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PURPOSE OF REVIEW: Obesity has increased worldwide recently and represents a major global health challenge. This review focuses on the obesity-associated cellular senescence in various organs and the role of these senescent cells (SnCs) in driving complications associated with obesity. Also, the ability to target SnCs pharmacologically with drugs termed senotherapeutics as a therapy for these complications is discussed. RECENT FINDINGS: Several studies have shown a positive correlation between obesity and SnC burden in organs such as adipose tissue, liver, and pancreatic-β-cells. These SnCs produce several secretory factors which affect other cells and tissues in a paracrine manner resulting in organ dysfunction. The accumulation of SnCs in adipocytes affects their lipid storage and impairs adipogenesis. The inflammatory senescence-associated secretory phenotype (SASP) of SnCs downregulates the antioxidant capacity and mitochondrial function in tissues. Senescent hepatocytes cannot oxidize fatty acids, which leads to lipid deposition and senescence in β-cells decrease function. These and other adverse effects of SnCs contribute to insulin resistance and type-2 diabetes. The reduction in the SnC burden genetically or pharmacologically improves the complications associated with obesity. SUMMARY: The accumulation of SnCs with age and disease accelerates aging. Obesity is a key driver of SnC accumulation, and the complications associated with obesity can be controlled by reducing the SnC burden. Thus, senotherapeutic drugs have the potential to be an effective therapeutic option.

The aging of the immune system, or immunosenescence, was recently verified to have a causal role in driving the aging of solid organs, while the senolytic elimination of senescent immune cells was found to effectively delay systemic aging. Our recent study also showed that immune cells in severely dystrophic muscles develop senescence-like phenotypes, including the increased expression of senescence-associated secretory phenotype (SASP) factors and senescence markers. Here we further investigated whether the specific clearance of senescent immune cells in dystrophic muscle may effectively improve the function of muscle stem cells and the phenotypes of dystrophic muscle. We observed increased percentage of senescent cells in macrophages from

MicroRNAs (miRNAs) have been demonstrated to modulate life span in the invertebratesC. elegansandDrosophilaby targeting conserved pathways of aging, such as insulin/IGF-1 signaling (IIS). However, a role for miRNAs in modulating human longevity has not been fully explored. Here we investigated novel roles of miRNAs as a major epigenetic component of exceptional longevity in humans. By profiling the miRNAs in B-cells from Ashkenazi Jewish centenarians and 70-year-old controls without a longevity history, we found that the majority of differentially expressed miRNAs were upregulated in centenarians and predicted to modulate the IIS pathway. Notably, decreased IIS activity was found in B cells from centenarians who harbored these upregulated miRNAs. miR-142-3p, the top upregulated miRNA, was verified to dampen the IIS pathway by targeting multiple genes includingGNB2, AKT1S1, RHEBandFURIN. Overexpression of miR-142-3p improved the stress resistance under genotoxicity and induced the impairment of cell cycle progression in IMR90 cells. Furthermore, mice injected with a miR-142-3p mimic showed reduced IIS signaling and improved longevity-associated phenotypes including enhanced stress resistance, improved diet/aging-induced glucose intolerance, and longevity-associated change of metabolic profile. These data suggest that miR-142-3p is involved in human longevity through regulating IIS-mediated pro-longevity effects. This study provides strong support for the use of miR-142-3p as a novel therapeutic to promote longevity or prevent aging/aging-related diseases in human.

Given the pro and anti-geronic roles of the TGF-β superfamily in aging, we hypothesized that human longevity involves genetic variation in TGF-β signaling genes. Here we utilized a candidate functional genomic approach to identify and characterize functional variants in TGF- β signaling associated with human longevity. Targeted sequencing of 113 genes involved in aging- associated TGF- β signaling in an Ashkenazi Jewish centenarian cohort identified genetic variants robustly associated with human longevity. In particular, a centenarian-enriched intronic variant residing in a cell-type specific enhancer in SMAD3, a critical receptor-regulated TGF- β signal transducer, was identified. This non-coding SMAD3 variant (rs8040709) altered binding of ELK1, a member of the ETS family of transcription factor important for enhancer activity in certain cell types, resulting in reduced SMAD3 expression. Analysis of the variant in cell types derived from gene edited iPSCs demonstrated the variant reduced SMAD3 expression, senescence and inflammation in endothelial cells. In addition, heterozygosity in SMAD3 improved healthspan and reduced senescence in theErcc1-/Δprogeroid mouse model of accelerated aging. Taken together, these experiments demonstrate that variants in a cell type specific enhancer of SMAD3 resulted in reduced expression, senescence and inflammation and contributes to human longevity. Thus, SMAD3 represents a validated targeted for drug development for extending human healthspan.

