Unit: Lymphocyte Cell Biology
Director: Andres ALCOVER
The research of the unit focuses on the study of the mechanisms of T lymphocyte polarization, which is triggered by antigen recognition and leads to the formation of the immunological synapse. We study the role of the actin cytoskeleton and of polarized intracellular vesicle trafficking in the formation of the immunological synapse and in T cell activation. We also investigate how viruses that infect T lymphocytes hijack the mechanisms of T cell polarization to modulate T cell responses and to spread more efficiently from cell to cell.
T cell polarization and the formation of the immunological synapse.
A crucial step of the immune response is the recognition by T lymphocytes of antigens on the surface of antigen presenting cells. Following antigen recognition, T cells polarize towards the antigen presenting cell. This polarization is characterized by morphological changes of the T lymphocyte, and by the concentration at the cell-cell contact zone of a number of molecules, such as the T cell receptor, adhesion molecules, signaling effectors and cytoskeleton components. This organized cell-cell junction has been called the immunological synapse, since ensures the complex communication between the T cell and the antigen presenting cell. We study the molecular mecanisms that generate this synapse, as well as its role in T cell activation. We analyze the role of the actin cytoskeleton and of intracellular vesicle trafficking. We also study how lymphotropic viruses, like human immunodeficiency virus, hijack the mechanisms of T cell polarization to modulate the immune response and to propagate from cell to cell.
1) Role of the actin cytoskeleton in the formation of the immunological synapse and in T cell activation (S. Charrin, C. Cuche and A. Alcover).
We have shown that ezrin, a protein that links the membrane with the actin cytoskeleton transiently polarizes towards the antigen presenting cell (Figure 1). Moreover, we observed that over-expression of a truncated form of ezrin inhibited T cell receptor clustering at the immunological synapse, as well as later events of T cell activation, such as IL-2 gene activation. These data provide evidence for an important role of ezrin in T lymphocytes. By linking membrane and actin cytoskeleton components, ezrin may help molecular clustering at the immunological synapse and modulate T cell activation. We are currently studying the involvement of ezrin in T cell receptor signaling pathways.
2) Role of intracellular vesicle trafficking in T cell receptor polarization at the immunological synapse (V. Das, M. I. Thoulouze and A. Alcover).
We have shown that intracellular vesicular trafficking is a key process in the formation of the immunological synapse. By using confocal microscopy, time-lapse digital imaging and quantitative image analysis, we showed that recycling endosomes can transport T cell receptors to the immunological synapse, and that this transport is necessary for their efficient accumulation at the synapse (Figure 2). Moreover, we showed that SNARE proteins that control the fusion between recycling vesicles and the plasma membrane are required for this process. These results show that intracellular vesicular transport is a key mechanism to transport T cell receptors, and to promote its polarization at the immunological synapse. This mechanism likely involves other molecules that cluster in the immunological synapse.
3) Modulation of T cell polarization and immunological synapse formation by human immunodeficiency virus (M. I. Thoulouze and A. Alcover, in collaboration with N. Sol-Foulon, F. Blanchet and O. Schwartz from the Virus and Immunity Group).
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 infection modulates surface expression and intracellular trafficking of several surface receptors, as well as signaling pathways. Since T cell polarization and immune synapse formation depends on signaling and intracellular trafficking processes, we studied the effect of HIV-1 infection of T lymphocytes in the formation of the immunological synapse. By means of confocal microscopy and quantitative image analysis, we showed that HIV-1-infected T lymphocytes display a reduced capacity to form immunological synapses. Thus, the number of conjugates between T cells and antigen presenting cell is reduced and the immunological synapses formed are abnormal. The intracellular trafficking of the T cell receptor and the protein tyrosine kinase Lck is altered leading to the retention of these molecules in the recycling endosomal compartment, and to their impaired clustering in the synapse. These phenomena are due to Nef, a viral protein known to affect intracellular trafficking and signaling processes. Finally, infected T lymphocytes have a reduced activation capacity, as depicted by the lower levels of protein tyrosine phosphorylation. Alteration of intracellular trafficking and signaling processes at the immunological synapse likely influences the function and fate of HIV-1-infected T lymphocytes.
Figure 1. Interaction of a T lymphocyte (Tc) with a B lymphocyte presenting a bacterial superantigen (APC) observed by confocal microscopy. The actin cytoskeleton associated protein ezrin (red) and the T cell antigen receptor (green) are polarized towards the antigen presenting cell (immunofluorescence, right). On the left, the morphology of the two cells is observed by differential interference contrast.
Figure 2. Polarized transport of T cell receptors towards the immunological synapse via endosomal vesicles. Human T lymphocyte (Tc) encountering a cell presenting a bacterial superantigen (APC). T cell receptors present in endosomal vesicles were traced using a fluorescent anti-T cell receptor antibody. The picture shows the merge between the fluorescence image (green) and the differential interference contrast image (gray). Endosomes containing T cell receptors appear polarized and closely apposed to the cell-cell contact zone where the immune synapse forms.
Figure 3. HIV-1-infected T lymphocyte. Immunofluorescence and confocal microscopy image showing the presence of the viral protein Nef (green) and the cellular tyrosine kinase Lck (red), in a tubulo-vesicular intracellular compartment. Both proteins co-localized in the pericentriolar region (co-localization is depicted by the yellow color in the immunofluorescence image (right image). Ce
Keywords: T cell antigen receptor, immunological synapse, polarisation, endosomes, intracellular traffic, SNARE, ezrin, T cell activation, actin cytoskeleton, cell biology, immunology, virology, AIDS