2.1.1 Genomic instability and telomere attrition

Genomic instability: Cells frequently encounter DNA damage factors, comprising endogenous factors (e.g., DNA replication errors) and exogenous factors (e.g., irradiation, oxidative stress, and chemical mutagens). DNA-related alterations including damage response and repair, mutations, replication stress, translocations, chromosomal aberrations, micronuclei, and fragment changes, could have effects on genes and transcriptional pathways, ultimately leading to defective biological functions. Additionally, DNA repair networks functionally decline with advancing age.¹¹ DNA, as a vehicle for genetic information, accumulates damage over time, disrupting regular cell activity and tissue equilibrium, posing the consequence of immunosenescence. Large-scale DNA transformations have been utilized to indicate a cell's biological state.⁷

Telomere attrition: Telomeres, nucleoprotein complexes situated at the ends of lineage chromosomes, are momentous for preventing DNA degradation, damage, and unwanted recombination in eukaryotes.⁷ They modulate telomere replication regulation, telomere capping, and higher-order structural determination of telomeric chromatin. Derecapping of protective protein complexes from telomeres triggers DNA damage responses and undesirable DNA repair.^{12, 13} During DNA replication, the replication machinery is incapable of replicating telomeric DNA entirely, causing telomeres to shorten each time, namely the end-replication problem.¹⁴ It could lead to DNA breakage and replicative senescence as telomeric DNA is shortened below a certain threshold.¹⁵ Therefore, telomere attrition could be utilized as a read-out for assaying immunosenescence.¹⁶

2.1.2 Epigenetic alterations and loss of proteostasis

Epigenetic alterations: Multicellular organisms’ cells are genetically homogeneous but heterogeneous in structure and function by virtue of gene expression differences are epigenetic since they are temporarily heritable without entailing DNA mutations.¹⁷ Epigenetic mechanisms critically manipulate proprietary biological processes by controlling gene transcription and translation, leading to increased transcriptional noise and abnormal mRNA production and maturation. Epigenetic dysregulation, including altered genomic DNA methylation (e.g., hypermethylated tumor suppressor genes), aberrant histone modifications (e.g., reduced H3K9 or H3K27 trimethylation), heterochromatin deletions, three-dimensional genome structural reorganization, and deregulated RNA modifications (e.g., dysregulated non-coding RNA), is broadly explored to be an immunosenescence hallmark.¹⁸

Loss of proteostasis: Immunosenescence expands the risk of aberrant protein aggregation due to progressive proteostasis loss.¹⁹ The proteostasis network consists of following core members: (1) molecular chaperones and co-chaperones facilitate highly efficient folding and prevent misfolded proteins from aggregating. (2) Autophagy–lysosome and ubiquitin–proteasome systems, which are primary quality control pathways.²⁰ (3) Protein misfolding mobilizes stress responses such as heat shock responses, endoplasmic reticulum unfolded protein response (UPR), and mitochondrial UPR.²¹ Immunosenescence, linked to decreased proteostasis network function, contributes to accumulated ubiquitinated, misfolded, and oxidized protein.²⁰ It has been demonstrated that chaperone status, autophagy–lysosome system function, ubiquitin–proteasome system activity, as well as UPR in the endoplasmic reticulum and mitochondria, could be evolved into markers of immunosenescence.⁷

2.1.3 Disabled autophagy and mitochondria dysfunction

Disabled autophagy: Autophagy refers to the process where cytoplasmic material is enclosed in a double-membrane vesicle, the autophagosome, which merges with lysosomes for digestion.²² Accordingly, autophagy, related to protein homeostasis and impacting non-proteinaceous macromolecules (e.g., glycogen), whole organelles, and pathogens, is a prominent factor in the decline of immune function during immunosenescence, affecting organelle turnover.⁹ Cassidy et al. have provided evidence that systemic autophagy inhibition induces premature manifestation of phenotypes and pathologies associated with immunosenescence in mammalian subjects.²³ Moreover, autophagy could boost cell resistance to harsh conditions, for example, autophagy in fibroblasts may potentially augment tropic support to malignant cells and even subvert immuno-surveillance. Collectively, disabled autophagy is justified as a characteristic feature of immunosenescence.²⁴

Mitochondria dysfunction: Mitochondria, integral organelles within cellular structures, are involved in most cell activities containing cell signaling, energy supply, apoptosis regulation, calcium homeostasis, and multiple biosynthetic pathways. The mitochondrial genome, known as mitochondrial DNA (mtDNA), encodes an array of genes including proteins, ribosomal RNAs, and transfer RNAs. Furthermore, reactive oxygen species (ROS), largely launched through mitochondrial aerobic respiration, mainly produced in mitochondrial respiratory chain complexes I and III, could sponsor oxidative damage to mtDNA.²⁵ The concomitant mtDNA mutations could trigger defective respiratory chain components that spark more ROS, which instigates a vicious spiral of ROS accumulation, mtDNA mutations, disequilibrium of mitochondrial functions, and acceleration of immunosenescence.²⁶

2.1.4 Deregulated nutrient-sensing and cellular senescence

Deregulated nutrient-sensing: The nutrient-sensing network, such as the insulin/insulin growth factor-1 (IGF-1) signaling pathway, acts as a central accommodator of cellular energy and metabolic homeostasis, as well as diverse biological processes incorporating mRNA biogenesis, protein synthesis, mitochondrial biogenesis, proteasomal activity, and glucose, nucleotide, and lipid metabolism.²⁷ The network primes metabolic or cellular defense pathways under stress and nutrient shortage circumstances. Nevertheless, it has been observed that a large number of nutrient sensors and targets undergo downregulation, a process correlated with immunosenescence, giving rise to a deregulated metabolism.²⁸ Furthermore, disorders in nutrient uptake mechanisms could perturb homeostasis at cellular level. Recent studies have revealed that a genetic reduction in activity of nutrient-sensing network components could potentially prolong a healthy lifespan in a spectrum of animal models.²⁹

Cellular senescence: Cellular senescence is a response elicited from stress and injury such as DNA damage, replicative stress, oxidative stress, oncogene activation, organelle stress, etc.³⁰ Cells will transition into a state of stable, permanent cell cycle arrest with flattened and enlarged macromolecular changes in culture.^{31, 32} Highly heterogeneous though, senescent cells share three key features: cell cycle arrest, resistance against apoptosis, and a hypersecretory, pro-inflammatory phenotype, commonly referred to as SASP.³³ Cellular senescence activation is saliently mediated through cell cycle proteins, cyclin-dependent kinases (CDKs), CDK inhibitors, and retinoblastoma oncogenes (RBs), respectively.^{30, 34} The downstream signaling cascades evoked by immunosenescence triggers eventually converge on p53/p21^CIP and/or p16^INK4a/pRB pathways, which dynamically interact with CDKs to suppress cell cycle, accordingly both p16^INK4a and p21^CIP1 could be applied for detection of immunosenescence in vivo.³⁵

2.1.5 Stem cell exhaustion and altered intercellular communication

Stem cell exhaustion: The phenomenon of stem cell exhaustion, both quantitative and qualitative decreases in stem cell function throughout an organism's lifespan, is considered a momentous contributor to organismal immunosenescence.³⁶ In the juvenile state, stem cells pertain to maintaining tissue homeostasis by facilitating tissue renewal and repair.³⁷ The potential exists for induction of cellular de-differentiation and plasticity by employing secreted cytokines, and growth factors to restructure microenvironment and extracellular matrix (ECM).³⁸ Inspiringly, immunosenescent phenotypes could be reversed by stem cell rejuvenation in vivo, which could be a robust tool with the possibility to boost stem cell function and shift anti-immunosenescence landscapes.³⁹

