T cell-directed anti-aging and anti-cancer vaccine against commensal cytomegalovirus

Abstract

An immune-based approach to reduce the risk, delay the onset, and / or slow the progression of aging and aging-related diseases by enhancing T-cell immunity against the commensal human cytomegalovirus (HCMV).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 441,390, filed January 26, 2023, the entire contents of which are incorporated herein by reference.

[0002] An immune-based approach to reduce the risk, delay the onset, and / or slow the progression of aging and aging-related diseases by enhancing T-cell immunity against the commensal human cytomegalovirus (HCMV). [Background technology]

[0003] Senescent cells develop in response to cellular stress, exhibit irreversible cessation of proliferation while resisting death, and can accumulate in the body with age. See, for example, He & Sharpless, Cell, 169, 1000-1011, doi:10.1016 / j.cell.2017.05.015 (2017) and Di Micco et al. Nat Rev Mol Cell Biol 22, 75-95, doi:10.1038 / s41580-020-00314-w (2021). Despite permanent cell cycle arrest, senescent cells are not inactive. Senescent cells actively communicate with their surroundings and influence the tissue microenvironment through multiple secreted molecules, including pro-inflammatory cytokines and tissue remodeling factors, collectively referred to as the senescence-associated secretory phenotype (SASP). See, e.g., Mahmoudi & Brunet Nat Cell Biol 21, 32-43, doi:10.1038 / s41556-018-0206-0 (2019). Thus, senescent cells can induce a chronic inflammatory state in tissues, which leads to the development of cancer and aging-associated degenerative disorders. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] He & Sharpless,Cell,169,1000-1011,doi:10.1016 / j.cell.2017.05.015(2017) [Non-patent document 2] Di Micco et al.Nat Rev Mol Cell Biol 22,75-95,doi:10.1038 / s41580-020-00314-w(2021) [Non-patent document 3] Mahmoudi & Brunet Nat Cell Biol 21,32-43,doi:10.1038 / s41556-018-0206-0(2019) Summary of the Invention

[0005] The accumulation of senescent cells is involved in the pathogenesis of aging-related diseases, including cancer. The mechanism that prevents the accumulation of senescent cells in aging organs is unknown. As shown herein, the commensal virus-immune axis controls the accumulation of senescent fibroblasts in human skin. Senescent fibroblasts were increased in aged skin compared with young skin. However, senescent fibroblasts did not increase with age in elderly individuals. CXCL9 and cytotoxic CD4 + The increase in T cells (CD4 CTL) was significantly associated with a decrease in the level of senescent fibroblasts in aged skin. Senescent fibroblasts expressed human leukocyte antigen class II (HLA-II) and human cytomegalovirus glycoprotein B (HCMV-gB), which are direct targets of CD4 CTL. Skin-resident CD4 CTLs expressed HCMV-gB in an HLA-II-dependent manner. +HCMV-gB protein activated human skin-derived CD4+ CTLs, eliminating senescent fibroblasts. Collectively, these findings demonstrate that HCMV reactivation in senescent cells allows CD4+ CTLs to directly eliminate senescent cells via recognition of the HCMV-gB antigen. We also identified the immune factor CXCL9, which controls the recruitment of cytotoxic CD4+ T cells, and the short-chain fatty acids (SCFAs), butyrate and pentanoate, as enhancing their function in senescent cell clearance. Because SCFAs are known to be microbiota-derived metabolic products, vaccine approaches containing HCMV-gB adjuvants (e.g., SCFAs) may be used to enhance cytotoxic CD4+ T cell function and reduce the risk of, delay the onset of, slow the progression of, and / or treat senescence, cancer, and other aging-related diseases.

[0006] Thus, provided herein are methods for delaying the onset or slowing the progression of aging in a subject. Also provided herein are methods for reducing the level of senescent fibroblasts in a subject. Also disclosed herein are methods for treating or reducing the risk of developing an aging-related disease or condition in a subject. In some cases, any of the methods described herein comprises administering to a subject an effective amount of a composition. In some cases, the composition comprises a plurality of (i) antigenic proteins derived from commensal human cytomegalovirus (HCMV), (ii) antigenic peptides derived from proteins derived from commensal human cytomegalovirus, or (iii) live or live-attenuated commensal human cytomegalovirus, and a T cell adjuvant that increases T cell responses to the plurality of antigenic proteins, antigenic peptides, or live or live-attenuated commensal human cytomegalovirus. The level of senescent fibroblasts is reduced in the skin of the subject.

[0007] In some cases, the subject has an aging-related disease. In some cases, the aging-related disease is one or more of cancer, cardiovascular disease, neurodegenerative disease, kidney disease, autoimmune disease, arthritis, osteoporosis, macular degeneration, chronic obstructive pulmonary disease (COPD), glaucoma, obesity, fibrosis, interstitial lung disease, cirrhosis, hepatic steatosis, and diabetes. In some cases, the cancer is one or more of osteosarcoma, breast cancer, prostate cancer, colorectal cancer, lung cancer, melanoma, kidney cancer, lymphoma, uterine cancer, pancreatic cancer, non-melanoma skin cancer, and bladder cancer. In some cases, the neurodegenerative disease is one or more of dementia, ataxia, Huntington's disease, motor neuron disease, or tau-mediated neurodegenerative disease, optionally Alzheimer's disease, Parkinson's disease, or progressive supranuclear palsy. In some cases, the cardiovascular disease is one or more of atherosclerosis, idiopathic pulmonary fibrosis, coronary heart disease, congestive heart failure, coronary artery disease, peripheral artery disease, valvular heart disease, arrhythmia, ischemic cardiomyopathy, hypertension, and stroke. In some cases, the autoimmune disease is one or more of multiple sclerosis, Crohn's disease, rheumatoid arthritis, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, and systemic lupus erythematosus. In some cases, the fibrosis is one or more of systemic sclerosis, scleroderma, idiopathic pulmonary fibrosis, and interstitial lung disease.

[0008] In some cases, at least one of the antigenic proteins, if present, is HCMV glycoprotein B. In some cases, at least one of the antigenic proteins, if present, comprises a sequence derived from HCMV glycoprotein B. In some cases, the sequence from HCMV glycoprotein B is or comprises a truncated sequence of HCMV glycoprotein B. In some cases, each antigenic peptide, if present, comprises a sequence of 9-30 amino acids derived from a protein derived from human cytomegalovirus. In some cases, each antigenic peptide, if present, comprises a sequence of 9-30 amino acids derived from HCMV glycoprotein B. In some cases, the commensal human cytomegalovirus, if present, comprises one or more of AD169, Towne, Toledo, PH, TR, FIX, VR1814, Merlin, and TB40 / E HCMV strains.

[0009] In some cases, the T cell adjuvant comprises a short-chain fatty acid. In some cases, the short-chain fatty acid comprises butyrate, pentanoate, or a combination thereof. In some cases, the T cell adjuvant comprises one or more of nanoparticles that enhance T cell responses, poly-ICLC (carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA), imiquimod, CpG oligodeoxynucleotides and formulations (IC31, QB10), AS04 (aluminum salt formulated with 3-O-desacyl-4'-monophosphoryl lipid A (MPL)), AS01 (MPL and saponin QS-21), MPLA, STING agonists, other TLR agonists, Candida albicans skin test antigen (Candin), GM-CSF, Fms-like tyrosine kinase-3 ligand (Flt3L), and / or IFA (incomplete Freund's adjuvant).

[0010] Also disclosed herein is a composition for use in a method of treating or reducing the risk of developing an aging-related disease or condition in a subject, the composition comprising a plurality of (i) antigenic proteins derived from commensal human cytomegalovirus (HCMV), (ii) antigenic peptides derived from proteins derived from commensal human cytomegalovirus, or (iii) live or live-attenuated commensal human cytomegalovirus, and a T cell adjuvant that increases T cell responses to the plurality of antigenic proteins, antigenic peptides, or live or live-attenuated commensal human cytomegalovirus. In some cases, the aging-related condition is one or more of graying hair, hearing loss, cataracts, frailty, and sarcopenia. In some cases, the aging-related disease is one or more of cancer, cardiovascular disease, neurodegenerative disease, kidney disease, autoimmune disease, arthritis, osteoporosis, macular degeneration, chronic obstructive pulmonary disease (COPD), glaucoma, obesity, fibrosis, cirrhosis, hepatic steatosis, and diabetes. In some cases, each antigenic protein, if present, is HCMV glycoprotein B. In some cases, each antigenic protein, if present, comprises a sequence derived from HCMV glycoprotein B. In some cases, the sequence from HCMV glycoprotein B is a cleavage sequence of HCMV glycoprotein B. In some cases, each antigenic peptide, if present, comprises a sequence of 9-30 amino acids derived from a protein derived from human cytomegalovirus. In some cases, each antigenic peptide, if present, comprises a sequence of 9-30 amino acids derived from HCMV glycoprotein B. In some cases, the commensal human cytomegalovirus, if present, comprises one or more of AD169, Towne, Toledo, PH, TR, FIX, VR1814, Merlin, and TB40 / E HCMV strains. In some cases, the T cell adjuvant comprises a short chain fatty acid. In some cases, the short chain fatty acid comprises butyrate, pentanoate, or a combination thereof.In some cases, the T cell adjuvant comprises one or more of nanoparticles that enhance T cell responses, poly-ICLC (carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA), imiquimod, CpG oligodeoxynucleotides and formulations (IC31, QB10), AS04 (aluminum salt formulated with 3-O-desacyl-4'-monophosphoryl lipid A (MPL)), AS01 (MPL and saponin QS-21), MPLA, STING agonists, other TLR agonists, Candida albicans skin test antigen (Candin), GM-CSF, Fms-like tyrosine kinase-3 ligand (Flt3L), and / or IFA (incomplete Freund's adjuvant).

