Therapeutic use of sting and tlrs agonists to induce p16 expression in immune cells

Abstract

The present invention relates to the use of a STING agonist, of a TLR5 agonist or of a TLR7 agonist for enhancing the number of p16high immune cells in vitro or in a patient in need thereof, in particular to extend health span and protect tissues against aging. It is also herein reported that p16high immune cells surprisingly play a key role in establishing disease tolerance, and can be useful for counteracting different lethal conditions, including LPS-induced sepsis, acute lethal SARS-CoV-2 infection, cancer and ionizing irradiation. The present invention thus relates to a pharmaceutical composition comprising autologous or heterologous p16high immune cells such as p16high CAR-T cells, and its use for various therapeutic purposes. These cells are preferably obtained by contacting them with a STING agonist, a TLR5 agonist and / or a TLR7 agonist.

Description

[0001] Therapeutic use of STING and TLRs agonists to induce p16 expression in immune cells

[0002] SUMMARY OF THE INVENTION

[0003] It is herein reported that p16highimmune cells surprisingly play a key role in establishing disease tolerance, and can be useful for counteracting different lethal conditions, including LPS-induced sepsis, acute lethal SARS-CoV-2 infection, cancer and ionizing irradiation. Mechanistically, it is shown that activation of the TLR7 and / or TLR5 and / or STING pathways induces an increase in p16highimmune cells subsets that, in turn, establishes a low adenosine environment and disease tolerance. Maintaining a beneficial level of p16highimmune cells by activating these pathways is shown here for the first time to advantageously delay organ deterioration upon aging and extend healthspan.

[0004] DESCRIPTION OF THE PRIOR ART

[0005] The ability of an organism to overcome infectious diseases has traditionally been linked to killing invading pathogens. Recent evidence, however, indicates that, apart from restricting pathogen loads, organismal survival is coupled to an additional yet poorly understood mechanism called “disease tolerance”. This defense mechanism relies on the concerted action of both innate and adaptive immunity but also is coupled to an important but poorly understood defense strategy that limits the extent of tissue damage. Instead of directly affecting the pathogen load, tolerance decreases the host susceptibility to tissue damage, or other fitness costs. Although tolerance mechanisms are largely unknown, they are expected to prevent, to reduce, or counter the pathological alterations caused by infections or other inducers of tissue damages including autoimmune diseases, stress or aging (Medzhitov, R., Schneider, D.S., and Soares, M.P. (2012). Science 335, 936-941).

[0006] Disease tolerance is known to vary across different ages, very young and very old people being the less tolerant to infections or other diseases. In the aged persons, frailty may be an extreme case of decline in tolerance, though its causes are not well understood. Antiaging interventions and treatments for aging-related diseases face great challenges. An important way to achieve healthy aging is through early intervention and prevention. Research on the latest therapeutic approaches, such as stem cell transplantation, elimination of senescent cells, promotion of anti-aging factors expression and inhibition of pro-aging factors expression, and tissue or organ’s regeneration provides new directions for treatments of aging-related diseases (Soares, M.P., Teixeira, L, and Moita, L.F. (2017). Nat. Rev. Immunol. 17, 83-96). Research on the mechanisms of aging and disease tolerance has thus an important role to play in improving human health and prolonging lifespan. Due to the aging of the global population, antiaging and healthy aging pursuits an undoubtedly important task for public health organizations, scientific research departments and drug research and development departments.

[0007] In this context, there is therefore a need to identify new compounds to enhance disease tolerance and to extend lifespan, so as to prevent or postpone the general decline of physiological fitness and loss of tissue homeostasis and the onset and progression of multiple diseases, including cancer, diabetes, Alzheimer's, and osteoarthritis (Guo J et al., 2022, Signal Transduction and Targeted Therapy, 16;7(1):391).

[0008] Different mechanisms have been proposed to contribute to this natural phenomenon such as telomere attrition, mitochondrial dysfunction, and enhanced inflammation (Lopez-Otin C et al., 2012, Cell 153, 1194-1217). Importantly, many of these mechanisms can be directly linked to the induction of cellular senescence (Hayflick L et al, 1961 , Exp Cell Res 25:585-621 ; Hernandez-Segura A et al., 2018, Trends Cell Biol. 28, 436-453 and LeBrasseur NK. Et al., 2015, Nestle Nutr Inst Workshop Ser. Epub 83:11-8), a well-characterized response that hinders cell cycle progression through the activation of the tumor suppressor genes CDKN1A (p21) or CDKN2A (p16) (Serrano M. et al., 1995, Science 267, 249-252; Noda A. et al., 1994, Exp. Cell Res. 211 , 90-98 ; Serrano M. et al., 1993, Nature 366, 704-707 ; Hara E. et al., 1996, Mol. Cell. Biol. 16, 859-867). Substantial experimental evidence suggests that the accumulation of senescent cells is an important factor in age-related tissue deterioration (Baker DJ. Et al., 2011, Nature 479, 232-236 and Childs BG. Et al., 2017, Nat. Rev. Drug Discov. 16, 718-735) as it is associated with the production of different molecules capable of restructuring the extracellular matrix, modifying the behavior of neighboring cells and systemically affecting the activity of immune system (Di Micco R. et al., 2021 , Nat. Rev. Mol. Cell Biol. 22, 75-95; Wang TW. et al., 2022, Nature 611, 358-364 and Tchkonia T. et al., 2013, J. Clin. Invest. 123, 966-972). For example, senescence cells accumulate in adipose tissue of patients with diabetes and age-related metabolic dysfunction, in osteoarthritic joints, in the aorta in vascular hyporeactivity and atherosclerosis and in the lungs in idiopathic pulmonary fibrosis. Moreover, the senescence-associated secretory phenotypes (SASPs), which are a mix of pro-inflammatory cytokines, proteases and growth factors, are upregulated and secreted by senescent cells and are part of the extrinsic arm of cellular senescence. In this context, selective elimination of p16+senescent cells was proposed to confer benefits in aging tissues and to extend lifespan (Baker DJ. et al., 2011, Nature 479, 232-236; Baker DJ. et al., 2016, Nature 530, 184-189). All these studies highlight the fact that p16 overexpression in certain tissues may reduce lifespan. Nevertheless, it has also been shown that the presence of p16+senescent cells are critical for tissue repair and favor wound healing (Demaria M. et al, 2014, Dev. Cell, 31 , 722- 733). Also, p16 regulates stem cell self-renewal processes in a variety of tissues, and its disruption has been shown to result in aging or tumor development (D’Arcangelo D. et al., 2017, Int. J. Mol. Sci., 18, 1591). Such studies highlight the fact that p16 overexpression may also have a beneficial effect on lifespan.

[0009] In front of these contradictory results, it remains a need to elucidate what is the role of p16 in immune cells in particular, and what can be the role of p16highimmune cells in human health.

[0010] DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention is based on the surprising realization that a number of different Pattern-Recognition Receptors (PRR) agonists can activate p16 expression in immune cells, and that p16highimmune cells play a key role in establishing an early and broad disease tolerance in response to multiple lethal conditions following severe inflammation and tissue damage, such as sepsis, SARS-CoV-2 infection and ionizing radiations.

[0012] More precisely, it is shown in the examples below that at least two PRR agonists leading to p16 increased expression in immune cells are able to play a key role in broad and early disease tolerance in response to lethal conditions following severe inflammation and tissue damage due to sepsis (example 5, example 15, example 18, example 19), radiations (example 7), viral infections (examples 7 and 12), cancer (examples 14 and 15), thereby promoting disease tolerance and extending health span (example 11), even after cell transplantation (example 16).

[0013] Pattern-recognition receptors agonists of the invention

[0014] Pattern recognition receptors (PRRs) are a class of receptors that can directly recognize the specific molecular structures on the surface of pathogens, apoptotic host cells, and damaged senescent cells. PRRs bridge nonspecific immunity and specific immunity. Through the recognition and binding of ligands, PRRs can produce nonspecific anti-infection, antitumor, and other immunoprotective effects. Most PRRs in the innate immune system of vertebrates can be classified into the following five types based on protein domain homology: Toll-like receptors (TLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene-l (RIG-l)-like receptors (RLRs), C-type lectin receptors (CLRs), and absent in melanoma-2 (AIM2)-like receptors (ALRs). These receptors are basically composed of ligand recognition domains, intermediate domains, and effector domains. They recognize and bind their respective ligands and recruit adaptor molecules with the same structure through their effector domains, initiating downstream signaling pathways to exert effects. In recent years, the increased researches on the recognition and binding of PRRs and their ligands have greatly promoted the understanding of different PRRs signaling pathways and provided ideas for the treatment of immune-related diseases and even tumors.

[0015] Nucleic acid sensors, which can detect extracellular or intracellular DNA or RNA as damage-associated molecular pattern signals, are the essential part of the PRRs as they can induce type I interferons (IFNs). The secreted Type I IFNs will act on producing and neighboring cells via IFNa / preceptor 1 or IFNAR1-IFNAR2 heterodimer and support cytotoxic T lymphocytes (CTLs) via several mechanisms. Three families of molecules are now well described to play an essential role in nucleic acid sensing: the retinoic acid-inducible gene-l (RIG- 1) like receptors including the melanoma differentiation-associated protein 5 (MDA5), tolllike receptors (including TLR7 and TLR5) and sensors of the cGAS-STING pathway (for more details, see the review of Chen M. et al., 2020, Signal Transduction and Targeted Therapy, 5:270).

[0016] Toll-like receptors (TLRs) are a class of pattern recognition receptors that play a bridging role in innate immunity and adaptive immunity. They can recognize both pathogen- associated molecular patterns and damage-associated molecular patterns such as lipopolysaccharide and free nucleic acids. Normally, they are expressed by macrophages, dendritic cells, natural killer (NK) cells and epithelial cells. Some TLRs are expressed in the intracellular endosomes (TLR3, 7, 8, and 9), while others are localized on the cell membrane (TLR1, 2, 4, 5, 6, 10, and 11). TLRs are activated by diverse agonists, e.g., TLR4 by lipopolysaccharide, and TLR3, 7 and 9 by nucleic acids. The binding of a TLR and its agonist generates an immune response, important for therapeutic research. Most of the TLR agonists are clinically tolerated and biologically active, even though some common adverse events have been observed (Chi H. et al, 2017, Frontiers in Pharmacology, 8:304).

[0017] TLR7 agonists

[0018] Among TLRs, TLR7 is an intracellular receptor expressed on endosomal membranes. TLR7 is closely related to TLR8, which also recognizes nucleosides and nucleotides from intracellular pathogens. There are two ligand-binding sites in TLR7. The first site for binding of small ligands is conserved in both TLR7 and TLR8. The second site differs from that of TLR8, and is used to bind with ssRNA to enhance activation of the first site. Activation of TLR7 can induce Type I interferon and inflammatory response, therefore targeting TLR7 is a promising strategy for both antiviral and antitumor therapy. The present invention relates to a new use of the toll-like receptor 7 (TLR7) agonist. The present inventors found for the first time that administration of a TLR7 agonist can increase the number of p16hi0himmune cells in animals, what plays a key role in broad and early disease tolerance in response to lethal conditions following severe inflammation and tissue damage (examples 4-8, and 12-19). Thus, TLR7 agonists are herein shown to be potent inducers of p16highimmune subsets in vivo.

[0019] As used herein, the term “TLR7 agonist” designates a compound (either a small organic molecule or a nucleic acid) that binds to the human TLR7 receptor and activates the signaling pathway through this receptor. Preferably, the interaction of the agonist of the invention with other human TLRs is weaker as compared to its interaction with human TLR7. Nucleotide and amino acid sequences of human and murine TLR7 are known (see, for example, GenBank Accession Nos. AF240467, AF245702, NM_016562, AF334942, NM_133211; and AAF60188, AAF78035, NP_057646, AAL73191 and AAL73192). TLR7 polypeptides include an extracellular domain having a leucine-rich repeat region, a transmembrane domain, and an intracellular domain that includes a TIR domain.

[0020] The agonist effect of a compound toward TLR7 can be detected by any conventional means, e.g., by measuring the activation of NF-KB and mitogen-activated protein kinases (MAPKs) and / or the expression of pro-inflammatory cytokines and costimulatory molecules and / or the translocation of the interferon-regulatory factor 7 (IRF7) into the nucleus, where it can induce the transcription of type I IFN genes in various immune cells, in particular in plasmacytoid dendritic cells (pDCs). Specifically, detection of IFNa or TN Fa can be used as an easy readout for TLR7 activation.

[0021] Preferably, the TLR7 agonist used in the invention is capable of increasing the number of p16hi0hmacrophages, p16hi0hT cells, p16hi0hB cells, p16hi0hneutrophils, p16hi0hmonocytes and / or p16hi0hdendritic cells, more preferably of p16highT cells and / or p16highmacrophages, in patients to which it is administered.

[0022] A number of TLR7 agonists have been already described, all of them being herewith encompassed. The most studied TLR7 agonists small molecules are imidazoquinolines and benzazepines. As preferred TLR7 small molecules that are useful in the context of the invention, one can cite : Imiquimod (also called Zyclara, Aldara or R-837), Resiquimod (also called R848, or EIK1001 or BDB001), Gardiquimod, 852A, loxoribine, bropirimine, 3M-011 , 3M-052, DSR-29133, SC1 , SZU-101 , SM-360320, and SM-276001 (the references of the studies describing these molecules are given in Table 1 of Chi H et al, 2017). Other TLR7 agonists have been disclosed and could also be used in the context of the invention: R07020531 (Yuen MF. et al. 2023, Lancet Infect Dis. 23(4) :496-507), DSP-0509 (Ota Y. et al., 2023, Front. Immunol. 14 : 1055671), 1 171CAN1012 (Yu H. , 2022, http: / / digitalcommons.psjhealth.org / publications / 6733), 1V209 (CAS number : 1062444-54-5), CL307 (CAS number: 1548551-79-6), CL075 (CAS number: 256922-53-9), CL097(CAS number 1026249-18-2), CL264 (CAS number : 1510712-69-2), poly(dT) (thymidine homopolymer ODN). These compounds are commercially available as research tools.

[0023] It is also possible to use the compound R837 or the compound R848 which have both been used in the examples below and were tested already in clinical trials in humans for cancer treatment, including basal cell carcinoma (R837) or cutaneous T cell lymphoma (R848), see https: / / www.cancer.gov / research / participate / clinical-trials / intervention / imiquimod or https: / / www.cancer.gov / research / participate / clinical-trials / intervention / resiquimod-hydrogel- based-sustained-release-formulation.

[0024] Another agonist to be considered is a formulated single-stranded RNA (ssRNA) like ssRNA40 (TLR7 / 8 agonist), which can be encapsulated.

[0025] Preferably, the TLR7 agonists used in the invention are chosen among imiquimod (1- (2-Methylpropyl)-1 H-imidazole[4,5-c]quinoline-4-amine, also called R837 or Zyclara) or resiquinod (1-[4-amino-2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol, also called R848), because they have been shown to trigger the expression of p16 in immune cells (figure 2G, figures 16 and 17). This TLR7 agonist is preferably administered orally, intramuscularly, intranasally or intravenously.

[0026] Single-stranded nucleic acids and in particular single-stranded RNAs (ss-RNAs) are also efficient TLR7 agonists (Heil F. et al, 2004, Science, 303 (5663): 1526-9) that can therefore be used as such in the present invention. These single-stranded nucleic acids can be of natural or synthetic origin. It is well accepted that any ss-RNA enriched with guanosine and uridine sequences can have the ability to activate TLR7 if they reach the cytoplasm. For example, R-1075 is a natural (HIV) derived sequence able to activate TLR7. Single stranded RNA oncolytic viruses are also efficient inducers of TLR7. All these ss-RNAs can be used as TLR7 agonists in the context of the invention.

[0027] In a particular embodiment, the single-stranded nucleic acid used in the invention as TLR7 agonist is a mRNA vaccine. As used herein, the term “mRNA vaccine” or “messenger RNA vaccine” refers to a type of vaccine that uses a small piece of genetic material, specifically messenger RNA, to stimulate an immune response in the body. An mRNA vaccine is a vaccine that employs messenger RNA to instruct cells in the body to produce a protein similar to a pathogen, triggering an immune response and the production of protective antibodies. This technology has been utilized in the development of vaccines for various infectious diseases, including COVID-19. Preferably, the mRNA vaccine is administered intramuscularly or intraperitoneally.

[0028] More preferably, the TLR7 agonist used in the invention is the BNT162b2 mRNA COVID-19 vaccine. Indeed, as demonstrated in the examples, the BNT162b2 vaccine is capable of efficiently inducing p16hi0himmune cells, particularly in peripheral blood, peritoneal cavity and liver. As shown herein, the BNT162b2 vaccine provides broad and rapid tissue protection and disease tolerance in order to counteract, for example, severe cases of inflammation and tissue damage. Moreover, BNT162b2 induces the expression of p16highin different populations of tumor-associated immune cells, such as T cells, neutrophils, macrophages, natural killers and natural killer T cells (example 4). Importantly, BNT162b2 vaccine treatment induces a reduction in the tumor burden and area (examples 14 and 15) and disease tolerance (examples 12 and 16).

[0029] As used herein, the expression “BNT162b2 mRNA COVID-19 vaccine” refers to an mRNA vaccine developed by the pharmaceutical company Pfizer in collaboration with the biotechnology company BioNTech. This vaccine has been widely used globally in the effort to combat the COVID-19 pandemic. The BNT162b2 vaccine is based on messenger RNA (mRNA) technology. It contains genetic instructions that encode a modified form of the spike protein found on the surface of the SARS-CoV-2 virus, which causes COVID-19. When a person receives the vaccine, their cells use these instructions to produce the modified spike protein. The immune system recognizes this protein as foreign and mounts an immune response, including the production of antibodies. In the future, if the vaccinated person is exposed to the actual virus, their immune system is better prepared to fight the infection. BNT162b2 has shown high efficacy in preventing COVID-19 in clinical trials and has been authorized for emergency use or fully approved for vaccination in many countries. It has played a significant role in global vaccination campaigns to help control the spread of the virus.

[0030] The composition of the invention preferably contains an “effective amount” of said TLR7 agonist. This “effective amount” is for example a dose that is capable to induce the secretion of IFNy but preferably not TNFa by in vivo immune cells. This effective dose is for example of 30pg for the BNT162b2 vaccine.

[0031] TLR5 agonist

[0032] Among TLRs, TLR5 is a transmembrane receptor expressed by keratinocytes and immune cells such as T cells, macrophages and monocytes. It is an extracellular sensor activated mostly by bacterial flagellin whose interaction activates NF-KB signaling and triggers an innate immune response to the invading pathogen.

[0033] The present invention relates to a new use of the toll-like receptor 5 (TLR5) agonist. The present inventors found for the first time that administration of a TLR5 agonist can increase the number of p16highimmune cells in animals (figure 2G), what is likely to play a key role in broad and early disease tolerance in response to lethal conditions following severe inflammation and tissue damage (as for the TLR7 agonist used in examples 4-8, and 12-15). As used herein, the term “TLR5 agonist” designates a compound that binds to the human TLR5 receptor and activates the signaling pathway through this receptor specifically. By “specifically”, it is herein meant that said agonist of human TLR5 does not bind to other human TLR receptors, or that the interaction of the agonist of the invention with other human TLRs is very weak as compared to its interaction with human TLR5. Amino acid sequences of human and murine TLR5 are known (see, for example. GenBank Accession Nos. NP_003259, NP_058624).

[0034] The TLR5 signaling cascade is commonly triggered by the binding of bacterial flagellum to TLR5 on the cell surface. Binding of flagellum induces the dimerization of TLR5, which in turn recruits MyD88 and Mal / TIRAP. The recruitment of MyD88 leads to subsequent activation of IRAK4, IRAKI , TRAF6, and eventually IKB kinases. Activation of IKB kinases contributes to the nuclear localization of NF-KB. NF-KB induces many downstream gene expressions, which initiates the canonical proinflammatory pathway. This TLR5 / flagellum interaction results in different responses in difference cell types. In epithelial cells, binding of flagellum to TLR5 induces IL8 production. In human monocytes and dendritic cells, this interaction results in the secretion of proinflammatory cytokines such as TNF.

[0035] Thus, the agonist effect of a compound toward TLR5 can be measured by any conventional means, e.g., by detecting the activation of NF-KB and IRAKs and IKB kinases and / or the expression of pro-inflammatory cytokines IL8 or TNF, depending on the cells which are used for the test.

[0036] Preferably, the TLR5 agonist used in the invention is capable of increasing the number of p16highmacrophages, p16highT cells, p16highB cells, p16highneutrophils, p16highmonocytes and / or p16highdendritic cells, more preferably of p16highT cells and / or p16highmacrophages, in patients to which it is administered.

[0037] A number of TLR5 agonists have been already described, all of them being herewith encompassed. As mentioned previously, the most well-known TLR5 agonist is flagellin. Alternative TLR5 agonist small molecules can however be used in the context of the invention, such as : Caveolin-1 (Lim JS., et al, Molecules and Cells. 38(12): 1 111 -7), CBLB502 (entolimod) and MAP1 S (the references of the studies describing these molecules are given in Table 1 of Chi H et al, 2017), or Heat Killed Salmonella typhimurium (HKST).

[0038] Preferably, the TLR5 agonist used in the invention is flagellin, because it has been shown to trigger the expression of p16 in immune cells (figure 2G).

[0039] As used herein, the term “flagellin” refers to a protein that makes up the filament of bacterial flagella, which are whip-like appendages that protrude from the surface of certain bacteria. These flagella are used by bacteria for locomotion, allowing them to move toward or away from stimuli in their environment. Flagellin is a crucial component of the bacterial flagellum structure.

[0040] The Flagellin used in the invention can be for example Flagellin from Salmonella typhimurium (FLA-ST), Flagellin from Bacillus subtilis (FLA-BS), or Flagellin from Pseudomonas aeruginosa (FLA-PA). It can be either recombinant or non-recombinant. This TLR5 agonist can be for example administered intranasally.

[0041] In the context of the invention, the TLR5 agonist can also be a flagellin derivative like the CBLB502 peptide (Bai H. et al, 2019, Biol. Reprod. 100(1):281-291).

[0042] STING agonist

[0043] Stimulator of interferon genes (STING), also known as “transmembrane protein 173” (TMEM173) and “MPYS”, “MITA”, “ERIS”, “NET23”, “SAVI”, “STING1”, “hMITA”, “hSTING”, “Stimulator of interferon genes”, “STING-beta”, or “stimulator of interferon response cGAMP interactor 1” is a transmembrane protein that in humans is encoded by the STING1 gene, which plays an important role in innate immunity. STING works as both a direct cytosolic DNA sensor (CDS) and an adaptor protein in Type I interferon signaling through different molecular mechanisms. It has been shown to activate downstream transcription factors STAT6 and IRF3 through TBK1, which are responsible for antiviral response and innate immune response against intracellular pathogen (Burdette DL et al, 2013, Nature Immunology. 14(1):19-26). This protein is localized intracellularly, in the endoplasmic reticulum membrane of hematopoietic cells in peripheral lymphoid tissues, including T lymphocytes, NK cells, myeloid cells and monocytes.

[0044] As the TLR receptors described above, STING mediates the type I interferon production in response to intracellular DNA and a variety of intracellular pathogens, including viruses, intracellular bacteria and intracellular parasites. More precisely, the STING protein is activated by the cyclic GMP-AMP synthase (cGAS) that binds to double-stranded DNA (dsDNA) directly. After binding to DNA directly, cGAS convert GTP and ATP into cyclic GMP- AMP (cGAMP) to form the second messenger 2’3’-cGAMP which will activate STING. Once bound to 2’3’-cGAMP, STING translocates from ER to Golgi to form a complex with TBK1 or IKK. STING-activated TBK1 is able to phosphorylate IRF3, promoting IRF3 dimerization and translocation to the nucleus where it induces the transcription of many inflammation genes especially IFN|3. On the other hand, STING-activated IKK could phosphorylate IKBO, which leads to the translocation of NF-KB to the nucleus, and then activates the transcription of pro- inflammatory cytokines (Chen et al., 2020).

[0045] The present invention relates to a new use of a STING agonist. The present inventors found for the first time that inhibition of the STING pathway impairs the increase of p16high immune cells in animals treated with TLR7 agonist (example 8). They also show that direct activation of STING with the specific agonist 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is sufficient to increase the number of p16Hi0himmune subsets including within Tregs as well as PD1- and PD-L1-positive T cells in different tissues (Figure 4E) and to protect mice from LPS-induced sepsis (figure 4G), when administered at low doses in animals.

