• E-mail :[email]
  • Phone : +33 4 78 78 29 64
  • Location : LYON, France
Last update 2011-10-05 15:40:11.471


Course and current status

January 2011 : HDR

Since 01/01/2007   : Chargée de Recherche 1ére classe -INSERM
Centre Léon Bérard- UMR INSERM 1052-CNRS  5286
Centre de recherche en Cancérologie de Lyon
Dép. Immunité Microenvironnement et Virus
Equipe Ciblage thérapeutique de la tumeur et de son environnement immunitaire. Team leaders : C CAUX and JY Blay

2002-2007 :Chargée de Recherche 2ème classe –INSERM
INSERM- U346, Hôpital Edouard Herriot-Pavillon R
69437 LYON Cedex. Team Leader : Colette DEZUTTER-DAMBUYANT

2001 :  Post-doctoral position at Institut Curie-INSERM U520. Team leader: S. Amigorena, Paris(France) 
Grant from"La ligue Nationale contre le Cancer"                                                  

1995-2000:  DEA and PhD in Immunology
Université UCBL  Lyon I, Laboratoire de Recherches Immunologiques, Schering-Plough, Dardilly (France)

Scientific summary

Following a change in my position in January 2007 from the ex-U346 (Hospital Edouard Herriot) to my current unit (U590), these two last years (2007 and 2008) were first devoted to complete and finish my previous project relating to mechanisms implicated in the recognition and presentation of microbial antigens by human cutaneous and mucosal dendritic cells (DC). For this aim, we had successfully isolated human DC populations of the skin and manage to study expression and function of various innate immunity receptors such as Toll like Receptors (JID 2009 and JI 2006). In this context, we have also developed an in-vitro model of vaginal mucosa in which Langerhans cells (LC) have been integrated in order to clarify the early steps of HIV entry into intact vaginal mucosa. For the first time, we have documented that, within 4h following contact with HIV-infected cells, translocation of free HIV particles across this pluristratified mucosa was not detectable and not increased by the presence of LC. We have thus provided a useful model for the development of strategies preventing HIV entry into the female genital tract, especially for testing the efficiency of various microbicides (AIDS 2008). In paralleled, as the Langerin lectin specifically expressed by LC, binds HIV-gp120, in collaboration with F. Fieschi (Grenoble) we have performed structural analysis. Since Langerin carbohydrate recognition domain (CRD) is crucial for HIV interaction and Birbeck granule formation, we have produced the CRD of human langerin and solved its structure at 1.5 A resolution. On this basis, gp120 high-mannose oligosaccharides binding have been evaluated by molecular modelling and using mutated and deleted langerin, the role of the different langerin domains deciphered (Biochemistry 2009).


Now, the project of the team I joined named Cytokine and Cancer, and directed by C. CAUX and Pr. J.Y. BLAY, is driven by the objective to develop anti-tumoral therapeutic strategies, in particular in breast carcinoma, combining a drug selectively inducing immunogenic tumor cell apoptosis with appropriate immune stimulation which may lead to therapeutic anti-tumour immunity.

In this context, the major aim of my project is to define the effect of different canonical apoptotic pathways induced through well characterised anti-tumoral drugs, or new targeted therapy, on tumour antigen presentation and antitumor immunity. Indeed, efficient anti-tumour immune responses require tumour-associated antigen presentation by DC to CD8+ cytotoxic T lymphocytes (CTL) by a mechanism known as cross-presentation. Dying tumour cells provide an interesting source of tumour antigens allowing responses against multiple known as well as unknown epitopes following antigen capture, processing and presentation by DC. Cross-presentation and the resulting immune response – cross priming or cross tolerance – critically depend on the cell death pathway. In our project, we are particularly interested by drugs targeting oncogenic pathways such as Her2 amplification, p53 mutations, p53 functional alterations (Twist) or dependence receptor (DCC/netrin) and we also address the immunological consequences of cell lyses by cytotoxic effectors such as natural killer (NK) cells compared to other apoptotic/necrotic pathways.

More precisely, we address the capacity of DC to uptake antigen from various dying cells, to be activated and to cross-present a model CMV epitope (N9V) to CD8+ T cells.

For this aim, during these two past years, we have first evaluated several apoptotic pathways on breast cancer cell line (MCF7, SKBR3, CAL51). Second, we have set up an in vitro human model for cross-presentation by :    

(i) Constructing lentivirus vector expressing CMV model antigen i.e pp65 whole protein or N9V CD8 epitope (NLVPMVATV) fused with OVA (collaboration with Jacqueline Marvel, CERVI, Lyon, FR)

(ii) Establishing breast cancer and K562 cell lines stably expressing this CD8+ epitope

(iii) Generating a large number of the specific N9V CD8+ T cell clone (collaboration with H VIE, Nantes)

(iv) Set up the experimental procedure for analysing CD8 activation (by intracytoplasmic IFNg and CD107a flow cytométrie analysis)

For cytotoxic effector cell-death, we have established the ménage à trois co-culture system with monocyte-derived DC, NK cells isolated from peripheral blood and the naturally targeted K562 cell line.

-For antigen capture, PKH67 or CFSE labelled tumour cells and DC were co-cultured overnight and then stained with a DC-specific marker allowing quantification of Ag uptake as a percentage of green DC on the total DC population. Phenotypic characterization was done also on DC in the same experimental conditions.

Using those assay, we found a good correlation between cell death and capture by DC. We observed antigen capture by DC following necrosis (heat shock), apoptosis induced by the Her2neu tyrosine kinase inhibitor Lapatinib, anthracyclin or NK-induced cytolysis. Partial DC maturation (CD86) was only observed with necrotic bodies (heat shock) or with NK-mediated cell death.

In various independent experiments, we observed that DC were able to cross-present N9V epitope to CTL following mixed culture with K562 and NK cells compared with alive or necrotic K562 cells This seems to be independent of an additional maturation process by TLR engagement following polyIC or LPS stimulation. In those experiments, we have also controlled that the responses were antigen specific and dependent on DC. Indeed, no cross-presentation was observed with K562 tranfected with a control lentivirus expressing only the GFP without N9V epitope or using a mismatched DC HLA-A2-.

Thus, we are currently evaluating the role of the NK-DC cross-talk in this phenomenon (soluble and cellular interactions), and we would like to determine molecular pattern specific of this NK-cell death pathway.

In this context, a collaborative Biopole project with Innate-Pharma, Transgène, and bioMérieux, named DEMINAP is dedicated to this aim in our team. Indeed, we study the presence and implication of endogenous TLR ligands released during the apoptosis process using cell lines expressing a NFKB-luciferase reporter gene transfected with TLR1, TLR2, TLR3, TLR5, TLR6, TLR7, TLR8 or TLR9. This will enable us to measure TLR triggering by a simple luminometric assay. In paralleled, we are also constructing other form of model Ag in order to better quantify the epitope (directly fused with GFP), and to better follow it during the apoptotic process by fusing this epitope-GFP with a plecksin domain which target proteins to the plasma membrane.

Finally, we evaluate the immunogenicity of various apoptotic pathways by their capacity to induce PBMC cytokine secretion and preliminary observations were made. Notably, among the "classic" chemotherapies tested (doxorubicin, methotrexate, cisplatin, etoposide, mitoxantrone or Bortezomib) on breast cancer cell lines, we observed a preferential IL1b secretion by the proteazome inhibitor Bortezomib compared to other apoptotic pathways. Thus, we are currently addressing which population is secreting IL1b and which mechanisms are involved.

Image d’exemple