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0012] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0013] [Figure 1A]The accumulation of senescent cells in aged human skin does not correlate with increasing age. Representative immunofluorescence (IF) staining of p16INK4a in young and aged human skin samples. Arrowheads point to p16INK4a-positive cells in the epidermis, and arrows point to p16INK4a-positive cells in the dermis. Nuclei are stained with 40,6-diamidino-2-phenylindole (DAPI). The dotted line in the IF image indicates the epidermal basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. [Figure 1B] Accumulation of senescent cells in aged human skin does not correlate with increasing age. Quantification of p16INK4a-positive senescent cells in the epidermis per high-power field (hpf) (Mann-Whitney U test). Nuclei are stained with 40,6-diamidino-2-phenylindole (DAPI). Cells are blindly counted and averaged across 10 randomly selected hpf per skin sample. Bars represent the mean + SD. n = 23 for the young skin group and n = 31 for the aged skin group. Scale bar, 100 μm. [Figure 1C] Accumulation of senescent cells in aged human skin does not correlate with increasing age. Quantification of p16INK4a-positive senescent cells in the dermis per high-power field (hpf) (Mann-Whitney U test). Nuclei are stained with 40,6-diamidino-2-phenylindole (DAPI). Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bars represent mean + SD. n = 23 for the young skin group and n = 31 for the aged skin group. Scale bar, 100 μm. [Figure 1D] The accumulation of senescent cells in aged human skin does not correlate with increasing age. Representative IF staining of p16INK4a and vimentin in young and aged skin samples. Arrows point to p16INK4a+vimentin+ fibroblasts in the dermis. Nuclei are stained with 40,6-diamidino-2-phenylindole (DAPI). The dotted line in the IF image indicates the epidermal basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. [Figure 1E]Accumulation of senescent cells in aged human skin does not correlate with increasing age. Quantification of p16INK4a+vimentin+ fibroblasts per hpf (Mann-Whitney U test). Nuclei are stained with 40,6-diamidino-2-phenylindole (DAPI). Cells are counted blindly and averaged across 10 randomly selected hpf per skin sample. Bars represent mean + SD. n=23 for the young skin group, n=31 for the aged skin group. Scale bar, 100 μm. [Figure 1F] Senescent cell accumulation in aged human skin does not correlate with increasing age. Correlation between the number of dermal senescent cells and age across young and aged skin samples (Student's t-test with Pearson correlation coefficient). Nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI). Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bars represent the mean + SD. n=23 for the young skin group, n=31 for the aged skin group. Scale bar, 100 μm. [Figure 1G] The accumulation of senescent cells in aged human skin does not correlate with increasing age. Correlation between the number of dermal senescent cells in aged skin samples and age (Student's t-test with Pearson correlation coefficient). Nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI). Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bars represent the mean + SD. n = 23 for the young skin group, n = 31 for the aged skin group. Scale bar, 100 μm. [Figure 2A] Senescent fibroblasts constitute the majority of senescent cells in the dermis of human skin. (A) Vimentin-positive / -negative dermal p16INK4a+ cells in each young and aged human skin sample. Nuclei are stained with DAPI. Bars represent mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 2B]Senescent fibroblasts constitute the majority of senescent cells in the dermis of human skin. Representative immunofluorescence (IF) staining of platelet-derived growth factor receptor α (PDGFRα) and vimentin in aged human skin. The dotted line in the IF image indicates the epidermal basement membrane. Nuclei are stained with DAPI. Nuclei are stained with DAPI. Bars represent the mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 2C] Senescent fibroblasts constitute the majority of senescent cells in the dermis of human skin. Pie chart showing the percentage of vimentin+ cells among PDGFRα+ cells in the dermis (n=10 aged skin samples, female skin donors, mean age: 61.3 years). Nuclei are stained with DAPI. Bars represent mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 2D] Senescent fibroblasts constitute the majority of senescent cells in the dermis of human skin. Pie chart showing the percentage of PDGFRα+ cells among vimentin+ cells in the dermis (n=10 aged skin samples, female skin donors, mean age: 61.3 years). Nuclei are stained with DAPI. Bars represent mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 2E] Senescent fibroblasts constitute the majority of senescent cells in the dermis of human skin. Percentage of p16INK4a-positive senescent fibroblasts among all fibroblasts per skin sample. Cells were blindly counted and averaged across 10 randomly selected high-power fields (hpf) per skin sample (n = 23 for the young skin group and n = 31 for the aged skin group). Nuclei were stained with DAPI. Bars represent mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 2F] Senescent fibroblasts constitute the majority of senescent cells in the dermis of human skin. Representative IF staining of p21+, p16INK4a+, and vimentin in aged human skin. Nuclei are stained with DAPI. Nuclei are stained with DAPI. Bars represent mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 2G] Senescent fibroblasts constitute the majority of senescent cells in the dermis of human skin. (G) Pie chart showing the percentage of p21+ cells among p16INK4a+vimentin+ cells and the percentage of p21+ cells among p16INK4a-vimentin+ cells in the dermis (n = 10 aged skin samples, female skin donors, mean age: 61.3 years, chi-square test). Nuclei are stained with DAPI. Bars represent mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 2H] Senescent fibroblasts constitute the majority of senescent cells in the dermis of human skin. Quantification of p21+ fibroblasts in young and aged skin samples (n=10 for the young skin group and n=10 for the aged skin group, female skin donors, mean age: young: 19.4 years and aged: 61.3 years). Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Nuclei were stained with DAPI. Bars represent mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 2I] Senescent fibroblasts constitute the majority of senescent cells in the dermis of human skin. Representative IF staining of SenTraGor (GL13), p16INK4a, and vimentin in aged human skin. Nuclei are stained with DAPI. Bars represent mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 3A]Epidermal thickness and blood / lymphatic vessel density decrease with age but do not correlate with the number of senescent dermal cells in aged skin. Representative hematoxylin and eosin (H&E) staining of young and aged skin samples. Green brackets highlight epidermal thickness. Dermal CD3+ cells per hpf represent T cells; CD3+CD4+ cells represent CD4+ T cells; CD4+T-bet+ cells represent Th1 cells; CD4+GATA3+ cells represent Th2 cells; CD4+ROR-γt+ cells represent Th17 cells; CD4+Foxp3+ cells represent regulatory T cells (Tregs); CD3+CD8+ cells represent CD8+ T cells; CD3+TCRγδ+ cells represent γδ T cells; and CD3+CD56+ cells represent NKT cells. The numbers of CD11c+CD141+ cells, indicative of cDC1, CD1c+CD11c+CD207- cells, indicative of cDC2, CD1a+CD207+ cells, indicative of Langerhan cells (LC), CD68+CD86+ cells, indicative of M1 macrophages, CD68+CD206+ cells, indicative of M2 macrophages, mast cell treptase+ cells, indicative of mast cells, neutrophil elastase+ cells, indicative of neutrophil cells, and CD3-CD56+ cells, indicative of NK cells, are shown as heat maps in Figure 4A. Nuclei were stained with DAPI, and the dotted line in the IF images indicates the epithelial basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bar graphs show the mean + SD. Young skin group, n=23; old skin group, n=31; *p<0.05, **p<0.01, and ***p<0.0001; ns: not significant, Mann-Whitney U test. Scale bar, 100 μm. [Figure 3B]Epidermal thickness and blood / lymphatic vessel density decrease with age but do not correlate with the number of senescent dermal cells in aged skin. Quantification of epidermal thickness (Mann-Whitney U test). Per hpf, dermal CD3+ cells represent T cells, CD3+CD4+ cells represent CD4+ T cells, CD4+T-bet+ cells represent Th1 cells, CD4+GATA3+ cells represent Th2 cells, CD4+ROR-γt+ cells represent Th17 cells, CD4+Foxp3+ cells represent regulatory T cells (Treg), CD3+CD8+ cells represent CD8+ T cells, CD3+TCRγδ+ cells represent γδ T cells, and CD3+CD56+ cells represent NKT cells. The numbers of CD11c+CD141+ cells, indicative of cDC1, CD1c+CD11c+CD207- cells, indicative of cDC2, CD1a+CD207+ cells, indicative of Langerhan cells (LC), CD68+CD86+ cells, indicative of M1 macrophages, CD68+CD206+ cells, indicative of M2 macrophages, mast cell treptase+ cells, indicative of mast cells, neutrophil elastase+ cells, indicative of neutrophil cells, and CD3-CD56+ cells, indicative of NK cells, are shown as heat maps in Figure 4A. Nuclei were stained with DAPI, and the dotted line in the IF images indicates the epithelial basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bar graphs show the mean + SD. Young skin group, n=23; old skin group, n=31; *p<0.05, **p<0.01, and ***p<0.0001; ns: not significant, Mann-Whitney U test. Scale bar, 100 μm. [Figure 3C]Epidermal thickness and blood / lymphatic vessel density decrease with age but do not correlate with the number of senescent dermal cells in aged skin. Correlation between epidermal thickness and the number of dermal p16INK4a+ cells in aged skin samples (Student's t-test for Pearson correlation coefficient). Dermal CD3+ cells represent T cells, CD3+CD4+ cells represent CD4+ T cells, CD4+T-bet+ cells represent Th1 cells, CD4+GATA3+ cells represent Th2 cells, CD4+ROR-γt+ cells represent Th17 cells, CD4+Foxp3+ cells represent regulatory T cells (Treg), CD3+CD8+ cells represent CD8+ T cells, CD3+TCRγδ+ cells represent γδ T cells, and CD3+CD56+ cells represent NKT cells per hpf. The numbers of CD11c+CD141+ cells, indicative of cDC1, CD1c+CD11c+CD207- cells, indicative of cDC2, CD1a+CD207+ cells, indicative of Langerhan cells (LC), CD68+CD86+ cells, indicative of M1 macrophages, CD68+CD206+ cells, indicative of M2 macrophages, mast cell treptase+ cells, indicative of mast cells, neutrophil elastase+ cells, indicative of neutrophil cells, and CD3-CD56+ cells, indicative of NK cells, are shown as heat maps in Figure 4A. Nuclei were stained with DAPI, and the dotted line in the IF images indicates the epithelial basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bar graphs show the mean + SD. Young skin group, n=23; old skin group, n=31; *p<0.05, **p<0.01, and ***p<0.0001; ns: not significant, Mann-Whitney U test. Scale bar, 100 μm. [Figure 3D]Epidermal thickness and blood / lymphatic vessel density decrease with age but do not correlate with the number of senescent dermal cells in aged skin. Representative IF staining of CD31 (a marker for blood endothelial cells) in young and aged skin samples. Arrows point to CD31+ blood endothelial cells in the dermis. Dermal CD3+ cells per hpf represent T cells; CD3+CD4+ cells represent CD4+ T cells; CD4+T-bet+ cells represent Th1 cells; CD4+GATA3+ cells represent Th2 cells; CD4+ROR-γt+ cells represent Th17 cells; CD4+Foxp3+ cells represent regulatory T cells (Tregs); CD3+CD8+ cells represent CD8+ T cells; CD3+TCRγδ+ cells represent γδ T cells; and CD3+CD56+ cells represent NKT cells. The numbers of CD11c+CD141+ cells, indicative of cDC1, CD1c+CD11c+CD207- cells, indicative of cDC2, CD1a+CD207+ cells, indicative of Langerhan cells (LC), CD68+CD86+ cells, indicative of M1 macrophages, CD68+CD206+ cells, indicative of M2 macrophages, mast cell treptase+ cells, indicative of mast cells, neutrophil elastase+ cells, indicative of neutrophil cells, and CD3-CD56+ cells, indicative of NK cells, are shown as heat maps in Figure 4A. Nuclei were stained with DAPI, and the dotted line in the IF images indicates the epithelial basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bar graphs show the mean + SD. Young skin group, n=23; old skin group, n=31; *p<0.05, **p<0.01, and ***p<0.0001; ns: not significant, Mann-Whitney U test. Scale bar, 100 μm. [Figure 3E]Epidermal thickness and blood / lymphatic vessel density decrease with age but do not correlate with the number of senescent dermal cells in aged skin. Number of dermal CD31+ blood endothelial cells per hpf (Mann-Whitney U test). Dermal CD3+ cells representing T cells, CD3+CD4+ cells representing CD4+ T cells, CD4+T-bet+ cells representing Th1 cells, CD4+GATA3+ cells representing Th2 cells, CD4+ROR-γt+ cells representing Th17 cells, CD4+Foxp3+ cells representing regulatory T cells (Treg), CD3+CD8+ cells representing CD8+ T cells, CD3+TCRγδ+ cells representing γδ T cells, and CD3+CD56+ cells representing NKT cells. The numbers of CD11c+CD141+ cells, indicative of cDC1, CD1c+CD11c+CD207- cells, indicative of cDC2, CD1a+CD207+ cells, indicative of Langerhan cells (LC), CD68+CD86+ cells, indicative of M1 macrophages, CD68+CD206+ cells, indicative of M2 macrophages, mast cell treptase+ cells, indicative of mast cells, neutrophil elastase+ cells, indicative of neutrophil cells, and CD3-CD56+ cells, indicative of NK cells, are shown as heat maps in Figure 4A. Nuclei were stained with DAPI, and the dotted line in the IF images indicates the epithelial basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bar graphs show the mean + SD. Young skin group, n=23; old skin group, n=31; *p<0.05, **p<0.01, and ***p<0.0001; ns: not significant, Mann-Whitney U test. Scale bar, 100 μm. [Figure 3F]Epidermal thickness and blood / lymphatic vessel density decrease with age, but do not correlate with the number of senescent dermal cells in aged skin. Correlation between the number of dermal CD31+ blood endothelial cells and the number of dermal p16INK4a+ cells in aged skin samples (Student's t-test for Pearson correlation coefficient). Dermal CD3+ cells represent T cells, CD3+CD4+ cells represent CD4+ T cells, CD4+T-bet+ cells represent Th1 cells, CD4+GATA3+ cells represent Th2 cells, CD4+ROR-γt+ cells represent Th17 cells, CD4+Foxp3+ cells represent regulatory T cells (Treg), CD3+CD8+ cells represent CD8+ T cells, CD3+TCRγδ+ cells represent γδ T cells, and CD3+CD56+ cells represent NKT cells per hpf. The numbers of CD11c+CD141+ cells, indicative of cDC1, CD1c+CD11c+CD207- cells, indicative of cDC2, CD1a+CD207+ cells, indicative of Langerhan cells (LC), CD68+CD86+ cells, indicative of M1 macrophages, CD68+CD206+ cells, indicative of M2 macrophages, mast cell treptase+ cells, indicative of mast cells, neutrophil elastase+ cells, indicative of neutrophil cells, and CD3-CD56+ cells, indicative of NK cells, are shown as heat maps in Figure 4A. Nuclei were stained with DAPI, and the dotted line in the IF images indicates the epithelial basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bar graphs show the mean + SD. Young skin group, n=23; old skin group, n=31; *p<0.05, **p<0.01, and ***p<0.0001; ns: not significant, Mann-Whitney U test. Scale bar, 100 μm. [Figure 3G]Epidermal thickness and blood / lymphatic vessel density decrease with age but do not correlate with the number of senescent dermal cells in aged skin. Representative IF staining of lymphatic endothelial hyaluronan receptor 1 (LYVE-1) (a marker for lymphatic endothelial cells) in young and aged skin samples. Arrows point to LYVE-1 lymphatic endothelial cells in the dermis. Per hpf, dermal CD3+ cells represent T cells; CD3+CD4+ cells represent CD4+ T cells; CD4+T-bet+ cells represent Th1 cells; CD4+GATA3+ cells represent Th2 cells; CD4+ROR-γt+ cells represent Th17 cells; CD4+Foxp3+ cells represent regulatory T cells (Tregs); CD3+CD8+ cells represent CD8+ T cells; CD3+TCRγδ+ cells represent γδ T cells; and CD3+CD56+ cells represent NKT cells. The numbers of CD11c+CD141+ cells, indicative of cDC1, CD1c+CD11c+CD207- cells, indicative of cDC2, CD1a+CD207+ cells, indicative of Langerhan cells (LC), CD68+CD86+ cells, indicative of M1 macrophages, CD68+CD206+ cells, indicative of M2 macrophages, mast cell treptase+ cells, indicative of mast cells, neutrophil elastase+ cells, indicative of neutrophil cells, and CD3-CD56+ cells, indicative of NK cells, are shown as heat maps in Figure 4A. Nuclei were stained with DAPI, and the dotted line in the IF images indicates the epithelial basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bar graphs show the mean + SD. Young skin group, n=23; old skin group, n=31; *p<0.05, **p<0.01, and ***p<0.0001; ns: not significant, Mann-Whitney U test. Scale bar, 100 μm. [Figure 3H]Epidermal thickness and blood / lymphatic vessel density decrease with age, but do not correlate with the number of senescent dermal cells in aged skin. Per hpf, dermal CD3+ cells represent T cells, CD3+CD4+ cells represent CD4+ T cells, CD4+T-bet+ cells represent Th1 cells, CD4+GATA3+ cells represent Th2 cells, CD4+ROR-γt+ cells represent Th17 cells, CD4+Foxp3+ cells represent regulatory T cells (Treg), CD3+CD8+ cells represent CD8+ T cells, CD3+TCRγδ+ cells represent γδ T cells, and CD3+CD56+ cells represent NKT cells. The numbers of CD11c+CD141+ cells, indicative of cDC1, CD1c+CD11c+CD207- cells, indicative of cDC2, CD1a+CD207+ cells, indicative of Langerhan cells (LC), CD68+CD86+ cells, indicative of M1 macrophages, CD68+CD206+ cells, indicative of M2 macrophages, mast cell treptase+ cells, indicative of mast cells, neutrophil elastase+ cells, indicative of neutrophil cells, and CD3-CD56+ cells, indicative of NK cells, are shown as heat maps in Figure 4A. Nuclei were stained with DAPI, and the dotted line in the IF images indicates the epithelial basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bar graphs show the mean + SD. Young skin group, n=23; old skin group, n=31; *p<0.05, **p<0.01, and ***p<0.0001; ns: not significant, Mann-Whitney U test. Scale bar, 100 μm. [Figure 3I]Epidermal thickness and blood / lymphatic vessel density decrease with age, but do not correlate with the number of senescent dermal cells in aged skin. Correlation between the number of dermal LYVE-1 lymphatic endothelial cells and the number of dermal p16INK4a cells in aged skin samples (Student's t-test for Pearson correlation coefficient). Dermal CD3+ cells represent T cells, CD3+CD4+ cells represent CD4+ T cells, CD4+T-bet+ cells represent Th1 cells, CD4+GATA3+ cells represent Th2 cells, CD4+ROR-γt+ cells represent Th17 cells, CD4+Foxp3+ cells represent regulatory T cells (Treg), CD3+CD8+ cells represent CD8+ T cells, CD3+TCRγδ+ cells represent γδ T cells, and CD3+CD56+ cells represent NKT cells per hpf. The numbers of CD11c+CD141+ cells, indicative of cDC1, CD1c+CD11c+CD207- cells, indicative of cDC2, CD1a+CD207+ cells, indicative of Langerhan cells (LC), CD68+CD86+ cells, indicative of M1 macrophages, CD68+CD206+ cells, indicative of M2 macrophages, mast cell treptase+ cells, indicative of mast cells, neutrophil elastase+ cells, indicative of neutrophil cells, and CD3-CD56+ cells, indicative of NK cells, are shown as heat maps in Figure 4A. Nuclei were stained with DAPI, and the dotted line in the IF images indicates the epithelial basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bar graphs show the mean + SD. Young skin group, n=23; old skin group, n=31; *p<0.05, **p<0.01, and ***p<0.0001; ns: not significant, Mann-Whitney U test. Scale bar, 100 μm. [Figure 3J-1]Epidermal thickness and blood / lymphatic vessel density decrease with age, but do not correlate with the number of senescent dermal cells in aged skin. Immune cell quantification in human skin. Per hpf, dermal CD3+ cells represent T cells, CD3+CD4+ cells represent CD4+ T cells, CD4+T-bet+ cells represent Th1 cells, CD4+GATA3+ cells represent Th2 cells, CD4+ROR-γt+ cells represent Th17 cells, CD4+Foxp3+ cells represent regulatory T cells (Treg), CD3+CD8+ cells represent CD8+ T cells, CD3+TCRγδ+ cells represent γδ T cells, and CD3+CD56+ cells represent NKT cells. The numbers of CD11c+CD141+ cells, indicative of cDC1, CD1c+CD11c+CD207- cells, indicative of cDC2, CD1a+CD207+ cells, indicative of Langerhan cells (LC), CD68+CD86+ cells, indicative of M1 macrophages, CD68+CD206+ cells, indicative of M2 macrophages, mast cell treptase+ cells, indicative of mast cells, neutrophil elastase+ cells, indicative of neutrophil cells, and CD3-CD56+ cells, indicative of NK cells, are shown as heat maps in Figure 4A. Nuclei were stained with DAPI, and the dotted line in the IF images indicates the epithelial basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bar graphs show the mean + SD. Young skin group, n=23; old skin group, n=31; *p<0.05, **p<0.01, and ***p<0.0001; ns: not significant, Mann-Whitney U test. Scale bar, 100 μm. [Figure 3J-2]Epidermal thickness and blood / lymphatic vessel density decrease with age, but do not correlate with the number of senescent dermal cells in aged skin. Immune cell quantification in human skin. Per hpf, dermal CD3+ cells represent T cells, CD3+CD4+ cells represent CD4+ T cells, CD4+T-bet+ cells represent Th1 cells, CD4+GATA3+ cells represent Th2 cells, CD4+ROR-γt+ cells represent Th17 cells, CD4+Foxp3+ cells represent regulatory T cells (Treg), CD3+CD8+ cells represent CD8+ T cells, CD3+TCRγδ+ cells represent γδ T cells, and CD3+CD56+ cells represent NKT cells. The numbers of CD11c+CD141+ cells, indicative of cDC1, CD1c+CD11c+CD207- cells, indicative of cDC2, CD1a+CD207+ cells, indicative of Langerhan cells (LC), CD68+CD86+ cells, indicative of M1 macrophages, CD68+CD206+ cells, indicative of M2 macrophages, mast cell treptase+ cells, indicative of mast cells, neutrophil elastase+ cells, indicative of neutrophil cells, and CD3-CD56+ cells, indicative of NK cells, are shown as heat maps in Figure 4A. Nuclei were stained with DAPI, and the dotted line in the IF images indicates the epithelial basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bar graphs show the mean + SD. Young skin group, n=23; old skin group, n=31; *p<0.05, **p<0.01, and ***p<0.0001; ns: not significant, Mann-Whitney U test. Scale bar, 100 μm. [Figure 3J-3]Epidermal thickness and blood / lymphatic vessel density decrease with age, but do not correlate with the number of senescent dermal cells in aged skin. Immune cell quantification in human skin. Per hpf, dermal CD3+ cells represent T cells, CD3+CD4+ cells represent CD4+ T cells, CD4+T-bet+ cells represent Th1 cells, CD4+GATA3+ cells represent Th2 cells, CD4+ROR-γt+ cells represent Th17 cells, CD4+Foxp3+ cells represent regulatory T cells (Treg), CD3+CD8+ cells represent CD8+ T cells, CD3+TCRγδ+ cells represent γδ T cells, and CD3+CD56+ cells represent NKT cells. The numbers of CD11c+CD141+ cells, indicative of cDC1, CD1c+CD11c+CD207- cells, indicative of cDC2, CD1a+CD207+ cells, indicative of Langerhan cells (LC), CD68+CD86+ cells, indicative of M1 macrophages, CD68+CD206+ cells, indicative of M2 macrophages, mast cell treptase+ cells, indicative of mast cells, neutrophil elastase+ cells, indicative of neutrophil cells, and CD3-CD56+ cells, indicative of NK cells, are shown as heat maps in Figure 4A. Nuclei were stained with DAPI, and the dotted line in the IF images indicates the epithelial basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bar graphs show the mean + SD. Young skin group, n=23; old skin group, n=31; *p<0.05, **p<0.01, and ***p<0.0001; ns: not significant, Mann-Whitney U test. Scale bar, 100 μm. [Figure 4A] CD4 CTLs are prominent cytotoxic lymphocytes in human skin, and their numbers inversely correlate with the number of dermal senescent cells in aged skin. Heatmap of aging-related changes in the number of cutaneous immune cells, determined by multiplex immunostaining on tissue sections. Intensity represents the relative change compared to the young group. n = 23 for the young skin group and n = 31 for the aged skin group. Nuclei are stained with DAPI. The dotted line in the IF images indicates the epidermal basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bars represent the mean + SD. Scale bar, 100 μm. [Figure 4B]CD4 CTLs are the predominant cytotoxic lymphocytes in human skin, and their number is inversely correlated with the number of dermal senescent cells in aged skin. (B) Correlation between the number of dermal senescent cells and dermal CD4 CTLs in aged skin samples (n = 31 aged skin samples, Student's t-test for Pearson correlation coefficient). Nuclei are stained with DAPI. The dotted line in the IF images indicates the epidermal basement membrane. Cells were blindly counted and averaged over 10 randomly selected hpf per skin sample. Scale bar, 100 μm. [Figure 4C] CD4 CTLs are the predominant cytotoxic lymphocytes in human skin, and their numbers inversely correlate with the number of dermal senescent cells in aged skin. Representative IF staining of CD4 and perforin in young and aged skin samples. Note that CD4+ cells are CD3+ T cells. Nuclei are stained with DAPI. The dotted line in the IF image indicates the epidermal basement membrane. Cells were blindly counted and averaged over 10 randomly selected hpf per skin sample. Scale bar, 100 μm. [Figure 4D] CD4 CTLs are the predominant cytotoxic lymphocytes in human skin, and their numbers are inversely correlated with the number of dermal senescent cells in aged skin. Quantification of cutaneous CD4+ perforin+ CD4 CTLs per hpf (n=23 in the young skin group, n=31 in the aged skin group, Mann-Whitney U test). Nuclei are stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bars represent the mean + SD. Scale bar, 100 μm. [Figure 4E] CD4 CTLs are the predominant cytotoxic lymphocytes in human skin, and their numbers inversely correlate with the number of dermal senescent cells in aged skin. Pie chart showing the percentage of perforin+ cells in aged dermis (n=31 aged skin samples). Nuclei are stained with DAPI. The dotted line in the IF image indicates the epidermal basement membrane. Cells were blindly counted and averaged over 10 randomly selected hpf per skin sample. Scale bar, 100 μm. [Figure 4F]CD4 CTLs are the predominant cytotoxic lymphocytes in human skin, and their numbers are inversely correlated with the number of dermal senescent cells in aged skin. Volcano plot of RNA-seq data displaying gene expression patterns in aged versus young human skin samples. Significantly upregulated genes are to the left of 0 on the x-axis, and downregulated genes are to the right of 0 on the x-axis (p<0.05 considered significant). Selected immune-related genes based on cluster analysis are shown. Nuclei are stained with DAPI. The dotted line in the IF image indicates the epidermal basement membrane. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Scale bar, 100 μm. [Figure 4G] CD4 CTLs are prominent cytotoxic lymphocytes in human skin, and their numbers inversely correlate with the number of dermal senescent cells in aged skin. (G) Representative IF staining of CXCL9 in aged skin samples. Nuclei are stained with DAPI. The dotted line in the IF image indicates the epidermal basement membrane. Cells were blindly counted and averaged over 10 randomly selected hpf per skin sample. Bars represent the mean + SD. Scale bar, 100 μm. [Figure 4H] CD4 CTLs are the predominant cytotoxic lymphocytes in human skin, and their numbers inversely correlate with the number of dermal senescent cells in aged skin. (H) Quantification of dermal CD4 CTLs per hpf in the CXCL9high vs. CXCL9low groups of aged skin samples (n=11 per group, Mann-Whitney U test). Nuclei are stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bars represent the mean + SD. Scale bar, 100 μm. [Figure 4I]CD4 CTLs are the predominant cytotoxic lymphocytes in human skin, and their numbers inversely correlate with the number of dermal senescent cells in aged skin. Quantification of p16INK4a-positive dermal senescent cells per hpf in the CXCL9high vs. CXCL9low groups of aged skin samples (n=11 per group, Mann-Whitney U test). Nuclei are stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per skin sample. Bars represent the mean + SD. Scale bar, 100 μm. [Figure 5A] scRNA-seq analysis of immune cells isolated from aged human skin. Illustrative schematic of the workflow for scRNA-seq analysis on human skin samples. PI, propidium iodide (indicates dead cells). [Figure 5B] scRNA-seq analysis of immune cells isolated from aged human skin. Uniform manifold approximated projection (UMAP) plot of lymphocyte-enriched CD45+ cells sorted from aged human trunk skin samples. A total of 17,624 cells from three independent donors (female skin donors, ages 50, 65, and 67 years) were analyzed. [Figure 5C-1] scRNA-seq analysis of immune cells isolated from aged human skin. Violin plot showing the distribution of gene expression levels of T cell-defining markers and selected cytotoxic T cell-associated genes in conventional CD4+ T cells, CD4 CTLs, and CD8+ T cells. DESeq2 was used for DE analysis and p-value calculation. [Figure 5C-2] scRNA-seq analysis of immune cells isolated from aged human skin. Violin plot showing the distribution of gene expression levels of T cell-defining markers and selected cytotoxic T cell-associated genes in conventional CD4+ T cells, CD4 CTLs, and CD8+ T cells. DESeq2 was used for DE analysis and p-value calculation. [Figure 6A]CXCL9 promotes migration of human skin-resident CD4 CTLs. Representative flow cytometry plots showing the gating strategy for identifying CD4+ and CD8+ T cells in human skin. The percentage of cells within each gate is indicated on the flow cytometry plot. Zombie fixable viability dye (viability) indicates dead cells. Skin samples from two independent donors were used to isolate skin-resident immune cells (female skin donors, ages: 60 and 73 years). Bar graphs show mean + SD, Mann-Whitney U test. [Figure 6B] CXCL9 promotes migration of human skin-resident CD4 CTLs. (B) Representative flow cytometry plots of CXCR3 expression on CD4+ and CD8+ T cells in human skin. Numbers on the flow cytometry plots represent the percentage of cells within each gate. Skin samples from two independent donors were used to isolate skin-resident immune cells (female skin donors, ages 60 and 73 years). [Figure 6C] CXCL9 promotes the migration of human skin-resident CD4 CTLs. (C) Experimental scheme for transwell migration assay using immune cells isolated from human skin. Skin samples from two independent donors were used to isolate skin-resident immune cells (female skin donors, ages 60 and 73 years). Bar graphs show mean + SD, Mann-Whitney U test. [Figure 6D] CXCL9 promotes the migration of human skin-resident CD4 CTLs. Effect of CXCL9 recombinant protein (585 ng / mL) treatment on the migration of human skin CD4 CTLs. Skin-resident immune cells were isolated using skin samples from two independent donors (female skin donors, ages: 60 and 73 years). Bar graphs show mean + SD, Mann-Whitney U test. [Figure 6E]CXCL9 promotes the migration of human skin-resident CD4 CTLs. The effect of CXCL9 recombinant protein (585 ng / mL) treatment on the migration of human skin CD8+ T cells. Skin-resident immune cells were isolated using skin samples from two independent donors (female skin donors, ages 60 and 73 years). Bar graphs show the mean + SD, Mann-Whitney U test. [Figure 7A] Skin-resident T cells eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative senescence-associated b-galactosidase (SA-β-Gal or X-gal) staining of normal and senescent dermal fibroblasts. Fibroblasts were preincubated with pan-HLA-DR blocking or isotype control antibodies. Skin immune cell-to-fibroblast ratio of 50:1 (n = 4 female skin donors, age range: 43-65 years, mean age: 48.8 years, one-way ANOVA). Skin samples used in coculture experiments were pan-HCMV antigen-positive. Nuclei are stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpfs per sample. Bars represent the mean + SD. Scale bar, 100 μm. [Figure 7B] Skin-resident T cells eliminate senescent fibroblasts in an HLA-II-dependent manner. (B) Heatmap showing the expression of immune cell activation ligand genes in senescent fibroblasts versus normal fibroblasts from RNA-seq data. (D) indicates downregulation (log2 fold change < -0.4, *p < 0.05) and (U) indicates upregulation (log2 fold change > 0.4, *p < 0.05) in senescent fibroblasts (n = 3) versus normal fibroblasts (n = 4). Fibroblasts were pre-incubated with pan-HLA-DR blocking or isotype control antibodies. A skin immune cell-to-fibroblast ratio of 50:1 was used (n = 4 female skin donors, age range: 43-65 years, mean age: 48.8 years, one-way ANOVA). Skin samples used in coculture experiments were pan-HCMV antigen-positive. Nuclei are stained with DAPI. Cells were counted blindly and averaged over 10 randomly selected hpf per sample. Bars show the mean + SD. Scale bar, 100 μm. [Figure 7C]Skin-resident T cells eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative flow cytometry histograms of ULBP2 on the surface of normal and senescent fibroblasts (n = 7 pairs of aged human dermal fibroblasts [female skin donors, age range: 43-70 years, mean age: 53.7 years], paired t-test). Fibroblasts were pre-incubated with pan-HLA-DR blocking or isotype control antibodies. A 50:1 skin immune cell-to-fibroblast ratio was used (n = 4 female skin donors, age range: 43-65 years, mean age: 48.8 years, one-way ANOVA). Skin samples used in coculture experiments were pan-HCMV antigen-positive. Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpfs per sample. Bars represent the mean + SD. Scale bar, 100 µm. [Figure 7D] Skin-resident T cells eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative relative fluorescence intensity (RFI) of ULBP2 on the surface of normal and senescent fibroblasts (n = 7 pairs of aged human dermal fibroblasts [female skin donors, age range: 43-70 years, mean age: 53.7 years], paired t-test). Fibroblasts were pre-incubated with pan-HLA-DR blocking or isotype control antibodies. Skin immune cell-to-fibroblast ratio of 50:1 (n = 4 female skin donors, age range: 43-65 years, mean age: 48.8 years, one-way ANOVA). Skin samples used in coculture experiments were pan-HCMV antigen-positive. Nuclei are stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpfs per sample. Bars represent mean + SD. Scale bar, 100 µm. [Figure 7E]Skin-resident T cells eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative flow cytometry histogram (E) of HLA-II on the surface of normal and senescent fibroblasts (n = 7 pairs of aged human dermal fibroblasts [female skin donors, age range: 43-70 years, mean age: 53.7 years], paired t-test). Fibroblasts were pre-incubated with pan-HLA-DR blocking or isotype control antibodies. Skin immune cell-to-fibroblast ratio of 50:1 (n = 4 female skin donors, age range: 43-65 years, mean age: 48.8 years, one-way ANOVA). Skin samples used in coculture experiments were pan-HCMV antigen-positive. Nuclei are stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpfs per sample. Bars represent mean + SD. Scale bar, 100 µm. [Figure 7F] Skin-resident T cells eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative RFI of HLA-II on the surface of normal and senescent fibroblasts (n = 7 pairs of aged human dermal fibroblasts [female skin donors, age range: 43-70 years, mean age: 53.7 years], paired t-test). Fibroblasts were pre-incubated with pan-HLA-DR blocking or isotype control antibodies. Skin immune cell-to-fibroblast ratio of 50:1 (n = 4 female skin donors, age range: 43-65 years, mean age: 48.8 years, one-way ANOVA). Skin samples used in coculture experiments were pan-HCMV antigen-positive. Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpfs per sample. Bars represent the mean + SD. Scale bar, 100 µm. [Figure 7G]Skin-resident T cells eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative IF staining of HLA-II, p16INK4a, and vimentin in young and aged skin samples. Arrows point to HLA-II-high senescent fibroblasts in the dermis. The dotted line indicates the epidermal basement membrane. Fibroblasts were preincubated with pan-HLA-DR blocking or isotype control antibodies. A 50:1 skin immune cell-to-fibroblast ratio was used (n = 4 female skin donors, age range: 43-65 years, mean age: 48.8 years, one-way ANOVA). Skin samples used in coculture experiments were pan-HCMV antigen-positive. Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpfs per sample. Bars represent the mean + SD. Scale bar, 100 μm. [Figure 7H] Skin-resident T cells eliminate senescent fibroblasts in an HLA-II-dependent manner. Quantification of HLA-II-high senescent fibroblasts in skin per hpf (n = 23 in the young skin group and n = 31 in the old skin group, Mann-Whitney U test). Fibroblasts were preincubated with pan-HLA-DR blocking or isotype control antibodies. Skin immune cell-to-fibroblast ratio of 50:1 (n = 4 female skin donors, age range: 43-65 years, mean age: 48.8 years, one-way ANOVA). Skin samples used in coculture experiments were pan-HCMV antigen-positive. Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpfs per sample. Bars represent the mean + SD. Scale bar, 100 μm. [Figure 7I]Skin-resident T cells eliminate senescent fibroblasts in an HLA-II-dependent manner. An exemplary experimental scheme for assaying autoimmune cell-induced cytotoxicity against senescent fibroblasts. Fibroblasts were pre-incubated with pan-HLA-DR blocking or isotype control antibodies. A skin immune cell-to-fibroblast ratio of 50:1 was used (n = 4 female skin donors, age range: 43-65 years, mean age: 48.8 years, one-way ANOVA). Skin samples used in coculture experiments were pan-HCMV antigen-positive. Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpfs per sample. Bar graphs show the mean + SD. Scale bar, 100 µm. [Figure 7J] Skin-resident T cells eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative immunocytochemistry (ICC) staining of cleaved caspase-3 and vimentin in normal and senescent fibroblasts after coculture with skin-derived autoimmune cells. Arrows indicate cleaved caspase-3+ apoptotic fibroblasts. Fibroblasts were preincubated with pan-HLA-DR blocking or isotype control antibodies. Skin immune cell-to-fibroblast ratio was 50:1 (n = 4 female skin donors, age range: 43-65 years, mean age: 48.8 years, one-way ANOVA). Skin samples used in coculture experiments were pan-HCMV antigen-positive. Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpfs per sample. Bars represent the mean + SD. Scale bar, 100 μm. [Figure 7K]Skin-resident T cells eliminate senescent fibroblasts in an HLA-II-dependent manner. Frequency of cleaved caspase-3+ normal and senescent fibroblasts cocultured with skin-derived autoimmune cells. Fibroblasts were preincubated with pan-HLA-DR blocking or isotype control antibodies. Skin immune cell-to-fibroblast ratio of 50:1 (n = 4 female skin donors, age range: 43-65 years, mean age: 48.8 years, one-way ANOVA). Skin samples used in coculture experiments were pan-HCMV antigen-positive. Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpfs per sample. Bars represent mean + SD. Scale bar, 100 μm. [Figure 8A] ULBP2 and HLA-II are expressed on the surface of senescent fibroblasts. (A) Gene set enrichment analysis plot of cellular senescence in replicatively induced senescent dermal fibroblasts (n=3) compared to normal dermal fibroblasts (n=4) from RNA-seq data. n=7 paired adult human dermal fibroblasts, female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test. Scale bar, 100 μm. [Figure 8B] ULBP2 and HLA-II are expressed on the surface of senescent fibroblasts. Representative IF staining of p16INK4a, ULBP2, and vimentin in aged human skin. Arrows point to ULBP2+ senescent fibroblasts in the dermis. Nuclei were stained with DAPI, and the dotted line indicates the epithelial basement membrane. n=7 pairs of adult human dermal fibroblasts, female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test. Scale bar, 100 μm. [Figure 8C]ULBP2 and HLA-II are expressed on the surface of senescent fibroblasts. Quantification of ULBP2+p16INK4a+vimentin+ fibroblasts in young and aged skin samples (n=15 in the young skin group and n=15 in the aged skin group, female skin donors, mean age: young: 20.7 years and old: 62.3 years). Cells were blindly counted and averaged across 10 randomly selected hpf per sample (Mann-Whitney U test). n=7 pairs of adult human dermal fibroblasts, female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test. Scale bar, 100 μm. [Figure 8D] ULBP2 and HLA-II are expressed on the surface of senescent fibroblasts. Quantification of dermal CD4 CTLs per hpf in HLA-II high-positive (HLA-II high senescent fibroblasts / hpf R 0.2) vs. HLA-II high-negative (HLA-II high senescent fibroblasts / hpf = 0) skin samples (n = 22 in the HLA-II high-positive group and n = 19 in the HLA-II high-negative group across young and old skin samples, Mann-Whitney U test). n = 7 pairs of adult human dermal fibroblasts, female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test. Scale bar, 100 μm. [Figure 8E] ULBP2 and HLA-II are expressed on the surface of senescent fibroblasts. Representative X-gal staining of human dermal fibroblasts irradiated with 5 J of UVA vs. sham. n=7 pairs of adult human dermal fibroblasts, female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test. Scale bar, 100 μm. [Figure 8F] ULBP2 and HLA-II are expressed on the surface of senescent fibroblasts. Representative flow cytometry histogram of ULBP2 on the surface of dermal fibroblasts 1 day after 5 J of UVA vs. sham irradiation. n=7 pairs of adult human dermal fibroblasts, female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test. Scale bar, 100 μm. [Figure 8G]ULBP2 and HLA-II are expressed on the surface of senescent fibroblasts. Representative relative fluorescence intensity (RFI) of ULBP2 on the surface of dermal fibroblasts 1 day after 5 J of UVA vs. sham irradiation. n=7 pairs of adult human dermal fibroblasts, female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test. Scale bar, 100 μm. [Figure 8H] ULBP2 and HLA-II are expressed on the surface of senescent fibroblasts. Representative flow cytometry histogram of HLA-II on the surface of dermal fibroblasts 1 day after 5 J of UVA vs. sham irradiation. n=7 pairs of adult human dermal fibroblasts, female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test. Scale bar, 100 μm. [Figure 8I] ULBP2 and HLA-II are expressed on the surface of senescent fibroblasts. Representative RFI (I) of HLA-II on the surface of dermal fibroblasts 1 day after 5 J of UVA vs. sham irradiation. n=7 pairs of adult human dermal fibroblasts, female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test. Scale bar, 100 μm. [Figure 8J] ULBP2 and HLA-II are expressed on the surface of senescent fibroblasts. Representative flow cytometry histogram of ULBP2 on the surface of dermal fibroblasts 1 day after 10 J of UVA vs. sham irradiation. (n=7 paired adult human dermal fibroblasts, female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test. Scale bar, 100 μm.) [Figure 8K] ULBP2 and HLA-II are expressed on the surface of senescent fibroblasts. Representative RFI of ULBP2 on the surface of dermal fibroblasts 1 day after 10 J of UVA vs. sham irradiation. (n=7 pairs of adult human dermal fibroblasts, female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test. Scale bar, 100 μm.) [Figure 8L]ULBP2 and HLA-II are expressed on the surface of senescent fibroblasts. Representative flow cytometry histogram of HLA-II on the surface of dermal fibroblasts 1 day after 10 J of UVA vs. sham irradiation. n=7 pairs of adult human dermal fibroblasts, female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test. Scale bar, 100 μm. [Figure 8M] ULBP2 and HLA-II are expressed on the surface of senescent fibroblasts. (L and M) Representative RFI of HLA-II on the surface of dermal fibroblasts 1 day after 10 J of UVA vs. sham irradiation. n=7 pairs of adult human dermal fibroblasts, female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test. Scale bar, 100 μm. [Figure 9A] Allogeneic skin-resident T cells selectively eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative ICC staining of cleaved caspase-3 and vimentin in normal and senescent human dermal fibroblasts after coculture with skin-derived allogeneic immune cells. Nuclei are stained with DAPI. Arrows point to cleaved caspase-3+ apoptotic fibroblasts. The percentage of IFNγ+ CD4+ T cells in the gate is shown on the flow cytometry plot. Skin samples from two independent donors were used to isolate skin-resident immune cells (female skin donors, ages 60 and 73 years). Bar graphs show the mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 9B]Allogeneic skin-resident T cells selectively eliminate senescent fibroblasts in an HLA-II-dependent manner. Frequency of cleaved caspase-3+ normal and senescent fibroblasts after 6 hours of coculture with skin-derived allogeneic immune cells using skin immune cell-to-fibroblast ratios of 0:1, 10:1, and 50:1 (n = 5 female skin donors, mean age: 50.4 years, Mann-Whitney U test). The percentage of IFNγ+ CD4+ T cells in the gated area is shown on the flow cytometry plot. Skin samples from two independent donors were used to isolate skin-resident immune cells (female skin donors, ages: 60 and 73 