Prophylactic and long-lasting efficacy of senolytic CAR T cells against age-related metabolic dysfunction

Senescent cells, which accumulate in organisms over time, contribute to age-related tissue decline. Genetic ablation of senescent cells can ameliorate various age-related pathologies, including metabolic dysfunction and decreased physical fitness. While small-molecule drugs that eliminate senescent cells (‘senolytics’) partially replicate these phenotypes, they require continuous administration. We have developed a senolytic therapy based on chimeric antigen receptor (CAR) T cells targeting the senescence-associated protein urokinase plasminogen activator receptor (uPAR), and we previously showed these can safely eliminate senescent cells in young animals. We now show that uPAR-positive senescent cells accumulate during aging and that they can be safely targeted with senolytic CAR T cells. Treatment with anti-uPAR CAR T cells improves exercise capacity in physiological aging, and it ameliorates metabolic dysfunction (for example, improving glucose tolerance) in aged mice and in mice on a high-fat diet. Importantly, a single administration of these senolytic CAR T cells is sufficient to achieve long-term therapeutic and preventive effects.


Article
https://doi.org/10.1038/s43587-023-00560-5uPAR-positive and uPAR-negative cells sorted by fluorescence-activated cell sorting (FACS) from the liver, fat and pancreas (Fig. 1b-m and Extended Data Figs. 2 and 3).Using unsupervised clustering and marker-based cell labeling 20,21 , we identified the major uPAR-positive cell types and cell states present in each of the three organs (Fig. 1b-d and Extended Data Fig. 2).Of note, some minor cell types (for example, hepatic stellate cells in the liver, and beta cells in the pancreas) require specialized isolation procedures and were not captured using our protocol 22,23 .
Analysis of the different populations for uPAR expression indicated that endothelial and myeloid cells were the most prominent uPAR-expressing populations in the liver (Fig. 1e and Extended Data Fig. 2b), whereas in adipose tissue uPAR was expressed mainly in subsets of preadipocytes, dendritic cells and myeloid cells (Fig. 1f and Extended Data Fig. 2d).In the aged pancreas, uPAR expression was prominent in subsets of endothelial cells, fibroblasts, dendritic cells and myeloid cells (Fig. 1g and Extended Data Fig. 2f).Compared to uPAR-negative cells, uPAR-positive cells were significantly enriched in gene signatures linked to inflammation, the complement pathway and the coagulation cascade as well as transforming growth factor-beta signaling (Extended Data Fig. 3a-c).
Importantly, when senescent cells present in these tissues were identified using two independent transcriptomic signatures of senescence 17,24 , we observed that the main senescent cell types present in aged tissues were distinct: endothelial and myeloid cells in the liver (Fig. 1h and Extended Data Fig. 3d,g-i), dendritic cells, myeloid cells and preadipocytes in adipose tissue (Fig. 1j and Extended Data Fig. 3e,j-l) and endothelial cells, fibroblasts, dendritic cells and myeloid cells in the pancreas (Fig. 1l and Extended Data Fig. 3f,m-o).Thus, uPAR-positive cells constituted a significant fraction of the senescent cell burden in these tissues (67-90% in liver, 92-66% in adipose tissue and 76-63% in pancreas; Fig. 1i,k,m and Extended Data Fig. 3h,k,n).Note that while our analysis could not evaluate pancreatic beta cells, analysis of published data revealed that expression of Plaur was significantly upregulated in senescent beta cell populations isolated from aged animals and subjected to bulk RNA-seq 25 .
Finally, to ascertain whether uPAR was expressed in senescent cells that accumulate with age in human tissues, we analyzed available datasets of human pancreas collected from young (0-to 6-year-old) and aged (50-to 76-year-old) individuals 26 .While we were limited to an analysis of PLAUR transcript abundance in these settings, we found that the fraction of PLAUR-expressing cells was substantially greater in older individuals (Fig. 2).
Overall, these results indicate that the levels of uPAR-positive senescent cells increase with age and that most senescent cells present in aged tissues express uPAR.The fact that we can identify settings in which an increased expression of uPAR protein expression does not correlate with Plaur mRNA levels indicates that the absence of an induction of Plaur transcript levels does not exclude the possibility of an increase in uPAR protein expression.

Effect of uPAR CAR T cells in naturally aged mice
To determine the tolerability and therapeutic activity of uPAR-targeting CAR T cells on physiologically aged mice, we intravenously infused damage and decreased function of the immune system leads to the accumulation of senescent cells 4,5 , thereby generating a chronic proinflammatory milieu that leads to a range of age-related tissue pathologies [6][7][8][9] .As such, senolytic strategies to eliminate senescent cells from aged tissues have the potential to dramatically improve healthspan.
Most efforts to develop senolytic therapies have focused on the development of small-molecule drugs that target poorly defined molecular dependencies present in senescent cells and that must be administered repeatedly over time 10 .In contrast, CAR T cells are a form of cellular therapy that redirects T cell specificity toward cells expressing a specific cell-surface antigen 11 .Unlike small molecules, CAR T cells only require that the target antigen is differentially expressed on target cells compared to normal tissues; moreover, as 'living drugs', these therapeutics have the potential to persist and mediate their potent effects for years after single administration 12 .We have shown that CAR T cells targeting the cell-surface protein uPAR, which is upregulated on senescent cells, can efficiently deplete senescent cells in young animals and reverse liver fibrosis.Here, we explore whether CAR T cells could safely and effectively eliminate senescent cells in aged mice and modulate healthspan.

