An aged immune system drives senescence and ageing of solid organs

Abstract

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

Extended Data Fig. 1 |. Molecular changes in Vav-iCre^+/−;Ercc1^−/fl mice.

a, Expression of Ercc1 was measured in tissues from 8–10-month-old Vav-iCre^+/−;Ercc1^−/fl and Ercc1^−/fl control mice (n = 5–7 Vav-iCre^+/−;Ercc1^−/fl; n = 3–5 Ercc1^−/fl, depending on the tissue (see Supplementary Table 3 for sample size details). b, Detection of ERCC1 in splenic lysates from a 9-month-old Vav-iCre^+/−;Ercc1^−/fl mice and littermate control by immunoblot. c, Superoxide anion levels were measured by electron paramagnetic resonance (EPR) in splenic tissue from 6–8-month-old Vav-iCre^+/−;Ercc1^−/fl and littermate control mice (n = 5 mice per group). d, e, Expression of the transcription factor NRF (Nfe2l2) and its downstream targets (Cat, Nqo1, Hmox1) measured by qRT–PCR in spleen (d) and bone marrow (e) of Vav-iCre^+/−;Ercc1^−/fl and littermate control mice at several ages (n = 3 at 3- and 5-months-old; n = 5 8–10-months-old) and in two-year-old wild-type mice (n = 5). f, Catalase activity measured in splenic tissue from 8–10-month-old Vav-iCre^+/−;Ercc1^−/fl (n = 6) and Vav-iCre^+/− (n = 3) mice (Methods). g, Catalase activity in 4-month-old (n = 3) and 24-month-old (n = 6) wild-type mice. h, i, The ratio of reduced to oxidized glutathione (GSH/GSSG) (Methods) (h) and levels of HNE protein adducts (i) measured by ELISA in splenic lysates of Vav-iCre^+/−;Ercc1^−/fl and littermate control mice at 8–11 months of age (n = 6 mice per group). j, Levels of four cyclopurine adducts in splenic tissue from 8–10-month-old Vav-iCre^+/−;Ercc1^−/fl mice and littermate controls (n = 4–5 Vav-iCre^+/−;Ercc1^−/fl; n = 5 Vav-iCre^+/−; see Supplementary Table 3 for sample size details) measured by liquid chromatograph–tandem mass spectrometry (LC–MS/MS/MS) (Methods). k, Total splenocyte cell counts from 8–10-month-old Vav-iCre^+/−;Ercc1^−/fl (n = 14) mice and Vav-iCre^+/− (n = 12) mice. l, The absolute number of T (CD4⁺, CD8⁺) and B (B220⁺CD19⁺) cells in spleens from the same mice (n = 10/4 Vav-iCre^+/−;Ercc1^−/fl; n = 8/3 Vav-iCre^+/− for CD4⁺ or CD8⁺/ B220⁺CD19⁺ measures, respectively) (Methods). m, Total splenocyte cell counts from young (7-month-old; n = 10) and old (24-month-old; n = 17) wild-type mice. n, The absolute number of CD4⁺, CD8⁺ and B220⁺CD19⁺ cell in spleens from the same mice (n = 8/3 young WT; n = 17/7 old wild-type mice for CD4⁺ or CD8⁺/B220⁺CD19⁺ measures, respectively). o, Analysis of CD8⁺ splenocytes from 8–10-month-old mice for memory (CD44⁺CD127⁺), exhaustion (PD-1⁺) and apoptosis (VAD-FMK⁺) markers (n = 6 mice per group). p, Thymic weight normalized to total body weight (n = 3 at 3 months old; n = 4–5 at 8–10 months old per group). q, Histology images (20×) of spleen and lymph nodes from 8–10-month-old Vav-iCre mice. Scale bar, 100 μm. GC, germinal centres. Data are mean ± s.d. *P < 0.05, ∞P < 0.01, ∇P < 0.001, #P < 0.0001, unpaired two-tailed Student’s t-test (a, c–e for the 3- and 5-month-old mice, g–p), one-way ANOVA (d, e for the 8–10-month-old mice) or two-way (f) with Tukey’s test.