years). Bar graphs show mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 9C] Allogeneic skin-resident T cells selectively eliminate senescent fibroblasts in an HLA-II-dependent manner. Frequency of cleaved caspase-3+ normal or senescent fibroblasts after 6 hours of coculture with skin-derived allogeneic immune cells. Fibroblasts were preincubated with pan-HLA-DR blocking or isotype control antibodies for 18 hours. Skin-derived immune cell to fibroblast ratio was 50:1 (n = 3 female skin donors, mean age: 50.3 years, one-way ANOVA). Cells were blindly counted and averaged across 10 randomly selected hpfs per sample. The percentage of IFNγ+ CD4+ T cells in the gate is shown on the flow cytometry plot. Skin samples from two independent donors were used to isolate skin-resident immune cells (female skin donors, ages: 60 and 73 years). Bar graphs show mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 9D]Allogeneic skin-resident T cells selectively eliminate senescent fibroblasts in an HLA-II-dependent manner. RFI (Mann-Whitney U test) of HLA-II on the surface of control versus CIITA siRNA-transfected senescent fibroblasts for 48 hours. The percentage of IFNγ+ CD4+ T cells in the gate is shown in the flow cytometry plot. Skin samples from two independent donors were used to isolate skin-resident immune cells (female skin donors, ages 60 and 73 years). Bar graphs show the mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 9E] Allogeneic skin-resident T cells selectively eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative ICC staining of cleaved caspase-3 and vimentin in senescent fibroblasts after coculture with skin-derived immune cells. Nuclei are stained with DAPI. The percentage of IFNγ+ CD4+ T cells in the gate is shown in the flow cytometry plot. Skin samples from two independent donors were used to isolate skin-resident immune cells (female skin donors, ages 60 and 73 years). Bar graphs show the mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 9F] Allogeneic skin-resident T cells selectively eliminate senescent fibroblasts in an HLA-II-dependent manner. Frequency of cleaved caspase-3+ normal and senescent fibroblasts cocultured with skin-derived immune cells. Fibroblasts were transfected with control versus CIITA siRNA for 48 hours before coculture with immune cells. Skin immune cell to fibroblast ratio of 50:1 (one-way ANOVA). Skin samples from two independent donors were used to isolate skin-resident immune cells (female skin donors, ages: 60 and 73 years). The percentage of IFNγ+ CD4+ T cells in the gate is shown in the flow cytometry plot. Skin samples from two independent donors were used to isolate skin-resident immune cells (female skin donors, ages: 60 and 73 years). Bar graphs show mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 9G]Allogeneic skin-resident T cells selectively eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative flow cytometry plots of CD107a+ cells among skin-resident (CD69+) CD4+ T cells after coculture with normal versus senescent fibroblasts. The percentage of CD107a+ CD4+ T cells in the gate is shown on the flow cytometry plot. The percentage of IFNγ+ CD4+ T cells in the gate is shown on the flow cytometry plot. Skin-resident immune cells were isolated using skin samples from two independent donors (female skin donors, ages: 60 and 73 years). Bar graphs show mean + SD, Mann-Whitney U test, scale bar, 100 μm. [Figure 9H] Allogeneic skin-resident T cells selectively eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative quantification of CD107a+ cells among skin-resident (CD69+) CD4+ T cells after coculture with normal versus senescent fibroblasts. The percentage of CD107a+ CD4+ T cells in the gate is shown on the flow cytometry plot. The percentage of IFNγ+ CD4+ T cells in the gate is shown on the flow cytometry plot. Skin samples from two independent donors were used to isolate skin-resident immune cells (female skin donors, ages: 60 and 73 years). Bar graphs show mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 9I] Allogeneic skin-resident T cells selectively eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative flow cytometry plots of CD137+ cells among skin-resident CD4+ T cells after coculture with normal versus senescent fibroblasts. The percentage of CD137+ CD4+ T cells in the gate is shown on the flow cytometry plot. The percentage of IFNγ+ CD4+ T cells in the gate is shown on the flow cytometry plot. Skin-resident immune cells were isolated using skin samples from two independent donors (female skin donors, ages: 60 and 73 years). Bar graphs show mean + SD, Mann-Whitney U test, scale bar, 100 μm. [Figure 9J]Allogeneic skin-resident T cells selectively eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative quantification of CD137+ cells among skin-resident CD4+ T cells after coculture with normal versus senescent fibroblasts. The percentage of CD137+ CD4+ T cells in the gate is shown on the flow cytometry plot. The percentage of IFNγ+ CD4+ T cells in the gate is shown on the flow cytometry plot. Skin-resident immune cells were isolated using skin samples from two independent donors (female skin donors, ages: 60 and 73 years). Bar graphs show mean + SD, Mann-Whitney U test, scale bar, 100 μm. [Figure 9K] Allogeneic skin-resident T cells selectively eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative flow cytometry plots of IFNγ+ cells among skin-resident CD4+ T cells after coculture with normal versus senescent fibroblasts (K). The percentage of IFNγ+ CD4+ T cells in the gate is shown on the flow cytometry plot. Skin samples from two independent donors were used to isolate skin-resident immune cells (female skin donors, ages 60 and 73 years). Bar graphs show mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 9L] Allogeneic skin-resident T cells selectively eliminate senescent fibroblasts in an HLA-II-dependent manner. Representative quantification of IFNγ+ cells among skin-resident CD4+ T cells after co-culture with normal versus senescent fibroblasts. The percentage of IFNγ+ CD4+ T cells in the gate is shown on the flow cytometry plot. Skin samples from two independent donors were used to isolate skin-resident immune cells (female skin donors, ages: 60 and 73 years). Bar graphs show the mean + SD, Mann-Whitney U test. Scale bar, 100 μm. [Figure 10A]HCMV DNA and RNA are upregulated in aged human skin. Quantitative PCR for HCMV DNA detection in HCMV-infected versus mock-infected human fetal fibroblasts using HCMV UL83 primers. Data are expressed as the ratio of GAPDH expression (n=4 per group). Bar graphs show mean + SD, Mann-Whitney U test. [Figure 10B] HCMV DNA and RNA are upregulated in aged human skin. Quantitative PCR for HCMV DNA detection in young versus aged human skin samples using HCMV UL83 primers. Data are presented as the ratio of GAPDH expression (n=21 for the young skin group and n=30 for the aged skin group, female skin donors, mean age: young: 23.3 years and aged: 62.5 years). Bar graphs show mean + SD, Mann-Whitney U test. [Figure 10C] HCMV DNA and RNA are upregulated in aged human skin. Quantification of HCMV RNA in situ hybridization (ISH) numbers in young versus aged human skin samples. HCMV RNA ISH-positive signals were blindly counted and averaged across three hpf with HCMV RNA ISH signals if any were detected per skin sample (n=21 in the young skin group and n=30 in the aged skin group, female skin donors, mean age: young: 23.3 years and aged: 62.5 years). Bar graphs show mean + SD, Mann-Whitney U test. [Figure 10D] HCMV DNA and RNA are upregulated in aged human skin. Representative images of HCMV RNA ISH (arrows) immunohistochemical staining and vimentin IF staining in the dermis of young and aged skin (scale bar, 100 μm). Bar graphs show mean + SD, Mann-Whitney U test. [Figure 10E]HCMV DNA and RNA are upregulated in aged human skin. Representative images of HCMV RNA ISH with IF staining of vimentin in dermal fibroblasts from young and aged skin. Arrows point to HCMV RNA+ signals in fibroblasts (scale bar, 10 μm). The HCMV RNA ISH probe was designed to detect UL123 (IE1) transcripts. Nuclei were stained with DAPI. Bar graphs show mean + SD, Mann-Whitney U test. [Figure 10F] HCMV DNA and RNA are upregulated in aged human skin. Representative IF staining of pan-HCMV antigen (Ag) and vimentin in aged human skin (scale bar, 100 μm). Bar graphs show mean + SD, Mann-Whitney U test. [Figure 10G] HCMV DNA and RNA are upregulated in aged human skin. Pie chart showing the percentage of young and aged human skin samples with pan-HCMV Ag+ cells in the dermis (n=23 for the young skin group and n=31 for the aged skin group, chi-square test). Bar graphs show mean + SD, Mann-Whitney U test. [Figure 10H] HCMV DNA and RNA are upregulated in aged human skin. HCMV DNA (UL83) expression in aged skin samples with pan-HCMV Ag+ cells (pan-HCMV Ag positive, n=21) versus aged skin samples without detectable pan-HCMV Ag+ cells (pan-HCMV Ag negative, n=10). Bar graphs show mean + SD, Mann-Whitney U test. [Figure 11A]Senescent fibroblasts express HCMV-gB antigen. (A) Representative IF staining of p16INK4a, HCMV-gB, and vimentin in aged human skin. Arrows indicate HCMV-gB+ senescent fibroblasts. The dotted line indicates the epidermal basement membrane. Data are expressed as a ratio of GAPDH expression. Nuclei are stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Bar graphs show the mean + SD. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. n = 7 pairs of adult human dermal fibroblasts (female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test). Scale bar, 100 μm. [Figure 11B] Senescent fibroblasts express HCMV-gB antigen. Quantification of HCMV-gB+p16INK4a+ fibroblasts in the dermis per hpf (n = 23 in the young skin group and n = 31 in the aged skin group, Mann-Whitney U test). Data are expressed as the ratio of GAPDH expression. Nuclei are stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Bar graphs show the mean + SD. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. n = 7 pairs of adult human dermal fibroblasts (female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test). Scale bar, 100 μm. [Figure 11C] Senescent fibroblasts express HCMV-gB antigen. Representative ICC staining of HCMV-gB and vimentin in normal and senescent fibroblasts. Arrows indicate HCMV-gB+ fibroblasts. Data are expressed as a ratio of GAPDH expression. Nuclei are stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Bar graphs show the mean + SD. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. n = 7 pairs of adult human dermal fibroblasts (female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test). Scale bar, 100 μm. [Figure 11D]Senescent fibroblasts express HCMV-gB antigen. Frequency of HCMV-gB+ normal fibroblasts versus senescent fibroblasts. Data are expressed as the ratio of GAPDH expression. Nuclei are stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Bar graphs show the mean + SD. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. n = 7 pairs of adult human dermal fibroblasts (female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test). Scale bar, 100 μm. [Figure 11E] Senescent fibroblasts express HCMV gB antigen. Quantitative PCR for HCMV DNA detection in normal and replication-induced senescent fibroblasts using HCMV UL83 primers. Data are expressed as a ratio of GAPDH expression. Data are expressed as a ratio of GAPDH expression. Nuclei are stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Bar graphs show the mean + SD. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. n = 7 pairs of adult human dermal fibroblasts (female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test). Scale bar, 100 μm. [Figure 11F] Senescent fibroblasts express HCMV-gB antigen. Frequency of HCMV-gB+ fibroblasts 5 days after UVA vs. sham UVA irradiation. Data are expressed as the ratio of GAPDH expression. Nuclei are stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Bar graphs show the mean + SD. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. n = 7 pairs of adult human dermal fibroblasts (female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test). Scale bar, 100 μm. [Figure 11G]Senescent fibroblasts express HCMV gB antigen. Quantitative PCR for HCMV DNA detection in fibroblasts 1 day after 5-UVA vs. sham UVA irradiation using HCMV UL83 primers. Data are expressed as the ratio of GAPDH expression. Nuclei are stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Bar graphs show the mean + SD. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. n = 7 pairs of adult human dermal fibroblasts (female skin donors, age range: 43-70 years, mean age: 53.7 years, paired t-test). Scale bar, 100 μm. [Figure 12] Detection of HCMV-IE1 / IE2 in aged human skin. A, Representative IF staining of HCMV-IE1 / IE2(Ag) and vimentin in young and aged human skin samples. Nuclei are stained with DAPI. B, Pie chart showing the percentage of skin samples with HCMV-IE1 / IE2+ cells in the dermis. n = 23 in the young skin group and n = 31 in the aged skin group. Chi-square test. Scale bar, 100 μm. [Figure 13] HCMV-gB is localized to early endosomes in senescent fibroblasts. A. Representative ICC staining of HCMV-gB, Rab5, and vimentin in HCMV-infected versus mock-infected human fetal fibroblasts. The arrow indicates colocalization of HCMV-gB and Rab5 signals in fibroblasts. B. Representative ICC staining of HCMV-gB, Rab5, and vimentin in normal and replication-induced senescent fibroblasts. The arrow indicates colocalization of HCMV-gB and Rab5 signals in fibroblasts. C. Representative ICC staining of HCMV-gB, Rab5, and vimentin in fibroblasts 5 days after 5 J of UVA or mock UVA irradiation. The arrow indicates colocalization of HCMV-gB and Rab5 signals in fibroblasts. Nuclei were stained with DAPI. Scale bar, 100 μm. [Figure 14A]HCMV-gB antigen activates skin-resident CD4 CTLs. Pie chart showing gene usage of TRBV06-05-expressing T cell clones in six HLA-DRB1*07:01+ elderly skin samples (female skin donors, age range: 59-74 years, mean age: 63.3 years). The percentage of CD4+ T cells in each quadrant is shown on a flow cytometry plot. Stimulation with phorbol-12-myristate-13-acetate and ionomycin (PMA / ion.) was used as a positive control (n = 18 for negative control, n = 18 for poly(I:C), n = 18 for HCMV-gB, n = 18 for HCMV-gB + poly(I:C), n = 16 for PMA / ionomycin, n = 12 for HCMV-gH, and n = 12 for HCMV-gH + poly(I:C) groups; one-way ANOVA, Dunnett's multiple comparison test). Skin samples from five independent donors were used to isolate skin-resident immune cells (female skin donors, age range: 48-73 years, mean age: 57.6 years). Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. Bars represent the mean + SD. Scale bar, 100 μm. [Figure 14B]HCMV-gB antigen activates skin-resident CD4 CTLs. Representative flow cytometry plots of HCMV-gB tetramer + perforin + CD4 + T cells among skin-resident CD4 + T cells stained with HCMV-gB tetramer versus control tetramer. Skin-resident CD8 + T cells stained with HCMV-gB tetramer are shown as a negative control. The percentage of tetramer + perforin + cells is indicated by the gate on the flow cytometry plot. The percentage of CD4 + T cells in each quadrant is shown on the flow cytometry plot. Stimulation with phorbol-12-myristate-13-acetate and ionomycin (PMA / ion.) was used as a positive control (n = 18 for negative control, n = 18 for poly(I:C), n = 18 for HCMV-gB, n = 18 for HCMV-gB + poly(I:C), n = 16 for PMA / ionomycin, n = 12 for HCMV-gH, and n = 12 for HCMV-gH + poly(I:C) groups; one-way ANOVA, Dunnett's multiple comparison test). Skin samples from five independent donors were used to isolate skin-resident immune cells (female skin donors, age range: 48-73 years, mean age: 57.6 years). Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. Bars represent the mean + SD. Scale bar, 100 μm. [Figure 14C]HCMV-gB antigen activates skin-resident CD4 CTLs. Quantification of HCMV-gB+ perforin+ CD4+ T cells as a percentage of total skin-resident CD4+ T cells (n=9 skin samples per group, female skin donors, mean age: young: 19.3 years and elderly: 63.3 years, Mann-Whitney U test). The percentage of CD4+ T cells in each quadrant is shown on the flow cytometry plot. Stimulation with phorbol-12-myristate-13-acetate and ionomycin (PMA / ion.) was used as a positive control (n = 18 for negative control, n = 18 for poly(I:C), n = 18 for HCMV-gB, n = 18 for HCMV-gB + poly(I:C), n = 16 for PMA / ionomycin, n = 12 for HCMV-gH, and n = 12 for HCMV-gH + poly(I:C) groups; one-way ANOVA, Dunnett's multiple comparison test). Skin samples from five independent donors were used to isolate skin-resident immune cells (female skin donors, age range: 48-73 years, mean age: 57.6 years). Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. Bars represent the mean + SD. Scale bar, 100 μm. [Figure 14D]HCMV-gB antigen activates skin-resident CD4 CTLs. Representative ICC staining of HCMV-gB, cleaved caspase-3, and vimentin in normal and senescent fibroblasts after coculture with skin-derived autoimmune cells. Arrows indicate HCMV-gB apoptotic fibroblasts. The percentage of CD4 T cells in each quadrant is shown on the flow cytometry plot. Stimulation with phorbol-12-myristate-13-acetate and ionomycin (PMA / ion.) was used as a positive control (n = 18 for negative control, n = 18 for poly(I:C), n = 18 for HCMV-gB, n = 18 for HCMV-gB + poly(I:C), n = 18 for PMA / ionomycin, n = 16 for HCMV-gH, n = 12 for HCMV-gH + poly(I:C) groups, n = 12 for HCMV-gH, and n = 12 for HCMV-gH + poly(I:C) groups; one-way ANOVA, Dunnett's multiple comparison test). Skin samples from five independent donors were used to isolate skin-resident immune cells (female skin donors, age range: 48-73 years, mean age: 57.6 years). Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. Bar graphs show the mean + SD. Scale bar, 100 μm. [Figure 14E]HCMV-gB antigen activates skin-resident CD4+ CTLs. Frequency of cleaved caspase-3+HCMV-gB+(E) normal and senescent fibroblasts cocultured with skin-derived autoimmune cells. Fibroblasts were preincubated with pan-HLA-DR blocking or isotype control antibodies. Skin immune cell-to-fibroblast ratio was 50:1 (n=4 individual donors, female skin donors, age range: 43-65 years, mean age: 48.8 years, one-way ANOVA). The percentage of CD4+ T cells in each quadrant is shown on a flow cytometry plot. Stimulation with phorbol-12-myristate-13-acetate and ionomycin (PMA / ion.) was used as a positive control (n = 18 for negative control, n = 18 for poly(I:C), n = 18 for HCMV-gB, n = 18 for HCMV-gB + poly(I:C), n = 16 for PMA / ionomycin, n = 12 for HCMV-gH, and n = 12 for HCMV-gH + poly(I:C) groups; one-way ANOVA, Dunnett's multiple comparison test). Skin samples from five independent donors were used to isolate skin-resident immune cells (female skin donors, age range: 48-73 years, mean age: 57.6 years). Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. Bars represent the mean + SD. Scale bar, 100 μm. [Figure 14F]HCMV-gB antigen activates skin-resident CD4+ CTLs. Frequency of cleaved caspase-3-HCMV-gB+ (F) normal and senescent fibroblasts cocultured with skin-derived autoimmune cells. Fibroblasts were preincubated with pan-HLA-DR blocking or isotype control antibodies. Skin immune cell-to-fibroblast ratio was 50:1 (n=4 individual donors, female skin donors, age range: 43-65 years, mean age: 48.8 years, one-way ANOVA). The percentage of CD4+ T cells in each quadrant is shown on a flow cytometry plot. Stimulation with phorbol-12-myristate-13-acetate and ionomycin (PMA / ion.) was used as a positive control (n = 18 for negative control, n = 18 for poly(I:C), n = 18 for HCMV-gB, n = 18 for HCMV-gB + poly(I:C), n = 16 for PMA / ionomycin, n = 12 for HCMV-gH, and n = 12 for HCMV-gH + poly(I:C) groups; one-way ANOVA, Dunnett's multiple comparison test). Skin samples from five independent donors were used to isolate skin-resident immune cells (female skin donors, age range: 48-73 years, mean age: 57.6 years). Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. Bars represent the mean + SD. Scale bar, 100 μm. [Figure 14G]HCMV-gB antigen activates skin-resident CD4 CTLs. Representative flow cytometry plots of skin-resident IFNγ+ perforin+ CD4+ T cells after stimulation with recombinant HCMV-gB, HCMV-gH, and / or poly(I:C). The percentage of CD4+ T cells in each quadrant is shown on the flow cytometry plot. Stimulation with phorbol-12-myristate-13-acetate and ionomycin (PMA / ion.) was used as a positive control (n = 18 for negative control, n = 18 for poly(I:C), n = 18 for HCMV-gB, n = 18 for HCMV-gB + poly(I:C), n = 16 for PMA / ionomycin, n = 12 for HCMV-gH, and n = 12 for HCMV-gH + poly(I:C) groups; one-way ANOVA, Dunnett's multiple comparison test). Skin samples from five independent donors were used to isolate skin-resident immune cells (female skin donors, age range: 48-73 years, mean age: 57.6 years). Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. Bar graphs show the mean + SD. Scale bar, 100 μm. [Figure 14H]HCMV-gB antigen activates skin-resident CD4 CTLs. G and H) Representative quantification of skin-resident IFNγ+ perforin+ CD4+ T cells after stimulation with recombinant HCMV-gB, HCMV-gH, and / or poly(I:C). The percentage of CD4+ T cells in each quadrant is shown on the flow cytometry plot. Stimulation with phorbol-12-myristate-13-acetate and ionomycin (PMA / ion.) was used as a positive control (n = 18 for negative control, n = 18 for poly(I:C), n = 18 for HCMV-gB, n = 18 for HCMV-gB + poly(I:C), n = 16 for PMA / ionomycin, n = 12 for HCMV-gH, and n = 12 for HCMV-gH + poly(I:C) groups; one-way ANOVA, Dunnett's multiple comparison test). Skin samples from five independent donors were used to isolate skin-resident immune cells (female skin donors, age range: 48-73 years, mean age: 57.6 years). Nuclei were stained with DAPI. Cells were blindly counted and averaged across 10 randomly selected hpf per sample. Skin samples used for fibroblast isolation were pan-HCMV antigen positive. Bar graphs show the mean + SD. Scale bar, 100 μm. [Figure 15] Butyrate and pentanoate enhance CD4 CTL-mediated clearance of senescent cells. A. Frequency of cleaved caspase-3+ senescent fibroblasts after 6 hours of coculture with cutaneous T cells pre-incubated for 2 days with 5 mM of each SCFA, using a 50:1 cutaneous T cell to fibroblast ratio. All data are presented as mean + SD. n = 3 individuals. B. Effect of treatment with each SCFA (5 mM) on CD4+ T cell cytotoxicity. Flow cytometry analysis of perforin and CD107a expression on CD4+ T cells exposed to each SCFA for 24 hours. n = 2 individuals. All data are presented as mean + SD. DETAILED DESCRIPTION OF THE INVENTION

