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