Extended Data Fig. 2 |. Vav-iCre^+/−;Ercc1^−/fl mice maintain normal weight and body composition.

a, Body weight (BW) of three different age groups of mice by genotype. b–d, Percentage fat (b), lean mass (c) and fluid (d) measured by NMR at three ages of mice (n = 8–25 mice per group) (see Supplementary Table 3 for sample size details). Data are mean ± s.d. P values (not significant) were determined by one-way ANOVA with Tukey’s test.

Extended Data Fig. 3 |. Measurement of immune function and lymphoid organ senescence in Vav-iCre^+/−;Ercc1^−/fl mice.

a, Footpad swelling measurements at several time points after antigenic (KLH) challenge, separated by mouse genotype (n = 7 Vav-iCre^+/−;Ercc1^−/fl; n = 6 Vav-iCre^+/−; n = 5 old WT mice). b, Footpad swelling by genotype (n = 7 Vav-iCre^+/−;Ercc1^−/fl; n = 6 Vav-iCre^+/−; n = 5 WT mice). c, Quantification of anti-KLH antibodies by ELISA one month after antigenic challenge (n = 3/7 Vav-iCre^+/−;Ercc1^−/fl; n = 3/6 Vav-iCre^+/−; n = 3/3 Ercc1^−/fl for naive (uninjected) and KLH-challenged (injected) mice, respectively). d, DTH assay in 2-month-old mice Vav-iCre^+/−;Ercc1^−/fl (n = 5) and Vav-iCre^+/− (n = 8) controls. Data are mean ± s.d. ∞P < 0.01, ∇P < 0.001, #P < 0.0001, two-way ANOVA with Tukey’s test. e, Quantification of Ercc1 expression by qRT–PCR in flow-sorted immune cell populations isolated from spleen (T and natural killer cells) and bone marrow (B cells and macrophages) of 5-month-old Vav-iCre^+/−;Ercc1^−/fl mice and littermate controls (n = 4 Vav-iCre^+/−;Ercc1^−/fl; n = 3 Vav-iCre^+/−). Data are mean ± s.d. ∇P < 0.001, #P < 0.0001, two-tailed unpaired Student’s t-test. f, Senescence marker expression in CD3⁺ peripheral T cells from 8–11-month-old Vav-iCre and old wild-type mice (n = 3–9 Vav-iCre^+/−; n = 6–9; Vav-iCre^+/−;Ercc1^−/fl; n = 4–7 two-year-old wild-type, depending on the gene) (see Supplementary Table 3 for sample size details). g, Measurement of senescence marker expression by qRT–PCR in splenic tissue and bone marrow from 2–3-month-old Vav-iCre mice (n = 3). h, Expression of senescence markers in splenic tissue from 8–10-month-old Vav-iCre^+/−;Ercc1^−/fl and old wild-type mice relative to controls by gender (n = 3–4/3–4 Vav-iCre^+/−; n = 3–5/3–4 Vav-iCre^+/−;Ercc1^−/fl; n = 3–6/3–5 4-month-old WT; n = 5/5 two-year-old WT males/females, respectively) (see Supplementary Table 3 for sample size details). i, Expression of senescence markers in bone marrow of Vav-iCre^+/−;Ercc1^−/fl mice (n = 3–4/3–4 Vav-iCre^+/−; n = 3–4/4–5 Vav-iCre^+/−;Ercc1^−/fl males/females, respectively). Data are mean ± s.d. *P < 0.05, ∞P < 0.01, ∇P < 0.001, #P < 0.0001, unpaired two-tailed Student’s t-test (e, g), one-way ANOVA (f) or two-way ANOVA (a–d, h, i) with Tukey’s test.

Extended Data Fig. 4 |. Identification of immune cell types and senescent cells by CyTOF.

a, viSNE analysis of total CD45⁺ cells to identify immune cell types. Representative viSNE plots are from a Vav-iCre^+/− control mouse at 10–12 months of age. See Supplemental Fig. 5 for the gating strategy. b, The proportion of the indicated immune cell subsets that express p16, p21 or CENP-B from 10–12-month-old Vav-iCre^+/−;Ercc1^−/fl (n = 6), Vav-iCre^+/−; (n = 6), and >2-year-old wild-type (n = 7) mice was determined by CyTOF. Each dot is an independent mouse. Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal–Wallis test with Dunn’s correction for multiple comparisons.

