The role of immunity in elderly cancer

1. Introduction 

A considerable body of data suggests that the incidence of cancer increases exponentially with age [1]. Aging is a complex physiological process that involves a number of biochemical reactions, with molecular changes that are manifested in single cells as well as in the whole organism. The epidemiological studies indicate that about 55% of tumors have been detected in a population over 65 years. The most frequent sites in men over 65 years are represented by lung, colon, rectum, prostate and bladder [1], whereas the women show higher incidence of tumors in breast, lung, colon-rectum, bladder and pancreas as well as non-Hodgkin lymphoma [1]. The association between cancer and age can be explained by a more prolonged exposure to carcinogens in older individuals and an increasingly favourable milieu for the induction of neoplasms in senescent cells [2], as complex biological phenomena, susceptibility to cancer and its age-dependent increase is thought to include mixed genetic and environmental components [3]. These potential causes lead older humans to accumulate effects of mutational load, increased epigenetic gene silencing, telomere dysfunction, limitless replicative potential, altered stromal milieu, evading apoptosis, sustained angiogenesis, tissue invasion and metastasis [4]. These changes allow transformed cells to acquire a selective advantage of proliferating and growing. Therefore, aging reflects the sum of all changes that occur in living organisms with the passage of time that lead to functional impairment and increased pathologies. The progressive deterioration of the immune system with age [5] seems to be of fundamental importance on the increased incidence of cancer in the elderly, because cancer has an important inflammatory component. The microenvironment of a developing tumor is infiltrated by numerous host cells, such as inflammatory cells, endothelial cells and fibroblasts. The interaction between genetically “initiated” cells and host cells is inevitable for carcinogenesis. Interestingly, once tumors arise, they and their products are recognized by the adaptive immune system and rejected. Nevertheless, the tumor often coevolves with immunity, like a parasite, as demonstrated by findings that inflammation enhances tumorigenicity. Tumors coexist with immune defence systems over extended periods and interact chronically with T cells. A process called “immunesurveillance” is used by the host to protect itself by carcinogenesis and to maintain cellular homeostasis. Both innate and adaptive immunity are involved in the first attack to nascent transformed cells, trying to eliminate them. The effect of this phenomenon is potentially similar to other situations of chronic antigenic stress, particularly maintained by lifelong persistent viral infections.

At the moment the role of the immunosenescence on cancer incidence is an extremely debated argument. It has been suggested that the immunosenescence is not an inevitable and progressive decline of all immune functions, but rather the result of a continuous remodelling process in which several functions are reduced, others increased, while others remain unchanged [6]. Studies of the immune system of centenarians, spotlighted that one of the main factors of longevity may be represented by well functioning immune system which allows the prevention of the main age-related pathologies including cancer, as death from cancer may decline at very old age.

In this article we will discuss how the immunosenescence can be involved as an age-related risk in cancer incidence. Elucidation of the causes of increasing cancer incidence with ageing can help to design a strategy for primary cancer prevention.

2. Alterations leading to immunosenescence 

2.1. Primary lymphoid organs involution and immunosenescence 

Data obtained in the context of cross-sectional studies on healthy subjects, from young people to centenarians [7] have reported the involution of the primary lymphoid organs and defects in the production of early lymphoid precursors severely impact on the immune system. A remarkable example is the contribution of the thymus in restoring the periphery of freshly generated T lymphocytes, which decays rapidly over age [7] (Fig. 1). Despite a massive involution of the thymus during the first decades of life, the decline of CD4+ and CD8+ T cell number is relatively insignificant, and the discrepancy between thymic mass and the number of peripheral T cells is particularly evident in the oldest old [7]. The current literature suggests that a substantial amount of T cells is maintained into late middle-age until the extreme limits of the human life-span, probably as a result of different mechanisms, such as extra-thymic T cells production, and the peripheral expansion of long-lived, antigen-experienced T cells [8]. Without a doubt, an age-dependent decrease of antigen-not experienced (naïve) CD95− CD45RA+CD62L+ cell number, particularly within CD8+ T cells pool arises in humans, as a consequence and/or possibly as a compensatory mechanism, suggesting that the immune system in aged has a lowered capacity to neutralize antigens not previously encountered [9]. These alterations are paralleled by an age-dependent increase of CD4+ and CD8+CD95+ antigen-experienced T cells. In particular, an impressive expansion of effector/cytotoxic CD28− T cells is observed throughout life [10]. It is conceivable that the alterations observed in total lymphocytes are mainly attributable to the change of expression of CD95 (APO1/Fas) on CD4+ lymphocytes, whereas the CD8+ CD95+ population rose steadily throughout the entire age range (Fig. 1). It has been demonstrated that the increase of CD95 on both CD45R0+ and CD45R0− T lymphocytes and the increase of CD95+/CD28+ T cells concomitant with a decline of the number of CD28+ T lymphocytes [11]. As consequence, the enhanced expression of CD95 on lymphocytes of old subjects leads to an enhanced proneness to undergo apoptosis. So it has been hypothesized that although there is a large amount of memory-preactivated lymphocytes in the elderly, the inability to evoke a strong secondary response to vaccines may be attributable to enhanced apoptosis [12]. The activation of T cells requires costimulatory signals, and it is known that CD28 represents an important costimulatory molecule, the expression of which decreases with age [13]. CD28+ T cells decrease mainly in the last decades of life. In addition, the enhancement of expression of CD95 on CD28+ lymphocytes with age may be indicative of an involvement of CD95 in the age-related depletion of CD28+ T lymphocytes.

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Fig. 1. The consequences of ageing on the various components of the immune system. The thymus involution in elderly people can results in a higher prevalence of CD8+CD95+ antigen-experienced T cells. In particular, an impressive expansion of effector/cytotoxic CD28− T cells is observed throughout life. These alterations are paralleled by an age-dependent decrease of antigen-not experienced (naïve) CD95− CD45RA+CD62L+ cell number, particularly within CD8+ T cells pool arises. The functional decline of haematopoietic stem–cell functions, consisting in a decreased capacity to renewal and in a lower homing potential, affects both the lymphoid and myeloid lineage. The aged HSC compromises all downstream events that depend on their integrity, including production of immune cells and subsequent immune responsiveness. HSC: haematopoietic stem cell; CLP: common lymphoid progenitor; ROS: reactive oxygen species; DC: dendritic cell; NK: natural killer; IFN: interferon; TCR: T cell receptor; Th1/Th2: T helper 1/T helper 2; VEGF: vessel endothelial growth factor; IL: interleukin; Treg: T regulatory cells.

