key: cord-1028651-966ldi6o
authors: Goodier, Martin R; Riley, Eleanor M
title: Regulation of the human NK cell compartment by pathogens and vaccines
date: 2021-01-18
journal: Clin Transl Immunology
DOI: 10.1002/cti2.1244
sha: b4277ac485ab2ae3ace825824b469b043bc4dc8a
doc_id: 1028651
cord_uid: 966ldi6o

Natural killer cells constitute a phenotypically diverse population of innate lymphoid cells with a broad functional spectrum. Classically defined as cytotoxic lymphocytes with the capacity to eliminate cells lacking self‐MHC or expressing markers of stress or neoplastic transformation, critical roles for NK cells in immunity to infection in the regulation of immune responses and as vaccine‐induced effector cells have also emerged. A crucial feature of NK cell biology is their capacity to integrate signals from pathogen‐, tumor‐ or stress‐induced innate pathways and from antigen‐specific immune responses. The extent to which innate and acquired immune mediators influence NK cell effector function is influenced by the maturation and differentiation state of the NK cell compartment; moreover, NK cell differentiation is driven in part by exposure to infection. Pathogens can thus mould the NK cell response to maximise their own success and/or minimise the damage they cause. Here, we review recent evidence that pathogen‐ and vaccine‐derived signals influence the differentiation, adaptation and subsequent effector function of human NK cells.

NK cells differentiate from haematopoietic bone marrow precursors under the influence of innate cytokines and stromal cell-derived factors and, in humans, are defined by the expression of CD56, whilst lacking CD3epsilon. It has been estimated that there are more than 100 000 distinct NK cell phenotypes in human peripheral blood alone 1 ; lymphoid and tissue-resident and migratory NK cells add to this diversity. 2 The distribution of these different NK cell subsets in the circulation, their tissue residence or their capacity to home to different tissues undoubtedly influences their capacity to respond to damaged, cancerous or infected cells.

Initial observations pointed to a potential differentiation pathway for peripheral blood NK cells in which cells expressing high levels of CD56 were considered as less differentiated precursors of cells with lower CD56 expression. 3 Several important pieces of evidence support this broad phenotypic characterisation including the higher intrinsic proliferative capacity and greater telomere length of CD56 bright NK cells compared to CD56 dim NK cells. [4] [5] [6] However, any relationship between CD56 bright and CD56 dim NK cells is unlikely to be direct and the exact relationship remains uncertain. A recent single-cell transcriptomic analysis indicated the presence of eight major differentiation phenotypes of peripheral blood of NK cells; some of these have the potential to interconvert in response to cytokine signals whilst others may represent more terminally differentiated lineages. 7 More recently, cord blood ILC-1-like precursors were shown to differentiate into CD56 dim KIR + NKG2A À Peforin + GzB + NK cells on OP9 stromal cells in the presence of interleukin (IL)-2, IL-7 and IL-15. 8 However, NK cells derived under these conditions contained relatively low frequencies of CD16 + NK cells. 8 CD56 bright NK cells express high levels of IL-12R, IL-15R and IL-18R enabling them to respond very efficiently to these cytokines. CD56 dim NK cells express lower levels of these receptors, instead expressing markers associated with more advanced differentiation (CD57), functional education in the context of HLA-ligands [Killer cell immunoglobulinlike receptors (KIR)], antibody-dependent activation (CD16 and CD32) and cytotoxicity (granzyme B and perforin). 9 However, these different differentiation states do not necessarily correlate with a cell's ability to integrate innate or adaptive signals per se. A subset of CD56 bright NK cells, for example, expresses the high-affinity IL-2 receptor heterodimer (CD25/CD122), responds to picogram concentrations of IL-2 and interacts with CD4 + T cells in secondary lymphoid tissues, suggesting that these cells amplify the responses of antigen-specific memory T cells. 10 However, CD56 dim NK cells that express high levels of FccRIII (CD16) could be viewed as adapted for integration of antibody-dependent signals but also acquire the high-affinity IL-2R on activation. 11 Furthermore, detailed examination of transcription factor and transmembrane signalling adaptor expression of human blood NK cells reveals a polarisation of CD56 dim NK cell functional phenotypes from 'canonical' cells (expressing the proteomyelocytic zinc finger (PLZF) molecule) to 'adaptive' NK cells (lacking the expression of this transcription factor). 12, 13 Reduced expression of PLZF is associated with loss of FceR1c and associated signalling components including Syk and EAT-2 and alternative signalling via the CD3zeta chain. 12, 13 Paradoxically, in vitro cross-linking of NKG2C combined with long-term IL-15 stimulation not only induces expansion of highly differentiated 'adaptive' CD56 dim CD57 + NKG2C + NK cells but also promotes their co-expression of NKG2A and the checkpoint inhibitors PD1 and LAG3 and induces trans-differentiation from CD45RA to CD45RO isoform expression. 14 Hence, chronic stimulation may promote further differentiation of NK cells. Interestingly, CD56 bright NK cells and a subset of CD56 dim NK cells also lack PLZF and FceR1c suggesting that there is significant plasticity in signalling pathways across distinct NK cell differentiation stages. 13 In many ways, this plasticity of NK cell differentiation and adaptation should not be surprising considering the role of epigenetic changes in the diversification process. 'Adaptive' NK cells undergo gene silencing via methylation of loci associated with expression and responsiveness to innate cytokines whereas demethylation promotes a lower threshold for activation via alternative pathways including at the IFN-c locus. 13 

