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

Abstract

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.

Main

Cellular senescence is a stress response program characterized by stable cell cycle arrest^1,2 and the production of the senescence-associated secretory phenotype (SASP), which includes pro-inflammatory cytokines and matrix remodeling enzymes³. In physiological conditions in young individuals (for example, wound healing, tumor suppression), the SASP contributes to the recruitment of immune cells, whose role is to clear the senescent cells and facilitate restoration of tissue homeostasis³. However, during aging, the combination of increased tissue damage and decreased function of the immune system leads to the accumulation of senescent cells^4,5, thereby generating a chronic pro-inflammatory 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 time¹⁰. In contrast, CAR T cells are a form of cellular therapy that redirects T cell specificity toward cells expressing a specific cell-surface antigen¹¹. 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 administration¹². 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.

Results

uPAR is upregulated in physiological aging

uPAR promotes remodeling of the extracellular matrix during fibrinolysis, wound healing and tumorigenesis¹³. In physiological conditions, it is primarily expressed in certain subsets of myeloid cells and, at low levels, in the bronchial epithelium¹⁴. We recently described the upregulation of uPAR on senescent cells across different cell types and multiple triggers of senescence¹⁴ 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 tissues¹⁴. 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 tissues¹⁷. To explore the association with uPAR expression in aged tissues further, we surveyed RNA-sequencing (RNA-seq) data from the Tabula Muris Senis project¹⁸. 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 levels¹⁹, 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 single-cell RNA sequencing (scRNA-seq) on approximately 4,000–15,000 uPAR-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-seq²⁵.

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) individuals²⁶. 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….