1. Investigating the role of exosomes in plasmacytoid DC mediated tolerance Ulf Gehrmann, fil.mag., Biomedicine I.Hypothesis & Objectives II.Background III.Methods IV.Results V.Discussion

2. Hypothesis I.Hypothesis & Objectives II.Background III.Methods IV.Results V.Discussion „ Exosomes can be taken up by plasmacytoid Dendritic Cells and are thus capable of inducing exosome or pDC specific tolerance.“ Project objectives: 1.Show that exosomes can be internalised by plasmacytoid Dendritic Cells 2.Show that pDCs can internalise exosomes with copmparable efficiency as myeloid Dendritic Cells 3.Elucidate the molecular mechanism behind exosome uptake

3. What are exosomes? Data sheet: Size: Size: 40-100nm Producing cells: Producing cells: APCs (DCs, B-cells, Macrophages) Reticulocytes Epithelial 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

4. 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)

5. Exosomes as transport vesicles and in immune stimulation Dendritic cell 1 Dendritic cell 2 Legend: AllergenpMHC II complex ER-vesicle endosome late endosomeMultivesicular body T T

6. Project Plan I.Hypothesis & Objectives II.Backgrounds III.Methods IV.Results V.Discussion Blood, ”Buffy Coat” HMC-1 cell line Density centrifugation Isolation of PBMCs MACS Differential ultracentrifugation Lymphoprep© Isolation of plasmacytoid Dendritic Cells (DCs), myeloid DCs and Monocytes Isolation of exosomes Magnetic sorting Human breast milk Controls Incubation (5min to 4hrs) Analysis using confocal microscopy and flowcytometry

7. Isolated vesicles are exosomes 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

8. Exosomes are associated with pDCs… I.Hypothesis & Objectives II.Backgrounds III.Methods IV.Results V.Discussion After 4 hours, HMC-1 exosomes are associated with pDCs, following the same trend as monocytes.

9. monocytes Plasmacytoid DCs HMC-1 exosomesPKH67 controlLatex beads 1 2 45 6 4 … but do they adhere to the surface ? I.Hypothesis & Objectives II.Backgrounds III.Methods IV.Results V.Discussion PKH67: negative control Latex beads: positive control HMC-1 exosomes: associate with pDCs and monocytes

10. Conclusions I.Hypothesis & Objectives II.Backgrounds III.Methods IV.Results V.Discussion 1.What have we done: 2.What needs to be 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…)

11. Acknowledgements I.Hypothesis & Objectives II.Backgrounds III.Methods IV.Results V.Discussion Many thanks for intellectual, financial and moral support: Dr.Esbjörn Telemo, Department of Rheumatology and Inflammation Research, Sahlgrenska University Hospital Gothenburg University