**4.** *In vitro* **assessment of the effect of CNTs on splenic NK cells**

Studies described above used NK cell lines for assessing the toxicity of various nanoparticles *in vitro*. This was followed by a detailed investigation into the effect of AF-SWCNTs on mouse spleen-derived NK cells.

#### **4.1. NK cell cytotoxicity assay**

While a basal level of NK cells exists in the mouse spleen, proliferation and activation of these cells can be induced *in vitro* by culturing with IL-2, IL-12, IL-15, or IL-18 [27]. NK cells achieve maximal activation after 3 days of activation with IL-2, and the activity gradually subsidizes within 5 days [28, 29]. Effect of CNTs on basal as well as IL-2-induced NK response was examined *in vitro*.

Spleen cells (5 × 106 /mL) obtained from C57BL/6 mice were treated with tris ammonium chloride buffer (ACK lysis buffer) to remove red blood cells (RBCs). Splenocytes, devoid of RBCs, were cultured with 500 U/mL IL-2 in complete medium (RPMI1640 + 10% FCS), with or without SWCNTs or AF-SWCNTs for 3 days. Control and activated spleen cells were washed, counted, and used as effector cells (E). YAC-1, a NK-sensitive murine lymphoma cell line, was used to examine the cytolytic potential of NK cells by using a 4 h 51Cr release assay as described before [30, 31].

Lytic units (LU) per 107 effector cells were calculated from the E/T ratio versus percent lysis plots [32]. Briefly, E/T ratios corresponding to a 20% target lysis were determined from the E/T ratio versus percent lysis plots, and the number of lymphocytes corresponding to this E/T ratio in the assay well was taken as one lytic unit.

Results in **Figure 8B** show that addition of SWCNTs or AF-SWCNTs during the chromium release assay had no significant effect on the killing of target cells by NK cells indicating that SWCNTs and AF-SWCNTs did not directly interfere with the E/T interaction in chromium release assay. A significant decrease in the NK activity by treatment with AF-SWCNTs is shown in **Figure 8A**.

**4.** *In vitro* **assessment of the effect of CNTs on splenic NK cells**

spleen-derived NK cells.

170 Natural Killer Cells

**4.1. NK cell cytotoxicity assay**

Studies described above used NK cell lines for assessing the toxicity of various nanoparticles *in vitro*. This was followed by a detailed investigation into the effect of AF-SWCNTs on mouse

**Figure 7.** Estimation of mitochondrial potential in YT-INDY cells treated with SWCNT or AF-SWCNT. YT-INDY cells cultured with SWCNT or AF-SWCNT were harvested, centrifuged, and rinsed twice with PBS, resuspended in 5 mM JC-1 or 10 mM MitoTracker red in serum-free media. Fluorescence emission was analyzed flow cytometrically (JC-1 monomers, excitation wavelength 488 nm, emission filter 530/30 nm; JC-1 aggregates, excitation wavelength 488 nm, emission filter 585/42 nm). Decrease in red fluorescence or increase in green fluorescence is indicative of depolarization of mitochondria. Panel A shows the percent increase in green fluorescence of JC-1 in YT-INDY cells treated with SWCNT or AF-SWCNT. The uptake of mitochondrial dye, MitoTracker, depends on membrane potential. Data in Panel B shows the mean fluorescent intensity (MFI) of MitoTracker red in control and SWCNT- and AF-SWCNT-treated cells.

While a basal level of NK cells exists in the mouse spleen, proliferation and activation of these cells can be induced *in vitro* by culturing with IL-2, IL-12, IL-15, or IL-18 [27]. NK cells achieve maximal activation after 3 days of activation with IL-2, and the activity gradually subsidizes

**Figure 8.** Effect of SWCNT and AF-SWCNT on the generation of NK cell response *in vitro*. NK cells were activated *in vitro* by culturing spleen cells with IL-2 in the presence or absence of SWCNT or AF-SWCNT (50 μg/mL). Anti-YAC-1 cytotoxic activity was assessed in a 4 h chromium release assay (CRA) at E/T ratios of 100, 50, 25, and 12.5 and lytic units/107 cells calculated (Panel A). Data in Panel B denoted the effect of addition of SWCNT and AF-SWCNT, during chromium release assay, on the cytotoxicity of NK cell response *in vitro*. \*p < 0.05 by ANOVA.

#### **4.2. Effect of CNTs on the recovery of IL-2-activated NK1.1<sup>+</sup> cells** *in vitro*

Suppression of IL-2-induced NK cell cytotoxicity by AF-SWCNTs could be due to a possible interference with the NK cell proliferation and/or activation process or a loss of NK cells due to toxic effect of AF-SWCNTs or both. In order to assess the possible toxic effect of SWCNTs and AF-SWCNTs on NK cells, recoveries of NK1.1+ cells from control and IL-2-activated spleen cultures were examined. Results in **Figure 9** indicate that the recovery of NK cells (percentage of NK 1.1+ cells by flow cytometry) in IL-2-activated spleen cell cultures declined by 17% if AF-SWCNTs were added to cultures.

#### **4.3. Effect of SWCNTs and AF-SWCNTs on activated NK cells**

Spleen cells express early activation marker CD69 on the surface as early as 12 h after *in vitro* stimulation with IL-2 [33]. Results in **Figure 10** show that in IL-2-treated spleen cell cultures, 53% of NK1.1+ cells expressed CD69 marker. Treatment with AF-SWCNTs significantly reduced the expression of CD69 on NK1.1+ cells by 23%.

