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The Impact of Aging on Regulatory T-Cells

Age-related deviations of the immune system contribute to a higher likelihood of infections, cancer, and autoimmunity in the elderly. Senescence of T-lymphocytes is characterized by phenotypical and functional changes including the loss of characteristic T-cell surface markers, while an increase of stimulatory receptors, cytotoxicity as well as resistance against apoptosis is observed. One of the key mediators of immune regulation are naturally occurring regulatory T-cells (Tregs). Tregs express high levels of CD25 and the intracellular protein forkhead box P3; they exert their suppressive functions in contact-dependent as well as contact-independent manners. Quantitative and qualitative defects of Tregs were observed in patients with autoimmune diseases. Increased Treg activity was shown to suppress anti-tumor and anti-infection immunity. The effect of aging on Tregs, and the possible contribution of age-related changes of the Treg pool to the pathophysiology of diseases in the elderly are still poorly understood. Treg homeostasis depends on an intact thymic function and current data suggest that conversion of non-regulatory T-cells into Tregs as well as peripheral expansion of existing Tregs compensates for thymic involution after puberty to maintain constant Treg numbers. In the conventional T-cell subset, peripheral proliferation of T-cells is associated with replicative senescence leading to phenotypical and functional changes. For Tregs, different developmental stages were also described; however, replicative senescence of Tregs has not been observed yet.


INTRODUCTION
The immune system combats against infectious agents and depletes damaged or transformed cells, whereas intact selfcomponents are usually ignored. Nevertheless, clinical manifestations of autoimmunity occur in at least 5% of the general population. The exact causes of autoimmune diseases are elusive; however, genetic and environmental risk factors as well as an insufficient elimination of cells bearing autoreactive T-cell receptors (TCRs) in the thymus contribute to the evolvement of disease (1,2). To prevent autoimmunity, tolerance mechanisms including clonal deletion, induction of apoptosis, or anergy of self-reactive T-cells are essential. In addition, regulatory T-cells (T regs ) were identified as sentinels of the immune response keeping aberrant/exaggerated immune reactions in balance. Several distinct T-cell subsets with regulatory function have been identified so far including natural T regs , adaptive or induced T regs (iT reg ), type 1 regulatory T-cells (Tr1), T helper 3 cells (Th3), double-negative (dn) T-cells, γδ T-cells, and iNKT cells. In a number of autoimmune diseases a diminished prevalence and/or impaired function of T regs were observed (3). As several autoimmune disorders (such as rheumatoid arthritis or vasculitis) occur more frequently in the elderly, the question arises whether aging is linked to quantitative and/or qualitative defects of the T reg pool (4)(5)(6).
In this review we summarize current data about the effects of aging on T regs and highlight the possible mechanisms leading to senescence of T regs .

CHARACTERIZATION OF T REGS DEFINITION AND PHENOTYPE
Natural T regs develop in the thymus through recognition of selfantigen presented by thymic epithelial or dendritic cells. For this process CD28 co-stimulation is required, whereas IL-2 and TGF-β are less important as indicated by knock-out mice models (7).
Today, there is still no consensus on the reliable identification of T regs by flow cytometry. A variety of cell surface molecules have been proposed as specific T regs markers such as glucocorticoid-induced tumor necrosis factor receptor (GITR), cytotoxic T-lymphocyte associated antigen-4 (CTLA-4), the coreceptors Neuropilin-1 and PD-1, the adhesion molecule CD62L, major histocompatibility complex (MHC) class II DR, or CD45 isoforms. The type I cytokine receptor CD127 is a negative marker of T regs and the absence of this molecule is frequently used for T reg identification (8).
The forkhead transcription factor FoxP3 was proposed as the most specific marker of T regs as FoxP3 expression is essential for T reg development and function (9): T regs were unable to develop in a mouse receiving FoxP3-deficient progenitor cells from another animal (10) and retroviral expression of FoxP3 in human and murine T-cells enabled the conversion of non-regulatory naïve T-cells into a T reg -like phenotype with suppressive activity and surface expression of CD25 (9). A mutation of the FoxP3 gene in humans results in the fatal autoimmune syndrome IPEX (immune dysregulation, polyendocrinopathy, X-linked) (11). For experimental studies, however, FoxP3 appears not to be an optimal T reg marker because first, permeabilization of T-cells is necessary to stain FoxP3 and cells are thus not viable anymore and second, newer data indicate that human FoxP3 is up-regulated in activated T-cells without suppressive function as well (12).
The Ikaros family transcription factor Helios was proposed as an alternative indicator of human T regs with a higher specificity compared to FoxP3. Recent data, however indicate that Helios is also up-regulated in activated non-regulatory T-cells (13). In summary, there is currently no specific marker of human T regs available limiting the validity of studies investigating qualitative and/or quantitative changes of the T reg pool.

