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!