[0014] Accumulating evidence indicates that genetic or pharmacological approaches to eliminate senescent cells from aging tissues can restore tissue homeostasis and result in increased healthy lifespan in mice. 3、5 However, current genetic and pharmacological approaches produce substantial side effects and lack long-term durability. 1、4 Considering that senescent cells produce SASP, their immunogenic phenotype marks them as potential targets for surveillance and clearance by the immune system. However, in experimental mouse models, immune clearance of senescent cells is inhibited by immunomodulatory molecules expressed by senescent cells and immunosuppressive factors in their microenvironment. 6~9 More importantly, it is unclear how immunity to senescent cells is regulated in humans.

[0015] Senescent cell development and phenotype differ fundamentally between mice and humans, including the role of telomere shortening and oxidative stress in the induction of cellular senescence. 10~14 Notable limitations of experimental mouse models for studying aging relate to their inability to fully capture the range of immune responses against senescent cells due to their pathogen exposure and the lack of evolutionarily distinct immune surveillance mechanisms. 15~17 Therefore, the mechanisms of senescent cell clearance in humans have been explored, and a novel commensal virome-immune axis that prevents the accumulation of senescent cells in aging skin has been discovered.

[0016] These findings reveal a previously unknown role for CD4 CTLs in the clearance of senescent cells undergoing cytomegalovirus reactivation. These findings have important implications for understanding how immunity to senescent cells is regulated in humans. Senescent cells, primarily dermal fibroblasts, accumulate in elderly skin compared with younger skin; however, their numbers do not linearly correlate with aging. Among possible explanations for the age-independent accumulation of senescent cells, CD4 CTLs were identified as key regulators of senescent cell proliferation in aged skin. CD4 CTLs recognize and eliminate senescent fibroblasts, at least in part, by targeting the HCMV-gB antigen, highlighting the commensal-like function of HCMV in the immune surveillance of senescent cells in immunocompetent hosts.

[0017] Cytotoxic lymphocytes utilize the perforin / granzyme pathway to kill virus-infected and tumor cells 40 Unexpectedly, CD4 + Histological analysis revealed that T cells were a prominent population of perforin-expressing cytotoxic lymphocytes in aged skin, and their frequency in the dermis was negatively correlated with the accumulation of senescent cells in aged human skin. Increased expression of the CXCL9 chemokine by senescent keratinocytes explains the high frequency of dermal CD4 CTLs in aged skin, suggesting that the cutaneous chemokine milieu is a major determinant of CD4 CTL infiltration in aged skin.

[0018] As shown herein, replicative and UVA-induced senescent human fibroblasts upregulate HLA-II and HCMV-gB expression. Endogenous HCMV-derived gB is sorted into endosomes and is associated with CD4 expression on HLA-II. + Presented to T cells 41 Thus, human skin-derived CD4 CTLs express HCMV-gB in an HLA-II-dependent manner. + Senescent fibroblasts were specifically eliminated.

[0019] CD4 CTLs can be detected during viral infections in humans, and their direct antiviral effector functions help control infection. 27 28 In particular, the high presence of circulating CD4 CTLs is a characteristic of centenarians, who live longer than 110 years in good health due to delayed onset and reduced incidence of age-related diseases. 29 Without wishing to be bound by theory, in addition to fighting infection, CD4 CTLs contribute to the elimination of senescent cells and their associated diseases in humans over 110 years old, achieving exceptional longevity.

[0020] HCMV is an infectious cause of birth defects and can cause severe morbidity in severely immunocompromised individuals. 42、43 , which is a widespread herpesvirus that, in immunocompetent hosts, establishes a lifelong latent infection in a large portion of the human population without symptoms. 44 HCMV produces several immunodominant antigens that profoundly affect the adaptive immune repertoire in healthy individuals during aging. 45 HCMV-specific CD4 CTLs exhibit high cytotoxicity while producing low amounts of cytokines, allowing them to effectively combat HCMV reactivation while minimizing tissue inflammation. 30 Although HCMV is known to establish latency in bone marrow cells, fibroblasts have been found to predominate in the pool of HCMV-infected cells in vivo. 46、47 Thus, HCMV is detected in dermal fibroblasts of normal human skin.

[0021] As shown herein, HCMV is reactivated upon induction of cellular senescence. Without wishing to be bound by theory, this may explain how CD4 CTLs, and possibly CD8 CTLs and NK cells, can detect senescent fibroblasts in a mixture of healthy cells infected with HCMV as a commensal virus. This finding supports the notion that HCMV induces p16 INK4a This is supported by the observation that upregulating 31HCMV reactivation during replication and UVA-induced senescence, coupled with induction of HLA-II and stress ligands on senescent cells, allows HCMV-specific CD4 CTLs to effectively target and eliminate senescent cells. Macrophages, NK cells, and CD8 + Other innate and adaptive immune cell types, including T cells, also play important roles in the clearance of senescent cells 48、49、50~53 Considering that senescent fibroblasts highly express ULBP2, a ligand for activating the NKG2D receptor widely expressed on cytotoxic lymphocytes, it is likely that innate and adaptive immune cells, along with CD4 CTLs, are involved in the clearance of senescent cells in the skin.

[0022] The absence of HCMV-gB expression and lytic infection in senescent fibroblasts in the presence of low immediate-early 1 (IE1) / immediate-early 2 (IE2) expression may represent an interesting biology associated with abortive replication cycles. For example, senescent cells may persist in the face of HCMV reactivation and fail to enter the lytic phase despite the expression of late viral proteins, which may then inhibit lytic virus spread. HCMV-infected adult fibroblasts, like uninfected embryonic fibroblasts, upregulate HLA-II during cellular senescence, suggesting that cellular senescence can overcome the suppression of HLA-II by HCMV, which undergoes reactivation in senescent cells. Interestingly, previous reports have shown that herpesviruses preferentially downregulate HLA-I expression rather than HLA-II, resulting in a prominent role for CD4 CTLs in the clearance of virus-infected cells. 54、55 Thus, cellular senescence may function as a natural antiviral defense mechanism by inhibiting the spread of lytic viruses while creating immunogenic targets for clearance. 56、57 This protective function of cellular senescence as an antiviral mechanism highlights a prominent aspect of immune-virome interactions that promote homeostasis in virus-colonized organs.

[0023] These findings provide new insights into the complex human immune system-commensal virome interactome. Without wishing to be bound by theory, these results suggest that the human immune system has evolved to establish a symbiotic relationship with HCMV that prevents the accumulation of senescent cells during aging. Thus, HCMV may have beneficial effects on human health mediated by competent antiviral T cell immunity. Described herein are methods involving vaccination against commensal HCMV antigens to enhance anti-HCMV T cell immunity and reduce the risk, delay the onset, and / or slow the progression of aging and aging-associated diseases. Without wishing to be bound by theory, it is believed that the vaccine increases senescent cell clearance.

[0024] How to induce immunity The methods described herein include methods for treating or reducing the risk of developing aging-related diseases or conditions. Also described herein are methods for delaying the onset or slowing the progression of aging in a subject, as well as methods for reducing the level of senescent fibroblasts in a subject. The methods include administering one or more doses of the vaccine composition described herein to a subject, for example, a subject in need thereof. The vaccine aims to induce T cell immunity against commensal viruses that have already infected tissues, and not to prevent or eliminate infection, but rather to use the presence of the virus in all cells to enhance the detection of senescent cells and their elimination by T cells.

[0025] The composition is administered in an effective amount. An "effective amount" is an amount sufficient to achieve a beneficial or desired result. For example, an effective amount is an amount that achieves a desired therapeutic effect, e.g., the amount necessary to treat a disease or reduce the risk of developing a disease or disease symptoms (also referred to as a therapeutically effective amount or a prophylactically effective amount, respectively). An effective amount may be administered in one or more administrations, applications, or dosages. The therapeutically effective amount (i.e., effective dosage) of a therapeutic compound will depend on the therapeutic compound selected. The composition may be administered once or more times per week, including once or more times per day to once every other day. It will be apparent to one of skill in the art that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatment history, the subject's general health and / or age, and other diseases present, can affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a therapeutic compound described herein can include a single treatment or a series of treatments. For example, the method may involve administering a first dose, followed by administering a second dose at a later time (e.g., a "booster" dose), e.g., 1, 2, 4, 6, 8, 12, 18, 24, or 52 weeks later.

[0026] The dosage, toxicity, and therapeutic efficacy of therapeutic compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions that exhibit a high therapeutic index are preferred. Compositions that exhibit toxic side effects may be used, but care must be taken to minimize or reduce the side effects.

[0027] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for human use.The dosage of such compounds is preferably within a circulating concentration range that includes the ED50 with little or no toxicity.Dosages may vary within this range depending on the dosage form used and the route of administration utilized.For any composition used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays.Doses may also be formulated in animal models.Such information can be used to more accurately determine useful doses in humans.

[0028] Many conditions and diseases are associated with aging. For example, aging is considered a risk factor for cancer and other diseases. See, for example, Laconi, et al. Br J Cancer. 2020 Mar;122(7):943-952. Aging-associated conditions include graying of hair, hearing loss, cataracts, frailty, and sarcopenia. Aging-associated diseases can include cancer, cardiovascular disease, neurodegenerative disease, kidney disease (e.g., chronic kidney disease), autoimmune disease, arthritis (e.g., osteoarthritis), osteoporosis, macular degeneration, chronic obstructive pulmonary disease (COPD), glaucoma, obesity, fibrosis (e.g., liver fibrosis), cirrhosis (e.g., liver cirrhosis), hepatic steatosis, and diabetes. Non-limiting examples of such cancers include osteosarcoma, breast cancer, prostate cancer, colorectal cancer, lung cancer, melanoma, kidney cancer, lymphoma, uterine cancer, pancreatic cancer, non-melanoma skin cancer, and bladder cancer. Non-limiting examples of neurodegenerative diseases include dementia, ataxia, Huntington's disease, motor neuron disease, or tau-mediated neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, or progressive supranuclear palsy). Non-limiting examples of cardiovascular diseases include atherosclerosis, idiopathic pulmonary fibrosis, coronary heart disease, congestive heart failure, coronary artery disease, peripheral artery disease, valvular heart disease, arrhythmias (e.g., arterial fibrillation), ischemic cardiomyopathy, hypertension, and stroke. Non-limiting examples of autoimmune diseases include multiple sclerosis, Crohn's disease, rheumatoid arthritis, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, and systemic lupus erythematosus. Non-limiting examples of fibrosis include systemic sclerosis, scleroderma, idiopathic pulmonary fibrosis, and interstitial lung disease.

[0029] The methods can also include any suitable method for measuring the onset or progression of any of the diseases described herein, for example, any of the methods described herein can include any suitable method for measuring the onset or progression of cancer, fibrosis, cardiovascular disease, and neurodegenerative disease.

[0030] The present method may also include, for example, administering one or more other treatments known in the art for treating an aging-related disease or condition, or a treatment for reducing the risk of developing an aging-related disease or condition, to a subject with the disease or condition. For example, a combination treatment of the compositions described herein with standard treatment for any of the conditions or diseases described herein may be used in combination with the present method. In some embodiments, these agents enhance antigen presentation (innate signaling), while the compositions of the present invention enhance antigen recognition by T cells.

[0031] Commensal HCMV vaccine Also described herein are compositions that can be used to induce a T cell-based immune response against cytomegalovirus, thereby treating or reducing the risk of developing an aging-related disease or condition. Several CMV vaccines have been developed, including live-attenuated, plasmid DNA, viral vector, and subunit vaccines (see, e.g., Rieder and Steininger, Clin Microbiol Infect 2014;20(Suppl.5):95-102; McVoy Clin Infect Dis 2013;57(Suppl.4):S196-9). In some embodiments, the compositions may include a live CMV vaccine or a live-attenuated CMV vaccine. Many CMV vaccines include CMV glycoprotein B (gB) or an antigenic portion thereof, delivered either as a protein or as a nucleic acid encoding the protein (e.g., plasmid DNA or mRNA). Non-limiting examples of CMV vaccines include CMVPepVax, Chiron gB, ineffective infectious single-cycle (DISC) V160 vaccine, modified vaccinia virus Ankara (MVA) vaccine vectors for expressing glycoprotein B, phosphoprotein 65, and all five subunits of the pentameric complex, and mRNA platforms encoding gB, pp65, IE1, or the pentameric complex. See, e.g., Cui and Snapper, Hum Vaccin Immunother. 2019;15(11):2673-2683.