MECHANISM OF SUPPRESSION
The mechanisms of T reg mediated immunosuppression are still unclear. Most likely, T regs have multiple functions with direct and indirect inhibitory effects on antigen-presenting cells (APCs) and T-cells such as the following (14, 15): (a) expression of the surface molecule CTLA-4 directly suppressing the activity of Tcells, (b) indirect inhibition of effector cells by the induction of anti-inflammatory biochemical pathways in APC, (c) direct or indirect killing of effector cells and APCs, and/or (d) production of immunoregulatory cytokines such as TGF-β and IL-10 (16).
Interestingly, a recent study reported that human T regs are able to induce senescence of naïve and memory responder T-cells in vitro and in vivo. The resulting senescent T-cell subset had an altered phenotype and revealed potent suppressive functions. The mechanisms leading to senescence of non-regulatory T-cells were not completely understood; however, the phosphorylation of p38 and ERK1/2 signaling pathways inhibiting naïve T-cell growth and cell-cycle regulation appeared to play a role (17).

THE EFFECT OF AGING ON T REG PREVALENCES AND FUNCTION
A prevalence of approximately 0.6-15% out of the CD4 + T-cell pool has been reported for T regs in healthy adults and mice (4,18). The influence of aging on T reg prevalence in humans has been rarely studied so far and available reports suggest only minor changes of the circulating T reg pool through age (19). Higher proportions of T regs were only found in cord blood samples suggesting a pivotal role of T regs during homeostatic proliferation of naïve Tcells in the fetal life (20, 21). During the first 36 months of life T reg levels decline rapidly (22) and remain relatively stable thereafter.
Mouse studies showed increased T reg prevalences in lymphoid organs of aged compared to young animals, whereas frequencies in circulating blood and thymus were unchanged (23, 24). This finding led to the hypothesis that during aging T regs accumulate in lymphoid tissues; hypothetically explaining the increased susceptibility to infections and reduced vaccine response in elderly animals. The accumulation of T regs has further been observed in the skin of aged persons possibly resulting in a higher risk of skin cancer as T regs reduce local anti-tumor immune responses (25-27).
In animals, T reg function seems to decrease with advancing age. The transfer of CD25 + T regs from aged mice into young animals for example resulted in a lower suppression of delayed type hypersensitivity responses compared to the infusion of young T reg cells (23). Another study found that CD4 + CD25 high T regs from aged animals less efficiently inhibited the proinflammatory activity of IL-17 + T-cells compared to T regs from young mice (28). In human studies it was observed that T regs from young and elderly individuals similarly inhibited the proliferation of responder cells whereas the production of the anti-inflammatory cytokine IL-10 was reduced in cells from the older group. The phenotype of T regs including expression of CD25, FoxP3, IL-7Rα, or chemokine receptor expression, however, was unchanged (29). In conclusion T regs from aged individuals are less efficient in preventing the occurrence of autoimmunity, while their number remains unaltered.
On the other hand, cancer and infections occur more commonly in the elderly suggesting increased T reg responses (see also above) (29)(30)(31). One mouse study found an increase of T reg prevalences in aged animals correlating with a defective tumor clearance. CD25-depletion restored the anti-cancer immune response (32). Similarly, CD25-depletion in aged mice reduced the lesion size in a Leishmania major infection model (24). Others reported that the depletion of T regs with denileukin diftitox improved tumorspecific immunity only in young mice whereas tumor growth was unaffected in aged mice. This was explained by increased numbers of myeloid-derived suppressor cell (MDSC) in aged animals, and upon depletion of these cells tumor-specific immunity was restored (33).
In summary, current data on age-related changes of T reg prevalences and function are conflicting and do not completely explain the simultaneously increased risk of autoimmunity (suggesting lower T reg function), cancer, and infections (indicating increased T reg responses) in the elderly. Apart from the difficulty of a reliable identification of T regs the possible accumulation of T regs in lymphoid organs and/or tissues during aging might lead to an underestimation of the total T reg pool in current human studies. Future studies investigating tissue samples from immune-organs of elderly individuals would be desirable to better understand the role of T regs in the pathogenesis of age-related diseases.

