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Thermal- and oxidative stress causes enhanced release of NKG2D ligand-bearing immunosuppressive exosomes in leukemia/lymphoma T and B cells.

Hedlund M, Nagaeva O, Kargl D, Baranov V, Mincheva-Nilsson L - PLoS ONE (2011)

Bottom Line: Acting as a decoy, the NKG2D ligand-bearing exosomes downregulate the in vitro NKG2D receptor-mediated cytotoxicity and thus impair NK-cell function.Interestingly, thermal and oxidative stress enhanced the exosome secretion generating more soluble NKG2D ligands that aggravated the impairment of the cytotoxic response.The adverse effect of thermal and oxidative stress, enhancing the release of immunosuppressive exosomes, should be considered when cytostatic and hyperthermal anti-cancer therapies are designed.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Immunology, Department of Clinical Microbiology, Umeå University, Umeå, Sweden.

ABSTRACT
Immune evasion from NK surveillance related to inadequate NK-cell function has been suggested as an explanation of the high incidence of relapse and fatal outcome of many blood malignancies. In this report we have used Jurkat and Raji cell lines as a model for studies of the NKG2D receptor-ligand system in T-and B cell leukemia/lymphoma. Using real-time quantitative RT-PCR and immunoflow cytometry we show that Jurkat and Raji cells constitutively express mRNA and protein for the stress-inducible NKG2D ligands MICA/B and ULBP1 and 2, and up-regulate the expression in a cell-line specific and stress-specific manner. Furthermore, we revealed by electron microscopy, immunoflow cytometry and western blot that these ligands were expressed and secreted on exosomes, nanometer-sized microvesicles of endosomal origin. Acting as a decoy, the NKG2D ligand-bearing exosomes downregulate the in vitro NKG2D receptor-mediated cytotoxicity and thus impair NK-cell function. Interestingly, thermal and oxidative stress enhanced the exosome secretion generating more soluble NKG2D ligands that aggravated the impairment of the cytotoxic response. Taken together, our results might partly explain the clinically observed NK-cell dysfunction in patients suffering from leukemia/lymphoma. The adverse effect of thermal and oxidative stress, enhancing the release of immunosuppressive exosomes, should be considered when cytostatic and hyperthermal anti-cancer therapies are designed.

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Electron microscopy analyses of secreted exosomes by Jurkat and Raji cells.Negative contrast staining showing typical exosomal morphology and electron micrographs illustrating immunogold staining of the NKG2D ligands MIC, ULBP1 and 2 of exosomes isolated from A. Jurkat and B. Raji. Bars represent 100 nm.
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pone-0016899-g002: Electron microscopy analyses of secreted exosomes by Jurkat and Raji cells.Negative contrast staining showing typical exosomal morphology and electron micrographs illustrating immunogold staining of the NKG2D ligands MIC, ULBP1 and 2 of exosomes isolated from A. Jurkat and B. Raji. Bars represent 100 nm.

Mentions: Isolated exosomes from steady state and stressed culture conditions were subjected to negative contrast staining to assess their morphology and purification grade, and thereafter to immunogold staining for NKG2DL and the exosomal marker CD63. Similar results were obtained for both cell lines (Figure 2). The negative contrast staining showed a pure population of microvesicles with typical cup-shaped exosomal morphology, varying in size between 40–100 nm, the majority around 90–100 nm. Besides morphology and size, the exosomal nature of the microvesicles was confirmed by CD63 immunogold staining (not shown). Thermal and oxidative stress can cause cell death, thus, precautions were taken to use cells in excellent conditions throughout all experiments and to exclude cell debris and apoptotic bodies from the exosomal preparation by the use of sucrose gradient in the isolation procedure. Electron microscopy demonstrated a pure exosomal population that was not affected in morphology and size by the stress conditions (not shown). Staining with anti-NKG2DL antibodies revealed that exosomes produced by Jurkat and Raji cells expressed MICA/B and ULBP1 and 2 on their surface. The results of the electron microscopy are illustrated with representative photomicrographs of exosomes from Jurkat (Figure 2A) and Raji (Figure 2B) cells under steady state conditions.


