Introduction

Immunosenescence is a concept that describes the quantitative and functional changes that occur in the innate and adaptive compartments of the immune system during the course of normal human aging. A functional decline and remodeling in cellular subsets have significant impacts on the course of neuro-inflammatory conditions, risk of infections, tumor surveillance, and response to vaccines [1••]. These changes may be particularly relevant in patients whose immune systems are iatrogenically altered by immunosuppressive and immunomodulatory medications used to treat inflammatory conditions, such as MS, neuromyelitis optica (NMO), connective tissue disorders, or sarcoidosis. Over the past decade, the emergence of several novel immunotherapeutic agents and their association with infections highlighted the relevance of immunosenescence to the risk of infection in older patients.

This review will focus on the changes that occur in the CD4, CD8 T lymphocytes, B lymphocytes, and the innate compartment of the immune system during aging, in an attempt to explain how these alterations can be additive to or synergistic with the changes induced by the disease-modifying therapies in regard to the risk of infections.

Immunosenescence: a Risk Factor for Opportunistic Infections Associated with Disease-Modifying Therapies

Several MS therapies (natalizumab, fingolimod, and dimethyl fumarate) have been increasingly associated with a risk of infections, including progressive multifocal leukoencephalopathy (PML). Some epidemiologic trends suggest that older age may be an important risk factor in PML development and outcomes.

In one study of natalizumab-associated PML, the infection occurred significantly earlier (before 24 infusions) in patients who were older at the time of natalizumab’s initiation as compared to those who were diagnosed at a later time during therapy (43.5 ± 9.7 vs. 36.9 ± 8.3 years, p = 0.03) [2]. In addition, older age is considered to be a predictive factor of poorer outcome in PML: a concept that is ubiquitously true in the context of age and infection-associated mortality. The age of the U.S. PML patients was found to be mean 47 years (median 47 years) in survivors and 52 (median 55) in those who did not survive [3]. However, one must note that the average age of patients with PML with natalizumab was 45 (22–73), not significantly different from the average age of the patients on natalizumab without PML [4]. A development of PML case in a 70-year-old patient treated with natalizumab for MS whose JCV antibody seroconverted to positive only 2 weeks prior to the onset of the disease [5] suggests the possibility that age-associated changes in humoral immunity reduce the ability to mount an effective antibody response, thus, possibly diminishing the reliability of the interval-based JCV antibody surveillance in that age group. Likewise, in the rare instance in which PML occurs in the absence of an identifiable predisposing cause, the immunological changes associated with aging may be a substantial contributing factor. In the literature, PML cases in apparently non-immunocompromised non-MS patients occurred in patients of more advanced age than the average population (mean age 59.4, median 62) [6,7,8,9,10,11,12].

Fingolimod, another disease-modifying drug, recently associated with PML cases, appears to demonstrate a similar age-dependent trend for both PML and cryptococcal meningitis. For fingolimod-associated PML, the mean age was 54 and median 58 (range 34–63) [13]. For cryptococcal meningitis cases, average age reported in the literature cases was 56 [14]. These patients are substantially older than the average age of the patients with relapsing remitting MS. In one large cohort of MS patients, the median age of onset was 28.7 years with an average duration before the onset of secondary progressive MS of 8.7 years ± 8.6 years [15]. In patients on long-term fingolimod, absolute counts of peripheral circulating CD4 and CD8 T cells were remarkably low as expected, but there was also a striking CD4:CD8 ratio reversal of 1:2 [16], mimicking the immunosenescent changes observed in cohorts of octa- and nonagenarians with increased risk of mortality [17]. In addition, a slightly higher rate of Varicella zoster virus (VZV) reactivation in patients on fingolimod was observed in the clinical trials and in the post-marketing surveillance. One study showed that the frequency of VZV-specific and EBV-specific IFN-γ producing T lymphocytes was reduced in patients on fingolimod. In this cohort, 20% developed a subclinical VZV or EBV reactivation. In addition, upon stimulation with VZV, the absolute number of cells proliferating in response to the virus was reduced [18]. This effect may be particularly important, as VZV predominantly reactivates in those of older age [19, 20, and 21]. In the USA, annual incidence of VZV reactivation nearly doubles from 5 to 6.5 per 1000 in those of 60 years of age to about 8–11 patients per 1000 in those 70 years of age or older [22].

