1. NKG2D Triggers Cytotoxicity in Murine Epidermal γδ T Cells via PI3K-Dependent, Syk/ZAP70-Independent Signaling Pathway 
Atsuko Ibusuki, Kazuhiro Kawai, Shigeru Yoshida, Youhei Uchida, Ayano Nitahara-Takeuchi, Kimiko Kuroki, Mizuho Kajikawa, Toyoyuki Ose, Katsumi Maenaka, Masanori Kasahara, Takuro Kanekura 
Journal of Investigative Dermatology 
Volume 134, Issue 2, Pages (February 2014)
DOI: /jid
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Recombinant NKG2D ligand (NKG2DL) proteins stimulate degranulation but not cytokine production in dendritic epidermal T cells (DETCs). (a, b) DETCs were stimulated with indicated NKG2DL-Fc or control-Fc, and (a) degranulation (cell surface CD107a expression) and (b) intracellular IFN-γ production were analyzed by flow cytometry. (c, d) DETCs were stimulated with indicated recombinant extracellular domain proteins of NKG2DL (rNKG2DL) or control protein (BSA), and (c) degranulation and (d) intracellular IFN-γ production were analyzed by flow cytometry. Data are expressed as mean positive cells (%) and SD. Representative data from three independent experiments are shown. Significant differences as compared with control are denoted with asterisks (*P<0.05, **P<0.01, ***P<0.001).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
NKG2D ligand (NKG2DL) triggers phosphatidylinosital 3-kinase (PI3K) but not Syk/ZAP70 signaling in dendritic epidermal T cells (DETCs). DETCs were preincubated with DMSO or 10 μM LY294002, and stimulated with H60a-Fc or hydrogen peroxide (H2O2) (positive control for Syk/ZAP70 phosphorylation; Schieven et al., 1994; Haas et al., 2008). Intracellular expression levels of (a) phosphorylated Akt and (b) Syk/ZAP70 were analyzed by flow cytometry. Fold change in mean fluorescence intensity (MFI) was determined as geometric MFI at indicated time point/geometric MFI before stimulation. Data are expressed as mean fold change and SD. Significant increases in the phosphorylation on stimulation are denoted with asterisks (**P<0.01, ***P<0.001). Representative profiles at 5 minutes (open histograms) from three independent experiments are also shown. Shaded histograms indicate cells before stimulation.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
NKG2D ligand (NKG2DL) triggers degranulation in dendritic epidermal T cells (DETCs) via the phosphatidylinositol 3-kinase (PI3K)–dependent, Syk/ZAP70-independent signaling pathway. DETCs were preincubated with indicated concentrations of LY or piceatannol, and stimulated with 10 μg ml−1 of immobilized H60a-Fc or anti-NKG2D mAb (clone ). Degranulation and intracellular IFN-γ production were analyzed by flow cytometry. Inhibition is expressed as a percentage of CD107a+ and IFN-γ+ cells observed in the absence of the inhibitors (mean and SD). Significant inhibition of the degranulation and IFN-γ production as compared with those in the absence of the inhibitors is denoted with asterisks (**P<0.01, ***P<0.001). Representative profiles in the presence of 25 μM LY or piceatannol (open histograms) from three independent experiments are also shown. Shaded histograms indicate cells in the absence of the inhibitors.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
TCR signaling is not necessary for dendritic epidermal T cell (DETC)–mediated killing of NKG2D ligand (NKG2DL)–transduced target cells. (a) DETCs and indicated target cells were coincubated in the presence of isotype control or blocking anti-NKG2D mAb (clone ). (b) DETCs and H60c-Ba/F3 cells were coincubated at the effector/target ratio of 10 in the presence of isotype control or anti-H60c mAb (clone 5G6), or in the absence or presence of Fab fragments of anti-TCR mAb. Data are expressed as mean specific lysis (%) and SD. Representative data from three independent experiments are shown. Significant inhibition of the cytotoxicity as compared with control is denoted with asterisks (*P<0.05, **P<0.01, ***P<0.001). NS, not significant.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
TCR signaling is not crucial for dendritic epidermal T cell (DETC)–mediated killing of keratinocytes. (a) DETCs and keratinocytes were coincubated in the presence of isotype control or anti-NKG2D mAb (clone ), or in the absence or presence of Fab fragments of anti-CD3 mAb. (b) DETCs and keratinocytes were coincubated at the effector/target ratio of 10 in the absence or presence of anti-NKG2D mAb (191004) and/or Fab fragments of anti-TCR or antiCD3 mAb. Data are expressed as mean specific lysis (%) and SD. Representative data from three independent experiments are shown. Significant differences in the cytotoxicity are denoted with asterisks (*P<0.05, **P<0.01, ***P<0.001).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Syk/ZAP70 signaling is not crucial for dendritic epidermal T cell (DETC)–mediated killing of keratinocytes. DETCs were preincubated with indicated concentrations of LY or piceatannol, and coincubated with keratinocytes at the effector/target ratio of 10. Inhibition of the cytotoxicity is expressed as a percentage of specific lysis observed in the absence of the inhibitors (mean and SD). Representative data from three independent experiments are shown. Significant inhibition of the cytotoxicity as compared with that in the absence of the inhibitors is denoted with asterisks (***P<0.001).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions