Abstract
Cellular senescence is characterized by stable cell-cycle arrest and a secretory program that modulates the tissue microenvironment1,2. Physiologically, senescence serves as a tumour-suppressive mechanism that prevents the expansion of premalignant cells3,4 and has a beneficial role in wound-healing responses5,6. Pathologically, the aberrant accumulation of senescent cells generates an inflammatory milieu that leads to chronic tissue damage and contributes to diseases such as liver and lung fibrosis, atherosclerosis, diabetes and osteoarthritis1,7. Accordingly, eliminating senescent cells from damaged tissues in mice ameliorates the symptoms of these pathologies and even promotes longevity1,2,8,9,10. Here we test the therapeutic concept that chimeric antigen receptor (CAR) T cells that target senescent cells can be effective senolytic agents. We identify the urokinase-type plasminogen activator receptor (uPAR)11 as a cell-surface protein that is broadly induced during senescence and show that uPAR-specific CAR T cells efficiently ablate senescent cells in vitro and in vivo. CAR T cells that target uPAR extend the survival of mice with lung adenocarcinoma that are treated with a senescence-inducing combination of drugs, and restore tissue homeostasis in mice in which liver fibrosis is induced chemically or by diet. These results establish the therapeutic potential of senolytic CAR T cells for senescence-associated diseases.
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Data availability
The RNA-seq data have been deposited in the Gene Expression Omnibus under the accession number GSE145642. Source data are provided with this paper. All other data supporting the findings of this study will be made available upon reasonable request to the corresponding authors. Source data
Change history
21 February 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41586-024-07197-3
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Acknowledgements
We thank A. Lujambio and R. Brody and the Biorepository and Pathology Core at Icahn School of Medicine at Mount Sinai, and G. Askan and O. Basturk at the Department of Pathology at MSKCC, for tissue samples; L. Zender and H. Chen for sharing plasmids; N. Salgado, H. Chen, T. Baslan, S. Tian, A. Wuest, W. Luan and G. Gunset for technical assistance; and C. J. Sherr, E. de Stanchina, N. Kuhn, A. Dobrin, M. L. Sjöstrand and other members of the Lowe and Sadelain laboratories for insightful discussions. This work was supported by a grant from the National Institute of Aging (AG065396) to S.W.L., the Pasteur-Weizmann/Servier award to M.S. and a Memorial Sloan Kettering Cancer Center support grant (P30 CA008748) to both S.W.L. and M.S. laboratories. S.L.F. was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK56621), a grant from the Department of Defense (CA150272) and the P30 grant (CA165979); C.A. was supported by a postgraduate fellowship from La Caixa foundation and is the recipient of the Harold E. Varmus graduate student fellowship from the Gerstner Sloan Kettering graduate school; J.F. was supported by the Care-for-Rare Foundation and the German Research Foundation (DFG); J.L. was supported by a fellowship from the DFG and a Shulamit Katzman Endowed Postdoctoral Research Fellowship; J.F. and J.L. are part of the Experimental Medicine Program at the University of Tuebingen; D.A.-C. was supported by a postdoctoral fellowship from Fundación Ramón Areces; J.A.B. was supported by the Grayer postgraduate fellowship and the Geoffrey Been graduate student fellowship from the Gerstner Sloan Kettering graduate school; and A.K. was supported by a grant from the National Cancer Institute (U54 0D020355-01). S.W.L. is the Geoffrey Been Chair of Cancer Biology and a Howard Hughes Medical Institute Investigator. We thank the following MSKCC core facilities for support: SKI flow cytometry core facility, animal facility, antitumor assessment core, laboratory for comparative biology, bioinformatics core and integrated genomics operation core.
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Contributions
C.A., J.F. and J.L. conceived the project, designed, performed and analysed experiments and wrote the paper with assistance from all authors. Y.-J.H. analysed RNA-seq data. C.Z., D.A.-C., J.M.-S., J.A.B., X.L., A.K., S.H. and T.G. performed and analysed experiments. A.P. performed histopathological toxicity analysis. S.L.F. provided human liver samples, analysed data and reviewed the manuscript. E.P. provided human carotid endarterectomy samples and reviewed the manuscript. V.P. helped with T cell imaging studies. M.S. and S.W.L. conceived the project, supervised experiments and wrote the paper. All authors read and approved of the paper.
