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, 118 (11), 3019-27

Complex Regulation of Human NKG2D-DAP10 Cell Surface Expression: Opposing Roles of the γc Cytokines and TGF-β1

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Complex Regulation of Human NKG2D-DAP10 Cell Surface Expression: Opposing Roles of the γc Cytokines and TGF-β1

Yuk Pheel Park et al. Blood.

Abstract

Natural killer (NK) cells help protect the host against viral infections and tumors. NKG2D is a vital activating receptor, also expressed on subsets of T cells, whose ligands are up-regulated by cells in stress. Ligation of NKG2D leads to phosphorylation of the associated DAP10 adaptor protein, thereby activating immune cells. Understanding how the expression of NKG2D-DAP10 is regulated has implications for immunotherapy. We show that IL-2 and TGF-β1 oppositely regulate NKG2D-DAP10 expression by NK cells. IL-2 stimulation increases NKG2D surface expression despite a decrease in NKG2D mRNA levels. Stimulation with IL-2 results in a small increase of DAP10 mRNA and a large up-regulation of DAP10 protein synthesis, indicating that IL-2-mediated effects are mostly posttranscriptional. Newly synthesized DAP10 undergoes glycosylation that is required for DAP10 association with NKG2D and stabilization of NKG2D expression. TGF-β1 has an opposite and dominant effect to IL-2. TGF-β1 treatment decreases DAP10, as its presence inhibits the association of RNA polymerase II with the DAP10 promoter, but not NKG2D mRNA levels. This leads to the down-regulation of DAP10 expression and, as a consequence, NKG2D protein as well. Finally, we show that other γ(c) cytokines act similarly to IL-2 in up-regulating DAP10 expression and NKG2D-DAP10 surface expression.

