{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/371342"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/371342","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Deciphering the regulation of perforin-2-mediated endocytic escape in dendritic cells","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Laub, Marco"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Kozik, Patrycja"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-04-19","date_published":"2024-04-19","updated_at":"2026-07-22T22:24:10Z","subjects":["Cross-presentation","Dendritic cells","Endocytic escape","Perforin-2"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/27bb01ad-d13a-43e4-9bbd-93ed61dd80f0/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.110558","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Kozik, Patrycja"]},{"key":"dc:creator","label":"Author","values":["Laub, Marco"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2024-04-19"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/371342"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Cross-presentation","Dendritic cells","Endocytic escape","Perforin-2"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/27bb01ad-d13a-43e4-9bbd-93ed61dd80f0/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.110558"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/84e2a577-4640-454c-8ea6-d7c060e085a2/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["87eda9de84448d1f82354d60eee3eb5f","97c3164032e3d38393c4edeb33144ad4"]},{"key":"dc:title","label":"Title","values":["Deciphering the regulation of perforin-2-mediated endocytic escape in dendritic cells"]}]}],"canonical_facts":{"dc:contributor.advisor":["Kozik, Patrycja"],"dc:creator":["Laub, Marco"],"dc:date.issued":["2024-04-19"],"dc:description.abstract":["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."],"dc:format.checksum.md5":["87eda9de84448d1f82354d60eee3eb5f","97c3164032e3d38393c4edeb33144ad4"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.110558"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/84e2a577-4640-454c-8ea6-d7c060e085a2/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/371342"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/27bb01ad-d13a-43e4-9bbd-93ed61dd80f0/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["Cross-presentation","Dendritic cells","Endocytic escape","Perforin-2"],"dc:title":["Deciphering the regulation of perforin-2-mediated endocytic escape in dendritic cells"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:10Z"}