T cell aging as a risk factor for autoimmunity

Abstract

Immune aging is a complex process rendering the host susceptible to cancer, infection, and insufficient tissue repair. Many autoimmune diseases preferentially occur during the second half of life, counterintuitive to the concept of excess adaptive immunity driving immune-mediated tissue damage. T cells are particularly susceptible to aging-imposed changes, as they are under extreme proliferative pressure to fulfill the demands of clonal expansion and of homeostatic T cell repopulation. T cells in older adults have a footprint of genetic and epigenetic changes, lack mitochondrial fitness, and fail to maintain proteostasis, diverging them from host protection to host injury. Here, we review recent progress in understanding how the human T-cell system ages and the evidence detailing how T cell aging contributes to autoimmune conditions. T cell aging is now recognized as a risk determinant in two prototypic autoimmune syndromes; rheumatoid arthritis and giant cell arteritis. The emerging concept adds susceptibility to autoimmune and autoinflammatory disease to the spectrum of aging-imposed adaptations and opens new opportunities for immunomodulatory therapy by restoring the functional intactness of aging T cells.

1. Introduction

Advancing age is associated with a spectrum of morbidities and mortality, attributable to progressive restructuring of all organ systems. The immune system stands out amongst the aging tissues, as immune aging has profound consequences for the host: the aging immune system fails to protect against infections and malignancies and leaves the host unable to repair structural damage. With expansion of life expectancy, it has now become clear that older adults are also more likely to develop autoimmune disease. By the time adults reach the 6^th decade of life, their T cell and B cell responses against self-antigens should be firmly established, raising important questions as to the mechanisms underlying autoimmune disease during the second half of life. Recent studies are disclosing an active participation of the aged adaptive immune system in autoimmune diseases, highlighting a central role for T cells and T cell aging-associated phenotypes (TASP) [1, 2]. The T cell system has unique mechanisms of replenishment, with new T cell generation totally dependent on an intact thymus that involutes during adolescence and early adulthood [3]. Consequently, generation of brand-new T cells begins to dwindle during the third decade of life, the naïve T cell compartment shrinks, and the memory T cell pool expands. As adults reach middle age, thymic T cell generation is replaced by homeostatic proliferation of peripheral T cells and accumulative antigen exposure inflates the memory T cell compartment [4]. As a reflection of chronic antigenic stimulation, memory T cells in older adults exhibit highly differentiated phenotypes [5, 6]. Amongst the TASP [2], exhausted and senescent T cells appear to be most important. Characteristics of senescent and exhausted T cells include mitochondrial dysfunction and epigenetic remodeling [7]. In addition, there is increasing recognition that many tolerance checkpoints are failing in aged T cells, resulting in aberrant T cell-driven tissue inflammation and breakdown of T cell self-tolerance thereby inducing or amplifying autoimmune responses [8].

In this review, we discuss the mechanism underlying T cell aging and the evidence supporting the concept that T cell aging contributes to the onset and progression of autoimmune conditions.

2. Basic Hallmarks of T cell aging

One striking hallmark of the immune aging is the involution of the thymus, the major organ responsible for the generation of self-restricted, self-tolerant and functional T cells [9]. In humans, thymic activity rapidly declines during adolescence and early adulthood [3]. The generation of nascent T cells is entirely dependent on the thymus, forcing middle-aged and older adults to resort to homeostatic proliferation of post-thymic T cells as the major means to generate new T cells after the age of 30 years [10]. Human T cell turnover rates remain stable over adult lifetime, which is consistent with the notion that even in the young adult, the majority of T cells are produced in the periphery [11]. Only in late life, a pickup in proliferation rates has been noticed, possibly because of increased cell death and evolving lymphopenia [12]. Naïve T cells decline with age both in absolute and relative numbers, likely due to the inability to maintain naivety in homeostatically proliferating cells. This decline is only modest for naïve CD4⁺ T cells but very striking for naïve CD8⁺ T cells even in healthy elderly [13]. The reduction in absolute numbers of naïve CD8⁺ T cells is one of the most significant hallmarks of T-cell aging. If cellular quiescence cannot be maintained, naïve T cells will undergo differentiation and transition into the memory T cell pool. Again, aging CD8⁺ T cells are more prone to differentiate [13], leading to inflation of the memory pool and the population of end-differentiated T cells.

Within the memory T cell population, the accumulation of terminally differentiated effector T cells is most significant, again much more so for the CD8 than the CD4 compartment. Terminally differentiated antigen-specific memory T cells that re-express CD45RA (T_EMRA cells) have been identified in both CD4⁺ and CD8⁺ T cells. T cells lose the expression of CD28 and CD27 and regain the expression of CD45RA. Such terminally differentiated CD45RA effector T cells are expanded at the expense of central memory and normal effector cells and comprise more than 50 % of the CD8⁺ compartment in most older individuals. Individuals with expansion of CD4⁺ T_EMRA cells can also be found, however, increased frequencies of CD4⁺ T_EMRA cells are typical for patients with chronic-inflammatory disease [14, 15]. Oligoclonality is most prevalent in the subset of T_EMRA cells, which is indicative of chronic antigenic stimulation underlying this expansion, such as viral infection and responses to autoantigens. The T_EMRA cell compartment is higher in individuals with chronic cytomegalovirus (CMV) infection, rheumatoid arthritis and coronary artery disease [16–19]. The proliferative pressure associated with T cell end-differentiation leaves an imprint that can be captured as somatic mutations, such as mutations found in clonally expanded CD8⁺ T cells from rheumatoid arthritis patients [20]. Acquired mutations such as in TET2 and DNMT3A confer T cell clonal growth advantage, imposing rapid contraction of TCR repertoire diversity [21]. Thus, aged T cells are genetically “marked”, and genetic changes need to be considered as a driving factor in T cell aging.

