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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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Residual urea in uBZLF1 pulsed cell cultures reveals no significant cytotoxic effect on lymphocytes.PBMC of two healthy adult individuals, indicated by the light and dark grey bars, were incubated with increasing concentrations of urea. Cell viability was determined after 20 h by propidium iodide (PI) staining.
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ppat-1000198-g002: Residual urea in uBZLF1 pulsed cell cultures reveals no significant cytotoxic effect on lymphocytes.PBMC of two healthy adult individuals, indicated by the light and dark grey bars, were incubated with increasing concentrations of urea. Cell viability was determined after 20 h by propidium iodide (PI) staining.

Mentions: As a model antigen we used the EBV BZLF1 protein, since it has been previously described to harbor an immunodominant B8-restricted CTL epitope termed RAK [24], thus offering a suitable read-out system for the detection of BZLF-specific CTL reponses. Heparinized whole blood obtained from HLA B8-positive EBV-positive individuals was stimulated with either 10 µg/ml urea-adjuvated BZLF1 (uBZLF1) or dialyzed urea-free BZLF1, while the synthetic RAK peptide and a 0.04 M urea solution served as positive and negative controls, respectively. Exposure of blood cells with uBZLF1 induced the reactivation of substantial numbers of CTL as shown by intracellular IFN-γ staining, which was not seen in cells stimulated with BZLF1 or 0.04 M urea (Fig. 1), even after prolongued incubation times (data not shown). In addition, the frequencies and ratios of specifically reactivated CTL upon stimulation with uBZLF1 and the RAK peptide differed significantly in blood samples of individual donors. The removal of urea from uBZLF1 by extensive dialysis resulted in an almost complete loss of its capacity to stimulated CD8+ T cells. Furthermore, we observed only low or undetectable numbers of IFN-γ producing CD4+ Th cells in response to stimulation with uBZLF1 (Fig. S1). Comparable results were achieved using recombinant cytomegalovirus (CMV) pp65 protein (Miltenyi Biotec). Urea-formulated pp65 (upp65) but not urea-free dialyzed pp65 induced significant reactivation of pp65-specific CTL, whereas removal of urea did not significantly influence the capacity of (u)pp65 to restimulate CD4+ T cells (Fig. S2). Dose titration studies with urea revealed no significant cytotoxic effects at concentrations of 0.04 M urea present in uBZLF1-stimulated peripheral blood mononuclear cells (PBMC) (Fig. 2). Beyond that, the applied urea solution did neither induce the secretion of cytokines from PBMC (Fig. S3) nor activate immature dendritic cells for the upregulation of costimulatory (CD80, CD83 and CD86) and MHC class-II molecules (Fig. S4). These data indicate that urea formulation mediates an efficient transfer of EBV BZLF1 into the endogenous HLA class I presentation pathway.


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)

Residual urea in uBZLF1 pulsed cell cultures reveals no significant cytotoxic effect on lymphocytes.PBMC of two healthy adult individuals, indicated by the light and dark grey bars, were incubated with increasing concentrations of urea. Cell viability was determined after 20 h by propidium iodide (PI) staining.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000198-g002: Residual urea in uBZLF1 pulsed cell cultures reveals no significant cytotoxic effect on lymphocytes.PBMC of two healthy adult individuals, indicated by the light and dark grey bars, were incubated with increasing concentrations of urea. Cell viability was determined after 20 h by propidium iodide (PI) staining.
Mentions: As a model antigen we used the EBV BZLF1 protein, since it has been previously described to harbor an immunodominant B8-restricted CTL epitope termed RAK [24], thus offering a suitable read-out system for the detection of BZLF-specific CTL reponses. Heparinized whole blood obtained from HLA B8-positive EBV-positive individuals was stimulated with either 10 µg/ml urea-adjuvated BZLF1 (uBZLF1) or dialyzed urea-free BZLF1, while the synthetic RAK peptide and a 0.04 M urea solution served as positive and negative controls, respectively. Exposure of blood cells with uBZLF1 induced the reactivation of substantial numbers of CTL as shown by intracellular IFN-γ staining, which was not seen in cells stimulated with BZLF1 or 0.04 M urea (Fig. 1), even after prolongued incubation times (data not shown). In addition, the frequencies and ratios of specifically reactivated CTL upon stimulation with uBZLF1 and the RAK peptide differed significantly in blood samples of individual donors. The removal of urea from uBZLF1 by extensive dialysis resulted in an almost complete loss of its capacity to stimulated CD8+ T cells. Furthermore, we observed only low or undetectable numbers of IFN-γ producing CD4+ Th cells in response to stimulation with uBZLF1 (Fig. S1). Comparable results were achieved using recombinant cytomegalovirus (CMV) pp65 protein (Miltenyi Biotec). Urea-formulated pp65 (upp65) but not urea-free dialyzed pp65 induced significant reactivation of pp65-specific CTL, whereas removal of urea did not significantly influence the capacity of (u)pp65 to restimulate CD4+ T cells (Fig. S2). Dose titration studies with urea revealed no significant cytotoxic effects at concentrations of 0.04 M urea present in uBZLF1-stimulated peripheral blood mononuclear cells (PBMC) (Fig. 2). Beyond that, the applied urea solution did neither induce the secretion of cytokines from PBMC (Fig. S3) nor activate immature dendritic cells for the upregulation of costimulatory (CD80, CD83 and CD86) and MHC class-II molecules (Fig. S4). These data indicate that urea formulation mediates an efficient transfer of EBV BZLF1 into the endogenous HLA class I presentation pathway.

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