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Dendritic cell-mediated-immunization with xenogenic PrP and adenoviral vectors breaks tolerance and prolongs mice survival against experimental scrapie.

Rosset MB, Sacquin A, Lecollinet S, Chaigneau T, Adam M, Crespeau F, Eloit M - PLoS ONE (2009)

Bottom Line: Other effectors with potential to eliminate PrPSc-producing cells are cytotoxic T cells directed against PrP-derived peptides but their ability to protect or to induce deleterious autoimmune reactions is not known.Frequencies of PrP-specific IFNgamma-secreting T cells were low and in vivo lytic activity only targeted cells strongly expressing hPrP.The efficacy appeared to be associated with antibody but not with cytotoxic cell-mediated PrP-specific responses.

View Article: PubMed Central - PubMed

Affiliation: INSERM UMR 938, Paris, France. martine.rosset@inserm.fr

ABSTRACT
In prion diseases, PrP(c), a widely expressed protein, is transformed into a pathogenic form called PrP(Sc), which is in itself infectious. Antibodies directed against PrP(c) have been shown to inhibit PrP(c) to PrP(Sc) conversion in vitro and protect in vivo from disease. Other effectors with potential to eliminate PrPSc-producing cells are cytotoxic T cells directed against PrP-derived peptides but their ability to protect or to induce deleterious autoimmune reactions is not known. The natural tolerance to PrP(c) makes difficult to raise efficient adaptive responses. To break tolerance, adenovirus (Ad) encoding human PrP (hPrP) or control Ad were administered to wild-type mice by direct injection or by transfer of Ad-transduced dendritic cells (DCs). Control Ad-transduced DCs from Tg650 mice overexpressing hPrP were also used for immunization. DC-mediated but not direct administration of AdhPrP elicited antibodies that bound to murine native PrP(c). Frequencies of PrP-specific IFNgamma-secreting T cells were low and in vivo lytic activity only targeted cells strongly expressing hPrP. Immunohistochemical analysis revealed that CD3(+) T cell infiltration was similar in the brain of vaccinated and unvaccinated 139A-infected mice suggesting the absence of autoimmune reactions. Early splenic PrP(Sc) replication was strongly inhibited ten weeks post infection and mean survival time prolonged from 209 days in untreated 139A-infected mice to 246 days in mice vaccinated with DCs expressing the hPrP. The efficacy appeared to be associated with antibody but not with cytotoxic cell-mediated PrP-specific responses.

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T cell responses in vaccinated C57BL/6 wt mice.(A). Frequency of IFN-γ secreting T cells in the spleens of immunized mice evaluated by a ELISPOT assay. Spleen cells (106/well) from two individual mice receiving AdTA or AdhPrP were stimulated 18 h at 37°C with each of 30-mer peptides from mPrP at a concentration of 10 µg/ml. The frequency of peptide-specific IFN-γ secreting T cells was calculated after substracting the mean number of spots obtained in the absence of peptide. Each column shows the mean numbers of spots±SD per 1×106 spleen cells of triplicate cultures. (B–C). In vivo cytotoxic activity was measured by flow cytometry: spleen cells from mice vaccinated with AdTA or AdhPrP were collected 20 h after injection of 107 CFSElowPrnp−/− cells mixed with 107 CFSEhigh cells expressing either (B) mPrP (C57BL/6) or (C) hPrP (Tg650). Number of CFSElow and CFSEhigh cells in the spleen of individual immunized mice and percentage of specific lysis of PrP+ over Prnp−/− splenocytes were calculated as detailed in “Materials and Methods”.
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pone-0004917-g002: T cell responses in vaccinated C57BL/6 wt mice.(A). Frequency of IFN-γ secreting T cells in the spleens of immunized mice evaluated by a ELISPOT assay. Spleen cells (106/well) from two individual mice receiving AdTA or AdhPrP were stimulated 18 h at 37°C with each of 30-mer peptides from mPrP at a concentration of 10 µg/ml. The frequency of peptide-specific IFN-γ secreting T cells was calculated after substracting the mean number of spots obtained in the absence of peptide. Each column shows the mean numbers of spots±SD per 1×106 spleen cells of triplicate cultures. (B–C). In vivo cytotoxic activity was measured by flow cytometry: spleen cells from mice vaccinated with AdTA or AdhPrP were collected 20 h after injection of 107 CFSElowPrnp−/− cells mixed with 107 CFSEhigh cells expressing either (B) mPrP (C57BL/6) or (C) hPrP (Tg650). Number of CFSElow and CFSEhigh cells in the spleen of individual immunized mice and percentage of specific lysis of PrP+ over Prnp−/− splenocytes were calculated as detailed in “Materials and Methods”.

Mentions: The T cell response was evaluated by measuring by ELISPOT the precursor frequency of IFNγ-secreting spleen cells upon stimulation with the library of murine PrP peptides (Table 1). Figure 2A shows that only one mouse out of two responded and that the PrP-specific T cell response was directed against one epitope lying in the murine P8 region. The T cell response to hPrP peptides was weak (data not shown).