[0032] In some embodiments, the compositions can include virus-like particles containing multiple proteins, e.g., glycoprotein B from commensal human cytomegalovirus, e.g., CMV strains such as AD169, Towne, Toledo, PH, TR, FIX, VR1814, Merlin, and TB40 / E. See, e.g., Murphy et al. Proc Natl Acad Sci USA. 2003 Dec 9;100(25):14976-81 and Wilkinson et al. Med Microbiol Immunol. 2015;204(3):273-284. Non-limiting examples of antigenic portions of CMV glycoprotein B include antigenic domains (AD) 1, AD-2, AD-3, AD-4, AD-5, and AD-6. For example, AD-1 comprises approximately 80 amino acids between positions 560 and 640 of glycoprotein B (e.g., of HCMV strain AD169). In some embodiments, AD-2 comprises at least two distinct regions between amino acids 50 and 77 of glycoprotein B. AD-3 comprises the intraluminal / intraviral portion of glycoprotein B. AD-4 comprises the region between amino acids 133-343 of glycoprotein B. AD-5 comprises discontinuous domains from amino acids 121-132 and 344-438. AD-6 comprises amino acids 648-697 of glycoprotein B. See, e.g., Poetzsch, et al. PLoS Pathog. 2011 Aug;7(8):e1002172 and Gomes et al. at Commun. 2023 Feb 23;14(1):1041. Non-limiting examples of CMV vaccines comprising CMV glycoprotein B or an antigenic portion thereof include vaccines comprising soluble recombinant glycoprotein B, which is a truncated version of glycoprotein B lacking the transmembrane domain (e.g., Chiron gB), and vaccines comprising recombinant trimeric glycoprotein B. See, e.g., Cui and Snapper, Hum Vaccin Immunother. 2019;15(11):2673-2683.

[0033] In some embodiments, the composition comprises multiple antigenic peptides derived from (i.e., including fragments thereof, i.e., including consecutive amino acids thereof) a protein from commensal human cytomegalovirus, such as glycoprotein B. The peptides can be derived from any antigenic protein in the virus. In some embodiments, the peptides are derived from glycoprotein B (e.g., glycoprotein antigenic domains described herein). CMV glycoprotein B sequences include APB97351.1, ABQ23592.1, QTT59567.1, QTT59229.1, QTT59398.1, QTT59064.1, and APA45814.1. In some embodiments, the composition comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more different peptides (i.e., peptides with different sequences). In some embodiments, the composition contains at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more different peptides from each viral strain, and includes peptides from two or more viral strains.

[0034] In some embodiments, the peptides are of a length optimized for MHC1 / MHCII presentation, e.g., 9-30 amino acids, e.g., 12-25, 12-18, 12-16, 13-16, 14-16, or 15 amino acids. The peptide sequences can be, for example, synthetic long overlapping peptides, e.g., bioinformatically identified to predict antigenicity, and / or generated using moving windows of overlapping peptides to cover the entire protein, e.g., 15-amino acid peptides with 10-amino acid overlaps. In some embodiments, overlapping synthetic long peptides (SLPs) are used (Zom et al., Cancer Immunol Res. 2014 Aug;2(8):756-64). The composition can include multiple peptides derived from one or more (e.g., multiple) different viral strains. The peptides are preferably synthetic, and methods for synthesizing peptides, including solution-phase techniques and solid-phase peptide synthesis (SPPS), are known in the art. See, for example, Petrou and Sarigiannis, Ch. 1 - Peptide synthesis: Methods, trends, and challenges, In: Editor(s): Sotirios Koutsopoulos, Peptide Applications in Biomedicine, Biotechnology and Bioengineering, Woodhead Publishing, 2018, pages 1-21, and Chandrudu et al., Molecules 2013, 18, 4373-4388.

[0035] In some embodiments, the compositions may include multiple DNA plasmids and / or RNA replicons containing nucleotide sequences for expressing proteins, e.g., proteins or antigenic peptides derived from glycoprotein B (i.e., comprising a fragment of glycoprotein B, i.e., comprising consecutive amino acids), from commensal human cytomegalovirus, e.g., CMV strains such as AD169, Towne, Toledo, PH, TR, FIX, VR1814, Merlin, and TB40 / E.

[0036] In some embodiments, the compositions may comprise multiple viral vectors that have been engineered to express proteins, e.g., proteins or antigenic peptides derived from glycoprotein B (i.e., comprising a fragment, i.e., comprising consecutive amino acids), from commensal human cytomegalovirus, e.g., CMV strains such as AD169, Towne, Toledo, PH, TR, FIX, VR1814, Merlin, and TB40 / E. Viral vectors for use in the methods and compositions include recombinant retroviruses, adenoviruses, adeno-associated viruses, alphaviruses, and lentiviruses.

[0037] T cell adjuvants The composition may also contain an adjuvant to increase T cell response. For example, such an adjuvant may contain a short-chain fatty acid. Non-limiting examples of short-chain fatty acids include acetate, propionate, butyrate, valerate, formate, isobutyrate, isovalerate, and 2-methylbutanoate.

[0038] In some embodiments, an oil-in-water emulsion may be included, such as an oil-in-water emulsion containing squalene (4.3%) in citrate buffer with the stabilizing nonionic surfactants Tween 80 (0.5%) and Span 85 (0.5%) (e.g., MF59®). In some embodiments, nanoparticles that enhance T cell responses may be included, such as those described in Stano et al., Vaccine (2012) 30:7541-6 and Swaminathan et al., Vaccine (2016) 34:110-9. See also Panagioti et al., Front. Immunol., 16 February 2018; doi.org / 10.3389 / fimmu.2018.00276. Alternatively or additionally, adjuvants including Toll-like receptor agonists (e.g., Toll-like receptor 9 agonists such as PF03512676), poly ICLC (carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA), imiquimod, resiquimod (R-848), CpG oligodeoxynucleotides and formulations (IC31, QB10), AS04 (aluminum salt formulated with 3-O-desacyl-4'-monophosphoryl lipid A (MPL)), AS01 (MPL and saponin QS-21), MPLA, STING agonists, other TLR agonists, GM-CSF, Fms-like tyrosine kinase-3 ligand (Flt3L), and / or IFA (incomplete Freund's adjuvant) may also be used. See, for example, Khong and Willem, Journal for ImmunoTherapy of Cancer 4:56 (2016), Coffman et al., Immunity. 2010 Oct 29;33(4):492-503, Martins et al., BioMedicine 3:67-78, 2016, and Del Giudice, Seminars in Immunology, 2018, doi.org / 10.1016 / j.smim.2018.05.001.

[0039] composition Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration.

[0040] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration, examples of which include parenteral, e.g., intravenous, intradermal, subcutaneous, intratumoral, intramuscular, or subcutaneous administration.

[0041] Methods for formulating suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005 and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, intramuscular, or subcutaneous administration may contain the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate, and an agent for adjusting osmotic pressure such as sodium chloride or dextrose. pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0042] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble), or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0043] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the above-listed ingredients as needed, and then sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients that are required from the above-listed ingredients.For the sterile powder used to prepare sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, and obtain the powder of active ingredient plus any additional desired ingredients from the solution that has been previously sterilized and filtered.

[0044] In one embodiment, the therapeutic compound is prepared with a carrier that protects the therapeutic compound against rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may be used. Such formulations may be prepared using standard techniques or may be commercially obtained, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells using monoclonal antibodies against cellular antigens) may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0045] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0046] subject The vaccine compositions described herein can be used to enhance immunity against senescent cells in a subject. In some embodiments, the subject does not have a senescence-associated disease (e.g., does not have any of the cancers described herein). In some embodiments, the subject is at high risk (i.e., has a risk above that of the general population) for developing a senescence-associated disease. For example, the subject can be an adult. In some embodiments, the subject is 40 years of age or older. For example, the subject can be 45, 50, 55, 60, 65, 70, 75, 80, or 85 years of age or older.

[0047] In some embodiments, the subject is not an infant, e.g., the subject is not under the age of 1. In some embodiments, the subject does not have congenital CMV (cCMV) disease. In some embodiments, the subject is not pregnant. In some embodiments, the subject is not at risk of transmitting cytomegalovirus to a fetus.

[0048] In some embodiments, the subject has a senescence-associated disease (e.g., any of the senescence-associated diseases described herein). In some embodiments, the subject has one or more of cancer (e.g., any of the cancers described herein), cardiovascular disease (e.g., any of the cardiovascular diseases described herein), neurodegenerative disease (e.g., any of the neurodegenerative diseases described herein), renal disease (e.g., any of the renal diseases described herein), autoimmune disease (e.g., any of the autoimmune diseases described herein), arthritis (e.g., osteoarthritis), osteoporosis, macular degeneration, chronic obstructive pulmonary disease (COPD), glaucoma, obesity, fibrosis (e.g., liver fibrosis), cirrhosis (e.g., liver cirrhosis), hepatic steatosis, and diabetes. In some embodiments, the subject has a cardiovascular disease (e.g., any of the cancers described herein).

[0049] In some embodiments, the subject is not a recipient of a transplanted organ or hematopoietic stem cells. In some embodiments, the subject is not immunocompromised.

[0050] Subjects that can be treated using the present method include mammals, eg, humans, and non-human veterinary subjects. [Example]

[0051] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0052] method The following materials and methods were used in this study.

[0053] Isolation and culture of human dermal fibroblasts Dermal fibroblasts were isolated from discarded normal skin samples generated as part of surgery. Subcutaneous adipose tissue was removed from human skin tissue, and tissue fragments were incubated overnight at 4°C in dispase solution (Stemcell, Vancouver, Canada, 07913). After digestion, the epidermis was separated from the dermis. The resulting dermis was incubated overnight at 37°C in collagenase / hyaluronidase (Stemcell, 07912). Fibroblasts were collected through a 70 μm cell strainer and collected at 3–5 × 10 4 cells / cm 2 At a density of 75cm 2 The cells were seeded into cell culture flasks and cultured in DMEM medium (Thermo Fisher Scientific, Waltham, MA11-965-118) containing 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 1% glutamine at 37°C in an atmosphere of 5% CO2 in air. Human fetal dermal fibroblasts (ScienCell Research Laboratories, Carlsbad, CA, 2300) and neonatal dermal fibroblasts (Lonza, Basalt, Switzerland, CC-2509) were purchased from us. The cells were cultured as described above.

[0054] Isolation of human skin immune cells As mentioned above 32Immune cells were isolated from human skin. Briefly, discarded normal skin samples generated as part of surgery were obtained. Subcutaneous fat tissue was removed from the human skin tissue, and the remaining tissue was finely minced. The skin tissue was finely minced and digested at 37°C for 2 hours in RPMI 1640 medium (Thermo Fisher Scientific, 21-870-092) containing 0.05% DNase-I (Sigma-Aldrich, St. Louis, MO, 10104159001) and 0.2% collagenase-I (Thermo Fisher Scientific, LS004196). The cells were then collected through a 70 μm cell strainer and incubated in RPMI 1640 medium containing 20% ​​FBS, 1% penicillin / streptomycin, 1% glutamine, 0.00035% β-mercaptoethanol, and 2 ng / ml human interleukin (IL)-2 recombinant (BioLegend, San Diego, CA 589104).

[0055] histology Human skin samples were fixed with 4% paraformaldehyde (PFA) and embedded in paraffin. 5 μm sections were cut and deparaffinized. After permeabilization with 0.2% Triton-X (Thermo Fisher Scientific, BP151) in phosphate-buffered saline (PBS), antigen retrieval was performed for 20 minutes using a pressure cooker in antigen unmasking solution (Vector Laboratories, Burlingame, CA, H-3300-250). Slides were washed three times for 5 minutes each in PBS containing 0.1% Tween 20 (Sigma-Aldrich, P1379). Slides were blocked for 1 hour with 5% normal goat serum (Sigma-Aldrich, G9023) and 5% bovine serum albumin (Thermo Fisher Scientific, BP1600) in PBS. Slides were stained overnight at 4°C with diluted primary antibodies (Table 3A) in blocking buffer. After application of the primary antibody, slides were washed and incubated in diluted secondary antibody (Table 3A) in blocking buffer for 2 hours at room temperature. Slides were washed and stained with 4',6-diamidino-2-phenylindole (DAPI, Invitrogen, D3571, 1:4000) in PBS for 10 minutes at room temperature. Slides were washed and mounted with ProLong Gold Antifade Reagent (Thermo Fisher Scientific, P36930). Stained tissues were imaged with a ZEISS confocal microscope (Zeiss, Oberkochen, Germany). Manual counts were performed using ZEN Blue Software (Zeiss). Cell counts were reported as the average cell count across 10 randomly selected high-power fields (hpf, 200x magnification) per skin sample in each group. For hematoxylin and eosin staining, slides were stained according to standard procedures and mounted with Cytoseal XYL (Thermo Fisher Scientific, 8312-4). Imaging of whole slides was performed using a Zeiss Axio Scan.Z1 (Zeiss).

[0056] immunocytochemistry Cells were cultured on chamber slides (CELLTREAT Scientific Products, Pepperell, MA 229168), fixed with 4% PFA for 10 minutes at room temperature, and permeabilized with 0.2% Triton-X in PBS for 10 minutes at room temperature. Slides were washed three times in PBS for 5 minutes each. Slides were blocked with 5% normal goat serum and 5% bovine serum albumin in PBS for 30 minutes. Slides were stained overnight at 4°C with diluted primary antibodies (Table 3A) in blocking buffer. After primary antibody application, slides were washed and incubated with diluted secondary antibodies (Table 3A) in blocking buffer for 1 hour at room temperature. Slides were washed as described above and stained with DAPI in PBS for 10 minutes at room temperature. Slides were washed as described above and mounted with ProLong Gold Antifade Reagent. Stained cells were imaged using a ZEISS confocal microscope. Cell numbers were reported as the average cell number across 10 randomly selected hpf per well in each group.

[0057] Senescence-associated β-galactosidase (SA-β-Gal) staining Cells were stained with β-galactosidase at pH 6.0 using the Senescence β-galactosidase Staining Kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol, and the stained cells were imaged with a ZEISS confocal microscope.

[0058] Flow cytometry Cells were washed once with PBS containing 5% newborn calf serum (Thermo Fisher Scientific, 26010074) and 0.01% sodium azide (Sigma-Aldrich, S2002-100G) and stained with antibodies (Tables 3B and 3B) on ice for 30 minutes, followed by secondary antibody staining as needed (Table 3A). After surface marker staining, cells were fixed and permeabilized for intracellular staining using True-Nuclear Transcription Factor Buffer Set (BioLegend, 424401). Permeabilized cells were stained with antibodies (Table 3A) overnight at 4°C. Cells were washed and then examined using a BD LSRFortessa X-20 flow cytometer (BD Bioscience, Billerica, MA). Data were analyzed using FlowJo software (BD Life Sciences, Franklin Lakes, NJ). Relative fluorescence intensities were calculated by subtracting the median fluorescence intensity of the isotype controls from the median fluorescence intensity of the stained samples.

[0059] Cytotoxicity assay against senescent fibroblasts 5 × 10 normal and senescent fibroblasts 3 Fibroblasts were seeded into 24-well plates at a density of 100 cells / well and cultured overnight at 37°C in DMEM containing 10% FBS, 1% penicillin / streptomycin, and 1% glutamine. Fibroblasts were pretreated overnight at 37°C with 100 μg / ml HLA-II blocking antibody (Bio X Cell, Lebanon, NH, BE0306) or 100 μg / ml isotype IgG antibody in the absence of FBS. Subsequently, skin immune cells were added to each fibroblast-seeded well and co-cultured in the presence of 20 ng / ml human IL-2 recombinant and 20 ng / ml human IL-15 recombinant (BioLegend, 570304) in a 24-well plate (immune cell-to-fibroblast ratio of 50:1). After 6 hours of co-culture, the remaining adherent fibroblasts were fixed and stained as described for immunocytochemistry.

[0060] Stimulation of human cutaneous T cells with recombinant HCMV proteins Isolated human skin immune cells were treated with 10 μg / ml recombinant HCMV-gB (Abcam, Cambridge, UK, ab43040) or 10 μg / ml recombinant HCMV-gH (MyBioSource, San Diego, CA, MBS1138239) in the presence or absence of 1.67 μg / ml Poly(IC) (Thermo Fisher Scientific, tlrl-pic). After 20 h of incubation, brefeldin A was added at a concentration of 5 μg / ml, and cells were incubated for 4 h and collected for flow cytometry analysis. As a positive control, cells were treated with 81 nM phorbol 12-myristate 13-acetate (PMA) plus 1.34 μM ionomycin (BioLegend, 423301) for 30 min. Cells were stained for flow cytometry as described.

[0061] RNA in situ hybridization RNA in situ hybridization was performed as previously described. 37Briefly, RNA in situ hybridization was performed on PFA-fixed, paraffin-embedded tissue sections using the RNAscope 2.5 HD Detection Reagent protocol (Advanced Cell Diagnostics, Newark, CA) with simultaneous vimentin protein staining. Five-micrometer sections were baked at 60°C for 60 minutes. Slides were treated with xylene, followed by 100% ethanol and allowed to dry. Slides were treated with hydrogen peroxide for 10 minutes at room temperature and then washed with deionized water. Antigen retrieval was performed for 15 minutes using RNAscope Target Retrieval Reagent (Advanced Cell Diagnostics, 322000) in a pressure cooker. Slides were incubated with DNase-I (Sigma-Aldrich, D5319-500UG) in a HybEZ Oven II (Advanced Cell Diagnostics, 321720) for 30 minutes at 37°C and then washed. RNAscope Protease Plus (Advanced Cell Diagnostics, 322331) treatment was applied for 15 minutes at 40°C. After the target probe amplification and hybridization steps, sections were stained with Fast RED reagent (RNAscope 2.5 HD Detection Reagents-RED, Advanced Cell Diagnostics, 322360). For hematoxylin staining, slides were washed with deionized water and then stained with hematoxylin (Sigma-Aldrich, GHS132-1L) for 1 minute, followed by 0.02% ammonium hydroxide (Ricca Chemical Company, Arlington, TX, 642-16). For immunofluorescence staining, slides were washed with deionized water and then PBS containing 0.1% Tween 20. Slides were blocked with 5% goat serum and 5% bovine serum albumin in PBS containing 0.1% Tween 20 for 1 hour at room temperature. Slides were stained as described in the histology section.

