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.
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).
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.
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.
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
(firstname.lastname@example.org), 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.
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.
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
Rennes; 3- Department of Neurosurgery, CHU Pontchaillou, Rennes; 4- UMR 6061 –
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.
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
Division of Infection and Immunity, UCL, London UK
Department of Oncology, UCL, London UK
Department of Oncology, UCL Hospitals, London UK
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.
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.
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.
The maturation cocktail for DC generation influences induction of MUC1 specific
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).
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
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
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.
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;
Center for Cancer Immune Therapy (CCIT), Department of Hematology, 2Department of
Oncology, and 3Department of Pathology, University Hospital Herlev, Copenhagen,
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.
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
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
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
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.
Role of microRNA-155 in mature dendritic cells
Isabelle Dunand-Sauthier1, Leonardo Capponi1, Antoine Geinoz1, Emmanuèle Barras1,
Hans Acha-Orbea2 and Walter Reith1
Department of Pathology and Immunology, University of Geneva Medical School,
Department of Biochemistry, Faculty of Biology and Medicine, University of Lausanne,
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
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
Lucia Conti, Marco Cardone, Barbara Varano, Patrizia Puddu, Filippo Belardelli and
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.
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,
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,
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.
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
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
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,
Pathology and 5Clinical Oncology, Leiden University Medical Center, Leiden, The
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+
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.
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.
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.
*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
Querying a DC "omics" database according to a pathway based logic.
Department of Pharmacology, University of Florence.
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.
Pathway Based analysis allows comparison between different datasets and
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
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
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.
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.
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
Different forms of Saccharomyces cerevisiae elicit specific responses in dendritic
Lisa Rizzetto1, Giorgio Napolitani2, Mirela Kuka3, Ugo D'Oro3, Duccio Cavalieri1
Dept. of Preclinic and Clinic Farmacology, University of Florence, Italy
Institute for Research in Biomedicine, Bellinzona, Switzerland
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
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.
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.
A module network approach to the unraveling of the molecular pathways
underlying the physiological diversity of the in vivo malaria parasite Plasmodium
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.
Comparison of the transcriptomes of CD34-derived and human skin Langerhans
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
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.
Epithelial cells as modulator of intestinal immuno-homeostasis.
G. Matteoli1, Ilian Iliev1, E. Mazzini1, E. Mileti1 and M. Rescigno1
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
Use of ontologies in pathways and high-throughput data analysis
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
In this presentation we propose a way to exploit causal and compositional relations in
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
Improvement of AMDA: the platform for automated data analysis of gene
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
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
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.
A collaborative model for representing and managing microarray-related
Marco Brandizi (email@example.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:
Details about the project are available at: http://brandizi.webhop.net/mysite/phdthesis.
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
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.