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  Director : FREITAS Antonio (afreitas@pasteur.fr)


  abstract

 

The main scientific objectives of the UBPL are the study of :

  1. B and T cell homeostatis.
  2. The dynamics of the lymphocyte populations and the mechanisms of lymphocyte survival.
  3. The role of lymphocyte competition in lymphocyte selection and in the control of the immune responses particularly in the induction and persistence of memory.



  report

cale

I. Lymphocyte homeostasis.

B cells. (Fabien Agenes & Manuela Rosado).

In the adult mouse the number of B cells remains constant in spite of the continuous production of new cells in the bone marrow (BM). The mechanisms that determine the number of lymphocytes in an adult mouse are poorly understood. It is not known why in normal physiological conditions the number of lymphocytes is what it is. We used B-cell deficient mice, with a developmental arrest of B cell production at the pro-B to pre-B stage of B cell differentiation to study the ability of a limited number of normal B cell precursors to populate peripheral B cell pools. We showed that the number of B cell precursors does, not determine the physiological number of peripheral B cells. A similar conclusion was obtained after parabiosis between one normal and two or three B cell deficient mice. These results demonstrate that peripheral B cells number is not determined by the rates of BM B cell production, but it is limited at the periphery. In the same set of chimeras we also showed that the compartment of activated IgM-secreting B cells was controlled by autonomous homeostatic mechanisms since the number of cells it comprises was regulated independently of the size of the mature B cell pool. These results support a model of the immune system in which the size of the different B cell compartments, i.e. pre-B, resting B and IgM-secreting, is autonomously regulated.

In an immune system where there is a continuous excess of B cell production and the total number of B cells is kept constant each newly produced B cell can only establish itself upon loss of other cells. The persistence of B cells at the periphery could, however, be modified by the continuous arrival of the newly formed BM migrants while the fate of the latter at the periphery could also be altered by the presence or absence of resident B cell populations. We investigated the possible mutual influences between established resident populations of peripheral B cells and newly formed B cell migrants. When we studied the fate of mature lymph node (LN) B cells injected into immune-deficient hosts unable to produce B cells, we found that a fraction of the transferred population of LN B cells expanded and persisted for prolonged periods of time.In summary our results show that B cells remaining after two weeks of transfer in immune-deficient hosts represent a stable B cell population which persists for prolonged periods of time by self-renewing, resists replacement and can modify the fate of newly coming B cells.

These findings suggest a hierarchical organization in which the first priority is the maintenance of normal serum IgM levels to ensure both a first natural barrier of protection while keeping a maximum of repertoire diversity among the resting B cell compartment.

T cells. (Afonso Almeida, Jose Borghans, Nicolas Legrand & Alix de la Coste).

The mechanisms that determine the number of T lymphocytes in the peripheral lymphoid system are poorly understood. In young adult mice there is a continuous seeding of the periphery by newly formed thymus migrants. The number of peripheral T cells remains, however, constant. This implies that either 1) the migrant cells are rapidly lost without ever colonizing the periphery, or 2) there is a continuous replacement of the peripheral cells by recent thymus migrants. We developed a novel strategy that allows a quantitative assessment of the contribution of the thymus T cell production and export to the establishment and maintenance of the peripheral T cell pools. We studied the ability of a limited number of T cell precursors to populate the peripheral T cell pools. Our results have major implications to the understanding of the mechanisms of T cell restoration after BM transplantation or after tri-therapy of HIV infected individuals. We show here that although the size of both naive and memory peripheral T cell pools are regulated largely independently of thymus output, complete T cell recovery requires a minimally functional thymus and can only be ensured with a minimal number of competent DN precursors. In this case attempts to reconstitute thymus function are recommended when the peripheral T cell pool is grossly depleted. We also show that insufficient production of mature T cells in the thymus results in deficient peripheral T cell numbers, preferential selection of T cells into the memory/activated pool and oligoclonal repertoires i.e. a situation that mimics the evolution of the immune system during aging.

