1. Interaction between the Cytomegalovirus and the Langerhans cells
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) Langerin (CD207)
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 Introduction Prerequisite Hypothesis
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
Differentiation of Cells
Generation of MDLCs from frozen Monocytes: EFS, thawed (+FCS), TGFβ1 + GM-CSF + RPMI, 6-well plates, /well
Langerhans-like cells staining for FACS: centrifugation + count of cells + resuspension (FACS Buffer), V-bottom 96-well plate, /well, 50µL of Fluo Ab (30min at 4°C without light) + 3 washes
Keratinocytes Culture: pieces of surgery, thawed (+FCS), specific medium
Langerhans-Like Cell Culture with Keratinocytes contact: MDLCs+Ks+Kmed or MDLCs+Kmed or MDLCs+Lmed at day 5 to day 8
Cell Phenotyping by FACS: FlowJo, SSC-FCS
Interactions between MDLC & CMV
MRC5 cell culture: thawed embryonic human fibroblasts, fully permissive to CMV infection
MDLC Infection: MDLCs ( /well) + U937 (DC-SIGN +), Trt (50µL, 30min 37°C), TB40/E-GFP Sn (200µL, 2h 37°C)
Trans-Infection: 3 washes + resuspension, 24-well plates of subconfluent MRC5 cells, centri + 48h 37°C
Interaction gB-MDLC assay: gB-Alexa647 + titration, RPMI medium, CD207 staining, washed twice

7. Results Generation of Langerhans-Like Cells Phenotypic Analysis
Phenotypic Analysis: CD1a +, CCR6 + (DC, LC) CD68 – , CD163 – (MØ)
CD32 + (Fc R) CD16 –, CD64 – (Fc R)
TROP-2 +
Axl +

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

9. Langerin-Expressing Cells at day 5 and day 8 of incubation
Results
Generation of Langerhans-Like Cells
Phenotypic Analysis
Improvement of the langerin expression
Generation of Langerhans-Like Cells
Phenotypic Analysis
Improvement of the langerin expression: time-dep (with fresh medium every 2-3 days)
medium, no better/no worse with K medium
Langerin-Expressing Cells at day 5 and day 8 of incubation
(in the LC medium)

10. Langerin-Expressing cells at days 7 and 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
Interactions between MDLC & CMV
L lectin-dependant trans-infection: trans-infection by DC-SIGN+ U937 cells, no trans-infection by MDLCs, no conclusion cause very low
(pb langerin, pb nb of cell…)

12. Glycoprotein B binding to MDLCs
Results
Generation of Langerhans-Like Cells
Interactions between MDLC & CMV
Lectin-dependant trans-infection
Binding between MDLCs and gB assay
Interactions between MDLC & CMV
L lectin-dependant trans-infection
Binding between MDLCs and gB assay: + (now need to identify the langerin  anti-langerin, iso-Ab, mannan, EDTA)
Glycoprotein B binding to MDLCs

13. And langerin expression
Results - Conclusion
Generation of LCs
And langerin expression
LC-gB binding
Trans-infection
LC medium
K medium
Keratinocyte
EXPECTATIONS ↗ +
1. Appropriate positive control
2. No transmission of virions from LC-like cells to MRC5 cells
FINDINGS
No significant change
No conclusion
Prominent
Matter of time and not type of medium

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. Thank you for
your attention!