2.3. B cells in the elderly 

B cells play a critical role in the anti-tumor humoral responses by differentiating into antibody-secreting plasma cells. The produced antibodies (IgM and IgG) destroy tumor cells through either activation of complement or interaction with lytic cells which possess surface receptors for the Fc portion of the antibody-dependent cell mediated cytotoxicity (ADCC). Humoral immunity in the elderly is compromised as the result both of the decreased production of long-term immunoglobulin-producing B lymphocytes dependent to intrinsic and micro-environmental defects, and of the loss of immunoglobulin diversity and affinity caused by the disruption of germinal centre formation [27].

The data concerning the B lymphocytes show that both quantitative and qualitative alterations affect these cells (Table 1; Fig. 1). In the first case, the number of B cells secreting immunoglobulins or antigen-specific immunoglobulins is decreased. Qualitative changes include alterations in the activity of B-cell subsets as well as shifts in the antibody repertoire with respect to the specificity, isotype and idiotype [28]. Paradoxically, while the antibody response to foreign antigens is lower in old individuals compared to the youngest, the number of B cells secreting antibodies is enhanced [29]. This paradox can be explained by the increased life-span and self-renewal of peripheral B cells in old mice, so the number but not the diversity of the B cell repertoire is maintained. This restricted diversity of the B cell repertoire may be associated with the appearance of B cell clonal expansions that can be precursors of late life B cell lymphomas [30]. Furthermore, some studies in murine models have shown that the antibodies produced by old mice have a lower affinity for their targets and so, are ineffective in preventing infection [31], [32]. Despite the significant decline in B-cell production in the aged mice, peripheral B-cells number remains relatively constant. One reason might be that the peripheral B-cell pool is already ‘saturated’, in a manner that is similar to what happens in T-cell homeostasis in the old [20]. However, another possible explanation is that peripheral B cells in the mouse reflect a decreased B-cell generation and an increase in peripheral B-cell longevity [33]. Furthermore, it has been reported that B cells from aged donors show a decreased expansion in response to antigens [34], whereas, the ability of aged B cells to differentiate into high-affinity, isotype-switched, antibody secreting cells is well preserved [28]. As a consequence of altered composition of B cell pool, it has been reported a shorter duration of humoral response in elderly than in the young. Recent studies have demonstrated a decline in B lymphopoiesis in aged mice which reflects the loss of very early B-lineage precursors [35] (Fig. 1). The decline in frequencies of pre-B cells it was presumed to be principally the consequence of the diminished capacity of Pro-B cells to differentiate. HSCs in the bone marrow give rise to early B cells from common lymphocyte precursors, which become Pro-B cells in the bone marrow by immunoglobulin heavy chain gene rearrangements and subsequently differentiate into Pre-B cells, which then migrate to the periphery [36]. Transition to Pro-B cell and Pre-B cell stages are dependent upon the activity of recombination activating gene 1 (RAG1) and RAG2 [36]. Several lines of evidence show that Pro-B cells in old mice are impaired in their capacity to rearrange the D to J gene segments and the V to DJ gene segments. Several studies have also underlined the loss of RAG mRNA in total bone marrow preparations from old mice [37]. Interesting studies using reciprocal bone marrow chimeras have led to hypothesize that RAG expression in Pro-B cells is controlled by the microenvironment itself rather than being an intrinsic defect of senescent B-cell progenitors [37]. Additional evidence supporting this hypothesis has come from stromal cell cultures, because cultures from old individuals are less efficient in supporting B-cell proliferation than those from young counterparts [15]. Nevertheless, other reports have suggested defects in aged B-cell precursor transcription factors. E2A and Pax-5 are crucial to B lymphopoiesis because they accompany differentiation, proliferation and survival of early B cells following interleukin-7 (IL-7) receptor interaction [38]. The reduced expression of the downstream products of E2A (E47 and E12) and Pax-5 (B-cell-specific activator protein; BSAP) have also been shown to accompany old age in Pro-B cells [38].

Table 1. B lymphocytes in elderly.

	Age-related alterations	Functional effects
	↓ production of Ab with high affinity and specificity	Lower efficiency of humoral immune response against external antigens
	↑ production of Ab with low affinity	Higher incidence of MGUS
	↑ production of autoantibody	Higher frequency of autoimmune disorders

Ab: antibody; MGUS: monoclonal gammopathies of uncertain significance.

Many other intrinsic B-cell defects have also been reported in aged mice and humans, including reduction of co-stimulatory molecules [39], defects in B-cell receptor signaling and lower titer and affinity of produced immunoglobulins which make them less protective [40]. These effects may be due to intrinsic defects of B cells, such as decreased expression of co-stimulatory molecules as B7 (CD 86) and defects on B cell receptor signalling. The receptor affinity of aged B cells is reduced, as they have defective somatic hypermutation [41].

The functional ability of B cells to mount specific antibody responses to primary and secondary antigens decreases in the elderly [27]. Such defects in T-cell helper function occurring during ageing [20], could have some influences on the diminished ability to produce strong high affinity and antibody response in the germinal centre [42]. The alteration in antibody production is the result of decline in T-cells function and in intrinsic B-cell function. T-cell/B-cell interactions appears to be disrupted both in aged humans and mice [39], [42] (Fig. 1).

