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  Director : Pierre-André CAZENAVE (cazenave@pasteur.fr)



Our laboratory focusses on immunophysiopathological processes during parasitic infections (Chagas disease and malaria), as well as fundamental studies of immune repertoires.



Study of the plasticity of the immune system (A. Six, P.A. Cazenave)

We study the plasticity of the immune system and more particularly of the B compartment (B lymphocytes and immunoglobulins) in extreme situations where the VH and VL regions germinal repertoire is very reduced. For this purpose, we derived a C57BL/6 congenic mouse, T15i.K-.l SEG. It is knocked-out for the expression of the k Ig light chains and possesses the M.spretus SEG mouse l locus which is characterized by a deletion of the l1-l3 unit of recombination and thus expresses only 2 Vl (Vl2 and Vlx); it is transgenic ("knock-in") for the VHT15 (this gene codes for the rearranged heavy chain of the TEPC15 myeloma presenting an anti-phosphorylcholin activity). We first characterized the lSEG locus and studied the repertoire of immunoglobulins and B lymphocytes (before and after immunization against two model antigens) of the B6.k-.lSEG mouse, intermediate of the B6.T15i.k-.lSEG "ultra-monoclonal" mouse. This study was carried out with a great variety of techniques: RIA, ELISA, SDS-PAGE, FACS, anchored PCR and Immunoscope. Having recently obtained the B6.T15i.k-.lSEG mouse, the same study is in course for this mouse.

The B6.T15i.k-.lSEG mouse model provides a remarkable tool to analyse the evolution of the lymphocyte repertoire, not only at the systemic level but also in various lymphocyte compartments. Indeed, a flux cytometry analysis allows to distinguish (and consequently to isolate) four B cell sub-populations characterized by the variable domains that they express at their surface: VHT15+, lx+; VHT15,l2+; VHT15-,lx+; VHT15-,l2+. The appropriate use of antibodies allows to sort out the B lymphocyte sub-populations according to their belonging to B1-a , B1-b or B2 groups, and this in various lymphocyte compartments. One can thus analyse the repertoire of these various lymphocyte compartments but also study their future in various situations following transfer into immunodeficient mice.

Tolerance of CD8+ T cells to the kappa light chains (Lk) of maternal immunoglobulins. (D. Rueff-Juy)

Analyzing T cell repertoire and functions in k-/- pups born to heterozygote k+/- knock-out mother (k+/-) we could show (1) that tolerance of Ck specific CD8+ T cells, which is strong until 5 weeks of age, is reversible and wanes with the decrease of Igk concentration in the pup's sera and suggest (2) that CD4+ regulatory T cells play a fundamental role in tolerance of CD8+ T cells since anti-CD4 mAb treatment abolishes this state of tolerance. The antigen recognized by regulatory T cells remains in most cases and particularly in non-transgenic models a large mystery. To investigate this important question we have introduced the k mutation in A or in CD1 knock-out mice (k-/- A-/- and k-/- CD1-/-). In k-/- A-/- mice, the CD4 population is strongly decreased and part of remaining CD4 cells are known to be selected on the CD1 molecule and to mainly consist of NKT cells.

Using both types of mice in conjunction with control antibodies or anti-CD4 mAb we show that the CD8+ anti-Ck T cells are tolerized by I-A- but not by CD1- restricted CD4 regulatory cells. These tools should allow us to direct our search towards the characterization of the antigenic structure recognized by the CD4 regulatory T cells.

Immunopathology of Trypanosoma cruzi infectieux process (P. Minoprio)

We have previously proposed that mitogens and superantigens, moieties that are essential components of micro-organisms can be responsible for the initiation of non-specific polyclonal lymphocyte responses and, per se, explain the strategy used by pathogens to avoid the host immune responses and to ensure persistence. We had shown that metacyclic infective forms of Trypanosoma cruzi secrete molecules responsible for such a mechanism of immune evasion. Using biochemical and molecular approaches we described a T-cell independent, B-cell mitogenic parasite protein (TcPA45) involved in polyclonal activation of B lymphocytes. The analysis of genomic organization and transcription of the gene encoding TcPA45 showed the presence of two alleles per haploid genome located in different parasite chromosomes and differential mRNA transcription in different parasite stages. Immunolocalization of TcPA45 protein confirmed that non-infective and infective parasite forms differentially express intracellular and secreted forms of TcPA45. Interestingly, we described that TcPA45 protein is also the first eukaryotic proline racemase, an enzyme that catalyses the inteconversion of L- and D-proline enantiomers. Both TcPA45 isoforms are co-factor independent proline racemases with similar Vmax and Kmax values, specific buffer and pH requirements that are compatible with the enzyme being functional in either internal or external cell compartments. Moreover, TcPA45 mitogenic activity is abolished or severely compromised whenever enzymatic activity is inhibited by specific inhibitors or substrate excess. We have also shown that engineered parasites that overexpress Tc45 gene/protein do better differentiate and are more invasive to host cells with greater pathological effect in vitro and in vivo. Conversely, parasites bearing antisense constructs of the Tc45 gene have severe growth difficulties, or are not viable, suggesting that proline racemase may be essential for parasite metabolism and virulence. These recent findings have vast implications for parasite biology and pathogenicity, as well as for the possible roles of D-amino acids in immune phenomena. Additionally, given the important immunoregulatory role mediated by natural antibodies, putative deviations in the pre-existing antibody repertoires in susceptible or resistant hosts could be important to facilitate or neutralize parasite induced mitogenic polyclonal activation. Our results thus suggest that the control of mitogenic/racemase parasite activities would be a major target for immune intervention and would favor and re-directs host immune reactions to the parasite resulting in a improved prognosis.((Laboratory web site)