Cellular senescence, a hallmark of aging, has been implicated in the pathogenesis of many major age-related disorders, including neurodegeneration, atherosclerosis, and metabolic disease. Therefore, investigating novel methods to reduce or delay the accumulation of senescent cells during aging may attenuate age-related pathologies. microRNA-449a-5p (miR-449a) is a small, noncoding RNA down-regulated with age in normal mice but maintained in long-living growth hormone (GH)-deficient Ames Dwarf (df/df) mice. We found increased fibroadipogenic precursor cells, adipose-derived stem cells, and miR-449a levels in visceral adipose tissue of long-living df/df mice. Gene target analysis and our functional study with miR-449a-5p have revealed its potential as a serotherapeutic. Here, we test the hypothesis that miR-449a reduces cellular senescence by targeting senescence-associated genes induced in response to strong mitogenic signals and other damaging stimuli. We demonstrated that GH downregulates miR-449a expression and accelerates senescence while miR-449a upregulation using mimetics reduces senescence, primarily through targeted reduction of p16 Ink4a , p21 Cip1 , and the PI3K-mTOR signaling pathway. Our results demonstrate that miR-449a is important in modulating key signaling pathways that control cellular senescence and the progression of age-related pathologies.

In addition to reducing fracture risk, zoledronate has been found in some studies to decrease mortality in humans and extend lifespan and healthspan in animals. Because senescent cells accumulate with aging and contribute to multiple co-morbidities, the non-skeletal actions of zoledronate could be due to senolytic (killing of senescent cells) or senomorphic (inhibition of the secretion of the senescence-associated secretory phenotype [SASP]) actions. To test this, we first performedin vitrosenescence assays using human lung fibroblasts and DNA repair-deficient mouse embryonic fibroblasts, which demonstrated that zoledronate killed senescent cells with minimal effects on non-senescent cells. Next, in aged mice treated with zoledronate or vehicle for 8 weeks, zoledronate significantly reduced circulating SASP factors, including CCL7, IL-1β, TNFRSF1A, and TGFβ1 and improved grip strength. Analysis of publicly available RNAseq data from CD115+ (CSF1R/c-fms+) pre-osteoclastic cells isolated from mice treated with zoledronate demonstrated a significant downregulation of senescence/SASP genes (SenMayo). To establish that these cells are potential senolytic/senomorphic targets of zoledronate, we used single cell proteomic analysis (cytometry by time of flight [CyTOF]) and demonstrated that zoledronate significantly reduced the number of pre-osteoclastic (CD115+/CD3e-/Ly6G-/CD45R-) cells and decreased protein levels of p16, p21, and SASP markers in these cells without affecting other immune cell populations. Collectively, our findings demonstrate that zoledronate has senolytic effectsin vitroand modulates senescence/SASP biomarkersin vivo. These data point to the need for additional studies testing zoledronate and/or other bisphosphonate derivatives for senotherapeutic efficacy.

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The concept of geroscience is that since ageing is the greatest risk factor for many diseases and conditions, targeting the ageing process itself will have the greatest impact on human health. Of the hallmarks of ageing, cellular senescence has emerged as a druggable therapeutic target for extending healthspan in model organisms. Cellular senescence is a cell state of irreversible proliferative arrest driven by different types of stress, including oncogene-induced stress. Many senescent cells (SnCs) develop a senescent-associated secretory phenotype (SASP) comprising pro-inflammatory cytokines, chemokines, proteases, bioactive lipids, inhibitory molecules, extracellular vesicles, metabolites, lipids and other factors, able to promote chronic inflammation and tissue dysfunction. SnCs up-regulate senescent cell anti-apoptotic pathways (SCAPs) that prevent them from dying despite the accumulation of damage to DNA and other organelles. These SCAPs and other pathways altered in SnCs represent therapeutic targets for the development of senotherapeutic drugs that induce selective cell death of SnCs, specifically termed senolytics or suppress markers of senescence, in particular the SASP, termed senomorphics. Here, we review the current state of the development of senolytics and senomorphics for the treatment of age-related diseases and disorders and extension of healthy longevity. In addition, the challenges of documenting senolytic and senomorphic activity in pre-clinical models and the current state of the clinical application of the different senotherapeutics will be discussed.

MicroRNAs (miRNAs) are potent regulators of multiple biological processes. Previous studies have demonstrated that miR-146a-5p increases in normal mice during aging, while long-living Ames dwarf (df/df) mice maintain youthful levels of this miRNA. The aim of this study was to elucidate the involvement of miR-146a-5p in modulating cellular senescence and apoptosis in visceral adipose tissue of df/df mice and cultured pre-adipocytes. To test the effects of miR-146a-5p overexpression on visceral adipose tissue, wild-type, and df/df mice, were treated with miRNA-negative control-base and df/df were transfected with 4 or 8 µg/g of a miR-146a-5p mimetic, respectively. Effects of miR-146a-5p overexpression were also evaluated in 3T3-L1 cells cultured under high and normal glucose conditions. Treatment with miR-146a-5p mimetic increased cellular senescence and inflammation and decreased pro-apoptotic factors in visceral adipose tissue of df/df mice. The miR-146a-5p mimetic induced similar effects in 3T3-L1 cells cultivated at normal but not high glucose levels. Importantly, 3T3-L1 HG cells in high glucose conditions showed significantly higher expression of miR-146a-5p than 3T3-L1 grown in normal glucose conditions. These results indicate that miR-146a-5p can be a marker for cellular senescence. This miRNA represents one of the significant SASP factors that if not precisely regulated, can accentuate inflammatory responses and stimulate senescence in surrounding non-senescent cells. The role of miR-146a-5p is different in healthy versus stressed cells, suggesting potential effects of this miRNA depend on overall organismal health, aging, and metabolic state. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11357-021-00490-3.

Aging of the skin is evidenced by increased wrinkles, age spots, dryness, and thinning with decreased elasticity. Extrinsic and intrinsic factors including UV, pollution, and inflammation lead to an increase in senescent cells (SnCs) in skin with age that contribute to these observed pathological changes. Cellular senescence is induced by multiple types of damage and stress and is characterized by the irreversible exit from the cell cycle with upregulation of cell cycle-dependent kinase inhibitors p16INK4a and p21CIP1. Most SnCs also developed an inflammatory senescence-associated secretory phenotype (SASP) that drives further pathology through paracrine effects on neighboring cells and endocrine effects on cells at a distance. Recently, compounds able to kill senescent cells specifically, termed senolytics, or suppress the SASP, termed senomorphics, have been developed that have the potential to improve skin aging as well as systemic aging in general. Here, we provide a summary of the evidence for a key role in cellular senescence in driving skin aging. In addition, the evidence for the potential application of senotherapeutics for skin treatments is presented. Overall, topical, and possibly oral senotherapeutic treatments have tremendous potential to eventually become a standard of care for skin aging and related skin disorders.

Senescent cells, which arise due to damage-associated signals, are apoptosis-resistant and can express a pro-inflammatory, tissue-destructive senescence-associated secretory phenotype (SASP). We recently reported that a component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface protein, S1, can amplify the SASP of senescent cultured human cells and that a related mouse β-coronavirus, mouse hepatitis virus (MHV), increases SASP factors and senescent cell burden in infected mice. Here, we show that SARS-CoV-2 induces senescence in human non-senescent cells and exacerbates the SASP in human senescent cells through Toll-like receptor-3 (TLR-3). TLR-3, which senses viral RNA, was increased in human senescent compared to non-senescent cells. Notably, genetically or pharmacologically inhibiting TLR-3 prevented senescence induction and SASP amplification by SARS-CoV-2 or Spike pseudotyped virus. While an artificial TLR-3 agonist alone was not sufficient to induce senescence, it amplified the SASP in senescent human cells. Consistent with these findings, lung p16INK4a+ senescent cell burden was higher in patients who died from acute SARS-CoV-2 infection than other causes. Our results suggest that induction of cellular senescence and SASP amplification through TLR-3 contribute to SARS-CoV-2 morbidity, indicating that clinical trials of senolytics and/or SASP/TLR-3 inhibitors for alleviating acute and long-term SARS-CoV-2 sequelae are warranted.

Extreme longevity in humans has a strong genetic component, but whether this involves genetic variation in the same longevity pathways as found in model organisms is unclear. Using whole-exome sequences of a large cohort of Ashkenazi Jewish centenarians to examine enrichment for rare coding variants, we found most longevity-associated rare coding variants converge upon conserved insulin/insulin-like growth factor 1 signaling and AMP-activating protein kinase signaling pathways. Centenarians have a number of pathogenic rare coding variants similar to control individuals, suggesting that rare variants detected in the conserved longevity pathways are protective against age-related pathology. Indeed, we detected a pro-longevity effect of rare coding variants in the Wnt signaling pathway on individuals harboring the known common risk allele APOE4. The genetic component of extreme human longevity constitutes, at least in part, rare coding variants in pathways that protect against aging, including those that control longevity in model organisms. In this whole-exome sequencing study of the largest centenarian cohort to date, Lin et al. demonstrate that conserved pathways—for example, IIS and AMPK signaling—are as relevant to human longevity and healthy aging as they are in worms, flies and mice.

The clinical severity of coronavirus disease 2019 (COVID-19) is largely determined by host factors. Recent advances point to cellular senescence, an ageing-related switch in cellular state, as a critical regulator of SARS-CoV-2-evoked hyperinflammation. SARS-CoV-2, like other viruses, can induce senescence and exacerbates the senescence-associated secretory phenotype (SASP), which is comprised largely of pro-inflammatory, extracellular matrix-degrading, complement-activating and pro-coagulatory factors secreted by senescent cells. These effects are enhanced in elderly individuals who have an increased proportion of pre-existing senescent cells in their tissues. SASP factors can contribute to a 'cytokine storm', tissue-destructive immune cell infiltration, endothelialitis (endotheliitis), fibrosis and microthrombosis. SASP-driven spreading of cellular senescence uncouples tissue injury from direct SARS-CoV-2-inflicted cellular damage in a paracrine fashion and can further amplify the SASP by increasing the burden of senescent cells. Preclinical and early clinical studies indicate that targeted elimination of senescent cells may offer a novel therapeutic opportunity to attenuate clinical deterioration in COVID-19 and improve resilience following infection with SARS-CoV-2 or other pathogens.

Sirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in aging and metabolism regulation. Targeted sequencing of SIRT6 locus in a population of 450 Ashkenazi Jewish (AJ) centenarians and 550 AJ individuals without a family history of exceptional longevity identified enrichment of a SIRT6 allele containing two linked substitutions (N308K/A313S) in centenarians compared with AJ control individuals. Characterization of this SIRT6 allele (centSIRT6) demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double-strand break repair, and more robustly kill cancer cells compared with wild-type SIRT6. Surprisingly, centSIRT6 displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD

Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly(1,2). To define the contribution of immune system ageing to organism ageing, here we selectively deleted Ercc1, which encodes a crucial DNA repair protein(3,4), in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence(5–7) in the immune system only. We show that Vav-iCre(+/−);Ercc1(−/fl) mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice(8–10). Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre(+/−);Ercc1(−/fl) or aged wild-type mice into young mice induced senescence in trans, whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre(+/−);Ercc1(−/fl) mice with rapamycin reduced markers of senescence in immune cells and improved immune function(11,12). These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing.

Cellular senescence, which is a major cause of tissue dysfunction with aging and multiple other conditions, is known to be triggered by p16

Parabiosis is a well-established method to facilitate a shared blood supply between two conjoined animals. In particular, the pairing of mice of dissimilar ages, termed heterochronic parabiosis, has been used extensively for differentiating cell autonomous and non-autonomous mechanisms of aging. Analysis of heterochronic parabionts also has helped to identify individual circulating factors that may act as either pro- or anti-geronics. Heterochronic parabiosis also has proven to be a valuable experimental system to evaluate the effects of specific hallmarks of aging on the process of aging. For example, heterochronic parabiosis was used recently to examine whether cellular senescence was driven via cell autonomous and/or non-autonomous mechanisms. As anticipated, markers of cellular senescence were elevated in old isochronically-paired mice relative to young controls. However, compared to old isochronically paired mice, the senescent cell burden was reduced in multiple tissues of old parabionts joined with young mice. This suggests that the rejuvenation of cells and tissues in old mice by exposure to young blood could be mediated, in part, through suppression or immune clearance of senescent cells. Conversely, young heterochronic parabionts showed increased markers of cellular senescence, demonstrating that exposure to an old circulation is able to drive senescence through a cell non-autonomous mechanism(s), likely contributing to accelerated aging in the young mice. Thus, heterochronic parabiosis is still an important methodology that should continue to be leveraged for evaluating other hallmarks of aging and their mechanisms.

Whether disc aging is influenced by factors beyond its local environment is an important unresolved question. Here we performed heterochronic parabiosis in mice to study the effects of circulating factors in young and old blood on age-associated intervertebral disc degeneration. Compared to young isochronic pairs (Y-Y), young mice paired with old mice (Y-O) showed significant increases in levels of disc MMP-13 and ADAMTS4, aggrecan fragmentation, and histologic tissue degeneration, but negligible changes in cellular senescence markers (p16INK4a, p21Cip1). Compared to old isochronic pairs (O-O), old mice paired with young mice (O-Y) exhibited a significant decrease in expression of cellular senescence markers (p16, p21, p53), but only marginal decreases in the levels of disc MMP-13 and ADAMTS4, aggrecan fragmentation, and histologic degeneration. Thus, exposing old mice to young blood circulation greatly suppressed disc cellular senescence, but only slightly decreased disc matrix imbalance and degeneration. Conversely, exposing young mice to old blood accelerated their disc matrix imbalance and tissue degeneration, with little effects on disc cellular senescence. Thus, non-cell autonomous effects of circulating factors on disc cellular senescence and matrix homeostasis are complex and suggest that disc matrix homeostasis is modulated by systemic factors and not solely through local disc cellular senescence.

