Ethics statement

All animal experiments were performed in accordance with and approved by the Institutional Animal Care and Use Committee of the University of Southern California. The maximal tumor size permitted by the approved animal protocol was 15 mm in diameter. Mice were euthanized when tumors reached this limit or when they became moribund and showed signs of cachexia, lateral recumbency, observable weight loss, or lack of response to noxious stimuli. The maximal tumor burden permitted by the institutional guidelines was not exceeded in any experiment.

Mice

Female and male CD45.1 or CD45.2 C57BL/6 mice, as well as BALB/c mice, were obtained from Charles River Laboratories or The Jackson Laboratory. Rag2 knockout (Rag2–/–; B6.Cg-Rag2tm1.1Cgn/J) mice were obtained from the Jackson Laboratory and were bred in-house. Mice were maintained under specific pathogen-free conditions according to institutional guidelines. Based on established lifespan comparisons, mice at 8–12 weeks of age are generally considered young adults, corresponding to early adulthood in humans (approximately 20–30 years). In contrast, mice at 65–75 weeks are commonly used to represent a late-life or aging stage, broadly analogous to late middle to early elderly stages in humans (approximately 50–70 years). However, such cross-species comparisons are inherently non-linear and depend on the physiological parameter assessed52. In this study, C57BL/6 mice aged 8–12 weeks and 65–75 weeks were used as the young and aged cohorts, respectively. For BALB/c mice used in this study, animals aged ≥65 weeks were defined as aged based on experimental availability.

Cell lines

The E0771 cell line was purchased from the CH3 BioSystems. B16-F10 (B16), RAW 264.7, and EMT6 cell lines were purchased from the American Type Culture Collection (ATCC). The AT-3 cell line was a gift from Dr. Scott Abrams (Roswell Park Comprehensive Cancer Center, Buffalo, NY). E0771, B16, and RAW 264.7 cells were cultured in RPMI 1640 (Gibco) supplemented with 10% FBS (Sigma-Aldrich), 1% nonessential amino acid (NEAA; Gibco), 2 mmol/L L-glutamine (Gibco), 0.5% penicillin/streptomycin (Gibco), and 50 µmol/L 2-mercaptoethanol (Gibco). AT-3 and EMT6 cells were cultured in DMEM (Gibco) supplemented with 10% FBS, 1% NEAA, 2 mmol/L L-glutamine, 0.5% penicillin/streptomycin, and 50 µmol/L 2-mercaptoethanol. These cell lines were authenticated by morphology, phenotype, and growth, and routinely screened for Mycoplasma and maintained at 37 °C in a humidified 5% (E0771, B16, RAW 264.7, EMT6) or 7% (AT-3) CO2 atmosphere.

Plasmids

PmIL-6 FL and pmIL-6 mut NF-κB were gifts from Dr. Gail Bishop (Addgene plasmid # 61286; http://n2t.net/addgene:61286; RRID:Addgene_61286 and Addgene plasmid # 61293; http://n2t.net/addgene:61293; RRID:Addgene_61293, respectively)53.

Tumor model

E0771 (5 × 105), AT-3 (5 × 105), or EMT6 (2 × 105) tumors were surgically implanted under anesthesia with isoflurane into the fourth mammary gland of female mice as described54,55. B16 (5 × 105) tumor cells were injected subcutaneously on the left flank of male mice. Tumor growth was measured 3–5 times a week, and the volumes were calculated by determining the length of short (l) and long (L) diameters (volume = l2 × L/2) as previously described54,55.

In vivo treatment

For in vivo thymulin-conditioning, thymulin solution (1.5 mg/kg of body weight) was prepared from thymulin acetate (MedChemExpress, Monmouth Junction, NJ, USA) with an equimolar concentration of ZnCl2 (39059, Sigma-Aldrich, St. Louis, MO, USA) in PBS (Gibco), and was injected intraperitoneally (i.p.) starting on day 1 after tumor cell injection and continued daily for 4 weeks. Control mice received ZnCl2 in PBS (vehicle). For PD-L1 blockade, anti-PD-L1 Ab (clone 10 F.9G2, BioXcell) was given i.p. on day 7, 10, 13, 16, 19, 22 after the tumor cell inoculation at a dose of 200 μg/mouse54,55.

