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As mentioned above, NK cells are powerful protectors from virus-infected, tumor, and senescent cells [25, 65–68].

Proportions and numbers of NK cell subpopulations are affected by aging [18, 69–74] (Fig. 1). For example, the overall percentage of NK cells among peripheral blood lymphocytes is increased in healthy aging and centenarians but there is a decrease in the CD56^brightCD16⁻ NK cell subset and an expansion of CD56^dimCD16⁺ NK cells [75]. Considering that CD56^brightCD16⁻ NK cells have a high capacity to produce different cytokines and chemokines in response to cytokines released by other activated immune cells, their decreased proportion may be responsible for the defective overall production of cytokines and chemokines by NK cells stimulated with IL-2 or IL-12 observed in elderly individuals including nonagenarians [76, 77]. On the contrary, old individuals show an increased production of granzyme A and IFN by CD56^bright cells, potentially representing a compensatory mechanism to maintain the protective and immunoregulatory role of these cells in older individuals [70]. The decrease in old individuals of the CD56^bright subset is due to a decreased output of these immature NK cells from the bone marrow to the peripheral blood reflecting possible alterations in hematopoietic stem cells [78]. CD56^dim CD16⁺ NK cell subsets are associated with decreased granzyme A but no change in GrzB and perforin levels sustaining their cytotoxicity either directly or through CD16 which does not change with aging [71]. Therefore, the observed decrease in cytotoxicity at the single cell level is due most probably to the defect of the recognition of the target cells as a consequence of the decreased expression of the NK cell-activating receptors observed in aged individuals [79–81].

Fig. 1. Phenotype and functional changes of NK cells in relation to their maturation, aging, and influence of exposure to viruses (CMV) and vaccination (VZV). Major receptors defining NK subpopulations are shown. More intense color of some receptor symbols indicates higher expression in some subpopulations; aging-associated changes in the levels of expression of surface molecules are not shown. Thick arrows inside the cell symbols and in the boxes describing major functionalities indicate decreased or increased proportions (black) and functionalities (light grey) of specific populations associated with aging and/or CMV infection. Refer to the main text for detailed description.

Furthermore, the effect of aging on the expression (and, likely, function) of NK cell receptors has been recently described [82]. Although there was no alteration in the CD16 expression or function in the total NK cells, expression of the activating natural cytotoxicity receptors NKp30 and NKp46 and of costimulatory molecule DNAX accessory molecule (DNAM)-1 was decreased [17, 18, 71, 79, 83, 84]. Analysis of NK cell inhibitory receptors showed an age-related increase in KIR expression and a decrease in CD94/NKG2A expression, although discrepancies could be found in different studies [17, 73].

Concomitantly, CD57 expression is increased with aging, which highly suggests a shift towards NK cell maturity with aging; however, it is of note that these changes cannot be always distinguished from chronic viral infections especially from that caused by the presence of CMV [85–87]. These CD57⁺ terminally differentiated NK cells have low proliferative capacity and have low responses to cytokines but are highly cytotoxic and produce high levels of IFNγ in response to CD16 ligation but only in CMV-infected old individuals [18]. As a functional consequence, NK cytotoxicity against classic NK cell targets was impaired especially on a single cell level as correlated with the decrease in NCR receptors such as NKp46, but antibody-dependent NKCC was not affected by aging. The expression of these NCRs, especially NKp46, was inversely proportional to the total number of NK cells [18]. The NKp46 receptors may recognize not only the foreign epitopes but also autologous ones, mediating some autoimmune effect such as seen in type 1 diabetes [88]. This regulation seems to be maintained in aging as we witness an increase in the total NK cell number with a decrease in NKp46 expression reducing some autoimmune conditions in aging. In the meantime, these changes may explain the higher incidence of infectious diseases (especially viral infections), malignancies, and atherosclerosis-based cardiovascular diseases [17, 89–92]. The gender differences in old individuals are controversial, with some studies reporting an increased functionality in aged women and a higher maturation state and lower NK cell activity in elderly men, but others have demonstrated no changes with gender during aging [18, 76].

Functional changes of NK cells may be also important for the physiological aging process [25, 89, 93]. Numbers of senescent cells in different tissues increase with aging and are considered among the major causes of aging (either directly or indirectly by the process of inflammaging) [94, 95]. NK cells may be major players in the elimination of senescent cells [25, 96]. It was shown that NK cells directly eliminate senescent cells, and as such they may contribute to maintenance of what is called the health span. NK cells eliminate senescent cells through the granule exocytosis (perforin/granzyme) proapoptotic pathway [97]. As these cytotoxic functions decrease with age, the elimination of senescent cells is also decreased. Moreover, senescent cells express increased levels of the nonclassical MHC-class Ib molecule HLA-E [25]. HLA-E has the capacity to inhibit the NK cytotoxicity by interacting with the inhibitory receptor NKG2A. This receptor is increased on senescent cells by IL-6 [25].