[0046] This pathway is thus likely to play also a key role in broad and early disease tolerance in response to lethal conditions following severe inflammation and tissue damage (as the two TLR agonists disclosed above). Yet, importantly, the inventors’ results show that only low levels of STING activation could be beneficial in protecting mice from LPS-induced sepsis, because high doses of STING agonists are detrimental due to induction of excessive inflammation.

[0047] As used herein, the term “STING agonist” designates a compound that binds to the human STING receptor and activates the signaling pathway through this receptor. Preferably, the interaction of the agonist of the invention with other receptors is weaker as compared to its interaction with human STING. Amino acid sequences of human STING are known (see, for example. GenBank Accession Nos. NP_001288667; NP_938023; NP_001354187).

[0048] The agonist effect of a compound toward STING can be measured by any conventional means, e.g., by detecting the phosphorylation of IRF3, and / or its translocation to the nucleus, or the expression of I NFp, or the translocation of NF-KB to the nucleus or the expression of pro-inflammatory ctytokines in immune cells contacted with the compound. It is also possible to detect with adequate antibodies if the Ser366 of the STING protein is phosphorylated.

[0049] Preferably, the STING agonist used in the invention is capable of increasing the number of p16highmacrophages, p16highT cells, p16highB cells, p16highneutrophils, p16highmonocytes and / or p16highdendritic cells, more preferably of p16highT cells and / or p16highmacrophages, in patients to which it is administered.

[0050] A number of STING agonists have been already described, all of them being herewith encompassed. For example, the second messenger 2’3’-cGAMP, and a number of synthetic or natural double-stranded DNAs (dsDNAs) can be used in this respect. Preferably, the STING agonist used in the invention is 5,6-dimethylxanthenone-4-acetic acid (DMXAA), because it has been shown to trigger the expression of p16 in immune cells (figure 4).

[0051] The STING agonist is preferably administered subcutaneously, intramuscularly, intranasally or intravenously.

[0052] As shown in example 8, when 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is used, low doses should be administered to the patient, in order to avoid the induction of inflammation. Specifically, these doses would induce the secretion of IFNy but not of TNFa in the contacted immune cells. The p16 protein

[0053] As used herein, the term “p16” has its general meaning in the art and refers to the “p16INK4a” or “p16INK4” or “multiple tumor suppressor-1 (MTS-1)’’ or “cyclin-dependent kinase inhibitor 2a (CDKN2A)” protein, which is a 16 kDa protein encoded by the CDKN2A gene, within the INK4 / ARF tumor suppressor locus on Chromosome 9 (9p21.3). This protein (Ref Seq: NP_000068; Uniprot number P42771) plays a crucial role in the regulation of the cell cycle by inhibiting cyclin-dependent kinases (CDK4 and CDK6 and CDK2) that are required to phosphorylate the retinoblastoma protein (Rb). When p16 is expressed, the phosphorylation of Rb is impaired, what mainly prevents the cell transition from G1 to S phase and causes subsequent proliferation arrest (Safwan-Zaiter et al., 2022, Life 12, 1332). By this mechanism, p16 thus plays an important role in the initiation as well as in the maintenance of cellular senescence.

[0054] As used herein, the term “p16high” or “p16+” has its general meaning in the art and refers to “high” or elevated level or activity of the p16 protein as defined above, within the cells of interest for the present invention (namely, immune cells). As used herein, the term “p16low” or “p16'“ has its general meaning in the art and refers to low or reduced level or activity of the p16 protein as defined above, within the cells of interest for the present invention (namely, immune cells).

[0055] In particular, the designation “p16highcells” or “p16+cells” is used to describe immune cells exhibiting a high level or abundance or activity of the p16 protein as defined above. On another hand, the designation “p16lowcells” or “p16_cells” is used to describe immune cells exhibiting a low level or abundance or activity of the p16 protein as defined above.

[0056] The term “level” as used herein refers to the “expression level” of the p16 protein in the target cells of the invention. Specifically, it refers to the quantity, amount or concentration of the p16 protein expressed in the studied cells. The measurement of the level of p16 in cells can be carried out using standard protocols known in the art. For example, it can be performed by contacting the cells with a compound capable of selectively interacting with the p16 protein, for example with antibodies, such as, for example, monoclonal antibodies or even aptamers. The interaction may be detected by using a competitive immunoassay, a non-competitive assay system using techniques such as western blots, a radioimmunoassay, an ELISA (enzyme linked immunosorbent assay), a “sandwich” immunoassay, an immunoprecipitation assay, a precipitin reaction, a gel diffusion precipitin reaction, an immunodiffusion assay, an agglutination assay, a complement fixation assay, an immunoradiometric assay, a fluorescent immunoassay, a protein A immunoassay, an immunoprecipitation assay, an immunohistochemical assay, a competition or sandwich ELISA, a radioimmunoassay, a Western blot assay, an immunohistological assay, an immunocytochemical assay, a dot blot assay, a fluorescence polarization assay, a scintillation proximity assay, a homogeneous time resolved fluorescence assay, a lAsys analysis, and a BIAcore analysis. The aforementioned assays generally involve the binding of the interacting compound (e.g., antibody or aptamer) to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. Measuring the level of p16 (with or without immunoassaybased methods) may also include centrifugation based on the p16 molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on p16’s affinity for the particular solid-phase that is used. Alternatively, p16 may be detected and measured by, for example, a mass spectrometer.

[0057] “High” or “low” level of p16 is typically identified by comparing the p16-associated signal obtained in a defined amount of the studied cells with a pre-determined reference value. This pre-determined reference value is preferably obtained by measuring the p16-associated signal generated by the same amount of cells that are known to be expressing high levels of p16. Generally, a “reference value” is obtained from several subjects known to be free of the disease or, alternatively, from the general population. Yet, it can also be adjusted to specific subject populations in the context of the invention, e.g., to inflammatory patients or to young / aged subjects, in which p16 expression is known to be either high or low. The reference value or reference level can be an absolute value; a relative value; a value that has an upper or a lower limit; a range of values; an average value; a median value, a mean value, or a value as compared to a particular control or baseline value. A reference value can be based on an individual sample value such as, for example, a value obtained from a sample from the subject being tested, but at an earlier point in time. The reference value can be based on a large number of samples, such as from population of subjects of the chronological age matched group, or based on a pool of samples including or excluding the sample to be tested.

[0058] To define if immune cells of a tested sample I patient express “high” or “low” level of p16, one can compare the expression level of p16 in the tested sample with the expression level of p16 in a “reference sample” of immune cells known to have a high expression / activity of p16 (the number of immune cells in the samples should be obviously normalized).

[0059] It is well-known that p16hi0himmune cells are induced with age and in patients suffering from an inflammatory condition (see Safwan-Zaiter et al., 2022, Life, 12, 1332, and also example 1). Examples of “reference samples” containing p16hi0hcells are biological samples containing immune cells which have been obtained in patients older than 65, more preferably older than 68 or 70. Other “reference samples” can be for example collected from healthy persons at least 7 days after their vaccination with the BNT162b2 COVID-19 vaccine (Figure 7D). In this case, the reference value used in the context of the invention can be the mean value of p16 expression measured in the CD45+immune cells contained in these reference samples. If the p16 expression level in the CD45+immune cells of the patient is the same or higher than this reference value, then the immune cells of the patient would be qualified as “pi6high”. If the p16 expression level in the immune cells of the patient is lower than this reference value, then the immune cells of the patient would be qualified as “pi6low”. It is possible to determine the p16 expression level in target cells in reference to the expression of housekeeping genes (e.g., beta-actin) these expression levels being for example measured by western blot and immunofluorescence.

[0060] Assessing p16 activity in a tested sample can be performed by any conventional means, for example by measuring the activity of the cyclin-dependent kinases (CDK4 and CDK6 and CDK2) and / or the phosphorylation state of the retinoblastoma protein (Rb). As for p16 expression, the activity of p16 in a tested sample containing a define amount of immune cells can be compared to the activity of p16 in a reference sample containing the same amount of immune cells. This reference sample is, for example, a biological sample containing immune cells which have been collected in patients older than 65, more preferably older than 68 or 70, or a sample of immune cells collected from healthy persons at least 7 days after they have been vaccinated with the BNT162b2 COVID-19 vaccine (Figure 7D). If the activity of p16 in the immune cells of the patient is the same or higher than in the reference sample, then the immune cells of the patient would be qualified as “pi6high”. If the activity of p16 in the immune cells of the patient is lower than in the reference sample, then the immune cells of the patient would be qualified as “p16low”.

[0061] The designation “p16highpatients” or “p16+patients” is herein used to describe patients whose biological samples contain a high percentage of p16highcells as defined above. By “high percentage”, it is herein meant that the immune cells contained in the biological sample of the patient are predominantly p16high. Specifically, this means that more than 50%, 60%, 70%, 80%, or 90% of the immune cells contained in the biological sample of p16highpatients are p16highas defined above. Conversely, this means that less than 50%, 40%, 30%, 20%, or 10% of the immune cells contained in the biological sample of p16highpatients are p16lowas defined above.

[0062] Conversely, the designation “p16lowpatients” or “p16_patients" is used to describe patients whose biological samples contain a low percentage of these p16highcells. By “low percentage”, it is herein meant that few of the immune cells contained in the biological sample of the patient are p16high. Specifically, this means that less than 50%, 40%, 30%, 20%, or 10% of the immune cells contained in the biological sample of p16lowpatients are p16highas defined above. Conversely, this means that more than 50%, 60%, 70%, 80%, or 90% of the immune cells contained in the biological sample of p16lowpatients are p16lowas defined above.

[0063] An exemplary biochemical test for identifying the number of p16highimmune cells contained in a sample employs a standardized test format, such as ELISA test wherein the wells of a microtiter plate are coated with a set of antibodies which recognize immune cells. A sample containing p16highimmune cells (or not) is then added to the coated wells. After a period of incubation sufficient to allow the binding of the immune cells on the plate, the plate is washed to remove unbound cells. The lysis or permeabilization of the cells can be performed so as to permit the contact between a detectably labelled p16 binding molecule and intracellular p16. The plate is washed and the presence of the p16 binding molecule is detected using methods well known in the art.

[0064] As used herein, the term “immune cells” refers to natural killer (NK) cells, lymphocytes (e g., natural killer T (NKT) lymphocytes, T lymphocytes (also called T cells), and B lymphocytes (also called B cells)); phagocytes (e.g., macrophages, monocytes, and dendritic cells); granulocytes (e.g. basophils, eosinophils and neutrophils); mast cells, plasma cells, and memory cells. Preferably, the p16highimmune cells of the invention are p16highNK cells, p16highmacrophages, p16highT cells, p16highB cells, p16highneutrophils, p16highmonocytes or p16highdendritic cells. Immune cells can be detected with conventional surface markers such as CD3, CD14, CD33, CD19, CD20, CD66b, B220, CD11c, Ly6C, Ly6G, F4 / 80, CD45, CD4, CD8, CD25, PD1 or PD-L1. Preferably, they are detected, isolated and / or sorted by flow cytometry.

[0065] As shown in the examples below, the expression level and activity of p16 in immune cells can be increased by treating same with any of the pattern-recognition receptor agonists described above.

[0066] The NN MT protein

[0067] As shown in example 9 below, NNMT activation is a critical mechanism of p16 induction either directly or indirectly. The present inventors show here for the first time that the PRR agonists of the invention require a functional NNMT enzyme to favor the expression of p16 in immune cells, and its further beneficial effects. Therefore, to perform the various methods of the invention, it is important to ensure that the expression and / or activity of the NNMT (nicotinamide N-methyltransferase) enzyme is normal in the immune cells in which p16 should be overexpressed.

[0068] Nicotinamide N-methyltransferase (NNMT, NCBI Ref Seq is NP_006160) is an enzyme that in humans is encoded by the NNMT gene (gene ID: 4837). NNMT catalyzes the methylation of nicotinamide and similar compounds using the methyl donor S-adenosyl methionine (SAM-e) to produce S-adenosyl-L-homocysteine (SAH) and 1 -methylnicotinamide.

[0069] In another aspect, the present invention relates to an in vitro method for selecting patients that will benefit from a treatment involving a TLR7 agonist, a TLR5 agonist, ora STING agonist, said method comprising the steps of : a) Detecting the expression and / or activity of the nicotinamide N- methyltransferase (NNMT) enzyme in immune cells present in a biological sample of a patient, b) Concluding that said patient will benefit from a treatment involving a TLR7 agonist, a TLR5 agonist, or a STING agonist if the NNMT enzyme is expressed and functional in said immune cells, c) Optionally, administering to said patient a treatment involving a TLR7 agonist, a TLR5 agonist, or a STING agonist, if the NNMT enzyme is expressed and functional in the immune cells present in the tested sample.

[0070] The expression and / or biological activity of the nicotinamide N-methyltransferase (NNMT) enzyme can be detected for example by measuring the methylation of nicotinamide and similar compounds in the immune cells contacted with a TLR7 agonist, a TLR5 agonist, or a STING agonist. It is also possible to detect the level of S-Adenosyl methionine: a drop in its level will be indicative of an increased NNMT activity.

[0071] Pharmaceutical composition of the invention

[0072] The present invention targets a pharmaceutical composition comprising an effective amount of a STING agonist as defined above, of a TLR5 agonist as defined above or of a TLR7 agonist as defined above, or of a combination thereof.

[0073] An “effective amount” or “therapeutically effective amount” of an agent, e.g., of a PRR agonist(s) as in the present invention, refers to an amount effective, at dosages and for periods of time necessary, to elicit the desired biological response in a subject. Such response includes alleviation of the symptoms of the disease being treated, prevention, inhibition or a delay in the recurrence of symptom of the disease or of the disease itself, an increase in the longevity of the subject compared with the absence of the treatment, or prevention, inhibition or delay in the progression of symptom of the disease or of the disease itself. As used herein, an “effective amount” is in particular the amount of the agent effective to achieve the desired therapeutic or prophylactic result. More specifically, an “effective amount” as used herein is an amount of the PRR agonist of the invention that confers a therapeutic benefit. A therapeutically effective amount is also one in which any toxic or detrimental effects of the PRR agonist are outweighed by the therapeutically beneficial effects. It will be understood, however, that the total daily usage of the agent is decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific agent; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0074] An effective amount of the PRR agonist of the invention can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the agent, the route of administration, etc. In some embodiments, effective amount also refers to the amount of the cells provided herein to achieve a specified result (e.g., decrease of the tumor size, activation of T cells, etc.). In some embodiments, this term refers to the amount of a therapy which is sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder or condition and / or a symptom related thereto. This term also encompasses an amount necessary for the reduction or amelioration of the advancement or progression of the treated disease, reduction or amelioration of the recurrence, development or onset of said disease. In the context of cancer therapy, a therapeutic benefit means for example any amelioration of cancer, including any one of, or combination of, halting or slowing the progression of cancer (e.g., from one stage of cancer to the next), halting or delaying aggravation or deterioration of the symptoms or signs of cancer, reducing the severity of cancer, inducing remission of cancer, inhibiting tumor cell proliferation, tumor size, or tumor number, or reducing levels of biomarker(s) indicative of the cancer.

[0075] The pharmaceutical composition of the invention contains, as active principle, the PRR agonist described herein, and optionally a pharmaceutically acceptable excipient. The term “pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. Compositions according to the invention are usually be administered by parenteral, topical, intravenous, intratumoral, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means. A typical route of administration is intravenous or intratumoral, although other routes can be equally effective.

[0076] Typically, the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It 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. Sterile injectable solutions are prepared by incorporating the active ingredient at the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0077] For intravenous and / or intratumoral administration, the composition of the invention will be under liquid form. It will thus contain, apart from the PRR agonist, a pharmaceutically- acceptable diluent that does not affect the biological activity of the cells of the invention. Example of such diluents are physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. In a preferred embodiment, the composition of the invention is under a liquid form. Combining different pattern-recognition receptors (PRRs) agonists is an interesting alternative because a combined use of two small molecules is easier to deliver, to formulate and to produce than large molecules. Moreover, use of small molecules avoids the variability of large molecule due to modification such as glycosylation during the manufacturing process. The avoidance of such modification variability is interesting for safety and stability concerns.

[0078] In a particular embodiment, the pharmaceutical composition of the invention contains, apart from the optional pharmaceutically acceptable excipient, a combination of two of the pattern-recognition receptors (PRRs) agonists disclosed above, for example:

[0079] - an agonist of STING as disclosed above, and an agonist of TLR7 as disclosed above, or

[0080] - an agonist of STING as disclosed above and an agonist of TLR5 as disclosed above, or

[0081] - an agonist of TLR5 as disclosed above and an agonist of TLR7 as disclosed above.

[0082] In a particular aspect, the present invention concerns a composition comprising, apart from the optional pharmaceutically acceptable excipient, a STING agonist as disclosed above and a TLR7 agonist as disclosed above. Specifically, this composition can for example contain a low dose of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and an effective amount of the BNT162b2 vaccine, which have been successfully tested in the examples below. Alternatively, the composition of the invention may comprise a low dose of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and either the R837 or the R848 compound, as also tested in the examples below.

[0083] The composition may also advantageously comprise an anti-CD3 antibody, which is shown in the examples 17-18 below to maintain the viability of T cells.

[0084] When used in the treatment of aggressive tumors such as melanoma, the composition may also advantageously comprise an immune checkpoint inhibitor such as an anti-PD1 antibody. Example 15 below indeed demonstrates that the combination of these kinds of agent and TLR7 agonists can synergistically reduce melanoma development.

[0085] Inhibitory immune checkpoints include PD-1 , SIRPa, CD47, PD-L2, LAG3, Tim3, BTLA, and CTLA4, and immune checkpoint inhibitors according to the invention are therefore preferably selected from antagonist antibodies :

[0086] • PD-1 (e.g. those described in WO2004 / 004771; W02004 / 056875; W02006 / 121168; WO2008 / 156712; W02009 / 014708; W02009 / 114335; WO2013 / 043569; and W02014 / 047350, in particular nivolumab, pembrolizumab and cemiplimab),

[0087] • SIRPa (e.g. WO2019 / 023347),

[0088] • CD47 (e.g. W02020 / 019135),

[0089] • PD-L2 (e.g. WO2019 / 158645),

[0090] • LAG3 (e.g. WO2018 / 071500),

[0091] • Tim3, (e.g. W02020 / 093023)

[0092] • BTLA (e.g. W02010 / 106051), and

[0093] • CTLA4 (e.g. those described in US 8,491 ,895, W02000 / 037504, W02007 / 113648, WO2012 / 122444 and WO2016 / 196237 among others, and in particular ipilimumab marketed by Bristol Myer Squibb as Yervoy® (see e.g. US 6,984,720; US 8,017,114), MK-1308 (Merck), AGEN-1884 (Agenus Inc.; WO20 16 / 196237) and tremelimumab (AstraZeneca; US 7,109,003 and US 8,143,379) and single chain anti-CTLA4 antibodies (see e.g. WO97 / 20574 and WG2007 / 123737).

[0094] Immune checkpoint inhibitors are however not always requested. See e.g., examples 13 and 14 showing that for treating KRAS driven lung tumors or lung implanted melanoma cells, a TLR7 agonist alone is sufficient to reduce tumor size and improve mice survival.

[0095] A “low dose” of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is for example a dose of DMXAA that can enhance the expression of p16 in immune cells without inducing any inflammatory side effects on its own, when injected in the patient. The skilled person will be able to identify the appropriate dose by conventional staggering means. Exemplary appropriate doses to start with are comprised between 0.1 and 10 mg / kg. Specifically, this dose would induce the secretion of IFNy but not of TNFa in the contacted immune cells. In the context of the invention, low doses of DMXAA are for example comprised between 6 and 400 mg / m2. Of note, such a low dose would be effectively active in humans, because it has the capacity to induce STING pathway in humans (escalating the dose would not increase this property). Importantly, this low dose would not be deleterious to humans because it is below the pro- inflammatory range and remain in the “mild” sometimes called “tonic” range of STING activation.

[0096] On another hand, an “effective amount” of the BNT162b2 vaccine is for example a standard vaccination dose of 30pg.

[0097] In another particular embodiment, the pharmaceutical composition of the invention contains, apart from the optional pharmaceutically acceptable excipient, a combination of the three pattern-recognition receptors (PRRs) agonists disclosed above. Accordingly, it may contain an agonist of STING as disclosed above, an agonist of TLR7 as disclosed above, and an agonist of TLR5 as disclosed above. In this case, the composition of the invention can for example contain a low dose of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and an effective amount of the BNT162b2 vaccine, or of the R837 / R848 compound, or of an effective amount of flagellin, which have been successfully tested in the examples below. Specifically, these doses would induce the secretion of IFNy but not of TNFa in the contacted immune cells.

[0098] An “effective amount” of R837 in human for topical application is typically comprised between 2% and 10% (cream formulation), for example 5% cream formulation. For treatment of basal cell carcinoma, external genital warts, and bladder cancer, recommended application is once daily before bedtime, 5 times per week for a full 6 weeks.

[0099] An “effective amount” of R848 in human can be of 250 ng / mL as proposed in the art in human studies. In an injectable solution, it can be at 1.0 mg / mL as proposed in the AGADIR study combining resiquimod sulfate with atezolizumab (an immune checkpoint inhibitor) or in other clinical trials in combination with pembrolizumab. When used topically, R848 can be used between 0.01% and 0.05% in a cream (see NCT01583816).

[0100] An “effective amount” of Flagellin is for example the dose that induces p16 expression in immune cells. Specifically, this dose would induce the secretion of IFNy but not of TNFa in the contacted immune cells.

[0101] When a combination of two or three active principles is used, it is possible not to provide the active principles in the same composition, but to provide them separately. In this case, the active principles can be administered simultaneously or at different time intervals. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention.

[0102] Therapeutic uses and methods

[0103] The present invention is based on the surprising realization that p16hi9himmune cells play a key role in establishing an early and broad disease tolerance in response to multiple lethal conditions following severe inflammation and tissue damage, such as sepsis, SARS- CoV-2 infection and ionizing radiations. More precisely, it is shown in the examples below that various compounds leading to p16 increased expression in immune cells are able to play a key role in broad and early disease tolerance in response to lethal conditions following severe inflammation and tissue damage due to sepsis (example 5, example 16, example 18 and example 19), radiations (example 7), viral infections (examples 7 and 12), cancer (examples 13-15), thereby promoting disease tolerance and extending health span (example 11).

[0104] In a first aspect, the present invention relates on the pharmaceutical composition of the invention, as described above (i.e. comprising an effective amount of a STING agonist, of a TLR5 agonist or of a TLR7 agonist, or a combination thereof), possibly with a pharmaceutically acceptable carrier (as explained above), for use in enhancing the number of p16highimmune cells in a patient in need thereof.

[0105] In a preferred embodiment, said “immune cells” are NK cells, T lymphocytes and / or macrophages.

[0106] The “patient in need thereof” can be any human being exhibiting or likely to exhibit tissue inflammation or tissue lesions or tissue damages. This inflammation I lesion / damages can be for example due to a pathogen infection, a cancer, a chemical or physical treatment (e.g., irradiation, chemotherapy), or by a physical damage (e.g., wound, bruising, dislocation, sprain, strains, limb injuries, bone fracture, pealing, scarification, shaving, etc). It can also be due to frailty due to age, induced by the loss of tissue homeostasis and by the onset and progression of multiple diseases, including cancer, diabetes, Alzheimer's, and osteoarthritis. The “patient in need thereof’ is thus in particular any patient that is at risk of - oralready suffers from - a pathogen infection, a cancer or frailty due to aging. It can also be a human being that has undergone - or will undergo - a chemical or physical damage such as radiation or surgery, that may induce inflammation of the damaged tissues, for any reasons.

[0107] The “patient in need thereof” according to the invention is not necessarily suffering from a disease and might display no symptom of an illness. He / she may be an elderly person in non-pathological state or in good health. He / she may however suffer from (or likely to develop) symptoms of frailty due to aging.

[0108] The term “frailty due to aging” hereby designates at least one kind of symptom selected from the group consisting of: loss of muscle mass, decline in grip strength, feeling of fatigue, depression state, delirium, dementia, sleep disorder, anxiety disorder and social withdrawal, in an elderly person.