Flow cytometry

Single-cell suspensions of mouse peripheral blood and tumors were prepared for flow cytometric analysis. Red blood cells in blood were lysed using ACK Lysis Buffer (Life Technologies). Tumor tissues were weighed, minced, filtered through 70-μm filters, and stored at −80 °C in FBS (Hyclone, Waltham, MA) with 10% DMSO (Sigma) before analysis. Cells were blocked with anti-mouse CD16/32 (553142, BD Biosciences, San Diego, CA, USA) and surface-stained with indicated markers. Live/dead cell discrimination was performed using LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Life Technologies). To evaluate changes in the myeloid and lymphoid compartments of peripheral blood and the tumor microenvironment, we used the 23-color flow cytometry gating strategy (Supplementary Fig. 1) as described previously54. For analysis of cytokine expression, intracellular IL-1α, IL-1β, IL-6, TNF-α, and IFN-γ assays were performed using Fixation/Permeabilization Solution Kit (BD Biosciences) according to the manufacturer’s recommendations. Prior to intracellular staining, cells were stimulated ex vivo for 4 h at 37 °C in stimulation media composed as follows: RPMI 1640 medium with 10% FBS (Hyclone), 50 μg/ml of gentamicin (Gibco), 25 mM HEPES (Gibco), 2 mM L-glutamine (Gibco), 0.1 mM sodium pyruvate (Gibco), 0.1 mM NEAA (Gibco), LPS (100 ng/ml; LPS-SM ultrapure, InvivoGen), and 1× Cell Stimulation Cocktail with protein transport inhibitors (containing phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, and monensin; eBioscience, Thermo Fisher Scientific, Waltham, MA). Antibodies used in this study are listed in Supplementary Table S1 and were diluted using Brilliant Stain Buffer (566349, BD Biosciences). Samples acquired on Aurora (Cytek, San Diego, CA) cytometers were analyzed with FlowJo software (Treestar). Visualization of the multidimensional data was performed using the dimensionality reduction algorithm Uniform Manifold Approximation and Projection (UMAP), provided in FlowJo Plugin Exchange (v4.0.4, Treestar). Annotated pie charts were generated by SPICE software developed by the National Institutes of Health (v6.1).

Parabiosis

Parabiosis surgery was performed as previously described56. In brief, mirror-image skin incisions were made from the elbow to the knee in each mouse anesthetized with isoflurane. The peritoneal openings of the adjacent parabionts were sutured together. Forelimbs and hindlimbs were sutured together, and the skin of each incision was closed using continuous suture. Each mouse was injected subcutaneously with buprenorphine, as directed, for pain and monitored during recovery. Parabiosed pairs were analyzed more than 2 weeks after surgery.

Generation of bone marrow chimeras

Bone marrow and mixed bone marrow chimeras were generated as previously described54,57. First, recipient C57BL/6 mice were irradiated with 500 cGy followed by a second dose of 550 cGy 3 h apart. To obtain donor bone marrow, femurs and tibiae were harvested, and the bone marrow was flushed out. For bone marrow chimeras, 1 × 107 bone marrow cells from aged or young mice were injected into irradiated young or aged mice. For mixed bone marrow chimeras, 1 × 107 bone marrow cells from young and aged mice of a 1:1 mixture were injected into irradiated young or aged mice. After 8–12 weeks, the recipients were used for the experiments.

Generation of bone marrow-derived macrophages

Bone marrow-derived macrophages were generated as described previously58. Briefly, bone marrow cells from flushed marrow cavities of femurs and tibiae of >65 weeks old female C57BL/6 mice were cultured in RPMI 1640 with 10% FBS containing 10 ng/ml macrophage colony-stimulating factor (M-CSF, PeproTech, Rocky Hill, NJ, USA) with/without thymulin (1 µg/ml) and an equimolar concentration of ZnCl2. A control group was cultured in the same media with M-CSF and ZnCl2. Medium was changed daily. One week later, bone marrow-derived macrophages were harvested. Cells were stimulated with LPS (100 ng/ml; LPS-SM ultrapure, InvivoGen, San Diego, CA, USA) for 24 h before ELISA analysis.

ELISA

Levels of IL-1α, IL-1β, IL-6, and TNF-α secreted by aged bone marrow-derived macrophages were measured using ELISA MAX Deluxe Set Mouse IL-1α (BioLegend), Mouse IL-1β ELISA Kit (Sigma-Aldrich), Quantikine Mouse IL-6 ELISA Kit (R&D Systems), and BD OptEIA Mouse TNF ELISA Set II (BD Biosciences), respectively. The absorbance of each ELISA plate was measured using a microplate reader CLARIOstar Plus (BMG LABTECH, Weston Parkway, NC).

