New perspectives on T cell function: dissecting the roles of TSP-1 in cytotoxicity and CCDC28B in lysosome biogenesis

Cytotoxic T cells (CTLs) eliminate tumorigenic and virally infected cells through the release of cytotoxic granules and the Fas/FasL signalling pathway. Recently a novel killing mechanism has been identified, involving the release of cytotoxic particles known as SMAPs (Supramolecular Attack Particles). They consist of a core of granzymes, perforin and serglycin, surrounded by a glycoprotein shell of which thrombospondin-1 (TSP-1) is a predominant component. TSP-1 is a secreted matricellular glycoprotein mainly expressed by vascular cells. The expression of TSP-1 in CTLs, as well as its association within SMAPs, has revealed a novel function for this protein in lymphocytes, however the underlying mechanism remains to be elucidated. Preliminary studies on TSP-1 have highlighted in effector CTLs the presence of a 60 kDa form of TSP-1 corresponding to the C-terminal portion of the protein, which may be generated by cleavage of the full-length protein. We have shown that the 60 kDa TSP-1 portion is able to localize to lytic granules. The main goal of this project is to engineer the 60 kDa C-terminal portion of TSP-1 because of its presence in the outer shell of SMAPs and its expression in mature differentiated CTLs. TSP-1 engineering consists in the deletion or mutagenesis of the natural adhesive determinants of TSP-1 and the addition of a domain to confer SMAPs specificity for a selected cell type, such as a cancer cell. In this thesis I have worked on the first part of engineering by generating three constructs encoding mutants of 60 kDa TSP-1 lacking the adhesive sites. These include a 60-kDa mutant lacking the 8 amino acids corresponding to the CD47 receptor binding site, a 60-kDa variant with a mutated RGD motif that mediates integrin binding, and a double mutant. Immunofluorescence analysis on primary CTLs transfected with all the described TSP-1 mutants have shown that they correctly localise to lytic granules. The next step of this project is to determine if the SMAPs containing the engineered TSP-1 can be released by CTLs and kill target cells as efficiently as the ones containing the wild-type protein. The data are expected to pave the way for the development of a new, SMAPs-based immunotherapy treatment for cancer patients. In the second part of this thesis, we examined the role of a ciliogenesis protein in fundamental processes within the non-ciliated T lymphocytes. Over the past few years different ciliogenesis proteins have been documented in non-ciliated cells, such as T lymphocytes, where they play a fundamental role in immunological synapse (IS) assembly and function. In particular, the intraflagellar transport (IFT) system component IFT20 has been the first to be associated to several mechanisms beyond its crucial role in the assembly and maintenance of the primary cilium. Indeed, IFT20 is considered a key player not only in ciliogenesis but also in IS assembly and in the vesicular trafficking of membrane receptors and signaling proteins to IS. These findings opened a new field in the characterization of a large panel of ciliogenesis proteins involved in IS assembly such as other IFT proteins, Rab GTPases, SNARE proteins and others. Accordingly, recent findings have demonstrated that another ciliary protein, known as Coiled-Coil Domain Containing 28B (CCDC28B), participates in IS assembly by regulating polarized T cell receptor (TCR) recycling. Lately cellular degradation pathways have emerged as new extraciliary functions of the IFT system, with a key role of IFT20 in a central degradation step controlling lysosome function, by regulating the mannose-6-phosphate receptor (M6PR)-dependent lysosomal targeting of acid hydrolases. Indeed, IFT20 acts as an adaptor molecule coupling the cation-independent mannose-6-phosphate receptor (CI-MPR) to dynein for retrograde transport to the trans-Golgi network (TGN). Since the ciliary protein CCDC28B has a role in regulating the actin cytoskeleton by recruiting the actin regulator WASH and retromer complex components, CCDC28B could be implicated, similarly to IFT20, in the regulation of CI-MPR retrograde trafficking in T cells and contribute to lysosome biogenesis and functions. To assess our hypothesis, experiments were performed on Jurkat cells knockout (KO) for the gene encoding the CCDC28B protein. Colocalization analyses of the M6PR with the TGN were carried out, and the retrograde transport of the MPR was also evaluated by confocal microscopy. These analyses have shown a decrease in the colocalization of CI-MPR with the TGN in CCDC28B KO cells, associated with a failure of antibody-tagged recycling CI-MPR to reach the TGN. These preliminary results suggest a defect of retrograde transport in the absence of CCDC28B. Considering its interaction with the actin regulator WASH and the retromer complex component, CCDC28B could be involved in retrograde transport of CI-MPR. The results could provide new knowledge of ciliary proteins in important cellular processes in the non-ciliated T cells.