Analyse intégrative de l’activation des lymphocytes T

Publications de l’équipe

Année de publication : 2009

Alain Fischer, Capucine Picard, Karine Chemin, Stéphanie Dogniaux, Françoise le Deist, Claire Hivroz (2009 Oct 16)

ZAP70: a master regulator of adaptive immunity.

Seminars in immunopathology : 107-16 : DOI : 10.1007/s00281-010-0196-x En savoir plus
Résumé

The protein tyrosine kinase ZAP70 became the subject of intense scrutiny in the early nineties, when ZAP70 mutations were characterized in several young patients presenting with severe T cell immunodeficiencies. The association of a lack of expression of ZAP70 with an immunodeficiency consisting in a markedly reduced T lymphocyte-mediated immunity highlighted the crucial role of this tyrosine kinase in T cell development and function. This discovery was soon accompanied by the characterization of the substrates of ZAP70 and the signalling cascades that depend on ZAP70 activity. These studies demonstrated that ZAP70 was indeed at the crossroad of several signalling pathways that control T lymphocyte development and function. Recently, a revival of interest for this protein came again from studies associating abnormal ZAP70 expression with pathological conditions. Some chronic lymphocytic leukemia B cells were shown to express ZAP70, and this expression was correlated with bad prognosis. Mouse models also revealed that partial defects in ZAP70 activity can be associated with autoimmunity. These last results suggested that ZAP70 is involved in the fine balance between immunity and tolerance. In this review, we will discuss the role of ZAP70 in T cell activation and focus on what we learnt from pathological conditions associated with defective expression or activity of the ZAP70 kinase.

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Capucine Picard, Stéphanie Dogniaux, Karine Chemin, Zofia Maciorowski, Annick Lim, Fabienne Mazerolles, Frédéric Rieux-Laucat, Marie-Claude Stolzenberg, Marianne Debre, Jean-Paul Magny, Françoise Le Deist, Alain Fischer, Claire Hivroz (2009 Jun 24)

Hypomorphic mutation of ZAP70 in human results in a late onset immunodeficiency and no autoimmunity.

European journal of immunology : 1966-76 : DOI : 10.1002/eji.200939385 En savoir plus
Résumé

Complete lack of function of the tyrosine kinase ZAP70 in humans results in a severe immunodeficiency, characterized by a lack of mature CD8(+) T cells and non-functional CD4(+) T cells. We report herein an immunodeficiency with an inherited hypomorphic mutation of ZAP70 due to a single G-to-A substitution in a non-coding intron. This mutation introduces a new acceptor splice site and allows low levels of normal alternative splicing and of WT ZAP70 expression. This partial deficiency results in a compromised TCR signaling that was totally restored by increased expression of ZAP70, demonstrating that defective activation of the patient T cells was indeed caused by the low level of ZAP70 expression. This partial ZAP70 deficiency was associated with an attenuated clinical and immunological phenotype as compared with complete ZAP70 deficiency. CD4(+) helper T-cell populations including, follicular helper T cells, Th1, Th17 and Treg were detected in the blood. Finally, the patient had no manifestation of autoimmunity suggesting that the T-cell tolerogenic functions were not compromised, in contrast to what has been observed in mice carrying hypomorphic mutations of Zap70. This report extends the phenotype spectrum of ZAP70 deficiency with a residual function of ZAP70.

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Capucine Picard, Christie-Ann McCarl, Alexander Papolos, Sara Khalil, Kevin Lüthy, Claire Hivroz, Francoise LeDeist, Frédéric Rieux-Laucat, Gideon Rechavi, Anjana Rao, Alain Fischer, Stefan Feske (2009 May 8)

STIM1 mutation associated with a syndrome of immunodeficiency and autoimmunity.

The New England journal of medicine : 1971-80 : DOI : 10.1056/NEJMoa0900082 En savoir plus
Résumé

A mutation in ORAI1, the gene encoding the pore-forming subunit of the Ca(2+)-release-activated Ca(2+) (CRAC) channel, abrogates the store-operated entry of Ca(2+) into cells and impairs lymphocyte activation. Stromal interaction molecule 1 (STIM1) in the endoplasmic reticulum activates ORAI1-CRAC channels. We report on three siblings from one kindred with a clinical syndrome of immunodeficiency, hepatosplenomegaly, autoimmune hemolytic anemia, thrombocytopenia, muscular hypotonia, and defective enamel dentition. Two of these patients have a homozygous nonsense mutation in STIM1 that abrogates expression of STIM1 and Ca(2+) influx.

