Figure 1a. Antigen recognition triggers T lymphocyte polarization and the formation of the immune synapse

T lymphocytes dock on the antigen presenting cells and scan their surface (1). If the appropriate antigen is found, T cells trigger intracellular signaling and polarize towards the contact site (2). T lymphocyte polarization is characterized by the translocation of the microtubule organizing center and the redirection of vesicle traffic towards the antigen presenting cell contact site, as well as by strong actin rearrangements in that area. Moreover, T cells generate at the contact site abundant dynamic molecular clusters containing surface receptors and signaling molecules (3). Finally, the actin cytoskeleton retracts, the T cell rounds up again and some small clusters coalesce into bigger clusters, eventually segregating into central and peripheral supramolecular clusters of different molecular composition (4).

Figure 1b. Scanning electron microscopy showing a conjugate formed between a T lymphocyte and an antigen presenting cell. It is worth noting the long shape of the T cell (Tc) polarized towards the antigen presenting cell (APC) and the membrane protrusions that adhere the T lymphocyte to the antigen presenting cell.

 Figure 2. Role of ezrin in the reorganization of the actin and microtubule cytoskeleton
at the immunological synapse. Confocal microscopy.

A. Following antigen recognition, activated T lymphocytes (bottom) undergo cell shape changes and display strong relocalization of F-actin (red) and ezrin (pink) in the membrane protrusions that engulf the antigen presenting cell (APC). Clustering of T cell receptors (TCR) is observed in the center of the contact site (green). Cell morphology was monitored by differential interference contrast (DIC). (Roumier et al. Immunity 2001. 15 : 715-728. Photos reproduced with permission from Elsevier). B. The microtubule network finely rearranges at the immunological synapse. The microtubule organizing center (green denser zone) translocates close to the immunological synapse and the microtubule network is rearranges in a radial manner (green filaments). Signaling molecular complexes (microclusters, red puncta) align on microtubules and need them to migrate from the edges of the synapse to the center. This microcluster mouvement is key for the regulation of T cell receptor signaling. Ezrin and the polarity regulator Dlg1 are key for stabilizing microtubule networks at the synapse and to ensure T cell receptor signal regulation The picture shows a T cell activated on a microscope slide coated with an anti-T cell receptor antibody. This activatory surface generates the formation of « pseudo immunological synapses » large enough to allow the observation of the microtubule network and microcluster dynamics (Lasserre et al 2010). 


Figure 3. Model of regulation of signaling complexes stability and T cell activation by HPK1.

SLP76-GADS complexes (only one is depicted for clarity) are recruited to the phosphorylated transmembrane adaptor LAT to form signaling-competent macromolecular assemblies (microclusters) at the immunological synapse (a). When phosphorylated on Tyr379, HPK1 is incorporated into microclusters by interacting with the SH2 domain of SLP76 (b) and phosphorylates Ser376 of SLP76 and Thr262 of GADS (c). 14-3-3 proteins then bind to SLP76-GADS complexes through these phosphorylated residues (d), thus leading to dissociation of these complexes from phospho-LAT and terminating signaling (e).

Figure 4. Polarized transport of T cell receptors to the immunological synapse via recycling endosomes

T cells encountering activatory antigen presenting cells rapidly polarize their recycling endosomes (arrowheads) towards the antigen presenting cell, and accumulate them in the cell-cell contact area (arrows). A. T cell receptors in recycling endosomes were labeled by a fluorescent anti-CD3 antibody (green). Cells were then put in contact with activatory antigen presenting cells (APC) and filmed for the indicated times. B. T cells expressing GFP-tagged cellubrevin (Cb) were put in contact with stimulatory antigen presenting cells and filmed. Cellubrevin is a v-SNARE protein present in recycling endosomes, which is involved in the fusion of endosomes with the plasma membrane. (Das et al. Immunity 2004. 20 : 577-588 15 : 715-728. Photos reproduced with permission from Elsevier).

Figure 5. HIV-1-infected lymphocytes accumulate the protein tyrosine kinase Lck in endosomal vesicles

Localization of the intracellular vesicular compartment containing Lck (red labeling), with respect to recycling endosomes displaying transferrin receptor (green). A. Non-infected cells display Lck in an intracellular compartment localized close but being distinct from transferrin positive endosomes. B. In HIV-1-infected cells, Lck is accumulated in an intracellular compartment that co-localizes with trasferrin positive endosomes. Yellow color indicates the colocalization of both compartments. C. In non-infected cells, Lck clusters at the immunological synapse (arrow). D.  In HIV-1-infected cells, Lck accumulates in the endosomal compartment (arrowhead), and does not reach the plasma membrane. Therefore, Lck cannot cluster at the immunological synapse (arrow). (Thoulouze et al. Immunity 2006. 24 : 547-561. Photos reproduced with permission from Elsevier)

Figure 6. Schematic representation of polarized vesicular traffic to the immunological synapse  and its alteration by HIV-1 infection

A. Intracellular vesicular compartments, such as recycling endosomes, polarize upon antigen recognition towards the antigen presenting cell contact site. This transport leads to the concentration at the synapse of the T cell receptor and of signaling molecules like the tyrosine kinase Lck. B. HIV-1 infection alters the intracellular vesicle traffic of the T cell receptor and of Lck in the T lymphocyte leading to their accumulation in recycling endosomes. This traffic alteration impedes the normal concentration and clustering of these proteins in the immunological synapse and, as a consequence, inhibits T cell activation. 


Figure 7. HTLV-1-infected T lymphocytes displaying a « viral biofilm » at the cell surface
A. Confocal microscopy of a HTLV-1-infected T lymphocyte. The cell surface was stained with concanavalin-A (green), while the viral protein Gag was stained with antibodies (red). Viral components are localized outside the cell attached to the cell surface. (Pais-Correia et al., 2010). B. Scanning electron microscopy of the surface of a HTLV-1-infected T lymphocyte displaying viral particle clusters tightly adhered to the cell surface. The virus Env glycoprotein was labeled with colloidal gold (white spots). C. Transmission electron microscopy of a HTLV-1-infected T lymphocyte displaying viral particle clusters outside the cells. Mature viral particles are characterized by an electron-dense core surounded by un envelope. The mesh of electron-dense material present among viral particles likely represents the extracellular matrix.



Figure 8. The « viral biofilm », a new mode of cell to cell virus spread.
A. Confocal microscopy. Transmission of a « viral biofilm » (viral components stained in red) from a HTLV-1-infected T cell (left) to another T cell (right). Cells are surface labelled with concanavalin-1 (green). B. Schematic representation of virus cell-to-cell spread via a « viral biofilm ».