Immunotherapeutic approach to reduce senescent cells and alleviate senescence‐associated secretory phenotype in mice

Abstract Accumulation of senescent cells (SNCs) with a senescence‐associated secretory phenotype (SASP) has been implicated as a major source of chronic sterile inflammation leading to many age‐related pathologies. Herein, we provide evidence that a bifunctional immunotherapeutic, HCW9218, with capabilities of neutralizing TGF‐β and stimulating immune cells, can be safely administered systemically to reduce SNCs and alleviate SASP in mice. In the diabetic db/db mouse model, subcutaneous administration of HCW9218 reduced senescent islet β cells and SASP resulting in improved glucose tolerance, insulin resistance, and aging index. In naturally aged mice, subcutaneous administration of HCW9218 durably reduced the level of SNCs and SASP, leading to lower expression of pro‐inflammatory genes in peripheral organs. HCW9218 treatment also reverted the pattern of key regulatory circadian gene expression in aged mice to levels observed in young mice and impacted genes associated with metabolism and fibrosis in the liver. Single‐nucleus RNA Sequencing analysis further revealed that HCW9218 treatment differentially changed the transcriptomic landscape of hepatocyte subtypes involving metabolic, signaling, cell‐cycle, and senescence‐associated pathways in naturally aged mice. Long‐term survival studies also showed that HCW9218 treatment improved physical performance without compromising the health span of naturally aged mice. Thus, HCW9218 represents a novel immunotherapeutic approach and a clinically promising new class of senotherapeutic agents targeting cellular senescence‐associated diseases.

However, accumulation of SNCs also drives aging and age-related diseases like diabetes, osteoporosis, cardiovascular diseases, dementia, neurodegenerative disorders, renal failure, sarcopenia, and macular degeneration (Bennett & Clarke, 2017;McHugh & Gil, 2018;Triana-Martinez et al., 2020), while SNC removal improves health span and life span in experimental animal models (Baker et al., 2016;Ogrodnik et al., 2021). Thus, senolytic and senomorphic therapies that clear SNCs and lower SASP with targeted drugs are being vigorously pursued for a healthy longevity (Sedrak et al., 2021;van Deursen, 2019). It has been recently shown that an aged immune system drives senescence and aging of solid organs (Yousefzadeh et al., 2021). Signaling from the SASP transforming growth factor (TGF)β is also known to play a major role in cellular senescence and aging-related pathology (Tominaga & Suzuki, 2019). Therefore, we hypothesized that an immunotherapeutic agent that rejuvenates a dysfunctional immune system and neutralizes TGFβ would act as an effective SNC reducing and senomorphic drug. Previously, we showed that the bifunctional immunotherapeutic HCW9218, comprising TGFβ receptor II and IL-15/IL-15 receptor α domains, exhibited potent TGFβ neutralizing and immune cell-stimulating activities, resulting in enhanced metabolic and cytotoxic activities of immune cells and promoted natural killer (NK)-cell-mediated clearance of therapy-induced senescent (TIS) cells in tumors and normal tissues in vivo (Chaturvedi et al., 2022;Liu et al., 2021). In this study, we employed type-2 diabetic db/db mice, as a model for metabolicdysfunction-induced senescence, and naturally aged C57BL/6J mice for chronological age-induced cellular senescence, to assess whether HCW9218 can function as an SNC reducing and senomorphic drug under conditions where the stress inducers of cellular senescence are poorly defined or unknown.

| H C W9218 TRE ATMENT ENHAN CE S IMMUNE-MEDIATED B IOLOG IC AL AC TIVITIE S IN D IAB E TI C d b/d b MICE
It has been shown that metabolic reprogramming of immune cells in obesity limits antitumor responses (Michelet et al., 2018). Therefore, we first evaluated whether metabolically dysfunctional db/db mice retained immune cell stimulatory capability following HCW9218 treatment. Mice were injected subcutaneously with 3 mg/kg of HCW9218 or HCW9228 (a derivative of HCW9218 without IL-15 activity due to an IL-15D8N mutation; Liu et al., 2021), or PBS (negative control). Mice treated with HCW9218, but not HCW9228 or PBS, showed higher percentages of CD3 + CD8 + T cells, and NK cells, but not CD4 + T cells, in the spleen (Figure 1a (Ganeshan & Chawla, 2014;Jung et al., 2019), we further determined the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of splenocytes following HCW9218 treatment in db/db health span of naturally aged mice. Thus, HCW9218 represents a novel immunotherapeutic approach and a clinically promising new class of senotherapeutic agents targeting cellular senescence-associated diseases.

