NK cells develop from the common lymphoid progenitor (CLP) in the bone marrow (Lanier et al., 1986b; Kondo et al., 1997). Transcription factors T-bet and Eomesodermin (Eomes) regulate NK cell development: dysfunction in both results in severe NK cell defects (Townsend et al., 2004; Intlekofer et al., 2005). In normal development, NK cells undergo continuous changes in receptor expression, commit to the NK lineage, and enter circulation as immature NK cells (Reviewed in Yu et al., 2013). Unlike T and B cells, NK cells do not express a rearranged antigen receptor. Instead, they express a diverse array of germline encoded activation and inhibition receptors. The balance between signals from these receptors determines their activation state and effector function (Lanier, 2008).

NK cells perform a variety of anti-viral functions in humans and mice which can be characterized by direct killing of target cells and production of immunomodulatory cytokines. NK cells can kill target cells through natural and antibody-dependent cytotoxicity (ADCC). Natural cytotoxicity refers to NK cell killing of target cells in the absence of inhibitory signals from self MHCI molecules (Kärre, 1985; Kärre et al., 1986). ADCC is induced through binding of the NK stimulatory Fcγ receptor CD16 to the Fc region of IgG bound to a target cell (Trinchieri et al., 1975; Perussia et al., 1984). During natural and antibody-dependent cytotoxic responses, activated NK cells mobilize and release cytotoxic granules containing perforin and granzymes which initiate apoptosis in the target cell (Kägi et al., 1994a; Voskoboinik et al., 2006).

NK cell inhibitory receptors of the killer cell immunoglobulin-like receptor (KIR) (human) and Ly49 receptor (mouse) families bind self MHC I molecules and inhibit cytotoxicity toward healthy self cells; malignant or infected cells which downregulate MHC I are susceptible to NK cell killing (Karlhofer et al., 1992; Wagtmann et al., 1995b). KIRs are immunoglobulin-like receptors whereas Ly49 receptors are C-type lectin type II family homodimer proteins linked by a disulfide bond (Yokoyama, 1993; Wagtmann et al., 1995a). Both human and murine NK cells also express CD94/NKG2A, which recognizes non-classical MHC molecules and closely resembles mouse Ly49 receptor structure (Phillips et al., 1996). Murine NK cells express GP49 which is structurally and functionally homologous to human KIRs (Wang et al., 1997).

NK cells express a variety of other MHC-independent inhibitory and stimulatory receptors which contribute to the cell's activation state. As evidenced by KIRs and Ly49s, NK cell receptor repertoires vary between mice and humans. Therefore, the functions of various phenotypically distinct populations identified in murine models of disease may not always be physiologically relevant to humans. This is a challenge in translating NK cell research findings in mice to humans and should be considered in our discussion of NK cell function in mice.

MHC-independent inhibitory receptors limit potential NK cell-mediated immunopathology but can be the target of immune evasion mechanisms by infected or malignant cells. For example, programmed death pathway receptor PD-1 and its ligands PD-L1 and PD-L2 are expressed on NK cells, protecting against hyperinflammatory responses during infection (Quatrini et al., 2018). NK cells are increasingly credited as important contributors to the improved outcomes of patients receiving checkpoint blockade therapies targeting these immunosuppressive mechanisms (Wiesmayr et al., 2012; Beldi-Ferchiou et al., 2016; Hsu et al., 2018). Similarly, surface receptors Tim3, TIGIT, and Siglec-7 and−9 inhibit NK cell cytotoxic function when bound by their ligands on potential target cells (Stanietsky et al., 2009; Ndhlovu et al., 2012; Jandus et al., 2014). Therapeutic blockades of Tim3 and TIGIT have also shown efficacy as anti-cancer therapeutics in mice (Xu et al., 2015; Zhang et al., 2018). NK cell inhibitory receptors regulate NK cell cytotoxic responses which are vital for control of infection; more thorough characterizations of these receptors may be beneficial in the development of targeted therapies to enhance NK cell anti-viral functions.

To kill a target cell, NK cells also require activation signals which differ depending on the responding cell, target cell, or surrounding environment. NKG2D is an activating receptor in murine and human NK cells which binds to MICA, a stress ligand upregulated on malignant or infected cells (Bauer et al., 1999). Activating natural cytotoxicity receptors (NCRs) such as human NKp46 (murine NCR1), NKp30 and NKp44 also recognize a variety of pathogen-derived and stress-induced ligands (Mandelboim et al., 2001; Vitenshtein et al., 2016). The NKG2C/CD94 complex functions as an activating receptor which recognizes the non-classical MHC molecule HLA-E (human) or Qa-1^b (mice) (Lanier et al., 1998; Vance et al., 1999). 2B4 also serves as a conserved NK activating receptor which binds to CD48 (Mathew et al., 1993; Brown et al., 1998). These receptors, among others, are fundamental in activating NK cell cytotoxic functions.

NK cells also kill infected cells through perforin and granzyme-independent mechanisms: Fas and TRAIL. Fas receptor expression is altered in infected, malignant or otherwise stressed cells, and NK cells express its ligand CD94 (FasL). Binding to Fas by FasL induces apoptosis in the target cell through a caspase-dependent pathway (Arase et al., 1995; Hao and Mak, 2009). Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is another ligand expressed on the NK cell surface which induces apoptosis in sensitive target cells expressing DR4 or DR5 (Zamai et al., 1998). These mechanisms complement cytotoxic killing mechanisms and expand the protective effects of NK cells.

In addition to direct killing of target cells, NK cells secrete a variety of pro- and anti-inflammatory cytokines which directly affect the nature of the immune response. IFN-γ is crucial in their control of intracellular pathogens (Dunn and North, 1991; Orange et al., 1995). NK cells are recruited to the lymph nodes during infection where their secretion of IFN-γ has two functions in T cell activation: promote Th1 differentiation and enhance DC antigen presentation. In the absence of IFN-γ, protective T cell responses are significantly impaired (Huang et al., 1993; Gerosa et al., 2002; Martín-Fontecha et al., 2004). TNF-α secreted by NK cells also contributes to activation of DCs and macrophages, further enhancing antigen presentation (Bancroft et al., 1989; Walzer et al., 2005).

Alternatively, NK cells secrete anti-inflammatory cytokine IL-10 during systemic inflammation (Mehrotra et al., 1998; Perona-Wright et al., 2009). NK cell derived IL-10 has been reported to regulate CD8 T cell responses and to be protective against excessive tissue damage in chronic infections such as murine cytomegalovirus (MCMV) and Hepatitis C Virus. However, this could contribute to persistent infection and inability to clear the virus (De Maria et al., 2007; Lee et al., 2009; Ali et al., 2019). NK cell cytokine production is essential for their control of viral infections, but the downstream effects of each cytokine varies significantly depending on the nature of the infection.

NK cells are often referred to in terms of phenotypically distinct subsets which exhibit different effector functions. NK cells in the mouse belong to one of four populations in order of maturity: CD11b⁻CD27⁻→CD11b⁻CD27⁺→CD11b⁺CD27⁺→CD11b⁺CD27⁻. CD11b⁻ NK cells are found in the bone marrow, lymph nodes and liver, whereas CD11b⁺ NK cells are dominant in the spleen, peripheral blood and lung. CD11b⁺ cells are defined by their efficient IFN-γ production and cytotoxicity in response to stimulus compared to CD11b⁻ NK cells. CD11b⁺CD27⁺ NK cells are considered the most functional subset. They show higher proliferative capacity, more IFN-γ production potential, and stronger cytotoxic function than the more mature CD11b⁺CD27⁻ subset (Kim et al., 2002; Chiossone et al., 2009).

In humans, CD56^brightCD16⁻ NK cells are similar but not homologous to CD11b⁻ murine NK cells. They are immature, abundant in lymphoid tissues and efficient cytokine producers. Alternatively, mature CD56^dimCD16⁺ NK cells are more abundant in the periphery and have higher cytotoxic activity (Lanier et al., 1986a). CD56^bright cells express high levels of KIRs, whereas CD56^dim cells lose KIR expression and gain CD94 expression. CD56^dimCD16⁺CD57⁺ NK cells are considered terminally differentiated. They have lower proliferative capacity and are unresponsive to cytokine stimulation, however they are sensitive to stimulation through CD16 (Lopez-Vergès et al., 2010). CD57⁺ NK cells modulate inhibitory receptor expression: as they differentiate they lose expression of NKG2A and gain expression of KIRs, increasing their threshold for stimulation (Björkström et al., 2010).

During infection, the phenotype of NK cells changes to reflect their activation state and effector function. Besides cytokine production and expression of cytotoxic granules, activated cells can be identified by a variety of activation markers. For example, CD69 is a marker of general NK cell activation (Borrego et al., 1993). CD107a (LAMP1) is upregulated in actively degranulating NK cells (Alter et al., 2004). CD38 is a marker of activation during viral infection: it is expressed on NK cells when they encounter influenza infected cells and is correlated with CD107a expression and IFN-γ production (Le Gars et al., 2019). These markers are commonly used to describe NK cell activation and function in various infections or disease.

NK cells in mice and humans are found in a variety of secondary lymphoid and non-lymphoid organs including lymph node, spleen, liver, skin, uterus, peripheral blood, and lung (King et al., 1991; Grégoire et al., 2007). The developmental stage, phenotype and function of NK cells varies by tissue. NK cells in the lymph nodes are mostly CD11b⁻ or CD56^brightCD16⁻ whereas in the peripheral blood they are predominantly CD11b⁺ or CD56^dimCD16⁺ (Ferlazzo et al., 2004; Chiossone et al., 2009). NK cell subsets can migrate from blood to tissues dependent on chemokine receptor expression. For example up to 50% of liver NK cells are tissue-resident and maintained in the liver by their expression of CXCR6 (Paust et al., 2010; Hudspeth et al., 2016). CXCR3⁺ and CCR5⁺ NK cells migrate to the lungs during influenza infection. This is correlated with increased expression of their ligands on lung epithelial cells (Carlin et al., 2018). Chemokine receptor expression is often used to determine tissue-specific residency. Other surface receptors—CD69, CD103, and CD49a—promote tissue retention and are used to describe tissue-resident NK cells in general (Shiow et al., 2006; Walzer et al., 2007; Björkström et al., 2016). Various circulating and tissue-resident NK cell populations may have unique and critical functions in host protection, but a variety of challenges have limited the understanding of trafficking and tissue-specific roles of NK cells (Reviewed in Ferlazzo and Carrega, 2012; Björkström et al., 2016).

NK cells are a prominent lymphocyte population in human and mouse lungs. In a healthy human lung, NK cells comprise 10–20% of all lymphocytes, most of which are CD56^dimCD16⁺. They are a highly differentiated population with a CD57⁺NKG2A⁻ phenotype and high KIR expression (Marquardt et al., 2017). Similarly, murine NK cells represent about 10% of lung lymphocytes (Grégoire et al., 2007). NK cells in mouse lungs are also dominated by a mature CD11b⁺CD27⁻ phenotype and show properties of terminally differentiated cells: higher levels of inhibitory receptor CD94 and lower levels of stimulatory receptors NKG2D and NKp46 compared to spleen and bone marrow NK cells (Hayakawa and Smyth, 2006; Wang et al., 2012). In the 1980s, human lung NK cells were described as “functionally impotent” due to unresponsiveness to stimulation; lung alveolar macrophages and epithelial cells had a suppressive effect on NK cell function (Robinson et al., 1984). Expression of inhibitory receptors on lung NK cells may contribute to their minimal responses to stimulus and serve as an important tolerance mechanism in the exposed lung microenvironment.

Recent analysis of human lung NK cells revealed a small but distinct subset of CD69⁺ NK cells characterized by a CD56^brightCD16⁻ phenotype and higher functionality (Marquardt et al., 2017). Another study characterized this population more specifically as CD56^brightCD49a⁺CD103⁺CD69⁺ lung-resident NK cells which upregulate CD107a, granzyme B and IFN-γ upon stimulation (Cooper et al., 2018). In mice there is evidence that NK cells accumulate in the lung and show increased function after infection, but it is unclear whether this is due to recruitment of blood NK cells, response of lung-resident NK cells, or some combination of the two (Stein-Streilein et al., 1988; Wang et al., 2012). Despite previous thinking that lung NK cells are not capable of significant cytotoxicity or cytokine production, more studies must be done to identify potential in vivo stimulatory factors which activate lung NK cells in respiratory infections.

T cells also develop from the common lymphoid progenitor (Kondo et al., 1997). Progenitor cells migrate from the bone marrow to the thymus where they commit to the T cell lineage (Miller, 1961; Ford et al., 1966). The T cell receptor (TCR)—a rearranged antigen receptor through which T cells recognize peptides presented on MHC of an infected cell—develops in the thymus. VDJ recombination, mediated by RAG1 and RAG2 enzymes, ensures a high diversity in TCR specificity (Reviewed in Schatz and Ji, 2011). Developing cells undergo positive selection ensuring functional TCR/MHC interactions and negative selection deleting self-reactive TCRs before committing to a single positive CD4 or CD8 lineage (Kisielow et al., 1988; Bill and Palmer, 1989).

During infection, viral antigens move through the lymphatic system to the lymph nodes where they are presented on MHC by antigen presenting cells (APCs). Naïve T cells also circulate through the lymphatics and are activated by APCs in the lymph nodes (Guermonprez et al., 2002; von Andrian and Mempel, 2003). CD4⁺ and CD8⁺ T cells recognize antigens presented on MHC II and I, respectively. Following initial proliferation and differentiation in the lymph node, effector T cells travel through the blood to the site of infection where they are activated to exert their effector function (Marelli-Berg et al., 2008). After a period of weeks, the effector T cell population contracts and a smaller memory T cell population in formed. Memory T cells can be tissue-resident or circulating and can respond immediately to control a second infection by the same pathogen (Reviewed in Seder and Ahmed, 2003; Chang et al., 2014).

CD4⁺ and CD8⁺ T cells are activated through similar mechanisms, but they play unique functional roles in infection. The CD4⁺ T cell response orchestrates both cell-mediated (Th1) and humoral (Th2) immunity in response to foreign pathogens. After initial activation, differentiation is driven by cytokine-dependent transcription factor expression (O'Shea and Paul, 2010). IFN-γ and IL-12 initiate Th1 responses characterized by T-bet expression and IL-2 and IFN-γ production. This induces a cellular response against intracellular pathogens characterized by enhanced CD8⁺ T cell cytotoxicity and development of memory CD8⁺ T cells (Mosmann et al., 1986). Notably, T-bet is a prevalent NK cell transcription factor and IL-2 is a potent NK cell activator; NK cell IFN-γ production in these conditions amplifies Th1 responses (Domzig et al., 1983; Townsend et al., 2004). GATA3 expression induces Th2 responses that produce IL-4, IL-5, and IL-15 and promote B cell antibody production and memory development (Mosmann et al., 1986). CD4 T cells can also differentiate into Tfh, Th17, and T regulatory cells (Tregs). Tfh cells are important costimulatory cells for B cell development (Reviewed in Vinuesa et al., 2005). Th17 cells are highly inflammatory cells regulated by Rorγt which produce IL-17, IL-22, and IL-27 and are associated with tissue homeostasis during infection (Park et al., 2005). Tregs are characterized by Foxp3 expression; they dampen the immune response and limit lung injury during influenza infection through secretion of TGF-β and IL-10 (Sakaguchi, 2000).

CD8⁺ T cells, or cytotoxic T cells, kill infected or “altered-self” cells (Zinkernagel and Doherty, 1974; Blanden et al., 1975). They release cytotoxic granules following recognition of a foreign antigen presented on MHC I. CD8⁺ T cells also express FasL and TRAIL through which they induce apoptosis in target cells (Kägi et al., 1994b; Jeremias et al., 1998). Viruses including herpesviruses, poxviruses, and adenoviruses evade CD8⁺ T cell immunity through downregulation of class I MHC molecules (Andersson et al., 1985; York et al., 1994; Byun et al., 2009). This would leave the virally infected cells susceptible to NK cell cytotoxicity, but they often employ other evasion mechanisms which affect expression of NK cell activating ligands on infected cells. For example, Kaposi's sarcoma-associated herpesvirus disrupts surface expression of ligands to NKG2D and NCR NKp80 while myxoma virus downregulates Fas (Coscoy and Ganem, 2000; Guerin et al., 2002; Thomas et al., 2008).

T cell cytotoxicity can be altered by inhibitory receptors or cytokines in the environment. T cell “exhaustion” is characterized by increased expression of inhibitory receptors and can prevent excessive inflammation but result in impaired viral clearance. T cell inhibitory receptors, similar to those discussed for NK cells, include PD-1, Lag3, Tim3, and TIGIT (Grosso et al., 2009; Yu et al., 2009; Jin et al., 2010). Treg cytokines IL-10 or TGF-β contribute to inhibitory receptor expression (Wherry, 2011). T cell dysfunction is primarily studied in cancer or chronic infections such a HIV, hepatitis B/C, and LCMV clone 13 (McLane et al., 2019). However, there is evidence of dysfunctional T cell activities due to inhibitory receptors in acute viral infections as well (Erickson et al., 2012). T cell expression of inhibitory receptors or immunosuppressive cytokines is implicated both in impairing viral clearance and protecting against tissue damage. For example, in influenza infection one study found enhanced Tim3 function improved survival outcomes whereas another found the opposite (Sharma et al., 2011; Cho et al., 2012). To understand T cell responses, it is important to acknowledge the balance between these features. Furthermore, the simultaneous regulation of both T cells and NK cell cytotoxicity through these mechanisms has not been addressed. It is possible that inhibitory receptors are necessary to limit pathological consequences of one response but impair the other cell type's control of the infection.

Both naïve and memory T cells circulate through the lymphatics and vasculature until they encounter cognate antigen, but some memory T cells reside in the tissues and are termed tissue-resident memory T (T_RM) cells. T_RM cells are generally identified by the expression of CD69 and CD103, but this method is not entirely accurate as CD69 can be transiently expressed on activated circulating cells and there are some T_RM cells which do not express one or both of these markers (Masopust and Soerens, 2019). Furthermore, CD103 is more commonly required for CD8⁺ T_RM cell maintenance in tissues than for CD4⁺ T cells (Casey et al., 2012). Chemokine receptor expression is also used to identify organ specific T_RM cells. An example of this in the lung is described in section Role in the Lung. Like tissue-resident NK cells, T_RM cells are poised to respond rapidly to infection and therefore are important for immune surveillance and host protection. The development and maintenance of T_RM cells is reviewed more thoroughly elsewhere (Masopust and Soerens, 2019).

In naïve mice, over 90% of T cells in the lung are circulating in the vasculature. During influenza and other infections or inflammatory responses, T cells accumulate transiently in the lung parenchyma (Turner et al., 2014). A multitude of chemokine receptors, adhesion molecules and their ligands have been proposed as regulators of T cell homing to the lung. CXCL16 expression in the lung is important in CXCR6⁺ T cell homing; this is responsible for the recruitment and maintenance of T_RM cells in the airways following influenza infection in mice (Morgan et al., 2005; Wein et al., 2019). Murine T cells are imprinted with lung-homing receptor CCR4 upon activation by lung DCs and elicit protective immune responses against influenza (Mikhak et al., 2013). Alternatively, the expression of CCR4 ligands CCL17 and CCL22 correlate with harmful lung inflammation in WT and humanized mouse models of asthma (Perros et al., 2009; Zhang et al., 2017). CCR5 and CXCR6 were similarly implicated in CD8⁺ T cell recruitment and related lung pathology in COPD patients (Freeman et al., 2007). Chemotaxis of T cells into the lung could play an important role in both T cell-mediated protection and pathology during lung inflammation.

Following respiratory virus infections, the population of T cells which persist as resident memory cells in the lung are correlated with protection upon secondary exposure. Both CD4⁺ and CD8⁺ memory cells exist in the lung of recovered mice, but the population is skewed to favor CD8⁺ T cells (Hogan et al., 2001a,b). A study of influenza infection in mice demonstrated influenza-specific lung-resident memory T cells were CD69⁺, with CD4⁺ T_RM cells co-expressing CD11a and CD8⁺ T_RM cells co-expressing CD103. In humans, lung-resident T cells showed similar expression patterns following influenza infection (Turner et al., 2014). During murine infection with Mycobacterium tuberculosis, CD4⁺CD69⁺CXCR3⁺ T_RM cells increase in the lung parenchyma. These cells show similar features to previously described hypofunctional lung-resident NK cells: decreased IFN-γ production and increased PD-1 expression. However, they are associated with decreased bacterial load upon infection (Sakai et al., 2014). Stimulation during infection could overcome the expression of inhibitory receptors or they could have another uncharacterized protective role.

The lung has three functionally distinct compartments which are important to analyze separately: tissue, airways, and vasculature. Many studies characterize responses of the lung in general, but do not look further into cells in the airways or vasculature. This potential limitation should be considered when interpreting data of lung immune cells. To be more thorough, the airways can be studied through bronchoalveolar lavage and cells circulating in the vasculature can be distinguished from cells resident in the tissue through intravascular staining (Anderson et al., 2012). Better understanding of the regulation and phenotypic characteristics of distinct lung immune cell populations will broaden knowledge of their protective or pathogenic roles during respiratory infections.