3. The immune response against tumors 

Recent data suggest that both the innate and the adaptive arms of the immune system are involved in the prevention of tumors. The immune system counteract the tumor cells growth through different ways: (i) protection of the host from virus-induced tumors by eliminating or suppressing viral infections; (ii) suitable eradication of pathogens and rapid resolution of inflammation thereby preventing the establishment of an inflammatory environment favourable to tumorigenesis; (iii) specific identification and elimination of tumor cells on the basis of their expression of tumor-specific antigens or molecules induced by cellular stress. By means of the latter process, referred to as tumor “immunesurveillance”, the immune system identifies cancerous or precancerous cells and eliminates them before they can cause injury. Despite tumor immunesurveillance, tumors develop even in the presence of a functioning immune system. The recent theory of tumor immunoediting [43] is an additional explanation for the role of the immune system in tumor development. The tumor immunoediting process includes three phases which can function either independently or in sequence: elimination, equilibrium, and escape [43]. The elimination phase of cancer immunoediting is closely related to the concept of tumor immune surveillance, as the immune system detects and eliminates tumor cells developed as a consequence of failed intrinsic tumor suppressor mechanisms. The elimination phase can be complete, when all tumor cells are removed, or incomplete in the case of partial tumor elimination. In the latter event the immunoediting process consist in a temporary state of equilibrium between the immune system and the developing tumor. During this period it is possible that tumor cells either remain dormant or continue to evolve, accumulating additional changes (DNA mutations or alterations in gene expression) which can modulate the tumor-specific antigens and stress-induced antigens expressed by them. If this process continues, the immune system exerts a selective pressure by eliminating susceptible tumor clones. The equilibrium process is a component of cancer immunoediting because tumor cells in equilibrium are immunogenic, whereas those spontaneously exiting equilibrium become growing tumor and have attenuated immunogenicity. The immune selecting pressure mediated by T lymphocytes and IFN-γ removes tumor cells more immunogenic, but leaves cells able to resist to immune system, leading tumor size to grow up. During this stage, which may hold over many years, mutations occur in the original tumor clone, producing many variants of resistant tumor cells. It has been demonstrated that elimination and equilibrium phases can be mechanistically distinguished. The elimination requires the actions of both innate and adaptive immunity, while the equilibrium is maintained exclusively by adaptive immunity. Equilibrium and escape are two distinct processes because, whereas equilibrium represents a time of tumor cell persistence without expansion, escape is characterized by progressive tumor growth. This does not forecast that every tumor cell is obliged to pass through an elimination process before it enters equilibrium, neither that every progressively growing tumor must transit through equilibrium process. However, it can be supposed that the majority of clinically apparent tumors may progress through the sequential processes of ‘elimination-equilibrium-escape’. The notion of the existence of an equilibrium state provides additional support to the idea that immunity can influence cancer development both quantitatively and qualitatively. Several findings in mouse model indicate that maintaining cancer in an equilibrium state may represent a relevant goal of cancer immunotherapy in which augmentation of adaptive tumor immunity could result in improved tumor control [44]. Moreover, these data provide mechanistic keystones for the recent idea that the quality and quantity of the immune reaction within certain tumor types are reliable prognostic indicators of cancer patient survival [44]. It is possible that some tumor promoting actions of chronic inflammation, such as the presence of leukocyte infiltration; the expression of cytokines such as tumor necrosis factor-alpha (TNF-α) or interleukin (IL)-1; chemokines such as CCL2 and CXCL8; active tissue remodelling and neo-angiogenesis elevated NF-κB activity [45], [46], may be the result of the action of adaptive immunity to hold occult cancers in equilibrium [44]. The pressure exerted by the immune system during this phase is sufficient to control tumor progression, but, if the immune response remains ineffective on the removal of the transformed cells, the selection of resistant tumor cells suppress the anti-tumor immune response leading to the escape phase. This phase results from the alterations in transduction molecules, such as missing chain TCR-ζ of tumor infiltrating lymphocytes (TILs), which intervenes in their proliferation. TILs develop as manifestations of the defense against malignant cells by the host immune system. Although such cells can be found within the tumor tissues, sometimes, they fail to control the growth of tumor. Many are the mechanisms described for dysfunction of TILs with regard to the roles of immunesurveillance against cancer. Functional defects of TILs have been linked to abnormalities of signaling molecules, such as reduced ɛchain, p56lck, Zap-70, and p59fyn expression and/or their functions [47], [48], Some of these signaling defects, such as those regarding ζ chains, are reversible after in vitro treatment with IL-2 [49]. Both macrophage-derived reactive oxygen species and chronic antigenic stimulation without co-stimulatory effect have been linked to the loss CD3-ζ and other signaling molecules. It was further asserted that the loss of CD3-ζ/ɛ chains was actually one accompanying feature of tumor-induced apoptosis that shared the same FasL-mediated signaling pathway [50]. Another form of signaling defect that could potentially damage TIL proliferation/activation involves altered IL-2/IL-2R pathways [51]. In human cervical cancer was demonstrated that the failure of response to IL-2 by TILs is dependent to the downregulation of CD25 (IL-2Rα) [52]. A role of matrix metalloproteinases (MMPs) in cancer-mediated immunosuppression by IL-2Rα cleavage has also been proposed. These cancer-derived MMPs also correlated with tumor metastasis, lymphovascular invasion in addition to immunosuppression [53]. Moreover the intracellular signaling molecule, Jak3 appears to be the cause of unresponsiveness to IL-2 by TILs in renal cell carcinoma [54]. It is well known that hypoxia can result in a tumor with more aggressive growth characteristics and more malignant phenotype [55]. Hypoxia induces apoptosis of TILs providing another growth benefit to the tumor, favouring growth of more resistant tumor cells. It has been reported that Endothelial-monocyte-activating polypeptide-II (EMAP-II) may play a role in this mechanism in colorectal cancer [56]. Furthermore, TCR-ζ is responsible for the activation of apoptotic cascades in T lymphocytes, no longer protected by Bcl-2 [57]. Soluble factors derived from tumor may block NF-κB in haematopoietic cells directly down-regulating the anti-tumor response of immune cells, as well as induce release of IL-10 and TGF-β, anti-inflammatory cytokines and inhibitor factors of both DCs and T cells activity. The cancer establishes an immunosuppressive network that allows tumor cells to resist to the elimination by the side of effector cells. Therefore tumor immune evasion is the result both of immune ignorance, for the decreased number of tumor antigens (TAs) and immune tolerance, for the reduced activation of effector T cells. During the escape phase the immune system is inadequate to counterattack tumor growth, and therefore the disease evolutes. On the basis of these views it is conceivable that the immunosenescence is an added factor which further promotes the tumor escape mechanism.

