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


  abstract

 

During the past year, research activities carried out in the Unite de Biologie Moléculaire du Gène, INSERM U277, although dealing with as various aspects of immunology as intra-epithelial lymphocytes and anti-tumor responses, have dominantly be based on the description of T-cell repertoires. Over the past years, a set of techniques, the Immunoscope I and now II, have been destablished in the laboratory, taht enables the easy determination of even minor changes in the size of a T cell clone. This set of technique has therefore been applied to a variety of physiopathological conditions as well as to normal differenciation of T cells.



  report

cale

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

We used the immunoscope technique which consists of detecting the respective amount of all rearranged b chain transcripts present in a TCRab+ T cell population followed by sequencing of the expanded population and we showed 1) that CD8ab+ and CD4+ thymus dependent lymphocytes which populate the mouse gut wall are oligoclonal both in the lamina propria and the epithelium and that each subset share identical TCRb clonotypes in the same mouse 2) that clones of large CD8ab+ lymphocytes circulating in the thoracic duct lymph also share homologies with that populating the epithelium. These findings are in the line of old results demonstrating that thoracic blasts migrate in the gut wall and its lymphoid associated tissue and have interesting implications in the understanding of the nature of the stimulation, the localization and the succession of events which lead up to the appearance of gut lymphocytes with a restricted TCRb repertoire


Team 2 headed by Laurent Ferradini: Tumor immunology

The project of the tumor immunology group takes advantage of the expertise and the tools developped these last few years in the laboratory which allow the precise analysis of antitumoral T-cell responses. We are interested in studying antitumoral T cell responses both in mice and in humans by focusing on three different aspects:
- In mice, we are developping an in vivo inducible melanoma model in order to study the antitumoral immune response in a situation as physiological as possible.
- In humans, we are analysing the antitumoral immune response during melanoma by focusing more specifically on the MHC class II restricted immune response through the development of MHC class II tetramers, and on the study of antitumoral memory T-cell compartment.
- Finally, we are participating to the immunological follow-up of cancer patients treated by immunotherapeutic protocols using the immunological station, developped and optimized in the laboratory, aimed to precisely follow T lymphocyte immune responses.


The team headed by Gabriel Gachelin has worked along two main lines of research. In collaboration with scientists of the Museum National d'Histoire Naturelle, we have carried out an extensive study aimed at determining the phylogenetic relationships between cartilaginous fishes and cyclostomes. Indeed, cyclostomes do not have an adaptive immune system, whereas all components of the latter are already present in sharks and rays. A study on the origin of the immune system thus implied the phylogenetic relationships between these different species to be clarified. Studies carried out using mitochondrial DNA sequences of several chondrichtyes and cyclostomes has led to the conclusion that cyclostomes are monophyletic, despite the morphological similarities between lampreys and osteichtyes. Thus no present representative of cyclostomes could be used as a more appropriate material to study the origin of the adaptive immune system.
Most of the research activities of the team were devoted to the study of natural killer T cells (NKT cells). These cells express both markers of NK cells and markers of T cells, particularly a semi-invariant TCR characterized by a distinctive TCR alpha chain and a skewed usage of V beta segments. In addition, the monomorphic MHC1b CD1d molecule restricts most of these NKT cells. Studies carried out in 1999-2000 have dealt with an improved molecular definition of NKT cells, mostly based on the molecular analysis of the beta chain of the TCR of sorted cell populations. We could show that all populations possessed a highly diverse TCR without any particular bias in their CDR3 region, although the latter is expected to recognize only hydroxyl residues and the skeleton of carbohydrates. NKT cells of any organ origin could be DN, CD4+ and never CD8+ . Some are CD62L+, others are CD62L-: only CD62L- cells are restricted by CD1d1 molecules. We are in the process of defining whether this phenotypic heterogeneity is indicative of a functional heterogeneity. Since NKT cells are, in the mouse, involved in the response against non-peptide antigens of mycobacterial origin, we have studied skin lesions of either mycobacterium origin, such as leper, or of non bacterial origin, such as sarcoidosis or granulum annulare. We could show that NKT cells are found only in Mycobaterium leprae lesions. However, the most important finding of the year has been the discovery that NKT cells, although their TCR can recognize the carbohydrate moiety of glycolipids presented by CD1d molecules, do not accumulate in the lesions due to mycobacterial glycolipids due to antigen-driven proliferation, but rather non-specifically due to the ongoing inflammatory process. Thus NKT cells are early cells of acute inflammatory responses, but not of chronic inflammatory responses; they do not use the TCR-CD1d1 activation pathway to accumulate at inflammatory sites, and accumulation and most probably part of the functions of NKT cells are independent of CD1d1/TCR. The work of the team is row re-orientating towards the mechanisms at work in the migration of NKT cells from the organs where they are stored to inflammatory sites.

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

The set up of the platform for monitoring CD8+ T cell response in Human has now been completed. 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. Specific T cells are sorted out by means of magnetic beads coated with MHC-peptide complexes. The rearranged genes encoding the TCR chains are sequenced, which allows the design of clonotypic primers specific for each clone. The frequency of these clones can then be readily measured in samples or sorted T cell subsets by quantitative RT-PCR amplification using these primers. This platform is now utilized by team n2 for the monitoring of melanoma patients enrolled into an immunotherapeutical clinical trial headed by IDM biotech company.
With the ultimate goal to analyze 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 analyzed separately with the Immunoscope at various time points in several donors.
Finally, this team has continued its efforts for identifying epigenetics alterations occurring during differentiation of murine naive T cells into effector or memory T cells. This study is 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.



  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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