Dimethyl fumarate (DMF)-associated PML cases occurred in patients who were 54, 59, 61, 64, and 66 years of age (mean 60.8/median 61) [23]. The risk of PML with dimethyl fumarate in most (though not all) cases has been associated with absolute lymphocyte counts of <500 cells/cu mm and, similarly, PML with the use of monomethyl fumarate in the treatment of psoriasis has also been linked to lower lymphocyte counts. Older age may increase susceptibility to lymphopenia in patients on fumarates. A recent retrospective study of patients on DMF showed that among patients who were 60 years old or older, 24 (71%) developed lymphopenia, while only 62 (36%) of the 172 younger than 60 years of age developed lymphopenia (P = .0005). The study concluded that patient age of 60 or older and baseline absolute lymphocyte counts <2.0 were an independent risk factor for developing lymphopenia [24]. Another retrospective study of 221 patients on DMF showed that people of older age (>55) had an increased risk of developing moderate to severe lymphopenia [25]. Foley et al. compared absolute lymphocyte counts (ALC) in 30 multiple sclerosis patients younger than 30 and 30 patients older than 65 and showed that the mean baseline ALC was 2543 in the younger patient versus 2189 in the older; after DMF exposure, the ALC declined to 1844 (27% decrease) in the younger cohort and to 1279 (41% decrease) in the older cohort. Moreover, 67% of patients in the lowest quartile for lymphocyte count at baseline were those older than 65 [26]. CD8 T cells, critically important for viral control and clearance, are preferentially lowered by DMF [27]. CD4/CD8 ratio alterations, such as those occurring with DMF [28], long-term fingolimod [16], and in the CSF of natalizumab patients in the first 6 months [29], may impair the immune surveillance to various degrees and increase the risk of infections, such as PML. These alterations may be additive to the many changes in the CD4 and CD8 compartments in those of older age.

In non-MS non-transplant patients treated with rituximab, a similar trend for advanced age and PML was noted [30]. These immune-modulating therapies affect various subsets of the immune system in many complex ways beyond their basic mechanism of action, thus adding to or potentiating the subtle deleterious effects of immunosenescence.

West Nile virus (WNV) epidemiology offers another unique example of the relationship between age and infection. WNV meningoencephalitis is more common among people over the age of 50 and, in fact, is 40 to 50 times more common in people over 70 as compared to those younger than 40 [31].

Those of older age have a higher magnitude and frequency of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) reactivation [32].

This review will highlight age-related changes in the immune system that may be both additive and synergistic in the context of disease-modifying therapies’ quantitative and qualitative effects on the immune system.

Thymus and Aging

The thymus is a primary lymphoid organ that provides an environment for T cell differentiation, maturation, and education, resulting in a positive (functional) and negative (deletion of autoreactive T cells) selection of naive T cells and their output into the circulation. Studies suggest that beginning in the 1st year of life, the thymus begins to involute at a rate of approximately 3% per year [33]. As the thymus atrophies, thymic output of naïve T cells falls [34, 35]. This age-related difference in thymic output is best highlighted by the studies of immune reconstitution in HIV-infected adolescents versus older adults, showing a significantly more robust naïve T cell reconstitution in adolescents, when treated with HAART [36]. Murray et al. found that at the age of 25, 20% of naïve T cells emerge directly from the thymus, whereas by the age of 55, only 5% of naïve T cells arise from thymic output [34]. Other studies also confirmed that some level of thymopoiesis continues well into the 5th decade [37].

As thymic output of naïve T cells falls, homeostatic peripheral T cell proliferation acts as a compensatory mechanism to maintain normal peripheral T cell counts [38, 39]. This peripheral naïve T cell pool is aged and may have accumulated senescence-associated defects, passing it onto the newly derived naïve T cells. In addition, this peripheral T cell pool is limited in its diversity, which leads to a more restricted T cell receptor repertoire, eventually undermining immune systems’ ability to respond to novel antigens. Initially peripheral proliferative responses may be sufficient to maintain a diverse naïve T cell repertoire well into the 50s; however, after the age 65, the diversity of the T cell repertoire drops precipitously [40].

