Date post: | 18-Jan-2018 |
Category: |
Documents |
Upload: | arnold-goodman |
View: | 219 times |
Download: | 0 times |
Investigating the role of exosomes in Investigating the role of exosomes in plasmacytoid DC mediated toleranceplasmacytoid DC mediated tolerance
Ulf Gehrmann, fil.mag., BiomedicineUlf Gehrmann, fil.mag., Biomedicine
I. Hypothesis & Objectives
II. Background
III. Methods
IV. Results
V. Discussion
HypothesisHypothesis
I. Hypothesis & Objectives
II. Background
III. Methods
IV. Results
V. Discussion
„ „ Exosomes can be taken up by plasmacytoid Exosomes can be taken up by plasmacytoid Dendritic Cells and are thus capable of inducing Dendritic Cells and are thus capable of inducing
exosome or pDC specific tolerance.“exosome or pDC specific tolerance.“
Project objectives:Project objectives:
1.1. Show that exosomes can be internalised by Show that exosomes can be internalised by plasmacytoid Dendritic Cellsplasmacytoid Dendritic Cells
2.2. Show that pDCs can internalise exosomes with Show that pDCs can internalise exosomes with copmparable efficiency as myeloid Dendritic Cellscopmparable efficiency as myeloid Dendritic Cells
3.3. Elucidate the molecular mechanism behind exosome Elucidate the molecular mechanism behind exosome uptakeuptake
What are exosomes?What are exosomes?
Data sheet:
Size:Size: 40-100nm
Producing cells:Producing cells: APCs (DCs, B-cells, Macrophages)ReticulocytesEpithelial cells (gut, kidney…)
Functions:Functions: -shed obsolete proteins (reticulocytes)-cell to cell transfer of molecules-immunostimulatory-tolerance inducing
I. Hypothesis & Objectives
II. Background
III. Methods
IV. Results
V. Discussion
What are plasmacytoid Dendritic Cells?What are plasmacytoid Dendritic Cells?
•What we knew:•Make up ~ 0,5% of all perinpheral blood mononuclear cells (PBMCs)•Have an unclear cellular origin (probably lymphoid)•Were believed to be immature precursors of active DCs (no antigen uptake, no antigen presentation)•Cellular marker proteins: CD123, BDCA-2, BDCA-4
I. Hypothesis & Objectives
II. Background
III. Methods
IV. Results
V. Discussion
•Recent research showed:•Can be activated by IL-3 and CD40L•Could mediate allograft specific tolerance and
significantly delay graft rejection (Ochando et al., 2006)
Exosomes as transport Exosomes as transport vesiclesvesicles
and in immune stimulationand in immune stimulation
Dendritic cell 1 Dendritic cell 2
Legend:Legend:AllergenAllergen pMHC II complexpMHC II complex
ER-vesicleER-vesicle endosome endosome
late endosomelate endosome Multivesicular Multivesicular bodybody
TT
TT
Project PlanProject Plan
I. Hypothesis & Objectives
II. Backgrounds
III. Methods
IV. Results
V. Discussion
Blood, ”Buffy Coat” HMC-1 cell
lineDensity centrifugation
Isolation of PBMCs
MACS
Differential ultracentrifugatio
n
Lymphoprep©
Isolation of plasmacytoid Dendritic Cells (DCs), myeloid DCs and Monocytes
Isolation of exosomesMagnetic sorting
Human breast milk
Controls
Incubation (5min to
4hrs)Analysis using
confocal microscopy and flowcytometry
Isolated vesicles are Isolated vesicles are exosomesexosomes
I. Hypothesis & Objectives
II. Backgrounds
III. Methods
IV. Results
V. Discussion
Breast milk exosomes and HMC-1 exosomes express exosomal surface markers CD9, CD63,
CD81
CD9
Breast milk
HLA-DRCD81CD63
HMC-1
Exosomes are associated with Exosomes are associated with pDCs…pDCs…
I. Hypothesis & Objectives
II. Backgrounds
III. Methods
IV. Results
V. Discussion
0
1.0×102
2.0×102
3.0×102
4.0×102
5.0×102
6.0×102
6.0×103 4 h pDC4 h mono
MFI
After 4 hours, HMC-1 exosomes are associated with pDCs, following the same trend as monocytes.
monocytes
Plasmacytoid DCs
HMC-1 exosomesPKH67 control Latex beads
1 2
4 5 6
4
… … but do they adhere to the but do they adhere to the surface ?surface ?
I. Hypothesis & Objectives
II. Backgrounds
III. Methods
IV. Results
V. DiscussionPKH67: negative control
Latex beads: positive control
HMC-1 exosomes: associate with pDCs and monocytes
ConclusionsConclusions
I. Hypothesis & Objectives
II. Backgrounds
III. Methods
IV. Results
V. Discussion
1.1. What have we What have we done:done:
2.2. What needs to be What needs to be done:done:
• Shown, that isolated vesicles express exosomal marker proteins.
• Shown, that HMC-1 exosomes are associated with pDCs.
• Visualised, that HMC-1 exosomes are associated with pDCs.• Show and visualise, that HMC-1 exosomes are found
inside the cell.
• Show, that pDCs internalise exosomes comparably efficient as mDCs and monocytes. (preliminary results: they do!)
• Show, which membrane molecules are responsible for uptake. (e.g. CD54 & CD11a, CD61 & MFG-E8, CD9 &CD81…)
AcknowledgementsAcknowledgements
I. Hypothesis & Objectives
II. Backgrounds
III. Methods
IV. Results
V. Discussion
Many thanks for intellectual, financial and moral Many thanks for intellectual, financial and moral support:support:
Dr.Esbjörn Telemo, Dr.Esbjörn Telemo,
Department of Rheumatology and Inflammation Research,Department of Rheumatology and Inflammation Research,Sahlgrenska University HospitalSahlgrenska University Hospital
Gothenburg UniversityGothenburg University