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  Director : GACHELIN Gabriel, directeur par intérim (ggachell@pasteur.fr)


  abstract

 

Research carried out in 2001 in the "Unité de Biologie Moléculaire du Gène" (INSERM U277) mostly deals with fundamental immunological processes the results of which are whenever possible extended to human pathological immune processes. Though our work deals with aspects of immunology as different as the selection of intraepithelial lymphocytes or lymphocytes in the inflammatory reaction, most of our research has in common to use techniques developed in the laboratory and particularly the Immunoscope approach. These techniques are aimed at defining precisely changes, even minor changes, of the size of individual T and B cells clones. These techniques are routinely applied to the study of various physio-pathological processes in man and mouse, whatever they are merely observed or induced, or during the follow up of immuno-therapies in man. The study of memory T cells has recently been included in our research subjects. More recently genomic analysis of the regions of chromosomal DNA that become undermethylated during a differentiation process (such as Th1/Th2 or passage to the condition of memory cells) has been set up , and has yielded promising results.



  report

cale

Team 1 headed by Delphine Guy-Grand : Intra intestinal lymphocytes

ONTOGENESIS OF GUT INTRA-EPIPHELIAL LYMPHOCYTES (IEL).

Among T lymphocytes, IEL have special features: localization very close to the source of antigens; predominance of CD8 T cells with a heteregoneous phenotype (CD8ab TCRab, CD8aa TCRab or gd); persistence in athymic animals of CD8aa+ IEL (mainly TCRgd+), suggesting the contribution of an extrathymic differentiation pathway. Our present work is focused on the exploration of thymic or extrathymic pathways in the differentiation of CD8aa IEL (all CD8ab+ IEL originate from the " double positive " thymic pathway). By using euthymic or nude transgenic mice in which all T lymphocytes express the same autoreactive TCR (anti-HY abTCR transgene on a RAG-/- genetic background), we have shown that CD8aa IEL may have a thymic origin through a " double negative pathway ". This pathway appears to exist also in wild-type mice, especially for CD8aa CD8ab IEL, and to be quantitatively prevalent over the extrathymic pathway, in contrast to common concepts.

Team 2 headed by Laurant Ferradini : Tumor immunology

ANALYSIS OF ANTITUMOR T CELL REPONSES - FOLLOW-UP OF IMMUNOTHERAPY PROTOCOLS

The project of the tumor immunology group follows three main directions. The first one is to set up an in vivo inductible melanoma model in the mice in order to study the natural or induced antitumoral immune response directed against a known antigen (GP- LCMV used as a tumor model antigen) during the course of progressive tumor development. Such a transgenic model should allow us to follow and caracterize the specific antitumoral response using tetramers and Immunoscope but also recent multiparametric methods to study gene and protein expression (cDNA microarrays, ProteinChips®). Our aim is to caracterize the specific T cells involved in an efficient in situ antitumoral immune response and to precise their activation and differentiation status. The second axe of research is to analyze antitumoral T cell responses during the course of human melanomas and more specifically MHC class II restricted responses. For that, we have produced distinct MHC class II tetramers (DR4, DR7 et DP4) in order to follow CD4+ T cells specific for different known tumor antigen (MAGE-3, MelanA/Mart1, NY-ESO-1). On the other hand, we are studying the repertoire of memory T cells in order to understand the dynamic of memory cell populations in normal donnors or cancer patients. Finally, the last topic of our project consist in using the advanced immunoscopy platform to follow specific antitumoral T cell responses during the course of immunotherapeutic protocols in cancer patients.

Team 3 headed by Gabriel Gachelin : Lymphocytes MKT

MKT CELLS AND ACUTE INFLAMMATORY PROCESSES

Our group has dominantly worked on the functions of natural killer T cells (NKT cells) , restricted by the monomorphic histocompatibility molecule CD1d1. In earlier studies, we had shown that NKT cells were actively recruited at inflammatory sites caused by the injection of bacterial glycolipids or during local bacterial infections. They are not detected during chronic skin inflammatory processes. During the past year, we have shown that NKT cells behave as inflammatory cells and migrate to acute inflammatory sites, irrespectively of their restriction element. NKT lymphocytes are thus a novel class of inflammatory cells. Ongoing studies have shown that NKT cells are recruited within hours following the induction of the inflammatory process, and have defined which cytokines and chemokines are precociously released. The patterns of chemokine receptors of the different NKT cells subpopulations have been determined : they are organ specific and are likely to underlie different functional properties. The study of the in vivo activation of liver NKT cells by Kupffer cells has been undertaken. Fluorescent reagents have been synthesized that allow the follow up of the behaviour and the metabolism of CD1d-affine molecules such as alpha-galactosyl-ceramide. Finally a study concerning the origin of the immune system and dealing with the relationships between cyclostomes and fishes has concluded to the monophyly of cyclostomes.

Team 4 headed by Jean Kanllopoulois : Studies of murine T lymphocyte repertoires

Following up on previous work, we are studying the selection of mouse T cell repertoires and the role of thymic nurse cells (TNC) in the selection of thymocytes.

