Childs, Eleanor; (2024) Cytosolic translocation of endocytic cargo: regulation by DNGR-1 and function in immunity. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

DNGR-1 is a receptor expressed by type 1 conventional dendritic cells (cDC1s) that binds to F-actin exposed on necrotic cells. DNGR-1 signalling induces phagosomal rupture, resulting in the cytosolic translocation of necrotic cell-associated antigens to promote cross-presentation to CD8+ T cells. This occurs through activation of the tyrosine kinase SYK and production of phagosomal ROS. In this thesis, I first explored how DNGR-1 signalling links to NADPH oxidase activation in relation to the poorly characterised RhoGEF FGD2, which is recruited to DNGR-1+ phagosomes. Loss of FGD2 abrogated phagosomal ROS in RAW264.7 cells and reduced membrane damage in mixed primary cDC cultures fed latex beads targeted to DNGR-1. Cross-presentation experiments revealed cargo-specificity for FGD2- dependent responses downstream of DNGR-1 signalling, with FGD2 being partly required for cross-presentation of bead-associated antigens but dispensable for necrotic cell-associated antigens. A second aim of this thesis was to examine whether DNGR-1-mediated phagosomal rupture could facilitate cytosolic translocation of nucleic acids in addition to antigens from ingested necrotic cells, resulting in the engagement of cytosolic innate immune pathways. Proof-of-concept experiments demonstrated that UV-irradiated necrotic cells trigger interferon stimulated gene (ISG) induction in the MuTuDC-1940 cDC1 cell line, in a DNGR-1- dependent manner. This was mediated through the cGAS/STING pathway, indicating that DNGR-1 promotes sensing of necrotic cell-derived DNA within the cytosol. Further work will be required to establish if this pathway also occurs in primary cDC1s and its physiological relevance. Overall, the data presented in this thesis provide new insights on the molecular mechanisms by which DNGR-1 promotes the cytosolic translocation of endocytic cargo and innate sensing by cDC1s.

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