A few studies have addressed the mechanistic changes underlying the decreased NKCC with aging. Granule exocytosis is the predominant mechanism utilized by NK cells to eliminate their targets. It was proposed that a defective perforin secretion into the immunological synapse mediates the reduction in NKCC that accompanies physiological aging. This age-associated impairment in perforin secretion was associated with defective polarization of lytic granules towards the immunological synapse [98]. An earlier study investigated whether the modification of NK lytic activity could be related to differences in the metabolic pattern of activation of NK cells in the elderly. The early signaling events related to hydrolysis of inositol phospholipids were investigated following incubation with K562 target cells and/or CD16 mAb for different times. The data showed a pronounced age-related decrease in the ability to generate total inositol monophosphates and, particularly, inositol trisphosphates by NK cells following K562 stimulation (spontaneous cytolytic activity) together with an attenuated and delayed hydrolysis of phosphatidylinositol bisphosphate, while phosphoinositide turnover was preserved following Fc triggering (antibody-dependent cell mediated cytotoxicity, ADCC). These results confirm that, also in old subjects, different biochemical pathways of activation are involved in NK cells when target or antibody-mediated triggering occurs [99]. Furthermore, other NK studies one of us participated in had demonstrated that decreased cytotoxicity of human NK cells with aging is correlated with a significant decrease in the activity of acid phosphatase in these cells which, in turn, seemed important for maintenance of their cytotoxic activity [100, 101] even if the enzyme does not belong to protein phosphatases which, on the other hand, have an established role in regulating the signal transduction pathways originating with the KIRs [102]. These studies confirmed intracellular molecular changes in NK cells with aging lead to the decrease in cytotoxic activity.

One important aspect of immune aging is the constant antigenic challenge which is mediated by extrinsic and intrinsic stressors [103, 104]. Some extrinsic stressors become intrinsic, as is the case with chronic viral infections which in once infected humans become chronic [105] and whilst maintained under control for most of the lifetime have the tendency to reactivate when immunosurveillance decreases. This is the case for CMV [106]. Most elderly people are infected with CMV, with 80–90% of humans infected at the end of their lifespan. Usually, it is considered that the main defense against this chronic latent infection is the expanded effector memory CD8⁺ T cells, which may represent a significant percentage of the CD8 population at the end of human life and form part of an immune risk phenotype predicting early mortality in the oldest subjects [43, 106]. Until now, it seems very difficult to disentangle the effects of CMV infection and the aging process which makes CMV infection one of the driving forces of what is called the immunosenescence [106].

Recently, it was shown that NK cells may be influenced by CMV infection [38]. CMV may modulate NK cell functions by means of affecting inhibitory receptor expression whilst avoiding activating receptors. CMV infection induces various NK cell subtypes, but most remarkable is the expansion of mature and dysfunctional (CD56^dimCD16⁺) NK cells which express CD94 and NKG2C [87]. These cells accumulate not only in CMV-infected old individuals but in all CMV infected individuals independently of their age [107]. At the first appreciation, they seem to be protective for the host, especially when they progressively lose the FcRγ and become FcRγ^– [108]. These cells express inhibitory receptors for HLA class-I, among them KIR, ILT2 as well as downregulate the activating receptors such as NKp46, NKp30, and acquire CD57 [109]. In infected humans, CMV further induces the memory-like NK cells by the loss of the transcription factor PLFZ, which subsequently will precipitate the loss the SYK, EAT-2, and FcεRγ adapter molecules [110, 111]. Recently, an important role for Tim-3 has been demonstrated in relation to chronic infection and NK cell functions [12]. The loss of Tim-3 is associated with downregulation of IL-12 and IL-18 receptors, returning these NK cells to the quiescent state. These data suggest that CMV induces several progressive changes and adaptation/maturation in NK cells [76]. These adaptive-like NK cells are able to survive and be activated for efficient effector functions on a long-term basis [12]. When they may collaborate with the CMV-specific IgG, they may even more efficiently control CMV infection and in the meantime against other pathogens also [85]. Indeed, the NKG2C⁺ NK cells expanding as “memory” NK-cells under CMV infection, unlike memory T cells have broad specificity. NKG2C⁺ NK cells have been observed to expand in response to active hantavirus, chikungunya, HIV, and HBV infections, but only in individuals previously infected with CMV. Recently, a new NK cell subpopulation characterized by the absence of surface expression of CD56 (CD56^neg) was described in relation to CMV and EBV coinfections [112]. These cells show decreased cytotoxic activity and IFNγ production, and represent the mature phenotype characterized by low CD57 and KIR expression while lack characteristic features of cell senescence. It is possible that they contribute to the immune dysfunction in aging [112].