The spectrum of NK cell differentiation varies between individuals reflecting, in part, their prior infection history. 7 Pathogens express immuneactivating molecules (pathogen-associated molecular patterns) or induce tissue damageassociated 'danger' signals that contribute to NK cell activation either directly or via cytokines produced by intermediary 'accessory' cells such as monocytes and macrophages. The phenotypic and functional repertoire of NK cells responding to a distinct pathogen, and the magnitude of that response, will therefore vary according to the strength and duration of these pathogen-derived signals.

A role for infection in driving the differentiation and functional adaptation of human NK cells is particularly well documented for human cytomegalovirus (HCMV). Expansions of NK cells expressing CD57 and NKG2C (a receptor recognising cognate HLA-E stabilised on HCMV-infected cells by peptides from the UL40 viral protein) and lacking PLZF and FceR1c are frequently found in bone marrow transplant patients experiencing HCMV infection or reactivation and are elevated in frequency in HCMV seropositive compared to seronegative individuals. 12 However, expansions of NK cells lacking FceR1c, EAT-2 and Syk1 but with intermediate levels of CD57 expression are also observed in HCMV seronegative individuals, highlighting the potential for peripheral NK cell differentiation to be driven by other mechanisms. 15 The potential for a non-linear, HCMV-independent pathway for functional NK cell diversification is further supported by the observation of dynamic, intra-individual fluctuations in the frequencies of these cells over a protracted period of time in HCMV seronegative individuals. 15 HCMV-infected myeloid cells and fibroblasts are in many ways the archetypic example of pathogen-induced NK cell activation and differentiation, both directly via binding of HCMV-induced ligands on infected host cells to NK cell surface receptors including NKG2C, LILRB1 and activating KIR and indirectly through the action of pro-inflammatory cytokines, interleukin-2 and antibodies emanating from other immune cells. 16, 17 Human 'adaptive' NK cells (defined by the expression of NKG2C or loss of PLZF/Fcer1c associated pathways and induced by cognate interaction between NKG2C and HLA-E on HCMVinfected cells) share many features of murine 'memory' NK cells induced by binding of the murine cytomegalovirus (MCMV) m157 protein to NK Ly49h receptors. 18 In both cases, NK cell expansion and differentiation are supported by accessory cell secretion of IL-12 and leads to generation of effector cells with superior killing activity and control of CMV infection. 18, 19 Additionally, 'cytokine-induced-memory-like' (CIML) NK cells can be generated in vitro in both humans and mice with combinations of accessory cytokines (IL-12, IL-18 and IL-15); these cells respond to subsequent in vitro stimulation by enhanced cytokine secretion, and this enhanced activity is retained after adoptive transfer. 20, 21 CIML NK cells are distinguishable from human adaptive NK cells and murine memory NK cells in their lack of specificity for individual pathogens and their independence from cognate CMV-NKG2C/Ly49h interactions (reviewed in Pahl et al. 22 ).

In the 'real world' of complex individual infection histories, these categorisations may be less clear with adaptive/memory cells acquiring additional CIML-like properties after in vivo exposure to acute infection, inflammation or vaccination. CIML NK cells are likely to be further expanded and differentiated by subsequent CMV infection. Nevertheless, the dominant role of HCMV in promoting the differentiation and functional adaptation of human NK cells (especially after infection in early life) has the potential to directly influence responses to third party infections. HCMV-associated 'adaptive' NK cells in humans have enhanced capacity for antibody-mediated activation and killing of infected target cells (antibody-dependent cellular cytotoxicity, ADCC) 12,13,18 although their longterm fate during HCMV infection or reactivation may ultimately depend on the presence or absence of co-stimulatory signals.

Endemicity of HCMV infection is greatly influenced by environment, for which geographical location and ethnicity have been used as proxies. Generally speaking, HCMV seroprevalence increases with equatorial proximity, with the highest prevalence being reported in sub-Saharan Africa. 23 Rates of HCMV infection in infants and children are also highest in these settings with between 83% and 98% of individuals becoming infected by the age of 14 years. 23 In a UK population, higher seroprevalence of HCMV (and, incidentally, herpes simplex virus, HSV) was observed in children of Asian heritage than amongst children of white British heritage raising the possibility that immune differentiation, and thus disease susceptibility or presentation, may vary amongst communities of differing ethnicity within the same geographical location. 24 NK cell differentiation occurs noticeably more rapidly (i.e. in younger age groups) in settings of high HCMV endemicity with, for example, maximal expansion of CD57 + NKG2C + and CD57 + NKG2A + NK cells being reached by 10 years of age in an African population with near universal HCMV infection in the first year of life. 25 This is accompanied by loss of NK cell responsiveness to innate cytokines but maintenance of robust antibody-dependent activation 25 and emergence of high frequencies of 'adaptive' FceR1c À CD56 dim NK cells in childhood and early adulthood. 26, 27 Similar effects are observed in temperate areas of the northern hemisphere, but over a more protracted period, with advanced NK cell differentiation mostly occurring later in life in these lower HCMV seroprevalence settings. [28] [29] [30] 

setting the stage for subsequent responses, other pathogens also influence the expansion and differentiation of particular NK cell subsets. In experimental settings, several examples have emerged of direct activation of NK cells by binding of pathogen-encoded ligands to either invariant or polymorphic NK cell receptors. One recent, well-characterised example is that of a conserved peptide epitope from bacterial recombinase A (rec A) that is presented by HLA-C*0501 and recognised by the human NK cell receptor KIR2DS4 enabling NK cell activation by diverse bacteria including Brucella, Campylobacter, Chlamydia and Helicobacter species. 31 Similarly, a highly conserved epitope in the helicase of flaviviruses is presented by HLA-C*0102 and binds to KIR2DS2, inducing NK cell degranulation and cytotoxicity 32 and several HIVderived peptides have been identified that either provoke or inhibit NK cell responses in the context of KIR-HLA interactions. 33, 34 By contrast, the fungal pathogen Aspergillus fumigatus is reported to bind directly to NK cell CD56, resulting in NK cell activation, beta chemokine production and a reduction in CD56 expression. 35 Whilst A. fumigatus appears to interact with both CD56 bright and CD56 dim NK cell subsets, fungal activation reportedly affects expression of other receptors which may be unevenly distributed across the NK cell differentiation spectrum, including NKG2D and NKp46. 36 Furthermore, A. fumigatus-derived signals synergise with indirect, dendritic cell-derived signals to activate, and prevent exhaustion of, less differentiated NK cell subsets. 37 Direct interaction of influenza A virus haemagglutinin with NKp46, NTB-A and 2B4 has also been implicated in the direct activation of NK cells. 38 Certain viruses have the capacity to directly infect human NK cells, with evidence of tropism for and impact on NK cell differentiation phenotype. Varicella-Zoster virus (VZV), for example, targets CD56 dim NK cells, resulting in acquisition of CD57 but, paradoxically, reducing surface CD16 expression despite the absence of VZV-specific antibodies in the culture system. 39 Less differentiated NK cells can be infected during chronic active Epstein-Barr virus infection 40 

and Akt activation with consequences for viral latency. 40 Evidence is also accumulating of indirect changes in NK cell phenotype and function in people actively infected with a variety of, mostly viral, infections (summarised in Table 1 ); potential points of interaction between distinct pathogens and different NK cell differentiation subsets are summarised in Figure 1 . In many cases, these infections seem to augment the effects of underlying HCMV infection, although data on HCMV infection are lacking in some studies (Table 1) . For example, many of the effects of chronic HIV-1 infection on NK cells are indistinguishable from those associated with recurrent infection/reactivation of HCMV including loss of CD56 bright cells and emergence of CD57 + NKG2C + FceR1c À cells. [41] [42] [43] Indeed, reversal of NKG2A/NKG2C frequencies over a 24-month period of antiretroviral therapy likely reflects enhanced immune control of HCMV infection. 44 Accelerated acquisition of highly differentiated, 'adaptive' NK cell phenotypes is observed in HIV-1-infected individuals in Europe but these effects are less marked in HIV-1 + Africans amongst whom NK cell differentiation is typically already well advanced due to HCMV infection early in life. 45 However, persistent untreated HIV-1 infection further extends NK cell differentiation with accumulation of CD56 À NKG2C + cells. 46 Proteomic analysis suggests that these cells emerge from a CD56 dim precursor population 47 but it as yet unclear whether their differentiation is driven by HIV-1 infection itself (with or without HCMV coinfection), whether opportunistic infections (including with fungal pathogens such as A. fumigatus) also drive this NK cell differentiation, 35 or whether the activating signals are direct (pathogen ligands binding to NK cell receptors) or indirect (e.g. mediated by inflammatory cytokines).