Extended Data Fig. 5 |. Co-localization of p16 mRNA with parenchymal markers in non-lymphoid tissues from Vav-iCre^+/−;Ercc1^−/fl mice.

a, Measurement of senescence marker expression in the organs of 5-month-old Vav-iCre mice (n = 3 mice per group). b–e, Representative images of p16 in situ hybridization with immunostain or chemical stain for parenchymal markers of liver (b, c), kidney (d) and lung (e) sections from 8–11-month-old Vav-iCre^+/−;Ercc1^−/fl mice, littermate control Vav-iCre^+/− mice and 2-year-old wild-type mice stained for albumin (liver), phalloidin (liver and lung) or kidney-specific (KSP)-cadherin in the red channel, DAPI (blue), and p16 LNA probe (green). The full set of images from Fig. 3c is shown in b. Original magnification, ×40. Scale bar, 10 μm. f, Representative images of SA-β-gal staining on tissues from 8–10-month-old Vav-iCre^+/−;Ercc1^−/fl and littermate controls. Original magnification, ×20. Scale bar, 50 μm. g, Senescence marker expression in the livers of 8–11-month-old Vav-iCre^+/−;Ercc1^−/fl (n = 5 male and 4–5 female) (see Supplementary Table 3 for sample size details by gender and gene) and littermate control mice (n = 3 male and 3–4 female) as well as 4-month-old (n = 3 male and 3–4 female) and 2-year-old (n = 6 male and 4–5 female) wild-type mice. h, Levels of circulating SASP factor proteins measured by multiplex ELISA in serum from Vav-iCre^+/−;Ercc1^−/fl (n = 3 male and 3–4 female) and Vav-iCre^+/− (n = 3 male and 3 female) mice. Data are mean ± s.d. *P < 0.05, ∞P < 0.01, ∇P < 0.001, #P < 0.0001, unpaired two-tailed Student’s t-test (a) or two-way ANOVA with Tukey’s test (g, h).

Extended Data Fig. 6 |. Cell non-autonomous effects of an aged immune system in non-lymphoid tissues of Vav-iCre^+/−;Ercc1^−/fl mice.