Reduced thymic output is of particular interest in patients with MS, with some studies showing that in patients with MS, thymic involution occurs earlier. Duszczyszyn et al. showed that recent thymic emigrants (RTE) were reduced in patients with RRMS and PPMS as compared to the age-matched healthy controls. When compared to healthy controls, patients with RRMS showed an age-associated thymic output of progressively immature CD4 RTEs. Further analysis of Ki-67 (cell proliferation marker) showed important changes in the T cell proliferative responses, leading to failure to maintain naïve CD4 T cell numbers [41].

Effects on T Cell-Mediated Immunity

Both CD4 and CD8 T lymphocyte subsets are affected by aging, with the changes being most profound in the CD8+ compartment [42••]. In the elderly, there is a decrease in the naive CD8+ T cells, with a predominance of terminally differentiated late effector memory CD8+ T cells. These cells exhibit features of replicative immunosenescence such as reduced ability to proliferate, shortened telomeres, and reduced telomerase activity. Some studies report that an increase in cell negative regulatory cell receptors such as PD1 (programmed death 1), TIM-3 (T cell immunoglobulin and mucin-domain containing-3), and KIRs (killer inhibitory receptors) is responsible for the reduced activation and proliferation that occur with age [43, 44]. However, whether these receptors represent markers of immunosenescence or simply terminal differentiation remains a matter of debate [45]. Aged CD8 T cells have a decrease in their primary response to infections and vaccinations [46, 47] as well as reduced quality of target cell lysis [48, 49]. Thus, in the course of aging, T cell repertoire is profoundly affected by the shifts in the ratios of naïve T cells to memory T cells [50] with some suggesting a two- to fivefold decrease in the size of the naïve T cell subset [51]. Van Epps et al. showed that people of older age (median age 80) had markedly lower frequencies of CD8+ naïve T cells (11 vs 47%) as compared to those of younger age (median age 27). The same age-related trend was observed for CD4+ naïve T cells (33 vs 45%) [52] (Table 1). In addition, virus-specific CD8+ T lymphocytes from the aged individuals produce less antigen-specific IFN-γ [53].

Table 1 Age-associated CD8+ and CD4+ T lymphocyte changes
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While naive CD8+ T cells decrease in numbers and accumulate age-associated defects in their ability to proliferate in response to antigen stimulation [54•], some argue that naïve CD4+ T lymphocytes may be affected to a lesser extent [55••]. Shulz et al. showed that after a vaccination with a yellow fever virus, those of older age had a decrease in peak frequencies of CD8+ T lymphocytes, while CD4 peak response frequency was not affected [56].

However, other studies suggest that the CD4 compartment likewise undergoes many important changes (Table 1). CD4 cells have critical helper functions in activating B cells, promoting germinal center formation and high affinity antibody production [57]. An adoptive transfer model comparing antigen-specific antibody responses between CD4 deficient mice who were inoculated with naïve TCR transgenic CD4 from aged mice donors versus young donors showed a 3 log decrease in the antigen-specific antibody response and a reduced germinal center expansion in the setting of CD4 cells from aged mice [58]. In addition, CD4 T cells from aged individuals (both human and mice) have a lower proliferative capacity and IL-2 production [59], essential for T cell differentiation into effector and memory subsets after antigen stimulation. Some studies report an increase in CD4 FOXp3 regulatory subset that can make the immune response to pathogens less robust [60, 61]. With age, CD4+ T cells are more susceptible to apoptosis and have a weakened T cell receptor response [62].

