**5.** *In vivo* **assessment of the effect of CNTs on the NK cell in murine model**

Effect of AF-SWCNTs was also examined on NK cell activation *in vivo*. For *in vivo* studies, mice were treated with poly I:C, a RNA analogue, which activates splenic NK cells. Poly I:C induces NK cell activation through the release of interferons [35–39]. The maximum activity of splenic NK cells upon stimulation with poly I:C occurs after 3 days of exposure [40, 41]. Intravenous treatment of AF-SWCNTs resulted in suppression of NK1.1+ cells by 15% and reduction in NK cytotoxicity by 46% (data not shown). NK cell mediates cytolytic activity through release of cytokines-IFN-γ and TNF-α [42]. The effect of AF-SWCNTs was examined on expression of IFN-γ and TNF-α by coculturing splenocytes with YAC cells. Intracellular expression of IFN-γ and TNF-α in splenocytes obtained from mice treated with AF-SWCNTs was assessed flow cytometrically by coculturing with YAC cells *ex vivo*. Our results showed that treatment with AF-SWCNTs resulted in decline of IFN-γ and TNF-α in NK1.1<sup>+</sup> cells by 31 and 41%, respectively (**Figure 12**).

**Figure 12.** Intracellular expression levels of IFN-γ and TNF-α in NK cells. Splenocytes were obtained from mice administered with poly I:C and treated with AF-SWCNT. Splenocytes (1 × 106 ) were cocultured *ex vivo* with YAC cells (2 × 105 ) for 5 h and treated with brefeldin and monensin. Cells were stained with antimouse IFN-γ or antimouse TNF-α mAbs and counterstained with antimouse NK1.1 mAb. Percentages of NK1.1+ cells expressing IFN-γ or TNF-α (upper right quadrant) in the presence and absence of YAC-1 target cells are shown. \*\*p < 0.01 by Student's *t* test.