a, Cyclopurine adducts were measured in the liver and kidneys of 8–11-month-old Vav-iCre^+/−;Ercc1^−/fl (n = 5) and littermate control Vav-iCre^+/− (n = 5 for liver and n = 6 for kidney) by LC–MS/MS/MS (Methods). b, Markers of oxidative stress including HNE protein adducts and the ratio of reduced to oxidized glutathione (GSH/GSSG) measured in the kidneys from 8–11-month-old Vav-iCre^+/−;Ercc1^−/fl and littermate control Vav-iCre^+/− mice (n = 6 mice per group). HNE measure by ELISA. GSH/GSSG measured by chromogenic assay (Methods). c, 8-oxoguanine DNA adducts measured by ELISA in the spleen, liver and kidney of mice at various ages (n = 5–6/5–6/5 Vav-iCre^+/−;Ercc1^−/fl; n = 5–6/5–6/5 Vav-iCre^+/−; n = 5/5/10 old WT mice for spleen/liver/kidney, respectively) (see Supplementary Table 3 for sample size details by genotype and tissue). d, Urinary levels of pro-geronic factor β₂-microglobulin and MCP-1 measured by ELISA in 8–11-month-old Vav-iCre^+/−;Ercc1^−/fl and littermate controls (n = 9 mice per group). e, Renal Mcp1 (also known as Ccl2) expression in 8–11-month-old Vav-iCre^+/−;Ercc1^−/fl mice (n = 7 per group) measured by qRT–PCR. f, Representative Coomassie-stained gel of urine samples from 8–11-month-old Vav-iCre^+/−;Ercc1^−/fl and littermate control mice demonstrating increased proteinuria. Recombinant albumin (Alb) was loaded on the gel as a control (lanes 6, 12) and its approximate molecular mass denoted by a box (marker ladder lanes 1, 5, 7, 11, 13–14). Each lane represents a unique mouse. g, Representative images from tissue sections stained for aggrecan (red) and DAPI (blue) in the nucleus pulposus (NP) of intervertebral discs from 8–11-month-old Vav-iCre^+/−;Ercc1^−/fl and littermate control mice. h, Quantification of aggrecan staining (n = 4 Vav-iCre^+/−; n = 7 Vav-iCre^+/−;Ercc1^−/fl). i, Measurement of senescence marker expression in the intervertebral discs of 8–11-month-old Vav-iCre^+/−;Ercc1^−/fl and littermate control mice (n = 4 mice per group) by qRT–PCR. j, Representative images of sections of intervertebral discs from 9-month-old mice stained with haematoxylin and eosin (H&E) and safranin O to detect proteoglycans. Arrows point to the anulus fibrosus. k, Representative images of gastrocnemius muscle sections from 8–11-month-old Vav-iCre^+/−;Ercc1^−/fl and littermate control mice after cardiotoxin injury (Methods) stained with haematoxylin and eosin or immunostained for M1 (CD68, green) and M2 (CD163, red) macrophages. l, Quantification of the ratio of M2/M1 macrophages (n = 4 Vav-iCre^+/−; n = 8 Vav-iCre^+/−;Ercc1^−/fl mice). m, Body weight and grip strength of Vav-iCre^+/−;Ercc1^−/fl and littermate controls at the indicated ages (n = 8 Vav-iCre^+/−;Ercc1^−/fl; n = 4 Vav-iCre^+/− at both ages). Data are mean ± s.d. *P < 0.05, ∞P < 0.01, ∇P < 0.001, #P < 0.0001, two-tailed unpaired Student’s t-test (a, b, d, e, h, i, l, m) or two-way ANOVA with Tukey’s test (c).

Extended Data Fig. 7 |. Age-associated increase in serum SASP factors of Vav-iCre^+/−;Ercc1^−/fl mice and wild-type mice.

a, This is an extension of the data shown in Fig. 3k, including two younger ages of mice. Circulating SASP factors were measured by ELISA (n = 3 for 2–3-month-old, n = 4 for 5-month-old, n = 5–7 for 8–11-month-old, n = 7 for 24-month-old) (see Supplementary Table 3 for sample size details). Data are mean ± s.d. *P < 0.05, *P < 0.01, ∇P < 0.001, #P < 0.0001, two-way ANOVA with Tukey’s test. b, Haematoxylin and eosin sections of brain, kidney and liver from 17-month-old Vav-iCre^+/−;Ercc1^−/fl and littermate controls (n = 5 mice per group) were scored for age-associated histopathological lesions using the Geropathology Grading Platform to generate a composite lesion score for each organ (CLS). Data are mean ± s.d. P values (not significant) were determined by two-tailed unpaired Student’s t-test.

Extended Data Fig. 8 |. Time course of bioluminescence signal in p16-luciferase mice transplanted with splenocytes and tissue distribution of transplanted cells.

a, Splenocytes from 8–10-month-old Vav-iCre^+/−;Ercc1^−/fl, Vav-iCre^+/− controls, or 2-year-old wild-type mice were injected retro-orbitally into 3–4-month-old p16^Ink4+/Luc senescence reporter mice (n = 2 donor mice per genotype) as described in Fig. 4. Tissues were collected from recipient mice 2 weeks after the final imaging and the expression of p21 was measured by qRT–PCR. Data are mean ± s.d. *P < 0.05, ∇P < 0.01, one-way ANOVA with Tukey’s test. b, Splenocytes (5 × 10⁶ cells) from 9–10-month-old Vav-iCre^+/−;Ercc1^−/fl and Vav-iCre^+/− mice were injected retro-orbitally into 3–4-month-old p16^Luc/Luc senescence reporter mice (n = 2 donor mice per genotype; n = 4 p16^Luc/Luc recipient mice for Vav-iCre^+/−;Ercc1^−/fl splenocytes; n = 3 receiving Vau-iCre^+/− splenocytes). Weekly measurements of luminescence in recipient reporter mice. Data are mean ± s.d. *P < 0.05, ∞P < 0.01, two-tailed unpaired Student’s t-test. c, Splenocytes from 7- or 26-month-old male mice were injected retro-orbitally into female mice to track distribution of the transplanted cells (n = 2 donor mice and n = 3 recipient mice per age group; n = 2 uninjected controls). Tissues were collected 24 h after injection. Expression of the Sry gene on the Y chromosome measured by qRT–PCR in RNA isolated from tissues of recipient mice was used to track homing of immune cells to various recipient mouse organs. There was little difference in immune cell homing if the donor mice were young or old.