uPAR is upregulated in physiological aging
uPAR promotes remodeling of the extracellular matrix during fibrinolysis, wound healing and tumorigenesis 13 .In physiological conditions, it is primarily expressed in certain subsets of myeloid cells and, at low levels, in the bronchial epithelium 14 .We recently described the upregulation of uPAR on senescent cells across different cell types and multiple triggers of senescence 14 and showed that CAR T cells targeting this cell-surface protein could efficiently remove senescent cells from tissues in young mice without deleterious effects to normal tissues 14 .Given these results, we set out to test whether uPAR might serve as a target for senolytic CAR T cells in aged tissues.
Plasma levels of soluble uPAR positively correlate with the pace of aging in humans 15,16 and Plaur (the gene encoding uPAR) is a component of the SenMayo gene signature recently reported to identify senescent cells in aged tissues 17 .To explore the association with uPAR expression in aged tissues further, we surveyed RNA-sequencing (RNA-seq) data from the Tabula Muris Senis project 18 .Expression of Plaur was upregulated in several organs in samples from 20-month-old mice compared to 3-month-old mice (Extended Data Fig. 1a).Because mRNA levels are not linearly related to surface protein levels 19 , we performed immunohistochemistry and indeed confirmed an age-associated increase in uPAR protein in liver, adipose tissue, skeletal muscle and pancreas (Fig. 1a and Extended Data Fig. 1b).This increase in the fraction of uPAR-positive cells was paralleled by an increase in the percentage of senescence-associated beta-galactosidase (SA-β-gal)-positive cells (Extended Data Fig. 1c-f).Co-immunofluorescence revealed that a large majority of these SA-β-gal-expressing cells were in fact uPAR positive, whereas only a minority of these cells were macrophages as evidenced by coexpression of F4/80 (Extended Data Fig. 1g-j).
To add granularity to our understanding of the molecular characteristics of uPAR-positive cells in aged tissues, we performed singlecell RNA sequencing (scRNA-seq) on approximately 4,000-15,000 Fig. 1 | uPAR is upregulated on senescent cells in physiological aging.a, Immunohistochemical staining of mouse uPAR in liver, adipose tissue, muscle and pancreas from young (age 3 months) or old (age 20 months) mice (n = 3 per age).b-m, Single-cell analysis of uPAR expression and senescence.uPAR-positive and uPAR-negative cells were sorted from the liver, adipose tissue and pancreas of 20-month-old mice and subjected to single-cell RNA-seq by 10x chromium protocol (n = the sequencing of four mice where two females were combined into one replicate and two males were combined into another replicate).b, Uniform manifold approximation and projection (UMAP) visualization of liver cell types.c, UMAP visualization of adipose tissue cell types.d, UMAP visualization of pancreas cell types.e, UMAP visualization of hepatic uPAR-negative and uPARpositive cell types.f, UMAP visualization of adipose uPAR-negative and uPARpositive cell types.g, UMAP visualization of pancreatic uPAR-negative and uPARpositive cell types.h,j,l, UMAP visualizations with senescence signature scores 24 in each cell indicated by the color scale.i,k,m, Quantification of the proportion of uPAR-positive and uPAR-negative cells contributing to the respective senescence signature.h,i, Liver; j,k, adipose tissue; l,m, pancreas.Results are from one independent experiment (a-m).DC, dendritic cell; NK, natural killer; pDC, plasmacytoid dendritic cell; ASPC, adipose progenitor and stem cells.endogenous T cells and therefore monitored over time (Fig. 3a).As controls, parallel cohorts of sex-and aged-matched mice were infused with the same dose of either untransduced T (UT) cells or T cells expressing a mouse CAR targeting human CD19 (h.19-m.28z)that does not recognize the mouse CD19 protein but encompasses the exact same signaling structure thus controlling for nonspecific T cell cytotoxicity.We opted to test a dose of 0.5 × 10 6 CAR-positive cells, which we previously found to balance safety and senolytic efficacy in young animals 14 .
Mice infused with m.uPAR-m.28zCAR T cells, but not controls, showed a reduction in the proportions of SA-β-gal-positive and uPARpositive cells throughout the tissues examined, most notably in the pancreas, liver and adipose tissue (Fig. 3b and Extended Data Fig. 4).As has been previously reported, our aged mouse cohort displayed elevated levels of pro-inflammatory cytokines linked to the SASP in the peripheral blood, a phenomenon often referred to as 'inflammaging' 27 .Consistent with a reduction in senescent cell burden and/or improved organismal health, m.uPAR-m.28zCAR T cell-treated animals showed a decrease in the plasma levels of these factors (Fig. 3c).
Despite detectable expression of uPAR in some normal tissues, our previous work indicates that a dose of 0.5 × 10 6 m.uPAR-m.28zCAR T cells is well tolerated in young mice 14 .As was the case in young animals, the dose of 0.5 × 10 6 m.uPAR-m.28zCAR T cells was well tolerated in aged mice (18-20 months old), all of whom remained active without observable signs of morbidity, weight loss or relevant alterations in serum chemistry or complete blood counts (Fig. 4).In addition, microscopic evaluation of tissues did not reveal tissue damage secondary to toxicity in aged tissues obtained from whole-body necropsies of m.uPAR-m.28zCAR T cell-treated mice when compared to age-matched control-treated animals (Extended Data Fig. 5).
One prominent feature of aging in humans and mice is the emergence of age-related metabolic dysfunction, which is a collection of phenotypes linked to impaired glucose tolerance 25,28 and decreased exercise capacity 29,30 .Interestingly, we observed that aged m.uPARm.28zCAR T cell-treated mice had significantly decreased fasting glucose levels compared with UT or h.19-m.28z-treatedcontrols (Fig. 5a).Upon challenge with an intraperitoneal bolus of glucose (2 g per kg body weight), m.uPAR-m.28zCAR T cell-treated aged but not young mice presented significantly lower plasma glucose levels than controls for over 2 h after administration (Fig. 5b,c and Extended Data Fig. 6a,b).Furthermore, m.uPAR-m.28zCAR T cell-treated mice had lower basal insulin levels after fasting that was followed by a significant increase in insulin levels 15 min after the glucose load, indicative of improved pancreatic beta cell function (Fig. 5d).Of note, m.uPAR-m.28zCAR T cell-treated aged mice also presented improved peripheral insulin sensitivity, suggesting a coordinated multiorgan improvement in glucose homeostasis (Extended Data Fig. 6c,d).In addition, most aged mice with m.uPAR-m.28zCAR T cells showed improvements in their exercise capacity at 2.5 months after treatment compared to pretreatment levels (Fig. 5e,f).Importantly, the improvement in metabolic function noted in m.uPAR-m.28zCAR T cell-treated old mice was accompanied by an expansion of m.uPAR-m.28zCAR T cells and their trafficking to several organs such as liver and spleen as assessed by flow cytometry (Fig. 5g,h).These m.uPAR-m.28zCAR T cells were mostly cytotoxic CD8 + T cells in the livers and CD4 + T cells in the spleen and presented an effector phenotype indicative of their activated response (Extended Data Fig. 7a-d).Of note, this expansion did not occur in aged-matched UT or h.19-m.28z-treatedcontrols and was lower in m.uPAR-m.28zCAR T cell-treated young mice, results that were consistent with the lower fraction of uPAR-positive cells in younger animals (Figs.1a and 5g,h and Extended Data Fig. 1).
Collectively, these results show that uPAR CAR T cells can safely and effectively remove senescent uPAR-positive cells in the tissues of naturally aged mice and ameliorate age-dependent metabolic and physical dysfunction.