Naïve and memory T cells are usually in a state of quiescence, poised to proliferate and differentiate upon antigen stimulation. Maintaining quiescence is vital to retain self- renewal potential and differentiation plasticity throughout life. In the quiescent state, cell division and growth are coordinately downregulated, the cell enters reversible cell cycle arrest, adapts to low metabolic activity, reduced ribosome biogenesis and lowered protein synthesis [1]. Emerging evidence supports the concept that features of T cell aging are reflective of advanced cellular differentiation caused by the failure to maintain quiescence. Even in the absence of a cognate antigen, aged T cells tend to leave their quiescent state and accumulate as partially differentiated T cells. Since the traits of quiescence, self-renewal and proliferative capacity are tightly interlinked, aging T cells lose the ability to self-renew, identified as an important factor in the decline of CD8⁺ T cell responses with age [22, 23]. The failed regulation of balancing quiescence entry, maintenance, and exit has been implicated in the T cell aging process and has bene associated with changes in the epigenetic landscape [24, 25].

Not unexpectedly, progressive aging goes hand-in-hand with a breakdown of T cell intracellular activity and function, especially, regulation of proteostasis, maintenance of mitochondrial fitness and the handling of cellular “garbage”. The emerging common denominator is the transition of young T cells into pro-inflammatory effector T cells as they no longer can handle their bioenergetics and garbage disposal. T cells respond to persistent lifelong antigenic and non-antigenic stimulation by acquiring an exhausted or even senescent phenotype that restricts T cell competence for protective immunity and expands host-damaging immunity [2, 5]. Although senescent T cells are defective in proliferating properly after TCR activation, they show high transcriptional and lysosomal activity and secret a range of mediators, such as pro-inflammatory cytokines, a property often referred to as senescence-associated secretory phenotype (SASP) [26, 27]. The term SASP was originally defined for fibroblasts, which undergo irreversible cell cycle arrest and, thus, are truly senescent. The term is now often used for all cell types, although it is unknown which specific secreted products the respective cells release when they reach a senescent state. T cells do not enter irreversible cell cycle arrest; that would not be compatible with survival. Accordingly, the term SASP needs to be used with caution when applied to end-differentiated T cells. Nevertheless, it is believed that the release of secretory products from “old” cells results in increased circulating levels of certain cytokines, such as Interleukin-6 (IL-6) and tumor necrosis factor (TNF), contributing to inflammaging, the chronic, low-grade inflammation in the absence of infection that appears in aging adults [28, 29]. Recent studies have posited that T cells may be relevant contributors to inflammaging [30, 31]. T cells with dysfunctional mitochondria owing to mitochondrial transcription factor A (TFAM) deficiency act as accelerators of senescence and inflammaging [31, 32]. These studies have highlighted the intimate relationship of bioenergetics and T cell function, amplified in the setting of T cell aging (Figure 1).

Fig. 1. Basic hallmarks of the aging T cell system.

Aged-related changes occur at the level of the individual T cell as well as the T cell population. Throughout adult life, homeostatic T cell proliferation is the major mechanism for T cell replenishment rather than de novo generation by the thymus. Homeostatic T cell proliferation imposes proliferative stress and triggers adaptation mechanisms; self-renewal potential declines, naïve compartments shrink, and memory T cell compartments inflate. Genetic and epigenetic changes inflict functional differences in activation and differentiation. Mitochondrial fitness suffers due to insufficient mtDNA repair. Intracellularly, damaged proteins accumulate, amplifying deterioration of proteostasis and promoting progressive loss of mitochondrial function. With progressive age, T cells enter a state of end-differentiation and acquire pro-inflammatory, tissue-damaging effector functions. Through these mechanisms, T cell aging contributes to inflammaging and the loss of self-tolerance.

Here, we review the evidence supporting the idea that aged T cell contribute to the onset and progression of various autoimmune diseases and tissue inflammation.

3. Homeostatic proliferation as a risk factor for autoimmunity

Homeostatic proliferation is the major means of T cell replenishment in adults and is essential to maintain a functional T cell system for many years after the thymus has seized to generate meaningful numbers of new T cells [33]. While designed as a rescue mechanism to avoid depletion of the T cell compartment, it is becoming clear that homeostatic proliferation has pleiotropic effects, including exposing the host to the risk of autoinflammatory and autoimmune disease. Peripheral selection and T cell activation and differentiation induced by slow, but continuous T cell replication can generate a repertoire that is more difficult to control by peripheral tolerance mechanisms. Newly generated T cells may recognize self-antigen with above average affinity, recognize neoantigens (e.g. citrullination), have lowered TCR activation thresholds or be hyper-reactive to growth factors, all increasing their autoreactive potential [8]. Rheumatoid arthritis (RA) is one of the striking examples for the relevance of this model, where aging is an important risk factor for autoimmune disease. Epidemiological studies of rheumatoid arthritis have suggested that age is an important factor for both disease onset as well as disease severity [34, 35]. Even more significantly, accelerated immune aging by more than 20 years is one of the major immunological phenotypes of the adaptive immune system in RA patients. T cell receptor diversity is decreased in RA patients, which is consistent with the notion that T cells from RA patients have undergone a period of enhanced homeostatic proliferation, resulting in a peripheral selection of a contracted repertoire [36, 37].