Thermal- and oxidative stress causes enhanced release of NKG2D ligand-bearing immunosuppressive exosomes in leukemia/lymphoma T and B cells.

Hedlund M, Nagaeva O, Kargl D, Baranov V, Mincheva-Nilsson L - PLoS ONE (2011)

Electron microscopy analyses of secreted exosomes by Jurkat and Raji cells.Negative contrast staining showing typical exosomal morphology and electron micrographs illustrating immunogold staining of the NKG2D ligands MIC, ULBP1 and 2 of exosomes isolated from A. Jurkat and B. Raji. Bars represent 100 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045385&req=5

pone-0016899-g002: Electron microscopy analyses of secreted exosomes by Jurkat and Raji cells.Negative contrast staining showing typical exosomal morphology and electron micrographs illustrating immunogold staining of the NKG2D ligands MIC, ULBP1 and 2 of exosomes isolated from A. Jurkat and B. Raji. Bars represent 100 nm.
Mentions: Isolated exosomes from steady state and stressed culture conditions were subjected to negative contrast staining to assess their morphology and purification grade, and thereafter to immunogold staining for NKG2DL and the exosomal marker CD63. Similar results were obtained for both cell lines (Figure 2). The negative contrast staining showed a pure population of microvesicles with typical cup-shaped exosomal morphology, varying in size between 40–100 nm, the majority around 90–100 nm. Besides morphology and size, the exosomal nature of the microvesicles was confirmed by CD63 immunogold staining (not shown). Thermal and oxidative stress can cause cell death, thus, precautions were taken to use cells in excellent conditions throughout all experiments and to exclude cell debris and apoptotic bodies from the exosomal preparation by the use of sucrose gradient in the isolation procedure. Electron microscopy demonstrated a pure exosomal population that was not affected in morphology and size by the stress conditions (not shown). Staining with anti-NKG2DL antibodies revealed that exosomes produced by Jurkat and Raji cells expressed MICA/B and ULBP1 and 2 on their surface. The results of the electron microscopy are illustrated with representative photomicrographs of exosomes from Jurkat (Figure 2A) and Raji (Figure 2B) cells under steady state conditions.

Bottom Line: Acting as a decoy, the NKG2D ligand-bearing exosomes downregulate the in vitro NKG2D receptor-mediated cytotoxicity and thus impair NK-cell function.Interestingly, thermal and oxidative stress enhanced the exosome secretion generating more soluble NKG2D ligands that aggravated the impairment of the cytotoxic response.The adverse effect of thermal and oxidative stress, enhancing the release of immunosuppressive exosomes, should be considered when cytostatic and hyperthermal anti-cancer therapies are designed.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Immunology, Department of Clinical Microbiology, Umeå University, Umeå, Sweden.

ABSTRACT
Immune evasion from NK surveillance related to inadequate NK-cell function has been suggested as an explanation of the high incidence of relapse and fatal outcome of many blood malignancies. In this report we have used Jurkat and Raji cell lines as a model for studies of the NKG2D receptor-ligand system in T-and B cell leukemia/lymphoma. Using real-time quantitative RT-PCR and immunoflow cytometry we show that Jurkat and Raji cells constitutively express mRNA and protein for the stress-inducible NKG2D ligands MICA/B and ULBP1 and 2, and up-regulate the expression in a cell-line specific and stress-specific manner. Furthermore, we revealed by electron microscopy, immunoflow cytometry and western blot that these ligands were expressed and secreted on exosomes, nanometer-sized microvesicles of endosomal origin. Acting as a decoy, the NKG2D ligand-bearing exosomes downregulate the in vitro NKG2D receptor-mediated cytotoxicity and thus impair NK-cell function. Interestingly, thermal and oxidative stress enhanced the exosome secretion generating more soluble NKG2D ligands that aggravated the impairment of the cytotoxic response. Taken together, our results might partly explain the clinically observed NK-cell dysfunction in patients suffering from leukemia/lymphoma. The adverse effect of thermal and oxidative stress, enhancing the release of immunosuppressive exosomes, should be considered when cytostatic and hyperthermal anti-cancer therapies are designed.

Show MeSH
Related in: MedlinePlus