1. Martine Bruley Rosset UMR S 938 INSERM Hôpital St Antoine Paris France
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

2. 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

3. 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

4. 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 (P9) as the main CD4+ T cell epitope of murine PrP
Experimental model of murine scrapie

5. 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?

6. T cell response specific for P9 lasts longuer in normal than in infected mice after immunization
to P9
Immunisation infection or not
P A

7. Presence of infection before immunization inhibits antibody and T cell responses to PrP peptide P9
Immunization before
infection
Immunization infection Immunization P A P9
2 wks wks
Infection before immunization 2
Infection Immunization
139A P9
7 wks wks
Antibody response
to P9
T-cell response
to P9

8. The presence of prion infection do not interfere with the response to a foreign antigen
Peptide doses
1 mg/well
0,1 µg/well
0,01 µg/well
T cell response to OT2
CFA OVA CFA OVA
Normal mice infected mice
T cell response to OT2 (dominant
CD4+T-cell epitope of ovalbumin)
Antibody response to OVA

9. In prion-infected mice, the reduced response to P9 is restored when CD4+CD25+ Tcells were eliminated
CD25-depleted
CD25-depleted
CD4+
P=0.05
P<0.01 .

10. Regulatory T cells control autoreactive T cells
Thymus
Specific for foreign antigen
Periphery: response to pathogens T
Spécifique for self-antigen T
High affinity
apoptose
periphery
Medium affinity
Autoreactive T cell autoimmune aggression
CD4+Foxp3-
CD4+
T-cell receptor
Self-PrP

11. Regulatory T cells control autoreactive T cells
Thymus
Specific for self-antigen T
periphery
Medium affinity
Regulatory T cell
CD4+CD25+Foxp3+
Autoreactive T cell autoimmune aggression
CD4+Foxp3-
Tregs
FOXP3+
CD4+ X
T-cell receptor
Inhibition of
proliferation
CD25+
Self-PrP
Tregs = 10 % of peripheral CD4+ T cells

12. 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

13. Prion infection is associated with a moderate accumulation of CD4+CD25low Foxp3+ T cells in the spleen
SPLEEN LYMPH NODES BLOOD
15,4
5,58
12,7
1,86
0,74
8,5
17,3
13,8
14,1
2,41
0,82
11,4
Foxp3-GFP
mouse
CD25
SSC
CD4
Foxp3-GFP
infected mouse
GFP

14. Elimination of CD25+ Tregs leads to a higher accumulation
of pathogenic splenic PrPsc
WT 139A
infected mice
infected mice + PC61
p<0,001
Amount of PrPSc en WB
Anti-CD A
mAb infection
Day WT mice Day Day70
WT 139A
infected mice
+ PC61

15. Transfert of FoxP3+ Treg cells 3 days prior to infection reduces accumulation of pathogenic PrPsc
WT 139A
infected mice
+ FoxP3-GFP+ cells
p<0,001
Foxp3+GFP mice WT mice Recipient mice
T cell transfer infection
2.105 Foxp A
Amount of PrPSc en WB
WT 139A
infected mice
+ FoxP3-GFP+ cells

16. 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

17. 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

18. Acknowledgements UMRS 938 INSERM 580 Antoine Sacquin David A. Gross
Hôpital St Antoine Hôpital Necker
Paris Paris
Antoine Sacquin David A. Gross
PhD student Jean Davoust