Aging, immunity, and tumor susceptibility

Immunol Allergy Clin N Am 23 (2003) 83 – 102

Aging, immunity, and tumor susceptibility Huang-Ge Zhang, MD, PhDa,b, William E. Grizzle, MD, PhDc,* a

The University of Alabama at Birmingham, Department of Medicine, Division of Clinical Immunology and Rheumatology, Lyons-Harrison Research Building, Room 473, 701 South 19th Street, Birmingham, AL 35294, USA b Veterans Administration Medical Center, 700 South 19th Street, Birmingham, AL 35233, USA c The University of Alabama at Birmingham, Department of Pathology, Zeigler Research Building, Room 408, 703 South 19th Street, Birmingham, AL 35294, USA

Not all cancers increase with age The statement that there is an increased incidence of cancer with age is misleading and usually indicates that most adult tumors are more frequent after a certain age, but not that most tumors occur after, for example, 65 years of age. Clearly, the major cancers (ie, lung, colorectal, breast, prostate, and pancreatic) are responsible for most tumors that kill individuals in the United States; data from these five categories of tumors would control most conclusions and generalizations [1 –5]. When one states that cancer incidence increases with age, the patterns that are characteristic of colorectal, breast, or prostate cancer (Figs. 1,2) are envisioned. Many tumors, however, do not follow these patterns. In fact, the majority of deaths that are secondary to cancers in general occur in the age range of 55 to 74 rather than in patients older than 75; the overall number of cancers diagnosed shifts to even earlier ages [1,2]. If each type of cancer is made equivalent, that is, given a score of 1 (eg, squamous cell carcinoma of the lung represents one cancer and neuroblastoma represents one cancer), then the statement that cancers increase with age is not supported. There are a group of tumors that occur primarily in children, others that occur primarily in young age and in middle age, whereas

This work was supported by NIH grants R01 CA44968, R01, CA8635902, and a Birmingham VAMC Merit Review Grant. * Corresponding author. E-mail address: [email protected] (W.E. Grizzle). 0889-8561/03/$ – see front matter D 2003, Elsevier Science (USA). All rights reserved. PII: S 0 8 8 9 - 8 5 6 1 ( 0 2 ) 0 0 0 8 5 - 1

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Fig. 1. Cancer incidence with age. (Top) Colorectal cancer. (Bottom) Breast cancer.

six major cancers (breast, prostate, pancreatic, bladder, renal, and colorectal) occur primarily in old age [1– 5] (Box 1).

Age-related incidence of cancer caused by a decrease in at-risk population Box 1 groups many tumors as to their average age of occurrence, and in some cases, the age of maximum incidence. These two measures are linked, but not always directly. Because age-range populations rapidly decline after 70, a high incidence of a specific cancer after 70 does not indicate that most of these specific tumors occur after 70, but rather that a greater proportion of a small population has developed that specific tumor. In general, tumors for which at

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Fig. 2. Prostate cancer incidence with age.

least one half of the absolute number of cases occur after 65 will have a pattern similar to cancers of the colorectum, breast, pancreas, bladder, and prostate. Those with average ages of diagnosis at less than 60 years are likely to have peaks in incidence at less than 80 years. Thus, if a person is older than 70 there is a greater likelihood of developing cancer because the cancers whose peak incidences occur after greater the age of 70 make up a group of cancers with the greatest absolute number of cases of cancer (ie, cancers of the breast, prostate, colorectum, pancreas, bladder, and kidney); in addition, the age-range population is now much smaller. In the evaluation of whether the development of cancers increases with age, we ignore childhood cancers; most of those tumors occur secondary to identifiable gross chromosomal abnormalities and such neoplastic lesions would not be expected to be influenced by aging. Nevertheless, even when childhood tumors are not considered, for any tumor whose incidence increases with old age (eg, colorectal adenocarcinomas) there is a matching, nonchildhood tumor whose incidence does not increase with age (eg, thyroid papillary carcinoma). Thus, the general concept that cancers increase with age is not supported by overall number of types of tumors; however, this does not exclude that aging may influence adult cancers in general. The complicated nature of carcinogenesis and progression of specific cancers would block our ability to detect definite effects of aging. For example, tumors such as soft tissue sarcomas (eg, liposarcomas), whose peak incidence occurs at less than 60 years, may contain subgroups of tumors that occur later in life, in part because of aging; such subgroups cannot be recognized because of a peak in incidence at an earlier age during the overall development of these specific tumors. Similarly, because an increased incidence of tumors with aging cannot be demonstrated, the development and progression of tumors cannot be tied to a decline in the immune system with age. Thus, to identify the effect of aging on the etiological progression of specific tumors, we must rely on animal models.