4. The acting company of the immunosenescence 

The immune system may schematically be divided into an ancestral/innate part, represented by monocytes, natural killer (NK) and dendritic cells (DC), and into a phylogenetically newest part, represented by adaptive immunity (B and T lymphocytes). The aging process compromises both sections of the immune system, although, in different ways (Fig. 1). The innate immunity appears to be better preserved [58], while the adaptive immunity [59], being more sophisticated and proficient to recognize fine antigenic specificities, manifests severe age-dependent modifications, which are often unfavourable to the elderly health. Nascent transformed cells can initially be eliminated by an innate immune response such as by NK. During tumor progression, even though an adaptive immune response can be provoked by antigen-specific T cells, immune selection produces tumor cell variants that lose major histocompatibility complex class I and II antigens and decreases amounts of tumor antigens in the equilibrium phase. Furthermore, tumor-derived soluble factors facilitate the escape from immune attack, allowing progression and metastasis.

4.4. Dendritic cells in the elderly 

Dendritic cells (DCs) have a crucial role in both activation of antigen-specific immunity and maintenance of tolerance, providing a link between innate and adaptive immunity. Dendritic cells are involved in antigen presentation in vivo. Sallusto and Lanzavecchia [115] have shown that monocytes, derived from the peripheral circulation, can be readily activated into dendritic cells by culturing with GM-CSF (granulocyte-macrophage colony stimulating factor) and IL-4. A recent work reports that the frequency of PBDCs (peripheral blood dendritic cells) progressively declines with age, particularly the myeloid sub-population of DCs (Table 2), with a following decreasing of immune response against infections and tumors [116]. It also indicated that the frequency of CD34+ cells progressively declines with age, contributing to the reduced availability of DCs. It may be due to increased levels of vascular endothelial growth factor (VEGF), which impairs the differentiation of CD34+ into mature DCs [117] (Fig. 1), thus influencing the decline of protective immunity and the arising of autoimmune disease, and malignancies. The same work demonstrated that the ability of PBDCs to produce IL-12 upon lipopolisaccaride (LPS) stimulation progressively declined with age, while their ability to produce IL-10 remained unaffected [116]. The decreased production of IL-12 may contribute to the imbalance between T-helper 1 and T-helper 2 subsets, leading to a prevalence of Th2 cytokines observed in the elderly [118].

Table 2. Dendritic cells in elderly.

		mDC	pDC
	Cytokine production	Producing IL-12	Producing IFN-α
	Appearance	Monocyte-like	Plasma cells-like
	Markers	Presence of myeloid markers	Absence of myeloid markers
	Function	Driving TH1 responses	Involved on the pathogenesis of autoimmune diseases
	Changes with aging	Decreased	No decreased
	CD expression	CD11+	CD123+

pDC: plasmacytoid dendritic cell; mDC: myeloid dendritic cell.

5. Genetic alterations in the elderly 

Intense investigations have been focused on the nature and polymorphisms of genes that regulate immune responses and their relationship with longevity. Nuclear translocation of transcription factor NF-κB is regulated by targeted degradation of phosphorylated IκBα by the 26S proteosome via the ubiquitin-proteosome pathway. Chymotrypsin-like activity of the proteosome is largely responsible for the degradation of the NF-κB inhibitor, IκBα, and is decreased in elderly individuals. A defective proteosomal degradation of IκBα results in lowered induction of NF-κB which may contribute to immune dysfunction during ageing [131]. It has also been suggested that the proteosomal protein LMP2 codon 60 polymorphism modulates TNF-α responses. Comparisons between two alleles R (Arg) and H (His) in elderly and young individuals show that while there were no statistically significant differences in young individuals, H carriers in the elderly group were less susceptible to TNF-α-induced apoptosis. The differences between TNF-α-induced apoptosis of PBMC from young and elderly people could be relevant to the progressive increase of inflammatory status which characterizes human ageing [132]. The DNA-dependent protein kinase complex is involved in DNA damage recognition and repair and includes the Ku70/Ku80 heterodimer, or Ku, binding DNA termini of breaks without sequence specificity, and the catalytic subunit DNA-PKcs. Ku, is the central component of the non-homologous end joining (NHEJ) pathway of double strand break (DSB) repair. Because Ku forms a ring through which the DSB threads, it likely becomes topologically attached to DNA during repair. DNA-binding of nuclear Ku is enhanced in lymphocytes by an IL6-type cytokine in young subjects but not in elderly individuals. Ku is associated with the kinases involved in signal transduction events after gp130 triggering, such as Jak2 and Tyk2, in the cytoplasm of PBMC from young, but not from elderly subjects [133].

Differences in the frequencies of several cytokine alleles may also play a role in the remodelling of the immune response with ageing. There are some data indicating that elderly individuals show cytokine production patterns which differ from those of the young, characterized by decreased production of type 1 cytokines and increased production of type 2 cytokines. Relatively unexplored thus far are also differences in chemokine secretion and receptor expression, which may also be critical regulators of immune responses [134]. Some polymorphisms at promoter regions of TNF-α, IL10, and IFN-γ have also been associated with susceptibility to infections. In this regard, centenarians have a different frequency of the alleles defined by a polymorphism at the 5′ flanking region of the IL-10 gene coding sequence that results in a high IL-10 producer phenotype. IL-10 exerts powerful inhibitory effects on the action of pro-inflammatory cytokines as TNF-α. Recent data suggest that the −174C/G polymorphism exerts a cell-specific control on IL6 production and is associated with gender [135].

