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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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iDC, mDC, monocytes and B cells are capable to process uBZLF1 for epitope-presentation to CTL.iDC, mDC, monocytes, and B cells from EBV-seropositive, HLA B8-positive individuals were incubated for 2 h with 10 µg/ml uBZLF1, RAK or the E10F control peptide, washed and co-cultured with autologous PBMC at the indicated ratios. Numbers of positive cells were determined by ELISpot. The data show mean SFC values+s.d. of 5 replicate stimulations and are representative for 3 independent experiments with different donors.
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ppat-1000198-g006: iDC, mDC, monocytes and B cells are capable to process uBZLF1 for epitope-presentation to CTL.iDC, mDC, monocytes, and B cells from EBV-seropositive, HLA B8-positive individuals were incubated for 2 h with 10 µg/ml uBZLF1, RAK or the E10F control peptide, washed and co-cultured with autologous PBMC at the indicated ratios. Numbers of positive cells were determined by ELISpot. The data show mean SFC values+s.d. of 5 replicate stimulations and are representative for 3 independent experiments with different donors.

Mentions: We analyzed different subpopulations of APC for their ability to cross-present epitopes from uBZLF1. Therefore monocyte-derived immature (iDC) and mature DC (mDC), as well as monocytes and B cells were generated from PBMC of HLA B8-positive individuals, pulsed with uBZLF1, washed extensively and co-cultured with autologous PBMC. Activation of T cells was assessed by IFN-γ ELISpot. APC pulsed either with RAK or E10F peptides served as positive and negative controls. Dendritic cells pulsed with uBZLF1 showed a higher ability to restimulate antigen-specific T cells than monocytes and B cells. DC loaded with uBZLF1 were more efficient in the restimulation of IFN-γ producing T cells than DC incubated with the RAK peptide, while the opposite effect was observed for monocytes and B cells (Fig. 6). These results indicate that various APC subpopulations may account for cross-presentation of uBZLF1 and subsequent reactivation of BZLF1-specific T cells in PBMC or whole blood cultures.


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)

iDC, mDC, monocytes and B cells are capable to process uBZLF1 for epitope-presentation to CTL.iDC, mDC, monocytes, and B cells from EBV-seropositive, HLA B8-positive individuals were incubated for 2 h with 10 µg/ml uBZLF1, RAK or the E10F control peptide, washed and co-cultured with autologous PBMC at the indicated ratios. Numbers of positive cells were determined by ELISpot. The data show mean SFC values+s.d. of 5 replicate stimulations and are representative for 3 independent experiments with different donors.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000198-g006: iDC, mDC, monocytes and B cells are capable to process uBZLF1 for epitope-presentation to CTL.iDC, mDC, monocytes, and B cells from EBV-seropositive, HLA B8-positive individuals were incubated for 2 h with 10 µg/ml uBZLF1, RAK or the E10F control peptide, washed and co-cultured with autologous PBMC at the indicated ratios. Numbers of positive cells were determined by ELISpot. The data show mean SFC values+s.d. of 5 replicate stimulations and are representative for 3 independent experiments with different donors.
Mentions: We analyzed different subpopulations of APC for their ability to cross-present epitopes from uBZLF1. Therefore monocyte-derived immature (iDC) and mature DC (mDC), as well as monocytes and B cells were generated from PBMC of HLA B8-positive individuals, pulsed with uBZLF1, washed extensively and co-cultured with autologous PBMC. Activation of T cells was assessed by IFN-γ ELISpot. APC pulsed either with RAK or E10F peptides served as positive and negative controls. Dendritic cells pulsed with uBZLF1 showed a higher ability to restimulate antigen-specific T cells than monocytes and B cells. DC loaded with uBZLF1 were more efficient in the restimulation of IFN-γ producing T cells than DC incubated with the RAK peptide, while the opposite effect was observed for monocytes and B cells (Fig. 6). These results indicate that various APC subpopulations may account for cross-presentation of uBZLF1 and subsequent reactivation of BZLF1-specific T cells in PBMC or whole blood cultures.

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