T lymphocytes recognize antigens as molecular fragments presented at the surface of antigen presenting cells. Antigen recognition triggers the polarization of T lymphocytes towards antigen presenting cells. This polarization is characterized by morphological changes, the reorientation of the T cell cytoskeleton (microtubules and actin cytoskeleton) and the intracellular vesicle traffic, as well as by the clustering of a number of molecules, such as the T cell receptor, adhesion molecules, intracellular signaling effectors, etc. The interface between the T lymphocyte and the antigen presenting cell becomes an organized cell contact that was named the immunological synapse, since it mediates the complex communication between the T cell and the antigen presenting cell (Figure 1). 

Figure 1: click on the panels to see a larger version with legend

1. Role of the actin and microtubule cytoskeleton in the formation of the immunological synapse and in T cell activation

R.Lasserre, S. Charrin, C. Cuche, S. Aguera-Gonzales and A. Alcover

Figure 2 : click to see a larger version with legend

Antigen recognition by the T cell receptor triggers strong actin cytoskeleton rearrangements in the T lymphocyte, which in turn condition morphological changes and molecular reorganization at the immunological synapse (Figure 1 and 2). The dependence between the molecular reorganization at the synapse and the actin cytoskeleton prompted us to investigate the involvement of ERM (ezrin, radixin, moesin) proteins, since they link membrane components with the actin cytokeleton (Reviewed in Charrin et al., 2006, Lasserre et 2010b).
We have shown that ezrin, polarizes towards the antigen presenting cell (Figure 2A) in a way that depends on T cell receptor signaling. We investigated the functional importance of ezrin on the formation of the immunological synapse and on T cell activation by studying the effect of overexpressing wild type or mutated forms of ezrin, or of RNA interference. The over-expression of a truncated form of ezrin inhibited T cell receptor clustering at the synapse, as well as T cell activation events, such as the activation of NF-AT, a transcription factor controlling the interleukin-2 gene (Roumier et al. 2001, Das et al., 2002). Our more recent work shows that ezrin plays a key role in structuring microtubule networks at the immunological synapse (Figure 2B), which in turn are fundamental for key for the dynamics of signaling complexes at the immunological synapse. The interaction between ezrin and the cell polarity regulator Dlg1 are involved in the cross talk between ezrin and microtubules and in its effects on T cell activation. Therefore, the cross talk between the actin and microtubule cytoskeleton through ezrin and Dlg1 is crucial for the structure and function of the immunological synapse (Lasserre et al 2010a, b).

2. Dynamics of signaling protein complexes within immunological synapses and control of T cell activation.

R. Lasserre, C. Cuche and V. Di Bartolo 

Signals generated by the engagement of the T cell antigen receptor and co-stimulatory receptors are decoded and integrated by macromolecular complexes (Acuto et al., 2008) that are assembled within immunological synapses. The composition and the spatio-temporal dynamics of these complexes must be finely regulated since they affect the outcome of T cell activation. We are interested in understanding which molecular mechanisms underlie the positive and negative regulation of these macromolecular complexes and how they eventually modulate T cell responses.
We have recently focused on the role of the Hematopoietic Progenitor Kinase 1 (HPK1), a member of the Germinal Center Kinases family of Ser/Thr protein kinases. This enzyme, initially described as an upstream activator of the SAPK/JNK MAP kinase pathway, has been shown to negatively regulate T cell activation. It was known that HPK1 interacts with the scaffold protein SLP-76, a key component of T lymphocytes signaling complexes, in activated T cells, but the significance of this interaction was unclear. We have demonstrated that HPK1 phosphorylates SLP76 and induces its interaction with several proteins of the 14-3-3 family. We have also demonstrated that 14-3-3 binding to SLP-76 negatively regulates proximal signaling events as well as the stimulation-dependent activity of the interleukin-2 gene promoter (Di Bartolo et al., 2007). Further studies have allowed us to show that HPK1 also controls the phosphorylation of the adapter protein GADS, which constitutively binds to SLP76 and is responsible for the recruitment of SLP76 within signaling complexes at the immunological synapse. Our results indicate that HPK1-dependent phosphorylation of both GADS and SLP76 is required for optimal binding of these proteins with 14-3-3 molecules. This interaction leads to the release of the SLP76-GADS complex from TCR-induced signaling complexes and consequently downregulates T cell activation (Lasserre et al. J.Cell.Biol.; in press). One of our current goals is to understand whether specific stimuli or pathological conditions may induce this HPK1-dependent negative feedback loop to modulate T cell responses.