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Box 1. Peak incidences or average age at diagnosis of various human tumors Children (less than 10 years of age) Ganglioneuroblastoma Neuroblastoma Wilm’s tumor Acute lymphoblastic leukemia (90% chromosomal) Retinoblastoma Rhabdomyosarcoma Ewing’s sarcoma or primitive neuroectodermal tumor (PNET) Juvenile astrocytoma Medalloblastoma Ependynoma Teratomas Hepatoblastoma Young age (10 to 45 years) Burkett lymphoma Seminoma - typical [30 –40]a Dysgerminoma [20 –40] Embryonal carcinoma testis [20– 30] Osteosarcoma [10 –20] Hodgkin Disease [32] Pulmonary carcinoid tumors [ < 40] Hepatocellular carcinoma in endemic areas of infectious hepatitis Papillary carcinoma of thyroid - female [40– 41] male [44 –45] Cervical [40– 45] Middle age (46 to 65 years) Melanoma [45– 55] Lung carcinoma NSb [50 –60] Gastric carcinoma - diffuse [48] intestinal [55] Multiple myeloma [50 –60] Liposarcoma [50 –65] Esophageal carcinomas - adenocarcinoma [50 –60] Leukemia Chronic myelogenous [45– 85] Chronic lymphocytic [60 – 65] Endometrial carcinoma [59– 60] Uterine tumors

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Stromal cell sarcoma [42– 53] Adenosarcoma [57 – 59] Leiomyosarcoma [40 – 60] Follicular carcinoma of thyroid - female [48] male [58] Old age (older than 65 years) Colorectum [>80]c Prostate [>70] Breast [>80] Acute myelogenous leukemia [>80] Bladder [>70] Renal cell carcinoma [60 –70] Pancreas - adenocarcinoma Oral squamous cell [50 –70] Larynges/laryngeal squamous cell [60 –70] Hepatocellular carcinoma in United States [>70] Ovarian carcinoma - all [79] a

[___] Average age at diagnosis. NS includes all types of carcinoma - small cell, squamous cell, adenocarcinoma, and large cell. c [ ] Age of peak incidence. b

Environmental and genetic factors that predispose to age-related tumor incidence The increase in cancers of old age, such as colorectal adenocarcinomas, may be secondary to many etiological factors. For example, colorectal cancers may occur at older ages because of a required length of time for which colorectal epithelial cells/stem cells must be exposed to a carcinogenic environment before cancer develops. The concept that tumors develop following long exposures to a carcinogenic environment is characteristic of lung carcinomas. Specifically, the risk of developing lung carcinoma is many times that of controls for smokers who smoke more than two packs of cigarettes per day for at least 10 years; however, the risk of developing lung carcinoma is reduced almost 60% 10 years after the cessation of smoking and more with longer, smoking-free periods. Thus, length of exposure to smoking, not age or immunity, primarily controls the development of most lung carcinomas [2– 6]. Similarly, 25% to 35% of all cancers are estimated to occur because of smoking [4,5], and as would be expected, the incidence of smoking-related cancers is beginning to decline as the decline in smoking is becoming long-term [6]. Another situation that shifts the occurrence of tumors to a pattern of occurrence at older ages is noted in patients with prostate cancer (PCA). In this pattern (see

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Fig. 2) precursor lesions, such as prostatic intraepithelial neoplasia (PIN), occur as a leading pattern; the invasive lesion follows 15 to 20 years after the precursor lesions appear. Thus, if the incidence of precursor lesions peaks at 50 to 60 years, the actual incidence of tumor will peak after 70 years. Such a pattern is not necessarily a feature of aging because the time to PCA development is not different in the older population than in the younger population (ie, the number of PCAs that develop seem to depend upon the number of predecessor cases of PIN).