An increase in the burden of senescent cells in tissues with age contributes to driving aging and the onset of age-related diseases. Genetic and pharmacologic elimination of senescent cells extends both health span and life span in mouse models. Heterochronic parabiosis in mice has been used to identify bloodborne, circulating pro- and anti-geronic factors able to drive or slow aging, respectively. However, whether factors in the circulation also regulate senescence is unknown. Here, we measured the expression of senescence and senescence-associated secretory phenotype (SASP) markers in multiple tissues from 4- to 18-month-old male mice that were either isochronically or heterochronically paired for 2 months. In heterochronic parabionts, the age-dependent increase in senescence and SASP marker expression was reduced in old mice exposed to a young environment, while senescence markers were concurrently increased in young heterochronic parabionts. These findings were supported by geropathology analysis using the Geropathology Grading Platform that showed a trend toward reduced hepatic lesions in old heterochronic parabionts. In summary, these results demonstrate that senescence is regulated in part by circulating geronic factors and suggest that one of the possible mediators of the rejuvenating effects with heterochronic parabiosis is through the reduction of the senescent cell burden. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11357-020-00185-1) contains supplementary material, which is available to authorized users.

SUMMARYSirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in the regulation of aging and metabolism. Targeted sequencing identified a SIRT6 allele containing two linked substitutions (N308K/A313S) as enriched in Ashkenazi Jewish (AJ) centenarians as compared to AJ control individuals. Characterization of this SIRT6 (centSIRT6) allele demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double strand break repair, and more robust cancer cell killing compared to the wild type. Surprisingly, centSIRT6 displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD+concentrations and substrates. Additionally, centSIRT6 displayed a stronger interaction with Lamin A/C (LMNA), which correlated with enhanced ribosylation of LMNA. Our results suggest that enhanced SIRT6 function contributes to human longevity by improving genome maintenance via increased mADPr activity and enhanced interaction with LMNA.

Although cellular senescence is increasingly recognized as driving multiple age-related co-morbidities through the senescence-associated secretory phenotype (SASP), in vivo senescent cell identification, particularly in bulk or single cell RNA-sequencing (scRNA-seq) data remains challenging. Here, we generated a novel gene set (SenMayo) and first validated its enrichment in bone biopsies from two aged human cohorts. SenMayo also identified senescent cells in aged murine brain tissue, demonstrating applicability across tissues and species. For direct validation, we demonstrated significant reductions in SenMayo in bone following genetic clearance of senescent cells in mice, with similar findings in adipose tissue from humans in a pilot study of pharmacological senescent cell clearance. In direct comparisons, SenMayo outperformed all six existing senescence/SASP gene sets in identifying senescent cells across tissues and in demonstrating responses to senescent cell clearance. We next used SenMayo to identify senescent hematopoietic or mesenchymal cells at the single cell level from publicly available human and murine bone marrow/bone scRNA-seq data and identified monocytic and osteolineage cells, respectively, as showing the highest levels of senescence/SASP genes. Using pseudotime and cellular communication patterns, we found senescent hematopoietic and mesenchymal cells communicated with other cells through common pathways, including the Macrophage Migration Inhibitory Factor (MIF) pathway, which has been implicated not only in inflammation but also in immune evasion, an important property of senescent cells. Thus, SenMayo identifies senescent cells across tissues and species with high fidelity. Moreover, using this senescence panel, we were able to characterize senescent cells at the single cell level and identify key intercellular signaling pathways associated with these cells, which may be particularly useful for evolving efforts to map senescent cells (e.g., SenNet). In addition, SenMayo represents a potentially clinically applicable panel for monitoring senescent cell burden with aging and other conditions as well as in studies of senolytic drugs.