1) T cell repertoire studies in terminal deoxynucleotidyl transferase (TdT) KO animals. We have estimated the size of the  repertoireof T splenocytes in TdT°/° and found that it is decreased by a factor 10 to 15 compared to wild type animals (Cabaniols,J-P, Fazilleau, N, et al. J.Exp.Med. (2001) 194:1385). As TdT KO animals have normal immune responses and resist infections like wild type animals, we are comparing the  and rearrangements of C57Bl/6 TdT°/° and C57Bl/6 TdT +/+ in response to proteins presented by MHC class I and II molecules. We isolate epitope specific T lymphocytes using MHC class I or II tetramers and we sequence their rearrangements. We analyse and compare the different CDR3 observed in TdT°/° and wild type animals in primary and secondary immune responses.

2) Role of TNC in thymocyte selection : identification of TNC-specific genes

We have used the method called "Representational Difference Analysis" to identify and clone genes which are expressed differentially by a given cell sub-population. This method was applied to TNC and two different sequences were identified. One corresponding to synaptotagmin I and the second one to a "Cystein Rich Protein" (CRP). Synaptotagmin I is primarily expressed in the central nervous system, in adreno-medulla and pancreas. With anti-synaptotagmin I antibodies, we want to identify the thymic epithelial cells in which this protein is expressed. Furthermore, using synaptotagmin I KO mice, we would like to determine the impact of this protein on thymocyte development and function. The CRP which we have identified, is restricted to mouse thymus. With anti-CRP antibodies, we found that tCRP is present in TNC and in a sub-population of epithelial cells of the thymic cortex.

Team François Huetz : Analysis of human B/cell repertoire

Immunoscope technic, which allows the study of lymphocyte repertoire by CDR3 size determination, have been widely used with T lymphocytes. We have now set this technic to study human B cell repertoire. and coupsled it with a study of VH gene families by real time quantitative PCR and with the determination of the sequence of B cell rearrangements. This should give us a size approximation of the B cell repertoire in physiological conditions. The influence of somatic mutations (which only occur in B cell VH genes) on repertoire diversity and the repertoire diversity of mutated B cells will be evaluated. Then the same studies will be undergone in pathological situations. This work will be then carry on on mouse B lymphocytes and should allow us to gain some insight on B cells memory induction and maintenance.

Team 5 headed by Christophe Pannetier and Philippe Kourilsky : Analysis of memory and effector T cell populations and search of the molecular basis of their phenotype.

The platform for monitoring CD8+ T cell response in Human, cofunded by the French, has been used for the monitoring of a melanoma clinical trial directed by the IDM biotech company. Using this platform, it is now feasible to detect, and then follow up, during a therapeutic treatment for instance, individual T cell clones specific for given antigenic MHC-restricted peptides, provided that their frequency is higher than 10-5. The description of the whole process will be published in the Journal of Immunological Methods in 2002. This platform will then be transferred to the Center for Clinical Research of the Institute. With the ultimate goal to analyse the impact of cancer or cancer treatments on the CD8+ T cell memory subset, this team further studied the repertoire of this cell population in healthy donors. CD62L+ and CD62L- subsets have been analysed separately with the Immunoscope at various time points in several donors. Finally, this team has continued its efforts for localizing alterations of the DNA methylation pattern throughout the genome occurring during differentiation of murine naive T cells into effector or memory T cells. This study is being carried out in collaboration with the Laboratory of Immunology of NIAID, and INSERM U345. Potential markers identifying differentially methylated regions have already been localized with the use of the RLGS-M method (see figure) in two models : Th1 and Th2 cell lines committed in vitro, and CD8+ naïve and memory T cells committed in vivo. Cloning and validation of these markers is on-going.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

Viviane CAPUT, TCE CNRS – caput@pasteur.fr

Jos EVEN, CR1 CNRS

Laurent FERRADINI, CR1 INSERM

Gabriel GACHELIN, Chef de Labo IP

Delphine GUY-GRAND, DR0 INSERM

Jean KANELLOPOULOS, Professeur, Orsay

Philippe KOURILSKY, Professeur, Collège de France

Iris MOTTA, CR1 INSERM

David OJCIUS, Professeur, Paris VII

Christophe PANNETIER, CR DGA

Nathalie PARDIGON, CR IP

Véronique BARON, doctorante, bourse CIFRE

Cécile BOUNEAUD, doctorante, bourse DGA

Min-Sun CHO, post-doc, poste vert INSERM

Nicolas FAZILLEAU, doctorant, bourse MNERT

Martin MEMPEL, post-doc, bourse CE

Cahterine RONET, doctorante, bourse MNERT

Nathalie THIEBLEMONT, post-doc, bourse ARC

Viviane CAPUT, TcE CNRS

Armanda CASROUGE, IE INSERM

Sylvie DARCHE, IE INSERM

Christiane DELARBRE, IR CNRS

Sacha GARCIA, T INSERM

Fabrice LEMAITRE, AI INSERM

Annick LIM, Ingénieur IP


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