Blees, Hanna; (2019) Deciphering the function of DNGR-1 in cross-presentation through the characterisation of phagosomal compartments in cDC1. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Induction of antigen specific cytotoxic CD8+ T lymphocyte (CTL) responses by dendritic cells (DCs) is essential for clearance of infected or malignantly transformed cells. Antigens derived from such cells are presented to naïve CD8+ T cells in the form of short antigenic peptides associated with major histocompatibility complex I (MHC I) on the DC surface. This process, called cross-presentation, often involves transfer of antigens from dying infected or malignantly transformed cells to DCs and is facilitated by innate receptors that sense dead cell-derived damage-associated molecular patterns (DAMPs). These receptors include the C-type lectin receptor DNGR-1, which allows DCs to detect the presence of dead or dying cells by binding to filamentous actin (F-actin) exposed by dead cell corpses. DNGR-1 promotes cross-presentation of dead cell-associated antigens but the mechanism involved is still poorly understood. The aim of my PhD project was to dissect the mechanism by which DNGR-1 facilitates cross-presentation of dead cell-associated antigens. I found it involved proteasomal degradation and was enhanced by inhibition of lysosomal proteases. Further, the cytoplasmic tail of DNGR-1 and, therefore likely DNGR-1 signalling, was essential to promote cross-presentation post cargo uptake. Since DNGR-1 was recruited to antigen-bearing phagosomes, I studied the characteristics of those DNGR-1+ phagosomes. Combined analysis of antigen degradation and staining for DNGR-1 and LAMP-2 revealed two distinct phagosome populations with varying degradative potential and MHC I recruitment: a DNGR-1+LAMP-2-MHC I+ that showed strikingly lower degradative potential, in contrast to DNGR-1-LAMP-2+MHC I- phagosomes. However, DNGR-1+ phagosomes eventually acquired LAMP-2+ resulting in an increase in antigen degradation. To test whether DNGR-1 ligand engagement was shaping the phagosomal proteome in cDC1s, I analysed FACS-purified DNGR-1+ and LAMP-2+ phagosome populations by mass spectrometry. A strong enrichment of the calcium pump SERCA1 and the autophagy initiator beclin-1 was observed in DNGR-1+ phagosomes containing DNGR-1 ligand. Preliminary experiments further revealed that the phagosomal lumen became accessible for cytosolic galectins in a DNGR-1-dependent manner suggesting that DNGR-1 might be involved in antigen to cytosol transfer. In summary, this thesis offers novel insights into the mechanisms by which dead cell antigens are cross-presented by cDC1 through the engagement of DNGR-1, which potentially regulates the stability of antigen-containing phagosomes and thus, might mediate the transfer of antigen from the phagosome into the cytosol.

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