We have also investigated the mechanisms controlling the expansion of CD45RBhighCD25-CD4+ and CD45RBlowCD25+CD4+ T cells after transfer into T cell deficient CD3-/- hosts. We found that when the two T cell populations were co-injected, the presence of CD45RBlowCD25+CD4+ T cells limited the expansion of the CD45RBhighCD25-CD4+ T cells. In sequential cell transfer experiments we found that the transfer of a limited number (5x104) of CD45RBlowCD25+CD4+ T arrests the growth of an expanding population of 5x106 resident CD45RBhighCD25-CD4+ T cells. These results show that the suppressive effects have real physiological relevance and must be particularly efficient in local environments, in particular when the number of CD25+ cells surmount that of proliferating naive CD4 T cells. In conclusion these studies make evident the role of T cell interactions in the homeostatic control of the size of the peripheral CD4+ T cell pool. CD45RBlowCD25+CD4+ T cells act by limiting the accumulation of dividing CD45RBhighCD25-CD4+ naive T cells, i.e. they control differentiation of naive CD4 T cells into the activated cellular compartment.

II. Lymphocyte competition and survival.

In an immune system where new lymphocytes are continuously produced in excess but their total numbers are kept constant, newly generated cells have to compete with other newly produced or resident cells to survive. Competition can be defined as "an interaction between two populations, in which, for each, the birth rates are depressed or the death rates increased by the presence of the other population". There are two main established criteria accepted as evidence of competition among populations: 1. The presence of competitors should modify the equilibrium size of a population and 2. The presence of competitors should alter the dynamics, e.g. the life span of a population. The question of whether competition arises between B and T cells was addressed by comparing the development and the fate of BCR-Tg, TCR-Tg and non-Tg populations in several different lines of mouse BM chimeras. It was found that: a) when injected alone, Tg and non-Tg cell populations show an identical behavior and generate peripheral pools of similar size. b) When Tg and non-Tg cells are mixed in the same host they initially accumulate at the same rate. However, after reaching steady state numbers there is a preferential selection of the non-Tg cells at the periphery. These observations fulfil the first criteria for competition since they demonstrate that the presence of non-Tg populations modifies the number of the Tg cells. In these experiments it was also found that the life expectancy of the Tg B and Tg T cells varied according to the presence and the type of other competing cells. These latter findings fulfil the second main criteria required for the definition of competition as they prove that the presence of competitors alters the life span of a population. In an immune system where the total number of cells is limited, cell survival can no longer be a passive phenomenon, but rather a continuous active process where each lymphocyte must compete with other lymphocytes. It can be said that lymphocytes follow the Red Queen Hypothesis postulate "it takes all the running you can do to keep in the same place".

B cells. (Emmanuelle Gaudin & Manuela Rosado).

Lymphocyte repertoires are shaped in primary lymphoid organs at the early stages of B and T cell development by episodes of positive and negative selection. To have a positive selection event it may suffice that one lymphocyte meet its specific antigen. In contrast, complete negative selection requires that all lymphocytes from the same clone meet the antigen. Whatever the selection mechanism involved it is evident that the dose of antigen must play a major role in the establishment of both B and T cell repertoires. We studied the effects of varying quantities of self-antigen in B cell development. We studied the development of HEL-specific B cells expressing different numbers of Ig receptors in BM chimeras producing varying amounts of HEL neo self-antigen. In the chimeras expressing high concentrations of HEL, B cells are deleted or functionally impaired independently of the number of receptors expressed. In chimeras with low HEL levels, B cells expressing normal numbers of HEL-specific receptors escape negative selection, while B cells expressing fewer receptors with a decreased apparent affinity are positively selected. In these mice, the number of B cells is 20-fold higher than in control HEL- chimeras and a fraction of the cells is activated and secretes HEL-specific IgMs that form immune complexes with circulating HEL. These findings have important implications for the understanding of both Ig allelic exclusion and B cell homeostasis. They also suggest that the "natural" self-reactive serum IgMs result from the selection and activation of low avidity B cells.

T cells. (Nicolas Legrand & Sylvie Garcia).