Influenza infection is associated with reduced frequencies of CD56 bright NK cells, coincident with NK cell production of antiviral and inflammatory cytokines, 48 as well as upregulation of activation/functional markers (CD69, CD38 and granzyme B) and a tendency for increased proliferation of both CD56 bright and CD56 dim CD16 + NK cells. 49 Crucially, a small population of CD49a + CD16 À CXCR3 + NK cells with potential lung homing capacity has been identified during acute influenza infection and equivalent cells isolated from lung tissue have been shown to have antiviral activity in vitro. 49 Importantly, many of these influenza-induced phenotypic changes are short-lived, with reversal to pre-infection characteristics during convalescence, compatible with redistribution and regulation of the NK cell response. 49 The importance of infection in promoting activation of relatively undifferentiated, tissueresident NK cells and migration to distinct anatomical sites is also evident in recall responses to VZV and hepatitis B viruses. 50,51 NK cells with characteristics of liver-resident NK cells (CD56 high CXCR6 + CD16 + NKG2D + CD69 + CD62L + ) were enriched in skin blisters after challenge with VZV skin test antigen, 50 and CD49a + CD16 À human liver NK cells acquire epigenetic modifications and migrate to the skin after hepatitis B vaccination. 51 Hantavirus infection leads to rapid and persistent in vivo expansion of NK cells across the differentiation spectrum including CD57 + NKG2C + NK cells. 52 Such expansions are associated with elevated levels of IL-15 and with the Hantavirusdriven upregulation of HLA-E on infected cells. Furthermore, over 80% of infected individuals were HCMV seropositive in the study, consistent with the possibility that Hantavirus further expands cells initially expanded by HCMV infection. 52 In this context, the activation of more differentiated NK cells is supported by hantavirusinduced upregulation of IL-15-IL-15R on epithelial cells. 53 Acute dengue virus infection is associated with robust proliferation of all NK cell subsets, irrespective of their differentiation status, although CD56 bright and CD56 dim CD57 À NK cells dominate the response; this is associated with increased IL-18 concentrations in plasma (and in experimentally induced skin blister fluid) and activation of IL-18-induced signalling pathways. 54 In this case, the cells preferentially homing to the skin were CD56 bright and expressed a unique combination of chemokine receptors, including CLA-1 which is typically associated with skin homing. 54 Acute hepatitis C virus (HCV) infection leads to increased blood frequencies of CD56 bright cells, and correspondingly decreased frequencies of CD56 dim NK cells, although both subsets of NK cells appear activated. 55 However, NK cell frequencies subsequently normalised in patients who went on to clear their infections and in those who developed chronic infection and no long-term impacts on NK cells were reported. 55 Similarly, another study reported increased frequencies of CD56 À NK cells and concomitantly reduced frequencies of CD56 dim , NKG2D + , NKp30 + and NKp46 + NK cells during HCV infections. 56 Importantly, frequencies of NKG2A + and CD94 + NK cells were increased in acute and chronic infection but not in those whose infections resolved, whereas frequencies of NKp30 + , NKp46 + and NKG2D + cells were lower in those who resolved their infections than in those who became chronically infected. 56 Consistent with these observations, a recent study of HCMV seropositive patients reported that high frequencies of adaptive CD57 + FceR1c À NK cells and elevated expression of PD-1 were associated with high viral load in chronic HCV infection and were reduced after direct-acting antiviral therapy (DAA). 57 In a separate study, chronic HCV infection was similarly associated with higher frequencies of CD57 + and PD-1 + NK cells compared to uninfected control individuals. 58 However, although PD1 expression frequency reduced after DAA, proportions of CD57 + and KLRG1 + cells increased after treatment, suggesting ongoing expansion of highly differentiated NK cells. 58 The slightly different NK cell outcomes in these two studies may be explained by differences in HCMV status, HCV viral genotype, viral load and treatment regimen. 57,58

Human malaria infections provide insights into the impacts of chronic and repeated infection on the NK cell compartment. Blood stage malaria parasites can induce high concentrations of inflammatory and NK cell-activating cytokines with associated NK cell activation seen in animal models and human co-culture systems in vitro. 59 Experimental, controlled human malaria infections (CHMI) have given valuable insights into the activation of NK cells during primary infection in malaria-na€ ıve individuals. Surprisingly, despite the known role for inflammatory cytokines (IL-12, IL-18) in activating less differentiated NK cells, the primary acute response during CHMI is characterised by activation (CD69 expression) of NKp30 + NK cells distributed across the differentiation spectrum but with CD56 dim cells dominating over CD56 bright cells 60 ; interestingly, these expansions were closely linked to the course of parasitaemia and resolved after treatment. 60 It is likely that both expansion and homing of activated CD56 bright NK cells toand their retention insecondary lymphoid tissues contribute to redistribution of NK cell subsets during active malaria infection.

The potential for chronic or repeated malaria infections to influence NK cell phenotype and function is illustrated by the identification of a novel, activated CD38 + HLA À DR + CD45RO + NK cell population in individuals with sickle cell trait who have persistent low-density parasitaemia. 61 A possible explanation for this is that chronic in vitro stimulation of NK cells via NKG2C in combination with IL-15 induces CD45 isoform switching on CD57 + NKG2C + cells, resulting in a CD45RO + population with high proliferative potential, characteristic of central memory T cells. 14 Increased expression of checkpoint inhibitors including TIM3 and PD-1 has also been observed after repeated malaria exposure with increasing age in endemic populations, 62 again with parallels to the emergence of LAG3 and PD1 + adaptive NK cells after activating receptor (NKp30, NKG2D, NKG2C) or HCMV-mediated activation in vitro. 14 Interestingly, a CD25 + CD45RO + NK cell population has been identified in patients with metastatic melanoma treated with anti-PD-1 62,63 further supporting a potential for progressive differentiation of NK cells in a manner analogous to that seen in T cells. There is also evidence that persistent Mycobacterium tuberculosis infections, and the associated chronic inflammatory response, lead to the emergence of CD45RO + NK cells and enrichment of these cells in the pleural effusion fluid of pulmonary tuberculosis patients. 64 These cells are potent producers of both IL-22 and IFN-c that are implicated, respectively, in tissue repair and inflammation. 65 Whether these NK cell expansions rely on prior infection or co-infection with other pathogens is unknown although there is some evidence that the course of tuberculosis can be modulated by HCMV co-infection. 66, 67 EMERGING PATHOGENS AND NK CELLS As described above, an abundance of experimental and observational studies suggests a role for infections, especially repeated or persistent parasite or viral infections, in gradual and extensive NK cell differentiation. Furthermore, studies of emerging pathogens are providing additional insights into the relative impacts of innate and adaptive immune pathways in inducing NK cell differentiation. Notably, severe systemic inflammation is a hallmark of severe Ebola and SARS-CoV-2 (COVID-19) disease; this inflammation may in turn induce expansion and differentiation of NK cells with potential long-term consequences.