Altered intercellular communication: Immunosenescence is connected with incremental changes in intercellular communication that hamper homeostasis and hormonal regulation. As a result, immunosenescence engages deficiencies in neural, neuroendocrine, and hormonal signaling pathways, containing adrenergic, dopaminergic, insulin/IGF-1 system, renin–angiotensin system, among others. Importantly, modification in intercellular communication also encompasses short-acting extracellular molecules, soluble factors, cell-bound receptors, and cell-to-cell interactions during the process of immunosenescence.⁴⁰ Dysregulation of intercellular communication links chronic inflammatory response weakened immunosurveillance against pathogens and premalignant cells, and altered bidirectional communication between human genome and microbiome to dysbiosis.⁹ Besides, the nexus between intercellular communication derangements and ECM disruption during immunosenescence is still in non-stop exploration, intending to further elaborate communication systems between cells.

2.1.6 Chronic inflammation and dysbiosis

Chronic inflammation: During immunosenescence, inflammation is chronically exacerbated, as evidenced by the elevated levels of circulating inflammatory cytokines and biomarkers. It is typified by elevated serum concentrations of inflammatory agents such as tumor necrosis factor (TNF), interleukin (IL)-6, IL-8, and other pro-inflammatory cytokines.⁴¹ Chronic inflammation invokes intrinsic stimulus to most immune cells, thereby attenuating their immune responses.⁴² Additionally, chronic inflammation is common in immunosenescence-related diseases and phenotypes, tissue senescence across several species, and is linked to increased mortality and multimorbidity risk.⁴³ In head-to-head comparisons, lower peripheral blood levels of inflammatory cytokines are positively correlated with favorable fitness and diminished death risk in the elderly.⁴⁴

Dysbiosis: It is acknowledged that composition of each individual's gut microbiota grows more unique ascribed to immune regulation, inflammation, and immunosenescence. Recently, gut microbes have appeared as a pivotal component in a myriad of physiological processes, including pathogen resistance, essential metabolite production, nutrient digestion and absorption, etc. Gut microbes also communicate with central and peripheral nervous systems, together with additional remote organs, facilitating comprehensive maintenance of host fitness.⁴⁵ However, a breakdown in bidirectional communication between bacteria and the host could lead to dysbiosis and manifold immunosenescence-related diseases, including cancer.⁴⁶ Encouragingly, studies have verified that immunosenescent physiological transformations may have a far-reaching influence on gut microbes of older individuals, independent of dietary and lifestyle variations.⁹

2.2.1 Antigen exposure and oxidative stress

Antigen exposure: Studies suggest that viruses, particularly human cytomegalovirus, could induce latent and chronic infections, thereby significantly influencing T-cell compartments.⁴⁷ Notably, human cytomegalovirus seropositivity is associated with an inversion of CD4/CD8 T-cell ratio, primarily through chronic expansion of CD8CD28⁺⁻ effector T cells.⁴⁸ These pro-inflammatory cells, produce substantial amounts of interferon-gamma (IFN-γ) and TNF, but exhibit limited proliferative capacity upon antigenic stimulation and, consequently are classified as “senescent” cells.⁴⁹ As specialized effector cells, they exhibit an increased susceptibility to apoptosis, which correlates with elevated mortality rates among the elderly population.^50-52 Special attention has been paid to the fact that lifelong chronic antigen exposure exhausts T lymphocytes and diminishes T-cell repertoire, resulting in inflammation and poor responses to new antigens. This phenomenon further drives immune system toward immunosenescence in elderly individuals.⁵³

Oxidative stress: Many studies have demonstrated that oxidative damage increases with age across various tissues and species, and that adjusting respiration could extend lifespan in model organisms.^54-57 Research conducted by Pangrazzi et al. indicates that senescence modifies molecules in bone marrow that are required to maintain memory T cells and plasma cells. Elevated ROS levels in cells drive harmful senescence effects, including inflammaging as well as senescence-related diseases.^58-60 The accumulation of ROS results from impaired mitochondrial protein repair and degradation, which causes increased oxidative stress. This oxidative stress, coupled with reduced proteasome activity, exacerbates protein damage and functional decline, thereby giving rise to inflammaging and cellular senescence.^{61, 62}

2.2.2 Inflammaging and SASP

There is potential for any cell to senescence and secrete inflammatory signals that mark themselves for destruction due to mutation damage. However, with aging, excessive damage causes even immune cells to senesce, disrupting this wound-healing process. Thus, not only do these immunosenescent cells inadequately perform necessary cleanup, but also exacerbate inflammation as well as further damage to adjacent healthy tissue.⁶³ This phenomenon termed “inflammaging,” refers to elevated self-reactivity in older adults, resulting in a classic chronic, low grade, and systemic sterile inflammatory state.⁶⁴ Inflammaging is majorly triggered by stimuli such as stress, mechanical trauma, and ischemia, and occurs without pathogens, releasing danger-associated molecular patterns. Consequently, they are detected by innate immune receptors such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptor family, pyrin domain containing 3, activating signaling pathways that lead to inflammation.⁶⁵

Senescent immune cells release a plethora of soluble factors including pro-inflammatory cytokines, proteases, growth factors, angiogenic factors, chemokines, matrix metalloproteinases, and ECM components, collectively known as SASP, which contribute to inflammaging and immunosenescence. Inflammaging damages tissue microenvironments and reportedly plays a role in the pathogenesis of various age-related diseases.⁶⁶ SASP could positively influence immune system by aiding in recruitment of pre-cancerous lesions and promoting tissue repair.^67-69 However, it also secretes pro-inflammatory substances such as IL-6, IL-8, membrane cofactor proteins, and macrophage inflammatory proteins, which could increase proliferation, angiogenesis, and inflammation thereby having unfavorable implications.⁷⁰ SASP has been confirmed to launch spontaneous DNA damage in immune cells and higher senescence and SASP markers in various immune cells, such as monocytes/macrophages, B cells, NK cells, and T cells.^{71, 72} Additionally, inflammaging is triggered by increased visceral adiposity, oxidative stress, enhanced gut permeability, chronic infections, dysfunctional immune cells, and the presence of SASP.^{43, 73}

2.2.3 Altered signaling pathways

Caenorhabditis elegans (C. elegans)mutations in daf-2 gene were discovered to nearly double imaginal lifespan in 1993 by regulating switch between natural developmental processes and alternating larval diapause formation.⁷⁴ Subsequent research revealed that senescence-associated genes such as daf-2 are mammalian genes orthologues involved in insulin/IGF intracellular signaling pathway, which could be inhibited for enhanced lifespan in yeast and flies.^75-77 Additionally, rapamycin was first found to have strong antifungal effects and was further recognized for inhibiting cell growth and modulating immune system.^{78, 79} Its mechanism was understood through mutants in Saccharomyces cerevisiae that countered its cell cycle-arresting effects.⁷⁸ The target of rapamycin is a multifunctional protein that merges nutrients, energy, and stress signals, manipulating autophagy, transcription, mRNA translation, and mitochondrial function, all of which are linked to increased lifespan.^{80, 81} Nutrient-sensing pathways such as mammalian target of rapamycin (mTOR) pathway, may significantly affect T-cell activation, differentiation, and overall immune responses. Moreover, increasing evidence shows that nicotinamide adenine dinucleotide (NAD⁺) levels and sirtuin activity, both crucial for cellular function, decline with age, immunosenescence, or a high-fat diet, contributing to senescence process.⁸² In senescent T cells, activation of p38 mitogen-activated protein kinase (MAPK) obstructs autophagy protein nine through an mTOR-independent pathway, thereby impeding autophagic process.⁸³