Moreover, accelerated immune aging in RA is already evident prior to differentiation into lineages since shortened telomeres have been found in hematopoietic stem cells in RA patients [38, 39]. Such as a defect may contribute to defective generation of T cells at an earlier age in RA patients. Reduced expression of human telomerase reverse transcriptase (hTERT) in RA patients cause the defective induction of telomerase activity [40]. Reduced telomeric activity not only results in shortened telomere but is also associated with increased susceptibility to apoptosis as well as pyroptosis during T cell proliferation [41, 42]. RA T cells have low expression of the double-strand-break repair nuclease MRE11A, leading to telomeric damage and senescence marker upregulation [42, 43]. Notably, MRE11A is not only critical for the repair of nuclear DNA and telomeric ends, but also crucially involved in securing intactness of the mitochondrial genome, connecting genome instability with metabolic demise and age-inappropriate T cell loss [44–46].

As a functional consequence, these defects all converge in lymphopenia compensated by increased homeostatic proliferation, which then again, accelerates immune aging. Increased homeostatic proliferation inevitably will promote the selection of autoreactive T cells and the differentiation into memory-like cells, thereby provide a potential for overcoming peripheral tolerance mechanisms and inducing autoimmune tissue inflammation.

4. Genetic and epigenetic alterations with autoimmunity

As with any other cell type, one common determinant of T cell aging is the accumulation of genetic damage throughout life. Maintaining genome integrity and stability is one of the major challenges during aging [47]. Somatic mutations accumulate with age in various tissues, including in T cells that are under high proliferative pressure [21, 48]. Acquired mutations conferring clonal growth advantage have been identified in hematopoietic stem cells (HSCs), with strong age dependence of the mutational load. TET2 and DNMT3A are the most frequently reported genes which drive clonal hematopoiesis (CH) [49].

Not surprisingly, such mutations are associated with adult T cell malignancy [50]. As discussed above, thymic T cell generation in older adults is low. Thus, in healthy adults, T lymphocytes are largely spared from hematopoietic stem cell derived mutations, compared with other hematopoietic descendants, such as granulocyte and B lymphocytes [51]. However, older T cells also have an extensive replicative history due to homeostatic proliferation and antigen induced clonal expansion. Mutations accumulated in T cells during proliferation lead to the generation of clonal T cell populations that have growth advantage, imposing rapid contraction in T cell repertoire diversity [52]. Gain-of-function mutations of STAT3 have been found in abnormally expanded CD8⁺ cytotoxic T cells in large granular lymphocytic leukemia [53]. Interestingly, there is evidence suggesting that these mutations not only associate with malignancy but also with autoimmune conditions. Somatic mutations, such as in SLAMF6 and IRF1 have been discovered in clonally expanded CD8⁺ T cells in patients with newly diagnosed rheumatoid arthritis but not in CD4+ T cells [20].

Genomic instability of T cells may arise from several common sources, including spontaneous cytosine deamination, errors in DNA double-strand break repair, replication errors and large structural changes, all of which increase with progressive age [54]. In T cells, the dysfunction of the DNA repair machinery has been associated with autoimmune diseases. In naïve CD4⁺ T cells from patients with rheumatoid arthritis, the DNA damage load and the cell death rate are significantly higher than in age-matched healthy controls. DNA repair is blunted and delayed after radiation. RA T cells fail to produce sufficient transcripts and protein of the DNA repair kinase ataxia telangiectasia (AT) mutated (ATM). NBS1, RAD50, MRE11, and p53 are also repressed [41]. The downregulation of the double-strand-break repair nuclease MRE11A leads to telomeric damage, juxtacentromeric heterochromatin unfolding and upregulation of the senescence markers cyclin-dependent kinase inhibitor 1 (CDKN1A, p21) and cyclin-dependent kinase inhibitor 2A (CDKN2A, p16) [43]. In RA T cells, DNA repair and longevity are further diminished by insufficiency of the non-homologous-end-joining protein DNA-protein kinase catalytic subunit (DNA-PKcs) [55]. Specifically, DNA repair capacity in RA T cells is limited by the repression of the Ku70/80 dimer, disabling the functionality of DNA-PKcs and essentially jeopardizing genome stability.

The accumulation of broken DNA in aging immune cells is accompanied by a range of epigenetic alterations. An important factor during the aging process is the exposure to environmental stimuli which may leave long-lasting imprints in the immune system. Analysis of the epigenetic landscape has provided unequivocal evidence that environmental factors modify chromosomes without changing the underlying gene sequence, eventually resulting in altered gene expression [56]. Epigenetic mechanisms regulate the accessibility of transcriptional factors to certain DNA regions and ultimately direct gene transcription. Such regulations happen at multiple layers: alterations in chromatin DNA methylation, changes in histone modifications, variations in high-dimensional nuclear organization, all of which are dynamically changing with advancing age [57]. Some of these epigenetic alterations have clear functional consequences, such as altered expression of the IL-7 receptor (IL-7R) and age-dependent IL-7 signaling in memory CD8⁺ T cells [58].

Age associated DNA methylation changes in T cell may alter regulatory mechanisms and signaling networks that predispose to autoimmunity [59]. In giant cell arteritis (GCA), an autoimmune and autoinflammatory disease of the aorta and the large arteries, a genome-wide methylation array of vasculitic versus non-vasculitic arteries revealed hypomethylation related to increased activity of the calcineurin/nuclear factor of activated T cells (NFAT) pathway and increased pro-inflammatory cytokine expressions [60, 61]. Also, noncoding RNAs have been implicated in regulating processes relevant in T cell aging. Numerous microRNAs are subject to age-related change [62, 63]. One of the well-studied microRNAs in human T cells is miR-181a, which declines with age and has been mechanistically involved in controlling T cell responsiveness. The loss of miR-181a in older adults has been attributed to the decline of the transcription factor YY1. Low abundance of miR-181a blunts TCR signaling in aging CD4⁺ T cells by upregulating several negative regulators, including the phosphatase DUSP6 as well as the deacetylase SIRT1 [64, 65]. Conversely, microRNA miR-21 is typically upregulated with age. In activated CD4⁺ T cells, miR-21 biases the transcriptome of differentiating T cells away from memory T cells and toward inflammatory effector T cells [66]. Concordantly, miR-21 appears to be upregulated in actively inflamed GCA arteries, compatible with the accumulation of aged T cells in the vasculitic lesions [67].

More recent work has shed light on disease relevant mRNA modifications that occur in older adults. A note-worthy example are mRNA modifications in macrophages in patients with coronary artery disease (CAD). Atherosclerotic lesions underlying the stenotic process in the coronary arteries of CAD patients are densely populated by macrophages and T cells. Macrophages contribute to multiple pathogenic processes in the lesions, including the uptake of lipids and the presentation of antigen to T cells. Macrophages from CAD patients overexpress the methyltransferase METTL3, which controls the accumulation of N6-methyladenosine (m6A) on mRNA. METTL3-dependent methylation of CD155 mRNA results in stabilization of the transcript and high expression of CD155 protein. CD155 high expressing macrophages provide a stop signal to interacting T cells, essentially paralyzing T cell immunity [68]. Interestingly, in CAD patients T cell paralysis has relevance for the generation of anti-viral immunity, exemplified in the high risk for severe and fatal infection in SARS-CoV2-infected CAD patients.

Taken together, a diverse array of genetic and epigenetic alterations contributes to T cell aging. Together, aging-induced mutations and the shift in the epigenetic landscape render T cells from older adults susceptible to functional failure. This functional failure spans from insufficient anti-pathogen immunity to poor anti-tumor immunity, leaving older adults with a high risk for infection and malignancy. More interestingly, excessive inflammatory activity is now recognized as a manifestation of aging-imposed immunodeficiency and the spectrum of T-cell-aging-related morbidities now includes typical autoimmune diseases (Figure 2).

Fig. 2. Genetic and epigenetic changes during aging.

(A) With progressive age, proliferatively stressed T cells accumulate somatic mutations. (B) Genomic instability of T cells increases with age. In premature aging syndromes, such as rheumatoid arthritis, T cells fail to produce sufficient DNA repair kinase ataxia telangiectasia mutated (ATM) and the double-strand-break repair nuclease MRE11A, leading to DNA damage and excessive T cell loss. (C) Profiles of epigenetic alterations, including histone and DNA modifications have been described in aged T cells. (D) Altered abundance of noncoding RNAs has also been linked to the T cell aging process. Loss of miR-181a blunts TCR signaling in aged CD4⁺ T cells. MicroRNA miR-21 is upregulated with age and has been reported to accumulate in age-related autoimmune disease.

5. Defective proteostasis in T cell aging and in autoimmunity

Accumulation of intracellular damage is a universal hallmark of aging. Maintenance of protein homeostasis, or proteostasis is achieved by precisely coordinated proteolytic systems that contribute to destruction of misfolded, damaged and aged proteins [60][69]. The central mechanism that enables the recycling of large protein aggregates and of entire organelles is autophagy. In principle, autophagy involves the fusion of autophagosomes with lysosomes, promoting the degradation of the luminal cargo. Aging-induced dysregulation of autophagy has been identified as a critical process of advancing age, with relevance for a variety of cells and tissues, including T cells [70]. In older adults, autophagy tends to become insufficient due to numerous factors and the outcome is the loss of proteostasis. Functional autophagy is now recognized as a critical element in memory CD8⁺ T cell formation [71] and thus, the age-dependent defect in autophagy will lead to a decline in immunological memory. An important inhibitor of autophagy is mTORC1 [72], mechanistically linking mTORC1 signaling with disruption of proteostasis. miR-21 has been shown to be upregulated in old naïve CD4⁺ T cells undergoing activation, maintaining activity in the mTORC1 signaling pathway. This signaling event has been associated with inhibition of memory T cell development [66]. In naïve CD4⁺ T cells from older individuals, mTORC1 activation occurs at late endosomes and depends on the sensing of cytoplasmic amino acids [73]. The upstream signaling abnormality has been localized to FOXO1, a transcription factor that regulates lysosome function through the induction of TFEB. Notably, CD4+ T cells from older individuals rapidly downregulate FOXO1, triggering impairment of lysosomal activity and compensatory expansion of multivesicular bodies (MVBs). The failure of autophagic activity leaves older CD4⁺ T cells with an increased cell mass, which is now recognized as a senescence feature. One consequence of the expansion of MVBs is the acceleration of exosome release, endowing older T cells with additional means of cell-cell communication [74]. Taken together, the aged T cell is biased to differentiate into a short-lived effector T cell, that neglects autophagic activity, accumulates damaged proteins, expands exosome release and has all the means to function as a pro-inflammatory effector cell.

