NANOIMMUNOLOGY of T Lymphocyte activation and apoptosis.

Study of the molecular bases involved in the polarized secretory traffic of exosomes in T lymphocytes: role of FMNL1 formin and actin cytoskeleton reorganization (Lymphoexosomes)

Exosomes are extracellular vesicles (EV) of endosomal origin constitutively released by many different eukaryotic cells by fusion of multivesicular bodies (MVB) to the plasma membrane. However, inducible exosome secretion controlled by cell surface receptors is restricted to very few cell types and a limited number of cell surface receptors. Among these, exosome secretion is induced in T and B lymphocytes when stimulated at the immune synapse (IS) via T-cell receptor (TCR) and B-cell receptor (BCR), respectively. IS formation by T and B lymphocytes constitutes a crucial event involved in antigen-specific, cellular and humoral immune responses. Upon IS formation by T and B lymphocytes with antigen-presenting cells (APC) the convergence of MVB towards the microtubule organization center (MTOC), and MTOC polarization to the IS, are involved in polarized exosome secretion at the synaptic cleft. Actin cytoskeleton reorganization occurring at the IS but also at the centrosomal area around the MTOC regulates MVB/MTOC polarized traffic to the IS. This specialized mechanism provides the immune system with a finely tuned strategy to increase the specificity and efficiency of crucial secretory effector functions of B and T lymphocytes. In addition to inducible exosome secretion at the IS, constitutive exosome secretion of promotes directional cell motility by reinforcing otherwise transient polarization states and promoting adhesion assembly during cell migration. Since inducible and constitutive exosome secretion by lymphocytes are critical features occurring in the immune system, this project considers the study of the traffic events and some of the intracellular signals leading to polarized exosome secretion at the IS, and also in migrating T lymphocytes and some of their biological consequences.

Specific Objectives.

This proposal is divided in 4 specific aims:

1. To establish the contribution of the different FMNL1 isoforms to MTOC/MVB polarization to the IS in T lymphocytes and to the leading edge or the uropod in migrating T lymphocytes.

2. To study the role of FMNL1beta phosphorylation in MTOC/MVB polarization to the IS in T lymphocytes and in exosome secretion by T lymphocytes.

3. To analyse the contribution of PKCdelta to centrosomal area and cortical F-actin reorganization in T lymphocyte migration and to study the role of PKCdelta-induced FMNL1beta phosphorylation in F-actin reorganization in T lymphocyte migration and exosome secretion.

4. To elucidate the contribution of FMNL1 isoforms to centrosomal area and cortical F-actin reorganization in T lymphocytes forming IS and in migrating T lymphocytes.