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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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Effect of stress on NKG2DL expression in Jurkat and Raji shows cell line-specific differences.A. NKG2DL mRNA expression before and after thermal- and oxidative stress measured by real-time quantitative RT-PCR. The relative mRNA expression under stress conditions was normalized to the mRNA expression in steady-state culture ( = 1, dark staples). The efficacy of stress treatment was assessed by measurement of mRNA for HSP70. 18S rRNA was used as endogenous control. B. Immunoflow cytometry staining of untreated and stressed Jurkat and Raji cells with mAbs against MICA/B and ULBP1-2. Isotype matched mAbs were used as negative controls and the expression was normalized to the expression in untreated cells. C. Tables summarizing the number of immunoflow cytometry experiments with stress-induced up-regulation of NKG2D ligands. * = statistical significance, p<0.05.
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pone-0016899-g001: Effect of stress on NKG2DL expression in Jurkat and Raji shows cell line-specific differences.A. NKG2DL mRNA expression before and after thermal- and oxidative stress measured by real-time quantitative RT-PCR. The relative mRNA expression under stress conditions was normalized to the mRNA expression in steady-state culture ( = 1, dark staples). The efficacy of stress treatment was assessed by measurement of mRNA for HSP70. 18S rRNA was used as endogenous control. B. Immunoflow cytometry staining of untreated and stressed Jurkat and Raji cells with mAbs against MICA/B and ULBP1-2. Isotype matched mAbs were used as negative controls and the expression was normalized to the expression in untreated cells. C. Tables summarizing the number of immunoflow cytometry experiments with stress-induced up-regulation of NKG2D ligands. * = statistical significance, p<0.05.

Mentions: Messenger RNA and protein expression of MICA/B and ULBP 1–3 in Jurkat and Raji cells following stress was assessed by real-time quantitative RT-PCR and immunoflow cytometry. The results of mRNA assessment are summarized in Figure 1A. Up- regulation of mRNA for HSP70 was used as a control of the experimental stress conditions. Both cell lines constitutively expressed mRNA for MICA, MICB, ULBP1 and ULBP2 and up regulated the message after cellular stress. We did not find ULBP3 mRNA expression at steady state or after thermal and oxidative stress. These results are in line with the report by Nückel et al. [24] that cancer cells from chronic B cell leukemia patients lacked ULBP3 mRNA. Lanca et al. [25] reported similar results for ULBP3 mRNA in Jurkat cells but a low ULBP3 mRNA expression in Raji. Some cell line-specific differences could be noted. In Jurkat cells the NKG2DL mRNA expression was approximately equally up-regulated by both types of stress. Raji cells were generally more susceptible to NKG2DL mRNA up-regulation compared to Jurkat and reached significantly higher levels of mRNA under thermal stress compared to oxidative stress.


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)

Effect of stress on NKG2DL expression in Jurkat and Raji shows cell line-specific differences.A. NKG2DL mRNA expression before and after thermal- and oxidative stress measured by real-time quantitative RT-PCR. The relative mRNA expression under stress conditions was normalized to the mRNA expression in steady-state culture ( = 1, dark staples). The efficacy of stress treatment was assessed by measurement of mRNA for HSP70. 18S rRNA was used as endogenous control. B. Immunoflow cytometry staining of untreated and stressed Jurkat and Raji cells with mAbs against MICA/B and ULBP1-2. Isotype matched mAbs were used as negative controls and the expression was normalized to the expression in untreated cells. C. Tables summarizing the number of immunoflow cytometry experiments with stress-induced up-regulation of NKG2D ligands. * = statistical significance, p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016899-g001: Effect of stress on NKG2DL expression in Jurkat and Raji shows cell line-specific differences.A. NKG2DL mRNA expression before and after thermal- and oxidative stress measured by real-time quantitative RT-PCR. The relative mRNA expression under stress conditions was normalized to the mRNA expression in steady-state culture ( = 1, dark staples). The efficacy of stress treatment was assessed by measurement of mRNA for HSP70. 18S rRNA was used as endogenous control. B. Immunoflow cytometry staining of untreated and stressed Jurkat and Raji cells with mAbs against MICA/B and ULBP1-2. Isotype matched mAbs were used as negative controls and the expression was normalized to the expression in untreated cells. C. Tables summarizing the number of immunoflow cytometry experiments with stress-induced up-regulation of NKG2D ligands. * = statistical significance, p<0.05.
Mentions: Messenger RNA and protein expression of MICA/B and ULBP 1–3 in Jurkat and Raji cells following stress was assessed by real-time quantitative RT-PCR and immunoflow cytometry. The results of mRNA assessment are summarized in Figure 1A. Up- regulation of mRNA for HSP70 was used as a control of the experimental stress conditions. Both cell lines constitutively expressed mRNA for MICA, MICB, ULBP1 and ULBP2 and up regulated the message after cellular stress. We did not find ULBP3 mRNA expression at steady state or after thermal and oxidative stress. These results are in line with the report by Nückel et al. [24] that cancer cells from chronic B cell leukemia patients lacked ULBP3 mRNA. Lanca et al. [25] reported similar results for ULBP3 mRNA in Jurkat cells but a low ULBP3 mRNA expression in Raji. Some cell line-specific differences could be noted. In Jurkat cells the NKG2DL mRNA expression was approximately equally up-regulated by both types of stress. Raji cells were generally more susceptible to NKG2DL mRNA up-regulation compared to Jurkat and reached significantly higher levels of mRNA under thermal stress compared to oxidative stress.

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