2.2.4 Activation of immunosuppressive network

The immune system has extensive plasticity and is capable of responding and adapting to diverse kinds of microenvironmental insults. Persistent inflammation often triggers compensatory immunosuppression to defend tissues.⁸⁴ Inflammatory mediators facilitate myelopoiesis and induce immature myeloid-derived suppressor cells (MDSCs) generation that scale up immunosuppressive network. The immunosuppressive network comprises regulatory T (Tregs) cells, regulatory B cells, regulatory phenotypes of macrophages, dendritic, NK, type II NKT cells, and immature MDSCs.⁸⁵ These members are able to amplify each other's suppressive functions and promote the differentiation of additional immunosuppressive cells.^{86, 87} Immunosuppressive cells release immunosuppressive factors such as ROS, IL-10, transforming growth factor beta (TGF-β), indoleamine 2,3-dioxygenase, and arginase-1, which inhibit immune cell function. As proof, TGF-β exposure halts T-cell proliferation by upregulating CDK inhibitors, inhibits T-cell activation, disrupts helper T cell (Th) cell differentiation, reduces CD8 T-cell cytotoxicity, and disrupts ECM architecture.⁸⁸ To sum up, activated immunosuppressive network inhibits functions of immune cells through: (1) hindering immune cell growth and proliferation. (2) Reducing CD8 T and NK cell cytotoxicity. (3) Blocking antigen presentation and antibody production. (4) Dampening responses to inflammatory signals.

2.2.5 Energy and substance metabolism disorders

Immune function relies heavily on nutritional metabolism, known as immunometabolism.⁸⁹ Senescence disrupts basic nutrient metabolism (glucose, lipids, amino acids) in immune cells, affecting NAD⁺ metabolism, triggering inflammation as well as speeding up immunosenescence.⁹⁰ During immunosenescence, reduced glycolytic metabolism and abnormal mitochondrial energy impair T and B-cell activation.^{91, 92} NAD⁺, a coenzyme essential for cellular metabolism, suffers decline with senescence in response to decreased biosynthesis from chronic inflammation, oxidative stress, and increased depletion from DNA damage.^{93, 94} This reduction activates NOD-like receptor family, pyrin domain containing 3 inflammasomes, potentially accounting for inflammatory diseases.⁹⁵ Furthermore, proteostasis deteriorates with age, crucial for maintaining protein structure and function, impacting immune responses, particularly in T cells, accounting for protein accumulation in tissues. Furthermore, amino acid metabolism deteriorates and a wide range of proteins fail to degrade and accumulate in tissues, which affects immune responses, particularly that of T cells, ultimately leading to immunosenescence-related diseases.^{96, 97}

To sum up, we will refer to these hallmarks of immunosenescence could prolong healthspan and lifespan, but they follow a certain hierarchy. The primary hallmarks (i.e., genomic instability, epigenetic alterations, loss of proteostasis, disabled macroautophagy, and telomere attrition) injure genome, epigenome, proteome, organelles, and telomeres and build up over time and initiate immunosenescence at upstream level. Further downstream, the antagonistic hallmarks (i.e., deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence) reflect a maladaptive response to injury and play a more subtle role in the aging process. Ultimately, integrative hallmarks (i.e., stem cell exhaustion, altered intercellular communication, inflammaging, and dysbiosis) emerge when damage from primary and antagonistic hallmarks becomes irreparable, collectively leading to drive aging along with molecular mechanisms (Figure 2).

2.3.1 Innate immune cells

Neutrophils: Unlike other cell populations, neutrophils do not exhibit a distinct senescence phenotype, thereby underscoring the need for a comprehensive characterization of their role in the immunosenescence process.⁹⁸ Microbicidal activities of neutrophils in older adults have been reported to be massively curtailed, primarily due to hampered phagocytosis, degranulation, and ROS generation.⁹⁹ Furthermore, migration of neutrophils reacting to chemotactic stimuli is similarly diminished in the aged, namely evoking defective chemotaxis. This impairment affects capacity of neutrophils to reach injury sites, alters their distribution, and further hinders recognition and clearance of pathogens.¹⁰⁰ In addition, reduced chemotactic response may exacerbate tissue damage and inflammation due to neutrophil-released proteases, such as neutrophil elastase, which facilitates migration in tissues.¹⁰¹

Monocytes/macrophages: Monocytes take part in generating anti-inflammatory cytokines and lipidic mediators and empower them to participate in wound healing and tissue restructuring.¹⁰² These cells are heterogeneous and highly adaptive, responding to pathogens and cellular debris, and simultaneously able to engage with adaptive immune system. Three distinct subpopulations of monocytes could be differentiated based on expression of surface molecules: (1) classical CD14⁺⁺CD16⁻ monocytes; (2) intermediate CD14⁺CD16⁺ monocytes; and (3) non-classical CD14^dim/lowCD16⁺ monocytes. It is further noted that there is a transition from classical monocytes to intermediate monocytes and subsequently to non-classical monocytes. These subsets exhibit immunosenescence-like phenotypes characterized by shortened telomeres, eliciting inflammaging states.^{103, 104} Consequently, it is concluded that advancing immunosenescence seems to be correlated with elevation in non-classical monocytes and intermediate monocytes.¹⁰⁴

APCs: Dendritic cells (DCs), the most crucial APCs, are specialized in uptake, processing, and presentation of antigens to T cells. Human DCs could be categorized as plasmacytoid DCs (pDCs) and myeloid DCs (mDCs), each exhibiting distinct functional activities. pDCs are primarily involved in interferon-α/β production against bacterial and viral infections, whereas mDCs are responsible for IL-12 production as well as inducing Th1 and cytotoxic T lymphocyte (CTL) responses.¹⁰⁵ Frustratingly, immunosenescent DCs could damage migration and phagocytosis, possessing a diminished or delayed potential to migrate to lymphoid organs and peripheral tissues, and eventually bring about chilly antigen presentation.¹⁰⁶ Additionally, a spectrum of TLRs is differentially expressed and functionally handicapped in the elderly, resulting in altered TLR-mediated immune responses. Elderly mDCs and pDCs are pronounced dampened in their ability to participate in TLR-stimulated secretion of TNF-α, IL-6, and IL-12, attending to impaired vaccination responses in older people.¹⁰⁷

NK cells: NK cells are dissected to be guardians of innate immunity, playing an instrumental role in defense against viral infections and tumor targets. They contribute to immunosurveillance by releasing soluble factors and cell-to-cell contact-mediated interactions. The link between NK cell deficiency and substantially increased viral infection incidence unravels that NK cells are critical in the early clearance of virus and prior to cancer control, preceding activation of adaptive immune responses.¹⁰⁸ NK cells could also clear cells that multiply with immunosenescence and interplay with macrophages and DCs to modulate their activation and functions.¹⁰⁹ There are distinguished subpopulations of NK cells based on differential expression of phenotypic and functional markers: (1) CD56^highCD16^low NK cells which are more immature, and (2) CD56^dimCD16⁺ NK cells which consist of mature NK cells.¹¹⁰ Immunosenescence induces a redistribution of NK cell subpopulations, characterized by an enlarged proportion of mature NK cells and a notable decline in immature NK cell subpopulations. More significantly, immunosenescence also aggravates variations in NK cell functions, as represented by disrupted NK cell proliferation in response to IL-2 stimulation.¹¹¹ Ultimately, while immunosenescence does not alter overall NK cell cytotoxicity attributed to augmented frequency of mature NK cells, it does result in impaired NK cell cytotoxicity in individual cells owing to diminished activation receptor expression.¹¹²

2.3.2 Adaptive immune cells

B cells: Impact of immunosenescence on B cells could be summarized in the following five dimensions: (1) B-cell differentiation. A study conducted by Nunez et al. examining a human population found no obvious changes in pro-B cells, pre-B cells, and immature B-cell frequencies among individuals aged 24–88 years.¹¹³ On the contrary, linear regression analysis in bone marrow samples from several cancer or non-cancer patients revealed that B lineage precursors waddled down with the onset of immunosenescence.¹¹⁴ It has been proposed that immunosenescence impedes B-cell differentiation and output of mature B cells in bone marrow. (2) Peripheral B-cell subsets. Both percentage and absolute number of B cells have been documented to drop off dramatically with age, and proportions of disparate B-cell subpopulations also shift in humans.¹¹⁵ Four main subpopulations could be identified by expression of anti-CD19, anti-CD27, and immunoglobulin (Ig) D antibodies: naïve (IgD⁺CD27⁻) B cells, IgM or unswitched memory (IgD⁺CD27⁺) B cells, switched memory (IgD⁻CD27⁺) B cells and double negative (IgD⁻CD27⁻) B cells. Immunosenescence decreases proportion of switched memory B cells and significantly increases proportion of naïve and double-negative B cells. Even so, it manifests no change in IgM memory B cells. However, additional studies have illuminated reduced IgM memory B cells in older adults, which is postulated to hasten susceptibility to pneumococcal infections.¹¹⁴ (3) Antibody production. Compared to younger individuals, mitogen-stimulated B cells in old folks display reductive levels of E47 and activation-induced cytidine deaminase expression and secrete less amount of IgG. Furthermore, reduced influenza vaccine responses in older adults could be partly attributed to specific serum antibodies, switching of memory B cells, and long-lived plasma cells.^{116, 117} It is confirmed that capacity to elicit vaccine-specific antibody responses is inversely related to serum TNF-α level.¹¹⁸ The outgrowth is that human unstimulated B cells in the aged secrete greater levels of TNF-α than B cells in younger adults. (4) Signal transduction. B-cell immunosenescence could produce a harmful effect on cell activation and signaling. Moreover, a preclinical assay underlined that calcium/calmodulin-dependent protein kinase type IV (CaMKIV) is phosphorylated after in vitro irritation of influenza-vaccinated peripheral blood mononuclear cells from young adults.¹¹⁹ CaMKIV is a constituent of calcium/CaMK family, which may guide Ca²⁺-dependent cellular functions and inflammatory responses through TNF-α generation. It was found that phosphorylated CaMKIV levels were negatively proportional to serum antibody titers 3 days after vaccination, indicating that CaMKIV may play a part in ordering antibody responses to influenza vaccines. (5) B-cell repertoire. Some researchers have examined changes in antibody repertoire with immunosenescence. Using Ig heavy chain complementarity-determining region 3 spectrometry to analyze DNA samples of peripheral blood from 19 to 94-year-old ascertained a considerable paucity of diversity in older individuals’ antibody repertoire, coupled with oligoclonal amplification, the absence of diversity positively correlated with frailty.¹²⁰ Over-expansion of plasma cells recruits unspecified monoclonal gammopathy and additional monoclonal B-cell expansions, which are closely tied to immunosenescence.¹²¹ To provide inimitable insights, another study using spectra-type analysis and next-generation sequencing exhibited that without antigenic challenge, B-cell complexes in older adults displayed non-specific clonal expansion.¹²² Thus, we arrive at a conclusion that lack of specific B-cell diversity has an intimate association with impaired health.

T cells: Immunosenescence could affect diversiform spheres of T-cell functions, involving their clonality or specificity functions, degree of chronological differentiation, proliferative capacity, and deviations in their activity. The straitened circumstances of immunosenescence on time-dependent deterioration of T lymphocytes could be summarized in the following four aspects: (1) reduction of T-cell receptor (TCR) repertoire. TCR repertoire also varies with immunosenescence related to a vast majority of elements including thymic involution and so on. Shreds of evidence from longitudinal and cross-sectional population studies implied that both endogenous and exogenous T-cell factors may aggravate a hindrance in TCR diversity to scuttle immune functions and induct an over-expansion of (autoantigens or persistent viruses that encode antigen-specific T cells.¹²³ (2) Naïve-memory imbalance. Naïve cell population shrinks with immunosenescence in response to the buildup of highly differentiated memory cells, which may in turn, reflect depletion of stem cell-like populations in T-cell repertoire. Interestingly, Mold and colleagues take the attitude that CD8⁺ naïve T cells revealed more recessionary homologous proliferation in vivo compared with CD4⁺ naïve T cells.¹²⁴ Naïve T cells will differentiate toward memory T cells as a result of lifelong antigenic stimulations. In a nutshell, the available data are suggestive of increases in memory, of which some are antigenically inexperienced T cells and reductions in naïve cell pool arising from thymic involution, sustained antigenic irritations, and inflammatory milieus. (3) T-cell senescence. T cells obtain a senescent phenotype through continued antigenic stimulation, especially chronic virus infections which limits T-cell responses.¹²⁵ T-cell immunosenescence could secrete substantial pro-inflammatory factors such as TNFs and osteopontin, which are evocative of SASP. T-cell immunosenescence also exhibits a sea of other characteristics, for instance, poor telomerase activity, telomere shortening, DNA damage evidence, and β-galactosides. Furthermore, immunosenescent T cells de-express costimulatory molecules CD27 and CD28 as well as up-regulate the expression of terminal differentiation markers.¹²⁶ Collectively, immunologically weakened but pro-inflammatory T cells gradually multiply and accumulate along with immunosenescence. (4) Lack of effector plasticity. Differentiated CD4⁺ T lymphocytes are commonly categorized as different subtypes, which have recently been reassessed, suggesting that the Th lineage forms a continuously polarized phenotype determined by antigenic challenge.¹²⁷ Immunosenescent T cell shed their quiescent state and enter a stage of terminal differentiation, which may execute deviations in T-cell lineage commitment, resulting in a lack of plasticity and a decline in immune system's ability to address de novo antigenic challenges.