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Benoît Carpentier, Paolo Pierobon, Claire Hivroz, Nelly Henry (2009 Mar 11)

T-cell artificial focal triggering tools: linking surface interactions with cell response.

PloS one : e4784 : DOI : 10.1371/journal.pone.0004784 En savoir plus
Résumé

T-cell activation is a key event in the immune system, involving the interaction of several receptor ligand pairs in a complex intercellular contact that forms between T-cell and antigen-presenting cells. Molecular components implicated in contact formation have been identified, but the mechanism of activation and the link between molecular interactions and cell response remain poorly understood due to the complexity and dynamics exhibited by whole cell-cell conjugates. Here we demonstrate that simplified model colloids grafted so as to target appropriate cell receptors can be efficiently used to explore the relationship of receptor engagement to the T-cell response. Using immortalized Jurkat T cells, we monitored both binding and activation events, as seen by changes in the intracellular calcium concentration. Our experimental strategy used flow cytometry analysis to follow the short time scale cell response in populations of thousands of cells. We targeted both T-cell receptor CD3 (TCR/CD3) and leukocyte-function-associated antigen (LFA-1) alone or in combination. We showed that specific engagement of TCR/CD3 with a single particle induced a transient calcium signal, confirming previous results and validating our approach. By decreasing anti-CD3 particle density, we showed that contact nucleation was the most crucial and determining step in the cell-particle interaction under dynamic conditions, due to shear stress produced by hydrodynamic flow. Introduction of LFA-1 adhesion molecule ligands at the surface of the particle overcame this limitation and elucidated the low TCR/CD3 ligand density regime. Despite their simplicity, model colloids induced relevant biological responses which consistently echoed whole cell behavior. We thus concluded that this biophysical approach provides useful tools for investigating initial events in T-cell activation, and should enable the design of intelligent artificial systems for adoptive immunotherapy.

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Année de publication : 2008

Nathalie Demotte, Vincent Stroobant, Pierre J Courtoy, Patrick Van Der Smissen, Didier Colau, Immanuel F Luescher, Claire Hivroz, Julie Nicaise, Jean-Luc Squifflet, Michel Mourad, Danièle Godelaine, Thierry Boon, Pierre van der Bruggen (2008 Mar 18)

Restoring the association of the T cell receptor with CD8 reverses anergy in human tumor-infiltrating lymphocytes.

Immunity : 414-24 : DOI : 10.1016/j.immuni.2008.01.011 En savoir plus
Résumé

For several days after antigenic stimulation, human cytolytic T lymphocyte (CTL) clones exhibit a decrease in their effector activity and in their binding to human leukocyte antigen (HLA)-peptide tetramers. We observed that, when in this state, CTLs lose the colocalization of the T cell receptor (TCR) and CD8. Effector function and TCR-CD8 colocalization were restored with galectin disaccharide ligands, suggesting that the binding of TCR to galectin plays a role in the distancing of TCR from CD8. These findings appear to be applicable in vivo, as TCR was observed to be distant from CD8 on human tumor-infiltrating lymphocytes, which were anergic. These lymphocytes recovered effector functions and TCR-CD8 colocalization after ex vivo treatment with galectin disaccharide ligands. The separation of TCR and CD8 molecules could be one major mechanism of anergy in tumors and other chronic stimulation conditions.

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Année de publication : 2007

Nathalie Sol-Foulon, Marion Sourisseau, Françoise Porrot, Maria-Isabel Thoulouze, Céline Trouillet, Cinzia Nobile, Fabien Blanchet, Vincenzo di Bartolo, Nelly Noraz, Naomi Taylor, Andres Alcover, Claire Hivroz, Olivier Schwartz (2007 Jan 12)

ZAP-70 kinase regulates HIV cell-to-cell spread and virological synapse formation.