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Competing interests
A patent application (PCT/US2020/016290; filed on 02/01/2020) has been submitted based in part on results presented in this manuscript on the use of CAR T cells that target uPAR as senolytic agents. C.A., J.F., J.L., M.S. and S.W.L. are listed as the inventors. J.F. and M.S. hold other unrelated patents on CAR technologies.S.W.L. is an advisor for and has equity in the following biotechnology companies: ORIC Pharmaceuticals, Faeth Therapeutics, Blueprint Medicines, Geras Bio, Mirimus Inc., PMV Pharmaceuticals and Constellation Pharmaceuticals.
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Peer review information Nature thanks Jesus Gil, Stephen Gottschalk and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Fig. 1 Genes encoding surface molecules that are commonly upregulated in senescence.
a, Heat map of genes upregulated in therapy-induced senescence (TIS), oncogene-induced senescence (OIS) or replication-induced senescence (RIS) in HSCs. b, Venn diagram showing the number of common genes upregulated in the three datasets in a. c, Fold change (log2(expression in senescent cells/expression in non-senescent cells)) of the eight commonly upregulated genes in the three different datasets in a. d, Combined enrichment score of significantly enriched gene sets among the eight commonly upregulated genes in senescence. ECM, extracellular matrix; GPI, glycosylphosphatidylinositol. e, Heat map showing the expression profile of uPAR (PLAUR) in human vital tissues (as determined by the Human Proteome Map) compared to the expression profiles of other targets of CAR T cells in clinical trials. NK cells, natural killer cells. f, Immunohistochemical staining of mouse uPAR (m.uPAR) in vital tissues of C57BL/6J mice. Representative results of n = 2 independent experiments. g, Reads per kilobase (RPKM) of PLAUR mRNA in proliferating, quiescent (induced by serum starvation) or senescent (triggered by overexpression of HRASG12V) human IMR-90 fibroblasts. Results of one independent experiment with n = 3 replicates for proliferating, quiescent and senescent conditions. Data are mean ± s.e.m.; two-tailed unpaired Student’s t-test
Extended Data Fig. 2 uPAR is a cell-surface and secreted biomarker of senescence.
a, b, qPCR of SASP-associated gene expression in senescent versus proliferating mouse KP tumour cells (a) or human primary melanocytes (b) and representative SA-β-gal staining; a.u. arbitrary units. c, d, Co-immunofluorescence staining and quantifications of uPAR (red) and Ki-67 (green) (c) or uPAR (red) and IL-6 (green) (d). e, Immunohistochemical staining of uPAR or phosphorylated ERK (P-ERK) in serial sections of mouse livers six days after transfection by HTVI with a plasmid encoding NrasG12V. Representative results of two independent experiments (n = 3 mice per group). f–i, Mice expressing endogenous KrasG12D in pancreatic epithelial cells were treated with caerulein (Cr) and euthanized 21 weeks afterwards when they had developed pancreatic intraepithelial neoplasias. Age-matched C;RIK mice (expressing wild-type Kras) injected with PBS were used as controls. f, Co-immunofluorescence staining of KATE (red) and uPAR (green). Representative results of two independent experiments (n = 3 mice per group). g, Levels of suPAR in the mice in f. Representative results of two independent experiments (n = 2 mice per group). h, Co-immunofluorescence staining and quantification of uPAR (red) and Ki-67 (green). Representative results of two independent experiments (n = 3 mice per group). i, Representative SA-β-gal staining. Representative results of one independent experiment (n = 3 mice per group). j–m, Mice were treated with either vehicle or CCl4 twice weekly for six weeks to induce liver fibrosis. j, Fold change in serum levels of suPAR. Representative results of two independent experiments (vehicle, n = 4; CCl4, n = 9 mice per group). Two-tailed unpaired Student’s t-test. k, Co-immunofluorescence staining and quantification of uPAR (red) and Ki-67 (green). Representative results of two independent experiments (n = 2 mice per group). l, Co-immunofluorescence staining and quantification of uPAR (red) and IL-6 (green). Representative results of two independent experiments (n = 3 mice per group). m, Representative SA-β-gal staining. Representative results of two independent experiments (n = 3 mice per group). Data are mean ± s.e.m. (c, d, h, j, l).
