THERA Directory Michaela Guendel et al. The dc-thera directory aims to be a comprehensive resource of research expertise within the dc-thera network (but not necessarily limited to it). It will include information on materials, sources, protocols and datasets available within the network, represented in a consistent way. This project has two main objectives: to be a useful tool for the dc-thera community, and to serve as a reference for its information. In order to be a useful instrument to support collaborations between partners, the dc-thera directory will provide advanced query functionalities on its content, and will provide links between entities presented. For instance searching for a dataset by keywords will based on ontology terms (hence by-passing spelling variations and generalizations), will provide a description of available data, access informations, a preview of data (where available) and link to related datasets, techniques, tools and protocols used to generate it, and participants involved. In order to serve as a reference, information entered in the dc-thera directory will be structured in accordance with emerging standards, and will employ dictionaries largely shared in the bio-medical community (such as the Open Biomedical Ontologies). The necessary editing effort will be in part carried by specialized personnel, but specific interfaces will be developed to make input of structured data available to participants. 34