**Natural Killer Cells Interaction with Carbon Nanoparticles Nanoparticles**

**Natural Killer Cells Interaction with Carbon** 

DOI: 10.5772/intechopen.69731

Anwar Alam and Rajiv K Saxena Anwar Alam and Rajiv K Saxena Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69731

#### **Abstract**

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158 Natural Killer Cells

The increased use of nanomaterials for biomedical purposes has warranted the need to introspect their toxicological properties and assess their utility to human health, particularly the immune system. Natural killer (NK) cells hold a pivotal position in innate immunity and serve as first line of defense against foreign bodies. Acid functionalized Carbon nanotubes (CNTs) that easily polydisperse in aqueous solution and could be coupled with fluorescent molecules were used to study the effect of carbon nanoparticles on NK cells *in vitro* and *in vivo*. Flow cytometry-based assays were used to study the effect of CNTs on various physiological parameters of NK cells, such as cell recovery, apoptosis, cell cycle, and generation of reactive oxygen species. A downregulation of the cytotoxicity of IL-2-activated murine NK cells was observed in the presence of acid-functionalized CNTs. The mechanistic basis of this downregulation was studied by assessing markers of NK cell activation (CD69), generation (NLK1.1), degranulation (CD107a) and apoptosis (annexin V assay). This chapter provides a blueprint for assessing the effect of carbon nanoparticles on NK cells. The assays mentioned in this chapter can be extrapolated to study the effect of other nanoparticles on different cell types as well.

**Keywords:** NK cell cytotoxicity, carbon nanotube, flow cytometry, apoptosis, NK cell degranulation, YT-INDY

### **1. Introduction**

Carbon nanoparticles (CNPs) have size less than 100 nm in at least one dimension and can be engineered in allotropic forms, such as nanodiamonds, fullerenes, nanobuds, and nanotubes. Each of these CNPs exhibits unique physicochemical properties and by virtue of their extremely low size can effectively interact with cells and tissues. Carbon nanoparticles, specifically carbon nanotubes (CNTs), are being tested for their potential use in the field of

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

nanomedicine including medicinal chemistry, imaging, vaccine delivery, etc. [1]. How these CNPs affect the living systems and the risks or benefits associated with their environmental, occupational, or therapeutic exposure is a matter of active research [2]. This chapter focuses on the interaction of an important component of innate immunity, the natural killer cells, with carbon nanotube.

#### **1.1. Natural killer cells**

Natural killer (NK) cells are important effector cells of the innate immune system and constitute about 5–15% of peripheral blood lymphocytes [3]. NK cells originate from lymphoid progenitors in the bone marrow and require IL-15-mediated signaling for development and survival [4]. NK cells can be identified through a set of markers, such as NK1.1 in mice or CD56 and CD16 in humans [5, 6]. The absence of CD3 on NK cells is a useful marker to differentiate between NK and natural killer T cells (NKT) [7].

NK cells kill syngeneic or allogeneic cells through mechanisms that require neither a prior sensitization with target cells nor the presentation of antigen in association with MHC-I. NK cell functions through an array of germline-encoded inhibitory or activating receptors that recognize MHC-I expressed in steady state on normal cells or altered ligands on dysregulated cells, respectively. Killer inhibitory receptor (KIR) in humans, the lectin-like Ly49 molecules in mice, and CD94/NKG2A heterodimers in both species detect MHC-I molecules on normal cells [8]. Perturbations in expression of MHC-I molecules on viral-infected cells or malignant transformed cells lead to loss of inhibitory signals causing activation of NK cells. Additionally, NK cells also require signaling through activating receptors, such as NKp30, NKp44, NKp46, NKp65, and NKp80 to trigger effector functions, such as cytokine production and cytolytic activity. Stimulation with inflammatory cytokines, such as IL-2, IL-15, IL-12, or IL-18 evokes differentiation into effector NK cells. CD16 or constant Fcγ-receptor IIIa (FcγRIIIa) exerts antibody-dependent cell-mediated cytotoxicity (ADCC) against various antibody coated cellular targets, leading to the exocytosis of perforin and granzyme-loaded vesicles. By integrating activating and inhibitory signals, NK cells contribute to the elimination of stressed cells expressing modified motifs while sparing healthy cells [9].

Upon recognition, target cells can get killed by NK cells through one of two pathways: either via the perforin and granzyme secretion pathway or via membrane-bound death receptors. NK cells store preformed perforin and granzyme in secretory vesicles that when triggered by activating signals causes formation of microtubule-organizing center (MTOC) which guides the vesicles, containing perforin and granzyme, in a directed way toward the target cell to prevent damage to bystander cells. Perforin forms pores in the target cell membrane, disrupts membrane integrity, and allows the entry of the apoptosis-inducing granzymes. The importance of the lytic perforin/granzyme pathway is evident in perforin knockout animals that exhibit lesser efficiency in ADCC or tumor control after transplantation of tumor cells. Perforin-/granzyme-containing cytoplasmic vesicles express CD107a or lysosomal-associated membrane protein (LAMP-1). Upon degranulation, CD107a is transferred transiently on the surface of NK cells and protects NK cells from damage from their own perforin or granzyme release [10].

NK cytotoxicity via its membrane-bound death receptors occurs upon binding with ligands, expressed on target cells. Receptor-ligand pairs, such as Fas-FasL, TNFR-TNF, and TRAILR-TRAIL, induce recruitment of various adaptor proteins leading to the formation of the deathinducing signaling complex (DISC). Subsequently, the caspases 8 and 10 get activated via proteolysis and initiate apoptosis. In addition to the cytotoxic response, NK cells are important sources of various cytokines, such as IFN-γ, TNF-α, or IL-10 and of chemokines, such as CCL3/CCL4/CCL5 or CXCL8. Natural killer (NK) cells basally express high levels of the signal transducer and activator of transcription 4 (STAT4) and produce the cytokine gamma interferon (IFN-γ). Type 1 interferons could potentially activate STAT4 and promote IFN-γ expression; however, concurrent elevated expression of STAT1 negatively regulates access to this pathway. IFN-γ due to its pleiotropic functions is considered to be the signature cytokine of NK cells [11]. IFN-γ has been shown to have antiproliferative effects on tumor cells and exert anti-angiogenic activity. A combination of TNF-α and IFN-γ has been shown to trigger tumor senescence and activates macrophages and dendritic cells [12]. Cytokine secretion is mediated via recycling endosomes using a distinct pathway from cytolytic vesicles, which allows for differential regulation.
