Senescent cells express atypical MHC molecules

Human fibroblasts were derived from skin explants of healthy volunteers and exposed to ionising radiation (X-ray) to induce senescence, as previously described25,26,27. We confirmed that a low dose of radiation (10 Gy) effectively arrested cell growth and induced the expression of senescence-associated markers, such as p16INK4a, persistent γH2AX foci and senescence-associated-β-galactosidase (SA-β-Gal) activity (Supplementary Fig. 1). We then examined the expression of MHC-class I, class II and non-classical MHC molecules (HLA-E, -F and -G) alongside MHC-related proteins of the MICA/B and ULBP families. We found that senescent cells have a significantly increased expression of atypical MHC molecules, such as MICA/B and HLA-E (Fig. 1a). A time course of cell surface expression of these ligands showed maximal expression between 7 and 14 days after irradiation (Fig. 1b), suggesting that MICA/B and HLA-E expression is associated with the establishment of senescence and is not only a result of acute DNA damage.

Fig. 1 Senescent human fibroblasts express atypical MHC molecules. a Primary human fibroblasts were derived from the human skin and induced to senesce by ionising radiation (IR, 10 Gy X-ray). MHC expression by senescent fibroblasts (white bars) analysed at day 14 after IR using flow cytometry (n = 6 different donors for MHC-I, HLA-E and MICA/B, n = 4 for MHC-II, HLA-G and ULBP). Mean fluorescence intensity (MFI) values are shown as fold change compared with non-irradiated controls, set as one (black bars). b Time course of HLA-E and MICA/B expression at the indicated intervals after irradiation (n = 5). c Representative FACS plots of the total MHC-I, HLA-E and MICA/B expression in senescent fibroblasts induced by ionising radiation (DNA-damage induced senescence), H-RAS activation (oncogene-induced senescence) or continuous passaging (replicative senescence). MHC expression was compared between senescent (black lines), non-senescent (filled histograms) and isotype controls (dashed lines). Human umbilical vein endothelial cells (HUVECs) were irradiated (10 Gy), and MHC expression analysed by flow cytometry as previously described. d Flow-cytometry analysis of co-expression of HLA-E and Ki67 and p16INK4a on irradiated fibroblasts (day 14 after irradiation) and non-irradiated controls. Numbers indicate percentages of cells per quadrant. The data are representative of at least three independent experiments from distinct samples. Statistical significance calculated with Mann–Whitney U test (a) and repeated measures ANOVA with Bonferroni correction (b). The data presented as means ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Full size image

To assess whether this pattern of MHC expression was common to other forms of senescence, we monitored MHC expression in cells induced to senesce by oncogenic RAS activation (H-RASG12V; oncogene-induced senescence; Supplementary Fig. 2A–C) or continuous passaging to replicative senescence (Supplementary Fig. 2D, E). Independently of the senescence-inducing stimuli, cells undergoing senescence consistently upregulated HLA-E and MICA/B (Fig. 1c). This expression was not unique to fibroblasts because human umbilical vein endothelial cells (HUVECs) exposed to a senescence-inducing dose of X-ray (Fig. 1c, bottom panels) also increased expression of HLA-E and MICA/B.

While the expression of MICA/B by senescent cells has been previously described16,17, the increased expression of HLA-E in senescent cells has not been shown previously. We used flow cytometry to confirm that HLA-E was expressed by senescent cells with additional markers. Non-irradiated fibroblasts expressed low levels of HLA-E and p16INK4a (Fig. 1d, left panel). In contrast, the frequency of HLA-E-positive cells was significantly higher in irradiated cells frequently co-expressing p16INK4a and rarely positive for Ki67 (Fig. 1d, right panel), indicating that the majority of HLA-E expressing cells were in cell-cycle arrest and expressed markers of senescence.

HLA-E expression by senescent cells is regulated by p38

To understand the molecular mechanisms regulating the expression of HLA-E on senescent cells, we investigated two major pathways that regulate senescence: the DNA-damage response (DDR) and p38 signalling. The DDR was previously implicated in increasing MICA/B expression in human and mouse fibroblasts exposed to genotoxic stress28,29. We therefore asked whether the DDR was also involved in regulating HLA-E expression after irradiation. We pretreated fibroblasts with the ATM inhibitor KU-55933 (10 μM) or DMSO 12 h before irradiation, and continuously thereafter over a period of 7 days. We monitored MHC expression by flow cytometry in non-irradiated cells and in vehicle- or KU-55933-treated cells 1, 4 and 7 days after irradiation. Consistent with previous reports, inhibition of ATM significantly decreased MICA/B expression after irradiation (Supplementary Fig. 3A). By contrast, ATM inhibition did not decrease the expression of HLA-E on irradiated fibroblasts (Supplementary Fig. 3B). Although we cannot exclude that HLA-E expression on senescent cells may be affected by the DNA-damage response, the increased expression of HLA-E at day 7 and day 14 after irradiation, when the expression of γH2AX is decreasing (Fig. 2a), suggests that the DNA-damage response is not necessary for HLA-E expression. By contrast, the similar kinetics of HLA-E and p38 phosphorylation at Thr180/Tyr182 (Fig. 2a) suggested a role of p38 signalling in regulating HLA-E expression on senescent cells. To test this, we pretreated senescent cells with the p38 inhibitor BIRB79630,31 or DMSO before irradiation and continuously thereafter for 7 days. As indicated by the levels of phosphorylated heat-shock protein 27 (p-Hsp27), a downstream target of p38, BIRB796 inhibited the activation of p38 after irradiation (Fig. 2b). Strikingly, the inhibitor also prevented the upregulation of HLA-E after irradiation, suggesting that activation of p38 is required for the upregulation of HLA-E expression after irradiation. These findings were confirmed in the oncogene-induced model of senescence (Fig. 2c) and also with an alternative p38 inhibitor SB203580, which inhibits p38 by a mechanism that differs from that used by BIRB79630 (Fig. 2d). Interestingly, we found no significant effect of p38 inhibition on MICA/B expression (Fig. 2e), suggesting that different pathways regulate the expression of MICA/B and HLA-E in senescent cells.

Fig. 2 HLA-E expression on senescent cells is regulated by p38 and induced by IL-6. a Representative immunoblot of human fibroblasts at the indicated intervals after IR showing the kinetics of HLA-E, p38 (Thr180/Tyr182) and γH2AX (Ser139) phosphorylation. b Human fibroblasts were treated with p38 inhibitor BIRB796 (0.5 μM) or DMSO 12 h before, and for 7 days after IR. Immunoblot of HLA-E expression at the indicated intervals after IR compared with DMSO-treated controls. Undetectable levels of phospho-Hsp27 (p-Hsp27) confirm effective inhibition of p38. c IMR-90 ER:STOP/ER:RAS cells treated with 4-OHT to induce senescence (as demonstrated in Supplementary Fig. 2A–C) and treated with BIRB796 (0.5 μM) or DMSO over 7 days, followed by protein extraction and immunoblot analysis of HLA-E and p-Hsp27. GAPDH served as a loading control in a–c. Uncropped immunoblots are provided in the Source Data file. d The summary data of HLA-E and (e) MICA/B expression analysed by flow cytometry on irradiated fibroblasts treated with SB203580 (10 μM), compared with DMSO-treated and non-irradiated controls (n = 5). f Flow-cytometry analysis of HLA-E expression on fibroblasts exposed to the conditioned medium (CM) from senescent (SEN) or non-senescent (NS) cells for 48 h (n = 5). g Supernatant from irradiated senescent or early-passage fibroblasts analysed by cytokine-bead arrays to measure secreted cytokines (in pg/mL) (n = 12). h Fibroblasts were exposed to IL-6 (20 ng/mL), IL-8 (20 ng/mL), IL-1β (20 ng/mL) or IFNα (500 U/mL) or combinations of these for 48 h and analysed for HLA-E expression by flow cytometry (n = 6). The data are representative of at least three independent experiments from distinct samples. Comparison between groups performed with Kruskal–Wallis test in (d), (e) and Mann–Whitney U test in (f), (g) and (h). The data presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Full size image

HLA-E expression is induced by SASP cytokines

Since p38 is a key regulator of the SASP32, we asked whether SASP factors could increase HLA-E expression in a paracrine manner. We exposed early-passage fibroblasts to conditioned media (CM) from senescent or non-senescent cells. Non-senescent cells increased HLA-E expression within 48 h of exposure to CM from senescent cells (Fig. 2f).