Study of the TCRB repertoire during the mouse cerebral malaria (A. Six, S. Pied, P.A. Cazenave)

In B10.D2 mice, a T cell expansion was noted during infection by P.berghei ANKA in individuals developing cerebral malaria (CM+). We then started to study the repertoire of the T lymphocytes of the blood and spleen during P. berghei infection of B10.D2 mice to determine the nature of the T expansion and to study the evolution of the repertoire in CM+ mice by comparison to infected mice that do not develop cerebral malaria (CM-).

The Immunoscope technique was used to obtain a qualitative image of the TCRB repertoire by the description the CDR3 length profiles for each V gene segment. Due to the high number of samples to analyse, we developed a set of software (ISEAPEAKS®, Institut Pasteur) to allow computer analysis of the data. The first program does the automatic extraction, smoothing, sorting and formatting of Immunoscope raw data in an Excel sheet. The second programs allows to assemble the data from various samples and analyse this peak database. Various scoring methods of the disturbance of immune repertoires were implemented and used as well as a new method specifically dedicated to searching recurrent oligoclonal expansions.

We were able to determine that the V8+ cell expansion is polyclonal, excluding the possibility of a classical antigenic stimulation. On the other hand, this approach allowed us to choose the most perturbed V presenting the most recurrent oligoclonal expansions for a more detailed analysis by the study of the V-J CDR3 length distribution. Again, the most perturbed combinations, presenting significant recurrent oligoclonal expansions were chosen for sequencing. Recurrent rearrangements were found the association of which with cerebral malaria under scrutiny.

Genetic determinism of the resistance to malaria after infection of the mouse by Plasmodium berghei ANKA( S Pied, P.A. Cazenave)

The majority of the laboratory mouse strains infected by the lethal P.berghei ANKA line develop fatal cerebral malaria (CM), with the exception of the DBA/2 mouse which is resistant (CM). We showed that it is protected from CM due to the integration in its genome of a proviral sequence (mtv7) which includes an ORF region coding for a superantigen. The CM resistant mice die later from hemolytic anaemia due to hyperparasitemia (HP+).

Analysis of a series of 12 new mouse strains, derived from individuals trapped in very different Eurasian regions, allowed us to identify 6 strains that present the CM- phenotype of strong resistance to CM. As it was expected, these CM- mice die from the consequence of hyperparasitemia (HP+).

To determine the genetic bases of resistance to CM, we crossed one of these CM- mice (W line) with the C57BL/6 mouse which is particularly sensitive (CM+). The F1 individuals had the CM- phenotype of the W parental line. To screen loci which would contribute to the resistance, we analysed a cohort of mice resulting from back-crosses with the other C57BL/6 parental line. An analysis of the genome of this cohort led us to demonstrate a significant association of the CM- phenotype to two genetic regions: one on chromosome 1 (c 2= 18.99, p < 1.3x10-5), the other one on chromosome 11 (c 2 = 16.51, p < 4.8x10-5). In a second series of experiments, we analysed mice of the F2 generation. We observed in 17 % of the F2 individuals, an unexpected phenotype, until then unknown in the infection by P.berghei. These CM- mice also present a HP- phenotype of resistance to hyperparasitemia which they control perfectly and survive. We showed that this HP- phenotype is associated to two genetic regions: one on chromosome 9 (c 2= 14.53 , p=0.001), the other one on chromosome 11 (c 2= 14.78, p < 0.001).

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  Office staff Researchers Scientific trainees Other personnel

Berson Michèle, IP, (mberson@pasteur.fr)

MINOPRIO Paola, IP, (pmm@pasteur.fr)

PIED Sylviane, CNRS, (spied@pasteur.fr)

ROLAND Jacques, CNRS, (jroland@pasteur.fr)

RUEFF-JUY Dominique, CNRS, (rueffjuy@pasteur.fr)

SIX Adrien, Université Pierre et Marie Curie (Adrien.Six@pasteur.fr)

AMRANI Yacine, PhD student.

BAGOT Sébastien, PhD student.

CHAMOND Nathalie, PhD student.

COLLETTE Alexis, PhD student.

GREGOIRE Christophe, Post-Doctorant.

SOULARD Valérie, PhD student.

BARBIER Eliane, IP (ebarbier@pasteur.fr)

BERNEMAN Armand, IP (berneman@pasteur.fr)

CORRE Jean-Philippe, IP (jphc@pasteur.fr)

COSSON Alain, IP (acosson@pasteur.fr)

COTNOAN Nicolas, IP (coatnoan@pasteur.fr)

DRAPIER Anne-Marie, CNRS, (amdrapie@pasteur.fr)

GORGETTE Olivier, IP, (ogorgett@pasteur.fr)

VOEGTLE Danièle, CNRS, (dvoegtle@pasteur.fr)


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