Ebola virus is an example of a novel pathogen with only very limited adaptation to a human host. Ebola virus-induced overproduction of the NK cell-activating inflammatory cytokines IFN-a2 and IL-18, insufficiently counterbalanced by antiinflammatory cytokines (IL-10), is associated with poor outcomes in Ebola virus disease (EVD). 68 During EVD, there is a rapid reduction in the frequency of CD56 bright NK cells, within days of infection, and a subsequent increase in the frequencies of proliferating CD56 bright and CD56 dim cells. 69 In vitro, Ebola virus glycoprotein directly induces the secretion of both proinflammatory cytokines (including IL-18 which contributes significantly to NK cell activation, degranulation and IFN-c production) and antiinflammatory IL-10 (which restricts these responses). 70 In this scenario, NK cell responses are dominated by CD56 bright and CD56 dim CD57 À cells, raising the possibility that these less differentiated, cytokine-producing cells may act to further amplify the inflammatory cascade (and thus disease severity) in vivo. Interestingly, increases in the frequency of CD56 À CD16 + NK cells are observed up to 2 years after recovery from severe EVD, suggestive of persistent activation and/or terminal differentiation from CD56 dim CD16 + NK cells. 47, 71 However, in experimental studies in mice, adoptive transfer of dendritic cells exposed to virus-like particles expressing the Ebola virus glycoprotein can prime NK cells to protect against subsequent lethal Ebola virus infection, raising the possibility that NK cell activation by direct binding of a viral ligand together with cytokine-mediated costimulation could induce protective NK cell effector mechanisms. 72 Indeed, human NK cells expressing NKp30 have been implicated in the cytotoxic response to Ebola virus after interaction with infected dendritic cells. 73 More recently, NK cells have been implicated in both protection against and pathogenesis of SARS-CoV-2 infection (COVID -19) . As in other viral infections, and irrespective of outcome in terms of disease, innate inflammatory pathways are triggered early in SARS-CoV-2 infection. expression of perforin and granzyme B is associated with inflammatory markers of disease severity, in particular with high concentrations of IL-6. 74 In addition, reduced expression of CD16 on CD56 dim NK cells in people with COVID-19 75 is consistent with ongoing activation of this subset by SARS-CoV-2 antigen/antibody immune complexes. 76 Importantly, HCMV infection and the associated expansion of the subset of adaptive CD56 dim NKG2C + NK cells were associated with severe COVID-19. Over 90% of severe COVID-19 cases in this cohort were HCMV + (compared with 60% of controls and 80% of mild COVID-19 cases). 74 More importantly,~65% of people with severe COVID-19 had expansion of adaptive NK cells compared with only 10-20% of controls and those with moderate disease. 74 Whether these expansions of adaptive NK cells in HCMV + people were driven by SARS-CoV-2, or whether they predated SARS-CoV-2 infection, is not known but these data do raise the possibility that adaptive NK cells may contribute to tissue damage in COVID-19. In support of this hypothesis, in COVID-19 patients these adaptive cells also express Ksp37, a marker of cytotoxic lymphocytes associated with lung disease, 77 and, despite their lower intrinsic proliferative capacity, also express markers of recent proliferation. 74 This is reminiscent of the increased proliferative capacity of adaptive CD45RO + NK cells in chronic malaria infection as discussed above 61, 74 and is also consistent with reports of enrichment of highly differentiated CD56 dim CD57 + NK cells with high proliferative capacity in the peripheral blood in a patient with SARS-CoV-2 infection and acute respiratory distress syndrome (ARDS). 78 A cluster of genes enriched in terminally differentiated CD8 + T cells and CD56 dim CD57 + NK cells was downregulated in the blood of children with multisystem inflammatory syndrome after SARS-CoV-2 infection 79 suggesting that there is no straightforward relationship between NK cell subset activation and disease severity. However, a causal relationship between activation of highly differentiated NK cells and severe COVID-19 is, as yet, unproven and homing of less differentiated CD56 dim NK cells to the tissues during severe disease may also contribute to alterations in cell frequencies in peripheral blood.