Several lines of evidence support the concept that intact autophagy is anti-inflammatory. Genetic manipulations designed to abolish autophagy in CD4⁺ T cells are known to result in systemic inflammation [75]. Similarly, the activation of the insulin-like growth factor (IGF) receptor enhances the protein kinase B-mammalian target of rapamycin (AKT-mTOR) pathway, which in turn bolsters aerobic glycolysis and promotes Th17 cells differentiation over that of Treg cells [76]. These regulatory events connect intracellular energy flux and utilization to the activity of the lysosomal system and identify autophagy as an anti-inflammatory process.

Swaying T cell differentiation away from Treg cells and towards short-lived effector T cells is a typical event in autoimmune disease, where it is tightly interlinked with the immunometabolic control of such T cells. CD4 T cells from RA patients have a defect in autophagy and shunt glucose towards the pentose-phosphate pathway, a mechanism that enables cell building and pro-inflammatory behavior [77].

Unopposed mTORC activation, directly relevant in promoting differentiation of T cells into IFN-gamma-producing and IL-17-producing effector cells is also a characteristic of T cells in the autoimmune disease giant cell arteritis, where T cell activation occurs at the endothelial layer of microvessels [78].

Some of the molecular mechanisms that link abnormal T cell differentiation and autoimmune disease to defects in the intracellular vesicle trafficking system are now understood. Proteomic analysis of T cells from patients with rheumatoid arthritis have identified a defect in posttranslational modification of proteins, impacting metabolic and functional regulation. Impaired protein myristylation in RA T cells has been implicated in how the energy sensor AMPK distributes intracellularly and how it communicates with mTORC1 [77]. Low production of N-myristoyltransferase in RA T cells disrupts the lipidation of AMPK and prevents the trafficking of the enzyme to the lysosomal surface. Poor accumulation of AMPK on the lysosomal surface leaves mTORC1 unopposed and enables continuous activation of the mTORC1 signaling pathway, shifting the T cells towards cell building, tissue invasion and cytokine production [77].

Increased activity of the mTOR signaling pathway is a consistent finding in patients with autoimmune disease. In the autoimmune vasculitides GCA and Takayasu arteritis (TAK), CD4⁺ T cells have spontaneous activation of mTORC1 [79]. SIRT1 is a critical regulator of mTORC1 as a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase. Peripheral blood T cells from GCA patients have lower SIRT1 activity than age-matched controls, sharing the phenotype encountered in the older individuals [80, 81]. A functionally relevant immune phenotype in GCA patients derives from the aberrant signaling in the NOTCH pathways. Specifically, NOTCH4 signaling suppresses the release of exosomes and with it the vesicular secretion of the enzyme NADPH oxidase 2 (NOX2). NOX2-containing exosomes are the functional unit of CD8⁺ T regulatory T cells and inhibit the activation of neighboring CD4⁺ T cells [82], therefore functioning as an important tolerance mechanism. The molecular mechanisms leading to the failure of CD8+ T regulatory cells in autoimmune vasculitis have been clarified [82]. Aberrant NOTCH4 signaling transcriptionally controls several RAB GTPases, resulting in the rerouting of endosomal trafficking and the suppression of exosome production, thereby inactivating the anti-inflammatory and tissue-protective activity of CD8⁺ T regulatory cells][83]. The functional deficiency of immunosuppressive CD8 T regulatory cells is part of the aging-associated decline of the adaptive immune system that ultimately causes uncontrolled tissue inflammation [1, 82].

With data emerging that place the loss of proteostasis upstream of age-related immunodeficiency, and the misrouting of the intracellular vesicle trafficking as one of the manifestations of inappropriate protein handling, the question arises whether proteostasis can be restored. One possible approach is to supply the NAD precursor Nicotinamide, restoring NAD-dependent processes. Supplementation of spermidine has been proposed as an alternative strategy and pharmacological inhibition of mTORC1 activity may provide an elegant solution towards resetting abnormalities related to abnormal proteostasis [84–86]. Targeting proteostasis and autophagy opens new avenues towards counteracting T cell aging and developing novel immunomodulatory interventions for autoimmune disease (Figure 3).

Fig. 3. Failing proteostasis and dwindling of mitochondrial fitness during aging.

With age, fundamental processes of maintaining cellular health, such as autophagy and lysosomal function, tend to become insufficient, resulting in the loss of proteostasis. Dysfunctional mitochondria fall short in generating bioenergy and intermediate metabolites, undermining their role in regulating nuclear processes and communicating with subcellular organelles. One of the outcomes of impaired mitochondrial fitness is a shift towards pro-inflammatory effector functions.

