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

The Intraflagellar Transport Protein IFT20 Recruits ATG16L1 to Early Endosomes to Promote Autophagosome Formation in T Cells

Lymphocyte homeostasis, activation and differentiation crucially rely on basal autophagy. The fine-tuning of this process depends on autophagy-related (ATG) proteins and their interaction with the trafficking machinery that orchestrates the membrane rearrangements leading to autophagosome biogenesis. The underlying mechanisms are as yet not fully understood. The intraflagellar transport (IFT) system, known for its role in cargo transport along the axonemal microtubules of the primary cilium, has emerged as a regulator of autophagy in ciliated cells. Growing evidence indicates that ciliogenesis proteins participate in cilia-independent processes, including autophagy, in the non-ciliated T cell. Here we investigate the mechanism by which IFT20, an integral component of the IFT system, regulates basal T cell autophagy. We show that IFT20 interacts with the core autophagy protein ATG16L1 and that its CC domain is essential for its pro-autophagic activity. We demonstrate that IFT20 is required for the association of ATG16L1 with the Golgi complex and early endosomes, both of which have been identified as membrane sources for phagophore elongation. This involves the ability of IFT20 to interact with proteins that are resident at these subcellular localizations, namely the golgin GMAP210 at the Golgi apparatus and Rab5 at early endosomes. GMAP210 depletion, while leading to a dispersion of ATG16L1 from the Golgi, did not affect basal autophagy. Conversely, IFT20 was found to recruit ATG16L1 to early endosomes tagged for autophagosome formation by the BECLIN 1/VPS34/Rab5 complex, which resulted in the local accumulation of LC3. Hence IFT20 participates in autophagosome biogenesis under basal conditions by regulating the localization of ATG16L1 at early endosomes to promote autophagosome biogenesis. These data identify IFT20 as a new regulator of an early step of basal autophagy in T cells

NMR study of the secondary structure and biopharmaceutical formulation of an active branched antimicrobial peptide

The synthetic antimicrobial peptide SET-M33 is being developed as a possible new antibacterial candidate for the treatment of multi-drug resistant bacteria. SET-M33 is a branched peptide featuring higher resistance and bioavailability than its linear analogues. SET-M33 shows antimicrobial activity against different species of multi-resistant Gram-negative bacteria, including clinically isolated strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumanii and Escherichia coli. The secondary structure of this 40 amino acid peptide was investigated byNMRto fully characterize the product in the framework of preclinical studies. The possible presence of helixes or β-sheets in the structure had to be explored to predict the behavior of the branched peptide in solution, with a view to designing a formulation for parenteral administration. Since the final formulation of SET-M33 will be strictly defined in terms of counter-ions and additives, we also report the studies on a new salt form, SET-M33 chloride, that retains its activity against Gram-negative bacteria and gains in solubility, with a possible improvement in the pharmacokinetic profile. The opportunity of using a chloride counter-ion is very convenient from a process development point of view and did not increase the toxicity of the antimicrobial drug