The expansion of CD56 dim NKG2C + NK cells in COVID-19 patients also raises the possibility that increased expression of HLA-E in conjunction with inflammatory mediators may mediate this adaptive NK cell expansion. 74 Indeed, a recent study suggested that a peptide derived from the SARS-CoV-2 spike 1 protein can stabilise HLA-E on lung epithelial cells leading to activation of NKG2C + NK cells. 80 Interestingly, an in silico study also identified SARS-CoV-2 peptides predicted to bind HLA-C alleles recognising activating KIR. 81 

An indirect role for NK cells in regulating virusor vaccine-induced responses has been highlighted in several experimental systems. For example, NK cells have been shown to regulate CD4 + T-cell responses and follicular T helper cell (Tfh) activity, thereby influencing the breadth and depth of the antibody response and effector CD8 + T-cell response to vaccinia virus and LCMV infections in murine models. [82] [83] [84] Moreover, adaptive/highly differentiated NK cells, which express higher levels of the endosomal effector protein RAB111FIP5, impact on the generation of broadly neutralising antibodies in HIV-1-infected individuals. 85 However, mounting evidence also suggests that NK cell differentiation is also affected by vaccination (summarised in Table 2 ). Less differentiated CD56 bright and CD56 dim CD57 À NK cells are activated after influenza virus vaccination 48, 86, 87 with evidence suggesting that common c chain cytokines (IL-2, IL-12 and IL-15) prime myeloid dendritic cells to secrete IL-12 to support NK cell responses. 88, 89 Post-vaccination increases in the frequencies of these less differentiated CD56 dim CD57 À NK cells in HCMV + individuals are consistent with IL-2 family cytokines also supporting maintenance and expansion of less differentiated, cytokineresponsive subsets. However, more differentiated CD56 dim CD57 + cells are also enhanced postvaccination in the presence of influenza-specific antibody rather than relying on cytokines for their activation. 76 In line with this, increased frequencies of CD56 dim CD16 + NKG2C + (CD57 À and CD57 + ) NK cells are observed up to 14 days after seasonal influenza vaccination in individuals with high titres of haemagglutination inhibiting antibodies. 90 A recent study demonstrated increased cytotoxic and proliferative responses to antigen-pulsed monocyte-derived dendritic cells by highly differentiated CD56 dim CD57 + KLRG1 + NK cells after hepatitis B subunit vaccination. 91 Interestingly, these responses did not require the presence of immune serum. Yellow fever vaccine 17D induces proliferation and expansion of less differentiated CD56 bright and CD56 dim CD57 À NK cells; these cells also demonstrate enhanced responsiveness to in vitro restimulation with innate cytokines. 92 Similarly, increased absolute numbers of CD56 bright and CD56 dim NK cells are observed within 3 days of immunisation with the rVSV-ZEBOV Ebola virus vaccine; increased NK cell expression of CXCR6 is an independent correlate of rVSV-ZEBOV vaccine responsiveness whilst reduced frequencies of NKG2D + and increased frequencies of NKp30 and KIR + NK cells are inversely correlated with plasma cytokine and chemokine concentrations, indicating likely recirculation of NK cell subsets within 72 h of vaccination. 93 The Ad26.ZEBOV Ebola virus vaccine also activates CD56 bright NK cells as assessed by the induction of CD25 and Ki67 expression up to 14 days after the second dose. 70 The extent to which different vaccine vectors, delivery platforms and adjuvating systems impact on NK cell differentiation will likely depend on their primary cellular tropism, interactions with pattern recognition receptors and downstream production of NK cell-activating cytokines; this aspect of vaccination has as yet received very little attention.

There are also many examples of potent, Fc receptor-mediated degranulation and cytokine production by more highly differentiated NK cells in response to vaccination-induced antibody. 76, [94] [95] [96] Moreover, subunit, virally vectored and whole viral vaccines, with or without chemical adjuvants, will provide distinct cytokine signatures for costimulation of NK cell responses with type 1 interferons, IL-15 and IL-18, all described to support antibody-dependent responses of adaptive NK cells. 27, 95, 97, 98 However, it is not known whether these cytokine-and antibody-mediated signals contribute to further differentiation of these particular NK cells.

Exposure to an infection, be it in early life, with a novel pathogen or after vaccination, is accompanied by bystander, accessory-celldependent proliferation and expansion of the 'less differentiated' pool of NK cells. Cumulative exposure to diverse pathogens, exacerbated by chronic or recurrent infection, can lead to progressive activation and expansion of 'more differentiated' NK cells working in tandem with the adaptive immune response. Cytokines from antigen-specific T cells and pathogen-specific antibodies amplify these NK cell responses, thereby co-opting NK cells into the adaptive immune response. Important questions remain, however, as to how pathogens or vaccines influence NK cell differentiation and functional diversification and what this means for the ability of NK cells to then contribute to protection from, or susceptibility to, both infectious and non-communicable diseases. Amongst these are the potential for maternal antibodies to protect the infant from infection by non-neutralising mechanisms and to induce NK cell activation and differentiation in early life. Differentiation of CD3 À CD16 + cells from cord blood ILC1-like precursors provides an opportunity for antibody-dependent effector cells to contribute to immune responses, perhaps earlier in life than hitherto appreciated. The extent to which NK cell subsets re-equilibrate after a pathogen is controlled or eliminated, and the mechanisms whereby uncontrolled infections (e.g. Hantavirus, severe SARS-CoV-2) or chronic or repeated infections (e.g. hepatitis C and malaria) induce lasting effects on the NK cell repertoire, are other key issues that need further investigation ( Figure 1 ). The role, and the longer-term functional consequences, of IL-15 in the expansion and terminal differentiation of adaptive NK cells also merits further investigation in the context of different pathogens. In summary, individual experiences of infection and vaccination over the life course will shape not only the adaptive immune cell population but also the NK cell population, with consequences for susceptibility to subsequent infections and, potentially, noncommunicable disease such as cancers as inflammatory conditions.

Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry

Tissue determinants of human NK cell development, function, and residence

The biology of human natural killer-cell subsets

CD56 bright human NK cells differentiate into CD56 dim cells: role of contact with peripheral fibroblasts

Telomere length in human natural killer cell subsets

CD56 bright CD16 -killer Ig-like receptor-NK cells display longer telomeres and acquire features of CD56 dim NK cells upon activation

Diversity of peripheral blood human NK cells identified by singlecell RNA sequencing

Umbilical cord blood-derived ILC1-like cells constitute a novel precursor for mature KIR + NKG2A -NK cells

The broad spectrum of human natural killer cell diversity

CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2: a potential new link between adaptive and innate immunity

Differential activation of CD57-defined natural killer cell subsets during recall responses to vaccine antigens

Epigenetic modification and antibody-dependent expansion of memory-like NK cells in human cytomegalovirus-infected individuals

Cytomegalovirus infection drives adaptive epigenetic diversification of NK cells with altered signaling and effector function

Chronic stimulation drives human NK cell dysfunction and epigenetic reprograming

Dynamic changes in natural killer cell subset frequencies in the absence of cytomegalovirus infection

CMV and natural killer cells: shaping the response to vaccination

Modulation of human leukocyte antigen-C by human cytomegalovirus stimulates KIR2DS1 recognition by natural killer cells

Cytomegalovirus generates longlived antigen-specific NK cells with diminished bystander activation to heterologous infection

IL-12-producing monocytes and HLA-E control HCMV-driven NKG2C + NK cell expansion

Cytokine-induced memory-like natural killer cells

Cytokine activation induces human memory-like NK cells

Memory-like NK cells: remembering a previous activation by cytokines and NK cell receptors

Human cytomegalovirus (CMV) in Africa: a neglected but important pathogen

Epstein-Barr virus and varicella zoster virus infection in the first two years of life: a cohort study in Bradford

Rapid NK cell differentiation in a population with near-universal human cytomegalovirus infection is attenuated by NKG2C deletions

Adaptive NK cells in people exposed to Plasmodium falciparum correlate with protection from malaria

Differential IL-18 dependence of canonical and adaptive NK cells for antibody dependent responses to P. falciparum

CD62L expression identifies a unique subset of polyfunctional CD56 dim NK cells

Expression patterns of NKG2A, KIR, and CD57 define a process of CD56 dim NK-cell differentiation uncoupled from NK-cell education

Impaired NK cell responses to pertussis and H1N1 influenza vaccine antigens in human cytomegalovirus-infected individuals

Human NK cell receptor KIR2DS4 detects a conserved bacterial epitope presented by HLA-C

KIR2DS2 recognizes conserved peptides derived from viral helicases in the context of HLA-C

Sequence variations in HIV-1 p24 Gag-derived epitopes can alter binding of KIR2DL2 to HLA-C*03:04 and modulate primary natural killer cell function

HIV-1 induced changes in HLA-C*03: 04-presented peptide repertoires lead to reduced engagement of inhibitory natural killer cell receptors

CD56 is a pathogen recognition receptor on human natural killer cells

Human NK cells develop an exhaustion phenotype during polar degranulation at the Aspergillus fumigatus hyphal synapse

First insights in NK-DC cross-talk and the importance of soluble factors during infection with Aspergillus fumigatus

The human 2B4 and NTB-A receptors bind the influenza viral hemagglutinin and co-stimulate NK cell cytotoxicity

Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

Patients with natural killer (NK) cell chronic active Epstein-Barr virus have immature NK cells and hyperactivation of PI3K/ Akt/mTOR and STAT1 pathways

Switch from inhibitory to activating NKG2 receptor expression in HIV-1 infection: lack of reversion with highly active antiretroviral therapy

Human cytomegalovirus infection is associated with increased proportions of NK cells that express the CD94/NKG2C receptor in aviremic HIV-1-positive patients

Adaptive reconfiguration of natural killer cells in HIV-1 infection

Chronic HIV-1 viremia reverses NKG2A/NKG2C ratio on natural killer cells in patients with human cytomegalovirus coinfection

Subordinate effect of -21M HLA-B dimorphism on NK cell repertoire diversity and function in HIV-1 infected individuals of African origin

Short communication: NKG2C + NK cells contribute to increases in CD16 + CD56 -cells in HIV type 1+ individuals with high plasma viral load