6. Mitochondrial as guardians of the immune system

It goes without saying that healthy and self-tolerant T cells require easy access to bioenergy and therefore depend on functional mitochondria. Mitochondrial dysfunction is widely recognized as a cause and manifestation of the aging process in most tissues, including T cells. CD4⁺ T cells from older individuals have more abundant mitochondrial proteins involved in the assembly of the electron transport chain, but they exhibit reduced oxidative phosphorylation, indicative of declining mitochondrial fitness. Defective autophagy probably results in accumulation of dysfunctional mitochondria [87]. Besides their irreplaceable role in generating ATP and thus securing the energy supply of the cell, mitochondria produce a broad spectrum of metabolic intermediates that function as signaling molecules. Mitochondrial intermediates, such as succinate, citrate, alpha ketoglutarate etc are directly involved in communicating with other subcellular organelles (e.g. the endoplasmic reticulum) and the nucleus [88]. Through anaplerosis and cataplerosis, mitochondria supply biosynthetic intermediates and metabolize essentially all energy carriers. The central role of mitochondria in bioenergy production and as a platform of communication are progressively lost as T cells age [89]. Ultimately, old mitochondria promote a pro-inflammatory T cell phenotype, supporting inflammaging and frank autoimmune disease.

Mitochondrial failure as a disease relevant mechanism has been implicated in several inflammatory conditions but is best understood in the autoimmune disease rheumatoid arthritis [2, 44]. Glucose metabolism, one of the primary metabolic pathways providing bioenergy and biosynthetic molecules, refers to the process of breaking down glucose into ATP and intermediate metabolites, including glycolysis, aerobic oxidation, and processing in the pentose phosphate pathway (PPP). Naïve CD4⁺ T cells from rheumatoid arthritis patient exhibit diminished glycolytic activity [78][90]. They utilize glucose in a distinct manner when compared to healthy naïve CD4⁺ T cells: they avoid glycolytic breakdown into lactate and instead divert glucose into the PPP, driving the accumulation of NADPH and consumption of cellular reactive oxygen species (ROS) [79][91]. Production of the mitochondrial intermediate succinate is impaired, and the mitochondrial tricarboxylic acid cycle (TCA) cycle is paralyzed [92]. The GDP-forming β subunit of succinate-CoA ligase (SUCLG2) deficiency in RA T cells reverts the tricarboxylic acid (TCA) cycle from the oxidative to the reductive direction. As a consequence, α-ketoglutarate, citrate, and acetyl-CoA (AcCoA) accumulate and in vitro and in vivo studies have indicated that the metabolic footprint of RA T cells has direct relevance for pathogenic effector functions [92]. In succinate^low AcCoA^hi RA T cells, tubulin acetylation stabilizes the microtubular cytoskeleton and directly affects cellular behavior. Notably, excessive acetylation changes the positioning of mitochondria, pulling them close to the nuclear membrane therefore exposing nuclear DNA to oxidative stress. Also, cytoskeletal rearrangements support cellular polarization, uropod formation, T cell migration, and tissue invasion [92]. Disruption of the TCA cycle in RA T cells has profound implications for mitochondrial aspartate production, disrupting proper communication between mitochondria and the endoplasmic reticulum [93]. Functional consequences include the expansion of ER membranes and selective accumulation of ribosomes on the ER surface. Remarkably, these ER-docking ribosomes are geared towards production of the cytokine TNF, the marker cytokine of rheumatoid arthritis. Thus, mitochondrial insufficiency turns RA T cells into TNF “superproducers”, directly supporting disease-relevant pathology [93].

Pathogenic consequences caused by the loss of mitochondrial intactness are not only related to the generation of bioenergy and to the production of metabolic intermediates. An important aspect of how mitochondria regulate cellular function stems from the fact that mitochondria contain DNA. The cell assigns a significant proportion of its negeri production towards repairing and maintaining nuclear DNA, but mitochondrial DNA is even more prone to damage because it lacks protective histones and lies in close vicinity of reactive oxygen species. Declining efficiency of the DNA repair machinery is now recognized as an aging-associated process. T cells from RA patients have been reported to accumulate DNA double strand breaks, directly associated with the transcriptional repression of the DNA repair kinase ataxia telangiectasia mutated (ATM) [41, 94]. Repair pathways dependent on DNA-PKcs, Ku70 and Ku80 are equally impaired [55], exposing the T cells to continuous cellular stress and increasing the propensity for premature T cell death. Excessive T cell loss will trigger intensified homeostatic proliferation, which functions as a driver of T cell end-differentiation and exhaustion. Unrepaired mitochondrial DNA leaks out of the mitochondria into the cytosol, where it is recognized by DNA sensors. In RA T cells, leakage has been attributed to the premature loss of the nuclease MRE11A in mitochondria. The leakage of mtDNA fragments into the cytosolic space induces the activation of inflammasomes, giving rise to the release of pro-inflammatory cytokines, specifically, IL-1β and IL-18 [42]. Inflammasome-induced cleavage of caspase 1 results in T cell pyroptosis, a highly inflammatory T cell death and has bene shown to sustain synovial tissue inflammation [42]. Caspase 1 activation has been reported in lymph node T cells from RA patients, placing the mitochondrial defect into lymphoid organs and upstream of the peripheral tissue lesions.