K E Y W O R D S
aging, cellular immunology, circadian genes, immunotherapy, inflammation, physical performance, senescence, senescent cell reduction, senomorphic, type 2 diabetes F I G U R E 1 HCW9218 enhances immune-mediated biological activities in db/db micee. (a-d). Representative flow cytometry data showing increase in immune cell surface makers on splenocytes of HCW9218-treated mice at Day 4 compared to controls. Individual value plot show the mean (n = 6/group) from two independent experiments. (e, f) Representative flow cytometry data showing increase in central memory cell and effector memory cell numbers in splenocytes of mice treated with HCW9218 compared to PBS-treated controls at Day 4. Individual value plot show the mean (n = 6/group) from two independent experiments. (g) Killing of Yac-1 target cells by in vivo HCW9218 treated splenocytes compared to control splenocytes. Individual value plot shows the mean (n = 6/group) from two independent experiments. (h) Increase in interferon (IFN)γ released by CD3 + cells upon antigen-independent stimulation by in vivo HCW9218 treated and ex vivo α-CD3/α-CD28 beads stimulated splenocytes compared to PBS control. Individual value plots show mean (n = 6/group) from one experiment. (i, j) Representative data for increase in extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) data from splenocytes of HCW9218, HCW9228 or PBS-treated mice and analyzed by Seahorse XFe Bioanalyzer (Agilent). (k-m) ELISA data showing decrease in TGFβ1 and TGFβ2 but not TGFβ3 in plasma after HCW9218 or HCW9228 treatment. Individual value plot show the mean ± SEM (n = 5/group) from two independent experiments. p values were determined by ordinary one-way ANOVA with Tukey's multiple comparisons test.   Figure S1A-D). These findings indicate that HCW9218, but not HCW9228 or PBS, was able to stimulate the NK and T cells in an IL-15 dependent manner in metabolically dysfunctional db/db mice. As anticipated, both HCW9218 and HCW9228 significantly reduced plasma levels of TGF-β1 and TGF-β2 in treated db/db mice (Figure 1k-m).

| H C W9218 TRE ATMENT REDUCE S S ENE SCENT PAN CRE ATI C IS LE T B E TA CELL S AND SA S P FAC TOR S AND IMPROVE S T YPE-2 D IAB E TE S PROFILE OF d b/d b MICE
Metabolic dysfunction induces senescence of pancreatic β cells,  Since T2D is a metabolic disease and the liver is a key metabolic organ governing body energy metabolism, we also performed RNA-Seq analysis on the livers of db/db mice following HCW9218 treatment. Of the differentially expressed liver genes, one was upregulated and 32 were downregulated which together could be grouped (by STRING) into four clusters based on function ( Figure 2l; Table S1). The expression of 8 genes related to glucose, lipid, or amino acid metabolism was significantly reduced in the liver following HCW9218 treatment (Figure 2m). Among this group, HCW9218mediated downregulation of resistin (Retn), previously found to be mainly synthesized in adipocytes, was particularly interesting.
Resistin has been shown to induce insulin resistance in mice partially through the toll-like receptor 4 signaling pathway (Benomar & Taouis, 2019;Shi, Fan, et al., 2019;Shi, Hua, et al., 2019), and its downregulation by HCW9218 treatment could contribute to the re-