Several cytokine-deficient mice also develop spontaneous malignancies [136]. In one study, approximately 50% of IFN-γ-deficient C57BL/6 mice develop T cell lymphomas [137]. Furthermore, the spectrum of tumors observed in IFN-γ- and STAT1-deficient mice do not overlap, despite STAT1 being a crucial signaling molecule downstream of the IFN-γ receptor, indicating either that these molecules have some non-overlapping activities or that the background strain has a modifying influence on tumor type. In addition, C57BL/6 mice lacking both IFN-γ and perforin display accelerated B cell lymphoma onset compared with perforin-deficient mice [137], indicating that IFN-γ has an important role in modifying the progression to B cell lymphoma in perforin-deficient mice. B cell lymphomas also arise in mice lacking both perforin and beta-2 microglobulin β2m, and tumor onset is earlier and occurs with increased prevalence compared with mice lacking only perforin. In addition, B cell lymphomas derived from mice lacking both perforin and β2m are rejected by either NK cells or γδT cells following transplantation to WT mice, rather than by CD8+ T cells (as in tumors derived from mice lacking only perforin), demonstrating that cell surface expression of MHC class I molecules by tumor cells can be an important factor in determining which effector cells mediate immune protective effects [138]. Intriguingly, mutations in the gene encoding perforin have also been identified in a subset of lymphoma patients [139], although it is not clear whether this contributes to disease. Furthermore, mice deficient for both IFN-γ and GM-CSF have also been found to develop tumors with age; in this case, tumor development is associated with acute or chronic inflammatory lesions in a range of organs, and maintaining mice on the antibiotic enrofloxacin prevents, or at least delays, tumor onset [140]. Mice insensitive to IFN-γ and lacking of gene p53 developed tumors more rapidly than wild type mice. Further analysis demonstrated that IFN-γ controlled the growth of sarcomas, as well as the initiation, growth and spread of tumors in mice [43]. Collectively, these findings demonstrate that the immune system can suppress tumor development, but they do not constitute proof for tumor immunoediting per se. These studies confirm the possible link between tumor immunity and autoimmune or infection-induced inflammation. The finding that antibiotic treatment could prevent tumor development in Gm-csf−/−Ifng−/− mice raises the possibility that rather than directly eliminating tumor cells, the immune system might prevent tumor growth by the timely elimination of infections, thereby limiting inflammation, which is known to facilitate tumor development [141]. However, this finding cannot be generalized, as Rag2−/− and Rag2−/−Stat1−/− mice maintained on the same antibiotics and housed under strict specific pathogen-free conditions still display heightened tumor incidence despite testing negative for common pathogens with known links to malignancy and showing no signs of idiopathic inflammation [142]. Mice lacking the death-inducing molecule TNF related apoptosis-inducing ligand (TRAIL) or expressing a defective mutant form of the death-inducing molecule FASL have also been shown to be susceptible to spontaneous lymphomas that develop with late onset [143]. IL-12 and IL-18 are important IFN-γ-inducing cytokines; however, studies of aging have demonstrated that neither IL-12- nor IL-18-deficient mice display increased incidence of tumor development compared with WT mice [137]. These spontaneous lymphomas are of B cell origin, and, when transplanted into WT mice, are rejected by CD8+ T cells [144]. Curiously, with age, 50% of mice lacking the β2 subunit of the IL-12 receptor (IL-12Rβ2) develop plasmacytomas or lung carcinoma in the context of the autoimmune disease immune complex mesangial glomerulonephritis [136].

It is presently unclear why IL-12-deficient mice on the same genetic background as the IL-12Rβ2-deficient mice do not display either autoimmunity or spontaneous tumor development. These aging studies have clearly demonstrated a critical role for cytotoxic pathways in immunoregulation and/or immunosuppression of spontaneous tumor development in mice.

Mitochondrial (mtDNA) damage has been investigated as a biomarker using PCR-based methods in combination with reference strand conformational analysis (RSCA) to identify genotype associations within successfully aged populations using polymorphism in the genes of the KIR (killer immunoglobulin like receptors) involved in T and NK cell-mediated cytotoxicity, cytokine, and oxidative stress defence systems [145]. Since metallothioneins (MTs) participate in zinc homeostasis and influence the secretion of pro-inflammatory cytokines they have been examined as potential markers of immunosenescence. Metallothionein gene expression is transcriptionally induced by a variety of stressing agents to protect cells from reactive oxygen species. The level of MT mRNA is decreased in lymphocytes of centenarians as compared to old normal populations. Zinc supply in ageing corrects the defect and prevents metallothioneins from sequestering intracellular zinc. As a result, MTs regain a protective role [146]. Studies of insertional mutagenesis and cDNA library transfer aimed to the identification of genes involved in the control of cellular differentiation and apoptosis have identified the retinoic acid receptor alpha (RARα) and c-myb as important regulators of myeloid cell differentiation. Interestingly, the gene Ras-induced senescence (RIS-1) located at 3p22, a chromosomal region containing several tumor suppressor gene, has been proposed to participate in anti-tumor responses that resemble cellular senescence and that are elicited by oncogenes such as Ras [147]. Studies with transgenic mice carrying extra-genomic copies of the tumor suppressor p53 have been used to address a number of questions related to tumor suppression and ageing in vivo [148]. The DNA array technology in senescent T lymphocytes has indicated alterations in redox-sensitive signalling genes, such as AP-1 and B-ATF [149]. Other alterations in regulators of gene expression may include altered methylation in ageing and altered histone deacetylization, which also changes with age in peripheral blood mononuclear cells [150]. Ageing results in changes in neutrophil as well as T lymphocyte signal transduction pathways. Different patterns of PKC isoenzymes can be observed between young and elderly individuals. A decrease in exogenous and endogenous tyrosine kinase activities (ZAP70, p56lck) after CD3 stimulation is associated with ageing. IL2-mediated signalling was also found to be altered with ageing (Jak3 and STAT3 and 5). The changes observed in signal transduction with ageing may be due to alterations of cell membrane fluidity as a consequence of an increment of cholesterol and are liable to modulation [151]. In PMNLs the GM-CSF signal transduction pathway is altered in elderly individuals showing a diminished tyrosine phosphorylation of Jak2, STAT3 and 5 [152]. T cells of old mice and especially the Pgp+ CD4+ subpopulation have an increased activity of μ-calpain, a protease that participates in the regulation of cellular proliferation and signal transduction. In humans, however, calpain activity in general decreases with increasing age [153].