Another important change that occurs in the T cell compartment is a loss of CD28, an important co-stimulatory molecule necessary for the effector functions of CD4 and CD8 T cells. Loss of CD28 and expansion of CD28 negative cell subset is associated with a decrease in antigen receptor diversity, reduced antigen-induced proliferation, and shorter life span [63, 64]. Expansion of CD28 T cell subset appears to be proportional to aging and is seen widely in polyclonal expansion associated with CMV infection. At birth, almost all CD8+ T cells contain CD28; by the age of 20–30, CD28 expression is reduced by about 20% and in people over 80, the expression is reduced by 50–60% [65]. This reduction is thought to occur due to prior burden of repetitive antigen exposure and is associated with chronic infections, such as CMV and EBV. Life-long repeated bouts of CMV reactivation within the host result in a repeated CMV antigen presentation and memory cell boosting, leading to a clonal expansion of CMV-specific CD8 T memory T cells, now comprising a larger part of the T cell compartment at the expense of the smaller numbers of naïve T cells. These CMV-specific T memory cells have phenotypic and replicative features characteristic of the immunosenescent cells [66]. Some studies report that with age, naïve T cells are less likely to become effector cells and when they do, the response is preferentially Th2 and not Th1 (due to a shift towards type II cytokine responses) [67]. Th1 is necessary for cellular immunity response to pathogens such as viruses and fungi. As T cell compartment becomes increasingly clonal, some studies suggest a propensity for auto-reactivity [68].

A recent study showed a defect in the interferon type I signaling pathway in T cells in the aged individuals [69]. Type I interferons (IFN-I) (interferon-α and interferon-β) are critical in T cell responses to bacterial and viral infections, allowing for T cell survival and differentiation. In activated T cells, interferon-α upregulates expression of IL-2Rα, c-myc, and pim-1, a set of genes responsible for cellular proliferation [70]. Using gene expression arrays, older individuals were shown to have a decreased IFN-I-induced gene expression in naïve CD4 cells. This reduced responsiveness to IFN-I may represent yet another mechanism of immune impairment in those of older age [69].

Longitudinal studies of healthy octagenarians and nonagenarians reported specific immunologic changes associated with increased morbidity and mortality, constituting an immune risk phenotype, characterized by CD4:CD8 ratio reversal (less than 1), increased terminally differentiated CD28- CD8 T cells, CMV seropositivity, decreased proliferation of T cells to mitogens, clonal expansion of CMV and EBV CD8 T cells, and increased frequency of CD28-KLRG-1+ dysfunctional cells among CMV-specific CD8 T cells that have low ability to proliferate and produce IFN [17, 100].

Most studies of immunosenescence have focused on the mechanisms of T cells in circulation, which represent only 2% of the total lymphocytes. Several recent studies illustrated that a large number of T cells consist of T resident memory cells that reside in the epithelial barriers and do not recirculate [71, 72]. These cells travel through the extracellular matrix (ECM) of the non-lymphoid organs. The T cell’s ability to mobilize and navigate in these tissues is critical for the successful immune surveillance and response in tissues and organs [73]. Changes that occur in the ECM in the process of aging may be important, as ECM proteins and proteoglycans contribute to the cell behavior via interacting with secreted ligands and, importantly, transmembrane receptors, such as integrins. This study led to a novel hypothesis that with age, increased stiffness and cross-linking of ECM leads to a decreased mobility of T resident memory cells and thus may be in part responsible for the reduced immune competency we observe in aged individuals [74]. T cell passage through a stiff matrix may damage its membrane and/or nucleus, potentially resulting in cellular death [75, 76] or sending danger signals that trigger the inflammasome. This leads to increased levels of pro-inflammatory cytokines such as IL 1, IL 18, and upregulation of NF kB (nuclear factor kappa-light-chain-enhancer of activated B cells) [77••], a transcription factor that regulates genes associated with chronic inflammation and leads to cellular phenotypes associated with aging in the brain, skin, and spine [78, 79] termed senescence-associated secretory phenotype (SASP) [80].

Effects on B Cell-Mediated Immunity

The humoral compartment of the immune system also undergoes important changes in the process of aging (Table 2). This is best illustrated by the reduced ability of the elderly to mount an adequate antibody response to novel antigens and vaccines [81]. A decreased ability to mount a specific antibody response to various vaccines (influenza, tetanus, Salmonella, S. pneumoniae) has been demonstrated in multiple studies [82, 83]. Several studies report that the total numbers of peripheral B cells are reduced in the course of aging [83, 84•]. B cell subsets also undergo many important changes such as a loss of naïve follicular B cells, increasing number of antigen-experienced B cells and changes in the antibody production, both functionally and qualitatively [85].

