Pierre-André Cazenave

Regulation of TCR α and β gene allelic exclusion during T-cell development

Abstract Early in their development, most T cells become committed to the expression of one, and only one, TCR αβ combination. How do T cells achieve this TCR allelic exclusion? This article discusses the configuration and expression of TCR α and β genes in mature T-cell lines and TCR αβ transgenic mice, and proposes three nonexclusive models to account for the significant occurrence of T cells with two productive α gene rearrangements.

Total and functional parasite specific IgE responses in Plasmodium falciparum-infected patients exhibiting different clinical status

BackgroundThere is an increase of serum levels of IgE during Plasmodium falciparum infections in individuals living in endemic areas. These IgEs either protect against malaria or increase malaria pathogenesis. To get an insight into the exact role played by IgE in the outcome of P. falciparum infection, total IgE levels and functional anti-parasite IgE response were studied in children and adults, from two different endemic areas Gabon and India, exhibiting either uncomplicated malaria, severe non cerebral malaria or cerebral malaria, in comparison with control individuals.Methodology and resultsBlood samples were collected from controls and P. falciparum-infected patients before treatment on the day of hospitalization (day 0) in India and, in addition, on days 7 and 30 after treatment in Gabon. Total IgE levels were determined by ELISA and functional P. falciparum-specific IgE were estimated using a mast cell line RBL-2H3 transfected with a human Fcε RI α-chain that triggers degranulation upon human IgE cross-linking. Mann Whitney and Kruskall Wallis tests were used to compare groups and the Spearman test was used for correlations.Total IgE levels were confirmed to increase upon infection and differ with level of transmission and age but were not directly related to the disease phenotype. All studied groups exhibited functional parasite-specific IgEs able to induce mast cell degranulation in vitro in the presence of P. falciparum antigens. Plasma IgE levels correlated with those of IL-10 in uncomplicated malaria patients from Gabon. In Indian patients, plasma IFN-γ , TNF and IL-10 levels were significantly correlated with IgE concentrations in all groups.ConclusionCirculating levels of total IgE do not appear to correlate with protection or pathology, or with anti-inflammatory cytokine pattern bias during malaria. On the contrary, the P. falciparum-specific IgE response seems to contribute to the control of parasites, since functional activity was higher in asymptomatic and uncomplicated malaria patients than in severe or cerebral malaria groups.

Clusters of Cytokines Determine Malaria Severity in Plasmodium falciparum–Infected Patients from Endemic Areas of Central India

We investigated the role of interferon (IFN)- gamma , interleukin (IL)-1 beta , IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, tumor necrosis factor (TNF)- alpha , and transforming growth factor (TGF)- beta in clinically well-defined groups of Plasmodium falciparum-infected patients manifesting mild malaria (MM), severe noncerebral malaria (SM), or cerebral malaria (CM) and in control subjects from Gondia, a malaria-endemic site in India, as well as in healthy subjects from non-malaria-endemic areas. Two-way coupled cluster analysis revealed 2 clusters of cytokines relevant to clinical subgroups of disease. The first cluster was composed of IFN- gamma , IL-2, IL-5, IL-6, and IL-12, the levels of which were significantly increased during infection but were predominant in patients with MM and allowed us to distinguish them from patients with SM or CM. The second cluster was composed of TGF- beta , TNF- alpha , IL-10, and IL-1 beta , the levels of which were highly correlated with each other in the different clinical groups of patients and significantly increased with disease severity, particularly in CM. Discriminant analyses allowed us to propose a minimal model. Levels of cytokines such as IL-5, IL-1 beta , IL-10, and IL-2 increase with infection. Levels of IL-12, IL-5, and IL-6 discriminate severe forms of malaria from MM. Finally, levels of IL-1 beta , IL-12, and IFN- gamma are relevant for the discrimination of CM from SM: high IL-1 beta levels are associated with CM, and high IL-12 and IFN- gamma levels are associated with SM.

