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  Director : ACUTO, Oreste (oacuto@pasteur.fr)


  abstract

 

Our research Unit studies the molecular mechanisms driving T cell activation, a process contributing to adaptive immunity. This includes T cell antigen and ancillary receptor engagement, signal triggering and propagation leading to gene activation and cell differentiation. Signal transduction is governed by protein and lipid kinases, phosphatases, lipases, small GTPases and adapter proteins. We study the regulation of protein tyrosine kinase Lck, Zap-70 and Tec family and of their substrates which connect the TCR and co-stimulatory receptors to various signaling pathways.



  report

cale

Regulation of the protein tyrosine kinase Zap-70

(Vincenzo Di Bartolo, en collaboration avec B. et M. Malissen)

Zap-70 (Zeta chain-associated protein of 70 kDaltons) is a PTK critical for triggering T cell activation. Zap-70 binds to the TCR-associated ITAM (Immune-receptor Tyrosine-based Activation Motif) following engagement with the antigen/MHC. The interdomain B of Zap-70 (a region connecting the two SH2 domains to the kinase domain) contains regulatory tyrosines which become phosphorylated upon TCR triggering. In previous work, we described the TCR-directed phosphorylation of tyrosines 315 (Y315) and 319 (Y319) and studied their respective roles. We demonstrated that the latter exert a key role in Zap-70 activation by the PTK Lck. We have carried out a collaborative study with the group of Bernard and Marie Malissen in Marseille to clarify the role in vivo of these regulatory sites. Recombinant mice carrying a mutation of Tyr315 showed a slight defect in T cell development and activation. In contrast, mutation of Y319 was associated with a profound defect in thymic positive selection and confirms in vivo its importance as a regulatory site of Zap-70 activation. In a parallel study, we identified the functional defect associated to the mutation of Y315. Indeed, through an allosteric mechanism, this residue ensures an effective association of Zap-70 to the ITAM of the CD3-zeta TCR subunits.

A novel transgenic mouse model to study the regulation of naïve CD8 T cells. (Maria Elena Marquez-Campos, Vincenzo Di Bartolo)

From previous studies we generated a gain-of-function mutant of Zap-70 whose activity is strongly augmented only when the TCR is engaged. This finding led us to create a novel transgenic mouse allowing to test the effect of lowering the activation threshold on the survival and tolerance of the CD8 T cells. This mouse expresses the Zap-70 mutant (named " SuperZap ") only in the mature CD8 T cell subset thanks to the usage of an " enhancer " sequence derived from the CD8 gene. The analysis of this mouse shows no apparent change in the number of CD8 T cells, nor signs of activation or autoimmunity. Interestingly however, a drastic modification of TCR-induced cytokine expression pattern is observed in bona-fide naïve CD8 cells of this transgenic model. These results suggest an adaptative differentiation of the peripheral compartment of CD8 T cells hyper-reacting to the self.

Roles of Lck and Itk in T cell response to antigen recognition revealed by calcium imaging

and electron microscopy (Frédérique Michel, in collaboration with A. Trautmann)

Antigen recognition by a T cell is conditioned by cell-cell adhesion and by cytoskeletal remodeling. In collaboration with A. Trautman's group (Cochin Hospital, Paris) we have examined the role played in these processes by Lck and Itk, two protein tyrosine kinases essential for T cell signaling. Early T cell responses (membrane ruffling, Ca2+ response, Antigen presenting cells (APC)-T cell adhesion) were monitored in T cells overexpressing kinase-defective (KD) Lck and Itk mutants. Neither Lck nor Itk are involved in the antigen-independent formation of a labile contact between T cells and APCs. By contrast, the antigen-induced Ca2+ response in a cell population was blunted similarly in both KD transfectants. However, the major effect of Lck-KD was to reduce the probability of giving rise to quasi-normal Ca2+ responses, whereas overexpression of Itk-KD resulted in tuning down all single cell Ca2+ responses. Moreover, Lck, but not Itk, was required for the formation of stable T/APC conjugates and for T cell polarization. Thus, Lck plays an ignition role controlling all the downstream events tested here, whereas Itk amplifies the Ca2+ response but is dispensable for APC-induced adhesive and morphological responses.

Signaling contribution of CD28 to T cell activation.

(Frédérique Michel, Giorgio Mangino, Setsuko Mise-Omata)

By using physiological activation of T cells (e.g. primary T cells or T cell lines stimulated with APCs) we have previously made an entirely novel observation concerning the mechanism of CD28 co-stimulation. We demonstrated that one of the functions of CD28 is to amplify the very first signaling events controlled by the TCR and suggested a novel view of the molecular basis of T cell co-stimulation. By using genetic and biochemical approaches, we then investigated the mechanism by which CD28 modifies the efficiency of TCR signaling using a T cell line model and primary human CD4 T cells. Comprehensively, these studies have demonstrated that: i) CD28 extracellular region critically contributes to T cell/APC cell-cell contact which facilitates TCR engagement. This is likely to be a primary function of CD28 in providing a transient "sealing effect" between the opposing T cell/APC membranes. ii) CD28 intracellular region is essential for optimal Phospholipase C gamma-1 activation and therefore affects the generation of second messengers stimulating calcium influx, PKCs and also Ras activation, as recent studies have demonstrated in T cells. Our data also show that the PTK Itk activated via CD28 is responsible for PLC-g1 activation. iii) CD28 signal, however, is not required (but its adhesion function is) for TCR activation of Zap-70 and for the phosphorylation of the adapter Lat, a key organizer of the signaling cascade and resident in lipid microdomains (rafts/GEMs). In conclusion, our studies indicate that the CD28-generated "second signal" selectively "boosts" part of the signaling machinery activated by TCR engagement to amplify and sustain cellular activation.

Figure legend :

Molecular organisation of T cell signaling :: The diagram illustrates the protein components of the signaling pathways activated by the TCR (blue) and CD28 (orange), respectively. Our recent work indicates that CD28 provides quantitative and qualitative contributions to the activation signal initiated by the TCR. GEMs : Glycosphingolipid-Enriched Membranes. LAT, Linker for Activation of T cells, is an adapter protein resident in the GEMs that serves as the central " organiser " of the T cell signaling machinery.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

HOUSSIN, Wendy, whoussin@pasteur.fr

ACUTO, Oreste, IP (Unit Director, oacuto@pasteur.fr)

MICHEL, Frédérique, IP (Senior researcher, fmichel@pasteur.fr)

DI BARTOLO, Vincenzo, IP (Researcher, vbartolo@pasteur.fr)

MANGINO, Giorgio, Post-doc

MISE, Setsuko, Post-doc

BLANCHET, Fabien, PhD student

IRLES, Claudine, PhD student

MARQUEZ CAMPOS, Maria Elena, PhD student

ATTAL-BONNEFOY, Géraldine, technician, gattal@pasteur.fr

DUFOUR, Evelyne, technician, edufour@pasteur

SECHET Emmanuel, technician, esechet@pasteur.fr


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