Extended Data Fig. 3 uPAR is a marker of senescence in senescence-associated human pathologies.
a, Left, immunohistochemical expression of human uPAR (h.uPAR) and SA-β-gal in human samples of hepatitis-induced liver fibrosis (n = 7 patients). Right, co-immunofluorescence staining and quantification of uPAR (red) and p16 (green) or uPAR (red) and IL-6 (green) in human samples of hepatitis-induced liver fibrosis (n = 3). b, Left, immunohistochemical expression of uPAR and SA-β-gal in human samples from patients with eradicated hepatitis C virus (HCV) and residual liver fibrosis (n = 7 patients). Right, co-immunofluorescence staining and quantification of uPAR (red) and p16 (green) or uPAR (red) and IL-6 (green) in human samples of HCV-induced liver fibrosis (n = 3). Data are mean ± s.e.m. (a, b). c, Immunohistochemical staining of uPAR in human carotid endarterectomy samples (n = 5 patients). d, Immunohistochemical staining of uPAR in human pancreas bearing pancreatic intraepithelial neoplasia (PanIN) compared to normal pancreas controls (n = 3 patients).
Extended Data Fig. 4 m.uPAR-h.28z CAR T cells selectively target uPAR-positive cells.
a, Construct maps encoding human m.uPAR-h.28z and h.19-h.28z CARs or mouse m.uPAR-m.28z and m.19-m.28z CARs. b, Flow cytometry analysis showing the expression levels of CAR and LNGFR in m.uPAR-h.28z and h.19-h.28z CAR T cells compared to untransduced T cells. Representative results of n = 4 independent experiments. c, Flow cytometry analysis of mouse uPAR and human CD19 expression on wild-type NALM6 cells and NALM6-m.uPAR cells. Representative results of n = 3 independent experiments. d, Cytotoxic activity of m.uPAR-h.28z, h.19-h.28z and untransduced T cells as determined by 4-h calcein assay with firefly luciferase (FFL)-expressing NALM6 wild-type or NALM6-m.uPAR cells as targets. Representative results of n = 3 independent experiments performed in triplicate. Data are mean ± s.e.m. e, Granzyme B (GrB) and IFNγ expression of CD4+ and CD8+ m.uPAR-h.28z CAR T cells 18 h after co-culture with wild-type NALM6, NALM6-m.uPAR or senescent KP cells as determined by intracellular cytokine staining. Results of n = 1 independent experiment (no target and NALM6 WT, n = 2; NALM6-m.uPAR and KP senescent, n = 3 replicates). Data are mean ± s.e.m. f, Experimental layout for Fig. 2c–i. Mice were injected with a plasmid encoding NrasG12V-GFP-luciferase and treated with 0.5 × 106 m.uPAR-h.28z CAR T cells or untransduced T cells 10 days after injection. Mice were euthanized 15 days after CAR administration and livers were used for further analysis. Images were created with BioRender.com. g, Flow cytometry analysis of mouse uPAR and human CD19 expression on wild-type Eμ-ALL01 cells and Eμ-ALL01-m.uPAR cells. Representative results of n = 3 independent experiments. h, Flow cytometry staining of Myc-tag and mouse uPAR on m.uPAR-m.28z CAR T cells, m.19-m.28z CAR T cells and untransduced T cells as compared to FMO control. Representative results of n = 2 independent experiments.
Extended Data Fig. 5 Senolytic CAR T cells target senescent cells in a KrasG12D-driven model of lung cancer.