[0109] The results of the inventors show that p16 is naturally increased in the immune cells of old animals (figure 1A). As these cells already express p16, they may be less sensitive to the treatments of the invention (see also example 10). Therefore, in this aspect of the invention, the immune cells of the patient in need thereof are preferably p16lowimmune cells before the treatment occur. This low amount can be due to the fact that the p16 protein is underexpressed in the immune cells of the patient (due to an invalidating mutation affecting its expression, for example) or because its biological activity as defined above is impaired in these cells. It can also be due to the age of the patient, because it is known that p16 baseline expression is associated with aging. Therefore, in this aspect of the invention, the patient in need thereof is preferably young, typically between 0 and 65 years old.

[0110] In example 10, the inventors show that low adenosine levels are essential for PRR- agonists-induced protection from LPS-induced sepsis both in young and old animals. Thus, in this aspect of the invention, the patient in need thereof preferably expresses a low level of adenosine. The level of adenosine can be detected by using specific kits; however, since adenosine is not stable, it is preferred to measure SAM levels (using ELISA based technology) since SAM is more stable than adenosine. If it is not the case, it is possible to administer an adenosine inhibitor such as SCH58261 . As a matter of fact, in aged mice presenting a higher level of adenosine, the administration of 2-4 mg / kg of SCH58261 by day during acute phase of sepsis extends survival after LPS challenge (Figure 5N).

[0111] Thus, in a particular embodiment, the pharmaceutical composition of the invention is for use in preventing and / or treating tissue inflammation and / or tissue damage.

[0112] In a more particular embodiment, said tissue inflammation and / or tissue damage is associated with or induced by a pathogen infection, a cancer, a chemical or physical treatment, or by a physical damage.

[0113] As used herein, the term “pathogen infection” designates any infection from any pathogen, in particular from any bacteria, virus, parasite or fungi that is likely to induce inflammatory adverse effects. In a particular embodiment, it can be a viral infection due to an inflammatory virus chosen in the group consisting of: the influenza virus, the rabies virus, the Ebola virus, the smallpox virus, the Marburg virus, the Nipah virus, the hantavirus, the anthrax virus, the Human Immunodeficiency Virus (HIV) virus, the Epstein-Barr virus, the Zika virus, the Hepatitis A, B and C virus, and the SARS-COV-2 virus, that can trigger rapid and strong inflammation in the infected patient. In another particular embodiment, it can be a bacterial infection due to an inflammatory bacteria chosen in the group consisting of: Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bacteroides fragilis, Bordetella pertussis, Borrelia sp. (burgdorferi, garinii, afzelii, recurrentis, crocidurae, duttonii, hermsii etc), Brucella sp. (abortus, canis, melitensis, suis), Campylobacter jejuni, Chlamydia sp. (pneumoniae, trachomatis), Chlamydophila psittaci, Clostridium sp. (botulinum, difficile, perfringens, tetani), Corynebacterium diphtheriae, Ehrlichia sp. (canis, chaffeensis), Enterococcus (faecalis, faecium), Escherichia coli 0157. -H7, Francisella tularensis, Haemophilus influenza, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira sp., Listeria monocytogenes, Mycobacterium sp. (leprae, tuberculosis), Mycoplasma pneumoniae, Neisseria (gonorrhoeae, meningitidis), Pseudomonas aeruginosa, Porphyromonas gingivalis, Nocardia asteroides, Rickettsia rickettsii, Salmonella sp. (typhi, typhimurium), Shigella sp. (sonnei, dysenteriae), Staphylococcus (aureus, epidermidis, saprophyticus), Streptococcus sp. (agalactiae, mutans, pneumoniae, pyogenes, viridans), Tannerella forsythia, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.

[0114] As used herein, the term “cancer” designates any kind of cancer, preferably those that can induce inflammation. In particular, the patient of the invention can suffer from a cancer selected from the group consisting of oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, lung cancer (in particular KRAS lung cancer), bone cancer, pancreatic cancer, skin cancer, head cancer, cancer of the neck, skin cancer, melanoma, adenocarcinoma, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, perianal cancer, endometrial carcinoma, carcinoma of the vagina, Hodgkin's disease, esophageal cancer, bladder cancer, gall bladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, renal cancer, ureters cancer, renal cell carcinoma, a renal pelvic carcinoma, a central nervous system tumor, a primary central nervous system lymphoma, a spinal cord tumor, brainstem glioma and pituitary adenoma.

[0115] In example 10 below, the inventors show that p16 upregulation in immune cells protect these cells from apoptosis during severe inflammation, thereby reducing the cytokine storm and deleterious effects of inflammation in patients treated with the agonists of the invention. Thus, in a particular embodiment, the pharmaceutical composition of the invention is for use in inhibiting the apoptosis of immune cells during inflammation and / or for preventing a cytokine storm linked to inflammation.

[0116] In examples 5, 16, and 18-19, the inventors show that p16 upregulation in immune cells protect the treated animals against the deleterious effects of LPS-induced sepsis. Thus, in a particular embodiment, the pharmaceutical composition of the invention is for use in preventing and / or treating the deleterious inflammatory effects of sepsis.

[0117] In example 6, the inventors show that the agonists of the invention have a rapid and noticeable radioprotective effect, so that p16 upregulation in immune cells protect the treated animals against the deleterious effects of radiation-induced tissue damages. BNT162b2 vaccine-induced disease tolerance has a rapid and noticeable radioprotective effect that potentially could be improved by strategies that increase the presence of p16highimmune cells in the intestine. Thus, in a particular embodiment, the pharmaceutical composition of the invention is for use in preventing / treating the deleterious inflammatory effects of a radiation therapy.

[0118] In example 12, the inventors show that patients suffering from severe COVID-19 disease display a low level of p16hi0himmune cells. In examples 7 and 12, the inventors show that p16 upregulation in immune cells after treatment with a TLR agonist provide a rapid and broad tissue protection from SARS-COV-2 induced severe inflammation, before the development of inhibitory antibodies against SARS-COV-2 infection. Thus, in a particular embodiment, the pharmaceutical composition of the invention is for use in preventing and / or treating a SARS-COV-2 infection, in particular for preventing the deleterious inflammatory effects induced by a SARS-COV-2 infection.

[0119] In examples 13, 14 and 15, the inventors show that the TLR agonists of the invention induce the expression of p16hi0himmune cells associated to melanoma tumors, and that KRAS lung cancer progression can be inhibited by administering in the animals the TLR agonists of the invention. Thus, in a particular embodiment, the pharmaceutical composition of the invention is for use in preventing and I or treating cancer, in particular for preventing the tissue inflammation and I or lesions associated with or induced by cancer, such as melanoma or lung cancer.

[0120] In another aspect, the present invention refers to a method of preventing and / or treating tissue inflammation and / or tissue damage, preferably induced by a pathogen infection, a cancer, a chemical or physical treatment, or by a physical damage, said method comprising the step of administering an effective amount of a STING agonist, of a TLR5 agonist or of a TLR7 agonist, or of a combination thereof. All the embodiments disclosed above apply to this method.

[0121] In another aspect, the present invention refers to the use of a STING agonist, of a TLR5 agonist or of a TLR7 agonist, or of a combination thereof, for the preparation of a medicament that is intended to be used for preventing and / or treating tissue inflammation and / or tissue damage, preferably induced by a pathogen infection, a cancer, a chemical or physical treatment, or by a physical damage. All the embodiments disclosed above apply to this use.

[0122] In other aspects, the present invention refers to the non-therapeutic use of a STING agonist, of a TLR5 agonist or of a TLR7 agonist, or of a combination thereof, to prevent or improve age-induced frailty and other side effects of aging, to maintain the good health of elderly persons, and therefore to extend their health expectancy and their healthspan. All the embodiments disclosed above apply to this use. Ex vivo cell therapy

[0123] In Example 16 and example 18 below, the inventors have shown that treating animals with a p16highCD45+immune cells collected from animals that have been treated with a TLR agonist or from human PBMCs that have been treated with the composition of the invention become also tolerant against septic shock. In particular, Example 16 shows that adoptive transfer of p16Highimmune cells works effectively in mice that have already started to develop a severe inflammatory response, suggesting that this strategy could potentially be used to treat patients with already developed severe inflammation, such as sepsis. And Example 18 demonstrates that the adoptive transfer of human p16-positive PBMCs, achieved using the protocol described in Figure 16, can protect mice from severe inflammation, such as LPS- induced sepsis. These examples demonstrate that disease tolerance promoted by the PRRs agonists of the invention can be transplanted to other animals. This experiment paves the way to the use of adoptive cell therapies, implying boosting p16 expression in immune cells that have been collected from a patient or generated in vitro from progenitors or iPS cells, so as to favor their potential of beneficial effects before being administered to the same or to another patient.

[0124] Patients that will mostly benefit from such an adoptive cell transfer treatment are preferably those whose immune cells already express a high level of p16. As a matter of fact, when the level or activity of p16 in the immune cells of a patient is already high enough, the compositions and classical in vivo treatments disclosed above may not be effective, as the number of endogenous p16highimmune cells cannot be more increased in the patients. In this case, an alternative strategy is to perform an adoptive cell therapy, for enhancing the number of p16highcells in the patient without affecting the expression of p16 in the endogenous immune cells.

[0125] These patients are for example elderly or aged patients (typically aged 70 and over) and / or those who suffer from an inflammatory disease when the treatment of the invention is administered. As a matter of fact, it is well-known that the p16 level in both situations increases in immune cells (see also in example 1). In these patients, the adoptive cell therapies of the invention are very useful for inducing disease tolerance, for protecting tissues against deleterious effect of aging and / or for extending health span. Furthermore, the proposed protocol for adoptive cell transfer could be especially effective in patients who are unable to mount a p16-induction response in their immune cells (a condition that could be detected using the procedure described in Figure 16 A-D as a diagnostic test). In these cases, increasing the number of p16Highimmune cells in such patients could not only protect against tissue damage but also improve various treatments, including cancer and severe inflammation treatments (such as sepsis). These patients may also suffer from cancer, as adoptive immunotherapy with suitable recombinant immune cells has already been proposed for the treatment of a number of cancers in humans. Typically, immune cells can be purified from the circulating blood of patients, cultured ex vivo and activated to induce their differentiation and increase their tumoricidal power, then reinjected into the same or other patients. It is also possible, using suitable drugs and vectors, to treat the collected immune cells and / or to transfer genes in these cells, thereby enabling them to be endowed with superior properties as proposed herein. Importantly, the proposed adoptive transfer protocol for p16Highimmune cells represents a more physiological, less toxic yet equally efficient alternative for anti-cancer therapies when the anti-cancer drugs are limited by their systemic toxicity.

[0126] In this embodiment, p16 overexpression or activation in immune cells can be performed in vitro by any means, e.g., by using one, two or three of the PRR agonists disclosed above, or by genetic means with a p16-encoding vector (see below).

[0127] In a particular embodiment, the invention targets the use of a STING agonist, a TLR5 agonist and / or a TLR7 agonist or the use of the composition of the invention, which is as disclosed above (i.e., comprising a STING agonist, a TLR5 agonist and / or a TLR7 agonist or combinations thereof) for ex vivo or in vitro inducing the expression or enhancing the activity of p16 in immune cells.

[0128] In other words, the present invention thus relates to an in vitro method to induce the expression or activity of p16 in immune cells, said method comprising the step of in vitro contacting immune cells with an agonist of a PRR, said agonist being chosen from a STING agonist, a TLR5 agonist or a TLR7 agonist, as defined above, or with a combination thereof. These agonists would be provided in appropriate doses, as discussed above. Specifically, these doses would induce the secretion of IFNy but not of TNFa in the contacted immune cells.

[0129] All the details concerning the PRR agonists of the invention have been provided previously and need not being reproduced. Preferably, the immune cells are contacted in vitro with a combination of a STING agonist and a TLR7 agonist, more preferably with a combination of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and of an effective amount of the BNT162b2 vaccine. These two agonists would be provided in appropriate doses, as discussed above. Specifically, these doses would induce the secretion of IFNy but not of TNFa in the contacted immune cells.

[0130] In another particular embodiment, the in vitro overexpression of p16 in immune cells can be achieved by using a replication-defective recombinant virus encoding the p16 protein, under a promoter that is functional in lymphocytes or in monocytes. This virus can be in T1 particular an adenovirus or a lentivirus or an AAV that are viruses known to be able to efficiently transduce immune cells. This replication-defective virus would contain the human CDKN2A gene (NCBI gene 1029; one mRNA thereof being NM_000077.5), under the control of a promoter that is functional in these cells, e g., the promoter E2F1 (E2 promoter binding factor 1) or the promoter of EFS (elongation factor 1a short), SFFV (silencing-prone spleen focus forming virus), CMV (cytomegalovirus), RSV (Rous sarcoma virus).

[0131] In another aspect, the invention targets a pharmaceutical composition containing such a replication-defective recombinant virus. This pharmaceutical composition could be used either in vitro to transduce PBMCs or lymphocytes before these cells are administered to the patients (adoptive therapy), or in vivo to transduce endogenous immune cells in patients (direct gene therapy).

[0132] Enhancing the expression or activity of p16 would be very advantageous in immune cells expressing a chimeric antigen receptor (CAR), because these p16hi0hCAR immune cells would be less prone to apoptosis once administered in vivo. Also, these p16highimmune cells expressing CAR would induce the beneficial anti-cancer and anti-inflammatory effects observed when p16 is enhanced in immune cells in vivo (see examples 13, 14, 15), especially when the treatment is combined with radiotherapy (example 6).

[0133] The present invention thus concerns p16highimmune cells expressing a functional recombinant CAR molecule and their incorporation into pharmaceutical compositions that can be used in cancer therapy, more particularly, in cancer immunotherapy.

[0134] In a preferred embodiment, the immune cells used in such cancer immunotherapy are NK cells, T lymphocytes and / or macrophages. In a more preferred embodiment, they express a chimeric antigen receptor (CAR), e.g., they would be CAR T cells, CAR macrophages or CAR NK cells.

[0135] In immunotherapeutic methods, the immune cells can originate from the patient himself (the composition therefore contains autologous cells) or from a donor (the composition therefore contains allogeneic or heterologous cells). For heterologous cells, HLA compatibility and matching between the donor and the patient receiving the cells is required. More generally, the invention relates to the isolation, culture, activation and / or treatment of these cells of the immune system, and their use in cell therapy, for example in adoptive immunotherapy. It also relates to immunotherapeutic methods using these particular cells.

[0136] In a preferred embodiment, the present invention proposes to use a pharmaceutical composition comprising an effective amount of autologous or heterologous p16highimmune cells, said immune cells being preferably T cells or macrophages expressing CAR. In a particular embodiment, said pharmaceutical composition contains a pharmaceutically acceptable excipient and at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 95% of autologous or heterologous p16highimmune cells. This means that, in this pharmaceutical composition, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 95% of the cells are p16highimmune cells.

[0137] A typical pharmaceutical composition according to the invention contains for example at least 105, preferably at least 10s, p16highCD45+immune cells.

[0138] Example 17 below describes a particular protocol for the in vitro induction of p16Highhuman immune cells from PBMCs, including within the CD3 and CD11 b populations, following simultaneous stimulation of the TLR7 and STING pathways. Briefly, TLR7 and TLR7 / 8 agonists (R837 and R848, respectively), and a STING agonist (DMXAA) were used, in combination with anti-CD3, anti-CD28, and anti-CD2 stimulatory antibodies. Example 18 demonstrates that the adoptive transfer of human p16-positive PBMCs, achieved using the protocol described in Example 17, can protect mice from severe inflammation, such as LPS- induced sepsis.

[0139] Ex vivo generated autologous or heterologous p16highimmune cells as defined herein can be used to treat any patient in need thereof. As explained above, these patients are preferably aged patients, or patients suffering from a fibrogenic or an inflammatory disease, whose endogenous immune cells usually naturally express high levels of p16. In particular, they can be used for preventing and / or treating tissue inflammation and / or tissue damage in these patients, said tissue inflammation and / or tissue damage being preferably induced by a pathogen infection, a cancer, a chemical or physical treatment, or by a physical damage, as explained above. Also, they can be used for inducing disease tolerance, for protecting tissues against deleterious effect of aging and / or for extending health span in these patients.

[0140] By “inflammatory disease”, it is herein meant a disease resulting from or including an inflammation reaction. An inflammation reaction is the response of body tissues to harmful stimuli. Inflammatory disease can be classified as either acute or chronic. In particular, chronic inflammation is associated with various inflammatory diseases, such as hay fever, periodontal disease, atherosclerosis, and osteoarthritis. Examples of inflammatory disease include notably acute pancreatitis; amyotrophic lateral sclerosis; Alzheimer's disease; cachexia / anorexia; asthma; atherosclerosis; chronic fatigue syndrome, fever; diabetes (e.g., insulin diabetes); glomerulonephritis; graft versus host rejection; hemorrhagic shock; hyperalgesia, inflammatory bowel diseases; inflammatory conditions of a joint, including osteoarthritis, psoriatic arthritis and rheumatoid arthritis; ischemic injury, including cerebral ischemia (e.g., brain injury as a result of trauma, epilepsy, hemorrhage or stroke, each of which may lead to neurodegeneration); lung diseases; multiple myeloma; multiple sclerosis; leukemias; myopathies; osteoporosis; Liver diseases like Non-Alcoholic Fatty Liver Disease (NAFLD), Non-Alcoholic Steatohepatitis (NASH), and Acute-on-chronic liver failure (ACLF); Parkinson's disease; psoriasis; reperfusion injury and septic shock, side effects from radiation therapy, temporal mandibular joint disease, tumor metastasis; or an inflammatory condition resulting from strain, sprain, cartilage damage, trauma, orthopedic surgery, infection or other disease processes. Cancers that induce inflammatory side effects are also encompassed by the term “inflammatory diseases”.

[0141] When the autologous or heterologous p16highimmune cells express a CAR molecule, ex vivo generated autologous or heterologous p16highimmune cells as defined herein can be used to treat specifically patients suffering from cancer. In this case, combination with a radiotherapy treatment can be advantageously contemplated, since the adoptive p16highimmune cells of the invention will also prevent side effects to occur because of this secondary treatment (example 6).

[0142] In another aspect, the present invention refers to a method of preventing and / or treating tissue inflammation and / or tissue damage, preferably induced by a pathogen infection, a cancer, a chemical or physical treatment, or by a physical damage, in a patient in need thereof, said method comprising the steps of: a) contacting a STING agonist, of a TLR5 agonist or of a TLR7 agonist or a vector encoding p16 with blood or purified immune cells (iPS or cells obtained from a donor mammal); and collecting the p16highimmune cells thereby obtained; b) optionally, contacting said p16highimmune cells with a vector encoding a CAR molecule, so as to obtain CAR-expressing p16highimmune cells; c) administering said p16highimmune cells into a patient in need thereof.

[0143] In another aspect, the present invention refers to a pharmaceutical composition comprising an effective amount of autologous or heterologous p16highimmune cells, said immune cells being preferably T cells or macrophages expressing CAR, for preparing a medicament intended to be used to prevent and / or treat tissue inflammation and / or tissue damage in a patient in need thereof. A typical pharmaceutical composition according to the invention contains for example at least 105, preferably at least 106, p16highCD45+immune cells.

[0144] For the reasons explained above, patients in need of this adoptive treatment are preferably experiencing an inflammatory state, or they are suffering from an inflammatory disease, or from cancer, or from a pathogenic infection. They can be of any age. This adoptive treatment can be also administered in healthy human beings expressing high levels of p16, typically in healthy people older than 65, to whom it can prevent age-associated diseases and / or extend lifespan.

[0145] Prognosis and screening methods

[0146] The results presented below show that p16 expression or activity in immune cells can be used as a prognostic marker of disease tolerance in humans. This marker can be easily detected by analyzing a blood sample collected from a patient. As a matter of fact, when the percentage of p16highimmune cells is low in a blood sample of a patient, then it can be concluded that the patient has a poor disease tolerance and will be very sensitive to an infection or an aged-related disease.

[0147] In another aspect, the present invention relates to an in vitro method for prognosing the disease tolerance of a patient, said method comprising the steps of : a) Detecting the expression and / or activity of the p16 protein in the immune cells present in a biological sample of said patient, b) Determining the percentage of p16highimmune cells in said sample, c) Concluding that the patient has a poor disease tolerance if the percentage of p16highimmune cells in the tested sample is low.

[0148] As used herein the term “biological sample” designates any sample collected in a patient that might contain immune cells as defined herein. It can be, for example, a blood sample, a saliva sample, a tissue sample, a bone-marrow sample, etc. A blood sample is however preferred, as it is easily collected and contains many immune cells. By “blood sample”, it is herein meant a whole blood, serum, or plasma sample obtained from the patient. Preferably the blood sample according to the invention is a plasma sample. In a particularly preferred embodiment, the peripheral bone-marrow cells (PBMCs) contained in the blood sample are isolated or purified by conventional means, so as to be studied for p16 expression / activity. As explained above, “the percentage of p16highimmune cells is low” herein means for example that less than 50%, 40%, 30%, 20%, or 10% of the immune cells contained in the tested biological sample are p16high. Conversely, this means for example that more than 50%, 60%, 70%, 80%, or 90% of the immune cells contained in the tested biological sample are p16low.

[0149] To optimize this prognostic method, the “immune cells” on which p16 should be detected are preferably lymphocytes, granulocytes, natural killer (NK), and macrophages.

[0150] Preferably, this method is completed with a treatment step consisting in administering to the p16lowpatient the pharmaceutical compositions disclosed above, containing either the PRR agonist(s) of the invention, or heterologous / autologous p16hi0himmune cells obtained by contacting immune cells with the PRR agonist(s) of the invention. All the embodiments exposed above apply, mutatis mutandis, without needing to be repeated.

[0151] The inventors herein show that patients infected with SARS-COV-2 and having immune cells that are p16highdo not suffer from severe forms of the COVID-19 (figure 7G), as they did not require any oxygen therapy. Thus, the prognostic method of the invention preferably can be implemented to detect specifically if a patient infected with an inflammatory virus (e.g., the SARS-COV-2 virus) will suffer from severe symptoms, or not.

[0152] In a particular embodiment, the prognostic method of the invention thus comprises the steps of : a) Detecting the expression and / or activity of the p16 protein in the immune cells contained in a blood sample of a patient infected with a SARS-COV-2 virus, b) Determining the percentage of p16highimmune cells in said sample, c) Concluding that the patient will suffer from a severe COVID19 if the percentage of p16highimmune cells in the tested sample is low.

[0153] This method can be furthermore completed by an optional treatment step consisting of administering to the p16lowpatient, along with the pharmaceutical compositions of the invention as defined above, a strong anti-COVID19 treatment. This anti-COVID19 treatment can be chosen in the approved treatments such as nirmatrelvir plus ritonavir (paxlovid), sotrovimab (Xevudy), molnupiravir (Lagevrio), dexamethasone and / or remdesivir (Veklury). Also, supplemental oxygen may be needed, and / or immunomodulators (baricitinib, tocilizumab, abatacept, infliximab).

[0154] More generally, the present invention also relates to an in vitro screening method for identifying drug candidates enhancing disease tolerance and / or extending health span, said method comprising the step of detecting the expression or activity of the p16 protein in immune cells, in the presence or in the absence of said drug candidates. Said drug candidates can be chosen e.g., from dietary supplement, chemical drug, oligonucleotide drugs, etc. In a preferred embodiment, the drug candidates will be selected if they increase the number of p16highimmune cells, when present. In the context of this screening method, the immune cells are preferably T lymphocytes and / or macrophages.

[0155] In a final aspect, the present invention relates to an in vitro method for selecting patients that will benefit from a treatment involving a TLR7 agonist, a TLR5 agonist, ora STING agonist, based on all the embodiments disclosed above.

[0156] This method comprises the steps of : a) Detecting the expression and / or activity of the p16 protein in immune cells present in a biological sample of a patient, b) Determining the percentage of p16highimmune cells in said sample, c) Concluding that the patient will benefit from a treatment involving a TLR7 agonist, a TLR5 agonist, ora STING agonist if the percentage of p16highimmune cells in the tested sample is low.

[0157] Preferably, this method is completed with a treatment step consisting in administering to the p16lowpatient the pharmaceutical compositions disclosed above, containing either the PRR agonist(s) of the invention, or heterologous / autologous p16highimmune cells obtained by contacting immune cells with the PRR agonist(s) of the invention. All the embodiments exposed above apply, mutatis mutandis, without needing to be repeated.

[0158] Preferably, this method for selecting patients can contain the further steps of: i) Detecting the expression and / or activity of the nicotinamide N- methyltransferase (NNMT) enzyme in the immune cells present in a biological sample of said patient, and ii) Concluding that the patient will benefit from a treatment involving a TLR7 agonist, a TLR5 agonist, or a STING agonist if the percentage of p16highimmune cells in the tested sample is low and if the NNMT enzyme is expressed and functional in the immune cells present in the tested sample.

[0159] As explained previously, the expression and / or biological activity of the nicotinamide N-methyltransferase (NNMT) enzyme can be detected for example by measuring the methylation of nicotinamide and similar compounds or the drop in SAM levels in the immune cells contacted with a TLR7 agonist, a TLR5 agonist, or a STING agonist.

[0160] Definitions

[0161] The term “higher” or “increased”, as used herein, refers to a level of p16 or to an activity of p16 at least 1.5 folds greater (preferably 2, 3, 4, 5, 6, 7, 8, 9, 10 folds greater) than the level of p16 or the activity of p16 in the reference sample. The term “lower” or “decreased”, as used herein, refers to a level of p16 or to an activity of p16 at least 1 .5 folds lower (preferably 2, 3, 4, 5, 6, 7, 8, 9, 10 folds lower) than the level of p16 or the activity of p16 in the reference sample.

[0162] As used herein, the term "cancer" is not limited to any stage, grade, histomorphological feature, invasiveness, aggressiveness or malignancy of an affected tissue or cell aggregation. In particular stage 0 cancer, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, grade I cancer, grade II cancer, grade III cancer, malignant cancer, primary carcinomas, and all other types of cancers, malignancies etc. are included.

[0163] As used herein, the term “infection” refers to the invasion and multiplication of microorganisms, such as bacteria, viruses, parasites, or fungi, within a host organism's body tissues. Infections can lead to a variety of signs and symptoms, ranging from mild to severe, and they can affect various organs and systems in the body. The immune system typically responds to infections by initiating an inflammatory response and mobilizing immune cells to eliminate the invading pathogens.

[0164] As used herein, the expression “tissue damage” refers to harm, injury, or impairment to biological tissues that is either linked to the presence and progression of a specific disease or condition, or to an external event (wound, bruising, dislocation, sprain, strains, limb injuries, bone fracture, pealing, scarification, shaving, etc.). In this context, the tissue damage impacts the affected tissues at the cellular or structural level where inflammation is induced.

[0165] As used herein, the term “radiation therapy” refers to a treatment involving particles or electromagnetic waves with sufficient energy to ionize atoms or molecules by removing tightly bound electrons. This type of radiation includes alpha particles, beta particles, gamma rays, X-rays, and certain types of neutrons. Ionizing radiation has enough energy to produce ions when it interacts with matter, and it can cause damage to biological tissues, often leading to inflammatory side effects. As used herein, the term “SARS-CoV-2” designates not only the firstly identified SARS-CoV-2 but also any variant or mutant thereof. The firstly identified SARS-CoV-2 is the initially discovered strain of the virus and the latter is also known as 2019-nCoV, HCoV-19, SARS2, COVID-19 virus, Wuhan coronavirus, Wuhan seafood market pneumonia virus and Human coronavirus 2019. The complete genome of this coronavirus (29903 bp ss-RNA) is accessible from the NCBI (“National Center for Biotechnology Information”) site https: / / www.ncbi.nlm.nih.gov / under the reference sequence NC_045512.2.

[0166] As used herein, the terms “treat”, “treating” and “treatment”, are meant to include alleviating, attenuating or abrogating a condition or a disease, in particular, an inflammation, a cancer or a tissue damage and / or the signs, symptoms and / or complications associated therewith. The signs or symptoms associated with a condition or a disease may be biochemical, cellular, histological, functional or physical, subjective or objective ones. This includes the complications associated with a condition or a disease.

[0167] The terms “prevent”, “preventing” and “prevention” as used herein, are meant to include not only delaying or precluding the onset of a condition or disease, and / or the signs, symptoms and / or complications associated therewith but also barring a patient from acquiring a condition or disease, in particular, or reducing a patient’s risk of acquiring a condition or disease.

[0168] As used herein, the term "disease" refers to an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration, or quality of life. As used herein, the term “condition” refers to one or more symptoms / complications of a disease, such as inflammation or sepsis or of a trauma such as tissue damage, resulting for example from ionizing radiations.

[0169] The term “nucleic acid” as used herein, refers to DNA, RNA, single-stranded, doublestranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof. Modifications include, but are not limited to, those providing chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, peptide nucleic acids (PNAs), phosphodiester group modifications (e.g., phosphorothioates, methylphosphonates), 2'-position sugar modifications, 5-position pyrimidine modifications, 7-position purine modifications, 8-position purine modifications, 9-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases, isocytidine and isoguanidine and the like. Nucleic acids can also include non-natural bases, such as, for example, nitroindole. Modifications can also include 3' and 5' modifications such as capping with a BHQ, a fluorophore or another moiety.

[0170] As disclosed herein, the terms “in vitro" and “ex vivo" are equivalent and refer to studies or experiments that are performed using biological components (e.g. cells or population of cells) that have been isolated from their usual host organisms (e.g. animals or humans). In contrast, the terms “in vivo” or “in situ" refer to studies that are conducted on whole living organisms (e.g., humans), after administration of the composition of the invention in a living subject.

[0171] LEGEND OF FIGURES

[0172] Other features and advantages of the invention will become apparent from the following examples, given by way of illustration, with reference to:

[0173] Figure 1 : results of the analysis of the fraction of p16highcells in different populations of the immune cells.

[0174] A. Analysis of the fraction of p16Highcells in different populations of the immune cells. Single cell suspensions were prepared from peritoneal cavity, spleen, liver, stromal-vascular fraction of abdominal fat (SVF), bone marrow, and peripheral blood from 2-, 12- and 18- month- old p16-Cre / R26-mTmG mice. Cell suspensions were stained with fluorescent-conjugated antibody against the corresponding markers of immune cells (CD3 for T lymphocytes, B220 for B lymphocyte, Ly6C for monocytes, Ly6G for neutrophils, CD11c for dendritic cells and F4 / 80 for tissue-resident macrophages) and analyzed by flow cytometry. Data are mean ± S.D. Statistical significance was analyzed using ANOVA plus Tukey post hoc test. *p<0,001

[0175] B. Abundance and distribution of p16highcells in the previously analyzed tissues in 18- month-old mice. Each pie chart represents the total number of p16highcells and how they are distributed in the different cellular compartments.

[0176] C. Percentage of p16highcells in the populations expressing PD-1 (Programmed cell Death protein 1), PD-L1 (Programmed Cell Death Ligand 1) and PD-L2 (Programmed Cell Death Ligand 2). Peritoneal, liver, bone marrow and abdominal fat SVF single cell suspensions from 16-month-old mice were stained using fluorescent-conjugated antibody against PD-1 , PD-L1 and PD-L2 and analyzed by flow cytometry. Bar graph represents the percentage of p16highin populations expressing PD-1 and PD-L1. Pie charts show the distribution of the total p16highcells expressing PD-1 , PD-L1 and PD-L2. Data are mean. D. p16highT lymphocytes express PD-1 , PD-L1 , PD-L2 and Foxp3. The percentage of p16highcells was determined in T lymphocytes obtained from peritoneal cavity, liver, bone marrow and abdominal fat SVF defined as CD3+CD4+PD1+, CD3+CD4+PD-L1+, CD3+CD4+PD-L2 and CD3+CD4+Foxp3+(Bar graphs) in 16-month-old mice. Pie charts show abundance and distribution of PD-1 , PD-L1 , PD-L2 and Foxp3 in the total CD3+CD4+p16highpopulation. Data are mean ± S.D.

[0177] E. Senescence-associated p-galactosidase activity was analyzed in 2-month-old and 12-month-old p16Highand p16lowF4 / 80-positive macrophages. Pictures were made with bright field - fluorescence microscope. The percentage of positive cells was calculated. Data are mean ± SD. Significant differences were determined by t-test. *p<0,05; **p<0,01 and ***p<0,001.

[0178] F. p16Highperitoneal macrophages are not proliferative. Peritoneal macrophages from 12-month-old p16-Cre / R26-mTmG mice were incubated with 5-ethynyl-2’-deoxyuridine (EdU) for 24 h. The percentage of EdU positive cells was analyzed in p16Highand p16Lowpopulations. Data are mean ± SD. Significance was analyzed by t-test. **p<0,01 .

[0179] G. Immunostaining of different markers of senescence. Peritoneal macrophages from 12-16 month-old p16-Cre / R26-mTmG mice were stained for senescence markers ATM, 53bp1, and Y-H2AX. Pictures were obtained by high-definition or confocal microscopy. Cells were counted and the percentages of positive cells for p16Highand p16lowpopulations were calculated. Data are mean ± SD. Significant differences were determined by t-test. *p<0,05; **p<0,01 and ***p<0,001.

[0180] H. Peritoneal macrophages from 12-16 month-old p16-Cre / R26-mTmG mice and wild type littermates were stained for p21 and Y-H2AX. Pictures were obtained by high-definition or confocal microscopy. Cells were counted and the percentage of p16Highcells in p21 + population (left panel) and the percentage of positive cells for Y-H2AX in p21+ (right panel) were determined. Data are mean ± SD. Significant differences were determined by t-test. ***p<0,001.

[0181] I. Peritoneal F4 / 8CF macrophages from 12-month-old p16-Cre / R26-mTmG mice were isolated using magnetic cell sorting. Then, p16Highand p16lowpopulations were separated by fluorescence-activated cell sorting (FACS). RNA expression of both populations was analyzed by RNA sequencing. Kegg pathway analysis of downregulated genes from RNA-seq data set reveals a set of clusters of genes related with the negative control of cell cycle. Individually analyzed genes are shown. Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) were analyzed by t-test. Data are mean ± SD. *p<0,05; **p<0,01 and

[0182] ***p<0,001 . Representative 8 genes are shown. J. Kegg pathway analysis of upregulated genes from RNA-seq data set reveals a set of clusters related with the negative control of immune cells and T-cells. Immunosuppressive and regulatory representative genes were individually analyzed and shown. Data are mean ± SD. Differences were established with t-test. **p<0,01 and ***p<0,001.

[0183] Figure 2 : Results of the analysis of p16highimmune cells in young animals in response to inflammation and tissue damage.

[0184] A-B. Dextran sodium sulphate (DSS) induces p16Highcells in vivo in young mice. 2-3 month-old p16-Cre / R26-mTmG mice were treated with DSS 2.5% dissolved in drinking water for 7 days and the percentage of p16High cells in different populations of immune system was determined. Single cell suspensions from peritoneal cavity and liver were stained with fluorescent conjugated antibody and analyzed by flow cytometry. Abundance and distribution of p16Highcells in the previously analyzed tissues (panel A) were determined and are shown as pie charts (B). Data are mean ± SD. Statistical significance was determined using ANOVA plus Dunnett post hoc test. **p<0,01 and ***p<0,001.

[0185] C-D. p16HighT lymphocytes express higher level of Foxp3, PD-1 , and PD-L1 after DSS treatment. The percentage of p16Highcells was determined in T lymphocytes obtained from peritoneal cavity and liver defined as CD3+CD4+PD1+, CD3+CD4+PD-L1+, and CD3+CD4+Foxp3+in 12-month-old mice. Pie charts show abundance and distribution of Foxp3, PD-1, and PD-L1 in the total CD3+p16Highpopulation (D). Data are mean.

[0186] E-F. Different pattern of gene expression in the absence of p16Highcells. Control and p16-Cre / R26-DTA (DTA) were treated with 2.5% DSS for 7 days and RNA from peritoneal cells (E) and liver (F) were isolated. RNA was analyzed by quantitative SYBR-green based PCR (qPCR) to determine the level of expression of different genes. Data are mean + / - S.D. Difference between groups was analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001.

[0187] G-H. Specific TLRs induce p16Highstate in vivo. 2-3 months p16-Cre / R26-mTmG mice were treated intraperitoneal (I.P) with a panel of agonist to different toll-like receptors (TLRs). 48 h later, peritoneal cells were stained with fluorescent-conjugated antibodies against F4 / 80 (G) and CD3 (H). The percentage of p16Highcells was determined by flow cytometry. Data are mean ± SD. Statistical significance were determined using ANOVA plus Dunnett post hoc test. *p<0,05 and ***p<0,001. Figure 3: The BNT162b2 mRNA COVID-19 vaccine induces p16Highimmune cells and disease tolerance to protect against severe inflammation and tissue damage

[0188] A-B. BNT162b2 mRNA COVID-19 vaccine induces p16Highcells in vivo. 2-3 month- old p16-Cre / R26-mTmG mice were treated intraperitoneal (I.P) with 5 pg of BNT162b2 mRNA COVID-19 vaccine. After 2 and 15 days the percentage of p16High cells was determined in different immune subsets in peritoneal cavity (A). Abundance and distribution of different p16Highcells in analyzed tissues. Each pie chart represents the total number of p16Highcells and how they are distributed in different cellular compartments (B). Data are mean ± SD. Statistical significance was determined using ANOVA plus Dunnett post hoc test. **p<0,01 and ***p<0,001

[0189] C. The percentage of p16High positive cells in different populations of T cells was determined at the same time than experiment shown in A. Data are mean ± SD. Statistical significance were determined using ANOVA plus Dunnett's post hoc test ***p<0,001.

[0190] D. BNT162b2 vaccine induces p16Highcells that are sensitive to senolytic treatment. 2- 3 month-old p16-Cre / R26-mTmG mice were treated I.P with 5 pg of BNT162b2 vaccine. 5 days later, mice were either treated orally with a senolytic combination of dasatinib (5 mg / kg) and quercetin (50 mg / kg) or vehicle (Mock). After 24h mice were euthanized and single cell suspensions from peritoneal cavity and liver were stained with conjugated antibodies against F4 / 80 and CD3. Cells suspensions were analyzed by flow cytometry and the percentage of p16High cells in each population was determined.

[0191] E. Genetic ablation of p16Highcells modifies the expression of inflammatory genes after BNT162b2 treatment. Wild type and p16-Cre / R26-DTA (DTA) were treated with 5 pg of BNT162b2 vaccine. After 2 and 5 days, RNA from peritoneal cells was isolated. RNA was analyzed by qPCR to determine the level of expression of inflammatory genes. Data are mean + / - S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001.

[0192] F. p16Highcells protect against acute inflammation. Wild type and p16-Cre / R26-DTA (DTA) were either treated with 5 pg of BNT162b2 vaccine or saline (Mock). After 5 days, animals were injected with lipopolysaccharides (LPS) from Escherichia coli 055: B5 (LPS) 40 mg / kg. Animals were euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. *p<0,05 and **p<0,01.

[0193] G p-|6Highcells protect against Ionizing Irradiation. Wild type and p16-Cre / R26-DTA (DTA) were treated with either 5 pg of BNT162b2 vaccine or saline (Mock). Animals were exposed to 8 Gy of y-irradiation. Animals were observed daily and euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. *p<0,05.

[0194] H-l. BNT162b2 vaccine induces p16Highcells in the lung. 2-3 month-old p16-Cre / R26- mTmG mice were treated I.P with 5 pg of BNT162b2 vaccine. 2 and 15 days later, mice were euthanized and the percentage of p16Highimmune cells was determined in the lung. Single cells suspensions from lung were stained with fluorescent conjugated-antibody against different markers of immune cells and analyzed by flow cytometry (H). Abundance and distribution of p16Highcells in the lung. Each pie chart represents the total number of p16Highcells in the lung and how they are partitioned into different cellular compartments (I). Data are mean ± SD. Statistical significance were determined using ANOVA plus Dunnett post hoc test. **p<0,01 and ***p<0,001.

[0195] J. BNT162b2 vaccine increases p16Highcells and induces Foxp3+, PD-1+and PD-L1+in CD3+CD4+populations. 2-3 month-old p16-Cre / R26-mTmG mice were treated I.P with 5 pg of BNT162b2 vaccine and after 5 days, single cell suspensions were stained with fluorescent- conjugated antibodies and the levels of CD3+CD4+PD1+, CD3+CD4+PD-L1+, and CD3+CD4+Foxp3+populations and the percentage of p16Highcells were analyzed by flow cytometry. Data are mean ± SD. Statistical significance were determined using ANOVA plus Dunnett post hoc test. ***p<0,001.

[0196] K. 2-3 months Balb / c mice were either treated with BNT162b2 vaccine or mock-treated and 3 days later were infected with a mouse-adopted MA10 virus. Analysis of probability of survival in experimental group as assessed, dead and mice that lost 70% and more body weight were considered as deceased. Difference between groups was analyzed using Gehan- Breslow-Wilcoxon test. *p<0,05.

[0197] Figure 4. The BNT162b2 mRNA COVID-19 vaccine induces disease tolerance by activation of TLR7 and tonic STING response.

[0198] A. p16 expression is dependent of TLR7 and STING. 2-3 months wilt type mice were treated with the BNT162b2 vaccine. One day before, in the same day, and day aftertreatment with BNT162b2, some animals were treated I.P with TLR7 inhibitor (M5049, 1 mg / kg) or STING inhibitor (H 151 , 10 mg / kg). p16 mRNA expression was determined after treatment in peritoneal cells and liver (left panel) by qPCR. 2-3 months MDA5+ / +(wild type) and MDA5-'- (MDA5-KO) mice were treated with the BNT 162b2 vaccine. After 5 days of treatment p16 mRNA expression was determined after treatment in liver (right panel) by qPCR. Data are mean + / - S.D. Difference between groups were analyzed using ANOVA test and Tukey post hoc test.

[0199] ***p<0,001. B. 2-3 months p16-Cre / R26-mTmG mice were treated with the BNT162b2 vaccine. One day before, in the same day, and day after treatment with BNT 162b2, some animals were treated I.P with TLR7 inhibitor (M5049, 1 mg / kg) or STING inhibitor (H151 , 10 mg / kg). 5 days after BNT162b2 treatment, animals were euthanized and single cells suspensions were analyzed by flow cytometry to determine the percentage of p16High cells on different immune subsets (left panel) and inside of Foxp3, PD-1 and PD-L1 (CD3+CD4+) populations (right panel). Data are mean + / - S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001.

[0200] C-D. 4-6 months p16-Cre / R26-mTmG mice were treated with the BNT162b2 vaccine. 5 days. 5 days after BNT162b2 treatment, animals were euthanized and single cells suspensions from peritoneal cavity (C) and liver (D) were analyzed by flow cytometry to determine the percentage of activated STING (p-STING) (phosphorylated at Ser366) in CD45+p16High, F4 / 80+p16High, and CD3+p16Highpopulations (left panel). The percentage covered of F4 / 80+p16High, and CD3+p16Highin the total CD45+p16Highp-STING+is shown in the upper pie chart. The p-STING+p16HighF4 / 80+and CD3+abundance and distribution were determined in the total CD45+p-STING+ and is show in the lower pie chart. Data are mean + / - S.D.

[0201] E. STING activation induces p16Highsubsets. 2-3 months p16-Cre / R26-mTmG mice were treated I.P with the STING agonist DMXAA (10 mg / kg). 5 days after treatment, animals were euthanized and single cells suspensions from peritoneal cavity were analyzed by flow cytometry to determine the percentage of p16Highcells on different immune subsets (left panel) and inside of Foxp3, PD-1 and PD-L1 (CD3+CD4+) populations (right panel). Data are mean + / - S.D. Differences between groups were analyzed using ANOVA test plus Dunnett post hoc test. *p<0,05; **p<0,01 and ***p<0,001.

[0202] F. 2-3 months wild type mice were treated I.P with the STING agonist DMXAA (10 mg / kg) one time (low dose) or 2 consecutive days (high dose). 5 days after first treatment, animals were euthanized and RNA from peritoneal cells (P.C), and liver was isolated. RNA was analyzed by qPCR to determine the level of expression of inflammatory genes. Data are mean + / - S.D. Differences between groups were analyzed using ANOVA test plus Dunnett post hoc test. *p<0,05; and ***p<0,001 .

[0203] G. Tonic STING activation promotes disease tolerance and tissue protection against severe inflammation. Wild type mice were either treated with saline (Mock); BNT162b2 vaccine (5 pg by mouse); BNT162b2 vaccine plus H151 (10 mg / kg, 3 consecutive days starting one day before BNT162b2 treatment); and DMXAA (10 mg / kg) for one (low dose) or two consecutive days subcutaneously (high dose). After 5 days, animals were injected with LPS 055: B5 30 mg / kg. Animals were euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow- Wilcoxon test. **p<0,01 and ***p<0,001.

[0204] Figure 5. NNMT expression and low adenosine are key conditions to induce p16Highimmune subsets and disease tolerance.

[0205] A. The NNMT gene is overexpressed in p16Highcells. RNA-seq data reveals overexpression of NNMT gene in p16Highcells. Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) from p16Highand p16lowpopulations were analyzed by t-test. Data are mean ± SD. ***p<0,001.

[0206] B. The NNMT gene is induced after DSS treatment. 2-3 month-old control and p16- Cre / R26-DTA (DTA) were treated with 2.5% DSS for 7 days and RNA from peritoneal cells and liver was isolated on day 8. RNA was analyzed by a quantitative SYBR-green based PCR (qPCR) to determine the level of expression NNMT mRNA. Data are mean + / - S.D. Difference between groups was analyzed using ANOVA test and Tukey post hoc test. ***p<0,001.

[0207] C. BNT162b2 vaccine-induced NNMT expression depends of p16Highcells. 2-3 month- old control and p16-Cre / R26-DTA (DTA) were treated with 5 pg of BNT162b2 vaccine. After 2 and 5 days, RNA from peritoneal cells, liver and lungs was isolated and analyzed by qPCR to determine the level of expression of NNMT mRNA. Data are mean + / - SD. Difference between groups was analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001.

[0208] D. NNMT expression is dependent of TLR7 and STING. 2-3 months wilt type mice were treated with the BNT162b2 vaccine. One day before, in the same day, and day after treatment with BNT162b2 some animals were treated I.P with TLR7 inhibitor (M5049, 1 mg / kg) or STING inhibitor (H151, 10 mg / kg). NNMT mRNA expression was determined after treatment in peritoneal cells (P.O) and liver (left panel) by qPCR. Data are mean + / - S.D. Difference between groups were analyzed using ANOVA test and Tukey post hoc test. ***p<0,001

[0209] E. NNMT is required to maintain p16Highimmune cells. 2-3 month-old p16-Cre / R26- mTmG and p16-Cre / NNMT-cKO (NNMT' / _conditional to the expression of p16) mice were treated intraperitoneal with 5 pg of BNT162b2 vaccine. After 5 days single cell suspensions from peritoneal cavity were stained with conjugated antibodies against different immune cell types. Cells suspensions were analyzed by flow cytometry and the percentage of p16Highcells in each population was determined. Data are mean ± SD. Statistical significance were determined using ANOVA plus Tuckey post hoc test. ***p<0,001 . F. Distribution and abundance of total p16Highcells from peritoneal cavity in p16- Cre / R26-mTmG and p16 / NNMT-cKO after BNT162b2 vaccine treatment are shown.

[0210] G. Percentage of p16Highpositive cells in different T cell populations was determined during experiment show in E. Data are mean ± SD. Statistical significance were determined using ANOVA plus T ukey post hoc test. ***p<0,001 .

[0211] H. Mice with selective inactivation of the NNMT gene in p16High cells cannot mount full immune response. 2-3 month-old control and p16 / NNMT-cKO mice were treated with 5 g of BNT162b2 vaccine. 5 days after treatment, RNA from peritoneal cells was isolated and analyzed by quantitative PCR to determine the level of expression of NNMT, p16 and inflammatory genes. Data are mean + / - S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001.

[0212] I. NNMT is necessary to protect against acute inflammation. 2-3 months wild type and p16 / NNMT-cKO mice were treated with 5 pg of BNT162b2 vaccine. After 5 days, animals were injected I.P with LPS 055: B540 mg / kg. Animals were euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. *p<0,05.

[0213] J. Activation of p16Highprogram lower adenosine level. 2-3 months wild type mice were treated I.P with saline (Mock), BNT162b2 vaccine, one (low) or two doses (high) of DMXAA (10 mg / kg), and BNT162b2 (pg by mouse) plus orally supplemented L-methionine daily during 5 days (200 mg / kg). Additionally, 2-3 months p16 / NNMT-cKO mice (written as NNMT-KO in graph label) were treated with saline or BNT162b2 vaccine. After treatment, peritoneal cells were collected and lysed, levels of adenosine were determined in the supernatant. Data are mean + / - S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001.

