By Jess Sharrock PhD
Natural Killer cell overview
Natural Killer (NK) cells were first described in the mid-1970’s as a population of innate lymphocytes that were able to mediate cytotoxicity against tumour cells without prior exposure to a target (Herberman et al., 1975, I and II). NK cells are found in primary and secondary immune compartments, some mucosal tissues and are continually circulating through the vasculature to perform host immunosurveillance. They make up between 5-15% of human peripheral blood cells and 2-3% of splenocytes in mice (Mah and Cooper, 2016). Along with their role in tumour cell surveillance, they are also valued for their early defence against microbial infection. In recent years, NK cells have been shown to possess significant similarities to adaptive immune cells, such as T and B cells, including clonal expansion, self-renewal, long-lived memory responses and antigen specificity (Adams et al., 2016; O’Sullivan et al., 2015; Sun et al., 2011). NK cells are also a member of a family of lymphocytes known as innate lymphoid cells (ILCs). ILCs can be classified into three main groups, ILC1s, in which NK cells are a member of, ILC2s and ILC3s, based on their different transcription factor profiles, cytokine production and cell surface markers. NK cells, unlike other members of the ILC1 family, constituently express the transcription factor Eomesodermin (Eomes) and the integrin CD49b (also known as Alpha 2 Beta 1 or DX5) (Gordon et al., 2012).
NK cells, similarly to T and B cells, are derived from the common lymphoid progenitor (CLP) and like B cells they are thought to develop in the bone marrow (Geiger and Sun, 2016), although it has also been proposed that NK cells may develop in both the thymus and the liver (Sojka et al., 2014). However, unlike T and B cells, NK cell receptors are germline-encoded and do not require gene recombination by recombination-activating gene (RAG) recombinase (Lanier et al., 1986). Following the development of NK cells, they undergo terminal maturation in secondary lymphoid tissues, including the lymph nodes and spleen (Geiger and Sun, 2016). Mature peripheral NK cells can be identified by the upregulation of the cell surface marker CD11b and the downregulation of CD27, with the most immature NK cells being CD27-CD11b-, and the most mature NK cells being CD27-CD11b+. (Chiossone et al., 2009). Furthermore, in parallel with T and B cells, NK cells require common-gamma-chain-dependent cytokine signalling, particularly Interleukin-15 (IL-15), for development, survival, function and to maintain homeostasis (Cooper et al., 2002). Following disruption of IL-15 signalling, NK cell survival is reduced and the NK cells that are present become arrested in an immature state (Vosshenrich et al., 2005).
Molecular mechanisms of the NK cell response
Maintenance and turnover of all cell types is tightly controlled in all multicellular organisms and it is essential that tissue damage is avoided and homeostasis is upheld. NK cells are able to rapidly produce inflammatory cytokines, including interferon-gamma (IFN-gamma), following the ligation of their activating receptors (Krzewski and Strominger, 2008). Their ability to produce these cytokines is not only essential for host protection against pathogens and cancerous cells but is also important for the maintenance of healthy immune function (Lee et al., 2009).
NK cells use two main mechanisms to respond to inflamed tissues or damaged cells, firstly, by detecting the presence of stress signals released by infected or impaired cells, which in turn cues the NK cells to kill them by releasing cytotoxic granules containing perforin and granzymes (Topham and Hewitt, 2009). This is thought to be a type of ‘self-sacrifice’. The second mechanism is the recognition of cells that lack ‘self’ identifying markers, all nucleated cells, and some non-nucleated cells such as platelets, have specific cell surface proteins known as major histocompatibility complex (MHC) that allows for the differentiation of cells that are ‘self’ from those that are ‘non-self’ (Gonzalez et al., 2011). Interestingly, NK cells are equipped to kill cells that lack these surface markers and have the ability to kill target cells without prior training, unlike other immune cells that have to learn to recognise a threat. As T cells require MHC for activation, it has led to some viruses and cancer cells evolving to remove MHC molecules from the surface of infected cells to avoid detection (Hewitt, 2003). Furthermore, unlike T-cells, in which activation is triggered by antigens displayed by antigen-presenting cells (APCs; professional APCs, which present antigens to T-cells, include dendritic cells and macrophages), NK cells are not governed by single receptors but regulated by the integration of co-activating (including NKG2D, NKp46 and LFA-I) and co-inhibitory (including NKG2A, TIGIT and KLRG1) signals via cell surface receptors that recognise their corresponding ligands on healthy cells or target cells to determine the responsiveness of the NK cells (Figure 1).