Constitutive NF‐κB activation is associated with cellular senescence and stem cell dysfunction and rare variants in NF‐κB family members are enriched in centenarians. We recently identified a novel small molecule (SR12343) that inhibits IKK/NF‐κB activation by disrupting the association between IKKβ and NEMO. Here we investigated the therapeutic effects of SR12343 on senescence and aging in three different mouse models. SR12343 reduced senescence‐associated beta‐galactosidase (SA‐β‐gal) activity in oxidative stress‐induced senescent mouse embryonic fibroblasts as well as in etoposide‐induced senescent human IMR90 cells. Chronic administration of SR12343 to the Ercc1 −/ ∆ and Zmpste24 −/− mouse models of accelerated aging reduced markers of cellular senescence and SASP and improved multiple parameters of aging. SR12343 also reduced markers of senescence and increased muscle fiber size in 2‐year‐old WT mice. Taken together, these results demonstrate that IKK/NF‐κB signaling pathway represents a promising target for reducing markers of cellular senescence, extending healthspan and treating age‐related diseases., Constitutive IKK/NF‐κB activation is associated with cellular senescence and aging. Here we demonstrate that pharmacological inhibition of IKK/NF‐κB activation by the small molecule SR12343 reduced senescence and SAPS factors in cell culture, improved healthspan in mice with accelerated aging, and reduced markers of senescence and pathology in multiple tissues of both accelerated and naturally aged mice. Therefore, IKK/NF‐κB pathway is a promising target for aging interventions to treat age‐related diseases.

