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The effect of manipulating apoptotic cell uptake on their immunogenicity

The effect of manipulating apoptotic cell uptake on their immunogenicity
The effect of manipulating apoptotic cell uptake on their immunogenicity
Failure of the immune system to differentiate between apoptotic tumour cells and cells rendered apoptotic as part of homeostasis prevents a successful response being mounted against tumours. Apoptotic cells are cleared rapidly by professional phagocytes thus preventing the release of potentially inflammatory or immunogenic material into the surrounding environment. In addition, macrophages release immuno-suppressive cytokines such as tumour growth factor ? (TGF-?) and interleukin-10 (IL-10) that dampen the initiation of cytotoxic T cell (CTL) immunity and render T cells tolerant. It was hypothesised that manipulation of this clearance pathway, which is normally employed to prevent autoimmunity, may alleviate immuno-suppression of apoptotic tumour cells and promote the expansion of a tumour antigen-specific T cell response.
Milk fat globule – epidermal growth factor 8 (MFG-E8) is a glycoprotein secreted by activated macrophages and immature dendritic cells. It facilitates the uptake of apoptotic cells by acting as a bridging molecule between integrins on the phagocyte and phosphatidylserine (PS) on the apoptotic cell surface. Here, two recombinant dominant-negative MFG-E8 proteins were generated: one which is shown to inhibit PS-dependent apoptotic cell uptake by macrophages by over 40% (DN-MFG-E8), and a second which re-directed apoptotic cells through Fc? receptors and conferred enhanced phagocytosis by both macrophages and immature dendritic cells (DN-MFG-E8-Fc) in a dose-dependent manner.
Cross-presentation of cell-associated antigen by bone marrow-derived dendritic cells (BMDCs) was determined by CD8+ T cell proliferation assays in vitro and in vivo. Loading BMDCs with apoptotic cells via a PS-independent pathway or through Fc? receptors (Fc?Rs) reduced their ability to induce CD8+ T cell proliferation in vivo, suggesting the blockade of a mechanism which is intrinsic for DC maturation or migration. Similarly, the balance between activating and inhibitory Fc?Rs proved essential for effective DC maturation. Apoptotic cells treated with DN-MFG-E8-Fc protein resulted in upregulation of costimulatory molecules, CD86 and CD70, when BMDCs were deficient for the inhibitory Fc?R, Fc?RIIb.
Additionally, the immunosuppressive effect of apoptotic cells on antibody production proved dependent on the exposure of PS; whereby both DN-MFG-E8 and DN-MFG-E8-Fc proteins were shown to alleviate this suppression.
Attfield, Kathrine Elizabeth
838635f0-dbdc-494f-8881-3c6553aa16ef
Attfield, Kathrine Elizabeth
838635f0-dbdc-494f-8881-3c6553aa16ef
Al-Shamkhani, Aymen
0a40b3ce-9d71-4d41-9369-7212f0a84504
Glennie, Martin
9f6f0eff-4560-48c2-80cd-0ec116110ded

Attfield, Kathrine Elizabeth (2009) The effect of manipulating apoptotic cell uptake on their immunogenicity. University of Southampton, School of Medicine, Doctoral Thesis, 192pp.

Record type: Thesis (Doctoral)

Abstract

Failure of the immune system to differentiate between apoptotic tumour cells and cells rendered apoptotic as part of homeostasis prevents a successful response being mounted against tumours. Apoptotic cells are cleared rapidly by professional phagocytes thus preventing the release of potentially inflammatory or immunogenic material into the surrounding environment. In addition, macrophages release immuno-suppressive cytokines such as tumour growth factor ? (TGF-?) and interleukin-10 (IL-10) that dampen the initiation of cytotoxic T cell (CTL) immunity and render T cells tolerant. It was hypothesised that manipulation of this clearance pathway, which is normally employed to prevent autoimmunity, may alleviate immuno-suppression of apoptotic tumour cells and promote the expansion of a tumour antigen-specific T cell response.
Milk fat globule – epidermal growth factor 8 (MFG-E8) is a glycoprotein secreted by activated macrophages and immature dendritic cells. It facilitates the uptake of apoptotic cells by acting as a bridging molecule between integrins on the phagocyte and phosphatidylserine (PS) on the apoptotic cell surface. Here, two recombinant dominant-negative MFG-E8 proteins were generated: one which is shown to inhibit PS-dependent apoptotic cell uptake by macrophages by over 40% (DN-MFG-E8), and a second which re-directed apoptotic cells through Fc? receptors and conferred enhanced phagocytosis by both macrophages and immature dendritic cells (DN-MFG-E8-Fc) in a dose-dependent manner.
Cross-presentation of cell-associated antigen by bone marrow-derived dendritic cells (BMDCs) was determined by CD8+ T cell proliferation assays in vitro and in vivo. Loading BMDCs with apoptotic cells via a PS-independent pathway or through Fc? receptors (Fc?Rs) reduced their ability to induce CD8+ T cell proliferation in vivo, suggesting the blockade of a mechanism which is intrinsic for DC maturation or migration. Similarly, the balance between activating and inhibitory Fc?Rs proved essential for effective DC maturation. Apoptotic cells treated with DN-MFG-E8-Fc protein resulted in upregulation of costimulatory molecules, CD86 and CD70, when BMDCs were deficient for the inhibitory Fc?R, Fc?RIIb.
Additionally, the immunosuppressive effect of apoptotic cells on antibody production proved dependent on the exposure of PS; whereby both DN-MFG-E8 and DN-MFG-E8-Fc proteins were shown to alleviate this suppression.

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Published date: March 2009
Organisations: University of Southampton

Identifiers

Local EPrints ID: 72701
URI: https://eprints.soton.ac.uk/id/eprint/72701
PURE UUID: f60c5f02-8bab-46e5-89ee-3c767be187aa
ORCID for Aymen Al-Shamkhani: ORCID iD orcid.org/0000-0003-0727-4189

Catalogue record

Date deposited: 22 Feb 2010
Last modified: 31 Jan 2019 01:37

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