Figure 1: Mechanism of NK cell response in normal and target cells. NK cells recognise ‘self’ MHC on normal cells, when activating receptors bind, an inhibitory interaction will suppress NK cell activation and no killing will take place (top). If NK cells encounter tumour cells or virally infected cells that lack the ‘self’ MHC molecule it will promote NK cell activation, leading to cytotoxicity, and results in the killing of the unwanted cell (bottom).
Immunometabolic role of NK cells
Organisms rely on numerous highly conserved mechanisms for their survival; two in particular being the ability to fight off infection and the ability to utilise and store energy efficiently. Often extreme changes occur within immune cells following activation, such as robust cell growth and proliferation, which requires corresponding modifications to metabolic pathways (Loftus and Finlay, 2016). A tightly controlled equilibrium between these two systems has evolved over time due to immune responses being so highly energy-demanding and metabolic pathways having to respond by shifting the energy usage away from non-essential tasks (Schipper et al., 2012). Dysregulation of these immune-metabolic interactions can lead to a range of diseases including obesity, diabetes and chronic inflammation (Brestoff and Artis, 2015).
Immunometabolism, or the functional change in intracellular metabolic pathways within activated immune cells, has become a major focus in recent years (O’Neill et al., 2016). There are two main factors that should be considered when thinking about immunometabolism, one is the effect the immune response has on systemic metabolism, and the second is the metabolic processes that are occurring within resting and activated immune cells (Lee et al., 2018). In the context of metabolism and immunity, there are a number of pathways that are currently been studied in great depths, including the glycolytic metabolic pathway, the tricarboxylic acid (TCA) cycle, amino acid metabolism, fatty acid synthesis and others (all reviewed in O’Neill et al., 2016). Metabolites and enzymes, including citrate and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) respectively, have also been implemented in events during immune cell activation as well as during metabolic processes (Kim, 2018).
Much of the work investigating the roles and mechanisms of immunometabolism in health and disease has been done using macrophages, primarily adipose tissue macrophages (ATMs), in the setting of diet-induced obesity. For many years, a large number of research groups have observed an upregulation of inflammatory gene expression in adipose tissue of obese mice, which has often been associated with an influx of inflammatory macrophages (Boutens and Stienstra, 2016; Pedersen et al., 2015; Weisberg et al., 2003). Following on from these observations, it has also been shown that changes in adipose tissue homeostasis can contribute to a reduction in insulin sensitivity and in some cases resulting in the development of type-2 diabetes (McCurdy and Klemm, 2013; Schipper et al., 2012). T cells have been shown to dramatically alter their metabolic profile to meet an increase in metabolic demands for cell growth, proliferation and effector function (Pearce et al., 2013). Depending on the activation status of T cells, and potentially other immune cells including NK cells, they display distinct metabolic profiles (Figure 2).
Figure 2: Metabolic profile of T cells during an immune challenge. T cells, and potentially other immune cells, display different metabolic states depending on their activation state. Naïve T cells (TN, red) are metabolically quiescent and use OXPHOS as their main metabolic pathway for ATP production. Following immune challenge, effector T cells (TEFF, orange) shift their metabolic state to become more activated, they also switch from OXPHOS to glycolysis for ATP production. Once the immune challenge has been cleared, memory T cells (TM, grey) return to metabolic state similar to that of TN cells. Adapted from Pearce et al., 2013.
Under steady state conditions, naïve T cells (TN) are metabolically quiescent, they have a basal level of nutrient uptake and use oxidative phosphorylation (OXPHOS) as their preferred pathway for adenosine triphosphate (ATP) production. Following immune challenge, or antigen encounter, T cells become activated, undergo extensive proliferation and differentiate into metabolically active effector T cells (TEFF). Following the clearance of pathogens, many of the TEFF cells die out, leaving behind a small pool of metabolically primed, long-lived antigen specific memory T cells (TM). TEFF cells preferentially use glycolysis over OXPHOS for ATP production, whereas the TM cells adopt a metabolic profile to TN cells and are usually more reliant on OXPHOS over glycolysis (Pearce et al., 2013).