Figure 3 : click to see a
larger version with legend

3. Role of intracellular vesicle trafficking in molecular polarization at the immunological synapse. Inhibition by human immunodeficiency virus (HIV-1)

H. Soares, J. Bouchet, M. I. Thoulouze and A. Alcover, in collaboration with the Virus and Immunity Unit (O. Schwartz and coworkers) and the Imagopole, Institut Pasteur

In order to concentrate at the immunological synapse, T cell receptor and signaling molecules, evenly distributed in the cell, need to be transported towards the antigen presenting cell contact zone and retained there. Various types of transport can be envisaged, either on the cell surface or through the cell interior. We have shown that recycling endosomes (vesicles through which receptors that are internalized and recycled back to the plasma membrane transit) transport T cell receptors to the immunological synapse. By means of live cell imaging, we observed that endosomes containing T cell receptors rapidly polarized towards the cell-cell contact and accumulated at the synapse (Figure 4 A, Das et al 2004). This mechanism is not specific for the T cell receptor, it seems to involve the whole recycling endosomal compartment (Figure 4 B, Das et al 2004) and it can transport to the synapse other receptors present in this compartment, such as the transferrin receptor. Using a series of biological and pharmacological modulators of T cell receptor trafficking, affecting endocytosis, polarization, recycling, or vesicle fusion, we could demonstrate the importance of endosomal transport in T cell receptor accumulation at the synapse. In addition, we showed that SNARE proteins that control the fusion between recycling vesicles and the plasma membrane also concentrate at the cell-cell contact zone, indicating that the immunological synapse becomes an active zone for docking and fusion of intracellular vesicles. Altogether our observations show that intracellular transport of endosomal vesicles is a key mechanism to translocate T cell receptors to the immunological synapse (Das et al., 2004). It can also transport signaling molecules like the protein tyrosine kinase Lck, or the adapter LAT (Reviewed in Pais-Correia et al., 2007, Alcover and Thoulouze 2010).

Figure 4 : click to see a larger version with legend

Human immunodeficiency virus type-1 (HIV-1) is able to modulate a variety of cellular processes in order to ensure its survival and replication inside the host cell, and its transmission to other cells. HIV-1 infects and actively replicates into T lymphocytes, leading to profound alterations of T cell physiology and to a massive depletion of these cells in infected individuals. HIV-1 modifies signaling pathways, as well as trafficking of a number of membrane receptors. Therefore, we studied the consequences of HIV-1 infection on the formation of the immunological synapse. By means of confocal microscopy and quantitative image analysis, we observed that HIV-1-infected T lymphocytes displayed a reduced capacity to form immunological synapses. Thus, the number of conjugates between infected T cells and antigen presenting cells was reduced and the immunological synapses formed were abnormal. The intracellular trafficking of the T cell receptor and the protein tyrosine kinase Lck was altered, leading to the retention of these molecules in the recycling endosomal compartment, and to their impaired clustering in the synapse (Figures 5 and 6). These phenomena were due to Nef, a viral protein known to affect intracellular trafficking and signaling processes. Finally, infected T lymphocytes had a reduced activation capacity, as depicted by the lower levels of protein tyrosine phosphorylation (Thoulouze et al., 2006). Therefore, the alteration of intracellular trafficking and signaling processes at the immunolgical synapse influences the function and fate of HIV-1-infected T lymphocytes (Reviews, Fakler et al 2007 ; Alcover and Thoulouze, 2010). We are currently studying the nature of the vesicular compartments where Lck and T cell receptor transit, the molecular mechanisms involved in the retention of these proteins during HIV-1 infection and the consequence of this retention in T cell activation.

Figure 5 : click to see a larger version with legend

Figure 6 : click to see a larger version with legend

4. T lymphocyte polarization and cell-to-cell transmission of the human T cell leukemia virus (HTLV-1).

 A. M. Pais-Correia, V. Robbiati, Rémi Lasserre, A. Alcover and M. I. Thoulouze, in collaboration with the Unit of Oncogenic Virus Epidemiology and Physiopathology (A. Gessain and coworkers) and the Imagopole, Institut Pasteur.

It has been shown by several laboratories that the molecular mechanism of T cell polarization cand be subverted by viruses that infect T lymphocytes, such as HIV-1 or HTLV-1 to better spread from cell to cell. The cell-cell contact through which these viruses spread was called the virological synapse, because of its common characteristics with immunological synapses (Reviewed in Pais-Correia et al., 2007, Thoulouze and Alcover, 2010, 2011). We have recently shown the HTLV-1 cell-to-cell transmission occurs via extracellular viral assemblies that ressemble in structure, composition and function to bacterial biofilms. These « viral biofilms » are induced by the virus and formed by clusters of infectious viral particles held together and to the cell surface by a carbohydrate-rich extracellular matrix components and linker proteins (Figure 7 and 8). The removal of the « HTLV-1 biofilm » from the surface of virus-producing T cells strongly impairs their ability to infect other cells. Our findings unveil a novel virus transmission mechanism (Pais-Correia et al, 2010, reviewed in Thoulouze and Alcover, 2010, 2011). We are currently seeking to characterize the mechanisms of viral biofilm generation, and to determine whether viruses other than HTLV-1 form this kind of structure. For viruses forming biofilms, it would be useful to define new anti-viral therapeutic strategies, targeting not only the virus itself, but also the formation of these viral biofilms.

Figure 7: click to see a larger version with legend
figure 8: viral biofilm
Figure 8: click to see a larger version with legend