2.4.1 Central immune organs

Thymus: Thymus is main organ in charge of generating a diversified but selective population of T-cell repertoire. Meanwhile, thymus involution is considered among the optimal-documented hallmarks in human immune system.¹²⁸ Thymic involution begins in childhood and climaxes in adolescence. Subsequently, the adipose tissue gradually starts to replace cortex and medulla of thymus.¹²⁹ Immunosenescence-related degeneration of human thymus is a breakdown of tissue architecture, shrinkage in thymus size and mass, and a drop in thymocyte numbers. Thymic degradation due to immunosenescence may be a direct reason for decreasing naïve T-cell generation, compensatory amplification of memory T-cell clones, as well as attenuated diversity of peripheral T-cell pool. Thymic involution could also result in lower T-cell functional activity, leading to decreased immunity with potentially defective immune tolerance and enhanced autoimmune responses.¹³⁰ Ultimately, developing an understanding to revitalize thymic functions and T-cell production offers an alternative strategy for immunosenescence interventions.

Bone marrow: Immunosenescence arises from senescence of hematopoietic stem cells (HSCs) for virtually all of its cells originate from HSCs. HSC microenvironment in bone marrow provides a balance that assures hematopoiesis by managing the multiplication, self-renewal, polarization, and relocation of HSCs and progenitor cells in a stable state, as well as in response to contingencies.¹³¹ Most HSCs are in a stationary or dormant state, beneficial for preserving their functions and juvenile stemness, whereas multiplication contributes to depletion. However, immunosenescent bone marrow has more myeloid-dominant HSCs but produces fewer mature blood cells per HSC. Meanwhile, competitive transplantation trials have proven that immunosenescent HSCs could inhibit self-renewal, regenerative potential, and homing capacity.¹³² It has been performed that immunosenescence of endothelial cells is competent to actuate hematopoietic aging phenotypes among young HSCs.¹³³ Insights into bone marrow microenvironment's role in underpinning HSC functions may be rewarding in managing functional hematopoietic decline linked to immunosenescence.

2.4.2 Peripheral immune organs

Lymph nodes (LNs): Human body has 300−500 LNs with a total weight of about 100 g.¹³⁴ The structure and composition of the functional areas of the LNs become progressively disorganized with the accumulation of degenerative alterations. Additionally, quantity of lymphoid tissues in cortex and medulla of LNs descends, along with number and diameter of lymphoid follicular germinal centers decrease. Moreover, follicular DC cells shrink in number, LN homeostasis is progressively disrupted, and levels of relevant chemokines (e.g., CCL-19, CCL-21, and IL-7) decrease.^{135, 136} Ultimately, these structural and functional deficits in the LNs triggered by immunosenescence eventually result in scarce T and B cells’ cellular homeostasis, the diminished ability of immune cells to recognize antigens, and weakened immune responses.⁷ Understanding the reasons for these stromal and lymphoid microenvironment shifts in LNs that spur the immunosenescence-related dysfunction of immune system could optimize long-term immune responses and effectiveness of vaccines in the elderly.

Spleen: Immunosenescence decreases diameter and bulk of human spleen. Specifically, thickness of splenic capsule could also be affected by age: from birth to puberty, it progresses to its zenith and then begins slowly thin. What's more, follicles become abnormal, and somatic mutations in the splenic germinal centers lessen with age.¹³⁷ Marginal zone B cells are located in spleen to trap antigens and immune complexes in blood and convey them to follicular DCs in B-cell follicles. Interestingly, it has been concluded that marginal zone B cells have an impaired capacity to trap antigens and pass between the follicular and marginal zones in spleen of old mice.¹³⁸

Mucosa-associated lymphoid tissues (MALTs): MALTs, differing from other immune organs throughout human body, suffer virtually no roadblock to immune functions during immunosenescence and play an essential part in elderly immunity.¹³⁹ The obligatory role of commensal bacteria in advance of maximal mucosal immune system function has been substantiated and its components with potent immune-activating features are implicated in pleiotropic diseases.¹⁴⁰ During immunosenescence, gut-associated lymphoid tissues (GALTs) immune response is modified. An immunosenescence-induced decline in number and mass of GALT T and B cells along with their subpopulations largely account for suppressed intestinal mucosal immune responses.¹⁴¹ The implications of immunosenescence on the composition, diversity, and functions of the intestinal flora, as well as the persistent chronic inflammatory stimuli caused by aging, lead to immunosenescence of the gastrointestinal mucosal lymphoid tissues at length. Modulation of microflora components with probiotics and prebiotics puts forward an alternative strategy to develop mucosal immunity in parallel with preventing and treating a slice of diseases.¹⁴²

The hallmarks, molecular mechanisms, and ramifications of immunosenescence are intricately interconnected. Various forms of molecular damage, instigated by immunosenescent hallmarks and mechanisms, affect organelles, thereby altering cellular function and even cell fatality. These alterations subsequently influence organ homeostasis, resulting in significant repercussions for entire organism.

4.1.1 Repression of genomic instability and targeting immune epigenetics

Repression of genomic instability: Retrotransposable elements are prime contributors to genomic instability, epigenetic alterations, and cellular senescence during immunosenescence.¹⁹² Retrotransposable elements have also been implicated in activating and triggering IFN-I-mediated inflammation, serving as epigenetic drivers of inflammaging. Admittedly, the IFN-I response is a phenotype of late senescence and contributes to the maintenance of SASP. Thereby, treating aged mice with lamivudine, a nucleoside reverse transcriptase inhibitor, reduced IFN-I activation and inflammaging.¹⁹³ Such drugs could even diminish IFN-I responses in SIRT6-deficient cells and notably enhance health and lifespan in SIRT6-knockout mice, which grossly exhibit abbreviated lifespans, growth delays, and high type 1 long interspersed nuclear element activities.¹⁹⁴ What's more, increased repair of oxidative DNA lesions through small molecule activation of 8-oxoguanine DNA glycosylase 1 might show therapeutic effects on immunosenescence.¹⁵⁶ Likewise, nicotinamide mononucleotide supplementation boosts NAD⁺, stabilizes telomeres, decreases DNA damage, and improves liver fibrosis in a mouse model.¹⁵⁷ The connection between telomere dynamics and organismal senescence has been thoroughly studied for the sake of optimal therapeutic approaches.

Targeting immune epigenetics: Senescence leads to significant epigenetic alterations and buildup of epimutations as a consequence of histone modifications (global DNA hypomethylation and gene-specific DNA hypermethylation). Epigenetic clocks using DNA methylation could predict age and mortality risk and assess lifespan-extending interventions.¹⁹⁵ In addition, the most effective in vitro method to counteract epigenetic senescence involves Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), which reset epigenetic clock by converting somatic cells into pluripotent stem cells.¹⁹⁶ Wang et al. used human mesenchymal precursor cells with mutations causing two types of premature aging syndrome to perform a genome-wide CRISPR-Cas9 screen, identifying histone acetyltransferase gene KAT7 as an instrumental driver of senescence. Inactivating KAT7 in human stem cells reduced histone H3 lysine 14 acetylation, suppressed p15^INK4b transcription, and mitigated cellular senescence. Additionally, intravenous lentiviral vectors encoding Cas9/sg-Kat7 delivery could reduce hepatocyte senescence, slower liver aging, and extend lifespan in both naturally and prematurely aged mice.¹⁸⁷ CRISPR-Cas9 genetic screening effectively pinpoints senescence genes, offering potential targets for senescence therapies.

