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 (Yi Hao, Emmanuelle Gaudin): B cell tolerance or autoimmunity 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 demonstrate 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 importantly, we showed 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 showed 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 autoantibodies that form circulating immune complexes with self-antigen. These findings demonstrate that conventional B cells can 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 proceeded with studies aimed at the identification of the factors responsible for the homeostasis of the CD4+ CD25+ regulatory T cell sub-population and to the understanding of the pivotal role played by this sub-population in the maintenance of the overall peripheral T cell homeostasis. A careful examination of previous and recent results points for a tight regulation of the CD4+CD25+ regulatory T cell proportion. By the generation and transfer of IL2 deficient CD4+CD25+ regulatory T cells with or without IL2+ or deficient other T cells we were able to demonstrate that IL2 produced by either CD4+CD25+ cells themselves or by activated/memory contaminant cells is a key factor in their ability to suppress and expand. The sum of our studies leads us to consider the possibility of indexation of CD4+CD25+ regulatory T cell numbers to the number of IL2 producing activated CD4+ T cells as the mechanism explaining why the relative proportion of CD4+CD25+ regulatory T cell is so tightly regulated. We have also investigated the consequences for third-party sub-populations of lymphocytes of the CD4+CD25+ regulatory T cell activity. We have verified that apparent suppression can be observed through suppression of bystandard helper effects. By repeating the experiments with homogeneous sub-populations of polyclonal CD8 T cells (defined by the activation markers CD44 and CD62L), we were able to observe that not all sub-populations of CD8 T cells are affected the same way. The sum of these experiments lead us to consider the sub-division of the peripheral CD8+ T cell pool taking into consideration the probability of bystandard expansion and the possibility of indirect control by CD4+CD25+ regulatory T cells. We have also initiated studies that should help us to understand the similarities and differences that are apparent from the analysis of the phenotype of the scurfy, the IL2-/- and the CD25-/- mouse strains. Published studies where the forced expression of the transcription factor foxp3 in peripheral naïve CD4+ T cells led to the acquisition of regulatory function by these cells have also shown that the expression of CD25 was induced. We have decided to examine the possibility of an absolute requirement of the expression of CD25 for the function of foxp3 expressing CD4+ regulatory T cells. We have retrovirally infected CD25-/- BM precursors with foxp3 and reconstituted Rag2-/- mice with those cells. We were able to rescue these BM chimeras. While it seems apparent from this result that CD25 expression is not an absolute requirement for CD4+ regulatory T cell function, the experiment is in fact non-conclusive by itself. In order to clarify this result the rescued mice were sacrificed and the mature peripheral T cells or the BM precursors were used in other experiments. These experiments are ongoing and should shed light on the relevance of different molecules in the generation, survival and function of the CD4+CD25+ regulatory T cell subset.

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). The outcome of an immune response relies on the competitive capacities acquired through differentiation of CD4+ T cells into Th1 or Th2 effector cells. As Stat4 and Stat6 proteins are implicated in the Th1 vs Th2 generation and maintenance respectively, we here compare the kinetics of Stat4-/- and Stat6-/- CD4+ T cells during competitive bone-marrow reconstitution and lymphopenia driven proliferation. After bone-marrow transplantation both populations reconstitute the peripheral T cell pools equally well. After transfer into lymphopenic hosts, wild type and Stat6-/- CD4+ T cells show a proliferation advantage, which is early associated with the expression of an active phospho-Stat4 and the down-regulation of Stat6. In spite of these differences, Stat4 and Stat6-deficient T cells reach similar steady-state numbers. However, when both Stat4-/- and Stat6-/- CD4+ T cells are co-injected into the same hosts, the Stat6-/- cells become dominant and out-compete Stat4-/- cells. These findings suggest that cell activation through the Stat4 pathway and the down-regulation of Stat6, confers to the pro-Th1 T cells a slight proliferation advantage that in a competitive situation has major late repercussions, as it modifies the final homeostatic equilibrium of the populations and favors the establishment of Th1 CD4+ T cell dominance.

Competition and survival among CD4 memory T cell compartment (Sylvie Garcia, Catarina Leitao). The survival 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 of these mechanisms are currently explored: 1-The competition between different memory subsets. 2-The capacities of newly generated T cell memory subpopulation to compete with preexistent T cell memory subsets. In case of out-competition, this process would lead to the qualitative impoverishment of the memory pool (named attrition). To address these two issues, we are creating mice containing several TCR tg T cell CD4 and CD8 subsets and polyclonal T cells. This is achieved either by engraftments of a mixture BM precursors or adoptive transfers of mature T cells of T cell deficient hosts. In these mice, each subsets can be easily distingued by the differential expression of Vα, Vβ and Ly5 and Thy1 alleles. The first point is analyzed by co-transferring several types of TCR transgenic memory CD4 and CD8 T cells into adoptive immunodeficient hosts. The second point is assessed by the sequential immunization of the different TCR transgenic naïve T cell subsets contained in the hosts. In both cases, the fate of each memory subsets is followed regarding to the specificity, function (Th1 vs Th2 for CD4 memory) and age of memory cells. These studies should help 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 (Yi Hao). In collaboration with Nicolas Legrand we have compared the MHC requirements for the survival of several monoclonal CD8+ T cells expressing different TCR specificities and phenotypes, by following T cell fate upon transfer into T cell deficient mice lacking selective MHC class I molecules. We found that while the survival of naïve aHY and P14 CD8+ T cells strictly require the presence of the H-2Db restricting element, OT-1 can survive in absence of its H-2Kb restricting element. We also studied the possible role of MHC class I as a resource by following the fate of monoclonal CD8+ T cells in mouse BM chimeras containing limited numbers of MHC class I expressing cells. Our findings suggest that the number of MHC class I expressing cells may control CD8+ T cell survival, lymphopenia driven proliferation and competition.

Notch signaling in lymphocyte development and survival (Alix de la Coste). The role of Notch signaling in T cell commitment during lymphoid development is well established. However, the identity of the ligand that triggers this critical signal in vivo is still unclear. By over expressing Delta-1 and Delta-4 ligands in the hematopoietic cells of athymic nu/nu host mice, we demonstrate that in vivo and in absence of a thymus, Delta-1 or Delta-4 expression is sufficient to promote T cell development from the most immature progenitor stages to complete maturation of both CD8+ and CD4+ αβ T cells. The mature T cells developing in a Delta-1 or Delta-4 enriched environment express a diverse TCR repertoire, are able to proliferate upon in vitro TCR stimulation, but show different profiles of cytokine production after in vitro anti-CD3 stimulation.

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


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