We used cytometric-bead arrays to measure SASP factors secreted by senescent cells. We identified IL-6, IL-8 and MCP-1 as the most highly secreted SASP-related factors, as previously reported32 (Fig. 2g). Exposing non-senescent fibroblasts to these factors revealed a significant effect of IL-6, alone or in combination with other factors, in stimulating HLA-E expression (Fig. 2h). We cannot exclude, however, that other factors other than SASP-related pro-inflammatory cytokines may contribute to the upregulation of HLA-E in senescent cells.

HLA-E ligates NKG2A and NKG2C on NK cells and CD8+ T cells

HLA-E and MICA/B bind receptors belonging to the Natural Killer Group 2 (NKG2) family, which regulate NK cell function33. MICA/B, and ULBP antigens, bind the stimulatory NK cell receptor NKG2D29, whereas HLA-E binds both the inhibitory NKG2A and stimulatory NKG2C receptors34. We analysed the distribution of NKG2 receptors on peripheral blood mononuclear cells (PBMCs) from healthy human volunteers (n = 27; mean age 49.9; range, 27–83 years) and found that NKG2D was highly expressed on NK (included in the CD3− subset) and CD8+ T cells (Fig. 3a), as previously shown29. Expression of NKG2A and NKG2C, the cognate receptors for HLA-E, was not restricted to NK cells, and a proportion of CD8+ T cells also expressed these receptors (Fig. 3b, c). Human CD8+ T cells can be stratified by their relative expression of the co-stimulatory receptors CD28 and CD27, defining early (CD28+CD27+), intermediate (CD28−CD27+) and late (CD28−CD27−) stages of differentiation (Fig. 3d). The majority of NKG2A+ and NKG2C+ CD8+ T cells were highly differentiated (CD28−CD27−) cells (Fig. 3e, f), a subset that is expanded with age35. We found the proportion of NKG2A+ within the highly differentiated subset of CD8+ T cells to be significantly positively correlated with age (Supplementary Fig. 5A). This effect was not seen in the NKG2C+ compartment (Supplementary Fig. 5B). No association was found between the frequency of NKG2A+ cells and age within the CD3− compartment (Supplementary Fig. 5C). Although we did not investigate the expression of NKG2A directly on NK cells in the CD3− compartment, previous studies show that the expression of this receptor does not change on NK cells during ageing36, while others report an age-associated decrease in expression37.

Fig. 3 MICA/B and HLA-E bind NKG2 receptors expressed on NK and CD8+ T cells. Distribution of (a) NKG2D+, (b) NKG2A+ and (c) NKG2C+ cells on the indicated subsets of human lymphocytes from blood of healthy volunteers (n = 27; mean age 49.9; range, 27–83), analysed by flow cytometry. FACS sequential gating strategies represented in Supplementary Fig. 4. d Human CD8+ T cells were stratified according to CD27 and CD28 expression in early- (CD27+28+), intermediate- (CD27+28−) and late-differentiated (CD27−28) cells, as represented in the flow-cytometry plot. e, f The summary data of the distribution of NKG2A+ (e) and NKG2C+ (f) cells within early-, intermediate- and late-differentiated CD8+ T cells gated as in (d) in the same donors (n = 27; mean age 49.9; range, 27–83). Comparison between groups done with Friedman test with Dunn's correction for multiple comparisons in (e) and (f). The data presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Full size image

NK and CD8+ T cells target senescent fibroblasts via NKG2D

NK cells have been implicated in the surveillance and elimination of senescent cells through interaction between NKG2D and its ligands expressed on senescent cells12,15,16,17. Since CD8+ T lymphocytes also express the cognate receptors for MICA/B and HLA-E, we asked whether these cells could also participate in immune surveillance of senescent cells in analogy with NK cells. To avoid allogeneic reactions with T cells, we developed a co-culture system with skin-derived primary human fibroblasts and immune cells obtained from the same individuals (Fig. 4a). After inducing senescence with ionising radiation, we co-cultured senescent and non-senescent fibroblasts with freshly isolated autologous NK and CD8+ T cells.