Persistence and prophylaxis by uPAR CAR T cells in aging
Unlike small molecules, CAR T cells can persist in the organism and exert their effects over time 12 .Indeed, in human cancer patients cured of disease, the presence of CAR T cells has been noted as much as 10 years after the initial infusion 12 .Such persistence raises the question of whether the administration of uPAR CAR T cells in young animals would prevent or delay the development of age-triggered phenotypes later in life.To explore this possibility, we infused young mice (3 months old) with one dose of 0.5 × 10 6 m.uPAR-m.28zCAR T, h.19-m.28zCAR T or UT cells and monitored the mice over their natural lifespan (Fig. 6).Despite the initially lower numbers of uPAR-positive cells compared to aged animals (see above), uPAR CAR T cells were detectable in the spleens and livers of treated mice 12 months after the initial single infusion at substantially higher levels than the low number of persisting UT or h.19 CAR T controls (Fig. 6a,b).Consistent with their persistent activity, flow cytometry of the spleen and livers of uPAR CAR T cell-treated mice indicated that the persisting cells were mostly cytotoxic CD8 + T cells harboring a memory and effector phenotype in the spleens (Extended Data Fig. 7e-h).Therefore, uPAR CAR T cells persist and expand over the lifespan of the animal, presumably owing to increased antigen stimulation as the frequency of target uPAR-positive cells increases over time.
As was observed in aged animals upon therapeutic treatment, prophylactic uPAR CAR T cell administration in young mice limited metabolic decline in old age.Specifically, uPAR CAR T cell-treated mice had significantly lower fasting glucose levels (Fig. 6c), improved glucose tolerance (Fig. 6d,e) and enhanced pancreatic beta cell function as assessed by glucose-stimulated insulin secretion (Fig. 6f) than mice treated with either UT or h.19-m.28z.In terms of fitness, mice that in their youth had been treated with m.uPAR-m.28zCAR T cells, compared with control-treated mice, showed higher exercise capacity at 9 months of age (Fig. 6g,h), although this waned over time (Extended Data Fig. 7i,j).These phenotypes correlated with a significant decrease in both SA-β-gal-positive and uPAR-positive cells in pancreas, liver and adipose tissue (Fig. 6i and Extended Data Fig. 7k-p).Taken together, these results show that uPAR CAR T cells can not only treat, but also prevent, features of age-dependent metabolic decline.

uPAR CAR T cells to treat or prevent metabolic syndrome
Many of the features associated with metabolic syndrome in aged mice can be recapitulated in young animals given a high-fat diet Article https://doi.org/10.1038/s43587-023-00560-5 (HFD) 31 and, indeed, obesity has been described to accelerate the 'aging clock' 32 .As in aged animals, such treatment leads to the accumulation of senescent cells 25 (Extended Data Fig. 8a-d).To test the therapeutic potential of uPAR CAR T cells in this context, we modeled metabolic syndrome by feeding mice an HFD, which induces obesity and metabolic stress 33 .After 2 months on an HFD, mice were treated with 0.5 × 10 6 m.uPAR-m.28zCAR T or UT cells and continued on the diet (Fig. 7a).At 20 d after infusion, mice treated with uPAR CAR T cells displayed significantly lower body weight, better fasting blood glucose levels and improvements in both glucose and insulin tolerance compared to controls (Fig. 7b-g).This therapeutic effect persisted through the period of monitoring (2.5 m after cell infusion) and was accompanied by decreased senescent cell burden in pancreas, liver and adipose tissue as assessed by SA-β-gal (Fig. 7h,i and Extended Data Fig. 8e-h).Thus, uPAR CAR T cell therapy produced a similar improvement to metabolic dysfunction in the context of metabolic syndrome in young animals as was observed in naturally aged mice.Article https://doi.org/10.1038/s43587-023-00560-5 To test whether prophylactic administration of uPAR CAR T cells could impede the development of metabolic disorders in young mice given an HFD, we administered 0.5 × 10 6 m.uPAR-m.28zCAR T cells 1.5 months before placement on an HFD (Fig. 7j).Remarkably, m.uPARm.28zCAR T cells (but not treatment with UT cells) acted prophylactically to blunt the accumulation of senescent cells over time, an effect that was also associated with decreased weight gain and glucose levels 3.5 months after infusion (Extended Data Fig. 8i-l and Fig. 7k-n).At this time, m.uPAR-m.28zCAR T cells were detectable and enriched in the spleens and livers of treated mice, where they again were composed mostly of CD8 + T cells with an effector phenotype (Extended Data Fig. 9).This preventive effect on metabolic dysfunction was sustained for at least 5.5 months after cell infusion despite continuous exposure to an HFD (Fig. 7o,p).
Overall, these data highlight the contribution of uPAR-positive cells to metabolic dysfunction in aged and obese mice and raise the possibility that targeting these cells through CAR T cells could have therapeutic benefit in humans.

