Interaction between the Cytomegalovirus and the Langerhans cells

1. Raluca MACSIM (Cluj-Napoca) Madeline BREUGNON (Nantes) Supervisor: Franck HALARY 18/12/2012 Interaction between the Cytomegalovirus and the Langerhans cells Does the Human Cytomegalovirus interact with Monocyte-derived Langerhans cells in a Langerin dependent manner?

2. Introduction • Post-transplantation infections • Public health problem • 40-100% CMV infected but asymptomatic patients • Immunosuppressive regimen and CMV reactivation

3. Introduction • Human Cytomegalovirus • Herpesviridae family • Able to infect hematopoietic cells • Reservoirs: monocytes • Reactivation in DCs and macrophages upon pro-inflammatory cytokine or allogeneic stimulation

4. Introduction • Langerhans cells (LCs) • First line of defense (epidermis) • Subtype of dendritic cells (DCs) • Engoulf the antigens in order to present it to T cells • Langerin (CD207) • The most important surface molecule (C-type lectin) • Resembles DC-SIGN (CD209) expressed by DCs

5. Introduction • Prerequisite • CMV-DC interaction is mediated by DC-SIGN • Freshly isolated skin LCs are able to bind CMV • Hypothesis • Frozen monocytes can differentiate into LC-like cells • Langerin mediates the CMV-LC interaction ? gB langerin LC HCMV

6. Material & Methods • Differentiation of Cells • Generation of MDLCs from frozen Monocytes • Langerhans-like cells staining for FACS • Keratinocytes Culture • Langerhans-Like Cell Culture with Keratinocytes contact • Cell Phenotyping by FACS • Interactions between MDLC & CMV • MRC5 cell culture • MDLC Infection • MRC5 Trans-Infection • Interaction gB-MDLC assay

7. Results • Generation of Langerhans-Like Cells • Phenotypic Analysis

8. Results • Generation of Langerhans-Like Cells • Phenotypic Analysis Day 1 Day 5 CD14-Expressing Cells

9. Results • Generation of Langerhans-Like Cells • Phenotypic Analysis • Improvement of the langerin expression Langerin-Expressing Cells at day 5 and day 8 of incubation (in the LC medium)

10. Results • Generation of Langerhans-Like Cells • Phenotypic Analysis • Improvement of the langerin expression Langerin-Expressing cells at days 7 and 10

11. Results • Generation of Langerhans-Like Cells • Interactions between MDLC & CMV • Lectin-dependant trans-infection

12. Results • Generation of Langerhans-Like Cells • Interactions between MDLC & CMV • Lectin-dependant trans-infection • Binding between MDLCs and gB assay Glycoprotein B binding to MDLCs

13. Results - Conclusion

14. Discussion & Prospects • ‘in vitro’ model: frozen monocytes • Incubation period: >8 days • - >80% of langerin expression • Medium: TGF-β1 • LC medium • K medium • Keratinocytes

15. Discussion & Prospects • ‘in vitro’ model: frozen monocytes • MDLCs were able to bind recombinant CMV gB • 46,2% • MDLCs with purified CMV virions, in the presence of different langerin inhibitors (EDTA, mannan)

16. Discussion & Prospects • ‘in vitro’ model: frozen monocytes • MDLCs were able to bind recombinant CMV gB • Trans-infection experiment • Positive control • LC-like cells with higher langerin expression (langerin transfectants) • Equal number of cells • We expect LC-CMV interaction to play a protective role upon CMV infection

17. Thankyou for your attention!