The EMBO journal : 516-26 En savoir plus
Résumé

HIV efficiently spreads in lymphocytes, likely through virological synapses (VSs). These cell-cell junctions share some characteristics with immunological synapses, but cellular proteins required for their constitution remain poorly characterized. We have examined here the role of ZAP-70, a key kinase regulating T-cell activation and immunological synapse formation, in HIV replication. In lymphocytes deficient for ZAP-70, or expressing a kinase-dead mutant of the protein, HIV replication was strikingly delayed. We have characterized further this replication defect. ZAP-70 was dispensable for the early steps of viral cycle, from entry to expression of viral proteins. However, in the absence of ZAP-70, intracellular Gag localization was impaired. ZAP-70 was required in infected donor cells for efficient cell-to-cell HIV transmission to recipients and for formation of VSs. These results bring novel insights into the links that exist between T-cell activation and HIV spread, and suggest that HIV usurps components of the immunological synapse machinery to ensure its own spread through cell-to-cell contacts.

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Année de publication : 2006

Clara Ballerini, Pauline Gourdain, Véronique Bachy, Nicolas Blanchard, Etienne Levavasseur, Sylvie Grégoire, Pascaline Fontes, Pierre Aucouturier, Claire Hivroz, Claude Carnaud (2006 Jun 6)

Functional implication of cellular prion protein in antigen-driven interactions between T cells and dendritic cells.

Journal of immunology (Baltimore, Md. : 1950) : 7254-62 En savoir plus
Résumé

The cellular prion protein (PrPC) is a host-encoded, GPI-anchored cell surface protein, expressed on a wide range of tissues including neuronal and lymphoreticular cells. PrPC may undergo posttranslational conversion, giving rise to scrapie PrP, the pathogenic conformer considered as responsible for prion diseases. Despite intensive studies, the normal function of PrPC is still enigmatic. Starting from microscope observations showing an accumulation of PrPC at the sites of contact between T cells and Ag-loaded dendritic cells (DC), we have studied the contribution of PrPC in alloantigen and peptide-MHC-driven T/DC interactions. Whereas the absence of PrPC on the DC results in a reduced allogeneic T cell response, its absence on the T cell partner has no apparent effect upon this response. Therefore, PrPC seems to fulfill different functions on the two cell partners forming the synapse. In contrast, PrPC mobilization by Ab reduces the stimulatory properties of DC and the proliferative potential of responding T cells. The contrasted consequences, regarding T cell function, between PrPC deletion and PrPC coating by Abs, suggests that the prion protein acts as a signaling molecule on T cells. Furthermore, our results show that the absence of PrPC has consequences in vivo also, upon the ability of APCs to stimulate proliferative T cell responses. Thus, independent of neurological considerations, some of the evolutionary constraints that may have contributed to the conservation of the Prnp gene in mammalians, could be of immunological origin.

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Année de publication : 2005

Claire Hivroz (2005 Feb 5)

[Everything you ever wanted to know about ZAP-70].

Médecine sciences : M/S : 150-5 En savoir plus
Résumé

The ZAP-70 tyrosine kinase has been described more than ten years ago. Its key role in thymocytes development and mature T lymphocytes activation has been illustrated by the characterization of several human immunodeficiencies presenting with mutations in the zap-70 gene resulting in the absence of ZAP-70 expression. More recently, it has been shown that deregulation of ZAP-70 activity can induce autoimmune diseases. Finally, ZAP-70 expression has been shown in some B chronic lymphocytic leukaemia and correlated with bad prognosis of the disease. The diversity of pathologies associated with deregulation of ZAP-70 demonstrates its key role in immune responses. Research aiming at deciphering the different signalling pathways regulated by ZAP-70 will not only shed some lights on these pathologies, but will also help finding new pharmacological tools, targeting ZAP-70, designed to induce immunosuppression or tolerance.

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Année de publication : 2004

Claire Hivroz (2004 Jun 11)

[ZAP 70, a kinase indispensible for T lymphocyte activation, implicated in autoimmunity].