Discussion
Our study provides proof-of-principle evidence that senolytic cell therapies can ameliorate symptoms associated with physiological aging.We previously showed that uPAR-targeting CAR T cells could safely and effectively eliminate senescent cells in the livers of young animals 14 .
Here, focusing on metabolic dysfunction as one prominent age-related pathology, we show that: (i) the fraction of uPAR-positive cells increases with age; (ii) these cells significantly contribute to the senescence burden in aged tissues; (iii) uPAR-positive cells with senescence signatures consist of both immune and non-immune populations, the latter consisting of a range of cell types that are organ dependent; (iv) uPAR CAR T cells can be effective at eliminating uPAR-positive senescent cells; (v) their effect is not associated with pathology in tissues or alterations of hepatic and renal functional parameters in aged mice; and finally, (vi) the action of uPAR CAR T cells is associated with improved glucose homeostasis and metabolic fitness in both physiological aging and HFD feeding.Importantly, at doses used to produce these therapeutic benefits, we noted no overt toxicities of uPAR CAR T cells, which could persist and expand for over 15 months as mice progressed from a youthful to an aged state.
Perhaps the most striking observation of the current work was the ability of uPAR CAR T cells to act prophylactically to blunt age-induced and diet-induced metabolic decline.Unlike senolytic approaches based on small molecules, uPAR CAR T cells have long-lasting effects after the administration of a single low dose, causing a marked impairment in ageinduced or HFD-induced metabolic syndrome when mice were treated during youth or administration of HFD, respectively.Our findings are consistent with those of an earlier study that explored vaccination against GPNMB on senescent cells to address age-related pathology 34  Article https://doi.org/10.1038/s43587-023-00560-5 although with our cellular therapy, both effect sizes and duration were substantially larger.In fact, our results demonstrate a protective effect for over a year in the context of physiological aging in the laboratory mouse, a species with an average lifespan of around 2 years.Studies using genetic or pharmacological approaches to senolysis have been equivocal as to whether elimination of senescent cells will significantly extend longevity 29,30,35 .Our current studies are not sufficiently powered to draw conclusions on longevity at this stage.As senescent cells contribute to a range of age-related tissue pathologies, studying the impact of senolysis in aged animals provides an opportunity to interrogate multiple comorbidities under similar conditions.Future studies will evaluate the potential of uPAR CAR T cells (or other

Article
https://doi.org/10.1038/s43587-023-00560-5senolytic cell therapies) in additional aging and related tissue-damage pathologies, the latter disease contexts providing a more likely starting point for clinical implementation.It remains to be determined which of the uPAR-positive cell populations targeted by uPAR CAR T cells are responsible for the improved metabolic function we observe.In other senolytic studies, the elimination of senescent pancreatic beta cells has been linked to improved glucose tolerance 25 .However, there are reports suggesting that targeting senescent cells in adipose tissue 28 or even immune cell senescence 36 may also play a role.In this regard, recent studies suggest that the elimination of macrophage populations with senescent features can also improve tissue decline in mice 37,38 .Whether or not these macrophages are truly 'senescent' or have an alternative cell state is a topic of debate; regardless, given that we observe a fraction of uPAR-expressing macrophages that also coexpress SA-β-gal and senescence-associated transcriptional signatures accumulating in aged tissues, it seems likely that their elimination contributes to the phenotypes we observe.
While the mechanism of action of most current small molecules is often inferred or poorly understood, senolytic CAR T cells have a clear underlying rationale based on the expression of a specific surface antigen.While toxicity issues are invariably a concern, cellular therapy harbors the versatility to simultaneously target several surface antigens through AND gate approaches 11 , modulate persistence through different CAR designs 39 and/or incorporate safety switches 40 , all of which provide avenues to mitigate side effects that are not possible through vaccination strategies or small-molecule approaches 40 .Indeed, another recent report reveals that mice and primates tolerate CAR T cells that target a natural killer cell ligand that is upregulated on senescent cells and other cell types 41 .Taken together, these efforts could result in the identification of tissue-specific senolytic antigens that could be targeted with cellular therapy to treat different agerelated phenotypes.The persistence of the uPAR-targeted CAR T cells and the durability of the effects after a single low-dose treatment highlight the clinical potential of the senolytic CAR T cell approach for the treatment of chronic pathologies.

Mice
All mouse experiments were approved by the MSKCC and/or CSHL Internal Animal Care and Use Committee (animal protocol 11-06-011 at MSKCC and 21-4 at CSHL).All relevant animal use guidelines and ethical regulations were followed.Mice were maintained under specific pathogen-free conditions.Housing was on a 12-h-12-h light-dark cycle under standard temperature and humidity of approximately 18-24 °C and 40-60%, respectively.The following mice were used: 3-to 4-month-old C57BL/6 mice (purchased from Charles River), 18-monthold C57BL/6 mice (obtained from the National Institute of Aging) and 6-week-old B6.SJL-Ptrca/BoyAiTac (CD45.1)mice (purchased from Taconic).Mice of both sexes were used at 8-12 weeks of age and 18-20 months of age for the aging experiments, males of 8-12 weeks old for the HFD experiments and females of 6-10 weeks old for T cell isolation.Mice were kept in group housing.Mice had free access to food and water except during the starvation period before glucose or insulin tolerance testing.Aging mice were fed a normal diet (PicoLab Rodent Diet 20, LabDiet), mice on the HFD experiments were fed an HFD (TD.06414, 60% of kcal from fat; Envigo).