Thus, these adaptive-like NK cells can be polyfunctional and affect HSV-1- and influenza-infected cells [44, 113, 114]. They are even more efficient if they express NKp46 or CD2 [113, 115, 116]. These cells are unexpectedly long-lived and are very efficient to combat reactivation by CMV [117]. It was shown that NKG2C^hi CD57^hi NK cells are also more responsive to NKp46 cross-linking. Both CD16 and NKp46 share the same signaling adaptors, FcRγ and CD3ξ. Fcγ deficiency may enhance the signaling when CD16 and NKp46 have to exclusively use CD3ξ, which contains three immunoreceptor tyrosine-based activation motifs [111]. In the meantime, CMV may also induce NKG2C^– NK cells which will be FcRγ^– KIR B⁺ and are also capable of efficient ADCC activity by the activation of the CD3ξ receptor [38, 109, 113, 118, 119], thus sharing the same functional profiles with NKG2C⁺ NK cells [113]. The downregulation of CD57 in these long-lived mature NK cells is further increasing their reactivity before they lose it [74, 85]. Thus, these cells are highly adapted to combat CMV reactivation.

One important cytokine that modulates NK cell function is IL-2 [120]. This is very important as it has been shown that IFNγ produced by NK cells from vaccinated adults challenged with various viruses, including influenza virus are largely dependent on IL-2 secreted by specific T cells in PBMCs [121, 122]. In elderly, stimulation of NK cells with IL-2 resulted in decreased IFNγ production compared to young individuals [123]. This was reinforced by a decreased signaling in NK cells under IL-2 stimulation [124]. Ultimately, the production of IL-2 is also decreased with aging [125]. So, the decreased NK functions are also due to the extrinsic decrease in IL-2 and the intrinsic alteration of IL-2-initiated signaling; however, very few data exist on the changes with age of the CD25 on NK cells [126, 127].

Table 1. Age- and CMV-dependent phenotypic and functional changes in NK cells

Moreover, recently immunometabolism became an area of intense research. This is dependent on the possibility to switch from OXPHOS to aerobic glycolysis, exhibited by activated lymphocytes [128]. NK cells are not exempt from this effect on cell metabolism [120, 129]. The reactive NK cells show a hyperactive Akt-mTOR signaling pathway during steady state. Under stimulation through activating NK cell receptors, the Akt-mTOR pathway induces the increase in calcium influx and lymphocyte function associated antigen 1 integrin activation as a hallmark of reactive NK cells. mTORC1 signaling in activated NK cells is necessary to induce NK cell effector molecules such as IFNγ and GrzB [130, 131]. Concomitantly, this activation is fundamental to the glycolytic reprogramming of the NK cells, assuring effector functions by producing IFNγ and GrzB [129]; however, this is still controversial [132, 133]. During activation, the mitochondrial mass and mitochondrial membrane potential in human NK cells are increasing, which depends on the increase in the expression of the transcriptional cofactor PGC-1α under Il-2 stimulation [134]. It was shown that in NK cells of aged individuals, the impaired IL-2-triggered signaling pathway as well as the no increase in mitochondrial functions and no switch in metabolism is a feature of their senescence, explaining the decreased global effector functions of NK cells in aging [120]. These signaling alterations are due to the inhibition of PGC-1α in NK cells of elderly.

Concomitant to the memory-like NK cells, CMV may also induce the expansion of cytokine induced memory-like NK cells [135, 136]. Under IL-12, IL-18 and IL-15 stimulation, the memory-like NK cells manufacture high quantities of IFNγ and show increased cytotoxicity, but this functionality is very quickly lost [137, 138]. However, if they express IL-2R (CD25), they can be reactivated by very small amount of IL-2 as may be seen in an inflammatory milieu such as aging or during vaccination [38, 103, 104, 139, 140]. Thus, these cells may be an important player (booster) of the organismal defense system when the subjects are vaccinated with the same antigen that the subjects already encountered earlier in some way [14].

There are numerous studies addressing the question of the NK cell response to human CMV during aging [86, 87] (Table 1). As mentioned, under CMV infection around 50% of the NK compartment may be NKG2C^bright, while a proportion of NKG2C^dim NK cells may be also found as in CMV^–subjects [44]. However, it seems that this proportion does not increase with aging per se [44, 71, 141]. This is very important as this signifies that the proportion of these cells is already determined during the primary infection independently of the age of the subject and the repetitive CMV reactivation during aging [73, 74]. Moreover, it was found that the memory CD8⁺ T cells are inversely proportional to their NK cell counterpart [142]. This suggests that a vigorous NK cell response may slow the progression of CMV-driven immunosenescence, and autoimmunity by preventing the accumulation of CMV-specific and late-differentiated T cells can become functionally exhausted in old age [18]. Together, it was shown that accumulation of CMV-driven NKG2C⁺ NK cells is associated with increased function in the NK cell compartment.