Although both NK cell and T cell responses to influenza are characterized by cytokine production and direct killing of infected cells, their mechanisms of activation vary. NK cells in the lung vasculature and tissue are the first to encounter influenza-infected cells. These NK cells vary in phenotype and function—some are immature, circulating cells whereas others are mature, lung-resident cells—and are important for initial viral control and recruitment of immune effector cells. Within 3 days following low dose influenza infection in mice, CXCR3⁺ and CCR5⁺ NK cells accumulate in the lung, airways, and lung-draining lymph nodes (Ge et al., 2012; Carlin et al., 2018).

Early studies of lung NK cells suggested natural cytotoxicity receptors (NCRs) NCR1 (mouse), NKp44 and NKp46 (human) encoded by the Ncr1 gene bind to HA on the surface of infected cells. NCRs are traditionally viewed as stimulatory NK receptors. One study of influenza infection in Ncr1^−/− mice suggests they are necessary for protection (Arnon et al., 2001; Mandelboim et al., 2001; Gazit et al., 2006). However, the stimulatory potential of NCR1 and NKp46 binding to HA during influenza infection is debated in the field. One study challenged NKp46-dependent NK cell activation using a mouse model with a W32R substitution in Ncr1. The mutation disrupted NKp46 expression on the NK cell surface without deleting the whole gene. Lack of NKp46 expression was associated with increased Helios-dependent transcription factor expression and NK cell activation. The mutation resulted in higher levels of NK cell IFNγ production and significantly higher survival during influenza infection (Narni-Mancinelli et al., 2012). More work is needed to clarify conflicting data regarding NKp46/NCR1-dependent NK cell responses.

NK cell costimulatory receptors 2B4 and NTB-A bind to HA and enhance NK cell cytotoxicity, but cannot activate NK cells on their own (Duev-Cohen et al., 2016). Influenza virus has evolved immune evasion mechanisms for NK cell recognition of HA. For example, virally encoded NA glycoproteins remove sialic acid residues on NKp46/NCR1, disrupting human and murine NK cell recognition of HA (Bar-On et al., 2013). Alternatively, HA can internalize into NK cells and inhibit NKp44/46-induced cytotoxicity through disruption of the ζ signaling chain (Mao et al., 2010). Influenza can also directly infect mouse and human NK cells, removing potential cytotoxic cells from the environment (Guo et al., 2009; Mao et al., 2009). This could impair NK cell responses, but they possess alternative activation mechanisms.

CD16 (FCγRIII) binds to the Fc portion of influenza-specific antibodies and initiates ADCC. Studies in humans and macaques demonstrate seasonal influenza infections elicit cross-reactive antibodies to HA and NA epitopes of multiple influenza strains; these antibodies promote NK cell ADCC against infected target cells (Jegaskanda et al., 2013c, 2014). The presence of ADCC-promoting antibodies correlates with reduced disease severity in humans and could explain the trend during the 2009 H1N1 pandemic where older individuals—who were more likely to have cross-reactive antibodies in their serum—were not as affected as younger individuals (Jegaskanda et al., 2013b; Valkenburg et al., 2019). A study in immunocompromised HIV patients who had never received an influenza vaccine found they also had cross-reactive antibodies capable of mediating NK ADCC against pH1N1 and H3N2 strains, confirming these antibodies can emerge from natural exposure to seasonal influenza strains (Nehul et al., 2018). There is also evidence that humans produce antibodies to conserved internal proteins which activate NK cells in vitro (Jegaskanda et al., 2013a; Vanderven et al., 2016). Seasonal influenza infections promote the development of cross-reactive antibodies which promote NK cell ADCC during infection with various influenza strains. This is a physiologically relevant protection mechanism which should be considered in the development of influenza therapeutics and vaccines.

Stress-related mechanisms are also associated with changes in NK cell activation state. For example, NKG2D recognizes stress-induced ligands MICA/B and ULBP on infected macrophages and DCs, respectively (Sirén et al., 2004; Draghi et al., 2007). Additionally, ssRNA recognition through endosomal pattern recognition receptor TLR7 activates spleen and lung NK cells in response to influenza infection in mice (Stegemann-Koniszewski et al., 2018). Undoubtedly, there are more mechanisms through which influenza infection leads to NK cell activation and in order to fully understand the NK cell response we should work to understand mechanisms of activation beyond NCRs and CD16.

Over the last several years, extensive research established that adaptive-like NK cells exist, recognize viral antigens, and contribute to host protection from secondary infection with a variety of pathogens, including influenza. The details of NK memory responses are reviewed elsewhere (Cerwenka and Lanier, 2016; Paust et al., 2017). Briefly, memory NK cells localize to the liver due to expression of CXCR6 and mediate protective responses against secondary influenza infection in mice (Paust et al., 2010; Li et al., 2017). There is evidence that influenza vaccination in mice promotes development of memory NK cells which rapidly secrete large amounts of IFN-γ upon viral challenge (Dou et al., 2015; Goodier et al., 2016). The recognition mechanisms which mediate NK cell antigen-specific responses are unclear. Future studies which address this for influenza and other pathogens will help with efforts to target NK cell responses therapeutically.

Antigen-specific T cells recognize influenza-infected cells through TCR binding to virally encoded peptides presented on MHC. Naïve influenza-specific T cell clones are activated by DCs and undergo proliferation in the lymph nodes before they become functional effector cells (Lawrence and Braciale, 2004). CCR7^−/− mice with inefficient DC migration to the lymph node fail to mount a protective influenza-specific T cell response (Heer et al., 2008; Ho et al., 2011). Following initial activation and proliferation in the lymph nodes, T cells return to the lung where they are activated by infected cells (Román et al., 2002; Legge and Braciale, 2003).