In essence, mitochondrial intactness is critically important to maintain T cell tolerance. Progressive mitochondrial failure, resulting from the inability to repair mitochondrial DNA, to assemble functional electron transport chains, to process metabolites and remove unhealthy mitochondria, all impose intense cellular stress that transitions T cells into tissue-invasive effector cells that support pathogenic immune responses. The different aspects of mitochondrial deficiency are key processes linking aging to autoimmunity (Figure 3).

7. T cell senescence and autoimmune diseases

Over lifetime, the immune system is challenged by daily exposure to infectious pathogens and malignant cells. As a result of persistent antigenic stimulation, especially through chronic viral infections, T cells are driven into repetitive cycles of differentiation and eventually become exhausted and acquire a senescent-like phenotype. As a rule, exhausted and senescent T cells are less efficient in generating protective immunity but are biased towards inefficient and pro-inflammatory response patterns [5, 95]. Senescent T cells are distinct from exhausted T cells in several signaling pathways and in their secretory profile. It is important to note that human T cells do not acquire a classical senesce phenotype, which would require that they undergo irreversible cell cycle arrest. They slow in cell cycle activity but generally are still able to proliferate. Senescent T cells, like other senescent cell types, secrete a range of mediators, including pro-inflammatory cytokines, a property known as senescent-associated secretory phenotype (SASP) [27, 96]. T effector memory CD45RA cells (T_EMRA cells) exhibit many characteristics of cellular senescence, including heightened inflammatory responses [97]. However, the cell cycle arrest of T_EMRA cells is reversible, which distinguishes them from classical senescent cells [98]. In old T_EMRA cells, stress-sensing sestrin proteins can activate the ERK-JNK-p38 MAPK pathway independently of regulators of the upstream MAPK cascade [99, 100]. Sestrins also induce reprogramming of senescent-like CD8⁺ T cells to innate-like killers. They downregulate the signaling activity of the TCR and instead express a protein complex containing the agonistic natural killer (NK) receptor NKG2D and the NK adaptor molecule DAP12, which promotes cytotoxicity against cells that express NKG2D ligands [101]. Accumulation of clonally expanded cytotoxic CD4⁺ T cells have been found in supercentenarians (people who have reached 110 years old) and in aged mice. Cytotoxic CD4+ T cells produce high levels of pro-inflammatory factors, including granzymes and perforin, similar to cytotoxic CD8⁺ T cells [102, 103]. Such cytotoxic CD4⁺ T cells are highly enriched in patients with coronary artery disease, where they damage endothelial cells and smooth muscle cells and accumulate in the atherosclerotic vessel wall lesions [19, 104–107]. Such cytotoxic CD4⁺ T cells are a prime example how T cell aging renders individuals susceptible to harmful immune responses, indistinguishable from autoimmune disease.

Senescent T cells have been involved in the pathogenesis of age-related diseases and have been recognized as an effector T cells in autoimmune tissue inflammation. Discovery of CD4⁺ CD28^- T cells in the inflamed synovium was the first clue that patients with rheumatoid arthritis have premature immune aging [16]. NKG2D has been reported to enhance the pathogenicity of Th1 and Th17 cells and enhance the production of pro-inflammatory cytokines [108]. CD4⁺ CD28^- T cells with upregulation of the NK receptor NKG2D [109] have bene placed into the vascular lesions of GCA patients, similar to the enrichment of senescent T cells in rheumatoid arthritis [110]. T cell senescence is now accepted as a critical driver of inflammaging, directly relevant for a broad spectrum of chronic inflammatory conditions [28, 111].

Not unexpectedly, T cell senescence has emerged as an attractive target for potential therapeutic strategies to rejuvenate the immune system and, in parallel, provide novel immunomodulators to treat autoimmune disease. One approach has been to simply deplete senescent cells, thereby eliminating a source of pro-inflammatory cytokines. Several compounds have been used to deplete senescent cells by targeting their survival pathways and by utilizing immune-mediated clearance [112]. The p38 MAPK and mTORC signaling pathways are notoriously upregulated in senescent T cells, providing targets for inhibitor therapy. Blocking the p38 MAPK pathway can rescue some senescence-associated defects in T_EMRA cells and improve their proliferation, telomerase activity, mitochondrial function and autophagy [98, 100, 113]. Low-dose inhibition of both mTORC1 and mTORC2 using the ATP mimetic pan-mTOR inhibitor AZD8055 reversed major phenotypes of senescence in near-senescent fibroblasts [114]. Treatment of senescent preadipocytes with a pan-Janus kinase (JAK) inhibitor alleviates SASP and production of pro-inflammatory mediators in vivo, possibly by interfering with a positive feedback loop from secreted cytokines [115]. Phytochemicals such as polyphenols, probiotic microbes and omega-3 fatty acids have also been reported to reverse immunosenescence and cellular senescence [116].

In summary, chronically stimulated T cells that reach senescence are inferior when it comes to protecting the host but superior when it comes to pro-inflammatory effector functions. Given typical epigenetic, transcriptomic and proteomic patterns in such end-differentiated T cells, it is tempting to try rejuvenating them, re-educating them or re-using them as potent effector cells. Available data have identified such end-differentiated T cells as key drivers of disease, emphasizing the need to develop means to control their functional activity. On the other hand, they may still possess protective functions, e.g. suppressing reactivation of latent virus infections in the older adult and depleting them may have harmful consequences for the aging host.

