The main scientific objectives of the Lymphocyte Population Biology Unit are:

To investigate these different issues we have followed several lines of research. We summarize our most important observations during 2006:

Agonist driven development of CD4+CD25+Foxp3+ regulatory T cells requires a second signal mediated by Stat6. (V. Sanchez-Guajardo, S. Garcia & A. Freitas)
 The factors that induce Foxp3 expression and Treg cell development remain unknown. We studied the role of Stat4 and Stat6 in agonist-driven generation of antigen-specific Foxp3-expressing Treg cells. Our findings indicate that fully efficient induction of Foxp3 expression and development of antigen-specific Treg cells requires the synergistic action of two signals: a TCR-mediated signal and a second signal mediated by Stat6. Indeed, by comparing the development of wild-type, Stat4- and Stat6-deficient HA-specific T cells in the presence of HA-antigen, we found that the absence of Stat6 impaired the generation of antigen-specific CD4+CD25+Foxp3+ cells. Moreover, in transgenic mice expressing a constitutively active form of Stat6, we found that the fraction of CD4+Foxp3+ cells largely exceeds that of control wild-type littermates. Overall these findings support a role for the Stat6 pathway in Treg cell physiology.

Endogenous TCR recombination in TCR transgenic Rag-2 deficient mice (C. Montaudouin, S. Garcia & A. Freitas)
 The transfer of monoclonal TCR Tg T cells from H2k 5CC7 Rag-2-/- mice, which are specific for the pigeon cytochrome C, into allogenic H2b Rag-/-gc-/- hosts resulted in the accumulation in the host mice of donor T cells expressing non-Tg TCRs. Molecular analysis of the expressed TCRs by Immunoscope confirmed that these donor T cells expressed a broad diversity of recombined endogenous TCRs. Nucleotide sequence analysis of the expressed non-Tg TCR indicates that we are in presence of a mechanism of “classical” Rag-dependent recombination in spite of the Rag-2 deficiency of the 5CC7 donors. We found that T cells expressing a non-transgenic TCR pre-exist in a very limited number both in the thymus and at the periphery of the naive 5CC7 Rag-2-/- mice. These results have important implications for the studies using TCR Rag-/- transgenic mice.

TCR specificity and clonal competition (C. Leitao, A. Freitas & S. Garcia)
The involvement of TCR/MHC-peptide interactions in the survival of CD4 and CD8 naïve T cells has been well documented. It has been proposed lymphopenia driven proliferation (LDP) is also controlled by TCR/MHC-peptide interactions. In both cases, these interactions would be of weak affinity and similar of those involved during thymic positive selection. Nevertheless, the nature of these interactions and the overlapping between those involved in the survival vs. LDP of T cells is unknown.
We asked to which extend TCR specificity determines clonal competition for survival and/or lymphopenia driven proliferation (LDP). We found that resident monoclonal T cells in TCR Tg Rag-/- mice, or monoclonal LDP derived TCR Tg T cells in Rag-/- hosts, inhibit the survival and/or the proliferation of T cells presenting the same TCR, but not of TCR Tg T cells bearing a different specificity. Using different transfer approaches we extended this notion to polyclonal T cells. Our findings show that T TCR-specificity determines peripheral T cell fate and indicate that specific sp-MHC complexes are limiting resources shared between developing, surviving and proliferating T cells.

Bystander CD4+ T cell help to CD8+ T cells during lymphopenia driven proliferation (LDP) (B. Zaragoza & A. Freitas)
 Since a fully functioning immune system requires a variety of lymphocyte sub-sets,  lymphpocyte homeostasis should control both absolute numbers and relative sizes of each sub-population; otherwise, deregulation and disease may occur. We studied CD8:CD4 T cell interactions during LDP. We found that the co-transfer of CD8+ T cells sub-sets with naïve CD4+ cells results in the 10-fold increase of the number of CD8+ T cells recovered irrespectively of the CD8 T cell sub-set transferred. This “bystander helper” effect results in the preferential accumulation of cells with a TEM phenotype. The mechanisms that mediate the CD4 bystander helper effect are currently under investigation.