Flow cytometry
For in vivo sample preparation, livers were dissociated using the MACS liver dissociation kit (Miltenyi Biotec, 130-1-5-807), filtered through a 100-μm strainer and washed with PBS, and red blood cells were lysed by an ammonium-chloride-potassium (ACK) lysing buffer (Lonza).Cells were washed with PBS, resuspended in FACS buffer and either used for immediate analysis or fixed with Fixation Buffer (BD Biosciences, 554655) according to the manufacturer's instructions and used for later analysis.Spleens were mechanically disrupted with the back of a 5-ml syringe, filtered through a 40-μm strainer and washed with PBS and 2 mM EDTA; then red blood cells were lysed by ACK lysing buffer (Lonza).Gonadal adipose tissue was dissociated as described 42 .In short, adipose tissue was isolated and placed in a digestion solution consisting of 4 mg ml −1 collagenase, type II (Sigma) in DPBS (Life Technologies) supplemented with 0.5% BSA (Sigma) and 10 mM CaCl 2 digested at 37 °C for 20 min in a rotational shaker.Afterwards, samples were mechanically dissociated with a 10-ml serological pipette, filtered through a 40-μm strainer and washed with PBS and 2 mM EDTA; then red blood cells were lysed by ACK lysing buffer (Lonza).Pancreata were placed into cold DMEM with 10% FBS and penicillin and streptomycin.The pancreata were minced in this media on ice into 2-to 4-mm fragments so that they would pass through the end of a 1-ml pipette tip with ease.The minced tissue was collected in a 15-ml Falcon tube and dissociated in 100 mg ml −1 Dispase (Life Technologies, 17105041), 20 mg ml −1 collagenase P (Roche, 11249002001) and 1 mM EDTA for 20 min on a heated rocker at 37 °C (Eppendorf).After 20 min, 5 ml of DMEM with 10% FBS was added to quench the reaction.The supernatant was removed and filtered through a 100-μm filter (VWR).Next, 5 ml of dissociation media consisting of 100 mg ml −1 Dispase (Life Technologies, 17105041), 20 mg ml −1 collagenase P (Roche, 11249002001) and 1 mM EDTA was added before replacing the 15-ml tube into the heated rocker for 20 min.The reaction was quenched again after 20 min with media and filtered via a 100-μm filter.The dissociated cells were spun at 500 r.c.f. for 10 min in a swinging-bucket centrifuge.The supernatant was discarded, and the cells were resuspended in ACK lysis buffer for 2-4 min in ice.Cells were washed with PBS, resuspended in FACS buffer and either used for immediate analysis or fixed with Fixation Buffer (BD Biosciences, 554655) and used for later analysis.

Article
https://doi.org/10.1038/s43587-023-00560-5 Gene expression reads were aligned to the mouse reference genome version gex-mm10-2020-A, available from the 10x Genomics website.We kept cells using the following parameters: 'min.cells> 10, nFeature_RNA > 500, nCount_RNA > 2,500, percent.mt< 15'.Gene expression count data were normalized using SCTransform to regress out the percentage of mitochondrial RNA.The R package BBKNN was used to remove batch effects between mouse samples, and 0.5 was used as expression cutoff to define uPAR High cell populations.Clusters were identified using a resolution of 0.8, and cell types were annotated using R packages celldex, SingleR, Azimuth and custom gene sets 20,21 .Known markers for each cell type were plotted using the DotPlot function in Seurat.Senescence gene sets from refs.17,24 were used to calculate signature scores using the AddModuleScore function in Seurat, and a signature score cutoff of 0.05 was used to define Senescence High cell populations.Differential expression analysis and functional annotations of gene sets were analyzed in the following way: Differential gene expression analysis was performed by comparing all the uPAR-positive versus uPAR-negative cells using RunPresto in Seurat, and the differentially expressed genes (DEGs) were determined by >1.5-fold change in gene expression with adjusted P value < 0.1.Pathway enrichment analysis was performed using the msigDB Hallmark gene sets using enrichR 43 .Significance of the tests was assessed using combined score, described as c = log(P) × z, where c is the combined score, P is Fisher's exact test P value, z is the z-score for deviation from expected rank, and adjusted P values defined by enrichR.A lollipop plot was generated by plotting the top enriched/ depleted log 2 (combined.score)on the x axis (directional), and size and color of the dots represents by −log 10 (adjusted P value).

Genetic modification of T cells
The mouse SFG γ-retroviral m.uPAR-m28z plasmid has been described 14 .The mouse SFG γ-retroviral h.19-m28z plasmid 14 was constructed by stepwise Gibson assembly (New England BioLabs) using the amino acid sequence for the scFv specific for human CD19 of the SFG-1928z backbone 46 and cloned into the backbone of the SFG γ-retroviral m.uPAR-m28z plasmid 14 .In both constructs, the anti-mouse uPAR scFv or anti-human CD19 scFv is preceded by a mouse CD8A leader peptide and followed by the Myc-tag sequence (EQKLISEEDL), mouse CD28 transmembrane and intracellular domain and mouse CD3z intracellular domain 44,45 .Plasmids encoding the SFGγ retroviral vectors were used to transfect gpg29 fibroblasts (H29) to generate VSV-G pseudotyped retroviral supernatants, which were used to construct stable retrovirusproducing cell lines as described 44,46 .

Glucose tolerance testing
Blood samples from mice fasted for 8-12 h were collected at 0, 15, 30, 60 and 120 min after intraperitoneal injections of glucose (Sigma-Aldrich; 2 g per kg body weight for aging experiments and 1 g per kg body weight for HFD experiments).Insulin was measured from serum collected at the 0-min and 15-min time points.Concentrations were determined using the UltraSensitive Mouse Insulin ELISA kit (Crystal Chem, 90080).

Insulin tolerance testing
Blood samples from mice fasted for 4 h were collected at 0, 15, 30 and 60 min after intraperitoneal injections of insulin (Humulin R; Eli Lilly; 0.5 units per kg body weight).

Histological analysis
Tissues were fixed overnight in 10% formalin, embedded in paraffin and cut into 5-μm sections.Sections were subjected to H&E staining.Immunohistochemical staining was performed following standard protocols.The following antibodies were used: anti-mouse uPAR (AF534, R&D, DCL0521042; 1:50 dilution) and horse anti-goat IgG (30116; Vector Laboratories, ZH0526).Three fields per section were counted per sample with Fiji-ImageJ and averaged to quantify the percentage of uPAR-positive area per field.SA-β-gal staining was performed as previously described 47 at a pH of 5.5 for mouse tissues.Specifically, fresh frozen tissue sections were fixed with 0.5% glutaraldehyde in PBS for 15 min, washed with PBS supplemented with 1 mM MgCl 2 and stained for 5-8 h in PBS containing 1 mM MgCl 2 , 1 mg ml −1 X-gal, 5 mM potassium ferricyanide and 5 mM potassium ferrocyanide.Tissue sections were counterstained with eosin.Three fields per section were counted with ImageJ and averaged to quantify the percentage of SA-β-gal-positive area per field.