Similar to TN cells, both mouse and human NK cells rely on OXPHOS for ATP production and are able to increase rates of both glycolysis and OXPHOS in response to various stimuli (Pearce et al., 2013). Freshy isolated splenic NK cells from mice have low levels of metabolic activity with preference for OXPHOS over glycolysis (Keppel et al., 2015). Short-term activation (4-6 hours) with cytokines or receptor ligation does not alter metabolic pathway usage. However, following stimulation with cytokines for longer periods of time, much like an in vivo response to infection, leads to increasing changes in NK cell metabolism that are much more prominent than in resting states (Gardiner and Finlay, 2017; Keppel et al., 2015). Ex vivo, both T cells and NK cells quickly shift to aerobic glycolysis following culture with high doses of IL-15, which leads to the activation and proliferation of both these cell types (Mah and Cooper, 2016).
The activation of immune cells, including NK cells, T/B cells and monocytes, and changes in metabolic state is thought to be regulated, at least in part, by mammalian target of rapamycin (mTOR). mTOR is considered a master regulator of the metabolic response (Liu et al., 2015), which integrates an array of cellular processes, including growth, proliferation and metabolic state, in response to environmental changes. mTOR is the catalytic subunit of two distinct complexes, mTOR complex 1 and 2 (mTORC1 and mTORC2), which have the ability to sense amino acids and growth factors as well as promote lipid synthesis to support cell growth and mRNA translation (Watanabe et al., 2011). Many studies have shown that mTOR signalling is able to regulate events that are crucial for the differentiation of monocytes and T cells (Weichhart et al., 2015) and is a key player in driving metabolic reprogramming of T cells (Finlay et al., 2012). Studies have also shown that NK cells respond to elevated levels of IL-12, IL-15 and IL-18 via the activation of mTOR signalling, which is linked to an increased rate of glycolysis to meet the heightened biosynthetic requirement by metabolising glucose into lactate (Figure 3). Intriguingly, mTORC1 has also been shown to be important for the development and maturation of murine NK cells, and is strongly upregulated in mature NK cells stimulated with IL-2 or IL-15, two cytokines that are thought to activate NK cell function (Wu et al., 2017; Yang et al., 2018.). Additionally, it is thought that mTORC1 signalling promotes Eomes expression, allowing for efficient maturation of NK cells (Frank et al., 2015).
Figure 3: A metabolic pathway utilised by activated NK cells
Following an increase in IL-12, IL-15 and IL-18, NK cells are able to respond through the activation of mTOR signalling. This process is associated with a higher rate of glycolysis to meet higher energetic demand by metabolising glucose into lactate in a process called aerobic glycolysis. Aerobic glycolysis is required by cells to carry out proliferation and effector cell function. Adapted from O’Sullivan and Sun, 2017.
mTOR-deficient NK cells are unable to progress past an immature stage, suggesting mTOR is needed for optimal proliferation of immature NK cells to populate a pool of mature NK cells during development and homeostasis (Marçais et al., 2014). Furthermore, in the presence of mTOR inhibitors or glycolysis pathway inhibitors, injections of the Toll-like receptor 3 (TLR3) agonist, Poly (I:C), or mouse cytomegalovirus (MCMV), which both induce IL-12, IL-15 and IL-18 production, leads to a reduction in IFN-gamma production by NK cells (Donnelly et al., 2014; Nandagopal et al., 2014). These observations suggest pro-inflammatory cytokine induced mTOR signalling may increase aerobic glycolysis of NK cells to strengthen IFN-gamma production (O’Sullivan and Sun, 2017), in turn helping to defend the body against virally infected or cancerous cells. When NK cells become activated in response in viral infection, there is an increase in mTORC1 signalling allowing the cells to leave their quiescence state, expand and maintain an adequate level of oxidative metabolism (Frank et al., 2015). Moreover, it is known that NK cell mediated control of viral infection requires glucose metabolism and inhibiting glucose metabolism with 2-deoxy-D-glucose (2DG), a glucose metabolism inhibitor, decreases NK cell proliferation and cytotoxicity in vitro (Mah et al., 2017).
The cross-talk between the immune system and metabolism has been at the forefront of many research laboratories for a number of years now, with much of the data being accumulated predominantly from T cell studies. Although metabolic similarities between NK cells and other immune cells can be drawn, the metabolic mechanisms and signatures of NK cell subsets remain to be fully explored and characterised in depth. NK cells and their immunometabolic connections are becoming of increasing interest as a new opportunity for NK-cell based immunotherapies for the treatment of cancer and viral infection, however there is still much that needs to be understood.
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