8. Conclusion and perspectives

The classical paradigm has made the assumption that autoimmune disease is a manifestation of “hyperimmunity”, too much immunity to autoantigen. Consequently, autoimmunity would be a disease of the young. However, epidemiologic data show that autoimmunity is a problem in the older adult. In the granulomatous vasculitis giant cell arteritis, inflammatory disease of the medium and large arteries occurs exclusively after the age of 50 years [117, 118]. The risk to be diagnosed with rheumatoid arthritis is highest in postmenopausal women [2]. How can the immune system of an aging individual become autoimmune? Progressive age is associated with profound restructuring of the immune system. The daily challenge of the immune system by infections, malignancies and damaged tissues imposes enormous replication stress to keep the pool of innate and adaptive immune cells filled. The aging bone marrow produce myeloid cells with high inflammatory potential [119]. Involution of the thymus starting with the second decade of life enforces T cell repopulation through a different mechanism: the homeostatic proliferation of post-thymic T cells. With progressive age, T cells become exposed to increasing proliferative stress, resulting in reprogramming of the adaptive immune system and the accumulation of hyperreactive, tissue-invasive and pro-inflammatory T cells.

Progress in mechanistic studies have identified key processes that turn young and protective T cells into old and autoreactive T cells [1](Table 1 and Table 2):

Table 1.

T cell aging associated phenotypes (TASP) in Rheumatoid Arthritis

Phenotype	Functional outcome
Age-inappropriate telomere erosion	Premature T cell aging T cell exhaustion
Mitochondrial deficiency	T cell exhaustion Immunodeficiency
Mitochondrial deficiency	Insufficient lipid oxidation Excess generation of invasive membrane ruffles
TCA cycle deficiency	Excess acetyl-CoA cytoskeleton hyperacetylation cellular polarization tissue invasiveness
TCA cycle deficiency	Low aspartate, low NAD ER stress and expansion Excess TNF production
Defective protein myristoylation	Misplacement of AMPK Unopposed mTORC1 activation
Persistent mTORC1 activation	TH1 lineage commitment
Impaired glycolysis	Shunting into the pentose phosphate pathway Cell building program T cell exhaustion
Defective autophagy	Accumulation of dysfunctional mitochondria
Long-lived tissue macrophages with high HLA-DR expression	Continuous antigen-presentation and T cell activation in the synovial tissue

Table 2.

T cell aging-associated phenotypes (TASP) in giant cell arteritis

Phenotype	Functional outcome
Aberrant NOTCH1 signaling	Linage bias towards TH1 and TH17 effector differentiation
Aberrant NOTCH1 signaling	T cell-endothelial cell interaction via NOTCH-NOTCH ligand binding
Aberrant NOTCH4 signaling	Inactivation of immunosuppressive T regulatory cells
Deficiency of PD-L1-expressing antigen-presenting cells	Unopposed T cell activation
MMP9-producing monocytes and macrophages	Basal membrane digestion and T cell tissue invasion

• Proteostatic mechanisms fail, the cell is not able to get rid of its garbage. The outcome is “garbaging” [120] (Figure 4).

• Autophagy and mitophagy declines, allowing the accumulation of dysfunctional mitochondria.

• Proliferative stress endorses DNA damage, both in the nucleus and the mitochondria.

• Mitochondrial DNA leaks into the cytoplasm, where it is recognized by DNA sensors that trigger inflammasome activation and pyroptotic death

• The mitochondrial electron transport chain fails, ATP production dwindles and aged T cells become energy depleted.

• The mitochondrial TCA cycle hyper-produces citrate and under-produces malate. The cytosol is loaded with acetyl-CoA, the malate shuttle fails and aspartate concentrations are low. Functional outcomes include the hyperacetylation of the cytoskeleton, cellular polarization and tissue-invasive capabilities.

• Impaired signaling from the mitochondria to the ER causes expansion of ER membranes, biased co-translational translocation and hyperproduction of TNF.

• Overall, the aged T cell fails to take care of its garbage, adapts to a dysfunctional mitochondria and fundamentally changes its cellular metabolism [45].

• The TNF-producing, hyper-migratory, tissue-invasive T cell with rapid commitment to effector differentiation creates ideal conditions for autoreactivity and tissue inflammation.

Fig. 4. Failing proteostasis results in “garbaging”.

Proteostatic mechanisms fail, the cell is not able to get rid of its garbage, such as misfoled proteins and DAMPs. The outcome is “garbaging”.

The development of a therapeutic armamentarium permitting the reversal of aged into young T cells would hit two birds with one stone: render the old host immunocompetent in the fight against infection and cancer and protect the host from pathogenic immunity that manifests as autoimmune disease.

Highlights:

• Aged T cells have distinct genetic and epigenetic profiles, impaired mitochondrial function, insufficient proteostasis, and can reach a senescent-like state

• Age-related T cell dysfunction can lead to loss of immune tolerance

• Aged T cells differentiate into tissue-invasive, pro-inflammatory effector cells, competent to trigger and sustain tissue-damaging autoimmunity

• T cell aging emerges as an important risk factor for autoimmune disease