a, Experimental layout. C57BL/6N mice were intravenously injected with 10,000 KrasG12D;p53−/− cells. Treatment with combined MEK inhibitor (1 mg per kg body weight) and CDK4/6 inhibitors (100 mg per kg body weight) was started seven days later, followed by adoptive transfer of 2 × 106 CD45.1+ T cells (m.uPAR-m.28z CAR T cells, m.19-m.28z CAR T cells or untransduced T cells) one week later. A subset of mice received a second infusion of 1 × 106 m.uPAR-m.28z CAR T cells, m.19-m.28z CAR T cells or untransduced T cells seven days after the first injection of T cells. The images of the mouse, tumour cells and CAR T cells were created with BioRender.com. Cp, cyclophosphamide. b, Kaplan–Meier curve showing survival of mice (one-sided log-rank (Mantel–Cox) test). Results of two independent experiments (UT, n = 16; m.19-m.28z, n = 14; m.uPAR-m.28z, n = 18; UT reinjection, n = 6; m.19-m.28z reinjection, n = 7; m.uPAR-m.28z reinjection, n = 7 mice). c, d, Weight (c) and temperature (d) measured 24 h before and at different time points after CAR T cell infusion. P values (ns, not significant) refer to the comparison between untransduced and m.uPAR-m.28z injected mice at 48 h (weight, P = 0.9329; temperature, P = 0.1534). Results of one independent experiment (UT, n = 5; m.19-m.28z, n = 5; m.uPAR-m.28z, n = 8; UT reinjection, n = 5; m.19-m.28z reinjection, n = 7; m.uPAR-m.28z reinjection, n = 7 mice). e, Cell counts of CD45.1+ T cells and expression of the activation markers CD25 and CD69 (UT, n = 4; m.19-m.28z, n = 5; m.uPAR-m.28z, n = 5 mice) on CD45.1+ T cells in the lungs of mice seven days after administration of m.uPAR-m.28z CAR T cells, m.19-m.28z CAR T cells or untransduced T cells. f, Representative SA-β-gal staining and quantification in the lungs of mice seven days after treatment with m.uPAR-m.28z CAR T cells compared to mice that were treated with m.19-m.28z CAR T cells or untransduced T cells (n = 3 mice per group). Data are mean ± s.e.m.; two-tailed unpaired Student’s t-test. (c–f).
Extended Data Fig. 6 Senolytic CAR T cells show therapeutic activity in CCl4-induced liver fibrosis.
a, Layout for experiments performed using the CCl4-induced liver fibrosis model: C57BL/6N mice received intraperitoneal infusions of CCl4 twice weekly for six weeks and were intravenously infused with 0.5–1 × 106 (Fig. 3) or 2–3 × 106 (c–i) mouse m.uPAR-m.28z CAR T cells, m.19-m.28z CAR T cells or untransduced T cells 16–24 h after administration of cyclophosphamide (200 mg kg−1). Mice were euthanized 20 days after CAR T cell infusion to assess liver fibrosis. Images were created with BioRender.com. b, Expression of GFP-tagged click beetle red (CBR) luciferase and Myc-tag in m.uPAR-m.28z and m.19-m.28z CAR T cells that were used for T cell imaging experiments (Fig. 3g, h) compared to control T cells. Representative results of n = 2 independent experiments. c, Sirius red and SA-β-gal staining and quantifications in livers from treated mice (n = 6 mice per group). d, Co-immunofluorescence of uPAR (red) and SMA (green) or Myc-tag (red) and SMA (green) in the livers of treated mice. e, Fold change difference in serum levels of suPAR 20 days after compared to 1 day before (day −1) injection of CAR T cells (UT, n = 18; m.19-m.28z, n = 6; m.uPAR-m.28z, n = 17 mice). f, g, Levels of serum ALT (f) and AST (g) 20 days after CAR treatment (UT, n = 10; m.19-m.28z, n = 8; m.uPAR-m.28z, n = 10 mice). h, Co-immunofluorescence staining of desmin (red) and Ki-67 (green) in the livers of mice 15, 20 and 77 days after treatment with CAR T cells. CCl4 treatment was stopped 20 days after T cell infusion (n = 3 mice per group). i, Mice were treated with CCl4 for 10 weeks. Sirius red staining in the livers of mice before (day −1) and 20 days after T cell administration (UT, n = 4; m.uPAR-m.28z, n = 2 mice). Representative results of n = 2 independent experiments (c–i). Data are mean ± s.e.m.; two-tailed unpaired Student’s t-test (c, e–g).