Fig. 4 HLA-E/NKG2A blockade enhances senescent cell killing. a The experimental design of the autologous co-culture system to study immune surveillance of senescent cells: primary human fibroblasts from the skin of healthy volunteers were expanded and induced to senescence by ionising radiation. NK and CD8+ T cells from peripheral blood of the same donors (n = 5, age range 20–46) were used in co-culture experiments with autologous fibroblasts. b The summary data (n = 6) of active caspase 3 expression by non-senescent (black) and senescent fibroblasts (grey) after incubation with NK cells or CD8+ T cells, using different effector to target (E:T) ratios. Controls (CTR) indicate spontaneous activation of caspase 3 in fibroblasts cultured without effector cells. c NK and CD8+ T cells were pre-incubated with blocking antibodies to NKG2A (Z199), NKG2D (1D11) or isotype-matched controls, using an E:T ratio of 20:1. The summary data (n = 4) of degranulation of NK and CD8+ T cells towards senescent and non-senescent fibroblasts assessed by CD107a expression. d The cumulative data of CD107a expression in NK (n = 5) and CD8+ T cells (n = 4) after incubation with normal and senescent fibroblasts transfected with siRNA to HLA-E or a control siRNA. The data are presented as the index of degranulation (calculated as described in the Methods section) in c and d. Measurements were from distinct samples. Statistical analysis done with Mann–Whitney U test in (b) and one-way ANOVA with Bonferroni's multiple comparison test in c and d. The data presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Full size image

Consistent with previous reports11,12, NK cells preferentially targeted senescent cells, as shown by significantly higher levels of active caspase 3 (measuring cell apoptosis) in senescent versus non-senescent fibroblasts when cultured with NK cells (Fig. 4b). Notably, CD8+ T cells could also induce significant activation of caspase 3 in senescent cells compared with non-senescent controls (Fig. 4b). These results were confirmed using a CD107a-based degranulation assay as a surrogate marker of cytotoxicity38, indicating that both NK and CD8+ T cells can target senescent fibroblasts in vitro (Fig. 4c). Pre-incubation of effector cells with NKG2D blocking antibody (1D11) decreased both NK and CD8+ T-cell degranulation towards senescent fibroblasts as compared with cells pretreated with a matched isotype control (Fig. 4c). By contrast, pre-incubation with NKG2A blocking antibody (Z199) significantly boosted the response of NK cells (Fig. 4c) and CD8+ T cells (Fig. 4c) against senescent cells, as compared with matched isotype controls. Collectively, these findings indicate that both NK and CD8+ T cells can kill senescent cells by an NKG2D-dependent mechanism, and that positive signals delivered by NKG2D ligation by MICA/B may be blocked if the inhibitory receptor NKG2A is engaged with its ligand HLA-E.

NKG2A/HLA-E blockade enhances senescent cell surveillance

To directly investigate the role of HLA-E in inhibiting immune responses against senescent cells, we used RNA interference to inhibit HLA-E expression. At 36 h post transfection, the expression of HLA-E on senescent fibroblasts was decreased upon transfection with siRNA against HLA-E compared with the siRNA control (Supplementary Fig. 5D). This resulted in a significant increase in degranulation by autologous NK and CD8+ T cells (Fig. 4d), as compared with cells transfected with a scrambled siRNA control. These findings suggest that HLA-E expression on senescent cells decreases their susceptibility to elimination by NK and CD8+ T cells that express the inhibitory receptor NKG2A. The differences noted in the CD107a degranulation assay between antibody blocking experiments and experiments using siRNA transfection are most likely associated with the transfection protocol and incomplete silencing of HLA-E expression after transfection (Supplementary Fig. 5D).

