The human immune system evolved to protect and defend the organism against disease and potentially to protect the symbiotic gut microbiota. T-cells are a major component of adaptive immunity, with a high degree of specificity in response to a pathogen challenge, enabling the host to mount a specific immune response and generate immunological memory [1]. Therefore, for effective immune protection against the primary and subsequent challenges, these cells must be maintained in a unimpaired state and appropriately regulated [2]. However, on aging, the immune system undergoes profound changes, loosely termed immunosenescence, that can affect the outcome of the immune response [3]. The impact of these age-related changes on the immune system has been associated clinically with decreased efficacy of vaccines, an increase in the frequency and severity of infectious diseases, and an increased incidence of chronic inflammatory disorders [3,4]. These alterations are associated with phenotypical and functional changes affecting a variety of immune cells, especially T-cells [5,6]. It has been shown that chronic stimulation of the immune system, such as by persistent viral infections, associates with age-related alterations in the peripheral T-cell pool [7]. Chronic infection especially by cytomegalovirus (CMV) has a dramatic influence on the T-cell compartment, both on CD8+ and CD4+ T-cells [8,9]. CMV interferes with different aspects of immune responses, and HLA, KIR, and GM genes have been shown to play a crucial role in CMV control (for review, see [10]). Recent epidemics of emerging pathogens that have a differential immune response depending on age, such as SARS-CoV-2, illustrate the crucial importance to understand the basis of an adequate response to new antigens and the relevance of factors such as age or CMV infection.

In humans, repetitive replication of T-cells is associated with the loss of CD28 and the acquisition of CD57. Thus, CD28− and CD57+ T-cells are considered to be late- or terminally differentiated T-cells characterized by low telomerase activity and shorter telomeres compared with CD28+CD57− T-cells. At least some of these CD28-CD57+ T-cells may be senescent [32,33]. It has been shown that besides age, persistent CMV infection is also associated with the accumulation of these highly differentiated T-cells [34,35]. Accumulating evidence supports a detrimental role of senescent T-cells in several chronic inflammatory clinical conditions, including cardiovascular diseases such as atherosclerosis and myocardial infarction (for a review, see [36,37]).

Our results show that in middle-aged and older overtly healthy individuals, the main factor driving the expansion of CD57+ T-cells is CMV infection. However, from the fourth decade onwards, these cells do not accumulate further with age. In previous work, we showed that the percentage of CD8+CD57+ T-cells was similar between young and middle-aged CMV-seropositive individuals [25]. Therefore, here, we extend our previous findings [24,25,26,38] to show that CD57 expression by T-cells is not only a hallmark of CMV infection in young individuals but also at older ages. Accordingly, once CMV infection takes place, CD57+ T-cells will expand, and after that, their percentage will remain rather stable over time. Thus, our results argue against the consensus that the expansion of these cells is a sign of chronological aging.

Regarding CD57+ T-cell functional capacities, our data also indicate that CD57+ T-cells are more polyfunctional than CD57− T-cells at any age, with one exception, namely the DN T-cell subset, where the PI does not change with CD57 expression. Nonetheless, we did observe an increase in trifunctional DN T-cells with CMV seropositivity, and the total PI of DN T-cells increased with CMV infection in middle-aged individuals. In this subset, CD57+ T-cells also accumulate in CMV-seropositive individuals.

Our results regarding CD4+CD57+ T-cell expansions with CMV infection are also in agreement with the observation that CMV, but not aging, has a significant effect on the expansion of pro-atherogenic CD4+CD28− T-cells [39]. These cells (that also express CD57) are cytotoxic, capable of causing vascular damage, and their expansion is associated with autoimmune and cardiovascular disease (for a review, see [36,37]). Here, we show that similar to young individuals [24], at older ages, CD4+CD57+ T-cells are also more polyfunctional (CD107a, IFN-γ, and TNF-α) than their CD57− counterparts. Additionally, the percentage of these polyfunctional cells correlated with the percentage of total CD4+CD107a+ (i.e., cytotoxic) T-cells (Spearman correlation p-value = 0.03, Figure S4). Notably, CMV-seronegative individuals had very low or no percentages of both cytotoxic CD4+ T-cells (CD1017a+) and CD4+CD57+ T-cells, supporting the conclusion that the expansion of cytotoxic CD4+ T-cells only occurs with CMV infection.