Extended Data Fig. 7 Safety profile of m.uPAR-m.28z CAR T cells at therapeutic doses of T cells.
a–e, C57BL/6N mice received intraperitoneal infusions of CCl4 twice weekly for six weeks and were intravenously injected with 0.5–1 × 106 m.uPAR-m.28z CAR T cells, 1 × 106 m.19-m.28z CAR T cells or untransduced T cells 16 h after administration of cyclophosphamide (200 mg kg−1). Mice were euthanized 20 days after T cell administration to assess potential toxicities and lung histopathology. a, Kaplan–Meier curve showing survival of mice after treatment with m.uPAR-m.28z CAR T cells (n = 16 mice), m.19-m.28z CAR T cells (n = 6 mice) or untransduced T cells (n = 6 mice). b, c, Weight (b) and temperature (c) of mice measured before and at different time points after CAR T cell infusion (UT and m.19-m.28z, n = 6; m.uPAR-m.28z, n = 7 mice). The P value in b refers to differences in weight at 48 h. d, e, Representative H&E staining of lungs (d) and complete blood counts (e) of treated mice 20 days after T cell infusion (UT and m.19-m.28z, n = 3 or 4; m.uPAR-m.28z, n = 4 mice). An increased accumulation of macrophages was observed in the immunodeficient setting. Representative results of n = 1 independent experiment (a–e). Data are mean ± s.e.m. (b, c, e); two-tailed unpaired Student’s t-test (b, e).
Extended Data Fig. 8 Safety profile of m.uPAR-m.28z CAR T cells at supratherapeutic doses of T cells.
C57BL/6N mice received intraperitoneal infusions of CCl4 twice weekly for six weeks followed by intravenous infusion of 2–3 × 106 m.uPAR-m.28z CAR T cells or untransduced T cells 16–24 h after administration of cyclophosphamide (200 mg kg−1). A subset of mice (as specified in the figure) received additional treatment with IL-6R-blocking antibodies (IL6Ri) and the IL-1R antagonist anakinra (IL1Ri), starting 24 h before T cell infusion and continuing daily until 6 days after T cell infusion. Mice were euthanized 12 weeks after CAR infusion to assess potential toxicities. a, Kaplan–Meier curve showing survival of mice after injection of CAR T cells (UT, n = 19; UT + IL6Ri/IL1Ri, n = 7; m.uPAR-m.28z, n = 30; m.uPAR-m.28z + IL6Ri/IL1Ri, n = 19 mice). b, c, Temperature (b) and weight (c) of treated mice (UT, n = 7; UT + IL6Ri/IL1Ri, n = 8; m.uPAR-m.28z, n = 11; m.uPAR-m.28z + IL6Ri/IL1Ri, n = 10 mice). d, Weight of mice 120 h after infusion with either m.uPAR-m.28z or m.uPAR-m.28z CAR T cells and additional treatment with IL6Ri and IL1Ri (m.uPAR-m.28z, n = 11; m.uPAR-m.28z + IL6Ri/IL1Ri, n = 10 mice). e, Serum levels of IL-6, GM-CSF, G-CSF and IFNγ in mice that were treated with either m.uPAR-m.28z or untransduced T cells 72 h or 20 days after T cell infusion (UT, n = 5; m.uPAR-m.28z, n = 4 mice at 72 h; n = 5 mice at 20 days). f, g, Number of adoptively transferred CD45.1+ T cells (f) and number of macrophages, uPAR+ and iNOS+ macrophages (g) in the lungs of mice that were treated with m.uPAR-m.28z CAR T cells, m.19-m.28z CAR T cells or untransduced T cells alone or in combination with treatment with IL6Ri and ILR1Ri three days after T cell infusion (n = 4 mice per group). h, i, Number of macrophages (h) and uPAR+ macrophages (i) in the lungs, liver, bone marrow (BM) and spleen of untreated mice or mice treated with either m.uPAR-m.28z CAR T cells or untransduced T cells 12 weeks after T cell infusion (n = 3 mice per group). Representative results of n = 3 independent experiments (a–d) or n = 1 independent experiment (e–i). All data are mean ± s.e.m.; two-tailed unpaired Student’s t-test (d, e).