4.1.2 Eliminate senescent cells and regulate immune metabolism

Eliminate senescent cells: Senescent cells are engaged in cancer by persistently secreting low-level pro-inflammatory and tissue necrosis factors in large quantities.¹⁹⁷ Selectively eliminating senescent cells could preclude harmful implications of immunosenescent cells without damaging healthy cell populations.¹⁵⁹ Both tyrosine kinase inhibitor dasatinib and flavonoid quercetin exhibit their ability to eradicate senescent adipocyte progenitors and endothelial cells in vivo and in vitro.¹⁶⁰ These drugs administered to old mice ameliorate burden on senescent cells and immunosenescent pathologies. Navitoclax and its analogs trigger apoptosis by selectively eliminating senescent cells in competing for inhibitory binding slots of BCL-2, BCL-XL, and BCL-W.¹⁶¹ Besides, D-Retro-Inverso peptide, a Forkhead box O4 (FOXO4) isoform that primes apoptosis of senescent cells by inhibiting p53 axis, has been exploited.¹⁹⁸ Although most of data on such drugs are derived from zoological models, one highly promising human trial has also been launched to assess beneficial effects of integrating small-molecule drugs with vaccines.¹⁹⁹ Unlocking the activity of human body's immune system against senescent cells is the realm of potential therapy. Monalizumab, an anti-NKG2A monoclonal antibody, has been proven to have superior anti-tumor ability by enabling NK and CD8⁺ T cells to throttle tumor cells that express inhibitory receptor human leukocyte antigen (HLA)-E.^{200, 201} More recently, natural and synthetic therapeutic agents, as well as multiple immunotherapies have been utilized in senescent cell clearance.²⁰²

Regulate immune metabolism: A few compounds and drugs are available to orchestrate metabolism of cells in TIME. Metformin, a drug administrated in type 2 diabetes typically, stimulates adenosine 5′-monophosphate-activated protein kinase to guide cellular metabolism and suppress mTOR pathway, which disrupts nutrient supply to tumor cells and enhances immune cell functions in TIME.¹⁶² A small clinical trial combined metformin, recombinant human growth hormone, and dehydroepiandrosterone to reverse epigenetic age of healthy older adults, resulting in thymic regeneration, immune changes, and increased life expectancy.²⁰³ Moreover, resveratrol affects immune cells such as macrophages, T cells, B cells, and NK cells, facilitates production of immunomodulatory and anti-inflammatory cytokines, and augments number of Tregs, thus exerting an anti-inflammatory function.¹⁸⁰ Similarly, spermidine, an endogenous metabolite that induces autophagy, lengthens lifespan and attenuates B-cell senescence in mice.^{181, 182} Additionally, in TIME, 2-deoxy-D-glucose, a glucose analog, effectively represses glycolysis, a critical metabolic pathway essential for energy production in cancer cells. 2-Deoxy-D-glucose could modify metabolic paradigm of TIME to oppress tumor proliferation and facilitate infiltration and functions of immune cells via inhibiting glycolysis.²⁰⁴ Other compounds and drugs include dichloroacetate, indoleamine 2,3-dioxygenase inhibitors, adenosine receptor antagonists, CD73 inhibitors, etc. These are all directed at distinct facets of tumor cell metabolism to optimize anti-tumor responses.²⁰⁵

4.1.3 Regulation of mitochondrial function and oxidation prevention

Regulation of mitochondrial function: Interestingly, lifespan in model organisms could be extended by partially impairing mitochondrial function early in development. A recent study has demonstrated that inhibiting mitochondrial protein synthesis or import via mitochondrial UPR fraction extends lifespan in C. elegans.²⁰⁶ What's more, inhibitors of the mitochondrial electron transport chain, for instance, metformin could extend C. elegans lifespan through mitochondrial hormesis involving peroxiredoxin peroxiredoxin-2.¹⁶³ Besides, mitophagy renews mitochondria pool and hinders accumulation of injured ones. Mitophagy inducer, for example, urolithin A has been proven to boost insulin sensitivity in mice and extend lifespan in C. elegans and aged mice.^{164, 165} A first-in-human clinical trial showed that UroA injection modified skeletal muscle mitochondrial gene expression and regulated plasma acylcarnitines in sedentary healthy individuals over 4 weeks.²⁰⁷ Furthermore, exogenous NAD⁺ treatment increases mitochondrial, cytosolic, and nuclear NAD⁺ levels, while nicotinamide riboside administration specifically raises mitochondrial NAD⁺ levels.¹⁵⁸

Oxidation prevention: Chronic inflammation, accumulation of defective mitochondria and heavy metal, decreased endogenous antioxidants, lower mitophagy efficiency, and proteasomal degradation jointly increase oxidized macromolecules with immunosenescence. Thereby, free radical scavengers and antioxidants, both endogenous (e.g., superoxide dismutase) or exogenous are beneficial.²⁰⁸ In a recent study, higher intake of fruits, vegetables, and antioxidants (such as vitamin C, carotenoids, and α-tocopherol) was found to be linked to lower risk of cardiovascular disease, cancer, and mortality.¹⁷¹ However, these foods contain various active substances beyond antioxidants, and benefits are rarely seen in trials with pharmacological antioxidants.²⁰⁹ Similarly, ROS could cause lipid peroxidation, especially in polyunsaturated fatty acids.²¹⁰ Deuterated supplementation indeed diminished oxidative stress and lengthened lifespan of C. elegans.²¹¹

4.1.4 Inhibit dysregulated signaling pathways and modulation of proteostasis

Inhibit dysregulated signaling pathways: Combining different inhibitors targeting overlapping signaling pathways may be promising. Rapamycin acts as an inhibitor of mTOR1 and prolongs lifespan of model organisms.¹⁶⁶ Data suggest that rapamycin and its analogs have positive effects on senescence-related conditions in humans.⁷⁹ Drugs affecting TGF-β and IGF pathways doubled C. elegans’ lifespan.²¹² A combination of MAPK kinase inhibitor trametinib, glycogen synthase kinase-3 inhibitor lithium, and rapamycin promoted Drosophila longevity.²¹³ Likewise, short-term systemic treatment with p38 MAPK inhibitor losmapimod could block monocyte-derived cyclooxygenase-2-driven inflammation, weaken downstream inflammatory processes, and accelerate inflammatory remission.¹⁶⁸ Recently, senostatics offer a promising method to prevent damage from accumulated senescent cells by modulating their pro-inflammatory secretions. Initial therapeutic targets include nuclear factor kappa B (NF-κB), p38, GATA4, mTOR, BRD4, and cGAS/STING, but more specific senostatics are still required.²¹⁴

Modulation of proteostasis: The endosome–autophagosom–lysosome pathway is primary protein clearance system for cellular and organismal homeostasis, and therefore, stimulating autophagy is an effective method to eliminate these intracellular aggregates.^{215, 216} Several life-prolonging compounds work as autophagy inducers, including VPS34 PI3-kinase inhibitors (e.g., wortmannin), histone deacetylase inhibitors (e.g., trichostatin A), MAPK inhibitors (e.g., suberin), TORC1 inhibitors (e.g., rapamycin, everolimus), and adenosine 5′-monophosphate-activated protein kinase activators (e.g., metformin, resveratrol).^{217, 218} Notably, spermidine showed significant neuro- and cardioprotective effects in aging models and humans, suggesting its potential for future human applications.²¹⁹ In addition, a high-throughput chemical genetics screen of 2750 compounds using a proteasome activity probe identified over ten compounds that boost proteasome activity, among which p38 MAPK inhibitor PD169316 was notably potent.¹⁶⁹ Various genetic, nutritional, and pharmacological pro-longevity interventions could regulate adult ECM remodeling and delay senescence-related decline in collagen expression, generating whole-body advantages.²²⁰

