Unit: Molecular Biology of the Gene
Director: Philippe KOURILSKY - Marie-Lise GOUGEON
The unit is involved in the study of T and B lymphocyte repertoire diversity and in the functional characterization of various lymphocyte subpopulations in physiological and pathological situations.
1- Analysis of central and effector memory T lymphocyte subsets in mice and humans (Cecile Bouneaud and Christophe Pannetier)
It is generally accepted that memory T cells can be subdivided into two subsets, central memory (TCM) and effector memory (TEM) cells. However, their differentiation pathways are not well understood. In previous studies, we showed that total human CD8 TCM et TEM cells exhibit very different T cell receptor (TcR) repertoires. In order to analyse antigen-specific memory subsets in various lymphoid organs and to evaluate their respective efficacy in a secondary response, we developed a murine model.
To study CD8 TCM et TEM spécific for the male peptide Smcy3 in the context H2-Db , we transfered naive CD8 T cells transgenic for the β chain of H-Y TCR into a C57BL/6 female and immunized them with syngeneic male bone marrow cells injected i.v. This model allowed to study the TCRαrepertoire of the two memory subsets, either in various lymphoid organs at a given time, or at two time points in the spleen with or without antigenic restimulation. Our data show : 1) at a given time, the repertoires of the two memory subsets are distinct and the T cell clones normally recirculate; 2) Following antigenic restimulation, a fraction of TCM clones give rise to TEM clones, while the majority of clones existing in the TEM subset at timepoint 1 are no longer detectable at timepoint 2, suggesting that either they die or they recirculate to peripheral tissues ; 3) under physiological conditions, TCM clones are stables whereas a significant fraction of TEM are detectable in TCM population at timepoint 2 ; 4) transfer experiments of purified TCM et TEM subsets showed that, in contrast to CD8 TCM cells, CD8 TEM cells are unable to mount a secondary response, male cells being eliminated by a primary response from the host.
Finally, in collaboration with the team of A. Lanzavecchia, we settled a model to study the TCR repertoires of the three memory T CD4 subsets specific for tetanus toxoid in the blood of healthy donors: the two classical TCM et TEM subsets and the TFH " follicular helper " subset, responsible for the help given to B cells in the germinal centers. The repertoires of these three subsets before and after a restimulation are currently analysed..
2- Analysis of the size of the human B cell repertoire (François Huetz, Annick Lim)
Analysis of lymphocyte repertoire diversity, through the determination of the size of CDR3 (Immunoscope), was initially developed for T cells. We have recently adapted this method to human B cells. A precise quantification of VH and JH gene usage was performed by coupling real time PCR with Immunoscope analyses. With this approach we were able to determine the size of the B cell repertoire and its variations under physiological conditions. We found that the size of the VH gene repertoire is between 107 and 108 expressed rearrangements, its diversity being greater than the one of TCR-ß repertoire. The contribution of somatic mutations (characteristic of B cells) to B cell repertoire diversity has also been evaluated.
Recently, we began to characterize the repertoire of CD27+ memory B cells, which proportions are altered under pathological situations, such as in autoimmune diseases (lupus), or in the context of genetic immune deficiencies leading to agammaglobulinemia with normal numbers of B cells. This study should help in better characterizing B cell memory under physiological and pathological conditions.
3- Immunoscope in clinical studies (Annick Lim, Laurent Ferradini, Christophe Pannetier)
Since its conception (1992), Immunoscope has been improved and adapted to the clinical follow-up of T cells in immunotherapy trials and in pathological situations. The improvements include quantitative analyses of T cell repertoire diversity, clonotypic Immunoscope analyses on peptide-specific T cells sorted on the basis of peptide-HLA class I tetramers, and the design of protocoles adapted to a prspective and retrospective clinical follow-up on frozen blood samples. For example, Immunoscope has been used in an immunotherapy trials in cancer patients (in collaboration with Dr. J.P. Abastado, I.D.M.) to identify the emergence of specific CTLs in response to the treatment. The recent development of Immunoscope for B cells should extend its application to clinical and vaccinal situations.
4- Analysis of the T cell repertoire in terminal déoxynucléotidyl transférase (Tdt) ko mice (Nicolas Fazilleau, Jean Kanellopoulos)
T cell repertoire in Tdt+/+ or Tdt ko mice in response to antigens presented by MHC class I or class II molecules
We have shown that the size of the αβ T cell repertoire is decreased 15 to 20 fold in Tdt ko mice compared to Tdt+/+ mice. In order to determine what is the impact of this decreased diversity to public immune responses, we have compared Vα and Vβ rearrangements of TcR selected by protein antigens in C57Bl/6 et C57Bl/6 Tdt ko mice. We have isolated T cells specific for a defined peptide with peptide-tetramer complexes of MHC class I or class II molecules. T cell CDR3α and β have then been sequenced and compared. Our data show that Tdtko mice exhibit a public repertoire very similar to that of Tdt+/+ mice. Indeed, the usage of V-(D)-J segments is conserved and CDR3 are identical or very homologous in responses against proteic epitopes. We have analyzed the repertoire evolution during the secondary response against the immunodominant peptide of influenza virus nucleoprotein, and we showed that in both Tdt ko and Tdt+/+ mice there is a maturation in the avidity of this response.