| H C W9218 S TIMUL ATE S IMMUNE CELL AC TIVITIE S AND ME TABOLI C FUN C TI ON S WHILE REDUCING SA S P AND CELLUL AR S ENE SCEN CE MARK ER S IN NATUR ALLY AG ED MI CE
Although HCW9218 treatment has been shown to effectively reduce therapy-induced and metabolic dysfunction-induced SNCs and SASP in vivo (Chaturvedi et al., 2022), it is unknown whether HCW9218 could also eliminate the heterogeneous population of SNCs generated and accumulated during natural aging. The heterogeneity of accumulated SNCs in the aging process is the result of poorly defined cell and tissue context-dependence inducers over time (Calcinotto et al., 2019). Thus, we evaluated both SNC-reducing and senomorphic activities of HCW9218 in naturally aged mice.   First, we evaluated whether HCW9218 exhibited immune cell stimulatory activity in aged mice which are reported to exhibit immunosenescence (Budamagunta et al., 2021). To determine the impact of HCW9218 on the immune cells of aged and young mice, we performed mass cytometry utilizing an antibody panel (Table S3) that focuses on lymphocytes (B cells, CD4 + , CD8 + T cells) and group CD4 + T cells in aged and young mice also increased on D4 following HCW9218 treatment ( Figure S2J). There was no effect of HCW9218 on Treg frequency ( Figure S2K). These findings were further con- Collectively, these findings provide evidence that HCW9218 treatment effectively stimulates and promotes the proliferation of NK cells and CD8 + T cells and improves the fitness (i.e., metabolic functions) of these immune cells in lymphoid tissue and liver of naturally aged mice.