NF-κB DNA-binding assay

The NF-κB p65 DNA-binding activity was measured with the TransAM NF-κB p65 according to the manufacturer’s recommendations (Active Motif, Carlsbad, CA, USA). Nuclear extracts were prepared from 1 × 106 bone marrow-derived macrophages generated from aged mice. After cells are cultured with thymulin (1 μg/ml) and an equimolar concentration of ZnCl2 or ZnCl2 only (control) for 1 week, the cells were activated with LPS (100 ng/ml) for 1 h, and cell lysates were prepared in 200 μl lysis buffer [10 mM Hepes-KOH, pH 7.8, 10 mM KCl, 0.1 mM EDTA, pH 8.0, protease inhibitor mixture (Roche), and 0.1% Nonidet P-40], and nuclear extracts were obtained in 30 μl buffer [50 mM Hepes-KOH, pH 7.8, 420 mM KCl, 0.1 mM EDTA, pH 8.0, 5 mM MgCl2, protease inhibitor mixture (Roche), and 20% glycerol]58. Nuclear extracts (5 μl) in microwells were used to evaluate NF-κB DNA-binding activity.

Immunoblot analysis

Cells were lysed with 200 μl lysis buffer [10 mM Hepes-KOH, pH 7.8, 10 mM KCl, 0.1 mM EDTA, pH 8.0, protease inhibitor mixture (Roche), and 0.1% Nonidet P-40]. The cell lysates were separated by standard SDS-PAGE (Bioland Scientific, Paramount, CA) and analyzed by immunoblotting using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The following antibodies were used: anti-phospho-IκBα (Ser32/36; 5A5, Cell Signaling Technology), goat anti-mouse IgG-HRP (Poly4053, BioLegend), and anti-β-actin-HRP (2F1-1, BioLegend). The dilution ratios for anti-phospho-IκBα, goat anti-mouse IgG-HRP, and anti-β-actin-HRP were 1:1000, 1:2000, and 1:1000, respectively. The Western HRP Substrate (SuperSignal West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific) was used for the development of positive signals, and chemiluminescence was detected using an iBright 1500 (Thermo Fisher Scientific).

Luciferase reporter assay

Luciferase reporter assays were performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). For transfections, 1 × 105 RAW 264.7 cells were transfected with 0.8 μg of pmIL-6 FL (WT NF-κB) or pmIL-6 mut NF-κB plasmid (mut NF-κB), and 0.1 μg of pRL-TK using Lipofectamine 2000 (Invitrogen) and then plated in a 24-well plate at 1 × 105 cells/well. After 24 h, cells were activated by LPS (100 ng/ml, InvivoGen) and thymulin (1 μg/ml) with an equimolar concentration of ZnCl2 or ZnCl2 only (control) for 24 h. Following activation, cell extracts were prepared using Passive Lysis Buffer (Promega). Luciferase activity was determined from a 20-μl cell extract and measured on the CLARIOstar Plus (BMG LABTECH) microplate reader.

CTL killing assay

CTL killing assays were performed as previously described59. Cytotoxicity was assessed using a 7-AAD/CFSE cell-mediated cytotoxicity assay kit (Cayman Chemical, MI, USA) according to the manufacturer’s instructions. Briefly, whole splenocytes were isolated from AT-3 or E0771 tumor-bearing aged mice 2 weeks after tumor implantation, following in vivo thymulin or vehicle (control) conditioning as described above. A total of 4 × 10⁷ splenocytes were co-cultured with 2 × 10⁶ IFN-γ-treated (100 U/ml; PeproTech) AT-3 or E0771 tumor cells in the presence of thymulin (1 μg/ml) and an equimolar concentration of ZnCl₂ or ZnCl₂ alone (control). After 5 days, cells were harvested and used as effector cells (E). For target cell (T) preparation, AT-3 or E0771 tumor cells were treated with IFN-γ (100 U/ml) for 48 h prior to use. Target cells were labeled with CFSE (5 mM stock solution in PBS) for 10 min at 37 °C, washed twice with PBS, and immediately used in the assay. CFSE-labeled target cells were co-incubated with effector cells at the indicated effector-to-target (E:T) ratios (12.5:1, 25:1, and 50:1) for 4 h. Flow cytometric analysis was performed as described above. The percentage of specific lysis was calculated as follows: % specific lysis = 100 × (% sample lysis − % basal lysis) / (100 − % basal lysis), where % sample lysis and % basal lysis represent the percentage of CFSE⁺ 7-AAD⁺ target cells in the presence or absence of effector cells, respectively.

