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Urea-mediated cross-presentation of soluble Epstein-Barr virus BZLF1 protein.

Barabas S, Gary R, Bauer T, Lindner J, Lindner P, Weinberger B, Jilg W, Wolf H, Deml L - PLoS Pathog. (2008)

Bottom Line: Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules.Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro.The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, University of Regensburg, Germany.

ABSTRACT
Soluble extracellular proteins usually do not enter the endogenous human leukocyte antigen (HLA) I-dependent presentation pathway of antigen-presenting cells, strictly impeding their applicability for the re-stimulation of protein-specific CD8(+) cytotoxic T lymphocytes (CTL). Here we present for the Epstein-Barr virus (EBV) BZLF1 a novel strategy that facilitates protein translocation into antigen-presenting cells by its solubilisation in high molar urea and subsequent pulsing of cells in presence of low molar urea. Stimulation of PBMC from HLA-matched EBV-seropositive individuals with urea-treated BZLF1 but not untreated BZLF1 induces an efficient reactivation of BZLF1-specific CTL. Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules. Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro. The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

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uBZLF1 is processed by a cross-presentation pathway.Cross-presentation of uBZLF1 is not influenced by (A) inhibition of endolysosomal acidification using chloroquine or NH4Cl, (B) but is significantly abrogated in presence of the proteosomal inhibitors epoxomycine and lactacystine. The ELISpot data show mean SFC values+s.d. of 5 replicate stimulations and are representative of results performed with PBMC of 3 different individuals. None of the applied drugs exhibited cytotoxic effects on PBMC at the used concentrations, as shown by PI-staining. (C) BFA strongly inhibits cross-presentation of uBZLF1. Whole blood was stimulated with uBZLF, RAK-peptide or, as control, 0.04 M urea in presence or absence of BFA. IFN-γ production was analyzed by intracellular staining using flow cytometry. The data show the number of specifically reactivated IFN-γ positive CD8+ T cells and are representative of experiments performed with 3 different donors.
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ppat-1000198-g005: uBZLF1 is processed by a cross-presentation pathway.Cross-presentation of uBZLF1 is not influenced by (A) inhibition of endolysosomal acidification using chloroquine or NH4Cl, (B) but is significantly abrogated in presence of the proteosomal inhibitors epoxomycine and lactacystine. The ELISpot data show mean SFC values+s.d. of 5 replicate stimulations and are representative of results performed with PBMC of 3 different individuals. None of the applied drugs exhibited cytotoxic effects on PBMC at the used concentrations, as shown by PI-staining. (C) BFA strongly inhibits cross-presentation of uBZLF1. Whole blood was stimulated with uBZLF, RAK-peptide or, as control, 0.04 M urea in presence or absence of BFA. IFN-γ production was analyzed by intracellular staining using flow cytometry. The data show the number of specifically reactivated IFN-γ positive CD8+ T cells and are representative of experiments performed with 3 different donors.

Mentions: To examine the role of proteolytic processes within endolysosomal compartments in the processing of uBZLF1 for epitope presentation on HLA class I molecules, we pre-treated PBMC for 45 min with two inhibitors of lysosomal acidification, NH4Cl or chloroquine [25],[26], and subsequently stimulated cells for an additional 20 h with uBZLF1 or RAK peptide in presence of the indicated inhibitors. Treatment of PBMC of HLA B8-positive, EBV-seropositive donors with both inhibitors did not significantly influence the capacity of uBZLF1 and the RAK peptide to specifically stimulate T cells for IFN-γ production, strongly indicating that lysosomal protein degradation plays no significant role in the generation of HLA class I presentable epitopes (Fig. 5A).


Urea-mediated cross-presentation of soluble Epstein-Barr virus BZLF1 protein.

Barabas S, Gary R, Bauer T, Lindner J, Lindner P, Weinberger B, Jilg W, Wolf H, Deml L - PLoS Pathog. (2008)

uBZLF1 is processed by a cross-presentation pathway.Cross-presentation of uBZLF1 is not influenced by (A) inhibition of endolysosomal acidification using chloroquine or NH4Cl, (B) but is significantly abrogated in presence of the proteosomal inhibitors epoxomycine and lactacystine. The ELISpot data show mean SFC values+s.d. of 5 replicate stimulations and are representative of results performed with PBMC of 3 different individuals. None of the applied drugs exhibited cytotoxic effects on PBMC at the used concentrations, as shown by PI-staining. (C) BFA strongly inhibits cross-presentation of uBZLF1. Whole blood was stimulated with uBZLF, RAK-peptide or, as control, 0.04 M urea in presence or absence of BFA. IFN-γ production was analyzed by intracellular staining using flow cytometry. The data show the number of specifically reactivated IFN-γ positive CD8+ T cells and are representative of experiments performed with 3 different donors.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2572144&req=5

ppat-1000198-g005: uBZLF1 is processed by a cross-presentation pathway.Cross-presentation of uBZLF1 is not influenced by (A) inhibition of endolysosomal acidification using chloroquine or NH4Cl, (B) but is significantly abrogated in presence of the proteosomal inhibitors epoxomycine and lactacystine. The ELISpot data show mean SFC values+s.d. of 5 replicate stimulations and are representative of results performed with PBMC of 3 different individuals. None of the applied drugs exhibited cytotoxic effects on PBMC at the used concentrations, as shown by PI-staining. (C) BFA strongly inhibits cross-presentation of uBZLF1. Whole blood was stimulated with uBZLF, RAK-peptide or, as control, 0.04 M urea in presence or absence of BFA. IFN-γ production was analyzed by intracellular staining using flow cytometry. The data show the number of specifically reactivated IFN-γ positive CD8+ T cells and are representative of experiments performed with 3 different donors.
Mentions: To examine the role of proteolytic processes within endolysosomal compartments in the processing of uBZLF1 for epitope presentation on HLA class I molecules, we pre-treated PBMC for 45 min with two inhibitors of lysosomal acidification, NH4Cl or chloroquine [25],[26], and subsequently stimulated cells for an additional 20 h with uBZLF1 or RAK peptide in presence of the indicated inhibitors. Treatment of PBMC of HLA B8-positive, EBV-seropositive donors with both inhibitors did not significantly influence the capacity of uBZLF1 and the RAK peptide to specifically stimulate T cells for IFN-γ production, strongly indicating that lysosomal protein degradation plays no significant role in the generation of HLA class I presentable epitopes (Fig. 5A).

Bottom Line: Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules.Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro.The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, University of Regensburg, Germany.

ABSTRACT
Soluble extracellular proteins usually do not enter the endogenous human leukocyte antigen (HLA) I-dependent presentation pathway of antigen-presenting cells, strictly impeding their applicability for the re-stimulation of protein-specific CD8(+) cytotoxic T lymphocytes (CTL). Here we present for the Epstein-Barr virus (EBV) BZLF1 a novel strategy that facilitates protein translocation into antigen-presenting cells by its solubilisation in high molar urea and subsequent pulsing of cells in presence of low molar urea. Stimulation of PBMC from HLA-matched EBV-seropositive individuals with urea-treated BZLF1 but not untreated BZLF1 induces an efficient reactivation of BZLF1-specific CTL. Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules. Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro. The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

Show MeSH
Related in: MedlinePlus