| H C W9218 REDUCE S S N C s AND SA S P IN PERIPHER AL ORG AN S IN NATUR ALLY AG ED MI CE
We next examined long-term changes in the expression of inflammation and senescence-associated genes in aged mice (76 weeks) receiving either one or two subcutaneous doses of HCW9218 (3 mg/kg) or PBS (control). RNA-Seq analysis was performed on the liver isolated at 60 or 90 days after HCW9218 treatment to determine global changes in transcription. Significant differentially expressed genes were clustered by gene ontology, and enrichment of gene ontology terms was tested using the Fisher exact test (GeneSCF v1.1-p2). The liver of HCW9218-treated aged mice showed dramatic changes in gene expression with 539 differentially expressed mRNAs compared to PBS-treated mice ( Figure 4a).
Expression of gene sets associated with glucose and fatty acid metabolism (e.g., Angptl4, Gos2,C1qtnf12,Fads2,Sorbs1,Zbtb16,Mvd,Scd1,Acaca,Acacb,Abcb1c,Abca6,Acly,Eci3,Ugt1a5,Ugta6,Ugta9,Acsl3,Lss,Acss2os,and Plin4), fibrosis (e.g., Col4a3, Col20a1, Jund, and Thbs1), and other liver functions (e.g., Dbp, Tef, and Acss2) was also altered with HCW9218 treatment (Figure 4c). Unexpectedly, the RNA-seq analysis also found that transcripts of circadian molecular clock repressor genes (orchestrating circadian rhythms) were altered 60 days after HCW9218 treatment ( Figure 4d). Expression of circadian repressor genes Per, Cry, Nr1d1, Nr1d2, and Dbp was upregulated by the HCW9218 treatment ( Figure 4d). 60 days after two-dose HCW9218 treatment, the repressor genes were found to be upregulated similarly to a single-dose treatment, but expression of the activator genes Arntl and Npas2 was downregulated Reduction of IL-1α, IL-6, and IL-8 in the liver was also confirmed at protein levels by ELISA ( Figure 5). In a two-dose treatment regimen, we also found that HCW9218 lowered biomarkers PAI-1 and fibronectin (Figure 5f), suggesting that HCW9218 could reduce liver fibrosis in aged mice, consistent with significant downregulation of Col4a3 and Col20a1 expression observed in the above RNA-seq study. In addition to gene expression signatures in solid peripheral (k, l) Total CD8 + T-cell frequency in the liver (k) and spleen (l) at Day 4 and Day 10. (m, n) Representative data for increase in extracellular acidification rates (ECAR) (m) and oxygen consumption rates (OCR) (n) data from splenocytes stimulated in vivo with HCW9218 from young and aged mice by Seahorse XFe bioanalyzer compared to control. (o) Measuring the ex vivo cytotoxic activity on Yac-1 target cells by in vivo HCW9218-stimulated splenocytes from young and aged mice compared to controls (PBS or HCW9228) by flow cytometry. Individual value plot shows the mean (n = 6/group) from two independent experiments. (p, q) Increase in IFNγ and TNFα released by CD3 + cells upon antigen-independent stimulation by in vivo HCW9218 treated and ex vivo α-CD3/α-CD28 beads stimulated splenocytes compared to control aged and young mice measured by MAGPIX multiplexing system. Individual value plot show the mean (n = 5/group) from two independent experiments. (r) Representative flow cytometry data showing increase in percentage of intracellular Granzyme B markers in liver ILC-1 and NK cells in young and aged mice treated with HCW9218 or controls. Individual value plot shows the mean (n = 6/group) from two independent experiments. p values were determined by ordinary one-way ANOVA with Tukey's multiple comparisons test.
organs, we also found a long-lasting impact of HCW9218 on enhancing splenocytes' glycolysis and mitochondrial respiration, which continued 45 days after treatment with the second dose (Figure 5g,h, Figure S5A,B).
The durability of the SNC-reducing and senomorphic activities of HCW9218 treatment on gene expression was further evaluated using bulk RNA-seq analysis of liver from aged mice isolated at 120 days. Remarkably, we continued to observe significant downregulation (e.g., Cdkn1a) or upregulation (e.g., Tert) of senescence and inflammation associated (Figure 5i,j and Table S2) genes (e.g., cytokine: Il7, Il15, Il18, S100a1, S100a4, S100a6, S100a10, S100a16, S100g and Dbp, were still upregulated 120 days after HCW9218 treatment ( Figure 5k). However, following 120 days after two-dose HCW9218 treatment, the expression of the activator gene Arntl was upregulated and the effects on Npas2 expression became insignificant compared with the HCW9228 treatment ( Figure S6B).
To further evaluate whether the TGF-βRII component of HCW9218 exhibited SNC-reducing and senomorphic functions, we treated young mice with a single-dose of HCW9228 and conducted the RNA-Seq analysis on the livers 10 days after treatment.
As shown ( Figure S6A), HCW9228 treatment significantly lowered expression of Cdkn1a and many circadian clock genes in the liver.
A comparison of the impacts of HCW9218 and HCW9228 120 days after treatment was also performed ( Figure S6B). RNAseq analysis of liver from treated mice showed that HCW9218, but not HCW9228, maintained the downregulation of Cdkn1a expression ( Figure S6B), while both treatments continued to upregulate Tert gene expression compared with PBS treatment. Interestingly, HCW9228 treatment significantly increased circadian molecular clock activator genes Arntl and Npas2 compared to HCW9218treated or the control group ( Figure S6B). Since HCW9228 did not activate or promote the proliferation of immune cells ( Figure S2A,B Figure 6a-c, and Figure  Ang; see hepatocyte cluster 1, Figure 6c). Hepatocyte cluster 2 identified genes associated with the metabolic pathway were Atp6v1h, Cth, G6pc, Got1, Hal, Bach2, and Lipc genes which were downregulated by HCW9218 whereas the cholesterol metabolism genes Apoa1, Apoe, Cyp7a1, and Tomm40l were upregulated by HCW9218.
In addition, the PAR signaling pathway genes Apoa1 and Cyp7a1 were found upregulated by HCW9218. Similarly, Srsf7, G0s2, Tkfc, and Cebpb involved in transcriptional regulation were upregulated, whereas the Auts2, Bhlhe40, Map2k6, Arhgap24, and Trp53inp1 genes were downregulated by HCW9218 (Figure 6d). Genes grouped in the cluster 2 also overlapped with genes identified the hepatocyte cluster 0 and cluster 1 (Figure 6b,c). There was no significant difference in expression between genes detected in the hepatocytes cluster 4 for HCW9218 and PBS control (Figure 6a). However, in hepatocyte cluster 5 (Figure 6e), the metabolism associated gene