T cells recognize a variety of external and internal influenza proteins. Conserved surface matrix protein 1 (M1) and intracellular nucleoprotein (NP) are important for T cell-mediated heterosubtypic immunity to influenza. Significant research in the 1970s and 80s identified T cell immunity to NP and M1 were responsible for cross-reactivity between strains whereas T cell clones specific for HA or NA only mediated strain-specific protection (Braciale, 1977; Townsend et al., 1984). Cross-reactive T cells were identified in recent years to novel influenza strains: pH1N1, H5N1, and H7N9. In each case, cross-reactive T cells from healthy patients were characterized in vitro and their presence was further correlated with protection against morbidity and mortality in humans and mice (Lee et al., 2008; Guo et al., 2011; Sridhar et al., 2013; McMaster et al., 2015). In one study following pH1N1 patients, the presence of IFN-γ⁺IL-2⁻ cross-reactive memory CD8⁺ T cells specifically was strongly correlated with less severe disease (Sridhar et al., 2013). One universal influenza vaccine approach involves enhancing T cell-meditated heterosubtypic immunity to conserved epitopes. Although the presence of cross-reactive T cells seems to be protective, studies should address cytokine production of these cells to understand the potential effects of enhancing cross-reactive T cell responses on T cell-mediated lung immunopathology.

Unique functional populations of NK cells and T cells contribute significantly to influenza responses through cytokine production and/or direct killing of infected cells. In general, the cell phenotype, mechanism of activation, and surrounding cytokine milieu are determinants of each cell's effector function. Here, we analyze NK and T cell effector functions and their contributions to lung immunopathology during influenza infection. The main ideas from this section are visualized in Figure 1.

The extent to which NK cell and T cell responses are protective before they begin to become pathogenic is highly debated. We will attempt to dissect this in terms of influenza infection; other articles address all aspects of the immune response in mediating lung immunopathology during respiratory infections (Damjanovic et al., 2012; Newton et al., 2016; Tavares et al., 2017). Mechanistic studies in mice attempt to characterize contributions of NK cell and T cell responses to host protection and pathology. There are conflicting hypotheses as to the effect of different cell populations and soluble mediators on optimal host responses: efficient viral clearance while maintaining lung homeostasis. Studies of human infections attempt to connect hypotheses from mouse models to real patient outcomes, but there is no clear consensus as to which are the most beneficial or detrimental aspects of the host response to influenza. The ability to understand the host response lies in understanding how a host's overall health is affected by viral replication and immune defense mechanisms given external factors such as viral serotype, severity of infection, and stage of the immune response.

Mouse models of influenza infection have correlated NK and T cells with both protection and tissue damage. IL-15 knockout mice have defects in multiple cell types including CD8⁺ T cells, NK cells, NKT cells and intraepithelial lymphocytes and are protected from lethal dose of influenza infection. Their immune response is characterized by less myeloid cell infiltration, increased IL-10 expression and decreased levels of pro-inflammatory cytokines IL-6 and IL-12. One study of IL-15 knockout mice attributed improved outcomes to limited CD8⁺ T cell-mediated damage, whereas another claimed the absence of NK cells was protective (Nakamura et al., 2010; Abdul-Careem et al., 2012). Likely, both contribute in some way which should be analyzed further. Neither of these studies looked at doses which were not lethal in wildtype mice. The literature suggests NK cells are associated with pathology in high dose infection due to more NK cell recruitment to the lung; the same question has not been answered so clearly for T cells (Zhou et al., 2013; Carlin et al., 2018). This is worth investigating, as severity of lung damage is largely dose dependent.

In addition to the viral dose, many hypothesize that whether NK cells have a positive or negative effect on host immune responses to influenza is dependent on the mouse genetic background. IL-15 knockout mice do not show major strain-dependent responses; antibody depletion of NK cells resulted in slightly better protection in B6 mice than BALB/C, but the authors attribute this to a more complete NK depletion in B6 mice (Abdul-Careem et al., 2012). Another study compared several strains of mice receiving high-dose influenza infection. After NK depletion, they found that only 129 mice exhibited decreased ability to control viral replication. NK cells from 129 mice were characterized by increased IFN-γ expression compared to other strains, but no differences in CD107a (Abdul-Careem et al., 2012). This suggests the protective effects in these mice are due to higher IFN-γ expression whereas cytotoxic functions of NK cells do not affect the ability to control the infection.

The role of NK cells in severe disease should be considered from the perspective of human infections as well. In humans infected with pandemic H1N1, NK cell lymphopenia in the blood has been reported in association with severe disease. One patient with a complete absence of peripheral blood NK cells even had detectable viral RNA in the blood. In this context, the absence of NK cells is harmful for human outcomes and viral control. However, this data was obtained from blood samples, not lung or airway, so it is possible that decreased NK cells in the blood is due to increased migration to the lung (Denney et al., 2010; Fox et al., 2012). Despite significant efforts, there is still no consensus as to whether NK cell responses are protective or pathogenic in influenza infection.

The role of the influenza-specific CD8⁺ T cell response in lung immunopathology has been studied extensively. A fundamental study presented contradictory roles: CD8⁺ T cell-deficient mice have reduced lung damage at the expense of lower survival (Wells et al., 1981). Cell-intrinsic contributions by T cells can be analyzed through adoptive transfer of HA-specific CD8⁺ T cells into a mouse constitutively expressing HA on its respiratory epithelial cells. This model revealed CD8⁺ T cell responses alone can induce significant host damage: weight loss, pro-inflammatory cytokine accumulation, monocyte recruitment, and death (Enelow et al., 1998). A variety of regulatory mechanisms induce inhibitory receptor expression and are essential to prevent excessive CD8⁺ T cell responses to influenza infection (Zhou et al., 2008; Erickson et al., 2016). Nevertheless, T cell responses are necessary to clear infections and understanding the mechanisms through which they are responsible for immunopathology during natural infections is important for developing effective therapeutic interventions.