Exercise capacity testing
Exercise capacity was assessed using a motorized treadmill (model 1050 EXER 3/6; Columbus Instruments).For 3 d before testing, mice were acclimatized to the treadmill (the mice walked on the treadmill at 10 m min −1 for 10 to 15 min per day).Following acclimatization, all mice underwent exercise capacity tests on consecutive days.Tests began with mice walking at 10 m min −1 with speed increased by 2 m min −1 every 2 min until exhaustion (mice could no longer achieve treadmill running speed despite repeated encouragement).The primary end points were time to exhaustion and maximum speed.

Blood measurements
Complete blood counts with differentials were performed using an automated hematology analyzer (IDEXX Procyte DX).For serum chemistry, blood was collected in tubes containing a serum separator.The tubes were then centrifuged, and the serum was obtained for analysis.Serum chemistry was performed by the LCP on a Beckman Coulter AU680 analyzer (Beckman Coulter Life Sciences).For cytokine analysis, plasma was collected and samples were processed and measured by Eve Technologies.

Statistical analysis
Data are presented as the mean ± s.e.m.Statistical analysis was performed by Student's t-test or Mann-Whitney test using Prism v9.3.1 (GraphPad software).No statistical methods were used to predetermine sample size in the mouse studies, and no randomization method was used to allocate mice to experimental groups.Mouse conditions were observed by an operator who was blinded to the treatment groups in addition to the main investigator who was not blind to group allocation.Pathological analysis and exercise testing studies were performed in a blinded fashion.Data analysis was not performed in a blinded fashion.Data analysis was based on objectively measurable data (cell count, blood tests).No data were excluded except for histological assessment of HFD experiments, where we excluded OCT cassettes of samples containing adipose tissue or pancreas that were folded and presented a morphology that did not allow for successful slide generation; these were not further processed.Data distribution was assumed to be normal, but this was not formally tested.No adjustment for multiple comparisons was performed.The rationale for this was that to increase the rigor of select analyses, two control groups were compared to the experimental group, but it could have been biologically possible to just have one control group.Thus, for any given endpoint, there were two pairwise comparisons: the experiment group separately compared to each control.While two tests were evaluated, we only considered the analysis statistically significant if both tests had a P value less than 0.05.If only one of the two tests was significant, we did not claim the groups were significantly different; instead, we considered the analysis inconclusive and reported a trend.Viewing the analysis as significant only if both P values were less than 0.05 preserves the family-wise error rate at less than 0.05.Figures were prepared using BioRender.comfor scientific illustrations in Figs.3a and 7a,j, GraphPad Prism v9.3.1, and Microsoft Excel v16.77 and Illustrator CC 2022 (Adobe).

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.Extended Data Fig. 5 | uPAR CAR T cells are not associated with signs of tissue damage in aged tissues and do not exacerbate spontaneous agerelated histological changes in lung, liver and kidneys.Mice received cell infusions at 18-20 months and were sacrificed 20 days after infusion of the indicated T cells.Sections were stained with hematoxylin and eosin.Aged mice showed mononuclear leukocytic aggregates composed predominantly of lymphocytes and plasma cells in tissues in an age dependent manner.These leukocytic aggregates were more frequently observed in tissues from uPARm.28zCAR T-treated aged mice than tissues from control aged mice and were not associated with necrosis and/or degeneration in tissues from both experimental and control aged mice.These lymphocytic and plasmocytic aggregates in tissues are often observed in naïve aged mice and are considered spontaneous background findings in longitudinal aging studies in mice 50,51 .a, Representative sections of normal cerebral cortex and meninges at the level of the posterior hypothalamus (inset: hippocampus).b.Histology of normal cardiomyocytes and interstitium in myocardium (inset: ventricles and interventricular septum).c.Representative histology of normal lungs showed dense aggregates of lymphocytes and fewer plasma cells and macrophages around bronchioles or vasculature (inset: pulmonary lobes).d.The liver from aged mice showed accumulation of lymphocytic and histiocytic aggregates in portal to periportal regions (Inset: hepatic lobe).e. Histology of the kidneys showed accumulation of lymphocytes and plasma cells in the renal interstitium (n & o) and around blood vessels (inset: renal cortex, medulla, and pelvis).f.Representative sections of normal pancreatic acini (exocrine pancreas) and islets of Langerhans (endocrine pancreas; inset: pancreatic lobule).Images were captured at 4x (insets) and 40x magnifications.Results of 1 independent experiment (with n = 3 per group).

Fig. 2 |
Fig. 2 | Upregulation of uPAR and senescence signatures in aged human pancreas.scRNA-seq data of human pancreas of different ages from ref. 26 were analyzed.a, UMAP visualization of Plaur expression across pancreas cell types in young humans (0-6 years old) and old humans (50-76 years old).b, UMAP visualization of senescence signature expression 17 across pancreas cell

Fig. 5 |
Fig. 5 | uPAR CAR T cells revert natural age-associated phenotypes.Mice were treated with m.uPAR-m.28zCAR T cells, h.19-m.28zCAR T cells or UT cells as schematized in Fig. 3a.a, Levels of basal glucose (mg ml −1 ) after starvation 2.5 months after cell infusion (n = 11 mice for UT; n = 12 for h.19-m.28zand for m.uPAR-m.28z).b, Levels of glucose before (0 min) and after intraperitoneal administration of glucose (2 g per kg body weight) 2.5 months after cell infusion (samples sizes as in a).c, Area under the curve (AUC) representing the results from b.Each point represents a single mouse.d, Levels of insulin before and 15 min after intraperitoneal glucose administration (2 g per kg body weight) 2.5 months after cell infusion (n = 6 for UT; n = 5 for h.19-m.28z;n = 6 for m.uPARm.28z).e, Fold change in time to exhaustion in exercise capacity testing before cell infusion and 2.5 months after it (n = 7 for UT; n = 8 for h.19-m.28zand n = 8 for m.uPAR-m.28z).f, Fold change in maximum speed in capacity testing before cell infusion and 2.5 months after it (sample sizes as in e).g,h, Percentage of CD45.1 + T cells in the spleen (g) or liver (h) of 4-month-old or 20-month-old mice 20 d after cell infusion (n = 3 mice per age group for UT and for h.19-m.28z;n = 4 for m.uPAR-m.28z).The corresponding flow cytometry gating is shown in Extended Data Fig. 10.Results are from two independent experiments (a-c, e and f) or one experiment (d, g and h).Data are the mean ± s.e.m.; P values from two-tailed unpaired Student's t-test (a, c, d, g and h) or two-tailed Mann-Whitney test (e and f).