The Bw cells, a novel B cell population conserved in the whole genus Mus (A. Thiriot & D. Rueff-Juy)
Studying 9 inbred and 39 outbred wild-derived mouse strains as well as 7 laboratory mouse strains we found that the CD5+ Mac-1+ peritoneal B cell population is only present in Mus musculus domesticus subspecies. In contrast, a novel B cell population Bw is present in the whole genus Mus. We showed that this population is not restricted to the peritoneal cavity but is also present in spleen, lymph nodes and peripheral blood lymphocytes. This Bw population shares some features with but is distinct from the B-1 and B-2 populations. It is enriched in autoantibody specificities and in anti-PC antibodies but produces only low concentrations of IL-10 in contrast to B-1 cells. This population may play a key role in innate immunity.

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

2005

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 of IgM-secreting cells (Yi Hao).
We have studied the role of BM B cell production in the renewal of peripheral B cells and the feedback mechanisms that control the entry of newly formed B cells into the peripheral B cell pools. When resting lymph node B cells are injected into B cell deficient hosts a fraction of the transferred cells expands and constitute a highly selected population which survives for prolonged periods of time by continuous cell renewal at the periphery. Although the number of donor B cells recovered is low, a significant fraction shows an activated phenotype and the serum IgM levels are as in normal mice. This population of activated B cells is resistant to replacement by a new cohort of B cells and is able to feedback regulate both the entry of newly formed B cells into the peripheral pool and terminal differentiation. These findings suggest that peripheral B cell selection follows the rule “first come, first served” and that IgM-secreting cells are generated from a pool of stable activated B cells with an independent homeostasis.
We questioned whether Ig secretion by the first population was responsible for the feedback effect observed. To test this hypothesis we used B cells from mutant mice, which are unable to secrete IgM. Our findings indicate that IgM secretion is not determinant in the feedback effect. We are currently testing alternative mechanisms. These experiments were done in collaboration with Dr. M. Ehrenstein (London). 

Toll-like Receptor 9 controls key checkpoints of B lymphocyte development (Yi Hao).
Toll-like receptors are involved in the expansion of autoreactive B lymphocytes. In collaboration with Drs. J.P. Pereira and P. Vieira (U. Dev. Lymph., Inst. Pasteur) we showed that the immature B cell compartment of young, but not adult, TLR9-deficient mice developed earlier than that of C57BL/6 mice. By a competitive repopulation strategy we showed that TLR9 signaling is necessary at key checkpoints of the murine B cell developmental program, selection into secondary lymphoid organs, and differentiation into Ig-secreting plasma cells.

CD8 T cell survival (Yi Hao).
In collaboration with Nicolas Legrand we continued to compare 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 and even in complete absence of any MHC class I molecule. 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. We found that the number of MHC-expressing cells and the ability of the TCR to recognize different MHC molecules determine CD8+ T cell survival and the size of the peripheral pool. Since these different capacities control clone size and niche formation, they likely have a major role in the final composition of peripheral T cell repertoires. By comparing the fate of naive and activated/memory cell from diverse polyclonal CD8+ T cell populations in absence of MHC class I, we illustrate that cells expressing promiscuous TCRs are preferentially found in the “natural memory/activated pool”.