[0062] HCMV DNA quantification As mentioned above38、39 Quantitation of HCMV DNA in human skin samples and fibroblasts was performed by quantitative real-time PCR. DNA was isolated from fibroblasts using the Quick-DNA / RNA Microprep Plus Kit (Zymo Research, Irvine, CA, D7005) according to the kit's instructions. For human skin, DNA was isolated using the Direct-zol DNA / RNA Miniprep Kit (Zymo Research, R2080) according to the kit's instructions. Quantitative real-time PCR used a SYBR Green format, and HCMV primers detected the lower matrix phosphoprotein (UL83) gene. GAPDH was used as an internal control gene. Primer sets are listed in Table 3F. PCR was performed in a 7500 Real-Time PCR System (Applied Biosystems, Inc., Foster City, CA) in the presence of 5 μL of DNA sample, 12.5 μL of SYBR Green PCR MasterMix (Bio-Rad, Hercules, CA, 1725121), and 250 nM of each primer in a total volume of 25 μL. The temperature profile was 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 60 seconds for 40 cycles. Melting curve analysis was performed at the end of each run. The melting temperature range for HCMV DNA-positive samples was 81.5 ± 0.5°C. Relative expression of UL83 was calculated by the ΔΔCt method.

[0063] UVA irradiation Fibroblasts were irradiated with UVA (5 or 10 J) generated by a UVP XX-Series Bench Lamp, 115 V (Thermo Fisher Scientific, UVP95004208) through PBS and cultured for 1 or 5 days. Sham irradiation was used as a negative control. Irradiation intensity was measured using a light meter (International Light Technologies, Peabody, MA, ILT2400).

[0064] Human cytomegalovirus (HCMV) infection Human cytomegalovirus AD-169 was purchased from the American Type Culture Collection (Manassas, VA, VR-538). Virus concentrations were determined by plaque assay. Human fetal dermal fibroblasts were cultured at 2–3 × 10 4 cells / cm 2 Cells were seeded at a density of 1000 μg / ml and cultured as described for the culture of human dermal fibroblasts. One day after seeding, the culture medium was replaced with DMEM containing 0.1% bovine serum albumin, and the cells were infected at a multiplicity of infection (MOI) of 1. Five days after infection, DNA was isolated from the cells. Three days after infection, the infected cells were stained for immunocytochemistry as described above.

[0065] RNA-Seq analysis Human skin tissue was homogenized in RLT buffer (Qiagen, Hilden, Germany, 79216) supplemented with 1% β-mercaptoethanol (Thermo Fisher Scientific, 21-985-023). Full-length cDNA and sequencing libraries were prepared as previously described. 33 , prepared from 1 ng RNA using the Smart-Seq2 protocol. Libraries were sequenced on a Novaseq 6000 (Illumina) via the Broad Genomics Platform. FASTQ files were aligned to the human genome / hg19 (GENCODEv19) using STAR-2.5.1b. 34 Aligned transcripts were quantified using RSEM-1.2.3.1 35 Differentially expressed genes (DEGs) were analyzed by DESEq2. 36Cultured fibroblasts were prepared in TCL buffer (Qiagen, 1031576) supplemented with 1% β-mercaptoethanol. Each sample was then added to a 96-well Eppendorf TwinTec barcoded plate provided by Broad Institute (Cambridge, MA). Construction and sequencing of modified SmartSeq2 complementary DNA and Illumina Nextera XT libraries were performed at Broad Institute using an Illumina NextSeq 500 system. The quality of the FASTQ files was checked using FastQC-0.11.8. Sequences were mapped to the human genome / GRCh38 using STAR-2.5.3. 34 Sequences located in the transcripts were quantified using RSEM-1.3.1. 35 DEGs were analyzed using DESEq2-1.24.0 36 The original data are available on the NCBI Gene Expression Omnibus (GEO) under accession number GSE191055.

[0066] 10x Genomics Sample Processing and cDNA Library Preparation Single-cell suspensions were prepared using the 10x Genomics Chromium Next GEM Single Cell 30 Reagent Kits v3.1 (Dual Index) User Guide (support.10xgenomics.com / single-cell-gene-expression / library-prep / doc / user-guide-chromium-single-cell-3-reagent-kits-user-guide-v31-chemistry-dualindex). Cells were isolated from normal breast skin from young and elderly individuals using a Whole Skin Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, Cat. 130-101-540). Following the kit's instructions, enzyme P was excluded to confirm CD4 and CD8 epitopes. After cell isolation, single-cell samples were passed through a 40 mm cell strainer. Cells were stained and analyzed for CD45 using an SH800 Cell Sorter (Sony Biotechnology Inc., San Jose, CA). + PI - After sorting, the cells were counted using a Cellometer Auto 2000, Slide SD100 (Nexcelom), and VitaStain AOPI Staining Solution (Nexcelom, CS2-010-5ml). An appropriate volume of each sample was then diluted to 6,000-10,000 cells per well. +The cells were harvested. The single-cell suspension, gel beads, and oil were then added to a 10x Genomics Single Cell G Chip. After droplet generation, the samples were transferred to PCR tubes, and reverse transcription was performed using a C1000 Touch Thermal Cycler (Bio-Rad). After reverse transcription, cDNA was recovered using the recovery agent provided by 10x Genomics, followed by Silane DynaBead cleanup as outlined in the user guide. Before cleanup using SPRIselect beads, the cDNA was amplified for 11–12 cycles, depending on the number of target cell harvests. cDNA concentration was detected using a Qubit 4 Fluorometer (Invitrogen, Q32856) and a Qubit 1X dsDNA HS Assay Kit (Invitrogen, Q33230). The average peak size was obtained using an Agilent 4200 TapeStation (Agilent Technologies, Santa Clara, CA, G2991BA) and an Agilent High Sensitivity D5000 ScreenTape Assay (Agilent Technologies, 5067-5592, 5593). A skin cell cDNA library was prepared according to the Chromium Next GEM Single Cell 30 Reagent Kits v3.1 User Guide. After library preparation, the cDNA concentration was rechecked using the Qubit 1X dsDNA HS Assay Kit and the KAPA Library Quantification Kit (Roche, Basel, Switzerland, KK4835), and the average peak size was obtained using an Agilent High Sensitivity D1000 ScreenTape Assay (Agilent Technologies, 5067-5584, 5585).

[0067] ScRNA-seq analysis Libraries were sequenced using the NextSeq 500 / 550 Hi Output Kit v2.5 150 cycles (Illumina, 20024907). Libraries were sequenced using the NextSeq 500 / 550 High Output Kit v2.5 150 cycles (Illumina, 20024907) with the NextSeq 550 (Illumina). Raw binary base call (BCL) sequences were converted to FASTQ files using Cell Ranger-6.0 (10x Genomics) mkfastq with default parameters. FASTQ files were mapped with Cell Ranger-6.0 counts using a human reference (GRCh38). Matrix data were analyzed using Seurat-4.3.0 in R-4.2.2. 60 Low-quality cells with fewer than 20 genes, more than 3000 genes, or more than 15% mitochondrial genes were removed from further analysis. The "NormalizeData" followed by the "ScaleData" function was used to normalize and scale the sequencing reads. To remove batch effects between samples, the top 2000 highly variable genes were identified from each sample, and then recurrently variable genes across samples were integrated using "SelectIntegrationFeatures" for principal component analysis (PCA). Anchors were identified using "FindIntegrationAnchors" and integrated into the data using the "IntegrateData" function. Uniform Manifold Approximation and Projection (UMAP) plots were obtained using "ScaleData," "RunPCA," "RunUMAP," "FindNeighbors," and "Findclusters." CD4 + T cell clusters are identified by CD3D + TRAC + TRDC - CD8A - The criteria were defined as follows. Using "FindMarkers" with DEseq2 parameters, conventional CD4 (PRF1 -, CD8A - ) and CD4 CTL (PRF1 + CD8A - NCAM - The differentially expressed genes (DEGs) between the CD8 CTLs and PRF1 + CD8A + The original data are available in the NCBI Gene Expression Omnibus (GEO) under accession number GSE221232.

[0068] siRNA transfection Normal fibroblasts and senescent fibroblasts were cultured at 4 × 10 4 Cells were seeded in 10 cm dishes at a density of 2.5 × 10 cells. One day after seeding, cells were harvested and transfected with human CIITA siRNA SMARTPool (Horizon Discovery, Waterbeach, UK, L-011083-00-0005) using the NHDF Nucleofector Kit (Lonza, VPD-1001) and the Nucleofector 2b Device (Lonza, AAB-1001). Negative control siRNA (Qiagen, 1022076) was used as a negative control. After transfection, fibroblasts were cultured at a density of 2.5 × 10 cells. 3 The cells were replated onto chamber slides (CELLTREAT Scientific Products) at a density of 1000 cells / well. 48 hours after transfection, cells were harvested for flow cytometry analysis. For cytotoxicity assays, fibroblasts were co-cultured with skin-isolated cells (E:T ratio = 50:1) and incubated for 6 hours. After co-culture, fibroblasts were fixed and stained as described in immunocytochemistry.

[0069] Characterization of skin-isolated immune cells co-cultured with senescent fibroblasts Normal fibroblasts and senescent fibroblasts were cultured at 2 × 10 3 The skin-isolated cells were seeded into 48-well plates at a density of 3 × 10 cells / well. One day after seeding, the cells were cultured at a density of 3 × 10 cells / well. 4Cells were added at a density of 1000 cells / well. After 20 hours of incubation, brefeldin A was added at a concentration of 5 μg / ml, and the cells were incubated for 4 hours and collected for flow cytometry analysis. Cells were stained as described for flow cytometry to detect CD137 and IFNγ expression. Skin-isolated cells were used to detect CD107a expression. After 1 hour of co-culture, brefeldin A (BioLegend, 420601, 5 μg / ml) and monensin (BioLegend, 420701, 2 μM) were added along with CD107a-FITC antibody (dilution: 200). After 4 hours, cells were collected for flow cytometry analysis.

[0070] Transwell migration assay Skin isolated cells were collected at 1.5 x 10 4 Cells were seeded at a density of 1000 cells / well into the upper insert of a 24-well transwell plate (Corning, 07-200-149). The pore size was 5 μm. RPMI containing recombinant human CXCL9 (PeproTech, 300-26) at a concentration of 50 nM or PBS (carrier only) was added to the lower well. The skin-isolated cells were incubated for 60 minutes. Cells were then counted and stained as described in the flow cytometry section.

[0071] HCMV epitope-specific CD4 CTL detection HCMV-gB-specific DRB1*0701 DYSNTHSTRYV phycoerythrin (PE)-conjugated tetramer was used based on previous publications. 61~65 , generated by the National Institutes of Health (NIH) tetramer core facility at Emory University. NIH also provided the DRB1*0701 PE-conjugated control peptide (PVSKMRMATPLLMQA) tetramer. DYSNTHSTRYV is an HCMV-gB epitope sequence previously shown to be recognized by human CD4 CTLs. 66The peptide DYSNTHSTRYV was provided by GenScript (Piscataway, NJ). Cells were incubated with test or control tetramers at a concentration of 5.6 μg / ml for 3 hours at 37°C in the dark. After repeated washing, cells were stained as described in the flow cytometry section.

[0072] HLA-DRB1*07 typing DNA isolation from snap-frozen tissues was performed using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, 69504) according to the manufacturer's instructions. DNA quality and concentration were determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, ND-1000). HLA-DRB1*07 typing experiments were performed according to the manufacturer's instructions using Olerup SSP (CareDx, Brisbane, CA, 101.118-24u). Briefly, DNA, master mix, and Platinum Taq DNA Polymerase (Invitrogen, 10966-026) were added to the appropriate primer cocktail. The reaction mixture was placed on a thermocycler, and samples were run on a 1% agarose (Denville Scientific, Metuchen, NJ, GR140-500) gel and then imaged. The kit was Certified by the HLA Nomenclature Committee in April 2021, a total of 21 reactions and one control reaction were used to classify DRB1*07:01 to DRB1*07:123.

[0073] TCR sequencing and analysis DNA isolation, concentration, and purity determination were performed as described above. Adaptive Biotechnologies (Seattle, WA) performed high-throughput sequencing of the provided DNA and generated the raw data.

[0074] SenTraGor staining SenTraGor staining was performed on formalin-fixed, paraffin-embedded (FFPE) slides according to the manufacturer's instructions. Briefly, after deparaffinization and rehydration, SenTraGor reagent incubation was performed for 3 minutes at room temperature. After washing, slides were stained with p16 INK4a and mouse anti-biotin antibody (Abcam, ab201341) with vimentin antibody overnight at 4°C. Secondary antibody staining was performed as described above.

[0075] SCFA-mediated CD4+ T cell stimulation assay 2.5 x 10 T cells in a 60 mm dish 5 Cells were seeded at a density of 1000 ng / mL and incubated for 2 days in 5 mM SCFA containing butyrate (EMD Millipore), pentanoate (Ambeed Inc.), or propionate (Sigma-Aldrich) along with 20 ng / mL human IL-2 recombinant and 20 ng / mL human IL-15 recombinant. After 2 days of co-incubation, T cells were added to each well containing senescent fibroblasts and cultured in T cell medium containing 20 ng / mL human IL-2 recombinant and 20 ng / mL human IL-15 recombinant (50:1 T cell-to-fibroblast ratio) in 24-well plates. After 6 hours of co-incubation, the remaining fibroblasts were stained for vimentin and cleaved caspase-3 to examine the frequency of T cell-mediated cytotoxicity against senescent fibroblasts.

[0076] statistical analysis All bar graphs and dot plots show the mean + SD. The two-tailed Mann-Whitney U test was used as a significance test for cell counts in human skin histological analysis. A two-tailed paired t-test was used for the cultured dermal fibroblast assay. Student's t-test with Pearson correlation coefficient was used as a significance test for linear regression in scatter plots. A one-way ANOVA with Tukey's multiple comparison test was used for the co-culture cytotoxicity assay. A one-way ANOVA with Dunnett's multiple comparison test was used for the HCMV-gB stimulation assay. The chi-square test was used to test the significance of categorical variables. Prism9 was used for statistical analysis. A P value of <0.05 was considered significant. [Table 1-1] [Table 1-2] [Table 1-3] [Table 2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 4] [Table 5] [Table 6] [Table 7]

[0077] Example 1. Increased Senescent Cells in Aging Human Skin Over 800 human skin samples from various ages and anatomical sites were screed and cohorts of sun-protected trunk skin from young (n = 23, mean age: 23.1 years) and older (n = 31, mean age: 62.1 years) women were identified (Table 1). Reduced epidermal thickness in older versus younger skin demonstrated biological evidence of skin aging in the selected skin samples (Table 1). These skin cohorts allowed for the assessment of age-associated senescent cell accumulation in human organs while excluding confounding factors such as ultraviolet (UV) irradiation, anatomical site variation, gender differences, and hair follicle density (Table 1). To determine whether the number of senescent cells changes with age, human skin samples were analyzed for p16, a marker of cellular senescence. INK4a Stained with 1、18 p16 INK4a The number of positive cells increased significantly with age in both the epidermis and dermis, but the magnitude of this increase was significantly greater in the dermis (Figure 1A-C). INK4a More than 80% of the positive cells expressed vimentin, and most vimentin-positive dermal cells were also platelet-derived growth factor receptor α (PDGFRα)-positive, indicating that most of the senescent cells in aged human dermis were fibroblasts (Figures 2A-2D). INK4a+ Vimentin + The number and percentage of fibroblasts were significantly increased in aged skin compared with young skin (Figures 1D, 1E, and 2E). INK4a+ The majority of dermal fibroblasts express p21, which is upregulated during early cellular senescence. 67~70 , p21 expression, p16 INK4a- It was negligible in dermal fibroblasts (Fig. 2F and 2G). + Vimentin + The number of fibroblasts was significantly increased in aged skin compared with young skin (Figure 2H). INK4a+ Dermal fibroblasts were also positive for SenTraGor (GL13), a marker of lipofuscin accumulation associated with cellular senescence (Figure 2I). 71The number of dermal senescent cells was positively correlated with age across young and aged skin samples (r = 0.5898) (P < 0.0001, Figure 1F). However, the number of dermal senescent cells did not show a significant correlation with aging within the aged skin cohort (r = 0.2121) (P = 0.2520, Figure 1G). This unexpected finding suggests that biological factor(s) other than aging may govern the accumulation of senescent cells in the elderly. [Table 8]

[0078] Example 2. CD4 CTLs are the predominant cytotoxic lymphocytes in aging human skin To identify the factor(s) that regulate the accumulation of senescent cells in aged skin, we investigated whether epidermal condition, blood vessel density, or lymphatic vessel density affected the number of senescent cells. Epidermal thickness, blood vessel density, and lymphatic vessel density were significantly reduced in aged versus young skin samples; however, these factors did not significantly correlate with the number of dermal senescent cells in aged skin samples (Figures 3A-3I). Comprehensive skin immune cell profiling using multiplex immunostaining on tissue sections revealed that several innate and adaptive immune cell types were highly enriched in aged dermis (Figures 4A and 3J). Importantly, several cytotoxic immune cell types were negatively correlated with the number of dermal senescent cells in aged skin samples (Table 2). Among them, cytotoxic CD4 T cells, characterized by perforin protein expression, were significantly increased in aged skin samples. + The number of T cells (CD4 CTL) was most negatively correlated with the number of dermal senescent cells in aged skin samples (r = -0.6796) (p < 0.0001, Figure 4B). + The number of CD4 CTLs was significantly increased in aged compared with young dermis (Figures 4C and 4D). Notably, 83.7% of perforin-expressing dermal cells in aged skin samples were CD4 +The CD4 T cells were predominantly CD4+ T cells (Figure 4E), indicating that CD4+ CTLs were the predominant cytotoxic lymphocytes involved in immune surveillance in aged human skin. To further characterize CD4+ CTLs in human skin, single-cell RNA sequencing (scRNA-seq) was performed on lymphocyte-enriched single cells from aged human skin samples (Figure 5A). CD4 + Within the T cell cluster, we identified a population of perforin (PRF1)-expressing CD4 CTLs that highly upregulated the resident memory marker CD69 (Figures 5B and 5C). In addition, CD4 CTLs expressed RUNX3 72、73 , RAB27A 74、75 , and expressed cytotoxicity-related genes, including KLRK1 (encoding the natural killer [NK] group 2D [NKG2D] receptor) ( Fig. 5C ). 76 These findings suggest that CD4 CTLs are effector cells involved in the clearance of senescent cells in aged human skin.