Relevance of HLA-E expression on senescent cells in vivo

To determine whether HLA-E expression was also elevated on murine senescent cells in vivo, we used p16-3MR reporter mice model that allows the identification of p16INK4a-positive (senescent) cells via the p16INK4a promoter driven expression of luciferase (Fig. 5a). Importantly, it also enables the selective elimination of senescent cells by administration of ganciclovir (GCV) through p16INK4a promoter driven expression of the herpes simplex virus 1 (HSV-1) thymidine kinase (HSV-TK)2. We treated p16-3MR mice with the genotoxic drug bleomycin to induce senescence and fibrosis in the lung39, with or without ganciclovir to eliminate senescent cells (Fig. 5b). We harvested whole lungs and analysed them by quantitative polymerase chain reaction for genes encoding Qa-1b (H2-T23, the mouse homologue of HLA-E), p16INK4a (CDKN2A) and collagen (COL1A1) as a proxy marker of fibrosis. CDKN2A mRNA levels increased 14 days after treatment with bleomycin (Fig. 5c), as did H2-T23 mRNA levels (Fig. 5d). Furthermore, when mice were treated with GCV to eliminate p16Ink4a-positive cells, H2-T23 gene expression declined to control levels (Fig. 5d). Likewise, COL1A1 mRNA levels increased upon induction of senescence by bleomycin and declined after eliminating senescent cells with GCV. These results suggest that fibrosis is associated with the development of senescence and is alleviated when senescent cells are cleared (Fig. 5e).

Fig. 5 The expression of Qa-1b (mouse homolog of HLA-E) in p16-3MR mice. a Schematic of the p16-3MR (trimodality reporter) fusion protein, containing functional domains of a synthetic Renilla luciferase (LUC), monomeric red fluorescent protein (mRFP) and truncated herpes simplex virus 1 (HSV-1) thymidine kinase (HSV-TK) driven by the p16 promoter. b p16-3MR mice were treated with bleomycin (intra-tracheal injection, 1.9 UI/Kg), ganciclovir (GCV, 25 mg/kg; daily i.p. injections) or PBS; c–e qRT-PCR was used to quantify levels of mRNAs encoding p16INK4a (CDKN2A), Qa-1 (H2-T23) and collagen (COL1A1) in lungs from mice treated with PBS (white bar), bleomycin + vehicle control (black bars) and bleomycin + GCV (grey bars). mRNA levels for the indicated genes were normalised to tubulin and presented as fold difference relative to PBS-treated mice. Statistical analysis between groups performed with one-way ANOVA with Bonferroni's multiple comparison test. The data presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Full size image

To assess the biological relevance of HLA-E expression by senescent cells in humans, we studied congenital melanocytic nevi (Fig. 6). Previous studies showed that melanocytic nevi are enriched with cells expressing markers of senescence and thus are likely an example of oncogene-induced senescence in vivo40,41. We immunostained formalin-fixed, paraffin-embedded (FFPE) tissue arrays of congenital and acquired human melanocytic nevi for HLA-E expression (Fig. 6a). We analysed the staining using Image Analysis Software and scored HLA-E expression based on the percentage of HLA-E+ cells, ranking as negative (score 0: between 0 and 1%), mild (score 1: 1–5%), moderate (score 2: 5–10%) or intense (score 3: >10%). In each array, normal skin and melanoma samples were used as controls (Supplementary Fig. 6). Among 24 lesions studied, HLA-E expression was detectable in most (23/24) human melanocytic nevi. In 10/24 (41.7%) lesions, we observed strong positivity for HLA-E (score 3), with frequencies of HLA-E positive cells up to 30% (Supplementary Table 1). Sections were also stained for Ki67 as a marker of proliferation, which demonstrated that cells expressing HLA-E were rarely Ki67 positive (Fig. 6b). To determine levels of immune cell infiltration, the same tissue arrays were stained for CD8 (Fig. 6c) and the percentage of these cells was calculated using the same software (mean 5.29 ± 4.01%, range 0.05–16.5%). Co-staining for CD8 and HLA-E showed that CD8+ cells frequently co-localised with HLA-E+ cells (Fig. 6d). As NK cells can also stain positively for CD8, tissue arrays were co-stained with CD3 to distinguish NK from T cells. This analysis confirmed the presence of CD3+ CD8+ T lymphocytes, however, these cells did not account for all the immune cell infiltration (Fig. 6e). We conclude that both T cells and NK cells associate with HLA-E expressing cells in melanocytic nevi, and we observed a significant positive correlation between the frequency of HLA-E+ cells in melanocytic nevi and the percentage of CD8+ T-cell infiltration in melanocytic nevi (Fig. 6f). Therefore, HLA-E expression is common in human melanocytic nevi and may explain why these nevi persist for decades in tissues, despite the presence of immune cell infiltrates.