Higher frequencies of CD4+CD57+ T-cells have been associated with poorer prognosis in several diseases. In acute heart failure patients, high percentages of these cells are associated with the development of cardiovascular events (defined as heart failure-associated mortality, transplantation, or rehospitalization) [40]. In end-stage renal disease patients, the frequency of CD4+CD57+ T-cells is associated with atherosclerotic changes [41], and in multiple sclerosis, their frequency is associated with disease severity and poorer prognosis [42]. In addition, in acute heart failure patients, percentages of IFN-γ+ and TNF-α+cells are higher within the CD4+CD57+ than the CD57− T-cell subset, and CD4+CD57+IFN-γ+T-cells were increased in patients compared with healthy individuals in response to anti-CD3 stimulation [40]. In our hands, the PI of CD4+ T-cells overall and the CD4+CD57+ T-cell fraction increased with CMV-seropositivity, but not age, supporting a significant role of CMV in the development of cardiovascular disease that can be explained, at least in part, through the expansion of these proinflammatory and cytotoxic CD4+CD57+ T-cells. Consistent with this, a link between CMV infection, CD4+CD28− T-cell expansions, and autoimmune and cardiovascular disorders has been previously suggested [36,43].

Our data may be of some practical clinical importance because CMV infection can be treated, and the expansion of these cytotoxic proinflammatory cells could potentially be prevented. In this respect, the use of Valacyclovir as anti-CMV treatment in patients with Antineutrophil Cytoplasmic Antibody (ANCA)-Associated Vasculitis was shown not only to suppress CMV reactivation but was also associated with a reduction of the CD4+CD28− T-cell frequency. Moreover, in a different context, a lower frequency of these cells correlated with improved responses to pneumococcal vaccination [44]. These results strongly suggest that the CMV-driven expansion of CD4+CD28−T-cells, and by extension CD4+CD57+ T-cells, might have a detrimental effect on the immune response to vaccination. These results, together with the proof of principle of the potential benefit of using anti-CMV treatments in ANCA-Associated Vasculitis, support the possible application of anti-CMV therapy in any clinical situation where CD4+CD57+ T-cells are implicated, including impaired responses to infection and vaccination.

Our results show that CD8+CD57+ T-cells are also expanded in CMV-seropositive individuals, both middle-aged and older. As in CD4+ T-cells, the expression of CD57 was associated with higher polyfunctionality determined by the PI value, this increase in the polyfunctionality of CD4+ and CD8+ T-cells being a hallmark of CMV infection. Indeed, it has been shown that CMV-specific CD8+ T-cells that produce IFN-γ and TNF-α as well as express CD107a play an important role in controlling this viral infection in the context of allogeneic stem cell transplantation [45]. Similarly, CMV-specific CD107a+IFN-γ+CD8+ T-cells are important for controlling CMV infection in rhesus macaques [46].

The expansion of CD8+CD57+ T lymphocytes is associated with CMV viremia in solid organ transplantation. These cells were characterized by IFN-γ and granzyme B production, and their expansion was associated with CMV-specific immune responses in pediatric cardiac transplantation [47]. Moreover, in renal transplant recipients, CMV induced the expansion of highly functional memory CD57+ T-cells [48], and during active CMV replication, highly differentiated CD8+CD57+ T-cells acquire cytotoxic activity [48]. Additionally, the frequency of total CD8+CD28− T-cells has been related to the specific immune response to CMV one year after solid organ transplantation [49].

As has been pointed out before, the effects of CMV infection and age differ among T-cell subsets. As reported here, CD4+ and CD8+ T-cells display a functional shift associated with CMV seropositivity, while NKT-like and DN T-cell subsets do not. Specifically, NKT-like cell functionality was not affected either by age or CMV, while DN T-cells’ response to SEB and PI was mainly altered by age. In individuals over 65 years of age, the impact of CMV infection on DN T-cell functionality seems to be diluted by the effect of aging.

The increased PI of DN T-cells with age seems to be due to a gain in cytotoxicity (CD107a marker of degranulation) rather than to higher cytokine production. DN T-cells are mainly gamma-delta T lymphocytes, and studies on the effect of its aging have yielded disparate results [50,51,52,53]. CMV does not stimulate Vdelta2+ cells, but it does stimulate Vdelta2− T-cells. These are characterized by the accumulation of highly differentiated Vδ2− subsets with time, in contrast to Vδ2+ T-cells, which are decreased in old individuals independently of CMV serostatus and maintain a less differentiated phenotype [50]. Although a limitation of our study is that we have not characterized the different gamma-delta subsets within the DN T-cells, our results clearly show a higher capacity of degranulation of these cells in older adults, independent of CMV serostatus.