Tregs limit immunopathology but can suppress protective anti-viral responses in an antigen-specific manner. They accumulate in the mouse lungs, airways, and mediastinal lymph nodes before effector T cells, but after NK cells, in response to influenza infection (Betts et al., 2012). They directly inhibit CD4⁺ and CD8⁺ T cell proliferation and anti-viral responses (Haeryfar et al., 2005; Bedoya et al., 2013). Tregs also inhibit NK cell cytotoxicity when exposed to influenza antigens, although the interactions between NK cells and Tregs are not thoroughly characterized (Trzonkowski et al., 2004). There is evidence, but limited mechanistic understanding, of their effect on innate immune responses: transferring Tregs into Rag^−/− mice improves survival outcomes by preventing excessive myeloid cell infiltration in the lung (Antunes and Kassiotis, 2010). Tregs are fundamental in promoting recovery; inhibiting Treg function following viral clearance increases neutrophil infiltration, promotes pro-inflammatory cytokines in the airways, and impairs weight gain (Moser et al., 2014). Overall, Tregs seem to be more beneficial than harmful to the host response: preventing lung damage improves survival outcomes more than the increased cytotoxic responses in their absence.

Despite unclear contributions of NK and T cells overall, many efforts attempt to characterize their role in immunopathology based on cytokine production. Both cell types produce TNF-α following influenza infection. Inhibiting TNF-α signaling leads to increased survival outcomes and decreased lung damage following lethal dose influenza infection. Transcriptional analysis following TNF-α blockade revealed an overall decrease in pro-inflammatory cytokines and increase in CD4⁺ T cells and NK cells in the lung. These results implicate CD8⁺ T cell—not CD4⁺ T or NK cell—secretion of TNF-α and subsequent cytokine storm as a main mediator of lung damage during lethal influenza infections (Belisle et al., 2010; Shi et al., 2013). Other studies demonstrate CD8⁺ T cell-derived TNF-α contributes to lung pathology by promoting inflammation and inducing apoptosis in alveolar epithelial cells. However, it is not required for T cell-mediated viral clearance in mice (Liu et al., 1999; Xu et al., 2004). Notably, none of these studies used lower infectious doses so it is unclear if TNF-α mediates detrimental lung damage in all infections. In human studies of 2009 pH1N1 infection, TNF-α levels were higher in hospitalized patients than mild cases. Within severe cases, the patients in non-critical states showed higher expression of TNF-α than those with critical illnesses (Bermejo-Martin et al., 2009; Yu et al., 2011). TNF-α may be protective among patients who develop severe disease. This study did not address lung damage like those in mice, but it is possible that although TNF-α induces lung damage during infection, it is necessary for a fully functional host response.

IFN-γ production is a significant part of the NK cell and T cell response to influenza infection. It has been implicated as both helpful and harmful in influenza infection. Adoptive transfer of HA-specific IFN-γ deficient CD8⁺ T cells showed that IFN-γ is not essential for optimal protection but is important for recruitment of effector cells and control of hyperinflammatory responses (Wiley et al., 2001). Another study showed that IFN-γ has protective effects in mice with intact NK cell responses but not in NK deficient mice (Weiss et al., 2010). Most recently, IFN-γ was observed to suppress innate lymphoid cell function. IFN-γ deficiency protected mice challenged with a lethal dose of influenza H1N1 virus (Califano et al., 2018). Due to IFN-γ's various roles in infection, it is likely these studies yield different results due to distinct experimental conditions and procedures. The challenge, as with other questions of protection vs. pathology in influenza virus, is determining which information is physiologically relevant to humans. IFN-γ expression and correlation with patient outcomes has been analyzed in various studies. Like with TNF-α, levels of IFN-γ in hospitalized pandemic H1N1 patients are significantly higher than in mild cases. However, within hospitalized patients high levels of IFN-γ is associated with less severe disease (Bermejo-Martin et al., 2009; Yu et al., 2011). More work should be done to further analyze the effects of IFN-γ on patient outcomes, especially in relation to NK cells and the functionality of their IFN-γ production.

The role of IL-17 in human and murine influenza infection, like other inflammatory cytokines, is highly controversial. IL-17^−/− mice exhibit less weight loss, decreased lung pathology, and increased survival in response to influenza infection. This could be due to decreased neutrophil infiltration or cytokine production in the lung; IL-17^−/− mice have lower levels of IFN-γ, G-CSF, IL-6, IL-1B, and TNF-α compared to WT mice (Crowe et al., 2009). A mouse model of 2009 pH1N1 infection also showed that blocking IL-17 signaling enhances survival outcomes (Li et al., 2012). Alternatively, a study of IL-10 knockout mice observed increases in the Th17 response were protective against high-dose infection. The protection was attributed to IFN-γ production and cytotoxic activity of Th17 cells in addition to elevated IL-17 levels (McKinstry et al., 2009). Knowing IL-17 plays a dynamic role in infection outcomes, numerous studies address Th17 responses in humans and their correlation with disease outcome. Some studies observe higher levels of IL-17 in patients who effectively control the infection compared with patients who experience severe disease or death (Almansa et al., 2011). Other studies claim the presence of IL-17 is associated with more severe disease. One study of human pandemic H1N1 infection found, like TNF-α and IFN-γ, IL-17 expression was increased in patients with severe disease but within all hospitalized patients the presence of IL-17 was associated with higher survival (Bermejo-Martin et al., 2009). The overlap in expression characteristics of TNF-α, IFN-γ, and IL-17 in patients with severe influenza infection is an interesting observation. More studies should investigate potential redundancies in their responses and attempt to differentiate which are truly essential for the observed protection in severe disease.