We have investigated the conditions necessary in vivo for the survival and expansion of naive and memory antigen-specific CD8- T cells. To study the TCR interactions required for the survival or division of naive CD8 T cells we compared their fate after transfer into irradiated hosts, that differed in MHC class I and HY antigen expression. Naive T cells could survive in a resting state in female CD8-deficient mice: they did not incorporate BrdU and the number recovered was constant from day one up to two weeks after injection. Expansion of naive cells required stimulation with male antigen because they divided only after transfer into male CD8-deficient hosts. Naive T cells survival required the right MHC restricting element. In mice lacking H-2Db, or expressing no class I, (H2-Db-2m-) naive cells did not survive but decayed to an average of 3% of the injected cohort at one week, 1% at 13 days and were undetectable at 2 weeks. This decay correlated with the absence of interactions with the MHC restriction element, because naive H-2Db restricted Tg cells persisted after transfer into H-2Kb-deficient mice expressing H-2Db. Thus, as described during thymus positive selection a minimal state of cell activation may allow survival, in the absence of cell division. We next studied the TCR interactions required to maintain CD8+ T cell memory. When memory Tg cells were stimulated after transfer into male CD8- mice their rate of division (>90% BrdU+ cells) was higher than that of naive cells. In contrast to naive cells, memory cells transferred into female CD8- hosts also divided extensively (70% where BrdU+) and survived and divided in mice lacking the H-2Db restricting element (42% BrdU+). In mice lacking class I (H2-Db-2m-mice), about 30% of memory cells still incorporated BrdU, indicating a response to autocrine or environment growth factors, even in the absence of T cell stimulation. However, this response was not sufficient to maintain memory T cells; they disappeared progressively. Two weeks after T cell transfer, donor cells in class I deficient (H2-Db-2m-) host mice were barely detectable.

 

Activation of T lymphocytes in presence of cyclosporin A modifies the program of gene expression (Paolo Truffa-Bachi, Laurent Mascarell, Jean Kanellopoulos, Rodolphe Auger & Véronique Cadet-Daniel)

Cyclosporin A (CSA) is an immunosuppressive drug widely utilized in organ transplantation. Although extremely efficient, CSA does not induce an immunological tolerance towards the graft antigens. CSA should then be given for all life leading to an immunosuppressed state that facilitates the outcome of tumors. In addition, CSA may act on tumorogenesis by other mechanisms such as the induction of genes encoding for proto-oncogenes or growth and differentiation factors for a variety of tissues. During the analysis of the proteins synthesized by activated T-cells, we found that a large number (‰ 150) were found only in presence of CSA. The importance of such novel protein synthesis in tumorogenesis requires the characterization and the analysis of their role. We have presently identified a set of genes that are induced by CSA. The expression of two of these genes was increased 5 to 10 fold by the immunosuppressor. Although the role of these genes is not yet known, we have found that their expression is tissue specific: the corresponding mRNA is found in the spleen, lymph nodes, brain, kidney and bladder. Constructions allowing the expression of these two genes have been performed and their transfection in various cell lines should allow defining their eventual role in cell tumorogenesis.



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  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

VOUGNY, Marie-Christine (mcvougny@pasteur.fr)

FREITAS, Antonio, IP (Pr, afreitas@pasteur.fr)

GARCIA, Sylvie, IP (Assistant, sygarcia@pasteur.fr)

AGENES, Fabien, INSERM (CR2, fagenes@pasteur.fr)

DE LA COSTE, Alix, post-doct

BORGHANS, Josephina, post-doc

ALMEIDA, Afonso, PhD Student

GAUDIN, Emmanuelle, PhD Student

LEGRAND, Nicolas, PhD Student

MASCARELL, Laurent, PhD Student

SANCHEZ GUAJARDO, Vanesa, PhD Studient

MAILHE-LEMBEZAT, Marie-Pierre (tech Sup Labo, mpmailhe@pasteur.fr)

CADET-DANIEL, Véronique (Tech Sup Labo, vcadet@pasteur.fr)

AUGER, Rodolphe (Ingénieur CNRS)

VOUGNY, Marie-Christine (Sec. Direction, mcvougny@pasteur.fr)


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