Fig. 6 |
Fig. 6 | uPAR CAR T cells prevent natural age-associated phenotypes.Threeto four-month-old C57BL/6N mice were injected with 0.5 × 10 6 m.uPAR-m.28zCAR T cells, h.19-m.28zCAR T cells or UT cells generated from CD45.1 mice 16 h after administration of cyclophosphamide (200 mg per kg body weight).Mice were monitored over time and/or harvested at 15 months of age.a,b, Percentage of CD45.1 + T cells in the spleen (a) or liver (b) of 15-month-old mice 12 months after cell infusion (n = 3 mice per group).c, Levels of basal glucose after starvation 15-18 months after cell infusion (n = 11 mice for UT cells; n = 12 for h.19-m.28zand for m.uPAR-m.28z).d, Levels of glucose before (0 min) and after intraperitoneal administration of glucose (2 g per kg body weight) 15-18 months after cell infusion (sample sizes as in c).e, AUC representing the results

Fig. 7 |
Fig.7| uPAR CAR T cells are therapeutic and preventive in metabolic syndrome.a, Experimental scheme for b-i.Three-month-old C57BL/6N mice were treated with an HFD for 2 months followed by intravenous infusion with 0.5 × 10 6 m.uPAR-m.28zor UT cells 16 h after administration of cyclophosphamide (200 mg per kg body weight).Mice were euthanized 1 month later or monitored over time.b, Body weight 1 month after cell infusion (n = 10 mice per group).c, Basal glucose levels after starvation at 1 month after cell infusion (n = 10 mice per group).d, Glucose levels before (0 min) and after intraperitoneal administration of glucose (1 g per kg body weight) 1 month after cell infusion (n = 10 mice per group).e, AUC representing the results from d. f, Glucose levels before (0 min) and after intraperitoneal administration of insulin (0.5 units per kg body weight) 1 month after cell infusion (n = 4 mice per group).g, AUC representing the results from f.Each point represents a single mouse.h, Glucose levels before (0 min) and after intraperitoneal glucose administration (1 g per kg body weight) 2.5 months after cell infusion (n = 3 mice per group).i, AUC representing the results from h.Each point represents

Extended Data Fig. 1 |
Characterization of uPAR-positive cells in aging.a, RNA expression of Plaur in liver, adipose tissue (fat) and muscle of young (3 months) or old (21 months) mice.Data obtained from the Tabula Muris Senis project 18 .b, Quantification of immunohistochemical staining of mouse uPAR in liver, adipose tissue, muscle and pancreas from young (age 3 months) or old (age 20 months) mice (n = 3 per age).c, Hematoxylin and eosin staining and immunofluorescence staining of young (age 3 months n = 3 mice) or old (age 18-20 months n = 3 mice) livers.uPAR (green), β-gal (red), F4/80 (white), DAPI (blue).d, Percentage of SA-b-gal positive cells in young and aged livers in c. e, Hematoxylin and eosin staining and immunofluorescence staining of young (age 3 months n = 3 mice) or old (age 18-20 months n = 3 mice) pancreas.uPAR (green), β-gal (red), F4/80 (white), DAPI (blue).f, Percentage of SA-b-gal positive cells in young and aged livers in e. g,h, Correlation (Pearson's R value) of β-gal and F4/80 co-staining, β-gal and uPAR co-staining or uPAR and F4/80 co-staining in aged livers (g) and aged pancreas (h).i,j, Percentage of β-gal positive cells that costain for F4/80, uPAR or uPAR and F4/80 in aged livers (i) and aged pancreas (j).Data are mean ± s.e.m (a,b,d,f-h); values are derived from two-tailed unpaired Student's t-tests (a,b,d,f) one-way ANOVA with multiple comparisons (g,h).Results are from 1 independent experiment (a-j).Extended Data Fig. 2 | Single cell profile of aged tissues.a, Dot plot showing expression of 34 signature genes across the 12 lineages of the liver.The size of the dots represents the proportion of cells expressing a particular marker, and the color scale indicates the mean expression levels of the markers (z-score transformed).b, Fractions of uPAR-positive and uPAR-negative cells in the various lineages in liver (n= the sequencing of 4 mice where 2 females were combined into one replicate and 2 males were combined into another replicate).Error bars represent s.d.c, Dot plot showing expression of 40 signature gene expressions across the 13 lineages of the adipose tissue.The size of the dots represents the proportion of cells expressing a particular marker, and the color scale indicates the mean expression levels of the markers (z-score transformed).d, Fractions of uPAR-positive and uPAR-negative cells in the various lineages in adipose tissue (n= the sequencing of 4 mice where 2 females were combined into one replicate and 2 males were combined into another replicate).Error bars represent s.d.e, Dot plot showing expression of 40 genes across the 12 lineages of the pancreas.The size of the dots represents the proportion of cells expressing a particular marker, and the color scale indicates the mean expression levels of the markers (z-score transformed).f, Fractions of uPAR-positive and uPARnegative cells in the various lineages in pancreas (n= the sequencing of 4 mice where 2 females were combined into one replicate and 2 males were combined into another replicate).Error bars represent s.d.Data are mean ± s.d.; p values are derived from two-tailed unpaired Student's t-tests (b,d,f).Results are from 1 independent experiment (a-f).Extended Data Fig. 3 | Characteristics of senescent uPAR-positive cells in aged tissues.a-c, Molecular Signature Database Hallmark 2020 signatures that are significantly enriched in uPAR positive cells vs uPAR negative cells of liver (a), adipose tissue (b) and pancreas (c).d-f, quantification of the proportion of uPAR positive and negative cells by cell type contributing to the respective senescence signature in Fig. 1h (d), Fig. 1j (e) and Fig. 1l (f).g-o, UMAP visualizations with senescence signature scores 17 in each cell indicated by the color scale.Below: quantification of the proportion of uPAR positive and negative cells contributing to the respective senescence signature in total (h,k,n) and by cell type (i,l,o).g,h,i, liver; j,k,l, adipose tissue; m,n,o; pancreas.Results are from 1 independent experiment with (n = the sequencing of 4 mice where 2 females were combined into one replicate and 2 males were combined into another replicate) (a-m).Extended Data Fig. 4 | Effect of uPAR CAR T cells on aged tissues.a-c, Quantification of SA-β-Gal-positive cells in adipose tissue, liver and pancreas 20 days after cell infusion (n = 3 for UT; n = 3 for h.19-m.28z;n = 4 for m.uPARm.28z).d-f, Quantification of uPAR-positive cells in adipose tissue, liver and pancreas 20 days after cell infusion (n = 3 per group).g-j, Percentage of dendritic cells and uPAR + dendritic cells in the adipose tissue (g,h) or liver (i,j) 20 days after cell infusion (n = 3 for UT; n = 3 for h.19-m.28z;n = 4 for m.uPAR-m.28z).k-n, Percentage of macrophages and uPAR + macrophages in the adipose tissue (k,l,) or liver (m,n) 20 days after cell infusion (n = 3 for UT; n = 3 for h.19-m.28z;n = 4 for m.uPAR-m.28z).o-r, Percentage of monocytes and uPAR + monocytes in the adipose tissue (o,p) or liver (q,r) 20 days after cell infusion (n = 3 for UT; n = 3 for h.19-m.28z;n = 4 for m.uPAR-m.28z).Results of 1 independent experiment (a-r).Data are mean ± s.e.m.; p values from two-tailed unpaired Student's t-test (a-r).