[0214] K. 3-4 months CDKN2A' / _(CDKN2A-KO) and CDKN2A+ / +(wild type) mice were either treated with saline (Mock) or BNT162b2 vaccine (5 pg by mouse). Single cell suspensions were analyzed by flow cytometry to determine the percentage of cleaved-caspase-3 in CD45, F4 / 80 and CD3 populations. Data are mean + / - S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001.

[0215] L. High adenosine levels reduce BNT162b2 effect on survival. 2-3 months wild type mice were treated with 5 pg of BNT162b2 vaccine, or BNT162b2 plus daily supplementation with L-methionine (200 mg / kg). After 5 days, animals were injected I.P with LPS 055: B5 30 mg / kg. Animals were euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. *p<0,05. M. Adenosine level is increase during aging. 3-4 months and 20 months-old wild type mice were treated with saline or BNT162b2 vaccine (5 pg by mouse). 5 days after animals were euthanized and levels of adenosine were determined in CD45+liver cells. Data are mean + / - S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05 and **p<0,01.

[0216] N. Blocking adenosine receptor A2A increase survival during aging. 2 year-old wild type mice were treated with 5 pg of BNT 162b2 vaccine, or BNT162b2 plus adenosine receptor A2A inhibitor (SCH58261 , twice by day, 3 mg / kg) starting one day before LPS treatment. 5 days after BNT162b2 treatment, animals were injected I. P with LPS O55:B520 mg / kg. Animals were euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. **p<0,01

[0217] Figure 6. MDA5 downregulation promotes increase physiological fitness during naturally aging

[0218] A. Activated STING and TBK1 are increased in young MDA5-KO mice. 3-4 months MDA5+ / +(wild type) and MDA5' / _(MDA5-KO) littermates were treated with saline (Mock) or STING inhibitor (H151.10 mg / kg). 48 h before, animals were euthanized and single cells suspensions from liver were analyzed by flow cytometry. Percentage of p-STING-Ser366 (left panel) and p-TBK1-Ser172 (right panel) positive cells was determined in CD45, F4 / 80 and CD3 populations. Data are mean + / - S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001.

[0219] B. p16 is overexpressed in MDA5-KO mice. 18-20 months MDA5+ / +(wild type) and MDA5' / _(MDA5-KO) littermates were treated with saline or STING inhibitor H151 (10 mg / kg). 48h before, animals were euthanized and mRNA from livers was isolated and analyzed by qPCR. Data are mean + / - S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. **p<0,01 and ***p<0,001.

[0220] C. MDA5-KO mice exhibit reduced adenosine levels during aging. 3-4 months MDA5+ / +(wild type); and 20 months MDA5+ / +(wild type) MDA5' / _(MDA5-KO) littermates were treated with saline or BNT162b2 vaccine (5 pg by mouse). 5 days after animals were euthanized and levels of adenosine were determined in peritoneal cells. Data are mean + / - S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001.

[0221] D. MDA5-KO mice shown less basal inflammation during aging. Liver RNA from 3-4 months MDA5+ / +(wild type) and MDA5' / _(MDA5-KO) littermates was isolated and analyzed by qPCR. The level of expression of inflammatory genes was determined. Data are mean + / -S.D.

[0222] Differences between groups were analyzed using t-test. *p<0,05; **p<0,01 and ***p<0,001.

[0223] E. Liver and muscle samples isolated from 24-month old wild type and MDA5-KO mice, stained for the endothelial marker CD31 . Graph at the right represents mean ± S.D of CD31+area. Area was determined among 3 animals per group. Differences between groups were calculated by unpaired, nonparametric Mann-Whitney test. ***p<0,001. Scale bar - 100 mm.

[0224] F. Immunofluorescent analysis of fibrosis marker aSMA in liver and muscle samples (same as in E). Graphs at the right represent mean ± S.D of aSMA area between groups, Area was determined among 3 animals per group. Differences between groups were determined by unpaired, nonparametric Mann-Whitney test. *p<0,05; and ***p<0,001. Scale bars: 200 mm for liver, 100 mm for muscle.

[0225] G. Muscle strength in 18 and 24 months old wild type and MDA5-KO littermates mice was determined with a grip test. Data are mean + / - S.D. Differences between groups were analyzed using ANOVA test and Dunnett post hoc test. **p<0,01 and ***p<0,001.

[0226] H. MDA5-KO mice show better physical fitness during natural aging. Frailty was assessed in 18 and 24 months old wild type and MDA5-KO littermates using an clinically relevant index for frailty during aging (see material and methods). Data are mean + / - S.D. Differences between groups were analyzed using ANOVA test and Dunnett post hoc test.**p<0,01 and ***p<0,001.

[0227] I. A group of wild type and MDA5-KO animals were housed in the same conditions and followed during 25 months to determine survival during aging. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. **p<0,01.

[0228] Figure 7. The BNT162b2 vaccine induces p16+immune subsets in humans that are significantly reduced in severe COVID-19 patients.

[0229] A-B. Expression of p16 and NNMT before (Day 0) and after BNT162b2 vaccine treatment (Day 7). RNA were isolated from PBMCs and analyzed by quantitative-PCR. Data were analyzed as a bulk (left panel) and as matched samples (right panel), t-test and paired t- test were used to determine the differences between conditions. *p<0,05 and **p<0,01.

[0230] C. Levels of p16+cells in different immune populations before and after BNT162b2 treatment are shown. 7 volunteers (included in the Covlmmune 2 cohort) with different range of age were involved in the study. Volunteers were treated intramuscularly with 30 pg of the BNT162b2 vaccine. Blood samples were collected before vaccination and after 7 and 30 days of treatment. Blood samples were analyzed by flow cytometry. Granulocytes, monocytes and lymphocytes populations were localized through size and granularity. CD45+, Tregs (CD3+ CD4+Foxp3+), PD1+, and p16+populations were localized with fluorescent conjugated antibodies. Data were analyzed as a bulk. Differences were determined using ANOVA test and Dunnetts test. *p<0,05 and ***p<0,001 .

[0231] D. Flow cytometry data were also treated as matched samples. The level of p16+cells inside singles populations are shown in each plot. Paired t-test was used to determine the differences before (Day 0) and after (Day 7 and day 30) the BNT162b2 vaccine treatment for CD45+, granulocytes, Tregs and PD1+. For monocytes and lymphocytes, Shapiro-Wilk test was used to determine normality of the data. ANOVA plus Dunnett's test or Friedman plus Dunn's test were used to determine difference before and after. *p<0,05 and **p<0,01 .

[0232] E. The total number of cells in CD45+, lymphocytes, granulocytes and monocytes, was used to determine the distribution of p16+cells among these populations. The percentage of contribution calculated for each population is showed in the pay graph at day 7.

[0233] F. One cohort (Covlmmune 1) of 40 unvaccinated patients and non-COVID volunteers older than 55 years were classified in negative for COVID-19, moderate COVID-19 and severe COVID-19. IL6 concentration in serum was analyzed by ELISA. Data are mean + / -S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test.***p<0,001.

[0234] G.H. One cohort of 40 unvaccinated patients older than 55 years were classified in negative for COVID-19, moderate COVID-19 and severe COVID-19. RNA from PBMCs were isolated and analyzed by quantitative-PCR. Level of p16 (G) and NNMT (H) expression were determined. Differences among groups were determined using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001.

[0235] Figure 8. Schematic representation of disease tolerance induced by p16High immune cells

[0236] Figure 9. Percentage of tumor associated p16highimmune cells after treatments using BNT162b2 vaccine, a-PDL1 or combination.

[0237] Samples were analyzed by flow cytometry using Cytoflex system (Beckman-Coulter) and CytoExpert software. An increase of p16highimmune cells in the tumors of the BNT162b2 treated mice is observed (upper panel), specifically in CD3+(T Cells), Ly6C+(Neutrophils), F4 / 80+(Macrophages) and NK1.1+(Natural killers and NK-T cells) immune subpopulations.

[0238] Figure 10. Lesions counting in the tumorigenic KRASG12Dmouse model after treatments with BNT162b2 vaccine, a-PDL1 or a combination thereof.

[0239] The number of lesions was manually determined following the criteria: small (<1 mm), medium (between 2 and 3 mm) and large (more than 3 mm) lesions. A reduction in the number of medium and large lesions was observed in the animals treated with BNT162b2. By combining all size lesions, the ability of BNT162b2 treatment reducing the number of lesions is significant including in the combined treatments (right panel).

[0240] Figure 11. Lesions counting in the tumorigenic p16-cre / DTA / KRASG12Dmouse model after treatments with BNT162b2 vaccine.

[0241] The number of lesions was manually determined following the criteria: small (<1 mm), medium (between 2 and 3 mm) and large (more than 3 mm) lesions in a KRAS mouse model in which the p16 gene is not expressed (p16-cre / DTA / KRASG12Dmice). A reduction in the number of medium and large lesions was not observed in the animals treated with BNT162b2. By combining all size lesions, the ability of BNT162b2 treatment reducing the number of lesion is not significant (right panel).

[0242] Figure 12. Tumor area measurement in KRAS and p16-cre / DTA / KRASG12Dafter treatment with BNT162b2 vaccine.

[0243] Longitudinal sections (3 pm thick) of lung from KRASG12Dexperiments, including all major lobes, were stained with hematoxylin and eosin (H&E) and evaluated. Lung stained sections were scanned and the percentage of tumor-occupied area was measured. It was found a significant reduction of tumor area in the BNT162b2 KRASG12Dtreated group with respect to the control, which is not as significant in p16-cre / DTA / KRASG12Dmice and reversed when BNT162b2 is combined with a-PDL1.

[0244] Figure 13. Analysis of survival in KRASG12Dtreated with the BNT162b2 vaccine and / or with an a-PDL1.

[0245] The percentage of survival in the BNT162b2 group (n=4), O-PDL1 group (n=3) and the combination group (n=7) was determined in comparison to the control group (n=38). Differences between groups were calculated using Mantel-Cox test (*p<0.05, **p<0,01 and ***p<0,001).

[0246] Figure 14. Analysis of the proliferation of B16F10 cells expressing luciferase that have been intravenously injected in mice, with different treatments.

[0247] (A) Individual values at day 1 were used to draw the plot as fold change. Animals were treated with BNT162b2 (5pg / mouse) or saline (mock) at day 3. Differences were determined with t test at day 10 (p<0,05).

[0248] (B) Administration of the BNT162b2 in combination with anti PD-1 therapy halts tumor growth of melanoma. (C) Tumor volume of B16F10 melanoma cells treated with BNT162b2 in combination with an anti PD-1 antibody. 1X106B16F10 cells were injected subcutaneously in immune competent mice. 7 days after B16F10 injection, animals were treated with saline (mock condition), single I.P injection of 5 ug of BNT162b2 by mouse, or 2 injections of 100 ug of anti- PD1 by mice (1 per week) or a combination of both. Tumor volume was measured along the experiment time frame using a caliper and the formula TV = (W(2) x [_) / 2

[0249] (D) Tumor volume of B16F10 melanoma cells treated with BNT162b2 in combination with an anti PD-1 antibody, with or without a TLR7 inhibitor (M5049) or a STING inhibitor (H 151). 7 days after B16F10 injection, animals were treated with saline, single I.P injection of 5 ug of BNT162b2 by mouse plus 2 injections of 100 ug of anti-PD1 by mice (1 per week). A set of animals also was treated with TLR7 inhibitor (2 mg / kg of M5049) or STING inhibitor (10 mg / kg of H151) 12h before BNT162b2 treatment. Tumor size was measured along the experiment time frame using a caliper and the formula TV = (W(2) x L) / 2 where TV is Tumor Volume, W - width and L -length.

[0250] Figure 15. Analysis of the effect of transplanted CD45+cells in mice. The protocol is detailed on (A). Briefly, CD45+cells from donor mice (previously treated or not with the BNT162b2 vaccine) are isolated and administered to recipient mice prior to a LPS challenge. The results are provided in (B). (C) describes the protocol for adoptive transfer of CD45+immune cells from donor (wild-type (proficient for p16Highcells) or p16-cre / DTA (deficient for p16Hi0hcells) mice) that were vaccinated with the BNT162b2 vaccine (“Vax”) into wild-type recipient mice that have been submitted to LPS-induced inflammation. (D) 2-3- month-old wild type (WT) and p16 / DTA (DTA) mice were treated with vehicle or BNT162b2 (5 ug per mouse). 5 days later CD45+ fraction was isolated from the livers of 4 donor mice. In parallel, 2-3 month- old recipient WT mice were treated with 32 mg / kg of LPS to induce sepsis. 6 h later, CD45+ cells from donor mice were injected I.V. into the recipients. Animals were monitored to determine point limit and the probability of survival. Number of animals (n) is showed in the plot. **p<0.01 .

[0251] Figure 16. Simultaneous stimulation of TLR7, STING and CD3 induces p16-positive cells in human whole blood in vitro. Whole blood from healthy human donors was treated in vitro with different conditions to induce p16Highcells. TLR7 and TLR7 / 8 agonists were used at 1 pg / ml (R837 and R848, respectively), and a STING agonist (DMXAA) was used at 10 pg / ml, either alone or in combination with anti-CD3, anti-CD28, and anti-CD2 stimulatory antibodies. p16Hi0hinduction was analyzed by flow cytometry specifically in CD45-positive cells (A) and CD3 T cells (B). RIG-I and MDA5 agonists were further tested as alternatives to the STING agonist in both total CD45-positive immune cells (C) and T cells (D). Whole blood from two healthy donors was treated with R837 and an anti-CD3 antibody (“QFM”) and DMXAA to induce p16Highcells as described above, and the treated blood samples were analyzed by flow cytometry (E and F) in CD11 b+fraction (G).

[0252] Figure 17. p16-positive cell-enriched humans PMBCs induce strong protection against LPS-induced sepsis in mice. Whole blood obtained from healthy human donors was treated in vitro with a TLR7 / 8 agonist (R848) at 1 pg / ml, the STING partial agonist (DMXAA) at 10 g / ml, and with anti-CD3 stimulatory antibodies, to induce p16-positive immune cells. A similar condition, with the addition of 100 ng / ml of dexamethasone, was also tested. Twenty-four hours later, recipient mice were treated with LPS to induce sepsis. Six hours later, PBMCs were isolated and transferred intravenously into the LPS-treated mice (under sepsis development). The protocol is explained in (A). The survival probability of the treated animals is displayed in (B).

[0253] Figure 18. BNT162b2 -induced p16-positive immune cells reduce tissue damage in multiple organs. Mice were treated with the BNT162b2 vaccine to induce p16-positive immune cells. Five days later, 10 mg / kg of LPS was injected (A). After 16 hours of sepsis induction, blood was collected, and plasma was analyzed to determine the level of liver enzymes, aspartate transaminase (ASAT) and alanine transaminase (ALAT), as a readout of liver damage (B). Similarly, mice were tested for gut leakage after sepsis induction. After 12 hours of LPS treatment, animals were orally gavaged with 12 mg of dextran-FITC (4 kDa), and 4 hours later (for a total of 16 hours), blood was collected and fluorescence was measured in the plasma (C). Finally, hypothermia was investigated, sixteen hours after LPS treatment (D).

[0254] EXAMPLES

[0255] Material and Methods

[0256] The material and methods used for the following examples are detailed below.

[0257] Animals

[0258] The p16-Cre knock-in mice were generated by Ozgene (Australia) by introducing a F2A-Cre-T2A-TK-E2AtdTomato cassette at the end of the last exon of the p16lnk4 gene. C57BL / 6 Embryonic Stem (ES) cells were used for targeting and correct integration was confirmed by Southern blot analysis. The Frt-loxed Neo cassette was removed by crossing the wt / p16-Cre heterozygous line with a homozygous FLP deleter line. Mice were then bred with Rosa26-mTmG (mixed C57BL / 6J and 129 / SvJ background) and Rosa26-DTA (C57BL / 6 background) mice (purchased from the Jackson Laboratory). Mixed background offspring were backcrossed with C57BL / 6 line to obtain p16-Cre / R26-mTmG, and p16-Cre / R26-DTA animals with pure C57BL / 6 background. Experiments were performed in mixed and pure background animals.

[0259] For all experiments heterozygous mice were used for both p16-Cre knock-ins and the reporters (Rosa26-mTmG or Rosa26-DTA).

[0260] For the present invention, a new model of transgenic conditional knock out for NNMT gene depending of p16 expression was generated. A conditional knockout mouse model of NNMT gene was generated by flanking exon 1 with loxP sites via gene targeting in mouse C57BL / 6 ES cells (Ozgene, Australia). The Cre-mediated deletion of the "floxed" 1 exon after crossing with p16-Cre knock-in mice15 resulted in removal of the ATG coding exon specifically in p16highcells.

[0261] For the SARS-CoV-2 (Mouse adapted (MA) 10) experiments wild type Balb / c females were used. Balb / c mice were purchased from SLC Japan.

[0262] Human population

[0263] The participants of the COVID study were included from the Covlmmune 1 cohort (NCT04355351) between April 2020 and September 2021 in Nice University Hospital.

[0264] Participants were recruited during an emergency room consultation following COVID- 19 symptoms, or as contact of a diagnosed COVID-19 case or following hospitalization for COVID-19. Patients were eligible for inclusion in this study if (i) they did not receive a vaccination regimen; (ii) SARS-CoV-2 infection was confirmed by a nasopharyngeal PCR or an antigenic test; (iii) they did not receive any COVID-19 treatment. Demographic, clinical, biological, and outcome data were collected by the study investigators and centralized in a database. Mild disease was defined as not requiring hospitalization and symptoms that did not include dyspnea, and severe disease, as require hospitalization and oxygen therapy. The non- COVID group is constituted of comorbid patients matched in age and sex to the COVID cohort and recruited before the start of the pandemic (March 2020) (NCT03804359).

[0265] The participants of a vaccine study were included from the Covlmmune 2 cohort (NCT04429594). Covlmmune 2 is an epidemiological study in the context of COVID-19 that monitored periodically since July 2020 patients developing a SARS-CoV-2 infection or response to SARS-CoV2 vaccination. Demographic, clinical, biological data were collected by the study investigators and centralized in a database. The participants from the Covlmmune 2 cohort (NCT04429594) were included to evaluate the effect of BNT162b2 mRNA COVID-19 vaccine after treatment. Seven volunteers were treated following the standard protocol approved for EMA and FDA. In total 30 pg of BNT162b2 mRNA COVID-19 vaccine were injected intramuscularly in the arm. Blood samples were taken right before vaccination and 7 and 30 days after vaccination. In vivo treatments

[0266] Mice

[0267] To phenocopy the effect of aging on intestinal tissue homeostasis, 2-3 months animals were treated with 2.5% dextran sodium sulphate (Mr -40,000) (Sigma-Aldrich 42867) dissolved in drinking water for 7 days. To find the specific toll-like receptor capable to induce p16 expression, 2 months animals were injected intraperitoneally with Pam3CSK4 (TLR2 / 1 agonist) 50 pg by mouse (Invivogen tlrl-pms), Pam2CSK4 (TLR 2 / 6 agonist) (3 pg by mouse) (Invivogen tlrl-pm2s-1); Poly (l:C) (TLR 3 agonist) (150 pg by mouse) (Invivogen tlrl-picwlv); Lipopolysaccharides from Escherichia coli 055: B5 (TLR4 agonist) (1 mg / Kg) (Sigma-Aldrich L2880); flagellin from Salmonella typhimurium (TLR5 agonist) (5 pg by mouse) (Invivogen tlrl- stfla); R-837 (mod) (TLR7 agonist) (50 pg by mouse) (Invivogen tlrl-imq); and ODN1585 (TLR9 agonist) (50 pg by mouse) (Invivogen tlrl-1585), 48 h later, animals were euthanized for analysis. To induce severe sepsis, 2 months animals were treated with Lipopolysaccharides from Escherichia coli O55:B5 (40 mg / Kg) (Sigma-Aldrich L2880). To induce severe tissue damage, 2 months animals were exposed to 8 Greys (Gy) of y-irradiation. To induce lethal COVID-19 infection in mice, 10 weeks old animals were treated intranasally with the SARS- CoV-2 murine strain MA10 (4 * 105FFU), virus infections experiments were done in a biosafety level 3 facility at Osaka University. To induce an increase of p16highcells in young animals, 2- 3 old months animals were treated with BNT162b2 mRNA COVID-19 vaccine (5 pg by mouse) (Pfizer-BioNTech), and 2, 3, 5 or 15 days later, animals were euthanized for analysis, the specific day and full description for each experiment is described in Figure legends. For all the stromal-vascular cell fraction (SVF). Spleens were mashed in cold DMEM containing DNase I at 100 pg / mL and sieved with a 75 pm and 30 pm strainer to obtain single cell suspensions. For bone marrow isolation, muscles were removed from both legs. An incision was made between lesser and greater trochanter. Bones then were centrifuged at 10,000 g during 30 seconds. Bone marrow was resuspended in HBSS containing DNase I at 100 pg / mL and sieved with a 30 pm strainer to obtain single cells suspension. Peripheral blood was obtained mediated cheek puncture. Red blood cells were removed using lysis buffer. Cells were centrifuged at 1500 RPM during 5 minutes at 4 °C. Cells were resuspended in cytometry buffer (HBSS 1% BSA, 4% FBS, 2 mM EDTA) and stained with fluorescent conjugated antibodies (see list for specific cases) at 2 pg / mL during 25 minutes. Cells were washed 2 times with cytometry buffer. For FOXP3 staining, after membrane markers labeling, cells were fixed, permeabilized, stained and washed using the Foxp3 / Transcription Factor Staining Buffer Kit (Tonbo biosciences cat. TNB-0607). Samples were analyzed by flow cytometry using Cytoflex system (Beckman-Coulter). At least 3X104events were recorded in singlets populations. Data were obtained and analyzed using CytExpert software. Human samples

[0268] In humans, p16 expression in T cell populations was assessed after immunostaining with specific antibody in whole blood by flow cytometry. In COVID-19 patients, flow cytometry was performed in isolated PBMC that were stored at -80°C in storage solution. In whole blood samples, red blood cells were lysate with Pharm Lyse™ Lysing Buffer (BD Biosciences™).

[0269] Cell surface staining was performed in 1x PBS for 30 min at 4°C. Cells were fixed and permeabilized using Transcription Factor Staining Buffer Kit (Tonbo Bioscience, San Diego, CA). Intracellular staining (FOXP3 and p16) was performed in permeabilization buffer for 30 min at 4°C. Flow cytometry data was acquired on a BD FACSLyric™ and analyzed in BD FACSuite™ software. At least 1x106events were recorded in singlets populations.

[0270] Antibodies are listed below. Cell isolation and cell culture

[0271] Peritoneal macrophages were isolated using F4 / 80 magnetic beads (Mi Itenyi Biotec # 130-110-443) and LS columns (Miltenyi Biotec #130-042-401) following the manufacture's instructions. Macrophages were cultured in Roswell Park Memorial Institute medium(RPMI) 10% heat inactivated fetal bovine serum (GIBCO) and 100 U ml-1 penicillin / streptomycin (Sigma, P4333) at 37 °C with 5% CO2. VeroE6 / TMPRSS2 cells (JCRB, 1819) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (MP Biomedicals, 2917354H) and 100 U ml-1penicillin / streptomycin (Sigma, P4333) at 37°C with 5% CO2. It was regularly confirmed the absence of mycoplasma contamination in our cultured cells.

[0272] Healthy volunteers and COVID-19 patient PBMC were isolated by density gradient using Ficoll (Human PANCOLL, Pan Biotech™) and stored in foetal bovine serum 10% DMSO at -80°C.

[0273] Virus preparation

[0274] The SARS-CoV-2 strain, Mouse-adapted SARS-CoV-2 (MA10), amplification and titration were previously described (Tsuji S., 2022, Nat. Aging 2, 115-124). Titration was used as a TCID50 (median tissue culture infectious dose) assay by Vero / TMPRSS2 cells.