Table 2 Age-associated B lymphocyte changes
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In adults, B cells are continuously repopulated from the pool of the hematopoietic stem cells (HSC) that reside in the bone marrow. With age, the frequencies of B cell progenitors decrease [86], likely reflecting HSC’s decreased propensity for lymphoid differentiation secondary to downregulation of the genes responsible for lymphoid specification [87]. Younger adults have a higher proportion of HSC committed to lymphoid lineage, whereas the elderly have a balance shift towards myeloid lineage [87]. McKenna et al. evaluated bone marrow B cell precursors in several hundred patients and showed a decrease associated with age [88]. With aging, B cells accumulate defects that result in the reduced efficiency of somatic hypermutation (SHM) (responsible for generating high avidity antibodies) and class switch recombination (CSR) (changing effector functions of the specific antibody) as well as AID (activation-induced cytidine deaminase), critical for CSR and SHM [83, 89]. Decrease in somatic hypermutation of IgG genes reduces the specific affinity of the antibody to the antigen [90]. In addition, Gibson et al. studied peripheral blood B cells from adults between the age of 19 and 85 and showed that in some elderly adults, there was a significant reduction in the antibody repertoire, a finding that was associated with frailty [89]. There is a decrease in B cell responses to novel antigenic stimulation, which in part may be due to the changes in CD4 helper T cells [58].

Effects on the Innate Immunity

Aging also leads to variable changes to the NK cell [91], monocyte, and neutrophil compartments, resulting in the impairment of their phagocytic and chemotactic functions [92, 93]. NK cells are critical as a first-line defense against intracellular pathogens as well as tumor surveillance and control. Low function of NK cells in elderly people was found to be associated with an increased risk of infection [94], while those 85 and older with decreased numbers of NK cells were found to have an increased rate of mortality at 2-year follow-up [95]. NK cell compartment undergoes changes quantitatively, phenotypically, and functionally [96]. Immature NK bright cells decrease in numbers, while NK dim cells prevail, with a predominance of CD57+ phenotype, a marker of terminal differentiation, that are highly cytotoxic but have a decreased ability to respond to cytokines and proliferate [97]. NK cells from healthy elderly people have lower rates of proliferation and production (by 50%) [98] compared to healthy young people, yet the overall numbers are similar, likely reflecting a proportionally expanded NK dim subset [99]. Cytotoxicity may be reduced at the cellular level, but due to the expansion of the NK dim subset, overall cytotoxicity is preserved [100]. NKp30 activating receptor expression decreases with age [101]. This receptor is critical in the cross talk between NK and dendritic cells presenting a foreign ligand. Engagement of this receptor leads to production of TNFα, IFN-γ, and dendritic cell activation or to direct killing of the dendritic cell by the NK cell [96]. When activated, dendritic cells produce Th1 cytokines further activating NK cells and recruiting cytotoxic arm of the adaptive immunity [102]. Other activating receptors such as NKp46 and DNAM-1 are also reduced in the elderly donors [96].

To summarize, aging is associated with an overall increase in NK cells, perhaps as a compensatory mechanism for their reduced function (reduced cytotoxicity at a single cell level, decreased cytokine production, and decreased ability to respond to cytokines) and subset redistribution (decreased NK brights) and phenotype changes (decreased activating receptors) [1•]. Blood sample analysis from middle-aged and elderly groups showed an increase in neutrophil counts and C-reactive protein (CRP) in the elderly. Some studies suggest that the former reflect low-grade inflammation and are associated with frailty [103].

Inflammaging

In addition to the many changes that occur in the immune system cellular compartments and function, aging is associated with a low-grade pro-inflammatory state, termed inflammaging [104]. This important theory of aging first began with an observation that peripheral mononuclear cells from the elderly patients expressed higher levels of pro-inflammatory cytokines (IL-6, TNFα, and IL-1β) than the cells from their younger counterparts [105]. A series of further experiments led to a better characterization of the cytokines involved. TNFα levels were found to be increased in the elderly populations and positively correlated with IL-6 and CRP levels and were associated with general atherosclerosis and Alzheimer’s disease [106]; in another study, IL-6 was found to be elevated in the elderly and associated with increased morbidity and mortality [107].