Fig. 6 HLA-E expression by senescent cells in human melanocytic nevi. Formalin-fixed paraffin-embedded tissue arrays of human melanocytic nevi were analysed by immunohistochemistry using (a) HLA-E (MEM-E/02), (b) Ki67, (c) CD8 specific antibodies. d Double-staining with antibodies for HLA-E (brown) and CD8 (red) showing CD8+ infiltrates surrounding areas with strong HLA-E expression. e Double-staining with HLA-E (brown) and CD3 (red) showing that part of the CD8+ infiltrates are also positive for CD3, identifying them as CD8+ T cells. Scale bar = 50 μm. f Correlation between the frequency of HLA-E+ cells and CD8+ infiltrates in human melanocytic nevi (n = 24) assessed by Spearman test Full size image

HLA-E expression by senescent fibroblasts in human skin

Senescent cells have been shown to increase in many tissues during ageing, including in the skin42. To investigate whether senescent cells express HLA-E in healthy individuals in vivo, we analysed histological sections of normal human skin from young (<40 years) and old (>65 years) healthy donors using confocal microscopy. Firstly, we identified non-senescent (Fig. 7a, top panels) and senescent cells (Fig. 7a, bottom panels) by staining for telomere-associated DNA-damage foci (TAF; left hand panels) or p16INK4A (Fig. 7a, right panels). In sections that were stained for both TAF and p16INK4A, there was a significant correlation between both senescent markers (Fig. 7b). We subsequently used TAF staining alone to identify senescent cells. There was a highly significant correlation between senescent (TAF+) cells in the interstitial dermis and increasing donor age (Fig. 7c).

Fig. 7 HLA-E expression by senescent fibroblasts in human skin during ageing. a Histological sections from healthy donors were stained for DAPI (blue), TelC (red punctate intranuclear), γH2AX S139 (green) and p16INK4A (white). Telomere-associated γH2AX foci (TAF) are shown (white arrow heads) in non-senescent (NSEN; top panels) and senescent (SEN; bottom panels) cells in the dermis of the human skin. Original image shown in Supplementary Fig. 7E and provided in the Source Data file. Scatterplot showing the relationship between the frequency of p16INK4A+ cells (b) and donor age (c) with the frequency of TAF+ cells in the interstitial dermis of the human skin. d Skin sections were stained for DAPI (blue), TelC (red punctate intranuclear), γH2AX S139 (green) and HLA-E (white). Telomere-associated γH2AX foci (TAF) are shown (white arrow heads) in senescent HLA-E+ cells (yellow asterisks) of the human dermis. The signal intensity of TelC and γH2AX along lines (a) and (b) are represented in histogram format and both signals overlap in the senescent, but not the non-senescent cell. e Correlation of HLA-E+ cells and TAF+ cells in the interstitial dermis of human skin. f Correlation of HLA-E+ cells and p16INK4A+ cells in the interstitial dermis of the human skin. g The frequency of HLA-E+ cells present in the superficial dermis of young (n = 4) and old (n = 5) human skin. h The frequency of HLA-E+ cells in the TAF+ and TAF− populations in the dermis of young (n = 4) and old (n = 5) human skin. The data are represented as mean ± SEM. Statistical significance calculated with Mann–Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Full size image

As seen in representative confocal micrographs of interstitial dermal cells of the human skin (Fig. 7d), TAF+ cells co-stain for HLA-E confirming that HLA-E expressing cells display markers of senescence. There was a positive correlation between the frequencies of HLA-E+ and TAF+ cells (Fig. 7e) and HLA-E+ and p16INK4A+ cells in the same skin sections (Fig. 7f). Finally, we found a significant increase in the frequency of HLA-E+ cells in older donors as compared with young (Fig. 7g). A significantly higher proportion of senescent (TAF+) cells expressed HLA-E+ compared with non-senescent (TAF−) cells (Fig. 7h). The majority of TAF+ senescent cells were dermal fibroblasts, a subset of which we identified as staining positive for the fibroblast-specific protein-1 (FSP1)43 (Supplementary Fig. 7A). A similar proportion of FSP1+ cells were found in the interstitial dermis of young and old subjects (Supplementary Fig. 7B). There was a significantly increased frequency of TAF+ senescent cells in both FSP1+ and FSP1− populations in the skin of old donors compared with young (Supplementary Fig. 7C, D), indicating that other cells as well as fibroblasts contribute to the senescent cell population in older individuals.