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  Director : Oreste Acuto (oacuto@pasteur.fr)


  abstract

 

The Molecular Immunology Unit studies the mechanisms of T cell activation. The molecular information carried by activated antigen presenting cells (APC) loaded with antigen is relayed to the T cell through a complex device formed by membrane receptors and a vast array of intracellular signaling proteins (e.g., protein tyrosine and serine/threonine kinases ; adapter proteins, small G proteins and membrane lipids and cytoskeletal components). This machinery decodes the signals (positive and negative) coming from the external milieu and determines the destiny of T cells whether they become effectors of the immune response or memory cells, or anergized cells. These differentiation programs are shaped by cell proliferation, gene re-programming and cell death. Our research is focused on the understanding of the role of some key signaling components on these events.



  report

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Mechanism controlling the activity of Lck by CD45 (Claudine Irles and Frédérique Michel)

The protein tyrosine kinase (PTK) Lck is a key element in the controls of the T cell activation threshold. Lck is essential in the triggering of a cascade of biochemical events initiated by the T cell antigen receptor (TCR) engagement leading to clonal expansion and differentiation. Lck is positively controlled by the transmembrane protein tyrosine phosphatase CD45 and at the same time to a negative control by the PTK Csk. The latter is found at the plasma membrane concentrated in the special lipid micro-domains called GEMs (Glycosphyngolipid-enriched membranes) or "lipid rafts". The equilibrium between these two opposing controls is the primary mechanism that establishes the activation threshold in T cells. CD45 possesses an intracellular phosphatase domain and exists in different isoforms expressed at different stages of T cell thymic development and during antigen driven differentiation. These isoforms, of which CD45RAB or BC and CD45R0 are the most abundant, differ in the dimensions and glycosylation content of their extracellular portions (ectodomain). The functional role of CD45 ectodomain was investigated in our laboratory by generating protein chimeras whose ectodomains, borrowed from other surface proteins, were of different size and glycosylation content; some very different from CD45, others somewhat mimicking CD45 ectodomain. Our studies demonstrated that CD45 ectodomain finely regulates its function by determining the interaction of the entire protein and therefore of the intracellular phosphatase domain with the "lipid rafts". In so doing, CD45 ectodomain determines the activation state of Lck at the basal level and after TCR triggering. Our studies also indicate the existence of an in cis interaction of CD45 ectodomain with one or more components of the lipid rafts (see model proposed in Figure1). Future studies are oriented towards the identification of CD45 binding partners and of small molecules capable of inhibiting this interaction.

Regulation and role of the PTK ZAP-70 during the T cell activation process (Vincenzo Di Bartolo, in collaboration with Marie and Bernard Malissen, Marseille)

Zap-70 (zeta-associated protein of 70 kDaltons) is a PTK essential for triggering T cell activation and is regulated by Lck. Zap-70, which associates to the TCRs engaged in the binding to antigen/MHC, possesses a region (named interdomain B, I-B) containing regulatory tyrosines (Tyr) previously discovered in our laboratory to be phosphorylated upon TCR engagement. In collaboration with the group of Marie and Bernard Malissen we have studied in vivo the role of two of them, Tyr315 and Tyr319, using mouse strains homozygous for mutation at either of these tyrosines. We found that Tyr315 concurs to an allosteric mechanism regulating effective binding of Zap-70 to the ITAM motifs of the CD3-z chains of the TCR, but has a minor role in T cell development. In contrast, Tyr319 was found to be quasi-essential for thymic positive selection of T cells in agreement with our previous in vitro data indicating the key importance of this residue in regulating TCR-mediated signaling.

In another collaborative study with Claire Hivroz at the Curie Institute, it was found that ZAP-70 is essential for the recruitment of all major components of the TCR signaling machinery and for re-orientation of the microtubules towards the T cell-APC contact (a function likely required for the polarization of the secretion and endocytic processes). However, unexpectedly, at least part of the immunological synapse (such as the individual pattern of segregation of the receptor CD2 and CD45 in the form of supra-molecular activation clusters, SMAC) appeared normal. These data suggest that other kinases, in addition to Zap-70, concur to the formation of the mature immunological synapse.

Molecular mechanisms of CD28 co-stimulation: new signaling pathways (Fabien Blanchet )

We have previously reported that CD28, the co-stimulatory T cell specific receptor, required for optimal T cell proliferation and differentiation, acts as a direct molecular amplifier of the TCR signaling machinery. We have undertaken a study aimed at discovering novel pathways by which CD28 exerts this function. Our recent work conducted by mass spectrometry allowed us to identify at least two novel proteins involved in CD28 signaling and found to interact with CD28 intracellular tail. Interestingly, we found that one of these proteins connects CD28 to a previously unsuspected metabolic pathway needed for certain regulatory protein modifications and for cell growth.

Study of the function of the adapter SH3P7 in the formation of the immunological synapse (Setsuko Mise-Omata)

We have studied the function of the protein adapter SH3P7 (also called Hip55 or mammalian Actin binding protein 1, mAbp1), recently identified. SH3P7 possesses a SCAD domain that binds to polymerized actin and an SH3 domain. The latter was suggested to bind to Dynamin 1 (also called "pinchase) a protein involved in the mechanism responsible for the formation of endocytotic vesicles. However, the actual function of SH3P7 has not yet been identified. Towards this goal, we applied the technology of RNA interference (iRNA) to inhibit the expression of SH3P7 initially in human cells of epithelial origin. Studies by fluorescence microscopy revealed that cells with reduced expression of SH3P7 have a marked defect in the endocytotic process with disruption of the early endosomal compartment. However, other processes that involve active membrane re-modeling, such as ruffling consequent to Rac-dependent lamellipodia formation were apparently intact. We then obtained stable T cell lines with reduced expression of SH3P7 expression to study its impact on the formation and stability of the immunological synapse between the T cell and the APC.

Figure legend. Schematic representation of the mechanism by which CD45 positively regulates Lck activity (e.g. , by dephosphorylating the negative regulatory Tyrosine 505 at Lck C-terminus) by weakly interacting with lipid rafts. X represents the hypothetical partner that mediates this interaction.

Keywords: T cell activation, kinases, phosphatases, adapter proteins, immunological synapse



  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, (Permanent staff scientist, fmichel@pasteur.fr)

DI BARTOLO, Vincenzo, IP (Permanent staff scientist, vbartolo@pasteur.fr)

MISE-OMATA, Setsuko, (post-doctoral research scientist, mise@pasteur.fr)

BLANCHET, Fabien, PhD student

IRLES, Claudine, PhD student

MARQUEZ CAMPOS, Maria Elena, PhD student

FOULON, Eliane, DEA student

CADET-DANIEL, Véronique (technician, vcadet@pasteur.fr)

MONTAGNE, Benjamin (technician, bmontagn@pasteur.fr)

HOUSSIN, Wendy (secretary, whoussin@pasteur.fr)


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