TY  - THES
AU  - Castiblanco Gaitan, Daniela
Y2  - 2021/05/31
Y1  - 2021
UR  - http://hdl.handle.net/11343/274852
AB  - Cytotoxic secretory granules (CGs) are unique and specialised lysosomal organelles found specifically in cytotoxic lymphocytes: Natural Killer (NK) cells and CD8+ T cells or Cytotoxic T-Lymphocytes (CTLs). CGs precisely store and deliver perforin and granzymes; molecules that synergistically induce the controlled death of infected and neoplastic cells. The precise molecular mechanisms that govern CG formation remain poorly understood and this work aimed to explore the potential role of the MiT/TFE family of proteins in transcriptionally regulating this process. Three members of the MiT/TFE family (MITF, TFEB and TFE3) are largely recognised as the master regulators of lysosome biogenesis and as the main drivers in the formation of organelles with a lysosomal nature. Under this hypothesis the role of the microphthalmia associated transcription factor (MITF) was studied. CTLs expressing mutant MITF showed a reduced ability to expand and kill target cells in vitro in response to antigen-specific stimulation. However, it was determined that the defect was not intrinsic to the lymphocytes but associated to antigen presenting cells required for CTLs activation. Having ruled out MITF in the formation of CGs, the role of transcription factor EB (TFEB) and transcription factor E3 (TFE3) was explored next. By studying the subcellular trafficking of these two proteins during mouse CTL activation, this work described that TFEB, but not TFE3, translocated from the cytosol into the nucleus. Despite the nuclear TFEB presence, RNA-sequencing of CTLs did not conclusively showed upregulation of canonical TFEB target genes. To gain further understanding on the role of TFEB in CTLs, the lysosomal storage disorder (LSD) Niemann-Pick type C1 (NP-C1) was implemented as a model. This devastating disease, caused by mutations in the lysosomal cholesterol transporter, NPC1 proved to be suitable for this purpose, given that it was shown here that TFEB is retained in the cytosol of CTLs in a mouse model of the disease and this was accompanied by a significant reduction of their cytotoxic function. The implementation of various microscopy techniques led to the discovery that NPC1-defficient CTLs accumulate aberrant autophagic bodies that fuse, or embed, lipid-dense CGs. These observations were corroborated in CTLs derived from a cohort NP-C1 patients. Finally, it was established that reconstituting nuclear TFEB was sufficient to restore autophagic dysregulation, but it was not linked to cytotoxic improvement. Contrastingly, pharmacological depletion of aberrantly accumulated lipids within CGs of NPC1-defficient CTLs, completely restored their cytotoxic function. The work presented here shows that the mechanism behind it is linked to lipid inactivation of the pore forming protein, perforin, and the consequent inability of NPC1-defficient CTLs to kill target cells. Overall, the evidence presented here suggests that TFEB plays a critical role in CTL activation which might be unrelated to its canonical lysosomal biogenesis transcriptional regulation. Moreover, while studying this transcription factor in CTLs in the context of the LSD, NP-C1, a number of phenotypical abnormalities were discovered, including accumulation of autophagic bodies and reduced cytotoxicity. Importantly, the impairment in the autophagic flux in NPC1-defficient CTLs was linked to limited TFEB nuclear translocation while the diminished capacity to kill target cells was associated with lipid accumulation within CGs.
KW  - Cytotoxic T-Lymphocytes
KW  - Cytotoxic secretory granules
KW  - MiT/TFE family
KW  - Niemann-Pick type C1
T1  - The role of the MiT/TFE family of transcription factors in cytotoxic secretory granule formation in cytotoxic Tlymphocytes and the Niemann-Pick type C1 disease as a model
L1  - /bitstream/handle/11343/274852/01f52657-f567-eb11-94d1-0050568d0279_210528_DC_Thesis.pdf?sequence=1&isAllowed=n
ER  -