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

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 to P9 Immunisation infection or not P9 139A

Presence of infection before immunization inhibits antibody and T cell responses to PrP peptide P9 Immunization before infection Immunization infection Immunization P9 139A P9 2 wks 7 wks Infection before immunization 2 Infection Immunization 139A P9 7 wks 13 wks Antibody response to P9 T-cell response to P9

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

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 .

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

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

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 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

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-CD25 139A mAb infection Day-3 WT mice Day+3 Day70 WT 139A infected mice + PC61

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 Foxp3+ 139A Amount of PrPSc en WB WT 139A infected 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 Antoine Sacquin David A. Gross Hôpital St Antoine Hôpital Necker Paris Paris Antoine Sacquin David A. Gross PhD student Jean Davoust