Unit: Lymphocyte Population Biology - CNRS URA 1961

Director: FREITAS Antonio

The research objectives of the " UBPL" are:

a) The study of the homeostatic mechanisms that control B and T cell numbers.

b) The study of the mechanisms of lymphocyte survival: the rates of production, renewal and death.

c) The study of the role of lymphocyte competition in the selection and control of the primary and memory immune responses.

B cell homeostasis and selection (Emmanuelle Gaudin, Yi Hao): B cell tolerance or auto-immunity is determined by selective events. Negative selection of self-reactive B cells is well documented and proven. In contrast, positive selection of conventional B cells is yet to be firmly established. We demonstrated that developing self-reactive B cells are not always highly sensitive to the deletion mechanisms imposed by membrane-bound self-antigens. At low amounts, membrane-bound antigens allow survival of B cells bearing a single high affinity self-reactive B cell receptor (BCR). More important, we show that forced allelic inclusion modifies B cell fate: low quantities of self-antigen induce the selection and accumulation of increased numbers of self-reactive B cells with decreased expression of antigen-specific BCRs. By directly measuring antigen binding by intact B cells, we show that the low amounts of self-antigen select self-reactive B cells with a lower association constant. A fraction of these B cells is activated and secretes auto-antibodies that form circulating immune complexes with self-antigen. These findings demonstrate that conventional B cells undergo positive selection and that the fate of a self-reactive B cell depends on the quantity of self-antigen, the number of BCRs engaged and on its overall antigen-binding avidity, rather than on the affinity of individual BCRs.

CD4+CD25+ regulatory T cell homeostasis and function (Afonso Almeida). We have established that the IL-2-IL2R axis is crucial for the generation and/or maintenance of CD4+CD25+ regulatory T cells (Almeida, A. et al, J. Immunol, 2002). We now aim at determining the in vivo role of IL2 in CD4+CD25+ regulatory T cell function. We generated new IL2-/- mice, either T cell sufficient CD25-/-IL2-/-or IL2-/-Ly5.1 (to use as BM or mature CD4+ T cell donors) or lymphopenic Rag-/- or CD3-/- deficient (hosts), in order to investigate possible T or non-T cell sources of IL2. We have used the lymphopenic mice as recipients for BM reconstitution experiments or mature CD4+ T cell peripheral transfers. Our preliminary results regarding the mechanisms of suppression of naïve CD4+ T cell expansion by CD4+CD25+ regulatory T cells exclude the hypothesis of suppression by IL2 consumption. Regarding the homeostasis of CD4+CD25+ regulatory T cells by using BM chimera reconstitution strategies, we obtained results that suggest that T cell derived IL2 is necessary to rescue CD25-/- or IL2-/- BM chimeras from death. We are currently developing Single-Cell quantitative RT-PCR essays, to further investigate these issues.

Role of the IL- 7/ IL-7 receptor on T cell homeostasis (Jaime Franco). The mechanisms that control the size of the peripheral T cell pool are not yet fully understood. IL-7 has been shown to be involved both in the generation and survival of mature peripheral T. It is therefore possible that IL-7 may also play a role determining the size of the mature T cell pool. Yet, The quantitative relationship between IL-7 levels and peripheral T cell has not been established. We have now set up an experimental system that allow the generation of B and T cells in IL-7-/-Rag-/- hosts, based in the enforced expression and production of IL-7. This experimental system will allow us to finely study the relationships between the available dose of IL-7 and T cell production and pool size. Using this system we can also generate IL-7 producing mature T cells, which will be used to regulate the T cell expansion potential and homeostasis in Il-7-/- hosts.