Extended Data Fig. 9 |. Suppression of senescence in progeroid mice by transplantation of young immune cells.

a, Schematic of adoptive transfer: 3-month-old progeroid Ercc1^−/Δ mice (n = 4 mice/treatment group) were injected retro-orbitally with 5x10⁶ splenocytes from 2-month-old WT mice or vehicle only (PBS) (n = 6 donors). One month later, the recipient mice and uninjected age-matched wild-type mice were euthanized, and tissues collected. b, Expression of senescence markers in organs of recipient mice (n = 4 Ercc1^−/Δ + splenocytes; n = 3–6 Ercc1^−/Δ + PBS) (see Supplementary Table 3 for sample size details by organ/endpoint). Gene expression was normalized to that of uninjected, age-matched wild-type controls (n = 4–7) represented as horizontal dashed line. c, SASP factor proteins MCP-1 and TNF were measured in the serum of recipient mice by multiplex ELISA (n = 4 Ercc1^−/Δ + splenocytes; n = 6 Ercc1^−/Δ + PBS) and compared to untreated, age-matched wild-type mice (n = 4–6). d, Footpad swelling of mice describe above and in Fig. 5f at several time points after antigenic challenge (n = 3/7 Vav-iCre^+/−;Ercc1^−/fl or n = 3/6 Vav-iCre^+/−; mice +/− rapamycin, respectively). e, Expression of p21 in PBMCs, measured by qRT-PCR. f, Serum MCP-1 and TNF measured by multiplex ELISA (n = 3/5 +/− rapamycin, respectively). Data are mean ± s.d. *P < 0.05, ∞P < 0.01, ∇P < 0.001, #P < 0.0001 one-way ANOVA (b, c) or two-way ANOVA (d–f) with Tukey’s test. Mouse images in schematic were used with permission from BioRender.

Fig. 1 |. Deletion of Ercc1 in haematopoietic cells causes accelerated ageing of the immune system.

