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I NTERACTION BETWEEN THE C YTOMEGALOVIRUS AND THE L ANGERHANS CELLS Does the Human Cytomegalovirus interact with Monocyte-derived Langerhans cells in a.

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Presentation on theme: "I NTERACTION BETWEEN THE C YTOMEGALOVIRUS AND THE L ANGERHANS CELLS Does the Human Cytomegalovirus interact with Monocyte-derived Langerhans cells in a."— Presentation transcript:

1 I NTERACTION BETWEEN THE C YTOMEGALOVIRUS AND THE L ANGERHANS CELLS Does the Human Cytomegalovirus interact with Monocyte-derived Langerhans cells in a Langerin dependent manner? Raluca MACSIM (Cluj-Napoca) Madeline BREUGNON (Nantes) Supervisor: Franck HALARY 18/12/2012

2 Post-transplantation infections Public health problem % CMV infected but asymptomatic patients Immunosuppressive regimen and CMV reactivation I NTRODUCTION

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

4 I NTRODUCTION 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 gB langerin HCMV LC ? I NTRODUCTION 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

6 M ATERIAL & M ETHODS 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 R ESULTS Generation of Langerhans-Like Cells Phenotypic Analysis

8 R ESULTS Generation of Langerhans-Like Cells Phenotypic Analysis CD14-Expressing Cells Day 1 Day 5

9 R ESULTS 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 R ESULTS Generation of Langerhans-Like Cells Phenotypic Analysis Improvement of the langerin expression Langerin-Expressing cells at days 7 and 10

11 R ESULTS Generation of Langerhans-Like Cells Interactions between MDLC & CMV Lectin-dependant trans-infection

12 R ESULTS 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 Generation of LCs And langerin expression LC-gB binding Trans-infection LC medium K medium Keratinocyt e EXPECTATIO NS ↗↗↗+ 1. Appropriate positive control 2. No transmission of virions from LC-like cells to MRC5 cells FINDINGS No significant change No conclusion + 1.Prominent 2. No conclusion Matter of time and not type of medium R ESULTS - C ONCLUSION

14 D ISCUSSION & P ROSPECTS ‘in vitro’ model: frozen monocytes Incubation period: >8 days - >80% of langerin expression Medium: TGF-β1 LC medium K medium Keratinocytes

15 D ISCUSSION & P ROSPECTS ‘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 D ISCUSSION & P ROSPECTS ‘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

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