Dendritic cell-mediated-immunization with xenogenic PrP and adenoviral vectors breaks tolerance and prolongs mice survival against experimental scrapie.

Rosset MB, Sacquin A, Lecollinet S, Chaigneau T, Adam M, Crespeau F, Eloit M - PLoS ONE (2009)

T cell responses in vaccinated C57BL/6 wt mice.(A). Frequency of IFN-γ secreting T cells in the spleens of immunized mice evaluated by a ELISPOT assay. Spleen cells (106/well) from two individual mice receiving AdTA or AdhPrP were stimulated 18 h at 37°C with each of 30-mer peptides from mPrP at a concentration of 10 µg/ml. The frequency of peptide-specific IFN-γ secreting T cells was calculated after substracting the mean number of spots obtained in the absence of peptide. Each column shows the mean numbers of spots±SD per 1×106 spleen cells of triplicate cultures. (B–C). In vivo cytotoxic activity was measured by flow cytometry: spleen cells from mice vaccinated with AdTA or AdhPrP were collected 20 h after injection of 107 CFSElowPrnp−/− cells mixed with 107 CFSEhigh cells expressing either (B) mPrP (C57BL/6) or (C) hPrP (Tg650). Number of CFSElow and CFSEhigh cells in the spleen of individual immunized mice and percentage of specific lysis of PrP+ over Prnp−/− splenocytes were calculated as detailed in “Materials and Methods”.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654673&req=5

pone-0004917-g002: T cell responses in vaccinated C57BL/6 wt mice.(A). Frequency of IFN-γ secreting T cells in the spleens of immunized mice evaluated by a ELISPOT assay. Spleen cells (106/well) from two individual mice receiving AdTA or AdhPrP were stimulated 18 h at 37°C with each of 30-mer peptides from mPrP at a concentration of 10 µg/ml. The frequency of peptide-specific IFN-γ secreting T cells was calculated after substracting the mean number of spots obtained in the absence of peptide. Each column shows the mean numbers of spots±SD per 1×106 spleen cells of triplicate cultures. (B–C). In vivo cytotoxic activity was measured by flow cytometry: spleen cells from mice vaccinated with AdTA or AdhPrP were collected 20 h after injection of 107 CFSElowPrnp−/− cells mixed with 107 CFSEhigh cells expressing either (B) mPrP (C57BL/6) or (C) hPrP (Tg650). Number of CFSElow and CFSEhigh cells in the spleen of individual immunized mice and percentage of specific lysis of PrP+ over Prnp−/− splenocytes were calculated as detailed in “Materials and Methods”.
Mentions: The T cell response was evaluated by measuring by ELISPOT the precursor frequency of IFNγ-secreting spleen cells upon stimulation with the library of murine PrP peptides (Table 1). Figure 2A shows that only one mouse out of two responded and that the PrP-specific T cell response was directed against one epitope lying in the murine P8 region. The T cell response to hPrP peptides was weak (data not shown).

Bottom Line: Other effectors with potential to eliminate PrPSc-producing cells are cytotoxic T cells directed against PrP-derived peptides but their ability to protect or to induce deleterious autoimmune reactions is not known.Frequencies of PrP-specific IFNgamma-secreting T cells were low and in vivo lytic activity only targeted cells strongly expressing hPrP.The efficacy appeared to be associated with antibody but not with cytotoxic cell-mediated PrP-specific responses.

View Article: PubMed Central - PubMed

Affiliation: INSERM UMR 938, Paris, France. martine.rosset@inserm.fr

ABSTRACT
In prion diseases, PrP(c), a widely expressed protein, is transformed into a pathogenic form called PrP(Sc), which is in itself infectious. Antibodies directed against PrP(c) have been shown to inhibit PrP(c) to PrP(Sc) conversion in vitro and protect in vivo from disease. Other effectors with potential to eliminate PrPSc-producing cells are cytotoxic T cells directed against PrP-derived peptides but their ability to protect or to induce deleterious autoimmune reactions is not known. The natural tolerance to PrP(c) makes difficult to raise efficient adaptive responses. To break tolerance, adenovirus (Ad) encoding human PrP (hPrP) or control Ad were administered to wild-type mice by direct injection or by transfer of Ad-transduced dendritic cells (DCs). Control Ad-transduced DCs from Tg650 mice overexpressing hPrP were also used for immunization. DC-mediated but not direct administration of AdhPrP elicited antibodies that bound to murine native PrP(c). Frequencies of PrP-specific IFNgamma-secreting T cells were low and in vivo lytic activity only targeted cells strongly expressing hPrP. Immunohistochemical analysis revealed that CD3(+) T cell infiltration was similar in the brain of vaccinated and unvaccinated 139A-infected mice suggesting the absence of autoimmune reactions. Early splenic PrP(Sc) replication was strongly inhibited ten weeks post infection and mean survival time prolonged from 209 days in untreated 139A-infected mice to 246 days in mice vaccinated with DCs expressing the hPrP. The efficacy appeared to be associated with antibody but not with cytotoxic cell-mediated PrP-specific responses.

Show MeSH
Related in: MedlinePlus