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