Changes in tumor aggressiveness with older age The data do not lead to a generalization with respect to changes in tumor aggressiveness with older age. For example, the prognosis of women with breast cancer was reported to be better in the older, postmenopausal population. Breast cancers that occur in postmenopausal women may be biologically different from tumors that occur in premenopausal women; they are usually of lower grade and positive for estrogen and progesterone receptors and have a lower rate of proliferation [7]. Our study found that when pre- and postmenopausal breast tumors were compared in populations matched for tumor grades, there was no difference in clinical outcome between younger and older patients [7]. Actually, for many patients with adult cancers, especially those that have large subgroups with familial cancer, it is sometimes stated that the tumors that occur in older age patients usually have a better outcome. Our data indicate that this is the case for patients with colorectal cancers [8]. This pattern of clinical outcome was suggested to be the result, in part, of the familial cases of tumors that occur among younger patients and that are more aggressive. Familial adenomatous polyposis (FAP) and hereditary nonpolyposis colon cancer (HNPCC) occur at an earlier age. Whereas FAP tumors are frequently aggressive, HNPCC tumors are less aggressive per T subgroup of stage. Nevertheless, because multiple tumors may develop over a patient’s life with either of these syndromes, patients with the familial cancers usually have poorer outcomes than patients with sporadic cancers. Whereas prostate cancers that occur in younger patients were once thought to be more aggressive, our recent review of the literature suggests that there is no difference in the aggressiveness of prostate cancer based on age (unpublished results). Thus, the literature does not support that tumors that occur after age 65 are more aggressive than those that occur at a younger age.

Age and immunity in the development and progression of human neoplastic lesions An even more difficult concept to support is the belief that the incidence of cancer increases in humans with age secondary to a declining function of the immune system. The determination and separation of the effects of aging or immunity on the development and progression of neoplastic lesions in the human is very difficult to ascertain. Nevertheless, data are available on the relation of age

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on the development or progression or aggressiveness of specific types of neoplasia, as well as separate data on the effects of the immune system on tumors, including their aggressiveness. If a decline in the human immune system with age is responsible for an increase in the development of specific tumors with age, why are not the same tumors more aggressive in the older population in which the immune system would no longer control their progression? A second issue is that if a decline in the human immune system with age is responsible for an increase incidence in tumors, why do some tumors have a peak incidence before immune senescence? Immune senescence and tumor onset may be related for some tumors (breast, colorectal and prostatic adenocarcinomas) and for some subgroups of tumors; however, many types of tumors, including most lung carcinomas, occur before 60 years of age, an age which is not associated with a grossly deficient immune system. One explanation for these observations is that there are underlying mechanisms that coordinately regulate immune senescence and tumor incidence and aggressiveness. For example, the age-related decrease in cell cycle entry may occur in normal immune cells and tumor cells; therefore an aggressive tumor and an aggressive immune response to the tumor decline with aging. We propose that imbalances in this equation would lead to a better than average or worse than average outcome, depending on the immune response maintenance, and the type of genetic lesion that underlies tumor development.

Increased tumor incidence occurs in immune-suppressed patients The effect of the human immune system on tumors, in general, is more easily demonstrated than are the overall effects of age and the decline of immunity with age on tumor development and progression. Several types of evidence, some anecdotal, that are listed in Box 2 indicate that the human immune system modulates tumor development and progression. First, several subtypes of tumors with extensive lymphoid infiltrates have better clinical outcomes than similar subtypes of the same tumor without lymphoid infiltrates. Such tumor subtypes include medullary carcinoma of the breast (Fig. 3) [9 –12], medullary-type and lymphoepithelioma-like colorectal tumors [13 –15], and lesions like the melanoma with an irregular halo that is characterized by a dense inflammatory infiltrate and associated with spontaneous, partial or complete regression of melanomas [16 – 19]. In the case of the colorectum, the extent of lymphoid infiltrate of more standard colorectal tumors was used to characterize the aggressiveness of tumors and the clinical outcome as a prognostic marker [4]. It is not clear that medullarytype tumors are not different physiologically, independent of their inflammatory infiltrate. For example, lymphoepithelioma-like tumors of the colorectum have been associated with Epstein-Barr virus [20]. Wilson et al [21] reported that lung cancer cells were allografted with a kidney into a patient who subsequently developed foci of lung cancer; these nodules of lung cancer completely regressed following the removal of the transplanted kidney and cessation of immunosuppression, and the lung cancer did not recur

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Box 2. Evidence of immune effects on tumors in humans  



    