BackgroundAutologous patient T cells engineered to express antitumor T cell receptors (TCRs) and chimeric antigen receptors (CARs) have been effective for the treatment of certain cancer types,1–4 and tumor neoantigens encoded by cancer-specific mutations have emerged as major targets of CD4+ and CD8+ T cells in immune checkpoint blockade (ICB) and in adoptive cell therapy (ACT).5–9 However, only a minority of intratumoral T cells are reactive to cancer antigens while the majority represent bystander cells.10–12 Conventional approaches to isolate tumor-reactive T cells and identify their TCRs from tumors rely on T cell function and can be impaired due to T cell exhaustion and dysfunction.13 14MethodsWe performed single-cell RNA and T cell receptor (TCR) sequencing (scRNA/TCR-seq) on over 46,000 T cells isolated from eleven archival metastatic tumor samples whose primary cancer types included colon, rectal, breast, anal, and melanoma. From these samples, 15 CD8+ and 17 CD4+ neoantigen-reactive TCR clonotypes (NeoTCRs) were known. We then performed transcriptomic clustering of these cells and mapped known NeoTCR clonotypes onto the transcriptomic map. Subsequently we predicted NeoTCRs from prospective metastatic colon cancer samples based on their presence within clusters sharing gene expression with NeoTCR+ clusters in the archival samples.ResultsProjecting known NeoTCRs onto the TIL transcriptomic map, we observed 325 total T cells bearing these NeoTCRs, and the majority (>80%) of NeoTCRs were expressed by T cells within 2 clusters, one CD4+ and one CD8+, that included by expression of CXCL13, ENTPD1 (CD39), TOX, TIGIT, LAG3, and PDCD1 (PD-1), indicating a dysfunctional state. Reasoning that T cells sharing phenotypes with those within the NeoTCR clusters could be novel NeoTCRs, we developed gene signatures (NeoTCR4 and NeoTCR8) of CD4 and CD8 NeoTCR+ cells, respectively, and four prospective patients' TIL were analyzed by scRNA/TCR-seq and scored according to NeoTCR signatures. We expressed predicted NeoTCRs in healthy donor PBL and screened them with antigen presenting cells (APCs) expressing candidate neoantigens. 33/73 predicted NeoTCRs (including both CD4 and CD8) were reactive against patients' tumors or candidate neoantigens.ConclusionsThis study enabled successful detection of tumor-specific NeoTCRs in the sequenced TIL of 14/14 patients for whom reactivity was studied. Deconvolution of NeoTCRs from bystander TCRs within the tumor-immune microenvironment represents an important step in the development of personalized immunotherapeutics, and prospective NeoTCR isolation based on TIL transcriptional phenotypes will allow for rapid development of personalized immunotherapy in the form of lymphocytes expressing these tumor-specific TCRs.AcknowledgementsWe thank the Surgery Branch TIL Laboratory and clinical team for generating TIL, and patients enrolled in our clinical protocols. Support from CCR Single Cell Analysis Facility was funded by FNLCR Contract HHSN261200800001E. This work utilized the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov). We also thank NIDAP for providing additional computational support and the CCR Genomics Core for next-generation sequencing supportReferencesRobbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, Wunderlich JR, Nahvi AV, Helman LJ, Mackall CL, Kammula US, Hughes MS, Restifo NP, Raffeld M, Lee CCR, Levy CL, Li YF, El-Gamil M, Schwarz SL, Laurencot C, Rosenberg SA.Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol 2011;29:917–924.Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP, Zheng Z, Nahvi A, de Vries CR, Rogers-Freezer LJ, Mavroukakis SA, Rosenberg SA. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 2006;314:126–129.June CH, Sadelain M. Chimeric Antigen Receptor Therapy. N Engl J Med 2018;379:64–73.Kochenderfer JN, Yu Z, Frasheri D, Restifo NP, Rosenberg SA. Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells. Blood 2010;116:3875–3886.Tran E, Robbins PF, Rosenberg SA, “Final common pathway” of human cancer immunotherapy: targeting random somatic mutations. Nat Immunol 2017;18:255–262.Robbins PF, Lu YC, El-Gamil M, Li YF, Gross C, Gartner J, Lin JC, Teer JK, Cliften P, Tycksen E, Samuels Y, Rosenberg SA, Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nat Med 2013;19:747–752.Parkhurst MR, Robbins PF, Tran E, Prickett TD, Gartner JJ, Jia L, Ivey G, Li YF, El-Gamil M, Lalani A, Crystal JS, Sachs A, Groh E, Ray S, Ngo LT, Kivitz S, Pasetto A, Yossef R, Lowery FJ, Goff SL, Lo W, Cafri G, Deniger DC, Malekzadeh P, Ahmadzadeh M, Wunderlich JR, Somerville RPT, Rosenberg SA. Unique Neoantigens Arise from Somatic Mutations in Patients with Gastrointestinal Cancers. Cancer Discov 2019;9:1022–1035.Gubin MM, Zhang X, Schuster H, Caron E, Ward JP, Noguchi T, Ivanova Y, Hundal J, Arthur CD, Krebber WJ, Mulder GE, Toebes M, Vesely MD, Lam SSK, Korman AJ, Allison JP, Freeman GJ, Sharpe AH, Pearce EL, Schumacher TN, Aebersold R, Rammensee HG, Melief CJM, Mardis ER, Gillanders WE, Artyomov MN, Schreiber RD. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature 2014;515:577–581.van Rooij N, van Buuren MM, Philips D, Velds A, Toebes M, Heemskerk B, van Dijk LJA, Behjati S, Hilkmann H, el Atmioui D, Nieuwland M, Stratton MR, Kerkhoven RM, Keşmir C, Haanen JB, Kvistborg P, Schumacher TN. Tumor Exome Analysis Reveals Neoantigen-Specific T-Cell Reactivity in an Ipilimumab-Responsive Melanoma. Journal of Clinical Oncology 2013;31:e439–e442.Duhen T, Duhen R, Montler R, Moses J, Moudgil T, de Miranda NF, Goodall CP, Blair TC, Fox BA, McDermott JE, Chang SC, Grunkemeier G, Leidner R, Bell RB, Weinberg AD. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9:2724.Simoni Y, Becht E, Fehlings M, Loh CY, Koo SL, Teng KWW, Yeong JPS, Nahar R, Zhang T, Kared H, Duan K, Ang N, Poidinger M, Lee YY, Larbi A, Khng AJ, Tan E, Fu C, Mathew R, Teo M, Lim WT, Toh CK, Ong BH, Koh T, Hillmer AM, Takano A, Lim TKH, Tan EH, Zhai W, Tan DSW, Tan IB, Newell EW, Bystander CD8 T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature 2018;557:575–579.Scheper W, Kelderman S, Fanchi LF, Linnemann C, Bendle G, de Rooij MAJ, Hirt C, Mezzadra R, Slagter M, Dijkstra K, Kluin RJC, Snaebjornsson P, Milne K, Nelson BH, Zijlmans H, Kenter G, Voest EE, Haanen JBAG, Schumacher TN. Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers. Nat Med 2019;25:89–94.Blank CU, Haining WN, Held W, Hogan PG, Kallies A, Lugli E, Lynn RC, Philip M, Rao A, Restifo NP, Schietinger A, Schumacher TN, Schwartzberg PL, Sharpe AH, Speiser DE, Wherry EJ, Youngblood BA, Zehn D. Defining “T cell exhaustion.” Nat Rev Immunol 2019;19:665–674.van der Leun AM, Thommen DS, Schumacher TN. CD8 T cell states in human cancer: insights from single-cell analysis. Nat Rev Cancer 2020;20:218–232.