Role of the MHC-ligand/TCR interactions in the survival and LDO of CD8 T cells (Sylvie Garcia).
The involvement of TCR/MHC-peptide interactions in the survival of CD4 and CD8 naïve T cells is well documented. It has also been proposed lymphopenia driven proliferation (LDP) is controlled by specific TCR/MHC-peptide interactions. In both cases, these interactions would be of weak affinity and similar of those involved during thymic positive selection. Nevertheless, the nature of these interactions and the overlapping between those involved in the survival vs. LDP of T cells is unknown.
In order to answer this question, we have used a model consisting of injecting CFSE labeled polyclonal CD8 T cells into Rag-/- hosts or OT-1 Rag-/- hosts transgenic for a Class I-restricted OVA-specific TCR. While virtually all T cells resulted of 8 divisions or more (CFSE-) 4 to 5 weeks after transfer into Rag2-/- hosts, in OT-1 hosts a fraction of the donor cells did not proliferate and remained CFSE+ and CD44lo. We studied whether the absence of proliferation of the CFSE+ cells was to due to a lack of “unspecific” (cytokines for example) or/and “specific” (MHC-ligand interactions) resources. CD8 T cells which remained CFSE+ were sorted and re-transferred into different secondary hosts. We found that while they divided in rag2-/- hosts excluding an intrinsic proliferative defect, they were still unable to proliferate in OT-1 hosts. Interestingly, these cells divided after transfer into other P14 hosts expressing a transgenic TCR class I-restricted and specific for the gp33 of LCMV. In these hosts, expansion was similar to that observed in rag-/- hosts, suggesting that these T cells may require “specific” MHC-peptide/TCR interactions to proliferate similar to those required by OT-1 T cells to survive. When co-transferred together with OT-1 T cells into rag2-/- hosts, the CFSE+ CD8 T cells out-compete OT-1 T cells, suggesting than here again CFSE+ T cells require “specific” MHC-peptide interactions to proliferate similar to those required by OT-1 T cells for LDP. Altogether, these findings indicate that the TCR/MHC-peptide interactions involved in the survival and the LDP of OT-1 T cells overlap. They  provide a relevant molecular basis to the occurrence of “spontaneous” auto-immune” lymphoproliferative disease in lymphopenic hosts engrafted with mature T cells.

Differential role of STAT proteins in the selection of antigen-specific of CD4+ T cells (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. Because Stat4 and Stat6 proteins are implicated in the Th1 vs Th2 generation and maintenance, respectively, we compare in this study the kinetics of Stat4-/- and Stat6-/- CD4+ T cells.  We had previously found that during LDP, T cells activated the Stat4 pathway and down-regulated Stat6, which conferred to Stat6-/- T cells a slight proliferation advantage that in a competitive situation had major late repercussions, because it modified the final homeostatic equilibrium of the populations and favoured the establishment of Th1-like CD4+ T cell dominance.
To further investigate if the observed proliferative advantage was also seen when CD4+ T cells were primed by cognate antigen, we crossed our Stat deficient mice with TCR transgenic mice specific for virus' hemagglutinin (TCR HA, peptide 111-119).  Peripheral transfer of TCR HA+ Stat-/- cells into lymphopenic mice, lacking or expressing cognate antigen, revealed that the proliferative advantage seen in the Stat6-/- T cell population was kept when primed by its specific antigen.  TCR HA+ Stat6-/- cells attaining a higher plateau than their Stat4-/- counterparts and out-compete them when co-transferred.  We then proceeded to analyze the effect of antigen presence during thymus T cell development.  To this effect we made BM chimeras of irradiated hosts expressing or lacking the HA peptide and BM from Stat6-/- and/or Stat4-/- TCR HA Rag2-/-.  Under these conditions, TCR HA+ Stat6-/- cells only show a proliferative advantage, have higher number of peripheral CD4+ cells, in chimeras expressing antigen.  Furthermore, the presence of antigen results in the accumulation of CD25 FoxP3+ CD4+ T cells of Stat4-/- origin, suggesting that the Stat pathways are involved in CD4+ T cell development and implicating them for the first time in the modulation of Treg development.

Competition and survival among memory T cells (Catarina Leitao, Sylvie Garcia).
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. The capacities of newly generated T cell memory subpopulation to out-compete preexistent T cell memory subsets has been observed during LCMV infection. This out-competition was only shown for the CD8 compartment and not for the CD4 compartment. This process lead to the qualitative impoverishment of the memory pool (named attrition). We ask: 1- whether CD4 memory is also affected by attrition during bacterial infections. 2- the role of inflammatory signals in this process. To address these 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 into T cell deficient hosts. In these mice, each subsets can be easily distingued by the differential expression of Va, Vb and Ly5 and Thy1 alleles. Sequential immunizations of the different TCR transgenic naïve T cell subsets contained in the hosts are performed. 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.

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

Last update : October 1st, 2012