Download (PrP) during prion infection: role of regulatory T cells

Studies of immune responses to Prion Protein
(PrP) during prion infection: role of regulatory T
cells
Martine Bruley Rosset
UMR S 938 INSERM
Hôpital St Antoine
Paris
France
Creutzfeldt Jakob Disease
• Sporadic
• Genetic
• Infectious: Iatrogenic, new variant CJD
Triggering event can be established a posteriori when disease
emerges
Long period of incubation where disease is
asymptomatic before clinical signs develop in the CNS
Early detection is essential for
– presence of infectivity for transmission
– application of therapy
In conventional infectious diseases,
infection is usually detected by:
Identification of the pathogen
presence of an immune response specific to the pathogen
In the case of infectious CJD:
Infectious agent is normal protein (PrP), which acquires a pathological conformation
(PrPSc) :
• no antibodies can discriminate the normal form from the pathological form
• PMCA is a new method for the detection of the PrPSc in body fluids
No evidence of specific immune response to PrPSc :
no antibodies to PrP are spontaneously produced in the blood of CJD patients
precluding early detection of infection
Evidences that immune responses to PrP
control prion diseases
1. Antibodies to PrP control efficiently the disease when given early
after infection but not late
Importance of early detection
2. Implication of T cells specific for PrP:
Identification of peptide 158-187 (P9) as the main CD4+ T cell
epitope of murine PrP
Experimental model of murine scrapie
Questions
Can prion infection be detected and/or
interfere with the host immune system
during the asymptomatic period ?
And if so, what is the contribution of
innate or adaptive arms of the immune
system?
T cell response specific for P9 lasts longuer in normal
than in infected mice after immunization
Response of normal or infected mice
to P9
IFNg-spot units/106 cells
350
Normal
Infected
control
300
250
200
150
100
50
0
2w ks
Immunisation infection or not
P9
139A
6w ks
Weeks after im m unisation
8w ks
Number of spots/10 6 cells
Presence of infection before immunization inhibits antibody
and T cell responses to PrP peptide P9
Immunization before
infection
Immunization infection Immunization
P9
139A
P9
Infection
139A
Immunization
P9
250
2,4
200
2
1,6
150
1,2
100
0,8
50
0,4
0
0
P9
CpG
pre
P9
7 wks
300
2,5
250
2
200
1,5
OD
6
cells
2 wks
2
P9
2,8
CpG
Number of spots/10
Infection before
immunization
300
150
3-4
7-8 11-13 18
weeks after infection
1
100
0,5
50
0
0
CpG
P9
7 wks
CpG
P9
13 wks
T-cell response
to P9
pre 7
10 14 20
weeks after infection
Antibody response
to P9
ELISPOT IFNg
Peptide doses
0
1 mg/well
1,20
1,00
OVA/…
PBS/C…
OD
0,01 µg/well
CFA
OVA
CFA
OVA
WT
mice
139Ainfected
infected
mice
NormalWT
mice
mice
T cell response to OT2 (dominant
CD4+T-cell epitope of ovalbumin)
CFA
OVA N
OVA inf
1,40
0,1 µg/well
OVA/…
100
80
60
40
20
0
PBS/C…
Nb of IFNg spots
T cell response
/5.105… to OT2
The presence of prion infection do not interfere with
the response to a foreign antigen
0,80
0,60
0,40
0,20
0,00
dil 1/50
dil 1/500
dil 1/5000
serum dilution
Antibody response to OVA
In prion-infected mice, the reduced response to P9 is
restored when CD4+CD25+ Tcells were eliminated
.
Nb of spots/2 10 5 CD4+ cells
CD4+
CD25-depleted
CD25-depleted
CD4+
P<0.01
450
medium
400
CD4+DCP9
350
CD4+CD25-+DCP9
P=0.05
300
250
200
150
100
50
0
control
P9
Non-infected mice
control
P9
Infected mice
Regulatory T cells control autoreactive T cells
Thymus
Specific for foreign antigen
T
Periphery: response to pathogens
Spécifique for self-antigen
T
High affinity
Medium affinity
apoptose
periphery
Autoreactive T cell
CD4+Foxp3-
autoimmune aggression
CD4+
T-cell receptor
Self-PrP
Regulatory T cells control autoreactive T cells
Thymus
Specific for self-antigen
T
periphery
Medium affinity
Regulatory T cell
CD4+CD25+Foxp3+
Tregs
Autoreactive T cell
CD4+Foxp3CD4+
X
FOXP3+
CD25+
Tregs = 10 % of peripheral CD4+ T cells
autoimmune aggression
Inhibition of
proliferation
T-cell receptor
Self-PrP
Mice expressing Green Fluorescent Protein Foxp3
from Kiffenig/Bernard Malissen Marseille-France
Foxp3+GFP mice
Rapid quantification and purification to study:
1. Influence of Tregs on natural development of the disease
2. Induction of Tregs specific for PrP
• In infected mice
• After vaccination
Prion infection is associated with a moderate accumulation
of CD4+CD25low Foxp3+ T cells in the spleen
SPLEEN
LYMPH NODES
BLOOD
15,4
12,7
0,74
5,58
1,86
8,5
17,3
14,1
0,82
13,8
2,41
11,4
SSC
CD25
Foxp3-GFP
mouse
CD4
Foxp3-GFP
infected mouse
GFP
Elimination of CD25+ Tregs leads to a higher accumulation
of pathogenic splenic PrPsc
Anti-CD25
mAb
Day-3
WT mice Day+3
Day70
Amount of PrPSc en WB
WT 139A
infected mice + PC61
p<0,001
1600000
Pathogenic PrPsc
(arbitrary unit)
WT 139A
infected mice
139A
infection
1200000
800000
400000
0
WT 139A
WT 139A
infected mice infected mice
WTPC61
139A
WT 139A
+
infected mice
infected mice
+ PC61
Transfert of FoxP3+ Treg cells 3 days prior to infection reduces
accumulation of pathogenic PrPsc
T cell transfer
2.105 Foxp3+
Foxp3+GFP mice
infection
139A
WT mice
Recipient mice
Amount of PrPSc en WB
WT 139A
infected mice
+ FoxP3-GFP+ cells
p<0,001
2500000
Pathogenic PrPsc
(arbitrary unit)
WT 139A
infected mice
2000000
1500000
1000000
500000
WT 139A
0
WT 139A
infected
mice
WT 139A
FoxP3-GFP
infectedinfected
mice
139Atransfered
infected mice
with
FoxP3-GFP+
cell
mice
+ FoxP3-GFP+ cells
Conclusions:
Our results demonstrate that
1. The immune system reacts to prion infection
2. Regulatory T cells are important actors
They are able to:
- control the generation of anti-PrP specific responses
- reduce the accumulation of pathogenic PrPSc in the spleen during
the natural course of infection
Perspectives
•
Confirmation of the accumulation of Tregs in lymphoid organs
infected with PrPSc
Kinetics, phenotype and PrPSc-specificity of Tregs?
•
Mechanism of interaction between Tregs and PrPSc:
intervention of another intermediate actor: B cells, Dendritic cells
Acknowledgements
UMRS 938
INSERM 580
Hôpital St Antoine
Paris
Hôpital Necker
Paris
Antoine Sacquin
David A. Gross
PhD student
Jean Davoust