[0275] SA- beta-Gal staining

[0276] Peritoneal cells from 12 months old p16-Cre / R26-mTmG mice were isolated by washing peritoneal cavity with cold RPMI. Cells were incubated overnight in RPMI supplemented with 10% fetal bovine serum at 37°C and 5% CO2 conditions to allow them to attach to the plate. Cells were washed with PBS and fixed for 5 min at room temperature in 4% PFA, then cells were washed 3 times with PBS. Fixed cells were incubated during 8-12 hours at 37°C with staining solution containing: 40 mM Citrate-sodium phosphate pH 6; 5 mM K3[Fe(CN)6]; 5 mM K4[Fe(CN)6]; 2 mM MgCI2; 150 mM NaCI; and 1mg / ml X-gal.

[0277] Fluorescence microscopy

[0278] Peritoneal cells from 12-16 months old p16-Cre / R26-mTmG mice were isolated by washing peritoneal cavity with cold RPMI. Cells were incubated overnight in RPMI supplemented with 10% fetal bovine serum at 37°C and 5% CO2 conditions to allow them to attach to 10 mm slides. Subsequently, cells were fixed in 4% PFA (10 min, room temperature blocked (1 hour, 5% Goat Serum, 0.3% triton and 1 %BSA) and incubated in blocking solution with first antibody (1 hour, room temperature): chicken anti-GFP (1 / 1800, ab13970, Abeam); rabbit anti-Lamin B1 (1 / 300, ab16048, Abeam); Rabbit anti- phospho-gH2AX (1 / 250, 9718, Cell Signalling); Rat anti-p21 (1 / 150, ab107099, Abeam), and Rat anti-RAD51 (1 / 500, BJ12112008, Bioss). The secondary antibodies used were Alexa Fluor 488 anti-chicken; Alexa Fluor 488 anti-rabbit; Alexa Fluor 647 anti-rabbit, and Alexa Fluor 647 anti-rat (1 / 500, Life Technology). Nuclei were counter-stained with DAPI included in mounting medium (Vectashield, H-1200). Images were taken using an HD Zeiss Axio Observer Z1 Microscope and a confocal Zeiss LSM 880 microscope (Zeiss, Gottingen, Germany) and analyzed by using ZEN (blue edition) software (Zeiss).

[0279] Proliferation Assay

[0280] Peritoneal cells from 12 months old p16-Cre / R26-mTmG mice were isolated by washing peritoneal cavity with cold RPMI. Cells were incubated overnight in RPMI supplemented with 10% fetal bovine serum at 37°C 5% CO2 to allow them to attach to the plate. Proliferation capacity was assessed using the Click-iT® Plus Edll Assay kit (Invitrogen C10640) following the protocol provided by the manufacturer. Cells were incubated with Edll during 24 h.

[0281] Migratory capacity Assay

[0282] Freshly isolated F4 / 80+ cells from 12 months p16-Cre / R26-mTmG mice peritoneal cavity (Anti-F4 / 80 MicroBeads UltraPure, Miltenyi Order no. 130-110-443) were seeded in a 8 pm pore permeable insert (24 well plate; 3,2 mm diameter Transwells Corning) previously coated with matrigel in a migratory solution (DMEM 0,1 % BSA). A lower chamber was filled with complete medium containing 100 nM Phorbol 12-myristate 13-acetate (PMA). Cells were incubated at 37°C and 5% CO2 during 24 h. Cells were counted using fluorescence microscope (EVOS system, Thermofisher scientific). The percentage of migrating cells were determined using cells seeded directly in 5 mm diameter wells as a control.

[0283] Phagocytic activity Assay

[0284] Peritoneal cavity cells from 12 months old p16-Cre / R26-mTmG mice were isolated by washing peritoneal cavity with cold RPMI. Cells were incubated overnight in RPMI supplemented with 10% fetal bovine serum at 37°C and 5% CO2 to allow them to attach to the plate. Cells were incubated with beads (Invitrogen 11151 D). Number of beads inside the cells were counted at 1, 2, 5, 10, 15 and 30 min. RNA sequencing

[0285] Peritoneal cavity macrophages were isolated using F4 / 80 beads. p16highand p16lowpopulations were separated using fluorescence activated cell sorting (FACS) (FacsAria 3 Analyzer, BD Biosciences). RNA isolation was performed using RNeasy kit (Quiagen # 74104) according to the manufacturer’s protocol. RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). RNA integrity and quantitation were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total amount of 1 mg RNA per sample was used as input material for the RNA sample preparations. Sequencing manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligoattached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer (5X). First Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease / polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150_200 bp in length, the library fragments were purified with AM Pure XP system (Beckman Coulter, Beverly, USA). Then 3 ml USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using SR Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and 50 bp / 100 bp single-end reads were generated.

[0286] Gene expression

[0287] Murine total RNA from peritoneal cells, liver, lung, or human PBMCs was isolated with RNeasy Mini Kit (Quiagen # 74104) according to the manufacturer’s protocol. 1 g of total RNA was used for cDNA synthesis using RevertAid First Strand cDNA Synthesis Kit (ThermoFisher # K1621) and 30-60 ng cDNA was used for a PCR reaction. The quantitative PCR was performed with KAPA SYBR FAST qPCR Kit (KAPPA Biosystem # KR0389) using the StepOnePlus Real-Time PCR System with the following parameters: enzyme activation 2 min at 95°C, and 40 cycles of 3”- 95°C, and 45”- 60°C. Primers for PCR were designed using Primer-BLAST tool at NCBI with the condition of separation of primer pairs with at least one intron. The mRNA expression levels of each gene were calculated relative to p-actin expression levels. Primers sequences are listed below. Cytokine detection assay

[0288] Blood samples were collected for basal cytokine measure in serum. IL-6 basal level was measured in serum, with custom-designed enzyme-linked immunosorbent assay (ELISA) Multiplex Cartridges (Bio-Techne) on Automated ELISA system Ella (ProteinSimple).

[0289] RNA-Seq Data analysis

[0290] Raw data (raw reads) of fastq format were firstly processed through in-house perlscripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality. Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Bowtie v2.2.3 and single-end clean reads were aligned to the reference genome using TopHat v2.0.12. It has been selected TopHat as the mapping tool for that TopHat can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools. For unigene DGE, Bowtie vO.12.9 was used to aligned single-end clean reads to the unigene sequences. HTSeq vO.6.1 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels (Spychala J., 2000, Pharmacol. Ther. 87, 161-173). For unigene DGE (digital gene expression), it has been used RSEM (Relative Standard Error of the Mean) to count the reads numbers mapped to each unigene.

[0291] Data representation and statistical analysis

[0292] Data presented based on at least three independent experiments, values are means + / - SD. For survival experiments, the sample size from all experiments is presented in each figure. For the rest of experiments, including in vitro and in vivo, data from a representative experiment is shown. Normal distribution of data was calculated using the Shapiro-Wilk normality test. Comparison of mean values between groups was evaluated by 2-tailed Student’s t-test, Paired t-test, Wilcoxon matched pairs test, ANOVA, Tukey-Kramer test, Dunnett test, Kruskal Wallis test, Friedman test, Dunn's test, Gehan-Breslow-Wilcoxon test and Sidak’s multiple comparisons test using the GraphPad Prism program Version 8.2.1. The specific choice of test for each particular experiment is annotated in the Figure legends. Pvalues less than 0.05 were considered significant. Any P-value less than 0.05 was designated with one (*) asterisk; less than 0.01 with two (**) asterisks, less than 0.001 with three (***) asterisks. All p-values are reported in the Figure legends. Results are considered significant when p < 0.05.

[0293] Cell lines

[0294] B16F10 (ATCC® CCL-6475™) is a murine melanoma cell line from a C57BL / 6J mouse. B16F10 were maintained in DMEM medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U / ml of penicillin and 0.1 mg / ml of streptomycin (Gibco, Grand Island, NY, USA).

[0295] Immunophenotyping

[0296] Syngeneic melanoma tumor model was implemented to study immune subsets within tumor microenvironment. B16F10 melanoma cells were cultivated, collected, and resuspended in HBSS (Gibco, Grand Island, NY, USA) with the final concentration of 1.2x106per 100 pl. p16-Cre / R26-mTmG mice were injected subcutaneously in the neck area with 100 pl of the B16F10 cell suspension. Mice were treated with senescence inducer (SI, undisclosed substance), PDL1 blocking antibody (a-PDL1) purchase from BioXcell Catalog #BE0101 (200pg per mouse), or their combination when a tumor was visible (around 7-9 days), and retrieved from cages 3-5 days later.

[0297] Mice were euthanized by cervical dislocation. Tumors were collected and incubated with 1 mg / ml collagenase type A at 37°C during 45 min, sieved consecutively with two 100, and 30 pm strainers to obtain single cell suspensions. Red blood cells were removed using lysis buffer. Cells were then centrifuged at 1500 RPM for 5 minutes at 4°C. Cells were resuspended in cytometry blocking buffer (HBSS 1 % BSA, 4% FBS, 2 mM EDTA) and stained with fluorescent conjugated antibodies (see the list below) at 2 pg / mL during 25 minutes. Cells were washed 2 times with the cytometry buffer. Samples were analyzed by flow cytometry using CytoFlex system (Beckman-Coulter, California, United States). At least 3X104 events were recorded in singlets populations. Data were obtained and analyzed using CytExpert software KRAS Tissue analysis

[0298] KRAS mice were divided into three groups at the age of 2 months and injected with a drug of interest. First group was a control group. Second group was injected two times with BNT162b2 2 weeks apart and euthanized on week 8. Third group was subjected to four alternating injections of BNT162b2 and a-PDL1 (200pg per mouse) throughout first 4 weeks and euthanized on week 8 as well. Collected tissue samples (lungs) were fixed in 4% PFA overnight before alcohol dehydration, Number of tumor was determined manually by counting the number of lesions. After that tumors were embedded in paraffin. Samples were cut into 3 pm sections using Automated Rotary Microtome HistoCore AUTOCUT (Leica, France). Paraffin-embedded sections were deparaffinised in xylene and rehydrated in ethanol with increasing concentrations of water, stained with haematoxylin and eosin for visualization of cancerous lesions in lungs of KRAS mice.

[0299] For long term survival, animals of 4 months were treated with BNT162b2 (5 pg per mouse), anti PDL1 antibody, (200 pg per mouse), the combination or saline (mock) 1 time by month until animals reach defined point limit.

[0300] Transfer of disease resistance

[0301] To test the capacity to transfer BNT162b2 -induced disease tolerance from mouse to mouse, it was decided to test the effect of injecting CD45+cells isolated from previously BNT162b2 and mock treated mice. After 5 days of treatment, mice were sacrificed and tissue resident CD45+were isolated. 5x105cells were injected into recipient mice. 24 h after cell injection, animals were challenged with a lethal dose of lipopolysaccharide (O55:B5) (35 mg / kg I.P) (Figure 15A). Probability of survival was assessed. Differences between groups were calculated using Mantel-Cox test (p<0,01).

[0302] Statistical analysis

[0303] Statistics were determined using GraphPad Prism, version 9 (GraphPad Software Inc., San Diego, CA, USA). The results are expressed as mean values ± SDs. p-values were calculated using Student’s t-test when comparing two groups of continuous variables. The significance level was defined as p-values <0.05 (* p < 0.05). Probability of survival was assessed. Differences between groups were calculated using Mantel-Cox test (*p<0.05, **p<0,01 and ***p<0,001). Results

[0304] Example 1: p16highimmune cells analysis in different tissues with age

[0305] The development of different genetic mouse models is now facilitating the further identification and characterization of p16highcells in vivo (Baker DJ. Et al., 2011, Nature 479, 232-236 ; Demaria M. et al., 2014, Dev. Cell 31 , 722-733 ; Omori S. et al., 2020, Cell Metab. 32, 814-828; and Liu JY. Et al., 2019, Proc. Natl. Acad. Sci.'l'lB, 2603-2611). Among the different p16highsubtypes, cells of the immune system, including T cells and macrophages, have been identified and further analysis revealed that some express additional markers of senescence such as enhanced senescence-associated [3-galactosidase (SA-p-gal) activity and DNA damage (Yousefzadeh MJ. Et al., 2021 , Nature 594, 100-105). Furthermore, the frequency of such cells increases significantly in animals during natural and accelerated aging, which may highlights their potential importance (Yousefzadeh MJ. Et al., 2021 , Nature 594, 100-10 ; Chou JP. Et al., 2013, Curr. Pharm. Des. 19, 1680-1698 and Childs BG. Et al., 2016, Science 354, 472-477). On the other hand, it is worth mentioning that modest or even transient activation of p16, as well as excessive lysosomal activity (and thus higher SA-p-gal activity) in phagocytic cells such as macrophages has been observed under different conditions (Liu JY. Et al., 2019, Proc. Natl. Acad. Sci. 6, 2603-2611 and Hall BM. Et al., 2017, Aging 9, 1867- 1884).

[0306] The transgenic murine model p16-Cre / R26-mTmG was used and cells with high expression of p16 were selectively identified (henceforth designated p16highcells) by detecting the expression of an EGFP (Enhanced green fluorescent protein) reporter gene (Grosse L. et al, 2020, Cell Metab. 32, 87-99). A flow cytometry-based analysis of p16highcells resident was performed in different murine tissues at 2, 12, and 18 months-of-age, including the bone marrow, peripheral blood, spleen, peritoneal cavity, liver and the stromal-vascular fraction (SVF) of abdominal fat. Then, percentage of cells expressing high levels of p16 within populations of cells expressing common markers of immune cell subtypes, including CD3 (T cells), B220 (B Cells), CD11c (dendritic cells), Ly6C (monocytes), Ly6G (neutrophils), and F4 / 80 (macrophages) was determined.

[0307] Results

[0308] Overall, a relative increase in the numbers of p16highcells is observed with age in all the tested immune subsets (Figure 1 A). Further analysis of different immune subsets revealed their significant contribution to the total number of p16highcells in different tissues ranging from 35%-45% for stromal-vascular fraction of abdominal fat (SVF) in abdominal fat and liver, to more than 90% in the bone marrow (Figure 1 B). Thus, p16highcells are present both in very short-lived (Ly6G+, neutrophils) and very long-lived (F4 / 80+, tissue-resident macrophages) immune subsets and constitute a significant fraction of the total p16highpopulation, thus potentially representing senescent cells in different tissues of aging animals.

[0309] Recent data showed an enrichment of PD-1-PD-L1 markers in senescent cells (Wang TW. et al, 2022, Nature 611, 358-364); therefore, the fraction of p16hi0hcells was determined among the total PD1+, PD-L1+and PD-L2+cells in bone marrow, liver, peritoneal cavity, and SVF in 16-month-old mice. We found that the contribution of p16highcells in these populations varied significantly depending on the tissue analyzed, from 1.5 % in the bone marrow to 40 % in the liver (Figure 1C).

[0310] Analysis of the fraction of PD1+, PD-L1+and PD-L2+cells in the total number of p16highcells revealed a wide range from a small (bone marrow) to a significant (peritoneum) contribution (Figure 1 C, pie charts). Thus, the contribution of p16highcells to the total number of PD1+, PD-L1+and PD-L2+cells and vice versa varies significantly among different tissues, with PDL1+ cells representing a relatively higher proportion of the p16hi0hcell population (Figure 1 C, bottom charts).

[0311] Then, an analysis was performed for fractions of CD3+CD4+PD-1+, CD3+CD4+PD- L1+, CD3+CD4+PD-L2+and CD3+CD4+FoxP3+cells in the p16highcell populations (Figure 1 D). A strong enrichment is observed of T cells expressing PD-1 , PD-L1 but most importantly regulatory FoxP3 markers in the p16hi0hcell populations, suggesting that a significant fraction of the total PD1+and PD-L1+cells expressing high levels of p16 in any given tissue (Figure 1C) are indeed T cells. Further analysis of different subsets among the p16highT cells revealed a strong enrichment for Tregs. By contrast, PD-L2+cells were less enriched inside the p16highT cells (Figure 1 D). Overall, these analyses showed that a noticeable fraction of p16hi0hT cells express markers of regulatory subsets and thus potentially could play an important role in negative regulation of tissue inflammation and damage through well-established immunosuppressive and tissue remodeling activities of TregS.

[0312] Example 2: p16highperitoneal macrophages analysis

[0313] Tissue-resident macrophages, in comparison to other immune subtypes, are of embryonic origin and maintained for the entire life span of the organism. Based on their longevity, it was speculated that tissue-resident macrophages could be the best immune cell candidates to express multiple markers of senescence in addition to the high levels of p16 as an indicator of full senescence. To investigate this hypothesis in more detail, F4 / 80+peritoneal tissue-resident macrophages were focused on. Previous studies have shown that macrophages exhibit a modest increase in expression levels of p16 and SA-p-gal, both putative markers of senescence, with age and in response to the presence of senescent cells in the peritoneal cavity.

[0314] To investigate peritoneal F4 / 80+macrophages, the SA-|3-gal activity in these cells isolated from 2- and 12-month-old p16-Cre / R26-mTmG mice was analysed.

[0315] Results

[0316] It was found that while SA-p-gal activity was indeed significantly higher in the cells isolated from 12-month-old mice when compared to those isolated from to 2-month-old mice, surprisingly, no difference was found in the levels of activity between the p16high(EGFP expressing cells) and p16Low (tdTomato expressing cells) populations (Figure 1E). Analysis of additional putative senescence markers revealed an increase in the percentage of both p- ATM+ and 53bp1+ cells, while yH2Ax was reduced in the p16highpopulation (Figure 1G). To explore further this unexpected reduction in yH2Ax+ cells in p16hi0hpopulation, the expression of p21 protein was evaluated, another putative marker of senescence. Double-positive p21+ / p16+ cells were infrequent and further analysis revealed that the majority (approximately 80%) of the p21+cells were positive for yH2Ax (Figure 1 H). Thus, the results showed the presence of two populations of peritoneal F4 / 80+macrophages that carry senescent markers in aging mice - one, which is positive for p16 and p-ATM / 53Bp1, and another, which is positive for p21 and yH2Ax - with both populations expressing SA-p-gal. Importantly, the analysis of proliferation using Brdll labeling confirmed the absence of proliferation by p16hi0hperitoneal F4 / 80+ macrophages, which is a feature of a cell cycle arrest (Figure 1 F). In addition, it was found a reduction in the migratory capacity of p16hi0hF4 / 8CF macrophages (not shown), while there was no difference in phagocytic activity (not shown) between the p16Highand p16Lowpopulations. Thus, p16highperitoneal F4 / 80+macrophages do not express many classical markers of senescence and thus, should be more accurately referred to as p16highand not senescent or senescent-like.

[0317] To gain a better understanding of the changes that accompany p16 activation, RNA- sequencing (RNA-seq) on p16highand p16lowF4 / 80+ peritoneal macrophages was performed. First, it confirmed the upregulation of p16 mRNA in p16highcells and subsequent KEGG pathway analysis revealed that the most represented clusters of downregulated genes were related to cell cycle arrest and DNA replication (Figure 11). Next, the list of genes related to the senescence-associated secretory phenotype (SASP) was manually curated. Surprisingly, the number of over-expressed factors associated with the SASP was limited in p16highcells, while none of the classical inflammatory factors were overexpressed (not shown). KEGG analysis of the upregulated genes revealed a set of genes related to the regulatory functions of immune cells (not shown) including the Arg1, Clec4g, Vsig4, Timd4 and Gpnmb genes (Figure 1 J). Thus, this analysis of p16highperitoneal F4 / 80+macrophages revealed that these cells express high levels of p 16, resulting in profound cell cycle arrest, as well as the expression of regulatory factors that exhibit both immunosuppressive and tissue remodeling activities such as Arg1 and Clec4g. Overall, the macrophage analysis together with the observed expression of several regulatory markers by p16highT cells (Figure 1C&D) suggested that p16 activation is accompanied by the appearance of different regulatory immune subsets.

[0318] Example 3: p16highimmune cells analysis in young animals in response to inflammation and tissue damage

[0319] Since the data in T cells (Figure 1C&D) and macrophages (Figure 1 J) pointed towards regulatory nature of p16highimmune cells, next further confirmation of these observations at the tissue level was performed.

[0320] Removal of p16Highimmune cells with subsequent analysis of tissue pro- and antiinflammatory profiles allow to define more precisely their role. This is something that would be difficult to achieve in an old organism due to present of different p16High, including numerous types of senescent cells in multiple tissues. Thus, the conditions for the accumulation of p16Highimmune cells were sought in young organisms so their clearance would provide a clear confirmation of their functions at a tissue level.

[0321] Aging is accompanied by a noticeable intestinal deterioration with the development of a so-called “leaky gut”. If the process spreads further, this increased intestinal wall permeability can trigger a defensive inflammatory response in both the peritoneum and the liver. To mimic an age-induced deterioration of the intestine, it has been employed a model of dextran sodium sulfate (DSS)-induced colitis (Chassaing B. et al, 2014, Curr. Protoc. Immunol. 104) in 2- month-old p16-Cre / R26-mTmG mice and analyzed the accumulation of p16highimmune subsets in the peritoneal cavity and liver by flow cytometry.

[0322] Results

[0323] It was found an increase in the percentage of p16highcells in different immune subsets both in the peritoneum and liver (Figure 2A).

[0324] Analysis of absolute numbers of p16highcells revealed that their largest fraction consisted of CD3+ T cells and F4 / 80+ macrophages (Figure 2B). Among the CD3+ T cells, it was found a significant enrichment of p16highcells expressing the regulatory marker Foxp3, as well as PD1 and PD-L1 (Figure 2C&D). Thus, DSS-induced colitis was confirmed to trigger an efficient accumulation of p16highimmune subsets in young mice.

[0325] Next, investigation was performed to analyze the impact of their genetic ablation using a diphtheria toxin A (DTA)-dependent ablation mouse line, in which p16highcells are selectively eliminated through activation of the Cre recombinase (p16-Cre / R26-DTA). p16-Cre / R26- mTmG and p16-Cre / R26-DTA mice were either mock- or DSS-treated and RNA was isolated from peritoneal and liver cells for qPCR analysis. First, it confirmed an increase in the level of p16 mRNA in both tissues after DSS treatment of p16-Cre / R26-mTmG mice, something that was fully abrogated in p16-Cre / R26- DTA mice. The Arg1 and 1110 genes have been implicated in both immunosuppression and tissue remodeling (Fouda AY. Et al., 2018, Cell Death Dis. 9, 1-15 and Edwards JP. et al. ,2006, J. Leukoc. Biol. 80, 1298-1307), and their analysis showed strong accumulation in both peritoneal and liver cells after DSS treatment of p16-Cre / R26- mTmG, but not in p16-Cre / R26-DTA mice (Figure 2E&F). In contrast, it has been observed marked accumulation of the pro-inflammatory genes I L1 b and IL6 in the peritoneal cavity after selective ablation of p16highimmune cells (Figure 2E). As such, the analysis directly confirmed that at the tissue level, p16highimmune cells have an important regulatory functions and their depletion under certain conditions can trigger an enhanced inflammatory and tissue damage responses.

[0326] To gain potential mechanistic insights, it has been explored the ability of Toll-like receptor (TLR) stimulation, which also occurs in a DSS model of colitis, to induce p16highimmune subsets (Chassaing B. et al., 2014, Curr. Protoc. Immunol. 104 and Rakoff-Nahoum S. et al., 2004, Cell 118, 229-241). TLRs are the first-line sensors and responders to possible harmful factors including different pathogens, in the surrounding environment. These receptors act as defense and homeostatic regulators by recognizing both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). To systematically address the role of different TLRs, it has been administered different TLR agonists to 2-month-old p16- Cre / R26-mTmG mice by intraperitoneal injection and analyzed p16highimmune cells in the peritoneal cavity. It was found that agonists of TLR7 (R-837) and to a lesser extent TLR5 (flagellin) were the best inducers of p16highmacrophages (figure 2G), while agonists of TLR1-2 (Pam3CSK4) and TLR7 (R-837) were the best inducers of p16highT cells (Figure 2H). Overall, these data suggested that induction of p16highimmune cells could be a part of a physiological response to tissue damage and inflammation and can be triggered by different TLRs receptors. Among the TLRs, TLR7 appeared to be strongest inducer of p16highmacrophages and T cells, both of which are long-lived immune subsets.

