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

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.

Figure 1. 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.

Figure 2. 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.

Figure 3. Thermal- and oxidative stress increases the release of exosomes by Jurkat and Raji cells.

Exosomes were isolated with sequential centrifugations and sucrose gradient from supernatants from the same number of untreated and stressed cells. Measurement of isolated exosomes by A. BCA protein assay, B. fluorescence measurement of Vybrant DiI stainings of exosomal lipid membranes, C. western blot for the exosomal marker CD63. D. Densitometry for the exosomal marker CD63, the density of the bands was normalized to the bands from exosomes released by cells cultured at steady-state conditions ( = 1). * = statistical significance, p<0.05.

Figure 4. Stress-increased exosomal NKG2DL enhance the suppressive effect of Jurkat and Raji exosomes on NK-cell cytotoxicity.

Immunoflow cytometry of latex microbead-captured exosomes released from unstressed and stressed cells stained for NKG2D ligands or the exosomal marker CD63. Geo mean of fluorescence intensity is normalized to steady state culture conditions at 37°C ( = 1). * = statistical significance, p<0.05.

Figure 5. 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.