T REG DEVELOPMENT AND HOMEOSTASIS
Development of natural T regs in the thymus depends on a positive selection process including high affinity interactions of the TCR to cortically expressed host antigens. Thymic stromal lymphopoietin activated CD11c-positive dendritic cells (34), co-stimulatory molecules including CD28, PD-1, CD40L (35) as well as the cytokine IL-2 were all shown to be crucial for thymic T reg generation (36)(37)(38). Besides, the Nr4a nuclear receptors (involved in apoptosis, proliferation, DNA repair, inflammation, and others) were recently reported to contribute to T reg development. Mice lacking these receptors in T-cells were unable to produce T regs and died early from systemic autoimmunity (39).
During aging a progressive degeneration of the thymus occurs leading to a substantial loss of its capacity to generate and export new T-cells (40,41). Throughout middle age thymic epithelial space and the functional unit of thymopoiesis (and thus the production of T-cells) decline by approximately 3% per year until the age of 45 when only an irrelevant level of functional thymic tissue remains. The total number of T-cells in the periphery nevertheless is unchanged and peripheral mechanisms of T-cell renewal have to compensate for progressive thymic failure (42)(43)(44).
Parallel to the overall reduction of thymic T-cell output the production of thymically derived T regs decreases with age (45). Alternative mechanisms such as increased surveillance of T regs in the elderly (46) as well as peripheral T reg generation may compensate for the loss of thymic function to maintain a sufficient T reg pool (see Figure 1). Indeed, numerous studies indicate a possible conversion of non-regulatory CD4 + CD25 − T-cells into T regs in vitro and in vivo (47,48). Moreover, mouse studies showed that peripheral self-antigen-driven proliferation of T regs is a thymusindependent mechanism to maintain T regs (49)(50)(51). The proportion of conventional T-cells differentiating into T regs as well as the relative contribution of homeostatic T reg proliferation to the overall T reg pool in elderly individuals are unknown.
Peripheral mechanisms of T-cell renewal (particularly homeostatic expansion of existing T regs ) are probably not infinite. Normally, T-cells proliferate beyond the seventh decade of life. Thereafter, telomere lengths are usually contracted to levels known as the "Hayflick limit". At this stage, non-regulatory T-cells do not proliferate anymore and undergo phenotypical and functional changes such as down-regulation of CD28 and acquisition of cytotoxic potential (4,52,53). Due to the fact that T regs display even shorter telomeres than non-regulatory T-cells, it is conceivable that peripherally proliferating T regs reach the "Hayflick limit" even earlier (54). Impaired T reg homeostasis may then result in immune dysfunction with increased risk of immune-mediated disorders.
In addition to the shortened telomere length, TCR diversity is also contracted to at least 100-fold in elderly individuals (55). This has been explained by the observation that homeostatic proliferation of T-cells is antigen dependent. Thus, T-cells with a high affinity TCR to self-antigens or antigens deriving from chronic virus infections have a survival advantage over other T-cells (42,56). Given that similar mechanisms drive peripheral proliferation of non-regulatory T-cells and T regs , a reduction of T reg TCR diversity (with a skew to certain antigens) can be expected in the elderly. Consequently, T regs could mediate increased immunosuppression in response to specific self-(even if transformed) or viral antigens with increased incidence of malignancies and infections in the elderly. At the same time the reduced diversity of T regs could result in decreased protection from autoimmunity (3).