a, Immunoblot detection of γH2AX in splenic lysates from 9-month-old mice. Levels of 8-oxo-guanine (8-OHdG) in spleens from 8–10-month-old mice (n = 6 mice per group). WT, wild type. b, Peripheral white blood cell (WBC) counts of Vav-iCre^+/− and Vav-iCre^+/−;Ercc1^−/fl mice (n = 4, 7, 8 and 5 Vav-iCre^+/−; n = 3, 6, 8 and 6 Vav-iCre^+/−;Ercc1^−/fl, at 3, 5–6, 12 and 18 months, respectively). c, Flow cytometric quantification of peripheral blood B and T cells in 4–5-month-old mice (n = 15–16 Vav-iCre^+/−; n = 6 Vav-iCre^+/−;Ercc1^−/fl) (see Supplementary Table 3 for sample size details). d, e, Splenic (d) and bone marrow (e) lymphocyte populations in 8–10-month-old mice (n = 8–11/7 Vav-iCre^+/−;Ercc1^−/fl; n = 4/4 Vav-iCre^+/− mice, for spleen/bone marrow). f, Analysis of CD4⁺ splenocytes from 8–10-month-old mice for memory (CD44⁺CD127⁺), exhaustion (PD-1⁺) and apoptosis (VAD-FMK⁺) markers (n = 6 mice per group). g, Splenic weights normalized to body weight (n = 3 at 3 months; n = 7 at 8–10 months). h, KLH delayed-type hypersensitivity data after sensitization of 5-month-old Vav-iCre or ≥24-month-old wild-type mice. Footpad swelling at 48 h after challenge (n = 6 Vav-iCre^+/−; n = 7 Vav-iCre^+/−;Ercc1^−/fl; n = 5 WT). i, KLH antibodies measured by ELISA one month after challenge (n = 3/6 Vav-iCre^+/−; n = 3/7 Vav-iCre^+/−;Ercc1^−/fl; n = 3/5 2-year-old wild-type challenged with PBS/KLH). j, Cytotoxicity of splenic natural killer cells from 8–12-month-old Vav-iCre mice (n = 5 mice per group). k, Senescence marker expression in flow-sorted cell populations from spleens (T, natural killer cells) and bone marrow (B cells, macrophages) of 5-month-old Vav-iCre mice (n = 4 mice per group). Expression is normalized to age-matched Vav-iCre^+/− controls (blue-dashed line). l, Senescence marker expression in splenic tissue (top) and bone marrow (bottom) from 8–11-month-old Vav-iCre and old wild-type mice (n = 3–9 Vav-iCre^+/−; n = 6–9 Vav-iCre^+/−;Ercc1^−/fl; n = 4–7 two-year-old WT, depending on the gene). Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired two-tailed Student’s t-test (a, c–g, j, k), one-way ANOVA (h, i, l) or two-way ANOVA (b) with Tukey’s test.

Fig. 2 |. Identification of senescent immune cell types by CyTOF.

a, Representative viSNE plots identifying immune cells that express p16 (top), p21 (middle) and PD-1 (bottom) from 10–12-month-old Vav-iCre^+/− or Vav-iCre^+/−;Ercc1^−/fl mice, or a 2-year-old wild-type mouse. b, Quantification of p16- and p21-expressing immune cell subsets (n = 6 Vav-iCre^+/−; n = 6 Vav-iCre^+/−;Ercc1^−/fl; n = 7 2-year-old WT mice analysed in a single CyTOF experiment). NKT cells, natural killer T cells. Data are mean ± s.d. *P < 0.05, **P < 0.01, Kruskal–Wallis test with Dunn’s correction. See Extended Data Fig. 4 for additional data.

Fig. 3 |. An aged immune system drives senescence and loss of tissue homeostasis in non-lymphoid organs.

Several senescence and tissue damage markers were measured in 8–11-month-old Vav-iCre mice and old wild-type mice (see Supplementary Table 3 for sample size details). a, b, Expression of p16 (a) and p21 (b) (n = 5–10 mice) in mutant mice was normalized to Vav-iCre^+/− controls (blue line). GI, gastrointestinal; VAT, visceral adipose tissue. c, Representative images of in situ hybridization on hepatic sections for p16 mRNA (green), albumin (red) to detect hepatocytes, and DAPI (blue nuclei). See also Extended Data Fig. 5. d, Hepatic 8-oxo-guanine DNA adducts and HNE protein adducts measured by ELISA (n = 6–8 mice per group). Glutathione ratio (reduced glutathione (GSH) to oxidized glutathione (GSSG) ratio) in livers of Vav-iCre^+/−;Ercc1^−/fl mice (n = 6), Vav-iCre^+/− controls (n = 6) and aged wild-type mice (n = 7). e, Serum aminotransferase levels (n = 9 mice per group). ALT, alanine aminotransferase; AST, aspartate transaminase. f, Urinary protein levels from Vav-iCre (n = 18) and old wild-type (n = 9) mice measured by Bradford assay. g, Serum amylase levels in Vav-iCre^+/−;Ercc1^−/fl (n = 5), Vav-iCre^+/− (n = 6), and old wild-type (n = 4) mice. h, Glycosaminoglycan (GAG) levels in intervertebral discs of Vav-iCre^+/−;Ercc1^−/fl (n = 7) and Vav-iCre^+/− (n = 4) mice. i, Wound area after cardiotoxin injury of the gastrocnemius (n = 3 mice per group). j, Serum β₂-microglobulin (β₂M) levels determined by ELISA of Vav-iCre^+/−;Ercc1^−/fl and Vav-iCre^+/− mice (n = 9 mice per group) and aged wild-type mice (n = 5). k, Serum SASP protein levels in Vav-iCre^+/−;Ercc1^−/fl (n = 6–7, depending on the protein), Vav-iCre^+/− controls (n = 5–6) and old wild-type (n = 5–7) mice, measured by ELISA. l, Lifespan of Vav-iCre^+/−;Ercc1^−/fl (n = 9) and Vav-iCre^+/− (n = 13) mice. Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed unpaired Student’s t-test (a, b, h, i), one-way ANOVA with Tukey’s test (d–g, j, k), or log-rank (Mantel–Cox) test (l).

