INTERACTION BETWEEN THE CYTOMEGALOVIRUS AND THE LANGERHANS CELLSDoes 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

Post-transplantation infections Public health problem 40-100% CMV infected but asymptomatic

patients Immunosuppressive regimen and CMV

reactivation

INTRODUCTION

INTRODUCTION Human Cytomegalovirus

Herpesviridae family Able to infect hematopoietic cells Reservoirs: monocytes Reactivation in DCs and macrophages upon pro-

inflammatory cytokine or allogeneic stimulation

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

gB

langerin

HCMV LC

?

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

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

RESULTS Generation of Langerhans-Like Cells

Phenotypic Analysis

Neg C

trl A

647

Neg C

trl F

ITC

Neg C

trl P

E

CD1a FIT

C

CD14 FIT

C

CD33 P

E

CD16 P

E

CD32 P

E

CD64 P

E

HLA-D

R PE

CD80 P

E

CD83 P

E

CD86 P

E

CD209

FITC

CD207

PE

CD115

PE

CD68 P

E

CD163

PE

CD103

PE

TROP-

1 PE

CD324

A647

CCR2 A64

7

CCR6 PE

CXCR3 PE

0

40

80

120

LC markers expression on frozen MDLCs (day 5)

Neg C

trl F

ITC

Neg C

trl P

E

CD14

FIT

C

CD1a

FIT

C

CD20

7 PE

TROP-

2 PE

Axl F

ITC

0102030405060708090

100

LC markers ex-pression on frozen

MDLCs (day 7)

RESULTS Generation of Langerhans-Like Cells

Phenotypic Analysis

CD14-Expressing Cells

Day 1

Day 5

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)

RESULTS Generation of Langerhans-Like Cells

Phenotypic Analysis Improvement of the langerin expression

Langerin-Expressing cells at days 7 and 10

RESULTS Generation of Langerhans-Like Cells Interactions between MDLC & CMV

Lectin-dependant trans-infection

TB-G

FP S

n ED

TA

TB-G

FP S

n Man

nan

TB-G

FP S

n NT

TB-G

FP S

n an

tiLan

g

TBGFP

Sn

iso ctrl

noSn

NT

MRC5

alon

e

DCSIGN T

BGFPSn

DCSIGN n

oSn

P TB

GFPSn

P no

Sn0

1000

2000

3000

Mean of fluorescence of infected MRC5 cells after trans-infection by MDLCs

(day 7)

TB-G

FP S

n ED

TA

TB-G

FP S

n Man

nan

TB-G

FP S

n NT

TB-G

FP S

n an

tiLan

g

TBGFP

Sn

iso ctrl

noSn

NT

MRC5

alon

e

DCSIGN T

BGFPSn

DCSIGN n

oSn

P TB

GFPSn

P no

Sn0

0.51

1.52

2.53

3.54

4.5

Percentage of fluorescent MRC5 cells after trans-infection by MDLCs (day 7)

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

Generation of LCsAnd langerin expression LC-gB

binding Trans-infectionLC

mediumK

mediumKeratinocy

te

EXPECTATIONS

↗ ↗ ↗ + 1. Appropriate positive control2. No transmission of virions from LC-like cells to MRC5 cells

FINDINGS

No significant

change

No significant change

No conclusion + 1. Prominent

2. No conclusion

Matter of time and not type of medium

RESULTS - CONCLUSION

DISCUSSION & PROSPECTS

‘in vitro’ model: frozen monocytes Incubation period: >8 days

- >80% of langerin expression Medium: TGF-β1

LC medium K medium Keratinocytes

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)

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

THANK YOU FOR YOUR ATTENTION!