Il6
F I G U R E 5 Two-dose HCW9218 stimulates metabolic functions and reduces inflammation (SASP) and cellular senescence markers in naturally aged mice liver for an extended time. (a) Schema of two-dose HCW9218 treatment in naturally aged (76 weeks) female C57BL/6 mice that were subcutaneously injected with 3 mg/kg of HCW9218 (n = 5-8) or saline. Mice received second dose of HCW9218 at Day 60 and were euthanized at Day 120. (b, c) Relative mRNA expression of Il1α, Pai-1, Il6, and Tnfα in kidney and Il1β, Il1α, PAI-1, Il6, and Tnfα in liver was analyzed by quantitative PCR after treatment with HCW9218 compared to control at Day 10 and/or Day 60. Individual value plot shows the mean from two independent experiments. (d) Relative mRNA expression Il1α Cdkn1a, Pai-1, Il1b, and Il6 in liver after treatment with HCW9218 one or two doses compared to control at Day 120 determined by quantitative PCR. Individual value plot shows the means (n = 8/group). (e, f) ELISA data showing protein levels of IL-1α, IL-6, IL-8, PAI-1, and fibronectin in liver tissue by ELISA liver after treatment with HCW9218 one or two doses compared to control at Day 120. Individual value plot show the mean of 10 mice per group in an experiment. (g, h) Representative data for the increase in extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) data from splenocytes stimulated in vivo with day 60 (g) and day 120 (h) measured by Seahorse XFe bioanalyzer compared to control. p values were determined by Student's t tests for two group comparison and p values were determined by ordinary one-way ANOVA with Tukey's multiple comparisons test where there are more than two groups. (i-k) Two-dose HCW9218 stimulates metabolic functions and reduces inflammation (SASP) and cellular senescence markers in naturally aged mice liver for extended time by bulk RNA-Seq analysis. Heat maps of the differentially expressed inflammation (i), senescence (j), and circadian rhythm (k) associated genes in liver after treatment with HCW9218 compared to control treatment plotted in fold changes (adjusted p value <0.05).
potent immunotherapeutic senomorphic and SNC-reducing agent for reducing SASP and clearing senescent cells, respectively.

| H C W9218 AND H C W9228 SUPP ORT THE MAINTENAN CE OF PHYS IC AL PERFORMAN CE OF NATUR ALLY AG ED MI CE
Studies have shown that the elimination of accumulated senescent cells can improve the cognitive functions and alleviate sarcopenia of aged mice (Kirkland & Tchkonia, 2017 (Figure 7c), which allowed the mice to freely explore their environment while measuring ambulation, HCW9218 treatment was associated with a significantly improved distance traveled (p = 0.0064), while HCW9228 treatment yielded a nearly significant result (p = 0.0566) compared to PBS treatment. Likewise, there was a significant effect of HCW9218 treatment, but not HCW9228 treatment, on speed (total velocity; p = 0.0111). Our results collectively suggested that both HCW9218 and HCW9228 treatments provided better overall support for neuromuscular and motor performance than PBS over a long period of time in naturally aged mice.

| LONG -TERM SAFE T Y AND TOLER AB ILIT Y OF H C W9218 IN MI CE
Considering the diverse physiological roles of SNCs in tissue homeostasis, the potential adverse effects of their removal must be considered. Thus, we conducted short-term and long-term toxicity studies of HCW9218 treatment in mice. Similar to results described previously (Liu et al., 2021), subcutaneous administration of HCW9218 at 5-100 mg/kg in two doses at a 14-day interval was well tolerated in a GLP toxicity study in C57BL/6 mice with no ob- We could not demonstrate that the two-dose treatment regimen provided a significant increase in the lifespan of naturally aged mice.
HCW9218 contains human-derived components and is known to be immunogenic inducing high titer of anti-drug antibodies after two doses of treatment. This prohibited us from evaluating whether a more frequent dosing regimen could provide a survival advantage to naturally aged mice treated with HCW9218.
Overall, HCW9218 treatment was well tolerated by mice at dose levels significantly higher than the therapeutic level (3 mg/kg) we employed in this study, and no long-term overt adverse effects of HCW9218 treatment were observed on the healthspan of naturally aged mice.