[0327] Example 4: Effect of BNT162b2 mRNA COVID-19 vaccine on p16highimmune cells

[0328] Since TLR7 receptor activation induced regulatory p16highsubsets potentially creating disease tolerance by reducing the negative impact of the infection and tissue damage on host fitness, it has been focused on this signaling pathway. TLR7 is located intracellularly primarily in endosomes and detects single-stranded RNA targets, including those from different viruses such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), and SARS-CoV-2 (Diebold SS., et al., 2004, Science 303, 1529-1531 and Mantovani S. et al., 2022, genes Immun. 23, 51-56). For further analysis, it has been focused on the BNT162b2 mRNA COVID- 19 vaccine as an only FDA-approved TLR7 agonist. It has been assessed the efficiency with which this vaccine induced different p16highimmune subsets. To do so, it has been intraperitoneally injected 2-month-old p16-Cre / R26-mTmG mice with BNT162b2 and investigated the induction of p16 in different immune cells at days 2 and 15.

[0329] Results

[0330] It was found that BNT162b2 efficiently induced p16hi0himmune cell subtypes both in the peritoneum and liver, which was observed as early as day 2 and lasted beyond day 15 after treatment (Figure 3A&B). Further analysis of p16 induction in T cell subsets revealed an increase in cell populations positive for regulatory Foxp3, PD-1 and PD-L1 in the peritoneum (Figure 3C) and for Foxp3 in the liver (not shown) after vaccine treatment.

[0331] Having previously found that the liver p16highF4 / 80+ macrophages in 12-month-old mice are sensitive to a senolytic cocktail of dasatinib and quercetin (DQ) (Grosse L. et al., 2020, Cell Metab. 32, 87-99,), the effects of BNT162b2 were assessed with and without subsequent DQ treatment (Figure 3D). It was found that DQ efficiently reduced the numbers not only of p16hi0hmacrophages, but also of T cells in both the peritoneum and liver in mice treated with BNT162b2. Thus, it confirmed that DQ treatment has a broad inhibitory activity on different p16highimmune subsets in vivo.

[0332] To understand the significance of BNT162b2-induced accumulation of p16hi0himmune cell subsets on the expression of inflammatory factors, 2-month-old p16-Cre / R26-mTmG and p16-Cre / R26-DTA mice were treated with the vaccine or saline control before analyzing cells from the peritoneal cavity and liver. First, it confirmed a BNT162b2-induced increase in the expression of p16 in control, but not in DTA mice (Figure 3E). It was found increased expression of the regulatory factors 11-10 and Arg1 in the peritoneum and liver. In contrast, in DTA mice, ARG1 expression was diminished, while IL10 expression was completely abolished (Figure 3E).

[0333] Example 5: Effect of BNT162b2-induced p16highcells against severe inflammation (sepsis)

[0334] As data above show that p16highimmune subsets could play a role in establishing an immunosuppressive and tissue remodeling environment after treatment with BNT162b2, which in turn could be linked to establishment of disease tolerance (Soares MP. Et al, Nat Rev Immunol. 2017 Feb;17(2):83-96), functional confirmation of these findings was sought. For this purpose, it has been evaluated the ability of BNT162b2 to acutely protect against severe inflammation and tissue damage under different / / ? vivo conditions via p16highcells. Two-month- old control and p16-Cre / R26-DTA mice were primed with BNT162b2 or saline control and 5 days later, the mice were treated intraperitoneally with a lethal dose of Escherichia coli lipopolysaccharide (LPS) to induce sepsis.

[0335] Results

[0336] It was found that 75% of the saline-treated mice died within 48 h of sepsis induction, while only 10% of the BNT162b2-treated mice died. At the termination of the experiment on day 6, the survival rate was 10% and 80% for saline and BNT 162b2-treated mice, respectively. Genetic ablation of p16highimmune cells resulted in a significant reversal of the protective effect of the vaccine, with only 40% of p16-Cre / R26-DTA mice surviving on day 6 (Figure 3F). Thus, the results indicate that the BNT162b2 vaccine rapidly established disease tolerance by protecting against a lethal dose of LPS, with p16highcells contributing substantially to this phenomenon.

[0337] Example 6: Effect of BNT162b2-induced p16highcells against severe tissue damage (irradiation)

[0338] It was performed investigations to the role of BNT162b2-induced p16highcells in establishing disease tolerance against tissue damage. For this purpose, a lethal dose (8 Gy) of ionizing irradiation (IR) was delivered. An investigation of the ability of the BNT162b2 vaccine to increase the number of p16highcells in the intestine as the primary site of tissue damage after this dose of irradiation was performed.

[0339] Results

[0340] A small but significant increase in the number of p16highcells in the intestine at day 5 after vaccine treatment was found (not shown).

[0341] It was hypothesized that a strong increase in the number of p16highimmune cells in the peritoneum (Figure 3A) and in the liver could restrict intestinal tissue deterioration after irradiation, positively contributing to animal survival after an IR. To investigate this further, mice were irradiated with 8 Gy on day 5 after treatment with saline or BNT162b2 and their survival was monitored. All saline-treated mice died by day 15, while 40% of the vaccinated control mice were alive and this effect was largely dependent on the presence of p16highcells (Figure 3G). Thus, the BNT162b2 vaccine-induced disease tolerance has a rapid and noticeable radioprotective effect that potentially could be improved by strategies that increase the presence of p16highimmune cells in the intestine.

[0342] Example 7: Effect of BNT162b2 vaccine in disease tolerance by early protection from lethal infection with SARS-CoV-2 virus

[0343] The BNT162b2mRNA COVID-19 vaccine has been proven to be very effective in the induction of neutralizing antibodies against a spike protein of the SARS-CoV-2 virus. However, the antibody production starts around day 14 after the priming dose of vaccine both in humans and mice, reaching high levels only by day 21 or after a booster shot (Li C. et al., 2022, Nat. Immunol. 23, 543-555). Furthermore, antigen-specific CD8+ T cells and increased levels of interferon-y were detected in mice only after the second dose of the vaccine (Li C. et al., 2022, Nat. Immunol. 23, 543-555). If only neutralizing antibodies are considered, these data strongly suggest that the development of a protective immune response against SARS-CoV-2 needs considerable time. Since it was found that TLR7-driven induction of p16highimmune subsets had almost immediate protective effect against inflammation and tissue damage (Figure 2), the ability of the BNT162b2 vaccine to promote a rapid and early disease tolerance against lethal inflammation caused by SARSCoV-2 through induction of p16highimmune subsets was assessed.

[0344] The lungs are the primary site of tissue damage in response to severe SARS-CoV-2 infection (Winkler ES. et al., 2020, Nat. Immunol. 21 , 1327-1335). As such, the ability of intraperitoneal injection of BNT162b2 to induce accumulation of p16highimmune cell subsets in the lungs was explored. A strong increase in p16highexpressing immune cells was found at day 2 after vaccine treatment and this effect was sustained beyond day 15 (Figure 3H&I). A more detailed analysis of T cells revealed a significant accumulation of Tregs and PD-L1+ cells among the p16highT cell populations in the lung (Figure 3J). A strong p16highcell-dependent accumulation of regulatory 1110 and Arg1 was found in the lungs after the treatment with BNT162b2. Together, the data suggest that the BNT162b2 vaccine induces a strong and rapid accumulation of p16highregulatory subsets in the lung. This, in turn, could have an immediate tissue protective effect against lethal inflammation. To verify this, a model of viral infection after a lethal dose of a mouse-adapted strain of SARS-CoV-2 (MA10) was employed (Leist, S.R., et al. (2020). Cell 183, 1070-1085.). It has been shown that hypercytokinemia (also known as cytokines storm) can, if not contained in a timely manner, eventually lead to Multiple Organ Dysfunction Syndrome (MODS), which is accompanied by rapid weight loss and the eventual death of mice as rapidly as day 2 after infection (Winkler ES. et al., 2020, Nat. Immunol. 21 , 1327-1335). Therefore, the ability of the BNT162b2 vaccine to provide rapid protection against a lethal dose of SARS-CoV-2 through induction of p16highcells was analyzed and thus, independently of antibody production. For this purpose, a group of mice was pre-treated with BNT162b2 or PBS (mock) and on day 3, the mice were inoculated intratracheally with a lethal dose of the mouse-adapted SARS-CoV-2 strain MA10 (not shown). In accordance with previous reports, mice started to lose weight at day 2 after inoculation and this dynamic continued in both groups until day 4, when a rapid recovery began in the BNT162b2 -treated mice, while the mock-treated animals continued to lose weight (not shown). Analysis of the probability of survival showed a strong protection in the BNT162b2-treated group at 100% survival, while only 30% of the mock-treated mice were alive on day 14 (Figure 3K). Further analysis of lung cytokine gene expression showed a decreased IL6 and INFb mRNA expression on day 3 in the BNT162b2 -treated group compared to the levels in control mice (not shown). Thus, these results provide evidence that mRNA vaccines can almost immediately induce disease tolerance against severe SARS-CoV-2 infection.

[0345] Example 8: both TLR7 and STING pathways are required for induction of p16highimmune subsets

[0346] Next, the potential mechanism(s) of induction of p16Highimmune subsets in response to BNT162b2 were analyzed. It has been previously shown that that immunization with BNT162b2 stimulated potent antibody and antigen-specific T cell responses, as well as strikingly enhanced innate responses after secondary immunization. This mechanism of innate and adaptive immunity to BNT162b2 appeared to be TLR5 and 7-and STING-independent while relied on activation of MDA5 (Li, C., et al. (2022). Nat. Immunol. 23, 543-555). To systematically address the role of TLR7, STING and MDA5 pathways in response to BNT162b2, selective inhibitors and knockout mice were used to dissect the mechanism of p16Highimmune subset induction.

[0347] Results

[0348] Inhibition of TLR7 and STING (with M5049 and H151 respectively) as well as deletion of MDA5 in mice significantly reduced the expression of Isg15 mRNA after treatment with BNT162b2 (not shown), which is consistent with their role in induction of innate and adaptive immunity to BNT162b2 (Li, C., et al. (2022). Nat. Immunol. 23, 543-555). Inhibition of both TLR7 and STING reduced the expression of p16 mRNA (Figure 4A, left panel) after BNT162b2, while surprisingly, MDA5-'- mice showed a significant increase in the basal level of the p16 mRNA expression (Figure 4A, right panel). Because of that, in the first instance TLR7- and STING-dependent pathways were focused on. The use of TLR7 and STING inhibitors blocked the effect of BNT162b2 by reducing the percentage of p16Highimmune subsets including specifically in Tregs as well as PD1 and PD-L1 -positive T cells in analyzed tissues (Figure 4). Next, an activated and phosphorylated at S366 form of STING was evaluated in p16Highcells. A significant fraction of p16Highcells was positive for p-STING with most noticeable co-staining in F4 / 80+macrophages (Figure 4C&D right panel and upper pie chart). Similarly, F4 / 80 p16Highp-STING+positive cells represented the most significant fraction among CD45+p-STING+cells (Figure 4C&D bottom pie chart). Altogether, these results show that activation of STING after treatment with BNT162b2 coincides with induction of p16High immune subsets.

[0349] Next, a direct activation of STING with the specific agonist DMXAA was found to be sufficient to increase the number of p16Highimmune subsets including within Tregs as well as PD1- and PD-L1 -positive T cells in different tissues (Figure 4E). While STING activation was important in induction of p16High immune subsets, its activation has been broadly implicated in the regulation of numerous pro-inflammatory pathways which is in part due to its capacity to induce TNFa (Berger, G., et al. (2022). Proc. Natl. Acad. Sci. 119). Consistent with these reports, an increased dose of the STING agonist, in contrast to low doses, strongly induced the expression of pro- inflammatory factors including I11 b, IL6 and TNF (Figure 4F).

[0350] Next the role of a direct STING activation on protecting mice from LPS-induced sepsis was analyzed. It was found that, while low doses of the STING activator DMXAA phenocopied the effect of BNT162b2 on protecting mice from LPS-induced severe inflammation, significant STING activation or inhibition after BNT162b2 no longer had a protective effect (Figure 4G). Thus, low levels of STING activation could be beneficial while high - detrimental due to induction of excessive inflammation, in protecting mice from LPS-induced sepsis.

[0351] Example 9: The role of NNMT in the control of suppressive p16highimmune subsets

[0352] To gain further insights into the potential mechanism(s) of p16 induction in immune cells, it was evaluated genes that were found to be differentially expressed in p16highversus p16lDWF4 / 80+ peritoneal macrophages based on RNA-Seq. It was found that some of the most upregulated genes included nicotinamide N-methyltransferase (NNMT) (Figure 5A), which regulates numerous physiologically-relevant processes by controlling the availability of Sadenosyl methionine (SAM), the main substrate for all types of methylation. Furthermore, recently it was identified an important role of NNMT in the induction of p16highfibroblasts during iPSC reprogramming, providing further credence to the role of NNMT as a potentially important molecule relevant to a program controlling p16 activation. First, we determined the level of Nnmt after treatment with DSS in vivo and found its significant induction in the peritoneum and liver in wild-type mice, but not in p16-Cre / R26-DTA mice (Figure 5B). Similar results with upregulation of Nnmt mRNA were obtained after treatment with BNT162b2 in different tissues (Figure 5C) while this effect was dependent on both TLR7 and STING (Figure 5D).

[0353] To gain further insight into the role of NNMT in controlling p16hi0himmune subsets in vivo, and due to low bioavailability and activity of current Nnmt chemical inhibitors, next it was generated NNMT conditional knockout (Nnmt-cKO) mice. In these mice, the first exon of the NNMT gene was flanked by LoxP sites to produce an inactive gene after excision with the Cre recombinase. Subsequently, NNMT-cKO mice were crossed with mice expressing Cre under the control of the p16 promoter (p16-Cre). The resulting animals in which the removal of NNMT was induced specifically in p16highcells were used for further experimentation. It was found that the ability of BNT162b2 to induce p16hi0hsubsets was greatly diminished in the immune cells analyzed in the p16-Cre / NNMT-cKO mice (Figure 5E&F) including in Tregs, PD1- and PD-L1-positive T cells (figure 5G). This correlated with the lack of p16 mRNA induction after BNT162b2 treatment in NNMT-cKO mice (Figure 5H), indicating that NNMT activation is a critical mechanism of p16 induction either directly or indirectly. Moreover, and in contrast to control mice, the cells from the peritoneal cavity of p16-Cre / NNMT-cKO mice were unable to mount a full inflammatory response (Figure 5H).

[0354] As conditional deletion of NNMT produced a phenotype that resembled the in vivo phenotypes of p16-Cre / R26-DTA mice, including the reduced number of p16highimmune cells and attenuated inflammatory response (Figure 5H), it was hypothesized that these mice would also have a reduced disease tolerance in response to LPS-induced sepsis. To test this hypothesis, control and p16-Cre / NNMT-cKO mice were treated with BNT162b2 and after 5 days, injected a lethal dose of LPS. It was found that the ability of BNT162b2-treated mice to survive LPS induced sepsis was greatly reduced in p16-Cre / NNMT-cKO mice (Figure 5I), representing a phenocopy of the observations in p16-Cre / R26-DTA mice (Figure 3F). Overall, the data support a critical role of NNMT in inducing regulatory p16hi0himmune cell subsets and establishment of disease tolerance.

[0355] Example 10: p16High immune cells protect from severe inflammation by reducing the levels of adenosine.

[0356] Overexpression of the NNMT gene leads to reduced levels of S-Adenosyl Methionine (SAM) in p16Highcells, which could result in attenuated adenosine concentrations after SAH hydrolysis. Adenosine is an important regulator of the immune system, with strong immunosuppressive properties. In turn, adenosine build up could derail a balanced immune response leading to reduced survival of animals during severe inflammation. Consistent with the role of NNMT in controlling SAM levels and potentially adenosine, the BNT162b2 treatment significantly reduced adenosine concentrations in wild-type but not in p16-Cre / Nnmt-cKO mice (Figure 5J). Same effect was observed with low but not high STING activation. Importantly, supplementation with L-methionine, a main precursor of SAM, fully reversed the effect of BNT162b2 on reducing the levels of adenosine (Figure 5J). Thus, BNT162b2 lowers adenosine concentrations via NNMT and reduction of SAM levels.

[0357] While the level of adenosine could be controlled via SAM, one of the major sources of adenosine, and specifically extracellular adenosine, is the ATP. ATP is released to the environment from apoptotic cells in damaged and inflamed tissues, including regulatory T cells. In turn, inhibition of the cell cycle, including by upregulation of p16, has been reported to have anti-apoptotic properties. In addition, p16Highcells commonly express the high level of anti- apoptotic protein Bcl2 (Schmitt, C.A., et al. (2002). Cell 109, 335-346). Because of that, it was hypothesized that p16 upregulation may protect immune cells from apoptosis which in turn could lead to reduction of extracellular adenosine.

[0358] To verify that, 2-month-old wild type and Cdkn2a knockout (Cdkn2a-KO) mice were treated with BNT162b2 and 5 days later exposed them to sub-lethal doses of LPS.

[0359] Analysis of cleaved-caspase 3- positive cells 12h after LPS treatment showed that BNT162b2 effectively reduced the activation of caspase-3 in CD45, F4 / 80 and CD3 populations from wild type but not Cdkn2a-KO mice (Figure 5K). Thus, upregulation of p16 could be critical in counterbalancing apoptosis in immune cells during severe inflammation.

[0360] Next, it was checked whether controlling adenosine levels or adenosine signaling could have an impact on protecting mice from LPS-induced sepsis. For that, 2-month-old animals were treated with BNT162b2 with and without daily supplementation of L-methionine. It was found that L-methionine-supplemented animals showed a significantly lower survival rate in a model of LPS-induced sepsis (Figure 5L). Since it was found that maintaining low adenosine levels are critical for development of BNT162b2-induced protection from LPS-induced sepsis, old (24 months) animals were studied to evaluate their response to LPS. It is well established that during aging, adenosine levels are significantly increased most likely due to a much higher rate of cell death in aged tissues. We further confirmed an increase in adenosine levels in different tissues of old (24 months) versus young (2 months) animals (Figure 5M). Interestingly, while BNT 162b2 was able to lower adenosine in old animals, this reduction has never reached the level observed in young mice (Figure 5M). Consistent with inability to significantly downregulate adenosine levels after treatment with BNT162b2 in old mice, it was found that BNT162b2 completely failed to protect 24-month-old mice from LPS-induced sepsis (Figure 5M). In contrast, a simultaneous treatment with the adenosine receptor A2A inhibitor SCH58261 was sufficient to significantly improve the protective effect of BNT162b2 from LPS- induced sepsis in old mice (Figure 5N). Thus, low adenosine levels are essential for BNT162b2-induced protection from LPS-induced sepsis both in young and old animals.

[0361] Example 11 : Deficiency of MDA5 promotes health-span

[0362] The present findings show that the presence of p16Highimmune subsets is indispensable for animal survival in response to multiple lethal conditions such as LPS-induced sepsis, acute COVID-19 infection, as well as Ionizing irradiation (Figures 3F,G,K). In turn, this could argue that maintaining an increased number of p16Hi0himmune cells subsets from early age and thus before any tissue damage or inflammation could potentially provide benefits in extending health-span. As it was previously found a higher basal level of p16 expression in young MDA5-KO mice (Figure 4A) it was explored whether indeed such an increase could provide bases for improved health-span. Since deletion of MDA5 could potentially induce STING as a feedback mechanism due to lack of sensing of endogenous double-stranded RNAs including from repetitive and antisense sequences (Chen, Y.G., and Hur, S. (2022). Nat. Rev. Mol. Cell Biol. 23, 286-301), next it was verified whether higher p16 expression in the tissues of MDA5-KO mice was indeed dependent on STING. The analysis of an activated and phosphorylated form of STING as well as its downstream target TBK1 in different immune populations (CD45, CD3, F4 / 80) showed their increase in MDA5-deficient cells which was significantly attenuated in the presence of the STING inhibitor, H151 (Figure 6A). p16 expression in MDA5 KO mice was reduced after H 151 treatment (Figure 6B). Next, aged MDA5 KO mice were analyzed. The level of adenosine rises with age (Figure 5M and Figure 6C) while maintaining it low is a prerequisite for increased organismal survival in response to multiple conditions as we describe here. Consistent with upregulation of p16 mRNA, the analysis of adenosine concentrations in MDA5 KO cells showed its significantly reduced levels (Figure 6C). Indeed, these levels were close to the ones observed in wild type mice after treatment with BNT162b2 suggesting that MDA5 KO mice are potentially in a continuously primed state of heighten resistance to different tissue damaging conditions and thus their tissue deterioration with aging could occur at a slower pace. To test that, 24-month-old wild type and MDA5-KO littermates mice were analyzed and it was found a significant reduction in the level of expression of pro-inflammatory cytokines IL-1 (a, but not P), IL-6, TNF-a, and Cxcl13, while an increase in the expression of anti-inflammatory IL-10 (Figure 6D). Next it was checked whether such a delay in developing of tissue inflammation with aging correlated with improved histological characteristics of different tissues. Among such characteristics, the level of tissue fibrosis and blood vascularization were assessed. The latter is particularly critical since it is significantly reduced with aging while its improvement in VEGF transgenic mice is sufficient to extend health- and life-span (Grunewald, M., et al. (2021). Science 373). It was found that the level of aSMA, a marker of fibrosis, was significantly reduced while the presence of Cd31 -positive cells, a marker of vascular endothelium, was strongly increased in different tissues from MDA5-K0 when compared to wild type littermates (Figure 6F). To test further whether observed changes in MDA5 KO mice had functional and physiological significance, the muscle strength based on a grip test was determined and found it significant improvement in MDA5 KO mice when compared to wild type littermates (Figure 6G). Encouraged by these results, the general physical state of animals was evaluated based on a frailty score. It was found that aged MDA5-K0 in comparison to wild type littermates showed a significantly reduced frailty index (Figure 6H). Finally, both wild type and MDA5-K0 littermates have been followed for 26 months to evaluate the median survival. It was found that both male and female MDA5 KO mice showed a significantly improved median survival with aging (Figure 6I) further supporting a model of targeting MDA5 for health-span extension.

[0363] Example 12: Effect of BNT162b2 vaccine in severe COVID-19 patients

[0364] To confirm that the BNT161b2 vaccine could induce p16hi0himmune cells in humans, it was evaluated peripheral blood before and after standard vaccination with BNT162b2. Seven healthy donors (4 women and 3 men) were sampled prior vaccination, as well as 7 and 30 days later. The mean age of the cohort was 36 years [23; 42], Most had already received at least three doses of RNA vaccines (either BNT162b2 or mRNA-1273), with the last dose >6 months earlier (according to French recommendations for health care workers), and >1 SARS- CoV-2 infection in the past 3 years. The fourth dose of the vaccine (BNT162b2 only) was well- tolerated by all participants and did not induce an inflammatory syndrome (increased IL-1 [3 and IL-6), but produced a significant SARS-CoV2-specific T cell response 1 month later as shown in Table 1 below.

[0365] Table 1 : Biological characteristics of the participants in the vaccine study

[0366] To further validate the results, it was performed RT-PCR analysis of isolated PBMCs and confirmed an increase in the level of p16 mRNA after vaccination (Figure 7A). In the same way, the expression of NNMT, which it was found to be upstream of p16 activation in immune cells was also increased after vaccination in humans (Figure 7B).

[0367] Next, a flow cytometry analysis was performed in freshly sampled whole blood and found a strong increase in the percentage of cells expressing p16 within the CD45+cell populations at 7d which are significantly subsided by 30d after vaccination (Figure 7C&D). All of the analyzed immune cell subtypes, including granulocytes, monocytes, lymphocytes but most importantly Tregs, showed significantly increased expression of p16 protein at 7d (Figure 7D&E). It was further observed a strong induction of p16 protein in PD1-positive immune cells after vaccination (Figure 7C&D). These changes accompanied by a significant increase in the level of plasma regulatory IL10 after vaccination (Table 1).

[0368] Thus, standard vaccination with BNT162b2 in humans transitory induces p16highimmune cell subsets which in turn could be critical to provide a rapid and broad tissue protection from severe inflammation before development of inhibitory antibody.

[0369] To evaluate the possible association of p16highimmune cells with a protective response against uncontrolled inflammation caused by SARS-CoV-2 in humans, it was next analyzed different groups of COVID-19 patients. For this analysis, it was focused on samples collected during the first two waves of pandemic back in 2020 and prior to any vaccination. The analysis was carried out in hospitalized COVID-19 patients who did not require oxygen therapy (moderate cases, n = 8), those who did require oxygen therapy (severe cases, n = 19) as well as in non-COVID-19 patients (n = 15). All groups of patients were comparable in terms of age, sex ratio and comorbidities as shown in Table 2 below.