Adoptive cell therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from rare mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. Here, we describe a library of T cell receptors (TCRs) that can target TP53 mutations shared among 7.3% of patients with solid cancers. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner both in vitro and in vivo. Patients with chemorefractory epithelial cancers treated with ex vivo-expanded autologous tumor infiltrating lymphocytes (TILs) naturally reactive with mutant p53 experienced limited clinical responses (2 PRs/12 patients), and we detected low frequencies, exhausted phenotypes, and poor persistence of the infused mutant p53-reactive TILs. Alternatively, we treated one patient with a chemorefractory breast cancer with ACT by transducing autologous peripheral blood lymphocytes with an HLA-A*02-restricted anti-p53R175H TCR. The infused cells exhibited an improved immunophenotype and prolonged persistence compared to the TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these data demonstrate the feasibility of off-the-shelf TCR-engineered cell therapies targeting shared p53 neoantigens to treat human cancers.

The ability of virally suppressive antiretroviral therapy use to extend the life span of people with HIV (PWH) implies that the age of PWH will also increase. Among PWH, extended survival comes at a cost of earlier onset and increased rates of aging-associated comorbidities and geriatric syndromes, with persistent inflammation and immune dysregulation consequent to chronic HIV infection and to antiretroviral therapy use contributing to an overall decrease in health span. The geroscience hypothesis proposes that the root causes of most aging-related chronic diseases and conditions is the aging process itself. Hence, therapeutically targeting fundamental aging processes could have a greater impact on alleviating or delaying aging-associated comorbidities than addressing each disease individually. Extending the geroscience hypothesis to PWH, we speculate that targeting basic mechanisms of aging will improve overall health with age. Clinical features and pathophysiologic mechanisms of chronic diseases in PWH qualitatively resemble those seen in older adults without HIV. Therefore, drugs that target any of the pillars of aging, including metformin, rapamycin, and nicotinamide adenine dinucleotide precursors, may also slow the rate of onset of age-associated comorbidities and geriatric syndromes in PWH. Drugs that selectively induce apoptosis of senescent cells, termed senolytics, may also improve health span among PWH. Preliminary evidence suggests that senescent cell burden is increased in PWH, implying that senescent cells are an excellent therapeutic target for extending health span. Recently initiated clinical trials evaluating senolytics in age-related diseases offer insights into the design and potential implementation of similar trials for PWH.

Cellular senescence is a cell fate that occurs in response to numerous types of stress and can promote tissue repair or drive inflammation and disruption of tissue homeostasis depending on the context. Aging and obesity lead to an increase in the senescent cell burden in multiple organs. Senescent cells release a myriad of senescence-associated secretory phenotype factors that directly mediate pancreatic β-cell dysfunction, adipose tissue dysfunction, and insulin resistance in peripheral tissues, which promote the onset of type II diabetes mellitus. In addition, hyperglycemia and metabolic changes seen in diabetes promote cellular senescence. Diabetes-induced cellular senescence contributes to various diabetic complications. Thus, type II diabetes is both a cause and consequence of cellular senescence. This review summarizes recent studies on the link between aging, obesity, and diabetes, focusing on the role of cellular senescence in disease processes.

The burden of senescent cells (SnCs), which do not divide but are metabolically active and resistant to death by apoptosis, is increased in older adults and those with chronic diseases. These individuals are also at the greatest risk for morbidity and mortality from SARS‐CoV‐2 infection. SARS‐CoV‐2 complications include cytokine storm and multiorgan failure mediated by the same factors as often produced by SnCs through their senescence‐associated secretory phenotype (SASP). The SASP can be amplified by infection‐related pathogen‐associated molecular profile factors. Senolytic agents, such as Fisetin, selectively eliminate SnCs and delay, prevent, or alleviate multiple disorders in aged experimental animals and animal models of human chronic diseases, including obesity, diabetes, and respiratory diseases. Senolytics are now in clinical trials for multiple conditions linked to SnCs, including frailty; obesity/diabetes; osteoporosis; and cardiovascular, kidney, and lung diseases, which are also risk factors for SARS‐CoV‐2 morbidity and mortality. A clinical trial is underway to test if senolytics decrease SARS‐CoV‐2 progression and morbidity in hospitalized older adults. We describe here a National Institutes of Health‐funded, multicenter, placebo‐controlled clinical trial of Fisetin for older adult skilled nursing facility (SNF) residents who have been, or become, SARS‐CoV‐2 rtPCR‐positive, including the rationale for targeting fundamental aging mechanisms in such patients. We consider logistic challenges of conducting trials in long‐term care settings in the SARS‐CoV‐2 era, including restricted access, consent procedures, methods for obtaining biospecimens and clinical data, staffing, investigational product administration issues, and potential solutions for these challenges. We propose developing a national network of SNFs engaged in interventional clinical trials.