| DISCUSS ION
Cellular senescence, a state of irreversible arrested proliferation, plays a pivotal role in various physiological processes. However, the induction and accumulation of SNCs, caused by a variety of stressors promoting chronic inflammation through their accompanied SASP, are implicated in a wide spectrum of aging-related diseases.
Therefore, interest is rapidly growing in targeting SNCs to improve healthy aging and age-related diseases (Gasek et al., 2021). Senolytic Recently, chimeric antigen receptor T cell and antibody-based targeting approaches to extracellular targets for immune-mediated clearance of SNCs have been reported with promising results in animal models (Amor et al., 2020). However, significant challenges for these approaches include the highly heterogeneous transcriptome of SNCs due to a dependence on different stressors and cell types and temporally dynamic changes in gene expression profiles. As a result, so far there is no unique cell-surface marker identified for targeting SNCs. On the contrary, there is evidence showing that cellular senescence can be controlled by immune response leading to senescent cell elimination (Papismadov & Krizhanovsky, 2021). SNCs upregulate NKG2D ligands on their surface, which are recognized by NK cells and NKT cells for cell-mediated killing (Brighton et al., 2017;Kale et al., 2020).
In a recent study, we showed that HCW9218 promotes NKcell-mediated clearance of chemotherapy-induced cancer and normal tissue SNCs (Chaturvedi et al., 2022). Herein, we clearly demonstrate that subcutaneously administered HCW9218 potently activates and promotes the fitness of NK and CD8 + T cells in metabolically dysfunctional and naturally aged mice. of SASP by removing p21 + SNCs is expected to lessen the systemically deleterious paracrine effects of SASP in islet β cell dysfunction. Interestingly, we observed that the expression of Tert was upregulated by HCW9218 treatment in the aged mice. TERT is implicated in telomere attrition causing replicative senescence in aged mice. It should also be noted that, unlike in TIS cells, we did not observe downregulation of Cdkn2a expression, another major pathway leading to cellular senescence, with HCW9218 treatment in naturally aged mice. In human fibroblasts, p21 was decreased after senescence establishment and p16 is upregulated for senescent-cell-cycle arrest maintenance (Stein et al., 1999).
Thus, it seems puzzling why we observed p21 + but not p16 + -cell reduction 6 months after two doses of HCW9218 treatment in naturally aged mice. We hypothesize that this may be the result of a difference in the magnitude or duration of p21 + versus p16 + -cell clearance induced by HCW9218 treatment. Six months after the two-dose regimen of HCW9218, the levels of p16 + cells but not the p21 + cells accumulated back to the pre-HCW9218 treatment level. It has been shown that genetic ablation of p16 + cells in a transgenic mouse model could increase the lifespan of the mice versus their p16 wild-type counterparts (Baker et al., 2016). In our study, the two-dose regimen of HCW9218 treatment did not increase the lifespan of naturally aged mice compared to untreated mice, although HCW9218 treatment provided major benefits in the improvement of physical strength to naturally aged mice without compromising their healthspan. We are initiating additional studies to examine whether a prolonged multiple-dose regimen of HCW9218 could reduce p16 + cells in the organs and prolong the lifespan of naturally aged mice. Further studies also are needed to explore whether cellular senescence induction in naturally aged mice is similar to TGF-β1 induced cellular senescence in glomerular endothelial cells via CDKN2A (p16) translocation from cytosol to nucleus (Ueda et al., 2021). A recent study showed that clearance of p21-but not p16-positive senescent cells prevented radiationinduced osteoporosis and increases marrow adiposity (Chandra et al., 2022). Therefore, the relevance of p21 and p16 in cellular senescence induction in naturally aged mice should be thoroughly evaluated.
In naturally aged mice, activation of the immune system and neutralization of TGFβ by HCW9218 administration correlated with a durable reduction of SNCs and SASP in peripheral organs.
Group 1 ILCs are tissue-resident immune cells comprised of both NK cells and ILC-1s and are particularly enriched in the liver. IL-15activated NK and ILCs exhibit cell-mediated cytotoxicity against target cells through granule exocytosis (Chaturvedi et al., 2022;Nixon et al., 2022). In this study, we showed that HCW9218 potently stimulated, promoted expansion, and enhanced the cytotoxicity of group 1 ILCs in the livers of aged mice. It is known that these cells can positively and negatively influence adaptive immune responses (Bal et al., 2020). Thus, while we presume the granzymemediated cytotoxicity or the secreted effector molecules (e.g., IFNγ) of HCW9218-activated group-1 ILCs are responsible for the SNCreducing activities, ILCs could also play an anti-inflammatory role in SASP. For example, it has been recently shown that group-1 ILCs regulate T cell-mediated liver immunopathology induced in hepatitis by controlling local IL-2 availability (Fumagalli et al., 2022).