Figures

Figure 1
Figure 1
Opposing effects of IL-2 and TGF-β1 on NKG2D cell-surface expression. (A) NKG2D cell-surface expression in primary human NK cells. Cells were left untreated (medium) or were stimulated with IL-2, TGF-β1, or IL-2, and TGF-β1 for either 1 or 3 days and the surface expression of NKG2D was analyzed by flow cytometry. Gray-filled histograms represent isotype control staining while black line histograms show staining with anti-NKG2D antibody from a representative experiment (left panel). Summary of the data from 22 donors is shown on the right (note: primary NK cells are not viable if maintained for 3 days in medium plus TGF-β1 alone). (B) NKG2D cell-surface expression by NKL cells. NKL cells were cultured without IL-2 for 3 days, then stimulated as described in panel A and NKG2D expression was analyzed by flow cytometry. The left panel shows a representative experiment; gray-filled histograms represent isotype control staining, black line histograms show staining with anti-NKG2D antibody. Summary of the cell-surface expression of NKG2D on day 1 and day 3, after treatment with IL-2, TGF-β1, or IL-2 plus TGF-β1, is shown on the right; the data are from 5 independent experiments. The numeric values in the histograms indicate the median fluorescence intensity of NKG2D cell surface expression. Graphs in panels A and B represent the average values of median fluorescence intensity ± SEM. Asterisks indicate statistical significance (paired Student t test; *P < .05; **P < .01; ***P < .005).
Figure 2
Figure 2
Transcriptional and translational regulation of NKG2D and DAP10 by IL-2 and TGF-β1. Ex vivo isolated human NK cells were left untreated or were stimulated with IL-2, TGF-β1, or both IL-2 and TGF-β1 for either 1 or 3 days. (A) NKG2D (left panel) and DAP10 (right panel) transcripts were analyzed by qRT-PCR with specific primer sets and normalized to 18S rRNA. Scatter plots show mean values ± SEM. Each symbol represents a donor. Statistical significance was tested by paired Student t test and indicated by asterisks (*P < .05; **P < .01; ***P < .005). (B) Total cell lysates of primary NK cells were immunoblotted with anti-NKG2D (left panel) or anti-DAP10 (right panel) antibodies. (With commercially available antibodies, we are not able to detect NKG2D in the lysates of primary NK cells unless we concentrate the protein by some means, in this case TCA precipitation. The resultant high concentration of protein loaded on the gels leads to some distortion of the protein bands.) Actin was used as a loading control. The results are representative of 3 independent experiments with different donors. (C) The intensities of the upper band (left panel) and lower band (right panel) of DAP10 protein were quantified by densitometric analysis. Band intensities were normalized to actin and calibrated to values from untreated cells (value = 1). Data are shown as mean ± SEM from 3 different donors. Statistical significance is indicated by asterisks (*P < .05; ANOVA). (D) Ex vivo isolated human NK cells were stimulated for 3 days with either IL-2 alone or IL-2 with increasing amounts of TGF-β1. DAP10 transcripts were analyzed by qRT-PCR and normalized to 18S rRNA. Data from separate experiments with 7 different donors are presented as the percentage of DAP10 mRNA level in cells treated only with IL-2. Error bars represent SEM, statistical significance is indicated by asterisks (*P < .05; paired Student t test). (E) DAP10 protein levels in total cell lysates from ex vivo isolated human NK cells treated as in panel D were assessed by Western blot. A representative result of 3 different donors is shown. Actin was used as a loading control.
Figure 3
Figure 3
TGF-β1 blocks DAP10 transcription. ChIP assays. Primary human NK cells (A) or NKL cells (B) were left untreated (□), treated with IL-2 (■), or treated with IL-2 plus TGF-β1 (▒) for 3 days (primary cells) or 36 hours (NKL cells). Cells were fixed, sonicated, and immunoprecipitated with an anti-RNA pol II antibody or IgG isotype control. The amount of DAP10 DNA immunoprecipitated in each experiment was assessed by quantitative PCR using 2 different promoter-specific primer sets and calculated relative to a standard curve generated with serial dilutions of human genomic DNA. All reactions were done in triplicate and the average value of triplicate was used for calculating the relative levels of each mRNA species. Relative quantification of the target genes was made with the 2nd derivative maximum method using the Roche Lightcycler software and calculating the fold change over the 18S rRNA or actin level. Results are expressed as the mean of RNA pol II fold enrichment over isotype control antibody, normalized to values from untreated cells. For primary NK cells, a representative result from 3 separate experiments with different donors is shown. For NKL cells, n = 2, error bar represents SEM.
Figure 4
Figure 4
DAP10 stability, glycosylation and association with NKG2D in human NK cells. (A) Primary NK cells were cultured with IL-2 for 3 days, followed by treatment with the translation inhibitor, cycloheximide (CHX) or DMSO (vehicle) for the indicated times. DAP10 protein levels from the total cell lysates were visualized by immunoblotting and protein loading was verified by blotting with anti-actin antibody. (B) NKG2D was immunoprecipitated from NKL cell lysates. The immunoprecipitated proteins were subsequently immunoblotted with anti-NKG2D and anti-DAP10 antibodies as indicated. Isotype specific IgG (cIgG) immunoprecipitation was used as a negative control. (C) NKL cells were treated with an O-glycosylation inhibitor (benzyl-α-GalNAC) for either 1 or 2 days. The total cell lysates were resolved on a 10%-20% gradient gel, followed by anti-DAP10 immunoblotting. (D) NKL cells were treated with benzyl-α-GalNAC for 2 days and NKG2D was immunoprecipitated from the total cell lysates. The immunoprecipitated proteins were immunoblotted with anti-DAP10 and anti-NKG2D antibodies. Isotype specific IgG (cIgG) immunoprecipitation was used as a control.
Figure 5
Figure 5
DAP10 expression levels regulate the amount of NKG2D surface expression. Cells (293T) were transfected with either NKG2D (1.0 μg), DAP10 (1.0 μg), or 1.0 μg NKG2D and an increasing amount of DAP10 (0.3, 1.0 μg) cDNA. Forty-eight hours after transfection, the cells were analyzed for mRNA and protein levels of NKG2D and DAP10. (A) The NKG2D (left panel) and DAP10 (right panel) transcripts were analyzed by qRT-PCR with specific primer sets and normalized to actin RNA, and are presented in arbitrary units. Data shown are from 2 separate experiments. (B) The expression of NKG2D on the cell surface of 293T cells after transfection was analyzed by flow cytometry. The value of median fluorescence intensity of NKG2D is indicated in parentheses in each graph. Gray filled histograms represent isotype controls. Data are representative of 2 separate experiments. (C) NKG2D and DAP10 protein levels from total cell lysates of 293T transfectants were analyzed by Western blotting. Anti-actin immunoblotting was used as a loading control. The result shown is representative of 2 independent experiments.
Figure 6
Figure 6
The γc chain cytokines share their ability to regulate the expression of the NKG2D-DAP10 receptor complex. Ex vivo isolated human NK cells were left untreated (medium) or were stimulated with IL-2, IL-7, IL-15, TGF-β1, or combinations as indicated for either 1 or 3 days. (A) Gray-filled histograms represent isotype control staining while black line histograms show staining with anti-NKG2D antibody from a representative experiment. Values in each histogram correspond to the median fluorescence intensity of NKG2D cell-surface staining. (B) Summary of the cell-surface expression of NKG2D on day 1 and day 3, following the indicated treatments. Bar graphs represent the average values of median fluorescence intensity from at least 5 donors; error bars represent SEM. Asterisks indicate the statistical significance (*P < .05; **P < .01; ***P < .005 paired Student t test). (C) NKG2D (top panel) and DAP10 (bottom panel) transcripts were analyzed by qRT-PCR with primer sets specific for each transcript of interest. mRNA expression levels, normalized to 18S rRNA, are presented in arbitrary units. Scatter plots show values for individual donors with mean ± SEM. Each symbol represents a donor. Statistical significance was tested by ANOVA for NKG2D and paired Student t test for DAP10 and indicated by asterisks (*P < .05; **P < .01; ***P < .005). (D) Total cell lysates of primary NK cells, left untreated or stimulated as indicated for 3 days, were immunoblotted with anti-DAP10 antibody. Actin was used as a loading control. The result shown is representative of 4 separate experiments with different donors.

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