4.1.5 Gene therapy and stem cell therapy

Gene therapy: The use of gene therapy to alter senescence-associated genes to promote longevity and a healthy lifespan, with potential clinical applications. Specifically, Bär et al. investigated therapeutic potential of adeno-associated virus expressing TERT in two independent mouse models of aplastic anemia because of telomere shortening.¹⁸⁸ Another gene target is NF-κB, which plays a key role in inflammaging and SASP. A study indicated that delivering a lentivirus expressing dominant-negative IκBα, an NF-κB inhibitor, into hypothalamus increased mouse lifespan by about 10%.¹⁸⁴ Furthermore, since senescence is a complex, multi-stage process involving numerous internal and external factors, combinatorial gene therapy, may enhance protection against immunosenescence. To illustrate, researchers have developed a gene therapy using adeno-associated virus vectors to co-express fibroblast growth factor 21, TGF-β receptor 2, and αKlotho, which has shown synergistic benefits in addressing senescence-related organ issues such as hypertrophic cardiomyopathy, immune recruitment abnormalities, and disorganized ECM in high-fat diet-fed adult mice.¹⁸⁹ Recently, Koblan et al. utilized in vivo base editing to correct LMNA gene pathogenic mutations in cultured fibroblasts derived from children with progeria and in a mouse model of Hutchinson–Gilford progeria syndrome.¹⁹⁰ Overall, as for in vivo gene therapy, the paramount concern is to determine targets.

Stem cell therapy: The body's ability to repair and regenerate tissues declined in stem cell function and reservoir during immunosenescence. Accordingly, transplantation of healthy stem cells into older organisms could antagonize senescence and restore balance in aged organs. Because unfavorable microenvironment in aged organs tends to hinder stem cell maintenance, genetically enhanced stem cells have been designed for transplantation to furnish better regeneration and stress resistance. Namely, transplanting genetically engineered hypothalamic stem cells, expressing dominant-negative IκBα, has been verified to increase lifespan of 18-month-old mice by 10%.¹⁸⁵ Ensuring safety and effectiveness of these cells is crucial for their use in translational medicine. Thus, FOXO3, a longevity-linked transcription factor, has been chosen for stem cell engineering therapy. Yan et al. found out that human embryonic stem cell-derived vascular cells could be functionally enhanced by engineering them to express an activated form of FOXO3, exhibiting delayed senescence, greater oxidative stress, and tumor transformation resistance.¹⁸⁶

4.1.6 Nutritional and microbiome interventions

Nutritional interventions: There are interactions between nutrition, immune response, and inflammatory condition, which are closely related to the characteristics of immunosenescence.⁴⁸ Older individuals often experience nutritional deficiencies, consequently placing them at an increased risk of immunosenescence. Interestingly, dietary additions such as vitamins, micronutrients, and omega-3 fatty acids have been unraveled as paramount in innate and adaptive immunity.²²¹ Mediterranean diet, as an immunosenescence-delaying diet, sustained intake of which encourages longevity, affects immune homeostasis preservation, and bolsters directly to the decline of inflammation and metabolic disorders.¹⁷⁷ According to research different forms of dietary restriction such as calorie restriction seem to be quite rosy modulators of immune system.²²² Calorie restriction diets could down-regulate pro-inflammatory molecules related to inflammaging and shortened lifespan, including IL-6, TNF-α, and IL-1β.¹⁷⁸ Similarly, studies have attested that replenishment with prebiotics or probiotics accelerates antibody responses to vaccination and produces innate immune responses to infection.⁴⁸ To identify nutritional tactics that both favor immunity and minimize side effects in old characters. Numerous studies are currently underway to examine the role of multiple nutritional interventions in modifying immune system characteristics and functions in older folks. Research shows that omega-3 polyunsaturated fatty acids could lengthen leucocyte telomeres in the elderly, reducing multifold immunosenescence markers and further decreasing systemic inflammaging.^{172, 173} Nutrients interact uniquely with GALT in the intestines. Enterocytes in the intestinal barrier could detect antigens from nutrients and microbiota, passing them to the immune system in lamina propria, which then initiates immune responses. Nutrients influence innate immunity and inflammation by regulating TLRs and cytokines, affecting immune cell crosstalk and signaling. They also impact adaptive immunity by modulating B and T-cell differentiation, proliferation, activation, and antibody production.²²³

Microbiome interventions: The gut's commensal microflora significantly influences immune homeostasis.²²⁴ Thus, using probiotics, prebiotics, and their combination (symbiotics) to modulate immune system by affecting microbiota is common. Castro-Herrera et al. found that elderly subjects had better responses to seasonal influenza vaccination after a year of probiotic therapy (e.g., Lacticaseibacillus and Bifidobacterium).²²⁵ Additionally, giving healthy elderly individuals Lactobacilli and soluble corn fiber boosted NK cell responses and reduced systemic inflammaging.¹⁷⁴ Studies on pro- and prebiotic supplementation in older people indicate reduced inflammation, with lower TNF-α, IL-1β, and IL-6 levels and higher IL-10 levels. These pro- and prebiotics also enhance innate immune responses, improving phagocytosis, cytotoxicity against bacteria such as Staphylococcus aureus, increasing NK cell activity, and reducing CD25 expression in resting T lymphocytes.²²⁶

4.1.7 Exercise and physical activity

Until very recently, it offered a renewed direction that regular physical exercise could decrease blood inflammatory cytokines levels and augment innate immune responses, decrease exhausted T-cell number, and increase T-cell proliferation in the elderly.¹⁷⁹ Aggrandizing thymic mass and naïve T-cell output by stirring IL-7 and growth hormone synthesis could be a hidden mechanism by which exercise governs immunosenescence hallmarks. Furthermore, muscle is an instrumental regulator of immune system. IL-15 produced by muscle accelerates cytotoxicity and cytokine secretion by NK cells and sustains T and NK cell numbers in blood. There is also a skeletal muscle that generates a protein termed myokines that has anti-inflammatory and immune response-boosting properties. By employing a similar strategy, Shen et al. unveiled that osteoblast and lymphocyte niches exhausted due to immunosenescence around the bone marrow could be arrested through the mechanical load of exercise.²²⁷ Exercise could mobilize and redistribute effector T cells relying on a combination of catecholamines with β-2 adrenergic receptors to replenish immune responses.²²⁸ Older persons are encouraged to facilitate more exercise as far as their capabilities.²²⁹

4.2.1 Innate immune cells

Neutrophils: Several studies have proven the possibility of restoring immunosenescence-induced neutrophil disequilibrium. Pharmacological interventions such as statins which are directed at cell division control protein 42 (CDC42) have been explored to amend neutrophil chemotaxis and diminish neutrophil extracellular trap lesions.¹⁷⁰ Besides, regulation of nutrition intake such as vitamin E has been ensured to improve chemotaxis, while eicosapentaenoic acid intake appears to reduce ROS bursts in the aged.¹⁷⁵ Permanent physical exercise also enhances neutrophil functions by gaining resistance to immunosenescence.⁴⁸