T cell repertoire in Tdt+/+ or Tdt ko mice in response to MOG protein
It has been shown that the introduction of Tdt deficiency in mice which spontaneously develop autoimmune diseases has a protective effect. To study this phenomenon in an experimental model which allows a precise analysis of T cells specific for a defined autoantigen, we have immunised Tdt+/+ et Tdt ko mice with recombinant soluble MOG protein, which is responsible for EAE (experimental autoimmune encephalitis) (in collaboration with Dr Danielle Pham Dinh and Cécile Delarasse). Analysis of MOG-specific T cell repertoire in C57Bl/6, mice is facilitated by the fact that there is only one immunodominant MOG-derived peptide presented by a single MHC class II molecule.. Immunisation with MOG or its immunodominant peptide of Tdt+/+ et Tdt ko mice induced an EAE in both strains. However, Tdt ko mice developed a single relapse while Tdt +/+ mice developed two relapses. Comparison of MOG-specific T cell repertoires in Tdt+/+ and Tdt ko mice showed that public Vα and Vβ repertoires are homologous in both strains. We are currently analysing the T lymphocytes which infiltrate the brain of Tdt+/+ during the relapse. Two aspects are studied : 1- the Vα and Vβ répertoires and 2- the antigenic specificity of T cells found during the relapse. We also perform adoptive transfers of T lymphocytes from Tdt+/+ or Tdt ko mice to analyse the regulatory potential of T cells from Tdt ko mice.
5- Immunological study of mélanoma (Laurent Ferradini)
Immunological responses to tumors are still poorly understood, whether these responses are spontaneous or induced in vaccinotherapy or immunotherapy protocoles. In particular, there is not a strict correlation between the tumor regression and the development of specific CTL. This is true for melanomas but also for other cancers poorly documented on an immunological aspect.
Two different approaches have been followed. On one hand, the search for regulatory T cells in human samples showed that they are found but in variable amounts in different patients. Since these cells are suppressive, the control of regulatory T cells in addition to the stimulation of CTL may be important to in,duce an efficient anti-tumoral response. On the other hand, a murine model has been developped, characterized by transgenic mice in which oncogenes are induced in melanocytes by an inducer present in the food. With this model, it is now possible to study fundamental questions such as the joined emergence of CTL and regulatory T cells during the development of the tumor.
6- Study of gut epithelium (Delphine Guy-Grand, Orly Azogui, Suzanna Celli)
In the absence of thymopoiesis, T lymphocytes are nevertheless present, mainly in the gut epithelium. Ontogeny of the extrathymic pathway and the extent of its involvement in euthymic mice were controversial. These questions have been addressed by assessing the expression of recombinase-activating-gene (RAG) through the use of green fluorescent protein-RAG2 transgenic mouse models. In athymic mice, T lymphopoiesis occurs mainly in the mesenteric lymph node and less in the Peyer's patches; ontogenic steps of this lymphopoiesis resemble those of thymopoiesis, but with an apparent bias towards γT cell production and with a paucity of oligoclonal αβT cells possibly resulting from a deficit in positive selection. Whether in athymic or in euthymic mice, neither T intraepithelial lymphocytes (IEL) nor cryptopatch cells (reported to contain precursors of IEL) displayed fluorescence indicating recent RAG protein synthesis. Newly made T cells migrate from the mesenteric node into the thoracic duct lymph to reach the gut mucosa. In euthymic mice, this extrathymic pathway is totally repressed, except in conditions of severe lymphocytic depletion. Thus, in normal animals, all gut T intraepithelial lymphocytes, including CD8 αα+ cells, are of thymic origin, CD8αα+ TCR αβ+ IEL being the likely progeny of double negative NK1-1- thymocytes which show polyclonal Vα and Vβ repertoires.
Legend : in nude mice expressing a GFP gene placed under the control of the RAG2 promoter, fluorescent lymphocytes are seen in the medulla of the mesenteric lymph nodes (Ref : Extrathymic T cell lymphopoiesis: ontogeny and contribution to gut intraepithelial lymphocytes in athymic and euthymic mice. D. Guy-Grand et al. J. Exp. Med. 2003. 197, 1-10)
Keywords: T and B cell repertoires, memory, terminal deoxynucleotidyl transferase, mélanoma, gut