CHARACTERIZATION OF T CELL HOMEOSTASIS AND EFFECTOR FUNCTIONS

The orchestration of adaptive immune response crucially relies on homeostatic and effector functions of T lymphocytes. Among the array of pathways that concur to the regulation of T cell maintenance, activation and differentiation, an emerging leading factor is autophagy. Autophagy is a catabolic process that subserves the dual function of eliminating damaged substrates while providing endogenous energy sources and building blocks to maintain cellular functions. This mechanism depends on autophagy related (ATG) proteins and their interaction with the trafficking machinery that orchestrates the membrane rearrangements leading to autophagosome biogenesis. The intraflagellar transport (IFT) system, first identified for its role in the control of vesicular trafficking along the axonemal microtubules of the primary cilium, participates in the regulation of autophagy in both ciliated and non- ciliated cells. In the first part of this thesis, we investigated the mechanism by which IFT20, an integral component of the IFT system, regulates basal CD4+ T cell autophagy. We show that IFT20 interacts with the core autophagy protein ATG16L1 and mediates its association with the golgin GMAP210 at the Golgi apparatus and the small GTPase Rab5 at early endosomes. GMAP210 downregulation, while leading to a dispersed Golgi-associated ATG16L1 pattern, did not affect basal autophagy. Nonetheless, we found that IFT20 is required for ATG16L1 recruitment to early endosomes tagged for autophagosome formation, thereby promoting autophagosome biogenesis in T cells. Growing evidence indicates that IFT proteins participate in additional cilia-independent processes in the non-ciliated T cells. IFT20 plays a key role in T cell activation by regulating the assembly of the immune synapse (IS), the specialized membrane domain at the interface between the T lymphocyte and the antigen presenting cell (APC), controlling the intracellular traffic of T cell receptor (TCR) and Linker for Activation of T cells (LAT). More recently, the involvement of IFT20 in another vesicular trafficking-related process, the mannose 6-phosphate receptor (MPR)-dependent transport of acid hydrolases to lysosomes, on which lysosome biogenesis and function depend, opened the way to the second part of this work. CD8+ cytotoxic T cells (CTLs) contain specialized secretory lysosomes, named lytic granules, their main tool for target cell death delivery. We show that IFT20 controls MPR-mediated granzyme B targeting to lytic granules and CTL cytotoxicity. IFT20 depletion leads to a lytic granule phenotype associated to lysosomal biogenesis defect, a process orchestrated by the coordinated lysosomal expression and regulation (CLEAR) gene network and controlled by the master transcription factor EB (TFEB). Consistently, we found that IFT20 is involved in the TFEB-driven lytic granule biogenesis program, taking part to the main pathway of lethal hit delivery of CTLs. Effective CTL killing relies on the proper biogenesis of lytic granules and on their polarized delivery to the IS. The dynamic reorganization of the microtubule cytoskeleton and centrosome reorientation at the IS is required for T cell effector functions, facilitating the polarized release of cytokines and cytolytic factors. In the third part of this thesis, the recent discovery that the kinase Aurora-A (AurA) promotes T cell activation, as well as the microtubule-driven delivery of CD3z-bearing vesicles to the IS, led us to investigate the potential involvement of AurA substrate Polo-like kinase 1 (PLK1), a mitotic regulator whose activation is pivotal for centrosome dynamics during cell cycle, in the assembly of the IS of CTLs. Our results show that PLK1 inhibition impairs TCR signalling and centrosome translocation towards the IS in CTLs, as well as lytic granule polarization towards the T-APC contact point, leading to defective CTL cytotoxic capability. The altered microtubule dynamics due to PLK1 inhibition may be the cause of defective IS assembly in CTLs, proposing PLK1 as a novel determinant of CTL-mediated killing. All in all, this thesis deals with different aspects of T cell homeostasis and effector functions, shedding light on some missing points of the physiology of one of the main players of adaptive immunity