Over the past several years, many studies looked into the mechanisms that comprise inflammaging. As tissues age, cellular and organelle damage results in the accumulation of cellular debris. This cellular debris (for example, damaged DNA) contains damage-associated molecular patterns (DAMP) that are sensed by the pattern recognition molecules, such as the Nod-like receptor family and toll-like receptors [108], capable of activating the innate immune system as well as the inflammasome and NF-κB [109]). Free radicals from oxidative stress, components of the damaged cells and organelles, and metabolic by-products such as fatty acids, urate crystals, cardiolipin, peroxidized lipids, amyloid, advanced glycation end products, and altered N-glycans can be recognized by a series of sensors as “danger” signals that activate immune responses [110].

One of these sensors is a Nlrp3 inflammasome, a multi-protein complex, which upon sensing a danger signal, leads to the activation of pro-caspase, resulting in an increase of the pro-inflammatory cytokines such as IL-1β and IL-18. IL-1β is a potent activator of IL-6 and IL-8, important players in SASP (senescence-associated secretory phenotype) [111]. Rodier et al. showed that persistent DNA damage leads to senescence-associated inflammatory cytokine secretion [112].

As cellular damage accumulates with aging, this pro-inflammatory state becomes chronic [104].

Most of the signals that activate the inflammasome result in the generation of reactive oxygen species in the mitochondria. When mitochondria are damaged, they release damage-associated molecular patterns (mitochondrial DNA and formyl peptides), some of which are phylogenetically similar to bacteria-associated molecular patterns, which are powerful activators of the innate immune system [113] as well as Nlrp3 inflammasome. Both mitochondrial cardiolipin and fragments of mitochondrial DNA (due to its evolutionary similarity to bacteria) represent an endogenous pathogen-associated molecular pattern capable of activating the inflammasome and leading to chronic inflammation [114, 115]. Wikby et al. found that IL-6 levels were associated with an immune risk phenotype (described above) [107]. Another group showed that two inflammatory marker (IL-6 and TNFα soluble receptor 1) levels could be used to predict the 10-year mortality in the elderly [116]. Erschler et al. showed that elevated IL-6 levels in the elderly are associated with the increased frailty and chronic inflammation [117].

Cumulative cellular damage and stress lead to cellular senescence via a cell cycle arrest, a mechanism that is set to protect the organism from accumulating damaged, aberrant machinery that can potentially result in cancer. These senescent cells comprise a senescence-associated secretory phenotype (SASP), producing various inflammatory cytokines (Il-1, IL-6, IL-8, IL-7, IL-13, IL-15) [118, 119]. With age, senescent cells accumulate in many tissues, particularly in the adipose tissue, known to be responsible for a large source of pro-inflammatory cytokines [120], such as IL-6 [121].

Conclusion

The concept of immunosenescence has an important implication for how we view inflammatory diseases, risk of infections, and safety of immunomodulatory and immune suppressive treatments in the aging population. Immune system changes in the elderly impart a higher susceptibility to infection and are associated with mortality, as well as a poor response to vaccines. The use of disease-modifying treatments, which are either immunomodulatory or overtly immunosuppressive, may have a more profound impact on patients whose immune system is already compromised and, thus, their risk of infection, for example, PML, may be higher. A similar concern stands for the increased risk of cancers associated with the use of some disease-modifying agents (fingolimod: lymphoma, basal cell carcinoma; natalizumab: melanoma), as the risk may be higher in the aging population due to the immunosenescent changes impairing tumor surveillance and control. Additionally, the qualitative and quantitative changes that occur with aging may be responsible for the nature of expression of multiple sclerosis, perhaps explaining the higher incidence of primary progressive MS in the older population [122].

Clinicians should exercise a high level of vigilance in monitoring the risk of infections in patients of older age on immunomodulatory treatments. The effects of immunosenescence should be strongly considered in the individual risk stratification of infection and cancer.