Vβ17 gene polymorphism in wild-derived mouse strains: Two amino acid substitutions in the Vβ17 region greatly alter T cell receptor specificity

Abstract Of 41 wild-derived mouse strains analyzed, 14 contained T cells bearing Vβ17 receptors in splte of the concomitant expression of I-E antigens. Reciprocal F1 and F2 hybrids of one of these strains, PWK, with laboratory strains revealed different patterns of Vβ17 T cell deletions from those observed with Vβ17 T cells from SJL, implying that the two Vβ17 regions are associated with recognition of distinct superantigens. The structures of the Vβ17 alleles differ by two amino acid substitutions, which lie together in an area distant from the predicted site of T cell receptor interaction with peptide-MHC complexes but overlapping with that implicated in Vβ8.2 recognition of Mls-1 superantigen. This demonstrates that the self-superantigen leading to Vβ17 T cell deletion varies with the allele of the receptor gene and confirms that T cell deletions by such ligands involve interactions with a region of the Vβ domain that is distinct from the conventional combining site.

Liver CD4−CD8− NK1.1+ TCRαβ Intermediate Cells Increase During Experimental Malaria Infection and Are Able to Exhibit Inhibitory Activity Against the Parasite Liver Stage In Vitro

Experimental infection of C57BL/6 mice by Plasmodium yoelii sporozoites induced an increase of CD4−CD8− NK1.1+ TCRαβint cells and a down-regulation of CD4+ NK1.1+ TCRαβint cells in the liver during the acute phase of the infection. These cells showed an activated CD69+, CD122+, CD44high, and CD62Lhigh surface phenotype. Analysis of the expressed TCRVβ segment repertoire revealed that most of the expanded CD4−CD8− (double-negative) T cells presented a skewed TCRVβ repertoire and preferentially used Vβ2 and Vβ7 rather than Vβ8. To get an insight into the function of expanded NK1.1+ T cells, experiments were designed in vitro to study their activity against P. yoelii liver stage development. P. yoelii-primed CD3+ NK1.1+ intrahepatic lymphocytes inhibited parasite growth within the hepatocyte. The antiplasmodial effector function of the parasite-induced NK1.1+ liver T cells was almost totally reversed with an anti-CD3 Ab. Moreover, IFN-γ was in part involved in this antiparasite activity. These results suggest that up-regulation of CD4−CD8− NK1.1+ αβ T cells and down-regulation of CD4+ NK1.1+ TCRαβint cells may contribute to the early immune response induced by the Plasmodium during the prime infection.

The mouse Igh-1a and Igh-1b H chain constant regions are derived from two distinct isotypic genes.

Genetic and structural analyses of the mouse genes encoding constant region of immunoglobulin subclasses (Igh-C) have shown that recombination is rare within this cluster which is inherited as a set designated the Igh haplotype. Recent molecular analyses have demonstrated that either DNA exchanges or gene duplications have probably occurred during the evolution of this set of genes. In order to assess the generality of the duplication processes, the presence and expression of two allelic forms of the Igh-1 (γ2a) gene (Igh-1a and Igh-1b) were examined in a large panel of wild mice belonging to Mus musculus domesticus and Mus musculus musculus species. Our data indicate that certain M. m. domesticus animals and most animals in the M. m. musculus group coexpress the two allelic forms of Igh-1. Moreover, genetic studies show that these two immunoglobulin types are encoded by tandemly arranged genes. We propose that wild mice, from which laboratory mice are derived, carry three isotypic γ2 genes (Igh-1a, Igh-1b, Igh-3), and these have given rise to the two isotypes seen in laboratory strains by a deletion/insertion mechanism.

The joining of germ-line Vα to Jα genes replaces the preexisting Vα-Jα complexes in a T cell receptor α,β positive T cell line

Abstract To determine whether T cell receptor genes follow the same principle of allelic exclusion as B lymphocytes, we have analyzed the rearrangements and expression of TCRα and β genes in the progeny of the CD3 + , CD4−CD8− M14T line. Here, we show that this line can undergo secondary rearrangements that replace the pre-existing Vα-Jα rearrangements by joining an upstream Vα gene to a downstream Jα segment. Both the productively and nonproductively rearranged alleles in the M14T line can undergo secondary rearrangements while its TCRβ genes are stable. These secondary recombinations are usually productive, and new forms of TCRα polypeptides are expressed in these cells in association with the original Cβ chain. Developmental control of this Vα-Jα replacement phenomenon could play a pivotal role in the thymic selection of the T cell repertoire.