Monocytes/macrophages: p38 MAPK signaling pathway inhibitors or other cyclooxygenase-2 inhibitors seem to revive immune response of monocytes.²³⁰ Pharmacological options such as flavonoid-rich cocoa polyphenols have been found to reduce expression of adhesion molecules and pro-inflammatory markers on monocytes.¹⁷⁰ Long-term exercise could reduce the production of inflammatory monocyte subpopulations and pro-inflammatory cytokines, thereby counteracting immunosenescence.⁴⁸ In terms of macrophages, repolarization from M2 to M1 could generate pro-inflammatory implications to clear immunosenescent cells, appearing more profound in counteracting immunosenescence.²³¹ More recently, it has been demonstrated that IL-4 therapy could prevent macrophage senescence and improve healthy lifespan in aged mice, while type 2 cytokine signaling likewise has curative potency to amend healthy aging.²³²

APCs: The employment of TLR agonists as adjuvants appears to be a prospective tactic for DCs against immunosenescence. TLR agonists have been proven to facilitate T-cell activation by boosting antigen uptake, presentation, and DCs’ maturation, as well as cytokine secretion.²³³ To date, quite a few TLR agonists are already administered as adjuvants in licensed vaccines, and others are under development.

NK cells: NK cells are instrumental in filling gap between innate and adaptive immunity, and diverse studies have revealed an essential function for NK cells in eliminating senescent tumor cells.²³⁴ NK group 2, member D (NKG2D), a receptor has been intensively studied in these processes, and use of certain immunosenescence-inducing chemotherapeutic agents could reward the excessive expression of these NKG2D ligands in a wide range of tumor types.²³⁵ Likewise, physical activity and dietary interventions are candidates to minimize NK cell immunosenescence. These factors increase NK cell frequency and enhance NK cell differentiation and NK cytotoxic activity. Additionally, zinc supplementation could recover NK lysis activity, and supplementation with vitamins B9 and B12 could scale up the number and activity of NK cells.¹⁷⁶

4.2.2 Adaptive immune cells

B cells: One plausible strategy for B cells to counteract immunosenescence is to enhance autophagy. Immunosenescent B lymphocytes could induct decreased levels of autophagy and obstructed B-cell responses, but treatment with spermidine could restore their functions instead.²³⁶ Inspiringly, B-cell subpopulations, repertoire, cellular functions in vitro, and immune responses in vivo were analyzed using depletion of blood B cells in older and younger mice models. Results found that generating rejuvenated B cells in bone marrow could cover cyto-responsiveness to immune stimuli in vitro.²³⁷

T cells: An underlying immunotherapeutic tactic could target altered molecules to reverse T-cell senescence. The most obvious target for T-cell senescence reversal is p38 MAPK, given that CD4⁺ and CD8⁺ T cells are at the heart senescence signaling.²³⁸ Targeting the p38 MAPK pathway could also restore TCR functions and revert CD8⁺ T-cell immunosenescence through a mTOR-independent process.¹⁶⁷ Others also target changed molecules in immunosenescent T cells, genetically inhibiting dual-specificity phosphatase (DUSP) 6 or DUSP4 could revive T-cell signaling, etc.²³⁹

Another latent strategy is to reshape T-cell immunosenescence by targeting cell signaling. Encouragingly, suppressing mTOR upstream activator VPS39 could increase level of memory T cells and foster antigen-specific T-cell amplification.²⁴⁰ It has been elucidated that drug interventions, for example, using drugs that favor lipolysis or blocking phospholipase A2, and cytosolic to remodel lipid metabolism may prevent T-cell decline.²⁴¹ Exercise and nutrients respectively have been proven to reduce immunosenescent lymphocytes and improve the functions and responses to vaccination.^{242, 243} Moreover, adoptive T-cell therapy which amplifies and translocates stem cells, memory cells, or virus-specific T cells in vitro to older adults has also been considered as an underlying strategy to confront T-cell immunosenescence.²⁴⁴

4.3.1 Central immune organs

Thymus: Reduction of Forkhead box N1 (FOXN1), an indispensable transcription factor for thymocyte differentiation and progression, could cause thymic regression, and the transcription factor is adversely connected with age. It could decrease thymic deterioration through gene therapy or transplantation of thymocytes with a high FOXN1 level and restore thymic activity by indirectly augmenting the number of immature T cells simultaneously. Equivalently, FOXN1 has been applied as a pivotal target for reversing thymic senescence.²⁴⁵ Moreover, a preclinical assay in mouse models underlined efficacy in recovering thymic functions and amplifying peripheral immune cells after immune injury.²⁴⁶

Bone marrow: Studies were conducted on the shifts in phenotype and functions of adaptive immune system and large amounts of ROS fueled by HSCs senescence.²⁴⁷ CDC42 has been illuminated to be a dominating factor in HSC senescence. A confirmed potent ROS inducer is p38 kinase, which could be inhibited to restore HSC potency. Consequently, suppressing CDC42 and targeting p38 kinase is the principal avenue to reverse HSC senescence.²⁴⁸ Pleasantly, Kuribayashi et al. harbored the idea that transplantation of older HSCs into younger niches reinstated the immunosenescent transcriptional profile of HSCs.²⁴⁹ Moreover, the repopulation of bone marrow with autologous induced pluripotent stem cell-derived HSCs is a potentially promising method to revitalize a large part of immune system, particularly innate immune response.²⁵⁰

4.3.2 Peripheral immune organs

LNs: Despite some studies to restore B-cell compartment vigor, it remains overwhelmingly challenging to recover immunocompetence and vaccination responses in immunosenescent models. It has been hypothesized that NK cell immunity is largely restored by killer cell lectin-like receptor C1 (NKG2A) inhibition. In addition, recombinant IL-7 was manifested in clinical trials to recover a physiological level in T cells.²⁵¹ Anti-fibrotic or senolytic drugs may reduce LN fibrosis and reshape its degenerated structure. Furthermore, synthetic LNs could robustly capture the features in vivo to regain immune functions.²⁵² This method stands a chance of opening renewed potentials which could be coupled with other tactics for enduring immune function rejuvenation.

Spleen: Yousefzadeh et al. discovered that transplanting splenocytes from young mice reduces senescence and tissue damage in older mice.²⁵³ What's more, SIRPα DCs have been revealed to regulate homeostasis of fibroblastic reticular cells via TNF receptor ligands in white pulp of adult spleen.²⁵⁴ However, there is still a paucity of pertinent studies, and understanding the molecular causes of immunosenescence-related changes in the spleen and LNs could help develop new treatments.

MALTs: In the aged, tremendous immunosenescent alterations, namely, decreased barrier functions, epithelial cell proliferation, stem cell retention, increased intestinal leakiness, and pro-inflammatory dysbiosis take place in the gut, leading to further exacerbated inflammaging and pro-inflammatory states.^{255, 256} There are some approaches to combat or pre-empt the deterioration of microbiota, for instance, supplementing prebiotics which could boost beneficial microbiota and decrease immunosenescent microbiota.²⁵⁷ A secretory IgA antibody elicited in upper respiratory pathway together with mucosal delivery could be another approach for MALTs to compete against immunosenescence through the neutralization of the virus and cross-protection of mutant strains.²⁵⁸