The role of a pore-forming protein, Mpeg1, in cytosolic import

Cross-presentation is a process by which professional antigen presenting cells acquire exogenous antigens and present them on their surface on MHC class I molecules. This process is key for the initiation of CD8+ T cell responses. Two main pathways for cross-presentation have been described, cytosolic and vacuolar. In the cytosolic pathway, antigens need to be imported from the endo/lysosomal compartment (to which they localise after their uptake) to the cytosol for further processing by the proteasome, which then generates peptides that can be loaded onto MHC class I molecules. In the vacuolar pathway, these peptides are generated by lysosomal processing, circumventing the need for cytosolic import. The cytosolic pathway of cross-presentation has been proposed to be more relevant for the initiation of anti-viral and anti-tumour immune responses in vivo, although the genetic evidence for this is still missing. Similarly, the mechanisms underlying cross-presentation remain to be fully characterised. The main aim of my PhD was to identify proteins that play a role in the cytosolic import of antigens during cross-presentation. To do so we developed a new assay, which relies on the type I ribosome inactivating protein, saporin, which can inhibit translation upon entry into the cytosol. Using the saporin assay, we performed a CRISPR/Cas9 screen targeting genes enriched in the dendritic cell (DC) subset most efficient at cytosolic import. With this approach we identified Mpeg1, as a potential player in cytosolic import in DCs. Mpeg1 is a member of the membrane attack complex/perforin (MACPF) superfamily. We find that DCs deficient in Mpeg1 are less efficient in their ability to import saporin into the cytosol, and that the activity of Mpeg1 is regulated by low pH and potentially proteolytic cleavage. Furthermore, expression of Mpeg1 in primary splenocytes correlates with the ability to import saporin into the cytosol ex vivo. Mpeg1-expressing cells efficient at cytosolic import ex vivo were also depleted in vivo following injection of saporin. Finally, using CRISPR/Cas9 technology, we have generated an Mpeg1 knock-out mice, and demonstrated that knocking out Mpeg1 in primary cells also inhibits cytosolic import. Given the newly discovered role of Mpeg1 in cytosolic import, I went on to study its role in antigen cross-presentation. Surprisingly we found that Mpeg1-deificient cells did not have defective cytosolic cross-presentation, suggesting that inhibition of cytosolic import may not be sufficient to inhibit cross-presentation. We hope that further characterisation of the Mpeg1 knock-out mice generated will allows us to clarify the role of Mpeg1 and cytosolic import in modulation of immune responses in vivo.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Rodríguez, Pablo

Advisor dc:contributor.advisor

• Kozik, Patrycja

• Cross-presentation
• Dendritic cells
• Immunology
• Mpeg1
• Cytosolic escape

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.81743

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/334330