Extended Data Fig. 9 Therapeutic intervention with IL-6R and IL-1R inhibitors does not decrease the therapeutic efficacy of senolytic CAR T cells in CCl4-induced liver fibrosis.
a, Experimental layout. C57BL/6N mice received intraperitoneal infusions of CCl4 twice weekly for six weeks and were intravenously infused with 2–3 × 106 m.uPAR-m.28z CAR T cells or untransduced T cells 24 h after administration of cyclophosphamide (200 mg kg−1). IL-6R-blocking antibodies (IL6Ri) and anakinra (ILRi) were first administered 24 h before T cell infusion followed by daily (IL6Ri) or twice daily (IL1Ri) injections for the first six days until treatment was stopped. Mice were euthanized 20 days after T cell infusion to assess liver fibrosis. Images were created with BioRender.com. b, Fold change difference in serum levels of suPAR 20 days after compared to 1 day before (day −1) CAR T cell treatment (UT, n = 4; UT + IL6Ri/IL1Ri, n = 8; m.uPAR, n = 5; m.uPAR + IL6Ri/IL1Ri, n = 8 mice). c, d, Levels of serum ALT (c) and AST (d) in treated mice 20 days after T cell infusion (UT, n = 3; UT + IL6Ri/IL1Ri, n = 5; m.uPAR-m.28z, n = 5 (ALT) and n = 3 (AST); m.uPAR-m.28z + IL6Ri/IL1Ri, n = 5 mice). e, Representative levels of fibrosis evaluated by Sirius red staining and SA-β-gal staining in livers from treated mice and quantification of liver fibrosis and SA-β-gal+ cells in the respective livers 20 days after treatment (UT, n = 4; UT + IL6Ri/IL1Ri, n = 4; m.uPAR-m.28z, n = 4; m.uPAR-m.28z + IL6Ri/IL1Ri, n = 5 mice). f, Co-immunofluorescence staining of uPAR (red) and SMA (green) or Myc-tag (red) and SMA (green) in the livers of treated mice. Representative results of n = 1 independent experiment (b–f). Data are mean ± s.e.m.; two-tailed unpaired Student’s t-test (b–e).
Extended Data Fig. 10 Safety profile of senolytic CAR T cells at therapeutic doses in a mouse model of NASH-induced liver fibrosis.
a, Immunohistochemical expression of uPAR in samples from the ‘STAM’ model52,58 (n = 3 mice). b, Experimental layout for experiments performed using the model of diet-induced NASH (Fig. 4, this figure). C57BL/6N mice were treated with a chow or a NASH-inducing diet50 for three months, followed by intravenous infusion with 0.5 × 106 m.uPAR-m.28z CAR T cells or untransduced T cells 16 h after administration of cyclophosphamide (200 mg kg−1). Mice were euthanized 20 days after CAR infusion to assess liver fibrosis. Images were created with BioRender.com. c, Kaplan–Meier curve showing survival of mice after treatment with either m.uPAR-m.28z CAR T cells or untransduced T cells (m.uPAR-m.28z, n = 16; UT, n = 10 mice). d, e, Weight (d) and temperature (e) of mice 24 h before and at different time points after T cell infusion (m.uPAR-m.28z, n = 11; UT, n = 9 mice). Data are mean ± s.e.m. f, Representative H&E staining of the lungs of treated mice (m.uPAR-m.28z, n = 6; UT, n = 4 mice). Representative results of n = 2 independent experiments (c–f).
Extended Data Fig. 11 Gating strategies, summary and potential applications of senolytic CAR T cells.
a, b, Representative flow cytometry staining of m.uPAR-h.28z CAR T cells (a) or untransduced T cells (b) obtained from the livers of mice that had undergone HTVI (as depicted in Fig. 2). Representative results of one independent experiment (n = 4 mice per group). c, Summary of the key points of our findings. uPAR-28z CAR T cells (red) infiltrate fibrotic livers that contain senescent cells (blue) and efficiently eliminate them, leading to fibrosis resolution and improved liver function. The therapeutic action of senolytic uPAR-28z CAR T cells might be extended to other senescence-associated diseases such as atherosclerosis, diabetes or osteoarthritis. Images were created with BioRender.com.
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Amor, C., Feucht, J., Leibold, J. et al. Senolytic CAR T cells reverse senescence-associated pathologies. Nature 583, 127–132 (2020). https://doi.org/10.1038/s41586-020-2403-9
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DOI: https://doi.org/10.1038/s41586-020-2403-9
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