Fig. 6 |
Effect of uPAR CAR T cells in young and old tissues.a-b, Mice received cell infusion at 3 months old.a, Levels of glucose before (0 min) and after intraperitoneal administration of glucose (2 g/kg) 2.5 months after cell infusion (n = 13 for untransduced T cells; n = 12 for h.19-m.28zand n = 13 for m.uPAR-m.28z).b, Area under the curve (AUC) representing the results from a.Each point represents a single mouse.c-d, Mice received cell infusion at 18-20 months old.c, Levels of glucose before (0 min) and after intraperitoneal administration of insulin (0.5 units/kg body weight) 2.5 months after cell infusion (n = 10 for untransduced T cells and n = 10 for m.uPAR-m.28z).d, Area under the curve (AUC) representing the results from c.Each point represents a single mouse.Results of 2 independent experiments (a,b) or 1 independent experiment (c,d).Data are mean ± s.e.m.; p values from two-tailed unpaired Student's t-test (b,d).Extended Data Fig. 7 | Profile of and long-term effects of uPAR CAR T cells in aging.a,b, Percentage of CD4 + or CD8 + cells among CD45.1 + T cells from the spleen (a) or liver (b) of 4-month-old or 20-month-old mice 20 days after cell infusion (n = 3 mice per age group for untransduced T cells [UT] and for h.19m.28z;n = 4 for m.uPAR-m.28z).c,d, Percentage of CD45.1 + T cells expressing differentiation markers CD62L and CD44 in the spleen (c) or liver (d) of 4-monthold or 20-month-old mice 20 days after cell infusion (sample sizes as in a).e,f, Percentage of CD4 + or CD8 + cells among CD45.1 + T cells in the spleen (e) or liver (f) of 15-month-old mice 12 months after cell infusion (n = 3 mice per group).g,h, Percentage of CD45.1 + T cells expressing differentiation markers CD62L and CD44 on CD45.1 + T cells in the spleen (g) or liver (h) of 15-month-old mice 12 months after cell infusion (n = 3 mice per group).i, Time to exhaustion in exercise capacity testing 12 months after cell infusion (n = 8 for untransduced T cells; n = 6 for h.19-m.28z;n = 12 for m.uPAR-m.28z).j, Maximum speed (m/min) in capacity testing 12 months after cell infusion (sample sizes as in i).k-m, Quantification of SA-β-Gal-positive cells 12 months after cell infusion in (k) adipose tissue (n = 6 for UT; n = 5 for h.19-m.28z;n = 6 for m.uPAR-m.28z);(l) liver (n = 6 for UT; n = 5 for h.19-m.28z;n = 5 for m.uPAR-m.28z)and (m) pancreas (n = 6 for UT; n = 5 for h.19-m.28z;n = 6 for m.uPAR-m.28z).n-p, Quantification of uPAR-positive cells in (n) adipose tissue, (o) liver and (p) pancreas 12 months after cell infusion (n = 3 per group).Results of 1 independent experiment (a-h, n-p) or 2 independent experiments (i-m).Data are mean ± s.e.m.; p values from two-tailed unpaired Student's t-test (a-h, k-p) or two-tailed Mann Whitney test (i,j).Extended Data Fig. 9 | Profile and persistence of uPAR CAR T cells in metabolic syndrome.T cells were assessed in spleen (a-d) and liver (e-h) 3.5 months after cell infusion in the experimental scheme depicted in Fig. 7j.a, Percentage of CD45.1 + T cells in the spleen.b, Percentage of CD4 + cells among CD45.1 + T cells in the spleen.c, Percentage of CD8 + cells among CD45.1 + T cells in the spleen.d, Percentage of CD45.1 + T cells from the spleen expressing differentiation markers CD62L and CD44.e, Percentage of CD45.1 + T cells in the liver.f, Percentage of CD4 + cells among CD45.1 + T cells in the liver.g, Percentage of CD8 + cells among CD45.1 + T cells in the liver.h, Percentage of CD45.1 + T cells in the liver expressing differentiation markers CD62L and CD44.Results in each panel are from 1 experiment (n = 5 mice per group).Data are mean ± s.e.m.; p values from two-tailed unpaired Student's t-test.