IL-22 is generally studied for its protective effects during recovery from influenza infection. IL-22^−/− mice recover slower and have increased lung damage and lymphocyte infiltration in the lungs and airways (Pociask et al., 2013). While Th17 cells produce IL-22 in the lung in response to a variety of stimuli, their contribution in influenza infection is not as established as that of NK cells. One study found murine lung CD27⁻ NK cells produce IL-22 when stimulated with IL-23 but observed no significant changes in weight loss or survival. Following this, another group showed NK cell-derived IL-22 is protective against excessive weight loss by promoting regeneration of tracheal epithelial cells. Although the first NK cell study claimed no significant effects in the absence of IL-22 and the second saw the opposite, both sets of data show a similar trend of increased weight loss in IL-22 depleted mice. The lack of significance could be due to different methods of IL-22 depletion; The first study used an IL-22 blocking antibody whereas the second used IL-22 knockout mice (Guo and Topham, 2010; Kumar et al., 2013). Further investigation in mice revealed that in lethal infections, IL-22 does not have any impact on the outcome, but in sublethal infections it is important for limiting inflammation and initiating tissue repair (Ivanov et al., 2013). It is unclear whether IL-22 production by NK cells and T cells has a physiologically relevant protective role in human influenza infections.

IL-10 can be produced by NK cells and multiple T cell subsets, but the contributions of different IL-10 sources to the host response to influenza is unclear. Some studies show that IL-10 deficient mice have higher influenza survival rates. However, other studies claim deficiencies in IL-10 signaling can increase morbidity and mortality (McKinstry et al., 2009; Sun et al., 2009, 2010). These conflicting results could be due to stimulatory roles of IL-10 during early stages of influenza infection discussed in section Cytokine Production. They reported higher morbidity and mortality in mice with either IL-10 deficiency or overexpression. This suggests a homeostatic level of IL-10 which does not induce too strong of an initial inflammatory response—but is still strong enough to prevent excessive tissue damage —is ideal for host protection in influenza infection (Dutta et al., 2015). In humans, high levels of IL-10 have been associated with severe disease, however this is often attributed to IL-10 mediated immune suppression impairing the ability of the patient to control the infection (Yu et al., 2011). A potential role for IL-10 in harmful hyperinflammatory responses has not yet been studied in association with severe disease in humans. It is also unclear whether excessive IL-10 production is a byproduct of highly pathogenic infections or if increased IL-10 directly contributes to severe disease progression in humans. Surely, more work must be done to understand the role of IL-10 in lung homeostasis during murine and human influenza infections.

One study described IL-27 as an important regulator of previously described aspects of the T cell response to influenza. IL-27R^−/− mice had significantly worse outcomes in terms of viral load, lung pathology and survival; they concluded that IL-27 protects mice from severe pathology by inhibiting excessive CD4⁺ T cell IFN-γ, IL-17, and TNF-α production. They treated WT mice with recombinant IL-27 (rIL-27) following lethal dose influenza infection and found lower IL-17 levels and higher IL-10 levels correlated with increased protection without compromising viral clearance. Interestingly, they only saw these effects when they treated with rIL-27 at peak viral titer or later, compared with worse outcomes if they treated at early stages of infection. Likely, the early inflammatory response is necessary for favorable outcomes, but as adaptive immune cells accumulate in the lung increased inflammation mediates harmful tissue damage (Liu et al., 2014). This study ties together information discussed about positive and negative aspects of the T cell response and suggests that many of these functions are regulated by IL-27. Complementary studies in mice and humans are needed to fully understand the impact of IL-27 on the host response to influenza infection.

While studies in mice are limited in their physiological relevance to human infections, human studies also present challenges in drawing conclusions. Mechanistic studies of the human lung are logistically difficult and rare, but analysis of PBMCs of infected patients does not always represent the site of infection. Therefore, human host responses cannot be adequately compared to mice. Many human studies of hospitalized patients are biased toward young children or older adults. Although these populations represent the most accessible cases, they may not represent influenza responses from all demographics. Similarly, many studies focus on severe viral strains such as pandemic H1N1 or avian influenza infections rather than analyzing immune responses to more mild seasonal influenza cases. While our understanding of why some cases of influenza develop into severe life-threatening diseases has progressed, there is much to learn about individual immune responses and how they can be manipulated to affect patient outcomes.

Non-human primates (NHPs) are an alternative model which address some challenges associated with mouse and human studies. They have been successfully infected with numerous influenza strains including 1918 and 2009 pandemic H1N1 (Kobasa et al., 2007; Safronetz et al., 2011), H5N1 (Baskin et al., 2009), and H9N2 (Zhang et al., 2013). NHP immune responses and gene expression patterns following influenza infection closely mirror those of humans (Baskin et al., 2004). Therefore, they will be an essential tool to expand our knowledge of the mechanisms through which NK cells and T cells contribute to lung pathology in human-like infections.