DEVELOPMENT AND CELLULAR SENESCENCE OF T REGS FROM NAÏVE TO MEMORY CELL STATUS
Similar to the developmental stages known for non-regulatory Tcells (development form CD45RA + naïve to CD45RO + memory and finally to CD28 − memory effector T-cells), different cellular subsets of T regs were also observed. In humans, CD4 + foxP3 + T regs may have either a "naïve-like" phenotype characterized by the expression of CD25 + CD45RA + or a CD25 hi CD45RO + "memorylike" phenotype (54). In mice, naïve-like T regs were characterized by the expression of CD25, CD62L, and CCR7 and by preferential homing to antigen-draining lymph nodes, where they FIGURE 1 | Age-related changes of T reg homeostasis. In young individuals T regs are generated in the thymus and are released as "naïve-like" T regs into circulation. After antigen-contact, T regs develop into a "memory-like" phenotype. T reg homeostasis is supported by homeostatic proliferation of "naïve-like" and "memory-like" T regs as well as conversion of non-regulatory T-cells into T regs . Telomere length and T-cell receptor diversity is higher in naïve-like compared to memory-like T regs . After puberty thymic function is progressively lost and in aged individuals homeostatic proliferation of existing T regs as well as conversion of non-regulatory T-cells into T regs compensate for thymic failure to maintain T reg pool. Due to ongoing homeostatic replication telomere length and T-cell receptor diversity of T regs from elderly people are contracted compared to those from young individuals. Recurrent stimulation of T regs might then lead to a status of "terminal-differentiation" with altered phenotype and function. T reg regulatory T-cell, TCR . . . T-cell receptor.
In humans, the highest prevalence of naïve-like T regs were found in cord blood and it was assumed that these naïve-like T regs are produced in the thymus (20, 59). The prevalence of memory-like T regs increases rapidly during childhood and it was demonstrated that these memory-like T regs have shorter telomeres and a lower content of TCR excision circles (Trecs) compared to naïve-like T regs reflecting a longer replicative history (54). The mechanisms mediating the transition of a naïve-like T reg into a memory-like phenotype still have to be explored; however, it is believed that antigen experienced dendritic cells migrating to secondary lymphoid tissues are involved. T regs proliferate upon stimulation with autologous immature and mature dendritic cells (54,60). A low surface expression of CD45RB on memory-like T regs further supports the hypothesis of an antigen-driven development of naïve-like T regs . CD45RB is normally down-regulated after repeated antigen-contact (61).
Human adult peripheral blood usually contains both, naïvelike and memory-like T regs . Parallel to the reduction of total naïve T-cells, the quantity of naïve-like T regs declines with age whereas the prevalence of memory-like T regs increases (29,62). The total pool of circulating T regs ; however, remains unchanged as mentioned above (19). As naïve-like T regs exhibit a higher proliferative potential in vitro compared to memory-like T regs it can be expected that the capacity of the immune system to downregulate abnormal immune responses declines with age (54).

END-DIFFERENTIATED T REGS AND ASPECTS OF T REG SENESCENCE
Replicative senescence of T-cells is a prominent feature of aging resulting from homeostatic proliferation and repetitive antigen exposure (63). The most important phenotypic feature of senescent T-cells is the loss of the type I transmembrane protein CD28, a major co-stimulatory molecule (64). From the functional perspective, non-regulatory CD28 − T-cells produce large amounts of interferon γ, perforin, and granzyme B, providing them with the ability to lyse target cells (65). Another feature of CD28 − T-cells is their longevity and persistence that can be explained by defects in the apoptotic pathway with upregulation of bcl-2 and Fas-associated death domain like IL-2-converting enzymelike inhibitory protein (FLIP) (66,67). Terminally differentiated T-cells also acquire new stimulatory receptors including killer cell immunoglobulin-like receptors (KIRs) and Toll-like receptors (TLRs) (68,69). Thus, activation of CD4 + CD28 − T-cells no longer depends on professional antigen-presenting cells, rather it is promoted by stress molecules as well as bacterial and/or viral products (65). The frequency of terminally differentiated CD4 + CD28 − Tcells is increased in old individuals as well as in younger patients with autoimmune diseases such as rheumatoid arthritis or spondyloarthritis (70). Given that T regs proliferate in the periphery to maintain the total T reg pool after thymic failure it is plausible to hypothesize that T regs may undergo terminal-differentiation as well.
Interestingly, a proportion of T regs from aged mice showed decreased expression of CD25 (46, 71). These CD25 low T regs occurred predominantly in the spleen (24) but had comparable functional properties to CD25 + T regs . A similar CD4 + CD25 − foxP3 + T reg population has been observed in SLE patients. SLE patients are known to have a prematurely aged immune system (72) with accumulation of CD28 − T-cells. A detailed characterization of CD4 + CD25 − FoxP3 + T regs regarding the expression of naïve/memory T-cell markers or determination of telomere lengths was unfortunately not performed. Further evidence for the occurrence of T reg senescence was found in a study on healthy aged individuals reporting the occurrence of a CD8 + CD25 + T reg population lacking CD28 expression. These regulatory cells shared phenotypic and functional features with CD4 + T regs from the same population (73). The occurrence and possible characteristics of terminally differentiated CD4 + T regs is an interesting issue that has to be investigated by future studies.

CONCLUSION
Accumulating evidence suggests age-associated changes of T reg prevalence and/or T reg function. Due to involution of thymus after puberty peripheral mechanisms including homeostatic proliferation of T regs or conversion of non-regulatory T-cells into T regs compensate for the decreasing generation of new T reg cells. However, these peripheral mechanisms are limited; this leads to altered composition of the T reg pool. Age-related changes of T regs are suspected to increase the risk of autoimmunity, cancer, and infections in the elderly; however, the exact mechanisms are still poorly understood. Current studies are limited by the difficult identification of human T regs and the uncertainty whether circulating T regs reflect the total T reg pool or a cellular subset only. Future studies are required to investigate cellular senescence of T regs and possible therapeutic approaches targeting T regs in aged individuals.