Lymphopenia driven proliferation of CD4+ T cells: Differential regulation of STAT proteins.(Vanesa Guajardo). We used a lymphopenia driven proliferation model to follow the fate of CFSE labelled T cells obtained from Stat6-/- and Stat4-/- Balb/c donors transferred into T cell deficient Balb/c Rag2-/- hosts. We found that Stat6-/- T cells start to proliferate earlier and proliferate faster than Stat4-/- T cells. In spite of their differential growth rate both cell types reached similar plateaux levels in the host mice (around 2 x106 cells/host). When the two T cell types where co-transferred at different cells ratios into the same T cell deficient hosts, Stat 6-/- CD4+ T cells out competed T cells from Stat4-/- origin. We analyzed the state of phosphorilation of the Stat proteins during the first 4 days after transfer. We observed that in Stat6-/- T cells Stat4 is transiently phosphorilated, in contrast in stat4-/- T cells Stat6 was down regulated. Preliminary RT-PCR results show, that during the first 4 days of proliferation after adoptive transfer, both type of cells have increased levels of T-bet and IFN-γ mRNA while the levels of Gata3 mRNA expression were decreased as compared with ex-vivo levels. We also found that although IFN-γ was produced by both Stat6-/- and Stat4-/- T cells. These findings suggest that in a default situation a Th1 type of responses may be preferentially induced.

Competition and survival among CD4 memory T cell compartment (Sylvie Garcia). The competitive properties of memory T cells are one of the parameters that control the composition of the memory compartments. The aim of this project is to define and compare the rules governing the CD4 and CD8 memory pool. Two aspects will be explored: 1-The competition between different memory cell subsets. This will be studied upon the co-transfer of several types of TCR transgenic memory CD4 and CD8 T cells into adoptive immunodeficient hosts. 2-The capacities of newly generated memory T cells to replace preexistent T cell memory subsets. In case of replacement, this would lead to the qualitative impoverishment of the old memory pool (attrition). The sequential transfer of different TCR transgenic memory and naïve T cells into immunodeficient hosts will assess this process of replacement. The fate of each T cell memory subset will be studied regarding the specificity, function (Th1 vs Th2 for CD4 memory) and age of memory cells. These studies may contribute to understand the regulatory processes controlling the generation and maintenance of the memory T cell pool during acute (i.e. vaccination) or chronic (i.e. HIV infection) antigenic stimulations.

CD8 T cell survival (Nicolas Legrand, Yi Hao). In order to test the role of MHC class I on the survival and lymphopenia-driven proliferation of CD8+ T cells, we have developed a new adoptive transfer system of T lymphocytes into hosts with different MHC class I expression patterns. Donor T cells, isolated from mouse strains transgenic for the TCR in a RAG-deficient background (MoaHY, MoP14 and MoOT-1 mice), were separately transferred into different types of hosts: T-cell deficient class I+ mice (CD3-/-), or mice deficient for the expression of MHC class I (CD3-/-H-2Db-/- ; CD3-/-H-2Kb-/- and CD3-/-β2m-/-H-2Db-/-H-2Kb-/-). After transfer into MHC class I+ hosts, T cells show maintenance without division (MoaHY) or strong lymphopenia-driven proliferation (MoP14, MoOT-1), depending on the hierarchy of TCR promiscuity (MoOT-1>MoP14>MoaHY). After transfer into H-2Db-deficient mice, MoaHY and MoP14 T cells, both H-2Db-restricted, disappear in less than 2 days. In contrast, OT-1 TCR expressing CD8 cells (H-2Kb-restricted), transferred into H-2Kb-deficient mice maintain their numbers. The usage of "TetraKO" mice should allow us to precise the controversial role of MHC class I molecules in the survival of memory CD8+ T cells.

Notch signaling in lymphocyte development and survival (Alix de la Coste). During lymphoid development, Notch signaling has been shown to influence B versus T cell commitment. The critical role of Notch activation in T cell linage choice has been largely documented. However, the identity of the Notch ligand that triggers this critical signal is still unknown. To test Notch ligand Delta-1 activity in vivo, we reconstituted the immune system of lethally irradiated mice with fetal liver hematopoietic stem cells (e14,5) transduced with a retrovirus expressing Delta-1. We found that Delta-1 induces ectopic development of double positive CD4+CD8+ immature T cells in the bone marrow, spleen and lymph nodes of C57BL/6 mice, without causing lymphoproliferative disease. The ectopic T cell development in the bone marrow is associated with a concomitant inhibition of B cell development. Moreover, we found that, in athymic nu/nu mice, Delta-1 expression could provide appropriate signals for complete T cell maturation. Thus, our findings demonstrate that in vivo, Delta-1 expression in a bone marrow environment is sufficient to fully recapitulate T cell development from the most immature stages to complete maturation.

Keywords: B cells, T cells, lymphocyte homeostasis, lymphocyte survival, immunological memory

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