Similar to diabetic mice, we observed effects of HCW9218 treatment on the liver transcriptome of aged mice. Besides the traditionally considered SASP proinflammatory genes, we observed a significant reduction of Lcn2, S100a8, S100a9, S100a11, Mt1, and Mt2 expression by HCW9218 treatment. LCN2 is released by various cell types and acts as a master mediator of intestinal and metabolic inflammation. In animal models of metabolic inflammation, T2DM, or nonalcoholic steatohepatitis, increased LCN2 expression promotes inflammation through the recruitment of inflammatory cells and induction of proinflammatory cytokines (Moschen et al., 2017).
Proinflammatory cytokines S100A8/S100A9/S100A11 are calciumbinding proteins that are upregulated in human cancer. They are involved in tumor growth, metastasis, angiogenesis, immune evasion, metabolic diseases, neurological diseases, and vascular calcification. shown that S100A8/S100A9 and S100A11 are recruited to numerous promoters and enhancers to act as transcriptional coactivation molecules. These signal transduction pathways activated by S100A8/ S100A9/S100A11 are the key pathways for SASP induction and are the targets for senomorphic drug development   (Liu et al., 2020). DBP and TEF play a pivotal role in xenobiotic detoxication, and PER3 functions as a potent mediator of cell fate by altering the transcriptional activity of PPARγ (Costa et al., 2011;Gachon et al., 2006). Interestingly, TGFβ acts as zeitgeber for peripheral clocks and has been recently identified as a peripheral coupler that mediates paracrine-phase adjustment of molecular clocks through transcriptional regulation of core-clock genes in vitro (Finger et al., 2021;Kon et al., 2008 -1, SERPINE1). PAI-1 is not only a biomarker of cellular senescence but also is necessary and sufficient for replicative senescence downstream of p53 and is a key inducer of the senescence program (Hiebert et al., 2018;Kortlever et al., 2006). There is evidence for a PAI-1/TGF-β1-positive feed-forward mechanism that supports tissue levels of TGF-β1 during the emergence of the senescent phenotype stimulating expression of PAI-1 that, in turn, reinforces continued TGF-β1 synthesis (Seo et al., 2009) Zn finger transcription factor, to bind to the distal and proximal regions of Cdkn1a, respectively, activating p21 expression (Pardali et al., 2000;Seoane et al., 2004). However, the use of TGFβRII alone (HCW9228), as shown in RNA-seq analysis, may have a more limited impact on SNCs and SASP ( Figure S3C) when compared to HCW9218 treatment.
SNCs play an important role in a variety of biological functions. Therefore, one concern regarding senolytic and senomorphic drugs is that their activity could reduce the potential benefits of SNCs and SASP. In a dose-ranging GLP toxicology study in mice, two-dose subcutaneous administration of HCW9218 did not result in any overt adverse events even at high dose levels. In a long-term multidose study in 72-week-old mice, we also did not observe any adverse differences in the healthspan of HCW9218-treated versus un-treated animals more than 6 months after receiving treatment, although the fasting glucose level was significantly lowered in the treatment group. This is likely the result of the combined effect of HCW9218 treatment to modulate gluconeogenesis and lipidmetabolism genes and to lower Angptl4 expression. Hepatocytespecific suppression of Angptl4 expression was recently shown to improve obesity-associated diabetes and mitigate atherosclerosis in mice (Singh et al., 2021).
We have previously shown that IL-15/IL-15Rα agonists are potent activators of immune cells, particularly NK cells (Rhode et al., 2016). Therefore, IL-15 agonists are expected to support immune-cell-mediated clearance of senescent cells if these agonists exhibit similar pharmacokinetic profiles as HCW9218. This is consistent with our observations in this study that some of the beneficial effects of HCW9218 are ameliorated when mice were treated with HCW9228 which lacks IL-15 activities. However, since TGFβ is a potent immunosuppressive cytokine and the master regulator of SASP, HCW9218 with combined TGFβ neutralizing and immune-cell activation capabilities could be a more effective immunotherapeutic than IL-15 agonists for senescentcell removal and SASP alleviation.
In conclusion, we have shown that a non-targeted immuno-

ACK N OWLED G M ENTS
We like to thank University of Miami, Histology Lab for tissue section and embedding and University of Colorado for tissue staining.
We also like to thank Genewiz for bulk RNA-Seq and Singulomics for single nucleus cell RNA-Seq.  Unrelated to this work, TAF consults for Affimed (SAB) and advises (equity interest) Indapta and OrcaBio.

DATA AVA I L A B I L I T Y S TAT E M E N T
The proprietary materials are available in limited quantities at a reasonable cost to the scientific community for non-commercial pur-