6. Tumor-induced immunosuppression in the elderly 

Immunosenescence, influence the ability of aged subject to react against exogenous antigens and, in particular, reduce the capacity of anti-tumoral immune defences in the elderly [154]. Besides these, other factors may contribute to triggering spontaneous tumor growth without being rejected by the immune system: passive mechanisms, such as the lack of either distinctive antigenic peptides or the adhesion and co-stimulatory molecules needed to elicit a primary T-cell response; and active mechanisms through which tumors can avoid or evade immune attack. Some evidence suggests that at least some of the mechanisms used by cancer cells to escape immune clearance might be more effective in ageing. One of these is related to the Fas ligand/Fas receptor (FasL/FasR or CD95L/CD95) interaction. FasL is a key molecule in normal immune development, homeostasis, modulation, and function and acts by inducing apoptosis of sensitized cells through interaction with its own FasR receptor, expressed on their surface. To date, the expression of functional FasL has been reported in several distinct lineages of tumors [155]. As mentioned before, the increased FasR expression observed on aged leukocytes might facilitate the immune escape of tumors expressing FasL in elderly patients by promoting the apoptosis of TILs. Another mechanism which enables tumors to evade immune rejection is the release by tumor cells of immunosuppressive cytokines. Many tumors produce TGF-β, or IL-10 or other cytokines which tend to suppress inflammatory T-cell responses and cell-mediated immunity, which are needed to control tumor growth and to destroy tumor cells. In old subjects, these suppressive cytokines released by tumor cells may synergize with immunosuppressive cytokines (TGF-β, IL-10 and others) which are already overproduced by leukocytes up to elevated concentrations able to impair anti-tumor immune responses. Furthermore, IL-6, another cytokine overproduced in the elderly, has been reported to increase the expression of the TGF-β receptor, thus facilitating this mechanism of tumor immune escape [156].

Prostaglandins are other factors that have been involved in cancer-induced immune suppression. Tumor cells produce prostaglandins which can inhibit various immune functions. The above examples lend weight to the idea that immune suppression induced by tumor cell-derived prostaglandins may have particular implications in ageing, since lymphocytes from elderly subjects are now known to be sensitive to inhibition by prostaglandins in comparison with lymphocytes from younger individuals [157]. Several mechanisms of active immune escape have been proposed to explain the incapacity of the immune response in rejecting tumors. It seems that these mechanisms of immune escape play an important role in the aged host even though the exact relevance of tumor-induced immunosuppression in the early phases of tumor development in the elderly remains to be proven.

7. Inflammaging cytokines and tumorigenesis 

Ageing is accompanied by a low-grade chronic, systemic up-regulation of the inflammatory response. The inflammatory changes are common to most age-associated diseases this process is termed “Inflammaging” [9]. Inflammaging results in both decreased immunity to exogenous antigens and increased auto-reactivity. Thus, the beneficial effects of inflammation devoted to the neutralization of harmful agents early in life become detrimental late in life. Cancer also represents an immunologic challenge, which upregulates the systemic immune response. Thus, tumor-related hyperinflammation and inflammaging synergistically lead to the systemic priming of inflammatory mediators. A large number of evidence reports the relationship between inflammation and cancer, and the importance of soluble mediators in tumor development and progression. A major force able to drive a chronic pro-inflammatory state during aging may be represented by persistent viral infections by CMV, which has determined by the frequency and the absolute number of viral antigen-specific CD8+ T-cells in subjects older than 85 years, who were serologically positive for CMV. The majority of CMV+ T cells belong to the CD28− subpopulation. Therefore, the chronic antigenic stimulation induced by persistent viral infections during aging bring significant adaptations among CD8+ subsets, which are evident in the presence of CMV persistence [158]. The age-dependent expansions of CD8+CD28− T-cells comprising the majority of CMV-epitope-specific cells, highlights the importance of chronic antigenic stimulation in the pathogenesis of the foremost immunological alterations of aging and may favour the appearance of several degenerative pathologies (arteriosclerosis, cancer, dementia) all of which are characterized be an inflammatory process.

The inflammatory insults leads to up regulation of non-specific proinflammatory cytokines such as IL-1α/β, IL-6, interferon (IFN)-α, and tumor necrosis factor (TNF)-α [159]. These cytokines, subsequently, induce the expression of acute-phase protein (APP) and pro-inflammatory chemokines [159]. Such unresolved chronic inflammation is associated with increased conversion of normal cells to preneoplastic foci. Cytokines appears to play a paradoxical role for in the development of cancer [160]. To one side, cytokines induce tumor cells death, directly or through enhancing immune response. To the other side, cytokines contribute to carcinogenesis, as a consequence of autocrine production, due to chronic inflammation [161]. Therefore, chronic inflammation and over production of cytokines is an additional mechanism of carcinogenesis [162]. It has been suggested that, pro-inflammatory cytokines and chemokines could be increased by carcinogens contained in food, smoking and industrial products [162].

The inflammatory scenario that characterizes inflammaging constitutes a highly complex response to various slight internal and environmental inflammatory stimuli mediated mainly by the increased circulating levels of pro-inflammatory cytokines [159]. Inflammaging also generates Reactive Oxygen Species (ROS) causing both oxidative damage and eliciting an amplification of the cytokines’ release, thus perpetuating a vicious cycle resulting in a chronic systemic pro-inflammatory state where tissue injury and healing mechanisms proceed simultaneously and damage slowly accumulates asymptomatically over decades and is a major determinant both of the ageing process and of the development of age-associated diseases [160]. The cytokines contribute to tumor growth, progression and immuno-suppression, by regulating cell death, survival or differentiation, as well as angiogenesis. Studies on malignant cells of papillary thyroid cancer have been shown that pro-inflammatory cytokines are activated by oncogene expression, promoting the growth and the spread of the malignant cells [160]. The most abundant cytokines present in tumor sites and in the tumor microenvironment belong to the pro-inflammatory type, as confirmed by the proportion of Th1 and Th2 cells observed in patients with bladder and colorectal cancer compared to the healthy population. In fact, the Th1 cells, producing IFN-γ or IL-2, were significantly reduced; whereas, Th2 cells, producing IL-4, IL-6, IL-10, were markedly increased [163]. Furthermore, in human cervical carcinoma the CD3+ tumor infiltrating T lymphocytes displayed enhanced Th2 cytokine patterns and increased IL-4 production, whereas IFN-γ production was reduced [164] (Fig. 1).