[0370] Table 2: Clinical and biological characteristics of the COVID cohort

[0371] As previously described (Hadjadj J. et al., 2020, Science 369, 718-724 and Reis G. et al., 2023, N. Engl. J. Med. 388, 518-528), patients with severe SARS-CoV-2 infection exhibited an exacerbated inflammatory response with a significant increase in IL6 (Figure 7F) and an impaired interferon response (Table 2).

[0372] Analysis of PBMCs collected at the time of patient admission and before any specific treatment against SARS-CoV-2 revealed that the levels of both p16 and NNMT mRNA were significantly reduced in patients with severe COVID-19 (Figure 7 G&H). These results further confirmed the potential role of p16highimmune cell subsets in establishing disease tolerance and protection against severe inflammation and tissue damage induced by severe SARS-CoV- 2 infection in humans.

[0373] Example 13: BNT162b2 vaccine induces expression of p16highin different populations of tumor-associated immune cells

[0374] Induction of p16highimmune cells in a subcutaneously implanted tumor was analyzed.

[0375] 1 million cells from B16F10 syngeneic melanoma line were injected subcutaneously in a 2 months p16-cre / R26-mTmG mice. After 9 days, and tumors establishment, mice were treated intraperitoneal as follows:

[0376] - Vehicule (PBS) (Mock),

[0377] - BNT162b2 vaccine (5 pg by mouse),

[0378] - Anti-PDL1 antibody (a-PDL1)(200 pg by mouse); or

[0379] - A combination of both (BNT162b2 day 9 and a-PDL1 at day 11).

[0380] After 3 days of treatment, animals were euthanized, tumor were digested using collagenase A to obtain single cell suspensions. Cells were blocked (HBSS 1 % BSA, 4% FBS,

[0381] 2 mM EDTA) and stained with different fluorochrome-conjugated antibodies directed to different immune subtypes. Samples were analyzed by flow cytometry using Cytoflex system (Beckman-Coulter) and CytoExpert software.

[0382] Results

[0383] An increase of p16highcells was observed in the tumors of the BNT162b2 treated mice (Figure 9, upper panel), specifically in CD3+(T Cells), Ly6C+ (Neutrophils), F4 / 80+ (Macrophages) and NK1.1 (Natural killers and NK-T cells) immune subpopulations (Figure 9, lower panel). Even though mice treated with a-PDL1 showed a slight increase in the number of p16high, the use of a-PDL1 reduced the increase of p16highwhen used in combination with the BNT162b2 vaccine in whole set of populations analyzed.

[0384] Example 14: Effect of BNT162b2 vaccine treatment on tumor burden in a murine model of constant expression of oncogenic KRAS gene

[0385] KRASG12Dis a life-threatening model of tumorigenesis. Constant oncogenic stress from KRASG12Dexpression deteriorated health of mice early in life (between 4-6 months) until a terminal end point. To evaluate the effect of BNT162b2 in a model of oncogenic transformation, a set of mice overexpressing the oncogene form of KRASG12Dwas divided in 3 groups. The group control was treated with vehicle (PBS), the second group was treated with the BNT 162b2 vaccine (5 g by mouse), and the third group with a combination of BNT162b2 and a-PDL1 (200 pg by mouse). Treatment started when the mice were 2 months old. Single treatments were applied 1 time a month for 2 consecutive months. For the combined condition, treatments with single agents were separated for 1 week starting with the BNT162b2 vaccine. All mice were euthanized at 4 months old. Lungs were fixed in 4% PFA for 24 h at 4 °C and then kept in 70% ethanol for 3 days. After that, the number of lesions was manually determined following the criteria: small (<1 mm), medium (between 2 and 3 mm) and large (more than 3 mm) (Figure 10).

[0386] The p16-cre / DTA is a mouse model for trace and specifically eliminate cells that overexpress p16, since diphtheria toxin A expression is subrogate to p16 expression. Animals carrying this genotype were crossed with the KRASG12Dmouse. This new breeding, p16- cre / DTA / KRASG12Dwas used to compare the induction of tumorigenic lesion against the KRASG12Dline. In addition, this mouse was treated with BNT162b2 (5 pg by mouse 1 time a month for 2 consecutive months) (Figure 11).

[0387] Longitudinal sections (3 pm thick) of lung from KRASG12D experiments, including all major lobes, were stained with hematoxylin and eosin (H&E) and evaluated. Lung stained sections were scanned and the percentage of tumor-occupied area was measured (Figure 12).

[0388] Results

[0389] A reduction in the number of medium and large lesions was observed in the animals treated with BNT162b2, however the combination was only effective reducing the large lesions. When all size lesions are combined, the ability of BNT162b2 treatment reducing the number of lesions is significant but not in the combined treatments (Figure 10, right panel).

[0390] Importantly, no differences were observed between KRASG12Dand p16- cre / DTA / KRASG12D. In addition, p16-cre / DTA / KRASG12Danimals treated with BNT162b2 only show differences reducing the number of big lesions, but not medium or small size. Even more, when total number of lesions is compared among the groups, no significant difference is observed (Figure 11 . Right panel). Thus, the present data shows that tumor growth inhibition is at least in part due to the induction of p16highpopulations.

[0391] A significant reduction of tumor area was found in the BNT162b2 KRASG12Dtreated group with respect to the control. The combination of BNT162b2 and a-PDL1 treatment reduces the effect of the BNT162b2 increasing the number of lesions. The same reducing effect was observed in the p16-cre / DTA / KRASG12Dtreated with the BNT162b2 vaccine (Figure 12).

[0392] To know if the effect of BNT162b2 treatment is able to induce an increase on the survival from KRASG12Dgenotype, a group of 4 months old KRASG12Dmice were treated with the BNT162b2 vaccine 1 time by month during 4 months. At the end of eight month, the percentage of survival in the BNT 162b2 group was 75%, mean in the control group the survival was 0. Even more, at 6 months, only 25% of the animals remained alive (Figure 13).

[0393] Example 15: Effect of BNT162b2 vaccine treatment on metastatic growth

[0394] 5x105B16F10 melanoma cells were injected intravenous as a model of metastatic growth. Before injection, cells were modified to express luciferase. To track tumor growth during time, a sample of blood was obtained and luciferase activity measured. Individual values at day 1 were used to draw the plot as fold change. Animals were treated with BNT162b2 (5pg / mouse) or saline (mock) at day 3. Differences were determined with t-test at day 10. p<0,05. The results are shown on Figure 14. It is clearly observed that the proliferation of the B16F10 melanoma tumor cells is prevented during at least 10 days in mice treated with the BNT162b2 vaccine.

[0395] 1 x 106B16F10 melanoma cells were injected subcutaneously into immunocompetent B6 mice. After 7 days, the animals were treated with PBS (mock condition), the BNT162b2 vaccine, 100 pg of anti-PD1 antibody, or a combination of both. Tumor growth was measured, and after 17 days, it was observed that mice receiving the combination of both BNT162b2 and anti-PD1 antibody exhibited a synergistic reduction in tumor size, which was not observed in animals treated with single agents (Figure 14 B-C).

[0396] To determine whether the pathways inducing p16-positive immune cells, namely TLR7 and STING, are responsible for the tumor-suppressing effect of BNT162b2, this experiment was repeated in the presence of specific inhibitors for TLR7 (1 mg / kg M5049) or STING (10 mg / kg H151). It was observed that, in both cases, the anti-cancer effect of the simultaneous treatment with BNT162b2 and anti-PD1 antibody was significantly attenuated (Figure 14 D).

[0397] Example 16: Disease tolerance can be transplanted from mouse to mouse, even in conditions of severe inflammation.

[0398] Disease tolerance is an innate ability to reduce the negative impact of infections on host fitness. It was previously shown that BNT162b2 vaccine induce diseases tolerance against septic shock through the induction of p16hi0hcells. To test if the cells carrying the capacity to induce disease tolerance can be used to offer protection to other recipients, young mice were treated with BNT162b2 vaccine. 5 days later, CD45+fraction from liver and blood were isolated and injected intravenously into young animals (5x105cells from liver and 2,5x105cells from blood) (Figure 15A). In parallel, another group of mice received the same number of cells, but from untreated donor mice. 24 hours later the recipient animals were treated with 35 mg / kg of lipopolysaccharide (LPS) from Escherichia coli 055: B5. After 48 hours of LPS treatment, it was observed a greater survival in the animals that received cells from BNT162b2 treated mice (Figure 15B). Thus, CD45+cells coming from previously treated animals are effective carrying disease tolerance and are an effective way to transplant it.

[0399] This example demonstrates that disease tolerance promoting by the BNT162b2 vaccine can be transplanted to other animals.

[0400] Donor wild-type mice (proficient for p16Highcells) and p16-Cre / DTA mice (deficient for p16Highcells) were treated with the BNT162b2 vaccine. Five days later, recipient mice received a dose of 32 mg / kg of LPS to induce severe sepsis. Six hours after LPS treatment, recipient mice also received 5 x 105freshly isolated CD45-positive immune cells from the livers of donor mice from different groups (as shown in Figure 15C), and their survival was assessed. A strong protective effect was observed on survival in animals that received CD45-positive immune cells from wild-type vaccinated donor mice (proficient for p16Highcells), but not from either mock- treated wild-type or mock-treated or BNT162b2-treated p16-Cre / DTA (deficient for p16Highcells) donor mice (Figure 15D). This example demonstrates that p16Highimmune cells transferred through adoptive transfer from one animal to another could induce strong protection from a lethal dose of LPS in the recipient.

[0401] Importantly, this procedure works effectively in mice that have already started to develop a severe inflammatory response, suggesting that this strategy could potentially be used to treat patients with already developed severe inflammation, such as sepsis.

[0402] Example 17. Procedure for induction of p16Highcells in human peripheral blood mononuclear cells (PBMCs) in vitro.

[0403] Whole blood from healthy human donors was treated in vitro with different conditions to induce p16Highcells. TLR7 and TLR7 / 8 agonists were used at 1 pg / ml (R837 and R848, respectively), and a STING agonist (DMXAA) was used at 10 pg / ml, either alone or in combination with anti-CD3, anti-CD28, and anti-CD2 stimulatory antibodies. p16Highinduction was analyzed by flow cytometry specifically in CD45-positive cells (Figure 16A) and CD3 T cells (Figure 16B). It was found that the best combination for inducing an accumulation of p16Highcells among T cells (CD3-positive) and total immune cells (CD45-positive) was a simultaneous use of the TLR7 / 8 agonist (R848), STING agonist (DMXAA), and CD3 antibody. RIG-I and MDA5 agonists were further tested as alternatives to the STING agonist but it was found that only a combination with the STING agonist worked in both total CD45-positive immune cells (Figure 160) and T cells (Figure 16D). TLR7 and CD3 agonists are part of a routine test (QFM) used to evaluate interferon response and cytokine production. When the STING agonist DMXAA was added to the mix, it produced a strong accumulation of p16Highcells in granulocytes (CD11b-positive). As shown here, whole blood from two healthy donors was treated to induce p16Highcells as described above, and blood samples were analyzed by FACS analysis (Figure 16E-G).

[0404] This example describes a protocol for the in vitro induction of p16Hi0hhuman immune cells, including within the CD3 and CD11 b populations, following simultaneous stimulation of the TLR7 and STING pathways.

[0405] Example 18. Adoptive transfer of p16-positive human PBMCs protects mice from severe sepsis.

[0406] Whole blood obtained from healthy human donors was treated in vitro to induce piepositive immune cells as described in Example 17. TLR7 / 8 agonist (R848) at 1 pg / ml and the STING partial agonist (DMXAA) at 10 pg / ml, in combination with anti-CD3 stimulatory antibodies, were used (QFM = R848 + anti-CD3 antibodies). A similar condition, with the addition of 100 ng / ml of dexamethasone, was also tested. Twenty-four hours later, recipient mice were treated with LPS to induce sepsis. Six hours later, PBMCs from QFM + DMXAA treated human whole blood were isolated and transferred intravenously into the LPS-treated mice (under sepsis development) (Figure 17A). Animals were monitored to evaluate survival probability. At the end of the experiment, 50% of the animals that received human PBMCs treated with QFM and DMXAA had survived. In contrast, all the animals that received human PBMCs that were either mock-treated or QFM + DMXAA + dexamethasone-treated had died (Figure 17B).

[0407] This example demonstrates that the adoptive transfer of human p16-positive PBMCs, achieved using the protocol described in Figure 16, can protect mice from severe inflammation, such as LPS-induced sepsis. Example 19. p16-positive immune cells protect multiple organs during LPS-induced severe inflammation.

[0408] Mice were treated with the BNT162b2 vaccine to induce p16-positive immune cells. Five days later, 10 mg / kg of LPS was injected (Figure 18A). This dose is sufficient to induce a strong but non-lethal sepsis. After 16 hours of sepsis induction, blood was collected, and plasma was analyzed to determine the level of liver enzymes, aspartate transaminase (ASAT) and alanine transaminase (ALAT), as a readout of liver damage. Animals pre-treated with the BNT162b2 vaccine showed less accumulation of both liver enzymes in plasma, suggesting a protective function of p16-positive immune cells on liver tissues during strong inflammation (Figure 18B).

[0409] Similarly, mice were tested for gut leakage after sepsis induction. After 12 hours of LPS treatment, animals were orally gavaged with 12 mg of dextran-FITC (4 kDa), and 4 hours later (for a total of 16 hours), blood was collected and fluorescence was measured in the plasma. A significantly lower level of dextran-FITC was observed in the BNT162b2-treated animals, suggesting they had reduced gut permeability after sepsis induction and their intestines were better protected (Figure 18C).

[0410] Finally, hypothermia was investigated, which in mice is associated with poor prognosis under sepsis conditions. Sixteen hours after LPS treatment, it was observed that LPS-treated animals had significantly lower body temperatures than the animals pre-treated with the BNT162b2 vaccine (Figure 18D).

[0411] These examples collectively show that p16-positive immune cells may play an important role in tissue protection across different organs during strong or severe inflammation.

[0412] Discussion

[0413] The process of aging represents the gradual deterioration of organism functions with time, which affects all tissues and organs. In humans, aging is the most prevailing risk factor for the morbidity and mortality attributable to numerous acute and chronic diseases. Despite relentless deterioration of our organism with age, a defined set of mechanisms has evolved to limit the negative impact of tissue damage caused by multiple factors on homeostasis. Among such protective mechanisms that rely on the concerted action of both innate and adaptive immunity is an important but poorly understood defense strategy that limits the extent of tissue damage known as disease tolerance. The concept of disease tolerance was originally introduced to explain additional to resistance mechanisms to fight inflammation by decreasing the host susceptibility to tissue damage without having a direct impact on the pathogens. Currently, little is known about the full spectrum of tolerance mechanisms, but they seem to revolve around several evolutionary conserved stress- and damage-induced responses that confer tissue damage control in the host. By exploiting different lethal conditions of inflammation and tissue damage with a focus on LPS-induced sepsis, it is shown here that animal survival can be significantly improved by building disease tolerance. The mechanism of disease tolerance as the ability to withstand these conditions could be intimately linked with induction of a p16Highprogram in immune subsets and maintenance of a low adenosine environment (Figure 8). The latter appeared critical since reversal of a BNT2162b2 vaccine- induced downregulation of adenosine levels with dietary methionine fully blocks the development of disease tolerance in young mice while inhibiting adenosine receptors reconstitutes this protective mechanism in old mice (Figures 5L&M). In both cases, the low adenosine environment could provide optimal conditions for immune cells including Tregs and IL10-expressing macrophages to perform their immune-suppressive and tissue remodeling activities. In the case of Tregs, that we found to be one of the major p16HighT cell subtypes (Figure 1), it has been shown that apoptotic Tregs have much higher immunosuppressive functions than live Tregs. It was found that specifically adenosine, a metabolite of extracellular ATP that is released from apoptotic Tregs, is much more immunosuppressive than typical suppressive factors such as PD-L1 , CTLA-4, TGF-p, IL-35, and IL-10. In turn, reducing apoptosis through activation of a p16-dependent cell cycle arrest (Figure 5K) could limit the pool of extracellular ATP while at the same time, the NNMT-dependent consumption of SAM lowers the level of endogenous adenosine (Figure 5J and 8). This, in turn, contributes to a higher survival rate of mice in response to different types of severe inflammation and tissue damage. Indeed, we found that the presence of p16Highimmune subsets was indispensable for animal survival in response to multiple lethal conditions such as LPS-induced sepsis (90% mice survival after induction of p16Highimmune subsets compared to 10% in control group), acute COVID-19 infections (90% mice survival after induction of p16High immune subsets compared to 30% in control group), and Ionizing irradiation (40% mice survival after induction of p16Highimmune subsets compared to 0% in control group) (Figures 3F,G,K). Mechanistically, we identify TLR7 and STING as potent inducers of p16Highimmune subsets. It is shown here that vaccine-based mechanism of p16Highimmune subset induction has a strong protective effect from severe viral, bacterial and sterile inflammation. Importantly, it is shown that in humans a single standard dose of vaccination with the BNT162b2 mRNA COVID-19 vaccine is sufficient to induce p16 in different immune cells. From a clinical standpoint, it was found that p16-expressing immune subsets were significantly reduced in the blood of severe COVID- 19 patients further supporting their critical role in preventing lethal inflammation. The clinical efficacy of the BNT162b2 mRNA vaccine in reducing the risk of hospitalization was demonstrated as early as 10 days after the first dose when the specific immune response has not yet been established in terms of development of specific antibodies and the cellular responses. The present results could explain these early benefits of vaccination on the risk of hospitalization by temporarily inducing p16highimmune cell-dependent tolerance that would eventually limit the evolution towards an inflammatory form of COVID-19 thus reducing the risk of hospitalization for severe cases. Furthermore, this previously unacknowledged first line of defense in response to mRNA vaccines by building tolerance could play an important role in shielding against deadly pathogens while providing sufficient time to develop inhibitory antibody as lasting protection. The present discovery of an age-induced accumulation of different p16Hi0himmune cell subsets would further support the idea that it could be secondary to tissue damage and inflammation and thus, is controlled by the aged environment. Indeed, the present findings that even very short-lived immune subsets as such Ly6G+ neutrophils contain a significant fraction of p16Hi0hcells in 18- month-old mice (Figure 1 A&B) indicates that such a response to the aged environment is rapid and robust. In accordance with this prediction, a recent study suggested that the aging microenvironment drives rapid epigenetic changes in macrophages based on the observation that adoptive transfer of young macrophages into old mice led to a rapid establishment of chromatin marks resembling those of aged macrophages64. The present findings raise the possibility that building tolerance by maintaining increased number of p16Hi0himmune cell subsets from early age and thus before any tissue damage or inflammation could potentially provide benefits in extending healthspan, something that we now show for MDA5-deficient mice (Figure 4A&6B). It is further tempting to speculate that the mechanism of precautious activation of p16Hi0hstate in 18 immune subsets in order to extend health- and lifespan is evolutionary conserved and active in long-lived animals. For example, in long-lived bats, continuous presence of multiple viruses could trigger a TLR7 / STING-induced activation of a p16 program in immune cells and tissue protection against both infection and tissue damage. At the same time, significantly reduced DNA-sensing in bats could keep STING activation in its low and physiological or, as also referred to, “tonic” state while preventing a strong pro-inflammatory response that could be detrimental for disease tolerance (Figures 4F&G). In another long-lived animal, the naked mole-rat, a lack of response to Poly (l:C) reported in hematopoietic cells may suggest a strong attenuation of MDA5-dependent sensing. Importantly, it was found here that deletion of MDA5 in mice induces STING potentially as a feedback mechanism due to lack of sensing of endogenous double-stranded RNAs including from repetitive and antisense sequence. This, in turn, could contribute to tonic STING activation and subsequent induction of p16 in immune subsets resulting in improvement of health-span of the animals. Altogether, the present results provide evidence for direct and early benefits of mRNA vaccine-induced p16Highimmune cell subsets in building tolerance and counteracting severe cases of viral, bacterial and sterile inflammation. In turn, sustainable activation of a p16Highprogram in defined immune subsets that could be achieved through a TLR7 and / or tonic STING activation by inhibiting MDA5 could provide a novel strategy for improving health- and potentially lifespan.

Claims

CLAIMS1 . A pharmaceutical composition comprising an effective amount of a STING agonist, of a TLR5 agonist or of a TLR7 agonist, or of a combination thereof, for use in enhancing the number of p16highimmune cells in a patient in need thereof.

2. The pharmaceutical composition of claim 1 , for use in preventing and / or treating tissue inflammation and / or tissue damage.

3. The pharmaceutical composition for use according to claim 2, wherein said tissue inflammation and / or tissue damage is induced by a pathogen infection, a cancer, a chemical or physical treatment, or by a physical damage.

4. The pharmaceutical composition of claim 1 or 2, for use in preventing and / or treating the deleterious inflammatory effects of sepsis or of radiation therapy.

5. The pharmaceutical composition of any of claim 1 -3, for use in preventing and / or treating SARS-COV-2 infection or cancer.

6. The pharmaceutical composition for use according to any of claims 1-5, wherein said immune cells are T lymphocytes and / or macrophages.

7. An in vitro method to induce the expression of p16 in immune cells, said method comprising the step of in vitro contacting immune cells with an agonist of a pattern recognition receptor chosen from a STING agonist, a TLR5 agonist or a TLR7 agonist, or with a combination thereof.

8. The in vitro method of claim 7, wherein said immune cells are T lymphocytes and / or macrophages, preferably CAR-T cells.

9. A pharmaceutical composition containing autologous or heterologous p16hi0himmune cells.

10. The pharmaceutical composition of claim 9, comprising p16hi0hCAR-T cells.

11. The pharmaceutical composition of claim 9 or 10, for use in preventing and / or treating tissue inflammation and / or tissue damage in a patient in need thereof, wherein said tissue inflammation and / or tissue damage is preferably induced by a pathogen infection, a cancer, a chemical or physical treatment, or by a physical damage.

12. The pharmaceutical composition of claim 9, for use for inducing disease tolerance, for protecting tissues against deleterious effect of aging and / or for extending health span in a patient in need thereof.

13. A pharmaceutical composition comprising a STING agonist and a TLR7 agonist.

14. An in vitro method for selecting patients that will benefit from a treatment involving a TLR7 agonist, a TLR5 agonist, or a STING agonist, said method comprising the steps of : a) Detecting the expression and / or activity of the p16 protein in immune cells present in a biological sample of a patient, b) Determining the percentage of p16highimmune cells in said sample, c) Concluding that the patient will benefit from a treatment involving a TLR7 agonist, a TLR5 agonist, or a STING agonist if the percentage of p16highimmune cells in the tested sample is low.

15. The in vitro method of claim 14, further comprising the steps of i) detecting the expression and / or activity of the nicotinamide N-methyltransferase (NNMT) enzyme in the immune cells present in a biological sample of said patient and ii) concluding that the patient will benefit from a treatment involving a TLR7 agonist, a TLR5 agonist, ora STING agonist if the percentage of p16highimmune cells in the tested sample is low and if the NNMT enzyme is expressed and functional in the immune cells present in the tested sample.

16. A method of preventing and / or treating tissue inflammation and / or tissue damage, said method comprising the step of administering an effective amount of a STING agonist, of a TLR5 agonist or of a TLR7 agonist, or of a combination thereof.

17. The method of claim 16, wherein said tissue inflammation and / or tissue damage is induced by a pathogen infection, a cancer, a chemical or physical treatment, or by a physical damage.

18. The method of claim 16 or 17, comprising the steps of: a) contacting a STING agonist, of a TLR5 agonist or of a TLR7 agonist or a vector encoding p16 with blood or purified immune cells; and collecting the p16highimmune cells thereby obtained; b) optionally, contacting said p16highimmune cells with a vector encoding a CAR molecule, so as to obtain CAR-expressing p16highimmune cells, c) administering said p16highimmune cells into a patient in need thereof.

19. An in vitro method to detect if a patient infected with the SARS-COV-2 virus will suffer from severe symptoms, comprising the steps of: a) Detecting the expression and / or activity of the p16 protein in the immune cells contained in a blood sample of a patient infected with a SARS-COV-2 virus, b) Determining the percentage of p16highimmune cells in said sample, c) Concluding that the patient will suffer from a severe COVID19 if the percentage of p16highimmune cells in the tested sample is low.

20. A pharmaceutical composition containing a replication-defective recombinant virus comprising the human CDKN2A gene encoding the p16 protein, under a promoter that is functional in lymphocytes or in monocytes.

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