**6. Conclusions**

NK cells possess inherent ability to kill tumor cells without requiring a prior sensitization. NK cells and cytotoxic T lymphocytes (CTLs) both exhibit cytolytic activity involving secretory (perforin and granzymes) and nonsecretory mechanisms (Fas-FasL interaction). SWCNTs as such are insoluble and do not interact efficiently with cells. We have prepared an acid-derivatized form of SWCNTs by subjecting them to high pressure and temperature in the presence of concentrated sulfuric acid and nitric acid. Acid-functionalized SWCNTs (AF-SWCNTs) are not only polydispersed in aqueous solution but are also amenable to be attached with fluorescent ligands to their carboxyl groups created on the backbone of SWCNTs. As a result we could visualize the interaction of AF-SWCNTs with live NK cells. This was a significant step as it opened a vast arena to explore the activity of NK cells *in vitro* and *in vivo* without fixing them, as required for transmission electron microscopy. The various physiological parameters of NK cells, such as apoptosis, cell cycle, activation, generation, and degranulation have been studied using flow cytometry. This technique is superior to other conventional spectrometric techniques as the results obtained have higher reproducibility and even minor changes in subpopulation can be monitored.

AF-SWCNT treatment showed greater toxicity which was dose and time dependent. At higher dose of 50 μg/mL, AF-SWCNTs exerted toxic effects that led to decrease in cell proliferation and cell cycle arrest. Mechanistic details showed that AF-SWCNT treatment caused greater generation of ROS that led to fluctuations in mitochondrial potential and calcium concentration. These changes offset the homeostatic mechanisms of the cells, which led to their killing. Previous studies by our group had showed that AF-SWCNTs show significant inflammatory effects in mouse lungs induced anemia in mice and caused suppression of cytotoxic response *in vitro* and *in vivo* [43–46]. This chapter demonstrated the inhibitory effects of AF-SWCNTs on activated NK cells. AF-SWCNTs induced inhibition of NK activation by suppressing cellular proliferation, activation processes, and increased apoptosis. AF-SWCNT treatment led to decreased degranulation of NK cells, lower Fas-FasL interaction, and lower production of inflammatory cytokines, including IFN-γ and TNF-α. Taken together AF-SWCNT treatment led to downregulation of NK cell system and stipulates further research for their prospective use in autoimmune disorder or hypersensitive conditions.

### **Acknowledgements**

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

Intravenous treatment of AF-SWCNTs resulted in suppression of NK1.1+

that treatment with AF-SWCNTs resulted in decline of IFN-γ and TNF-α in NK1.1<sup>+</sup>

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].

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

NK cells possess inherent ability to kill tumor cells without requiring a prior sensitization. NK cells and cytotoxic T lymphocytes (CTLs) both exhibit cytolytic activity involving secretory (perforin and granzymes) and nonsecretory mechanisms (Fas-FasL interaction). SWCNTs as

**Figure 12.** Intracellular expression levels of IFN-γ and TNF-α in NK cells. Splenocytes were obtained from mice

) for 5 h and treated with brefeldin and monensin. Cells were stained with antimouse IFN-γ or antimouse TNF-α

administered with poly I:C and treated with AF-SWCNT. Splenocytes (1 × 106

quadrant) in the presence and absence of YAC-1 target cells are shown. \*\*p < 0.01 by Student's *t* test.

mAbs and counterstained with antimouse NK1.1 mAb. Percentages of NK1.1+

cells by 15% and

) were cocultured *ex vivo* with YAC cells

cells expressing IFN-γ or TNF-α (upper right

cells by 31

**model**

174 Natural Killer Cells

and 41%, respectively (**Figure 12**).

**6. Conclusions**

(2 × 105

The work was funded by the Department of Science and Technology (DST), Government of India, to RKS. AA received Senior Research Fellowship from Indian Council of Medical Research (ICMR), New Delhi.