It has been reported that the pro-inflammatory interleukin 1 alpha (IL-1 α) plays a role in tumorigenesis, in particular in the promotion of cervical carcinoma growth [165]. The paracrine secretion of IL-1β may influence the growth and the chemoresistance of pancreatic cancer [166] and induce the production of angiogenic factors in lung carcinoma in vivo [167]. Studies performed in mice models have shown that the lacking of IL-1β, may be responsible of the absence of metastasis both in melanoma cells models and in breast and prostate cancer [168].

Another cytokine playing an important role both in inflammatory diseases and in tumor promotion is the TNF-α [169], which may affect tumor cell survival through the induction of genes encoding NF-κB-dependent anti-apoptotic molecules [170]. Additionally, TNF-α plays an important role in the initiation of tumor by stimulating the production of nitric oxide (NO) and reactive oxygen species (ROS), which can lead to DNA damage and mutations [171]. Moreover, TNF-α favours tumor progression, promoting angiogenesis and metastasis and allows tumor escape by suppressing many T cell responses and macrophage cytotoxic activity [172]. Enhanced levels of TNF-α, due to genetic polymorphisms increase the risk of multiple myeloma, hepatocellular carcinoma, bladder, gastric and breast cancer, and then it correlates with poor prognosis of haematological malignancies [169]. Promising results come from treatment with antibody against TNF-α in hepatocellular carcinoma (HCC) during promotion stage. These specific antibodies led to apoptosis of transformed hepatocytes and a failure to the tumor progression [173].

IL-6 is a potent pleiotropic inflammatory cytokine playing a pivotal role in growth and promotion of tumor and behaving as an anti-apoptotic factor. The signaling pathway used by IL-6 (e.g. NF-κB and STAT3) controls tumor development through a direct effect on tumor cells. The paracrine secretion of IL-6 acts as a growth factor for multiple myeloma, non-Hodgkin's lymphoma, bladder, colorectal and renal cell carcinoma [174]. The signaling pathway of soluble IL-6 receptor appears to be responsible of the development of colon cancer [175], as enhances, remarkably, the production of IL-6 from T cells [175], contributing to T cell survival. Antagonist of IL-6 might be useful for the treatment of colon cancer.

The cytokines belonging to the Th2 pattern, such as IL-10, are involved in tumor growth and in angiogenesis induction. Further, both IFN-α and IFN-β have been considered as components of tumor surveillance system [176] and affect negatively tumor growth, since they participate to shape innate immune system, including dendritic cell maturation and NK-cells responses [177].

Many experimental evidences show a contradictory function of IL-10, which is in some cases pro-tumorigenic and in other cases anti-tumorigenic. The development of cancer is inhibited when IL-10 inhibits the production of pro-inflammatory cytokines, as IL-6, IL-12 and TNF-α [178], by blocking the activation of NF-κB [179]. Likely, IL-10 inhibits tumor growth, down-regulating the expression of MHC-class I on cell surface, thus enhancing tumor cells lysis mediated by NK cells [180]. The IL-10 is also involved in the modulation of apoptosis and in the suppression of angiogenesis during tumor regression. IL-10 could affect negatively the release of pro-angiogenic factors from tumor stroma cells [181]. In contrast, increased plasma levels of IL-10 have been found in patients bearing diffuse large B cell lymphoma, and it correlates with poor prognosis [182]. It has been reported that IL-10 promotes the development of Burkitt lymphoma through the production of a B cell-activating factor of the TNF family/B lymphocyte stimulator (BAFF/BLyS), which in turn, promotes B cells and lymphoma survival [183]. Moreover, patients with gastric cancer showed increased IL-10 expression, that correlates with attenuated CD8+ T cell infiltration and poor prognosis [184]. Likely, IL-10 favours tumor growth suppressing adaptive immunity, leading to the tumor escape from immunesurveillance [185].

Anti-tumor effect is played by IL-12, which promoting Th1 adaptive immunity and CTL responses, inhibits tumorigenesis and induce regression of established tumors [186]. The negative effects of IL-12 on tumor development might be strengthen by IFN-γ, that shows a direct toxic effect on tumor cells and inhibits neo-vascularization [186].

IL-13 carries out a suppressive action on cellular mediate immunity [187] and so, acting directly on neoplastic cells influences the tumor growth.

Recently it has been reported that also IL-17 has a pro-tumorigenic role, likely due to pro-angiogenic activity [188]. This cytokine induces pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α. Elevated expression of IL-17 has been found in human cervical carcinoma and non-small cell lung carcinoma (NSCLC) [188], as well as in C57BL/6 mice bearing fibrosarcoma [189].

IL-18 is linked with disease progression in large granular lymphocyte leukaemia [190].

A cytokine not generally considered as a Th2 cytokine, but with Th2-like cytokine activities, is the IL-23 which plays a contradictory role. To one hand, it enhances proliferation of memory T cells and the production of IFN-γ and IL-12 by activated T cells. On the other hand, it induces the production of IL-17, promoting inflammatory reactions [191]. In a murine model the absence of IL-23 leads to a marked decrease of local inflammation in tumor microenvironment and a higher presence of T cytotoxic cells, thus playing a functional defence against cancer development [192]. In addition, this cytokine favours a pro-tumor microenvironment [192].