Fig. 4 |. Adoptive transfer of splenocytes from Vav-iCre^+/−;Ercc1^−/fl and aged wild-type mice are sufficient to drive senescence in trans.

Splenocytes from 8–10-month-old Vav-iCre^+/−;Ercc1^−/fl mice, Vav-iCre^+/− controls, or two-year-old wild-type mice were injected retro-orbitally into 3–4-month-old p16^Ink4+/Luc senescence reporter mice (n = 2 donor mice per genotype). a, Representative images and weekly measures of luminescence in recipient mice (n = 3 recipient mice for Vav-iCre^+/−;Ercc1^−/fl or Vav-iCre^+/− splenocytes; n = 4 recipients for old WT splenocytes). b, Tissues were collected from recipient mice 2 weeks after the final imaging and the expression of the p16- and the p16-driven luciferase reporter were measured by quantitative PCR with reverse transcription (qRT–PCR). c, Levels of serum SASP factors MCP-1 (left) and TNF (right) in recipient mice 2 weeks after adoptive transfer. d, Lifespan of Ercc1^−/Δ mice after adoptive transfer of splenocytes from 19-month-old Vav-iCre^+/−;Ercc1^−/fl or Vav-iCre^+/− (n = 2 donors per group) mice. Recipient mice were 2 months of age when transplanted (n = 5 for mutant; n = 6 for control splenocytes). Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey’s test (b, c), or log rank (Mantel–Cox) test (d).

Fig. 5 |. Transplantation of splenocytes from young mice suppresses senescence and tissue damage in aged recipients, whereas rapamycin improves immune function of Vav-iCre^+/−;Ercc1^−/fl mice.

a, Adoptive transfer: 3-month-old Ercc1^−/Δ mice were injected retro-orbitally with 5 × 10⁶ splenocytes (SPL) from 2-month-old wild-type mice (n = 2 donors) or vehicle only (PBS) at days 1 and 4. At day 7, tissues were collected from recipient mice (n = 4 mice per group) and uninjected age-matched wild-type mice. b, Expression of senescence markers p16 and p21 measured by qRT–PCR. Gene expression was normalized to that of untreated, age-matched wild-type mice (horizontal dashed line). c, SASP proteins activin A, GDF-15, MCP-1 and IL-1β were measured in the serum of recipient mice by single- or multiplex-ELISA. d, Serum aminotransferase levels. e, Serum amylase levels. f, Mice were treated with or without rapamycin (Rapa; 4 mg kg⁻¹ intraperitoneally, three times per week) for 6 weeks starting at 3 months of age, followed by 1 week of no drug, then a DTH assay was initiated with KLH (Methods). Footpad swelling was measured 48 h after antigenic challenge (n = 3/7 Vav-iCre^+/−;Ercc1^−/fl or n = 3/6 Vav-iCre^+/− mice +/− rapamycin, respectively). g, One month after DTH challenge, anti-KLH antibodies were measured by ELISA (n values as in f). h, Peripheral WBC count 1 month after DTH challenge (n = 3/7 Vav-iCre^+/− and for Vav-iCre^+/−;Ercc1^−/fl +/− rapamycin, respectively). i, Expression of p16 in peripheral blood mononuclear cells (PBMCs), measured by qRT–PCR. Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA (b–e) or two-way ANOVA (f–i) with Tukey’s test.