Constitutive Endocytosis and Degradation of the Pre-T Cell Receptor

The pre-T cell receptor (TCR) signals constitutively in the absence of putative ligands on thymic stroma and signal transduction correlates with translocation of the pre-TCR into glycolipid-enriched microdomains (rafts) in the plasma membrane. Here, we show that the pre-TCR is constitutively routed to lysosomes after reaching the cell surface. The cell-autonomous down-regulation of the pre-TCR requires activation of the src-like kinase p56lck, actin polymerization, and dynamin. Constitutive signaling and degradation represents a feature of the pre-TCR because the γδTCR expressed in the same cell line does not exhibit these features. This is also evident by the observation that the protein adaptor/ubiquitin ligase c-Cbl is phosphorylated and selectively translocated into rafts in pre-TCR– but not γδTCR-expressing cells. A role of c-Cbl–mediated ubiquitination in pre-TCR degradation is supported by the reduction of degradation through pharmacological inhibition of the proteasome and through a dominant-negative c-Cbl ubiquitin ligase as well as by increased pre-TCR surface expression on immature thymocytes in c-Cbl–deficient mice. The pre-TCR internalization contributes significantly to the low surface level of the receptor on developing T cells, and may in fact be a requirement for optimal pre-TCR function.

Identification of two cerebral malaria resistance loci using an inbred wild-derived mouse strain

Malaria is a complex infectious disease in which the host/parasite interaction is strongly influenced by host genetic factors. The consequences of plasmodial infections range from asymptomatic to severe complications like the neurological syndrome cerebral malaria induced by Plasmodium falciparum in humans and Plasmodium berghei ANKA in rodents. Mice infected with P. berghei ANKA show marked differences in disease manifestation and either die from experimental cerebral malaria (ECM) or from hemolytic anemia caused by hyperparasitemia (HP). A majority of laboratory mouse strains so far investigated are susceptible to ECM; however, a number of wild-derived inbred strains show resistance. To evaluate the genetic basis of this difference, we crossed a uniquely ECM-resistant, wild-derived inbred strain (WLA) with an ECM susceptible laboratory strain (C57BL/6J). All of the (WLA × C57BL/6J) F1 and 97% of the F2 progeny displayed ECM resistance similar to the WLA strain. To screen for loci contributing to ECM resistance, we analyzed a cohort of mice backcrossed to the C57BL/6J parental strain. A genome wide screening of this cohort provided significant linkage of ECM resistance to marker loci in two genetic regions on chromosome 1 (χ2 = 18.98, P = 1.3 × 10−5) and on chromosome 11 (χ2 = 16.51, P = 4.8 × 10−5), being designated Berr1 and Berr2, respectively. These data provide the first evidence of loci associated with resistance to murine cerebral malaria, which may have important implications for the search for genetic factors controlling cerebral malaria in humans.

Comparative Study of Brain CD8+ T Cells Induced by Sporozoites and Those Induced by Blood-Stage Plasmodium berghei ANKA Involved in the Development of Cerebral Malaria

ABSTRACT To obtain insight into the mechanisms that contribute to the pathogenesis of Plasmodium infections, we developed an improved rodent model that mimics human malaria closely by inducing cerebral malaria (CM) through sporozoite infection. We used this model to carry out a detailed study on isolated T cells recruited from the brains of mice during the development of CM. We compared several aspects of the immune response related to the experimental model of Plasmodium berghei ANKA infection induced by sporozoites in C57BL/6 mice and those related to a blood-stage infection. Our data show that in both models, oligoclonal TCRVβ4+, TCRVβ6+, TCRVβ8.1+, and TCRVβ11+ major histocompatibility complex class I-restricted CD8 T cells were present in the brains of CM+ mice. These CD8+ T cells display an activated phenotype, do not undergo apoptosis, secrete gamma interferon or tumor necrosis factor alpha, and are associated with the development of the neurological syndrome.