Exosomes from red blood cell units
bind to monocytes and induce
proinflammatory cytokines, boosting
T-cell responses in vitro
Ali Danesh, Heather C. Inglis, Rachael P. Jackman, Shiquan Wu,
Xutao Deng, Marcus O. Muench, John W. Heitman, and Philip J.
Norris
Blood 2014;123:687-696
Danesh A et al. Blood 2014;123:687-696
Vienna 17.3.2014
Outline
•
Introduction
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–
•
•
•
Why is this paper important for our group?
What are exosomes?
Special methods used in this study
Key results
Discussion
Vienna 17.3.2014
Relevance
protein
supernatant
product
lipids
microRNA
extracellular
vesicles
Gambim et al. Critical Care 2007 11:R107
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Extracellular
Vesicles
non-radiated PBMC
György et al. Cellular and Molecular Life Sciences 2011; 68(16):2667-88.
radiated PBMC
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Extracellular
Vesicles
György et al. Cellular and Molecular Life Sciences 2011; 68(16):2667-88.
Vienna 17.3.2014
Extracellular
Vesicles
György et al. Cellular and Molecular Life Sciences 2011; 68(16):2667-88.
Vienna 17.3.2014
Extracellular
Vesicles
György et al. Cellular and Molecular Life Sciences 2011; 68(16):2667-88.
Vienna 17.3.2014
Study Aim
Do red blood cell units contain
extracellular vesicles?
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Results
n=6
EV relative size stored RBC units.
Danesh A et al. Blood 2014;123:687-696
Vienna 17.3.2014
Results
EV absolute count in
stored RBC units.
Source
Annexin +
Danesh A et al. Blood 2014;123:687-696
Vienna 17.3.2014
Results
Exosome vs. Microparticle
CD63
Danesh A et al. Blood 2014;123:687-696
Vienna 17.3.2014
Cytokine and growth factor
secretion by PBMCs
stimulated with EVs.
T helper cell
subset
NLRP3
Inflammasome
T cell growth
factors
Danesh A et al. Blood 2014;123:687-696
Vienna 17.3.2014
PBMC survival
Danesh A et al. Blood 2014;123:687-696
Cell population analysis
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EV-induced immune augmentation of T-cell response
Proliferation PHA+EV
CD8
CD4
Proliferation: EV
CFSE
Danesh A et al. Blood 2014;123:687-696
CFSE
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Mechanism of EV-induced
immune augmentation.
PHA + EV
PHA + EV + Monocytes
Danesh A et al. Blood 2014;123:687-696
Vienna 17.3.2014
EV act via monocyte
activation
CD4+
Danesh A et al. Blood 2014;123:687-696
CD8+
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3h
3h
24h
T-cells
24h
Monocytes
Interaction of PBMC
subpopulations with
EVs.
Danesh A et al. Blood 2014;123:687-696
TNF-α production
indicates monocytes
as primary interacting
cells with EVs
Monocyte TNF-α
Danesh A et al. Blood 2014;123:687-696
Exosomes stimulate TNF-α
release
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Exosome signaling via
CD40 CD40L
interaction
CD4+
Danesh A et al. Blood 2014;123:687-696
CD8+
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Summary
Packed red blood cells
1.
1.
2.
3.
4.
5.
6.
7.
8.
contain extracellular vesicles (EV)
EV originate mainly from red blood
cells and platelets
Modify cytokine secretion of PBMCs
EV effect PBMC survival
EV augment T-cell proliferation
EV mediate via fusion with monocytes
blockade CD40/CD40L interaction
attenuated EV mediated effects
biological active EV are <200 nm
Danesh A et al. Blood 2014;123:687-696
Vienna 17.3.2014
Thank you for
your attention!
Vienna 17.3.2014
Exosome
Exosomen
Mikrovesikel
Apo-Körper
Größe
30-100 nm
100-1000 nm
1-5 μm
Sekretion
Exozytose
Budding
Blasenbildung durch
Apoptose
Isoliering
Vorzentrifugation +
Ultracentrifugation mit
mind. 100,000 g
Zentrifugation mit ca.
20,000g
Keine definitive
Isolationsmöglichkeit,
meist Zellüberstand
von apo. Zellen
Detektion
TEM, Western-Blotting
FACS, Massenspek.
FACS
FACS
Marker
Annexin 5, CD63…
Integrine, Selektine,
CD40L
Histone
Vienna 17.3.2014