It has been reported that NKT-like cells expand with age and in certain disease states. Our previous work [26,38] showed that in CMV-seropositive individuals of up to 60 years of age, there was no effect of age on the expansion of these cells. We only observed an increase in NKT-like cell percentages when comparing young CMV-seronegative individuals and middle-aged CMV-seropositive individuals, indicating that CMV is an important factor for their expansion. However, there was no increase in NKT-like cell frequency with CMV in young individuals. To shed light on whether their accumulation is due to CMV infection alone or a combined effect with chronological aging, we analyzed the percentage of this cell subset at older age. Our results demonstrate that NKT-like cells expand in CMV-seropositive individuals over the age of 40 and onwards, and that age per se had no effect on their accumulation. Furthermore, we have also observed a significant expansion of NKT-like CD57+ cells in CMV-seropositive individuals, which is unrelated to aging. Earlier reports have shown heterogeneous results regarding the possible effect of aging on the number and function of NKT-like cells [54]. In the present study, we show that NKT-like cells accumulate from middle age onwards with CMV infection but not age alone, although no functional alterations were observed in this subset. These results are in contrast to other studies showing a higher functionality of CD56+ T-cells (determined by levels of CD107a and proinflammatory cytokines) in CMV-seropositive individuals [55]. These differences could be due to the fact that the age of the young group ranged from 23 to 60 years old in that study.

CMV infection induces the expansion of CD57+ T-cells, but whether this has a beneficial or detrimental or neutral role for immunity and long-term effects on health and its direct effect on response to pathogens and vaccines is still under debate. Nevertheless, there is mounting evidence that CMV-seropositivity is associated with a reduced response to both invasion with a novel pathogen and to vaccination Some studies have reported a beneficial effect of CMV infection in both young and older adults in response to vaccination [21,23], while other studies suggest that it can be detrimental [12,13]. The increased functionality of CD57+CD4+ and CD57+CD8+ T-cells shown can be considered beneficial, but under certain pathological conditions, CD57+ T-cells have immunosuppressive activity [56,57]. Thus, it is essential to further investigate the effects of CMV on the immune response at older ages. In this regard, our present study complements our previous investigations allowing us to dissect the effect of age and CMV in older individuals. This is especially important for vaccine development, as it has been shown for the influenza vaccines Fluad^® or Intanza^®, especially designed for elderly individuals [58,59]. A poor response to Intanza^® was associated with CMV seropositivity, but only in older individuals [58,60]. In addition, it has been shown that older CMV-seropositive individuals have lower frequencies of influenza specific memory T-cells than CMV-seronegative, but this was not observed in younger individuals [61]. However, despite the lower frequencies of influenza-specific T-cells found in CMV-seropositive older individuals, they exhibited a significantly higher IFNγ T-cell response to influenza virus in the acute phase of the disease compared to CMV-seronegative older individuals [61]. These results agree with our results showing an increased polyfunctionality of CD4 and CD8 T-cells with CMV-seropositivity in older donors.

Our results regarding CD57+ T-cells expansion in CMV-seropositive older donors are also of interest in the study of CMV infection in transplant recipients where CMV infection represents a significant complication. CD8+CD57+ TEMRA cells increased over time after transplant specifically in CMV-seropositive but not CMV-seronegative recipients, and changes in CD8+ T-cells compatible with accelerated immune aging have been observed in CMV-seropositive transplant recipients [62]. Our results showing that CD57+ T-cells are mainly expanded in CMV-seropositive individuals, and maintain cytokine production and polyfunctionality independently of age, suggest that CD57+ T-cells may contribute to the promotion of adverse inflammatory outcomes, such as late graft dysfunction, chronic kidney rejection, cancer, and atherosclerosis observed after kidney transplant associated with CMV infection.

The results presented also highlight the relevance of considering the heterogeneity of CMV seropositivity and aging in the design of clinical trials or new vaccines in some clinical conditions such in HIV patients. Thus, CMV-induced chronic immune activation and premature immunosenescence in people living with HIV must be taken into consideration not only when comparing trial outcomes between various populations exhibiting diverse CMV positivity rates but also for HIV vaccine development [63].

Therefore, immunological treatments should consider both age and CMV infection as a major factor. This strengthens the need for validation studies with not only the aim to present something novel but also to confirm findings in different populations.

Our results support the view that CMV is a major driving force for the expansion of CD57+ T-cells, and that these cells are more polyfunctional than their CD57-negative counterparts within the CD4+ and CD8+ subsets (including NKT-like cells). Age has no significant effect on either the frequencies of CD57+ T-cells or their polyfunctionality when only CMV seronegative individuals are considered. Thus, our results indicate that the CD57+ T-cell population might play an important role for antiviral control, which is in line with their high cytotoxic and polyfunctional activity. The association of these T-cell expansions with CMV infection and disease underlines the necessity of considering CMV serology in any study regarding immunosenescence and emphasizes that the price of immune protection is always some degree of immunopathology.