Médecine sciences : M/S : 505-6 En savoir plus
Résumé

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Année de publication : 2002

Nicolas Blanchard, Claire Hivroz (2002 Dec 26)

The immunological synapse: the more you look the less you know…

Biology of the cell / under the auspices of the European Cell Biology Organization : 345-54 En savoir plus
Résumé

Before T cells of the immune system can recognize pathogens, antigen presenting cells (APCs) must process pathogen-derived peptides and present them together with major histocompatibility complex molecules (MHC) to T lymphocytes. T lymphocytes then scan the surface of APCs and antigen-specific activation of the T cell will happen after interaction of T cell antigen receptor (TCR) with MHC-peptide complexes expressed at the membrane of APCs. This interaction takes place in a nanometer scale gap between the two cells, referred to as an immunological synapse. Recent three-dimensional fluorescence analysis of this synapse revealed a dynamic spatial organization of membrane receptors, cytoskeleton and intracellular signaling complexes on the T cell side showing specific patterns, which depend on the nature of the T cell:APC pair. Although it is obvious that establishment of an intimate contact between T cells and APCs will facilitate cell:cell communication it is not clear what is the role, if any, of this receptors patterning. This molecular reorganization has long been thought to enhance and/or sustain TCR signaling and thus T cell activation, but this is now a matter of controversy. Moreover, mechanisms controlling immunological synapse formation are still unraveled. Segregation of proteins may occur spontaneously as proposed by mathematical modeling taking into account membrane fluidity, protein size and receptor/ligand affinity. Alternatively patterning of the molecules at the cell:cell interface could be driven by active processes involving T cell signaling and/or specific features of the APC. These different questions will be discussed herein.

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Marie Granboulan, Danielle Lankar, Graça Raposo, Christian Bonnerot, Claire Hivroz (2002 Dec 3)

Phosphoinositide 3-kinase activation by Igbeta controls de novo formation of an antigen-processing compartment.

The Journal of biological chemistry : 4331-8 En savoir plus
Résumé

Antigens that bind B cell antigen receptor (BCR) are preferentially and rapidly processed for antigen presentation. The BCR is a multimeric complex containing a signaling module composed of Igalpha and Igbeta. Signaling pathways implicated in antigen presentation through the BCR are ill defined. Here we demonstrate that phosphoinositide 3-kinase (PI3K) inhibitors preclude antigen presentation induced by BCR or Igbeta but not Igalpha. Unraveling the mechanisms responsible for this inhibition, we show that PI3K inhibitors block neither antigen internalization nor degradation. Rather PI3K inhibitors block de novo formation of a multivesicular antigen processing compartment, which is induced by triggering of the BCR or Igbeta. Strikingly, we found using fluorescent probes binding specifically to PI3K products that BCR and Igbeta but not Igalpha induce PI3K activation in endocytic compartments wherein antigen is transported. Altogether, these results strongly suggest that Igbeta couples the BCR to PI3K activation that is instrumental for de novo formation of the antigen processing compartment and efficient antigen presentation.

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Nicolas Blanchard, Vincenzo Di Bartolo, Claire Hivroz (2002 Oct 22)

In the immune synapse, ZAP-70 controls T cell polarization and recruitment of signaling proteins but not formation of the synaptic pattern.

Immunity : 389-99 En savoir plus
Résumé

Recognition by T cells of their ligands at the surface of antigen-presenting cells (APCs) leads to T cell activation, polarization of the T cell toward the APC, and formation of an immune synapse. Using ZAP-70-deficient T cells expressing zeta-GFP, we show that ZAP-70 signaling drives the TCR-dependent reorientation of the microtubule-organizing center thus leading to relocation of a zeta-GFP(+) intracellular compartment close to the APC. ZAP-70 is also necessary to supply the synapse with the signaling molecules PKC-theta and LAT. In contrast, ZAP-70 is not required for clustering of zeta-GFP and CD2 or exclusion of CD45 and CD43 from the synapse. These data show that ZAP-70-dependent signaling is required for formation of a functional immune synapse.

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Céline Dumont, Nicolas Blanchard, Vincenzo Di Bartolo, Nathalie Lezot, Evelyne Dufour, Sébastien Jauliac, Claire Hivroz (2002 Aug 8)

TCR/CD3 down-modulation and zeta degradation are regulated by ZAP-70.

