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Deciphering the regulation of perforin-2-mediated endocytic escape in dendritic cells https://doi.org/10.17863/CAM.110558 Laub, Marco Kozik, Patrycja Dendritic cells (DCs) are important players in the initiation of cytotoxic CD8+ T cell-mediated immune responses against pathogens and tumours. DCs continuously sample their environment for antigens and load them on their MHC class I molecules to prime naïve CD8+ T cells in a process known as cross-presentation. Unlike most cell types, cross-presenting DCs have leaky endocytic compartments from where internalised proteins can escape into the cytosol for proteasome-mediated generation of MHC-I-binding peptides. Prior to my project, the Kozik lab had identified the pore-forming protein perforin-2 as a dedicated effector of endocytic escape that allows cytosolic entry of exogenous antigen during cross-presentation. Perforin-2 is a member of the membrane attack complex and perforin superfamily (MACPF) and, similar to perforin and complement, can form oligomeric pores on liposomes *in vitro*. However, the regulation of perforin-2 pore formation in the context of endocytic escape *in vivo* is still poorly understood. The aim of my PhD project was to decipher the mechanisms that enable DCs to assemble perforin-2 pores on their own membranes for endocytic escape without causing uncontrolled lysis of intracellular compartments. Analysis of its steady-state distribution showed that perforin-2 was proteolytically processed with full-length and cleaved forms present in early and late endocytic compartments, respectively. Since perforin-2 was recruited to antigen-containing phagosomes, I studied its stepwise proteolytic processing during phagosome maturation. Flow cytometry analysis of isolated phagosomes revealed that, in line with previous *in vitro* studies, perforin-2 processing was dependent on acidification. However, by developing an assay to monitor perforin-2 activity at the phagosome, I discovered that inhibiting acidification had no impact on the efficiency of perforin-2-mediated escape. In addition, perforin-2 activity had no detectable effect on the luminal pH or degradative potential of phagosomes. To better understand the role of pH-dependent processing of perforin-2 during phagosome maturation, I studied the proteases involved. The analysis showed that the pore-forming ectodomain was cleaved off the transmembrane domain by cysteine proteases and further processed by asparagine endopeptidase while the remaining membrane stub was removed by γ-secretase. Specific inhibition of these proteases had no impact on perforin-2-mediated endocytic escape suggesting that they are not directly required for pore formation. However, analysis of a perforin-2 trafficking mutant suggested that a currently unknown processing step may be necessary for efficient perforin-2 activity at the phagosome. In summary, this thesis offers novel insights into the regulation of perforin-2 which potentially releases antigens for cross-presentation early during phagosome maturation. University of Cambridge 2024-04-19 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/371342 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/27bb01ad-d13a-43e4-9bbd-93ed61dd80f0/download 87eda9de84448d1f82354d60eee3eb5f https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/84e2a577-4640-454c-8ea6-d7c060e085a2/download 97c3164032e3d38393c4edeb33144ad4 https://www.rioxx.net/licenses/all-rights-reserved/ Cross-presentation Dendritic cells Endocytic escape Perforin-2