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.