Journal of immunology (Baltimore, Md. : 1950) : 1705-12 En savoir plus
Résumé

TCR down-modulation following binding to MHC/peptide complexes is considered to be instrumental for T cell activation because it allows serial triggering of receptors and the desensitization of stimulated cells. We studied CD3/TCR down-modulation and zeta degradation in T cells from two ZAP-70-immunodeficient patients. We show that, at high occupancy of the TCR, down-modulation of the CD3/TCR is comparable whether T cells express or do not express ZAP-70. However, if TCR occupancy was low, we found that CD3/TCR was down-regulated to a lesser extent in ZAP-70-negative than in ZAP-70-positive T cells. We studied CD3/TCR down-modulation in P116 (a ZAP-70-negative Jurkat cell-derived clone) and in P116 transfected with genes encoding the wild-type or a kinase-dead form of ZAP-70. Down-modulation of the TCR at high occupancy did not require ZAP-70, whereas at low TCR occupancy down-modulation was markedly reduced in the absence of ZAP-70 and in cells expressing a dead kinase mutant of ZAP-70. Thus, the presence of ZAP-70 alone is not sufficient for down-modulation; the kinase activity of this molecule is also required. The degradation of zeta induced by TCR triggering is also severely impaired in T cells from ZAP-70-deficient patients, P116 cells, and P116 cells expressing a kinase-dead form of ZAP-70. This defect in TCR-induced zeta degradation is observed at low and high levels of TCR occupancy. Our results identify ZAP-70, a tyrosine kinase known to be crucial for T cell activation, as a key player in TCR down-modulation and zeta degradation.

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Nicolas Blanchard, Danielle Lankar, Florence Faure, Armelle Regnault, Céline Dumont, Graça Raposo, Claire Hivroz (2002 Mar 22)

TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex.

Journal of immunology (Baltimore, Md. : 1950) : 3235-41 En savoir plus
Résumé

We show in this study that human T cells purified from peripheral blood, T cell clones, and Jurkat T cells release microvesicles in the culture medium. These microvesicles have a diameter of 50-100 nm, are delimited by a lipidic bilayer membrane, and bear TCR beta, CD3epsilon, and zeta. This microvesicle production is regulated because it is highly increased upon TCR activation, whereas another mitogenic signal, such as PMA and ionomycin, does not induce any release. T cell-derived microvesicles also contain the tetraspan protein CD63, suggesting that they originate from endocytic compartments. They contain adhesion molecules such as CD2 and LFA-1, MHC class I and class II, and the chemokine receptor CXCR4. These transmembrane proteins are selectively sorted in microvesicles because CD28 and CD45, which are highly expressed at the plasma membrane, are not found. The presence of phosphorylated zeta in these microvesicles suggests that the CD3/TCR found in the microvesicles come from the pool of complexes that have been activated. Proteins of the transduction machinery, tyrosine kinases of the Src family, and c-Cbl are also observed in the T cell-derived microvesicles. Our data demonstrate that T lymphocytes produce, upon TCR triggering, vesicles whose morphology and phenotype are reminiscent of vesicles of endocytic origin produced by many cell types and called exosomes. Although the exact content of T cell-derived exosomes remains to be determined, we suggest that the presence of TCR/CD3 at their surface makes them powerful vehicles to specifically deliver signals to cells bearing the right combination of peptide/MHC complexes.

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Année de publication : 2001

E Meinl, T Derfuss, R Pirzer, N Blank, D Lengenfelder, A Blancher, F Le Deist, B Fleckenstein, C Hivroz (2001 Jul 21)

Herpesvirus saimiri replaces ZAP-70 for CD3- and CD2-mediated T cell activation.

The Journal of biological chemistry : 36902-8 En savoir plus
Résumé

The protein tyrosine kinase ZAP-70 plays a pivotal role involved in signal transduction through the T cell receptor and CD2. Defects in ZAP-70 result in severe combined immunodeficiency. We report that Herpesvirus saimiri, which does not code for a ZAP-70 homologue, can replace this tyrosine kinase. H. saimiri is an oncogenic virus that transforms human T cells to stable growth based on mutual CD2-mediated activation. Although CD2-mediated proliferation of ZAP-70-deficient uninfected T cells was absent, we could establish H. saimiri-transformed T cell lines from two unrelated patients presenting with ZAP-70 deficiencies. In these cell lines, CD2 and CD3 activation were restored in terms of [Ca(2+)](i), MAPK activation, cytokine production, and proliferation. Activation-induced tyrosine phosphorylation of zeta remained defective. The transformed cells expressed very high levels of the ZAP-70-related kinase Syk. This increased expression was not observed in the primary T cells from the patients and was not due to the transformation by the virus because transformed cell lines established from control T cells did not present this particularity. In conclusion, wild type H. saimiri can restore CD2- and CD3-mediated activation in signaling-deficient human T cells. It extends our understanding of interactions between the oncogenic H. saimiri and the infected host cells.

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