Proteome analysis of human CD56 -NK cells reveals a homogeneous phenotype surprisingly similar to CD56 dim NK cells

Changes in cytokine levels and NK cell activation associated with influenza

Influenza A virus infection induces hyperresponsiveness in human lung tissue-resident and peripheral blood NK cells

Human natural killer cells mediate adaptive immunity to viral antigens

A discrete subset of epigenetically primed human NK cells mediates antigen-specific immune responses

Rapid expansion and long-term persistence of elevated NK cell numbers in humans infected with hantavirus

NK cell activation in human hantavirus infection explained by virus-induced IL-15/IL15Ra expression

NK cells are activated and primed for skin-homing during acute dengue virus infection in humans

Activation of natural killer cells during acute infection with hepatitis C virus

Reduced frequencies of NKp30 + NKp46 + , CD161 + , and NKG2D + NK cells in acute HCV infection may predict viral clearance

Adaptive natural killer cell functional recovery in hepatitis C virus cured patients

The Authors

Hepatitis C virus eradication with interferon free, DAA-based therapy results in KLRG1 + , hepatitis C virus-specific memory natural killer cells

Differentiation and adaptation of natural killer cells for anti-malarial immunity

Activatory receptor NKp30 predicts NK cell activation during controlled human malaria infection

A novel population of memory-activated natural killer cells associated with low parasitaemia in Plasmodium falciparum-exposed sickle-cell trait children

PD-1 expression on NK cells in malaria-exposed individuals is associated with diminished natural cytotoxicity and enhanced antibody-dependent cellular cytotoxicity

Age-associated changes in the immune system may influence the response to anti-PD1 therapy in metastatic melanoma patients

Human natural killer cells expressing the memory-associated marker CD45RO from tuberculous pleurisy respond more strongly and rapidly than CD45RO -natural killer cells following stimulation with interleukin-12

Memory-like antigen-specific human NK cells from TB pleural fluids produced IL-22 in response to IL-15 or Mycobacterium tuberculosis antigens

Cytomegalovirus infection is a risk factor for tuberculosis disease in infants

Memory of Natural Killer Cells: A New Chance against Mycobacterium tuberculosis?

Immune parameters and outcomes during Ebola virus disease

Immunologic timeline of Ebola virus disease and recovery in humans

Ebola virus glycoprotein stimulates IL-18-dependent natural killer cell responses

Long-lasting severe immune dysfunction in Ebola virus disease survivors

Role of natural killer cells in innate protection against lethal Ebola virus infection

NKp30-dependent cytolysis of filovirus-infected human dendritic cells

Natural killer cell immunotypes related to COVID-19 disease severity

Comprehensive mapping of immune perturbations associated with severe COVID-19

Sustained immune complexmediated reduction in CD16 expression after vaccination regulates NK cell function

Increase in killerspecific secretory protein of 37 kDa in bronchoalveolar lavage fluid of allergen-challenged patients with atopic asthma

Immune alterations in a patient with SARS-CoV-2-related acute respiratory distress syndrome

Cytotoxic lymphocytes are dysregulated in multisystem inflammatory syndrome in children

SARS-CoV-2 spike 1 protein controls natural killer cell activation via the HLA-E/NKG2A pathway

Conserved HLA binding peptides from five non-structural proteins of SARS-CoV-2-An in silico glance

Weak vaccinia virus-induced NK cell regulation of CD4 T cells is associated with reduced NK cell differentiation and cytolytic activity

Generation of cellular immune memory and B-cell immunity is impaired by natural killer cells

Affinity maturation is impaired by natural killer cell suppression of germinal centers

RAB11FIP5 expression and altered natural killer cell function are associated with induction of HIV broadly neutralizing antibody responses

Influenza vaccination generates cytokine-induced memory-like NK cells: impact of human cytomegalovirus infection

Expansion of 2B4 + natural killer (NK) cells and decrease in NKp46 + NK cells in response to influenza

IL-15 Promotes polyfunctional NK cell responses to influenza by boosting IL-12 production

Influenza vaccination primes human myeloid cell cytokine secretion and NK cell function

Responsiveness to influenza vaccination correlates with NKG2C -expression on NK cells

HBV vaccination and HBV infection induces HBV-specific natural killer cell memory

The human NK cell response to yellow fever virus 17D is primarily governed by NK cell differentiation independently of NK cell education

Rapid dose-dependent natural killer (NK) cell modulation and cytokine responses following human rVSV-ZEBOV Ebolavirus vaccination

Antibody-dependent natural killer cell activation after Ebola vaccination

Fc functional antibody responses to adjuvanted versus unadjuvanted seasonal influenza vaccination in community-dwelling older adults

Contribution of NK cell education to both direct and anti-HIV-1 antibodydependent NK cell functions

Influenza virus infection enhances antibody-mediated NK cell functions via type I interferon-dependent pathways

Vaccineinduced antibodies mediate higher antibody-dependent cellular cytotoxicity after interleukin-15 pretreatment of natural killer effector cells

We thank Asia-Sophia Wolf for assistance with preparing the figure. 

The authors declare no conflict of interest. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.