Limits...
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.

Show MeSH

Related in: MedlinePlus

Stress enhances the immunosuppressive effect of NKG2DL-bearing exosomes.NK-cell cytotoxicity assay using PBMC from healthy donors and K562 targets at an E∶T ratio 40∶1. The cytotoxic effect was measured in the presence or absence of exosomes released from cells cultured under steady state or stressed conditions. The cytotoxic response of untreated or antibody-blocked effector and target cells are shown in blue staples. The suppression of cytotoxicity by native exosomes released from cells cultured in various conditions is shown in red staples. Gray staples underlayed under the red staples show reversal of cytotoxicity to normal levels when the exosomes were blocked with a cocktail of Abs against NKG2DL or with Abs against the exosomal marker CD63. Green staple shows the level of cytotoxicity in the presence of used supernatant after exosome isolation indicating that the suppressive effect was associated with the exosomal fraction. * and # indicates statistical significance, p<0.05.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3045385&req=5

pone-0016899-g005: Stress enhances the immunosuppressive effect of NKG2DL-bearing exosomes.NK-cell cytotoxicity assay using PBMC from healthy donors and K562 targets at an E∶T ratio 40∶1. The cytotoxic effect was measured in the presence or absence of exosomes released from cells cultured under steady state or stressed conditions. The cytotoxic response of untreated or antibody-blocked effector and target cells are shown in blue staples. The suppression of cytotoxicity by native exosomes released from cells cultured in various conditions is shown in red staples. Gray staples underlayed under the red staples show reversal of cytotoxicity to normal levels when the exosomes were blocked with a cocktail of Abs against NKG2DL or with Abs against the exosomal marker CD63. Green staple shows the level of cytotoxicity in the presence of used supernatant after exosome isolation indicating that the suppressive effect was associated with the exosomal fraction. * and # indicates statistical significance, p<0.05.

Mentions: It has previously been reported by other and us that NKG2DL-bearing exosomes can impair the cytotoxic function of NK cells [12]–[15]. Therefore, as a next step, we investigated if the increased secretion of NKG2DL-bearing exosomes had consequences for the cognate receptor-mediated killing in vitro. The experiments were done with the NKG2D ligand expressing target cells K562 in effector∶target ratio of 40∶1 and in the presence or absence of exosomes, which were isolated from equal number of cultured cell under thermal or oxidative stress conditions. PBMC from healthy donors, containing NKG2D-receptor expressing NK-, CD8+- and γδT cells were used as effector cells. Cytotoxicity was assessed in untreated effector cells or effector cells pretreated with native exosomes, Ab-blocked exosomes, Ab-blocked target- or Ab-blocked effector cells and supernatant after exosome isolation, as described in Material and Methods. The results are summarized in Figure 5. As can be seen, there was a significant downregulation of the cytotoxic response with reduction by approximately 50% in the presence of native exosomes isolated from Jurkat and Raji cells cultured under steady state conditions (Figure 5, red staples). Moreover, the suppression was enhanced when the exosomes were from cells cultured in stressed conditions. An interesting observation is that in Jurkat cells, enhanced suppression was observed in exosomes from oxidative stress conditions, which was the stress that caused the highest significant increase of exosome secretion as illustrated in Figure 3. In contrast, thermal stress caused significant increase of exosome secretion in Raji cells (Figure 3). Accordingly, we found the highest suppression of cytotoxicity when Raji exosomes produced under thermal stress conditions were used (Figure 5, red staples). The suppression of cytotoxicity was reversed when the exosomes were pretreated with blocking Abs as illustrated in the gray staples behind the red ones (Figure 5). No effect was observed when used supernatant after exosome isolation was tested, indicating that the specific suppression of cytotoxicity was found in the exosomal fraction (Figure 5, green staples).


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)

Stress enhances the immunosuppressive effect of NKG2DL-bearing exosomes.NK-cell cytotoxicity assay using PBMC from healthy donors and K562 targets at an E∶T ratio 40∶1. The cytotoxic effect was measured in the presence or absence of exosomes released from cells cultured under steady state or stressed conditions. The cytotoxic response of untreated or antibody-blocked effector and target cells are shown in blue staples. The suppression of cytotoxicity by native exosomes released from cells cultured in various conditions is shown in red staples. Gray staples underlayed under the red staples show reversal of cytotoxicity to normal levels when the exosomes were blocked with a cocktail of Abs against NKG2DL or with Abs against the exosomal marker CD63. Green staple shows the level of cytotoxicity in the presence of used supernatant after exosome isolation indicating that the suppressive effect was associated with the exosomal fraction. * and # indicates statistical significance, p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016899-g005: Stress enhances the immunosuppressive effect of NKG2DL-bearing exosomes.NK-cell cytotoxicity assay using PBMC from healthy donors and K562 targets at an E∶T ratio 40∶1. The cytotoxic effect was measured in the presence or absence of exosomes released from cells cultured under steady state or stressed conditions. The cytotoxic response of untreated or antibody-blocked effector and target cells are shown in blue staples. The suppression of cytotoxicity by native exosomes released from cells cultured in various conditions is shown in red staples. Gray staples underlayed under the red staples show reversal of cytotoxicity to normal levels when the exosomes were blocked with a cocktail of Abs against NKG2DL or with Abs against the exosomal marker CD63. Green staple shows the level of cytotoxicity in the presence of used supernatant after exosome isolation indicating that the suppressive effect was associated with the exosomal fraction. * and # indicates statistical significance, p<0.05.
Mentions: It has previously been reported by other and us that NKG2DL-bearing exosomes can impair the cytotoxic function of NK cells [12]–[15]. Therefore, as a next step, we investigated if the increased secretion of NKG2DL-bearing exosomes had consequences for the cognate receptor-mediated killing in vitro. The experiments were done with the NKG2D ligand expressing target cells K562 in effector∶target ratio of 40∶1 and in the presence or absence of exosomes, which were isolated from equal number of cultured cell under thermal or oxidative stress conditions. PBMC from healthy donors, containing NKG2D-receptor expressing NK-, CD8+- and γδT cells were used as effector cells. Cytotoxicity was assessed in untreated effector cells or effector cells pretreated with native exosomes, Ab-blocked exosomes, Ab-blocked target- or Ab-blocked effector cells and supernatant after exosome isolation, as described in Material and Methods. The results are summarized in Figure 5. As can be seen, there was a significant downregulation of the cytotoxic response with reduction by approximately 50% in the presence of native exosomes isolated from Jurkat and Raji cells cultured under steady state conditions (Figure 5, red staples). Moreover, the suppression was enhanced when the exosomes were from cells cultured in stressed conditions. An interesting observation is that in Jurkat cells, enhanced suppression was observed in exosomes from oxidative stress conditions, which was the stress that caused the highest significant increase of exosome secretion as illustrated in Figure 3. In contrast, thermal stress caused significant increase of exosome secretion in Raji cells (Figure 3). Accordingly, we found the highest suppression of cytotoxicity when Raji exosomes produced under thermal stress conditions were used (Figure 5, red staples). The suppression of cytotoxicity was reversed when the exosomes were pretreated with blocking Abs as illustrated in the gray staples behind the red ones (Figure 5). No effect was observed when used supernatant after exosome isolation was tested, indicating that the specific suppression of cytotoxicity was found in the exosomal fraction (Figure 5, green staples).

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