8. Chemokines and tumorigenesis 

The chemokines are cytokines of a chemoattractant family and play a central role in leukocyte recruitment to the sites of inflammation and in the host response against infection. To date have been identified more than 40 chemokines, which have been subdivided into four families on the basis of the position of their cysteine residues [193]. The α and β chemokines containing four cysteines appear to be the largest families. In the α-chemokines, one amino acid separates the first two cysteines residues (cysteine-x amino acid cysteine or CXC) whereas in the β-chemokines, the first two cysteine residues are adjacent to each other (cysteine-cysteine or CC). Two chemokines that do not fit into this classification are lymphotactin and fractalkine [194]. The chemokine are induced by early pro-inflammatory cytokines, such as IL-1 and TNF-α. In addition, IFN-γ and IL-4 can induce the production of chemokines and synergize with IL-1 and TNF-α to stimulate chemokines secretion [195]. The CXC chemokines are active on neutrophils and lymphocytes whereas CC chemokines act on several leucocytes subsets including monocytes, eosinophils, dendritic cells, lymphocytes and NK cells but not neutrophils [196]. Recent evidences report that aged T cells showed changes in chemokines receptor expression, such as increased CCR1, 2, 4, 5, 6, 8 and decreased CCR7 and 9 in CD4+ T cells [197]. On the basis of these changes, mRNA levels increased, thereby enhancing protein expression [197]. It is interesting notice that a reduced expression of CCR7 in animals, involved on cell homing to secondary lymphoid organs [198], may explain the age difference in T cell trafficking [197].

Also the chemokines are involved in tumor development, as they have a direct tumor growth factor effect, as well as they affect angiogenesis process and metastatic invasion (Table 3). In addition, they may act indirectly, recruiting macrophages in tumors and stimulating them to release other growth factors for cancer development. However, some chemokines and their receptors are involved in anti-tumor immunity. The CXCR3-ligands, such as CXCL9, CXCL 10, CXCL11 are able to attract Th1, CD8 and NK effectors to the tumor microenvironment. They could have also angiostatic effect binding with high affinity CXCR3+ microvascular endothelial cells [199].

Table 3. Chemokines receptors and tumorigenesis.

	Name of chemokines receptor	Properties pro-tumorigenesis
	CXCR 2	Angiogenesis
	CXCR 4	Metastatic spread of many tumors
	CCR 2	Recruitment of macrophages into tumor microenvironment
	CCR 7	Metastasis into sentinel lymph nodes

CCL5/RANTES is the first chemokine detected for mediating anti-tumor immunity in part through direct T cell effector recruitment [200]. It has been proposed that other CCR5-ligands were involved in T cell infiltrates of nasopharyngeal carcinoma [201] and epithelial ovarian tumors [202]. The expression of high levels of RANTES in breast and cervical carcinoma is found in advanced diseases [196]. Furthermore, experiments on mice models bearing tumor have identified CXCL 10/IP10, as a required factor for IL-12 mediated CD8-dependent anti-tumor immune responses [203]. The transfection of lymphotactin CXCL1 into tumor cells leads to CD4 and CD8 T cell infiltration and tumor rejection [204].

9. Concluding remarks 

Dysregulated function of immune system affecting older people, involves both the reaction against infectious pathogens and the anti-tumor defence, leading to a decrease of the activity in both circumstances. Since many mechanisms involved in the ageing process share molecular ways implicated in carcinogenesis processes [205], it is understandable the existence of relationships between ageing and risk of tumor development. Particularly, one of these risks consists with the impairment of apoptosis process. Normal cells are able of a finite number of mitotic cycles before entering a state of replicative senescence prior to cell death. Replicative senescence in fibroblast is associated with increased Bcl2 expression and resistance to apoptosis [206], [207]. The Bcl2 gene promotes malignancies by decreasing apoptotic cell death [208]. Bcl2 has been shown to protect cells against variety of cytotoxic stimuli including anticancer drugs. Spaulding et al. have shown that senescent CD8+ T cell cultures show reduced apoptosis in response to a wide variety of treatments and diminished caspase-3 activity compared with quiescent early passage cultures from the same donor [68]. Moreover, T cell replicative senescence is also associated with increased Bcl2 expression which may contribute to the prolonged survival of senescent CD8+ T cells in culture [68]. The nature of the immunological effectors, which induce apoptosis, is still debated. According to one hypothesis, interaction between antigen presenting cells and T cells leads to the remarkable increment of pro-inflammatory cytokines IL-1, TNF-α and IFN-γ, which in synergy induce apoptotic signalling cascades. A second theory implies that apoptosis is induced by Fas/Fas-ligand interaction and the perforin granzyme system.

Other studies reported the immunosuppressive role of some cytokines, such as IL-10 and TGF-β which increasing with ageing tend to suppress the cell-mediated activity, needed to control tumor growth and to destroy tumor cells [156]. An other pro-inflammatory cytokines over-expressed in the old people is IL-6, which influences the tumorigenesis mediated by TGF-β, by increasing the receptor of this last cytokine [156]. In conclusion the knowledge of alterations consistent with aging could influence the enhanced quality of life and life-span in this people. The immunosenescence is associated with a dramatic reduction in responsiveness as well as functional deregulation which contributes to the increased incidence among the older people of morbidity and mortality from infectious disease and cancer.

Since the carcinogenesis is an extremely complex phenomenon, it is very difficult to establish the exact role of the immunosenescence. Future studies and continuous effort in this field will help us to break off the vicious cycle that leads to large oligoclonal populations of dysfunctional cells and to restore a good function of the immune system in the elderly people. The elucidation and understating the links between innate and adaptive immunity in the regulation of cancer development will offer new therapeutics pathways to develop the most optimal immunotherapeutic strategies that can lead to total tumor eradication.

Reviewers 

Prof. Salvatore Musumeci, University of Sassari, Department of Pediatrics, Viale San Pietro 12, I-07100 Sassari, Italy.

Prof. Pierre-Yves Dietrich, University Hospital of Geneva, Department of Oncology, Rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland.

Dr. Anis Larbi, University of Tübingen, Centre for Medical Research, Tübingen, Germany.