Induction of apoptosis in NK cells activated by IL-2 in the presence of SWCNTs or AF-SWCNTs was also examined by using annexin V staining. Our results indicated that as compared to 11% apoptotic NK cells in control IL-2-activated spleen cells, the presence of SWCNTs and AF-SWCNTs increased the percentage of apoptotic cell to 17 and 22%, respectively (data not shown). Taken together, our results point to the possibility of decreased NK cell proliferation as well induction of apoptotic cell death resulting from exposure to AF-SWCNTs. In addition, expression of CD69, an early cell activation marker, was significantly lower in NK cells treated with AF-SWCNTs, indicating that the AF-SWCNTs interfered with NK cell activation process. Increased apoptosis of IL-2-activated NK cells indicates that activated NK cells are more prone to lysis than resting NK cells.

**Figure 9.** Effect of SWCNT and AF-SWCNT on recovery of NK1.1<sup>+</sup> cells *in vitro*. Spleen cells activated by IL-2 in the presence and absence of 50 μg/mL of SWCNT or AF-SWCNT were stained with NK1.1 mAb and analyzed on flow cytometer. Illustrative flow cytometry histograms for percent NK cell recovery in control and activated NK cells have been shown. \*p<0.05, by Student's *t* test.

**Figure 10.** Effect of SWCNT and AF-SWCNT on the expression of CD69 activation marker on NK cells. Splenocytes cultured in the absence or presence of 500 U/mL of IL-2 were simultaneously treated with 50 μg/mL of SWCNT or AF-SWCNT. After 12 h, the splenocytes were harvested, double stained with antimouse NK1.1 and CD69 mAbs, and analyzed on a flow cytometer.

#### **4.4. Effect of CNTs on effector functions of NK cells**

**4.2. Effect of CNTs on the recovery of IL-2-activated NK1.1<sup>+</sup>**

**4.3. Effect of SWCNTs and AF-SWCNTs on activated NK cells**

and AF-SWCNTs on NK cells, recoveries of NK1.1+

by 17% if AF-SWCNTs were added to cultures.

reduced the expression of CD69 on NK1.1+

more prone to lysis than resting NK cells.

**Figure 9.** Effect of SWCNT and AF-SWCNT on recovery of NK1.1<sup>+</sup>

been shown. \*p<0.05, by Student's *t* test.

(percentage of NK 1.1+

172 Natural Killer Cells

tures, 53% of NK1.1+

Suppression of IL-2-induced NK cell cytotoxicity by AF-SWCNTs could be due to a possible interference with the NK cell proliferation and/or activation process or a loss of NK cells due to toxic effect of AF-SWCNTs or both. In order to assess the possible toxic effect of SWCNTs

spleen cultures were examined. Results in **Figure 9** indicate that the recovery of NK cells

Spleen cells express early activation marker CD69 on the surface as early as 12 h after *in vitro* stimulation with IL-2 [33]. Results in **Figure 10** show that in IL-2-treated spleen cell cul-

Induction of apoptosis in NK cells activated by IL-2 in the presence of SWCNTs or AF-SWCNTs was also examined by using annexin V staining. Our results indicated that as compared to 11% apoptotic NK cells in control IL-2-activated spleen cells, the presence of SWCNTs and AF-SWCNTs increased the percentage of apoptotic cell to 17 and 22%, respectively (data not shown). Taken together, our results point to the possibility of decreased NK cell proliferation as well induction of apoptotic cell death resulting from exposure to AF-SWCNTs. In addition, expression of CD69, an early cell activation marker, was significantly lower in NK cells treated with AF-SWCNTs, indicating that the AF-SWCNTs interfered with NK cell activation process. Increased apoptosis of IL-2-activated NK cells indicates that activated NK cells are

presence and absence of 50 μg/mL of SWCNT or AF-SWCNT were stained with NK1.1 mAb and analyzed on flow cytometer. Illustrative flow cytometry histograms for percent NK cell recovery in control and activated NK cells have

cells by 23%.

cells by flow cytometry) in IL-2-activated spleen cell cultures declined

cells expressed CD69 marker. Treatment with AF-SWCNTs significantly

 **cells** *in vitro*

cells from control and IL-2-activated

cells *in vitro*. Spleen cells activated by IL-2 in the

NK cell cytotoxicity is mediated by release of cytotoxic granules like perforin or through Fas-FasL mechanism. Perforins are stored in preformed granules within the cytoplasm of NK cell and are released when NK cells are triggered by interaction with target cells. Granule release is correlated with the lysosomal marker CD107a (lysosomal-associated membrane protein or LAMP-1) [34].

Results in **Figure 11** show that addition of AF-SWCNTs to IL-2-activated culture resulted in downregulation in expression of CD107a on YAC cocultured NK1.1+ cells from 57 to 44% (22% decline) indicating that AF-SWCNT treatment impaired the process of degranulation of activated NK cells, which may be a contributing factor in suppressing cell-mediated cytotoxicity seen in AF-SWCNT-treated NK cells.

FasL expression on IL-2-activated NK cells was also examined. Our results showed that 74% of IL-2-activated NK1.1+ cells expressed FasL, and this significantly declined by 31% upon treatment with AF-SWCNTs (data not shown) (see **Figure 11**).

**Figure 11.** Effect of AF-SWCNT on NK cell degranulation. Splenocytes (2 × 10<sup>6</sup> ) cultured in the absence or presence of 500 U/mL of IL-2 were simultaneously treated with 50 μg/ml of AF-SWCNT. After 72 h, the activated splenocytes used as effector cells were harvested and cocultured with YAC-1 target cells to induce NK cell degranulation. The cells were stained with antimouse CD107a mAb and counterstained with antimouse NK1.1 mAb. \*\*p < 0.01 by Student's *t* test.