[0079] To determine the factors that recruited CD4 CTLs in aged skin, we performed RNA sequencing (RNA-Seq) of young and aged skin samples. Cluster analysis showed that immune-related genes, including CD69, CD96, CD276, CXCL9, KLRD1, IL2RB, IL17RC, IL36G, MB21D1, S100A9, and VTCN1, were among the genes significantly altered in aged versus young skin (Figure 4F). Among these, the CXCL9 chemokine, a ligand for CXCR3 expressed on CD4 CTLs, was significantly altered in aged versus young skin (Figure 4F). 19 CXCL9-expressing cells were mainly localized in the basal layer of the epidermis in aged skin samples (Fig. 4G). high and CXCL9 low By dividing the CXCL9 high Skin, CXCL9 lowWe found that keratinocytes contained significantly higher numbers of CD4 CTLs and fewer dermal senescent cells than keratinocytes (Figures 4H and 4I). Furthermore, CD4 CTLs in human skin express CXCR3, and CXCL9 significantly induced CD4 CTL migration in a transwell assay system (Figure 6). These findings suggest that CXCL9 expressed by keratinocytes recruits CD4 CTLs to the skin, which may lead to the clearance of dermal senescent cells. [Table 9]

[0080] Example 3. CD4 CTLs directly target senescent fibroblasts To investigate whether CD4 CTLs can target senescent fibroblasts, senescent human dermal fibroblasts were generated ex vivo through repeated passage-induced replicative senescence, which mimics cellular senescence in sun-protected aged skin. 20 Senescence-associated β-galactosidase (SA-β-Gal) staining indicates senescent fibroblasts (Figure 7A). RNA-Seq analysis further confirmed the enrichment of the cellular senescence gene set and revealed that several ligands recognized by cytotoxic lymphocytes were significantly altered in replicative senescence compared to normal fibroblasts (Figure 7B and Figure 8A). Among these, NKG2D, a ligand for activating the natural killer group 2D (NKG2D) receptor on cytotoxic lymphocytes, was significantly altered in replicative senescence compared to normal fibroblasts (Figure 7B and Figure 8A). 21 UL16-binding protein 2 (ULBP2) was highly upregulated in senescent fibroblasts (Figure 7B). Thus, ULBP2 was expressed on the surface of senescent fibroblasts, in contrast to its lack of expression on normal fibroblasts (Figures 7C, 7D). Similarly, ULBP2 +The number of senescent fibroblasts significantly increased in aged compared with young human skin (Figs. 8B and 8C). Human leukocyte antigen class II (HLA-II) surface expression is crucial for CD4 CTL-induced immunity. To determine whether senescent fibroblasts could be a direct CD4 CTL target, we examined HLA-II expression on senescent fibroblasts. Importantly, HLA-II was highly expressed on aged fibroblasts, whereas its expression was negligible on normal fibroblasts (Figs. 7E and 7F). Consistent with this finding, HLA-II high The number of senescent fibroblasts significantly increased in aged compared with young human skin (Fig. 7G, H). high Skin samples with senescent fibroblasts express HLA-II high Senescent fibroblasts contained significantly higher numbers of CD4 CTLs than those without (Figure 8D), a finding supporting the role of the CD4 CTL / HLA-II axis in immune surveillance of senescent fibroblasts.

[0081] Ultraviolet A (UVA) radiation is a significant cause of skin aging. 22 , known to induce senescence in fibroblasts 22 UVA irradiation generated senescent human dermal fibroblasts ex vivo (Figure 8E). ULBP2 and HLA-II levels were significantly increased on the surface of UVA-induced senescent fibroblasts (Figures 7A-7M). These findings indicate that senescent fibroblasts are highly immunogenic targets and can be directly recognized by CD4 CTLs in human skin.

[0082] Example 4. CD4 CTLs reduce senescent fibroblasts in an HLA-II-dependent manner To determine whether CD4 CTLs could reduce the level of senescent fibroblasts, normal and replication-induced senescent fibroblasts were cocultured with immune cells isolated from aged human skin. Skin-derived allogeneic T cells specifically triggered apoptosis in senescent fibroblasts in an HLA-II-dependent manner (Figures 9A-9C). Next, a coculture system was established in which normal and senescent fibroblasts were exposed to autologous T cells isolated from the same human skin (Figure 7I). Autologous T cells cleaved caspase-3. + Apoptotic senescent fibroblasts were induced at a significantly higher rate compared with normal fibroblasts (Figure 7J, K). Importantly, HLA-DR antibody blockade eliminated cutaneous T cell-induced apoptosis of senescent fibroblasts (Figure 7K). Furthermore, using small interfering RNA (siRNA) treatment against CIITA, a master regulator of HLA-II gene expression, we generated senescent fibroblasts in which HLA-II was significantly downregulated. 77 HLA-II downregulation significantly blocked skin immune cell-induced apoptosis of senescent dermal fibroblasts (Figures 9D-9F). Flow cytometry analysis demonstrated the induction of degranulation marker, CD107a, and activation markers, CD137 and interferon-g (IFNg), in skin-resident CD4+ T cells exposed to senescence compared with normal fibroblasts (Figures 9G-9L). These findings indicate that senescent fibroblasts are eliminated by T cells in an HLA-II-dependent manner.

[0083] Example 5. CD4 CTLs reduce the level of senescent fibroblasts by targeting the HCMV-gB antigen We next investigated which HLA-II-binding antigens induce CD4 CTLs to reduce the level of senescent fibroblasts (e.g., by killing senescent fibroblasts). The glycoprotein B (gB) from the commensal human cytomegalovirus (HCMV) induces CD4 CTLs. + It is the primary target of T cells and can activate CD4 CTLs to eliminate human host cells 23 Furthermore, gB-specific CD4 + T cells are found in the blood of 95% of healthy individuals 24HCMV DNA and RNA were detectable in normal human skin, and their levels were significantly increased in aged compared with young skin samples (Figures 10A-10D). HCMV RNA expression was particularly prominent in dermal fibroblasts of aged skin (Figures 10D, E). To further verify the HCMV infection status in skin samples, a clinical-grade antibody cocktail was used for pan-HCMV antigen detection. HCMV-positive dermal cells were detected in 67.7% of aged skin samples and 43.5% of young skin samples, which is consistent with the seropositivity prevalence in elderly and young individuals in the US population (Figures 10F and 10G). Notably, pan-HCMV antigen-positive skin samples had higher HCMV DNA levels (Figure 10H).

[0084] Senescent fibroblasts express HCMV-gB in human skin, and HCMV-gB is more prevalent in aged compared with young skin. + It was found that the number of senescent fibroblasts was significantly increased (Figs. 11A and 11B). On the other hand, HCMV immediate-early (IE)1 / IE2 proteins were only detectable in some aged skin samples (Figs. 12A and 12B). In line with in vivo findings, replicative and UVA-induced senescent fibroblasts highly upregulated HCMV DNA and HCMV-gB expression ex vivo (Figs. 11C-11G). HCMV induces the accumulation of early endosomes in infected cells. 25、26 HCMV-gB is sorted into endosomes and presented to HLA-II 23 Similar to HCMV-infected fetal fibroblasts, HCMV-gB replicated and was transported to endosomes in UVA-induced adult senescent fibroblasts (Fig. S13). These findings demonstrate that HCMV is activated in senescent fibroblasts and that HCMV-gB can be displayed as an endogenous antigen on HLA-II.

[0085] CD4 CTLs that primarily express the T cell receptor (TCR) beta chain TRBV6-5 gene segment are HLA-DRB1 * Recognizes the HCMV-gB epitope presented on 07:01 78,79 HLA-DRB1 *Using TCR sequencing on 07:01-positive aged skin samples, TRBV6-5-expressing T cell clones were detected in aged skin (Figure 14A). To further determine the presence of HCMV-gB-specific CD4 CTLs in human skin, T cells isolated from young and aged human skin were transfected with HLA-DRB1. * Stained with HLA-peptide tetramer containing the gB-derived DYSNTHSTRYV peptide conjugated to 07:01 (DR7) 80~84 This analysis demonstrated the presence of HCMV-gB-specific CD4 CTL in aged human skin (Figs. 14B and 14C).

[0086] Autologous T cell / fibroblast co-culture assays revealed that most apoptotic senescent fibroblasts expressed HCMV-gB (Figures 14D and 14E). Importantly, HCMV-gB + Senescent fibroblasts remained intact upon HLA-DR blockade (Fig. 14F). Finally, recombinant HCMV-gB inhibited ex vivo human skin-derived perforin + IFNγ expression was induced in CD4 CTLs (Fig. 14G and 14H). + CD4 CTLs were further increased in the presence of HCMV-gB + poly(I:C), which is known to enhance the activation of antigen-presenting cells in the skin (Figures 14G and 14H). 86、86 In contrast, HMCV glycoprotein H (gH) + poly(I:C) did not induce IFNγ expression in CD4 CTLs (Figures 14G and 14H). Together, these findings indicate that HCMV-gB-specific CD4 CTLs contribute to the clearance of senescent fibroblasts in aging human skin.

[0087] Example 6. Butyrate and pentanoate enhance CD4 CTL-mediated clearance of senescent cells Skin T cells were exposed to 5 mM short-chain fatty acids (SCFAs), propionate, butyrate, and pentanoate, for 2 days and then cocultured with senescent dermal fibroblasts. After 6 hours of co-incubation, fibroblasts were stained for vimentin and cleaved caspase-3 to examine the frequency of T cell-mediated cytotoxicity against senescent fibroblasts. T cells exposed to either butyrate or pentanoate, but not propionate, induced apoptosis in senescent cells to a greater extent (Figure 15A). In addition, skin CD4+ T cells exposed to the SCFAs, butyrate and pentanoate, exhibited more cytotoxic activity, as indicated by increased expression of perforin+ and CD107a+ on CD4+ T cells (Figure 15B). These results indicate that butyrate and pentanoate enhance CD4+ CTL-mediated clearance of senescent cells.

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[0089] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative, but not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method of delaying the onset or slowing the progression of aging in a subject, comprising administering to the subject: a plurality of (i) antigenic proteins derived from commensal human cytomegalovirus (HCMV), (ii) antigenic peptides derived from proteins derived from commensal human cytomegalovirus, or (iii) live or live-attenuated commensal human cytomegalovirus; and a T cell adjuvant that increases T cell responses to the plurality of antigenic proteins, antigenic peptides, or live or live-attenuated commensal human cytomegalovirus.

2. 1. A method of reducing the level of senescent fibroblasts in a subject, comprising administering to the subject: a plurality of (i) antigenic proteins derived from commensal human cytomegalovirus (HCMV), (ii) antigenic peptides derived from proteins derived from commensal human cytomegalovirus, or (iii) live or live-attenuated commensal human cytomegalovirus; and a T cell adjuvant that increases T cell responses to the plurality of antigenic proteins, antigenic peptides, or live or live-attenuated commensal human cytomegalovirus.

3. 3. The method of claim 2, wherein the level of senescent fibroblasts is reduced in the skin of the subject.

4. The method of any one of claims 1 to 3, wherein the subject has an aging-related disease.

5. 1. A method of treating or reducing the risk of developing an aging-related disease or condition in a subject, comprising administering to the subject: a plurality of (i) antigenic proteins derived from commensal human cytomegalovirus (HCMV), (ii) antigenic peptides derived from proteins derived from commensal human cytomegalovirus, or (iii) live or live-attenuated commensal human cytomegalovirus; and a T cell adjuvant that increases T cell responses to a plurality of said antigenic proteins, said antigenic peptides, or said live or live-attenuated commensal human cytomegalovirus.

6. 6. The method of claim 4 or claim 5, wherein the aging-related disease is one or more of cancer, cardiovascular disease, neurodegenerative disease, kidney disease, autoimmune disease, arthritis, osteoporosis, macular degeneration, chronic obstructive pulmonary disease (COPD), glaucoma, obesity, fibrosis, cirrhosis, hepatic steatosis, and diabetes.

7. 7. The method of claim 6, wherein the cancer is one or more of osteosarcoma, breast cancer, prostate cancer, colorectal cancer, lung cancer, melanoma, renal cancer, lymphoma, uterine cancer, pancreatic cancer, non-melanoma skin cancer, and bladder cancer.

8. 7. The method of claim 6, wherein the neurodegenerative disease is one or more of dementia, ataxia, Huntington's disease, motor neuron disease, or tau-mediated neurodegenerative disease, optionally Alzheimer's disease, Parkinson's disease, or progressive supranuclear palsy.

9. 7. The method of claim 6, wherein the cardiovascular disease is one or more of atherosclerosis, idiopathic pulmonary fibrosis, coronary heart disease, congestive heart failure, coronary artery disease, peripheral artery disease, valvular heart disease, arrhythmia, ischemic cardiomyopathy, hypertension, and stroke.

10. 7. The method of claim 6, wherein the autoimmune disease is one or more of multiple sclerosis, Crohn's disease, rheumatoid arthritis, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, and systemic lupus erythematosus.

11. 7. The method of claim 6, wherein the fibrosis is one or more of systemic sclerosis, scleroderma, idiopathic pulmonary fibrosis, and interstitial lung disease.

12. The method of any one of claims 1 to 11, wherein at least one of the antigenic proteins, if present, is HCMV glycoprotein B.

13. The method of any one of claims 1 to 11, wherein at least one of the antigenic proteins, if present, comprises a sequence derived from HCMV glycoprotein B.

14. 14. The method of claim 13, wherein the HCMV glycoprotein B-derived sequence is or comprises a truncated sequence of HCMV glycoprotein B.

15. 12. The method of any one of claims 1 to 11, wherein each antigenic peptide, if present, comprises a sequence of 9 to 30 amino acids derived from a protein from human cytomegalovirus.

16. 16. The method of claim 15, wherein each antigenic peptide, if present, comprises a sequence of 9 to 30 amino acids derived from HCMV glycoprotein B.

17. 17. The method of any one of claims 1-16, wherein the commensal human cytomegalovirus, if present, comprises one or more of the following HCMV strains: AD169, Towne, Toledo, PH, TR, FIX, VR1814, Merlin, and TB40 / E.

18. The method of any one of claims 1 to 17, wherein the T cell adjuvant comprises a short chain fatty acid.

19. 19. The method of claim 18, wherein the short chain fatty acid comprises butyrate, pentanoate, or a combination thereof.

20. 20. The method of any one of claims 1 to 19, wherein the T cell adjuvant comprises one or more of nanoparticles that enhance T cell responses, poly-ICLC (carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA), imiquimod, CpG oligodeoxynucleotides and formulations (IC31, QB10), AS04 (aluminum salt formulated with 3-O-desacyl-4'-monophosphoryl lipid A (MPL)), AS01 (MPL and saponin QS-21), MPLA, STING agonists, other TLR agonists, Candida albicans skin test antigen (Candin), GM-CSF, Fms-like tyrosine kinase-3 ligand (Flt3L), and / or IFA (incomplete Freund's adjuvant).

21. 1. A composition comprising: a plurality of (i) antigenic proteins derived from commensal human cytomegalovirus (HCMV), (ii) antigenic peptides derived from proteins derived from commensal human cytomegalovirus, or (iii) live or live-attenuated commensal human cytomegalovirus; and a T cell adjuvant that increases T cell responses to the plurality of antigenic proteins, antigenic peptides, or live or live-attenuated commensal human cytomegalovirus, for use in a method for treating or reducing the risk of developing an aging-related disease or condition in a subject.

22. 2. The composition of claim 1, wherein the aging-related condition is one or more of gray hair, hearing loss, cataracts, frailty, and sarcopenia.

23. 2. The composition of claim 1, wherein the aging-related disease is one or more of cancer, cardiovascular disease, neurodegenerative disease, kidney disease, autoimmune disease, arthritis, osteoporosis, macular degeneration, chronic obstructive pulmonary disease (COPD), glaucoma, obesity, fibrosis, cirrhosis, hepatic steatosis, and diabetes.

24. The composition of any one of claims 21 to 23, wherein each said antigenic protein, if present, is HCMV glycoprotein B.

25. The composition of any one of claims 21 to 23, wherein each of said antigenic proteins, if present, comprises a sequence derived from HCMV glycoprotein B.

26. 26. The composition of claim 25, wherein the sequence derived from HCMV glycoprotein B is a truncated sequence of HCMV glycoprotein B.

27. 24. The composition of any one of claims 21 to 23, wherein each antigenic peptide, if present, comprises a sequence of 9 to 30 amino acids derived from a protein from human cytomegalovirus.

28. 28. The composition of claim 27, wherein each antigenic peptide, if present, comprises a sequence of 9 to 30 amino acids derived from HCMV glycoprotein B.

29. 29. The composition of any one of claims 21-28, wherein the commensal human cytomegalovirus, if present, comprises one or more of the following HCMV strains: AD169, Towne, Toledo, PH, TR, FIX, VR1814, Merlin, and TB40 / E.

30. 30. The composition of any one of claims 21 to 29, wherein the T cell adjuvant comprises a short chain fatty acid.

31. 31. The composition of claim 30, wherein the short chain fatty acid comprises butyrate, pentanoate, or a combination thereof.

32. 30. The composition of any one of claims 21-29, wherein the T cell adjuvant comprises one or more of nanoparticles that enhance T cell responses, poly-ICLC (carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA), imiquimod, CpG oligodeoxynucleotides and formulations (IC31, QB10), AS04 (aluminum salt formulated with 3-O-desacyl-4'-monophosphoryl lipid A (MPL)), AS01 (MPL and saponin QS-21), MPLA, STING agonists, other TLR agonists, Candida albicans skin test antigen (Candin), GM-CSF, Fms-like tyrosine kinase-3 ligand (Flt3L), and / or IFA (incomplete Freund's adjuvant).

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