Human PBMC analysis

Written informed consent was obtained from 93 healthy donors aged between 21 and 87 with no personal history of cancer for the collection, storage, and analysis of blood samples under the Institutional Review Board of the University of Southern California (approval number: HS-22-00354) in accordance with the Declaration of Helsinki. Peripheral blood was obtained, and PBMCs were isolated using Lymphocyte Separation Medium (Corning) density gradient centrifugation and stored as previously described60,61,62,63. For the evaluation of the effect of thymulin, cells were cultured with thymulin (1 μg/ml) and an equimolar concentration of ZnCl2 or ZnCl2 only (control) for 24 h and were activated by LPS (100 ng/ml, InvivoGen) and 1× Cell Stimulation Cocktail with protein transport inhibitors (Thermo Fisher Scientific) for 4 h and were analyzed by flow cytometry.

IPA database exploration

To identify candidate factors associated with aging and inflammation, we performed a systematic exploration of the QIAGEN Ingenuity Pathway Analysis (IPA) database64. Within the “Diseases and Functions” category, the term “Aging” was used as a query. This search initially yielded 163 molecules, all of which were extracted for further analysis (Supplementary Dataset 3). These molecules were then filtered based on the “Causal or Correlated” annotation provided in IPA, and 35 molecules classified as “Causal” were selected. Among these, we further narrowed down the candidates by applying the criteria “Disease or Function = Longevity” and “Effect on Disease or Function = decreases/affects,” resulting in 9 molecules. Finally, from these 9 candidates, we identified molecules for which knockout mouse models have been reported to exhibit increased levels of pro-inflammatory cytokines, including IL-1, IL-6, and/or TNF-α. Based on this criterion, three factors—FOXO3, LMNA, and SIRT family—were selected for subsequent analyses.

Single-cell RNA sequencing

Count matrices, barcodes, feature data, and patient metadata for the eight oldest and eight youngest untreated primary breast tumors were downloaded from the Gene Expression Omnibus (GEO) (GSE176078). Cells with more than 200 detected genes (nFeature_RNA > 200), more than 250 total detected transcripts (nCount_RNA > 250), and less than 20% mitochondrial content were retained for downstream analyses. Doublets were removed using the DoubletFinder R package (v2.0.6)65. Briefly, each patient sample is preprocessed using Seurat (v5.4.0), and doublet detection was performed using the top 50 principal components. Artificial doublets were generated and integrated into each patient dataset at a defined proportion (pN = 0.25). The optimal neighborhood parameter (pK) was determined empirically using the paramSweep, summarizeSweep, and find.pK functions. Optimal pK was selected based on the maximum BCmetric score. The expected number of doublets (nExp) was estimated as 7.5% of the total cell number and adjusted for homotypic doublets based on cluster identities. Cells were then classified using DoubletFinder, and predicted doublets were removed from downstream analyses. Filtered datasets were merged, normalized using SCTransform, and subjected to principal component analysis (PCA) for dimensionality reduction. The ElbowPlot function was used to determine the optimal dimensionality of the dataset (PCs = 30). Data integration was performed using Seurat’s IntegrateLayers function with canonical correlation analysis (CCA) to minimize batch effects. A shared nearest neighbor graph was constructed using the FindNeighbors function, and cell clustering was performed using the FindClusters function at a resolution of 0.8. Differentially expressed genes (>25% cells, >0.25-fold change) were identified using the FindAllMarkers function and used for cluster annotation. Differential expression between annotated clusters was determined using the MAST method via the FindMarkers function, using a minimum expression proportion of 25% and a minimum fold change of 0.1. Single-cell gene set enrichment analysis (GSEA) was performed using VISION (v3.0.2) to calculate enrichment scores for curated gene signatures and biological pathways.

Statistical analysis

Statistical analysis was performed using a two-tailed Student’s t-test for comparisons between 2 groups, a one-way ANOVA with Tukey’s multiple comparisons test for comparisons among more than two groups, a two-way ANOVA with Bonferroni’s multiple comparisons test, the Mantel–Cox method (log-rank test) for survival analysis, or Pearson correlation analysis to assess relationships among variables, using GraphPad Prism 10.5.0 (GraphPad Software). p < 0.05 was considered statistically significant. Data are presented as mean ± SEM.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.