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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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Uptake of uBZLF1 occurs in temperature-dependent manner by clathrin-mediated endocytosis.(A) This uptake and cross-presentation of uBZLF1-derived epitopes is inhibited at 4°C. PBMC were pulsed for 2 h at 4°C or 37°C with uBZLF1 or RAK peptide. The number of BZLF1-specific lymphocytes was determined by ELISpot. (B) Cross-presentation of uBZLF1 is blocked by sucrose, an inhibitor of clathrin-mediated endocytosis. Donor PBMC were stimulated with either uBZLF1 or the RAK peptide in presence of the indicated concentrations sucrose and the frequency of IFN-γ secreting cells was analyzed. Sucrose did not exhibit cytotoxic effects on PBMC at the used concentrations, as shown by PI-staining. All ELISpot data show mean SFC values+s.d. of 5 replicate stimulations and are representative of results performed with PBMC of 2 different individuals.
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ppat-1000198-g004: Uptake of uBZLF1 occurs in temperature-dependent manner by clathrin-mediated endocytosis.(A) This uptake and cross-presentation of uBZLF1-derived epitopes is inhibited at 4°C. PBMC were pulsed for 2 h at 4°C or 37°C with uBZLF1 or RAK peptide. The number of BZLF1-specific lymphocytes was determined by ELISpot. (B) Cross-presentation of uBZLF1 is blocked by sucrose, an inhibitor of clathrin-mediated endocytosis. Donor PBMC were stimulated with either uBZLF1 or the RAK peptide in presence of the indicated concentrations sucrose and the frequency of IFN-γ secreting cells was analyzed. Sucrose did not exhibit cytotoxic effects on PBMC at the used concentrations, as shown by PI-staining. All ELISpot data show mean SFC values+s.d. of 5 replicate stimulations and are representative of results performed with PBMC of 2 different individuals.

Mentions: We used specific inhibitors of the HLA class I processing and presentation pathway to evaluate the molecular mechanisms underlying the urea-mediated uptake, processing and presentation of BZLF1. In order to study the energy dependency of uBZLF1 uptake into APC, PBMC were pre-incubated for 45 min at 4°C or 37°C and subsequently pulsed at the same temperature with uBZLF1 or the RAK peptide for an additional two hours. After removal of excessive antigen by washing, cells were incubated for an additional 20 h at 37°C and the numbers of IFN-γ producing T cells were determined by ELISpot. Pre-incubation and pulsing of cells with uBZLF1 at 4°C resulted in significantly reduced numbers of IFN-γ producing T cells compared to PBMC pulsed at 37°C, indicating a temperature sensitive and thus energy-dependent uptake of uBZLF1. In contrast, the efficiency of CTL reactivation by the RAK peptide was independent of the pre-incubation and pulsing temperature (Fig. 4A).


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)

Uptake of uBZLF1 occurs in temperature-dependent manner by clathrin-mediated endocytosis.(A) This uptake and cross-presentation of uBZLF1-derived epitopes is inhibited at 4°C. PBMC were pulsed for 2 h at 4°C or 37°C with uBZLF1 or RAK peptide. The number of BZLF1-specific lymphocytes was determined by ELISpot. (B) Cross-presentation of uBZLF1 is blocked by sucrose, an inhibitor of clathrin-mediated endocytosis. Donor PBMC were stimulated with either uBZLF1 or the RAK peptide in presence of the indicated concentrations sucrose and the frequency of IFN-γ secreting cells was analyzed. Sucrose did not exhibit cytotoxic effects on PBMC at the used concentrations, as shown by PI-staining. All ELISpot data show mean SFC values+s.d. of 5 replicate stimulations and are representative of results performed with PBMC of 2 different individuals.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2572144&req=5

ppat-1000198-g004: Uptake of uBZLF1 occurs in temperature-dependent manner by clathrin-mediated endocytosis.(A) This uptake and cross-presentation of uBZLF1-derived epitopes is inhibited at 4°C. PBMC were pulsed for 2 h at 4°C or 37°C with uBZLF1 or RAK peptide. The number of BZLF1-specific lymphocytes was determined by ELISpot. (B) Cross-presentation of uBZLF1 is blocked by sucrose, an inhibitor of clathrin-mediated endocytosis. Donor PBMC were stimulated with either uBZLF1 or the RAK peptide in presence of the indicated concentrations sucrose and the frequency of IFN-γ secreting cells was analyzed. Sucrose did not exhibit cytotoxic effects on PBMC at the used concentrations, as shown by PI-staining. All ELISpot data show mean SFC values+s.d. of 5 replicate stimulations and are representative of results performed with PBMC of 2 different individuals.
Mentions: We used specific inhibitors of the HLA class I processing and presentation pathway to evaluate the molecular mechanisms underlying the urea-mediated uptake, processing and presentation of BZLF1. In order to study the energy dependency of uBZLF1 uptake into APC, PBMC were pre-incubated for 45 min at 4°C or 37°C and subsequently pulsed at the same temperature with uBZLF1 or the RAK peptide for an additional two hours. After removal of excessive antigen by washing, cells were incubated for an additional 20 h at 37°C and the numbers of IFN-γ producing T cells were determined by ELISpot. Pre-incubation and pulsing of cells with uBZLF1 at 4°C resulted in significantly reduced numbers of IFN-γ producing T cells compared to PBMC pulsed at 37°C, indicating a temperature sensitive and thus energy-dependent uptake of uBZLF1. In contrast, the efficiency of CTL reactivation by the RAK peptide was independent of the pre-incubation and pulsing temperature (Fig. 4A).

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