Lymphoid infiltrates may improve the clinical outcome of specific subtypes of tumors. Allografted tumors in humans grew and progressed under immunosuppression but regressed completely following withdrawal of immunosuppression. Tumor regression has been associated with bacterial infections of patients and hence with an intense immune reaction; immunotherapy with BCG is successful in some cases of tumors. Tumor control is sometimes induced by the development of graft versus host disease. Increased incidence of specific tumors occur in patients with AIDS or patients with induced immunosuppression. Spontaneous regression of preinvasive neoplasia, primary cancers, and metastatic cancers have been reported. Specific metastatic spread of a tumor that is dormant to local irradiated areas in the humans has been described. Successful treatment of subgroups of metastatic melanomas, renal cell carcinomas, and multiple other tumors with immunoregulatory molecules (eg, IL-2 and interferon-n).

when a new regimen of immunosuppression was instituted with a second transplant after 9 months. A similar case occurred at the University of Alabama at Birmingham; immunosuppression was proposed as a permissive factor in other cases where tumors were inoculated and grew following renal transplantation [22]. Tumors sometimes undergo spontaneous regression. In animal models, tumors have undergone spontaneous regressions in association with bacterial infections/ sepsis [23]; this led to successful immunotherapies with BCG [24]. Similarly, recurrent malignancies that were previously treated with bone marrow transplants regressed after the development of graft versus host disease. Also, with the development of AIDS or with the induction of immunosuppression (transplant medications), there is an increase in the occurrence of specific tumors, including sarcomas, skin carcinomas, and cervical intraepithelial neoplasia (CIN) in patients with various organ transplants [25] and lymphomas, and anal carcinomas in patients with AIDS [26]. Because the immune system seems to inhibit the growth and progression of many tumors, do chemotherapeutic agents that suppress CD8 + , CD4 + , and other lymphocytes involved in cytotoxic immunity increase tumor progression? Again, this is a difficult question to answer in humans; however, if the chemotherapeutic agent is effective against the tumor, the tumor may not recur until the CD8 + and CD4 + cellular cells have returned to normal. This may require up to 6 months in humans [27]. If an agent is ineffective against a tumor yet suppresses the cytotoxic component of the immune system, then treatment with that agent

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Fig. 3. (A) Ductal adenocarcinoma of the breast with rounded groups of cells that do not form glands (H&E 400). (B) Medullary carcinoma subtype with a characteristic intense lymphoid and plasma cell infiltrate (hematoxylin and eosin, 400).

may stimulate tumor growth and progression. This concept is supported by animal studies in which treatment with cytophosphamide or anti-CD4 + monoclonal antibodies caused IL-2 to have an increased action to inhibit tumor growth and progression [28]. This might be a good reason to develop better methods to ensure that a tumor is sensitive to an immunosuppressive agent before it is used in treatment of neoplastic processes.

Successful forms of immunotherapy that implicate the immune system in the control of cancer Several forms of therapy for cancer suggest that the immune system can modulate the progression of specific cancers. For example, interleukin-2 was

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Box 3. Factors that affect the immune reaction to tumors Tumor factors Loss of antigen expression by tumor cells (eg, secondary to genetic instability). Tumor-associated proteins may be weak antigens (eg, oncofetal antigens expressed during late embryogenesis). Deficient antigen presentation to immunoregulatory cells. The dominant epitopes of major antigens may be masked by polysaccharides. Anatomic isolation of the tumor or its antigens. Inactivation by the tumor microenvironment of the effect on cells of immunocytotoxicity secondary to production/expression of: Inhibitory growth factors/cytokines such as transforming growth factor (TGF)-o Inhibitory metabolites such as NO Suppressor T cells Killer cell immunoglobulin-like receptors (KIRs) recognizing self MHC-I antigens A neoplastic microenvironment that modulates tumor associated macrophages (TAMs) to inhibit the immune system. Immune response factors Impaired host immune suppression secondary to: Age Chemotherapy Immunosuppressive therapy including steroids Transfusions of blood, especially cellular products Inadequate processing of major tumor antigens (eg, mucin 1 (MUC-1) by dendritic cells, p185erbB-2) Impaired response of T cells because of decreased ‘‘z’’chains of clusters of differentiation (CD)-3 or decreased p561ck Inadequate expression of immunomodulatory molecules such as the costimulatory factors B7, lymphocyte function-associated antigen (LFA)-3, intercellular adhesive molecule (ICAM)-1, o2-microglobulin or the immunostimulatory molecules IFN-n, IL-2, IL-4, IL-6, TNF-n or granulocyte macrophage-colony stimulating factor (GM-CSF).

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demonstrated to be effective against metastatic melanomas (7% complete remissions and 9% partial remissions) and metastatic renal cell carcinoma (9% complete remissions and 10% partial remissions). Similarly, interferon-a (INF-a) is an effective agent against several malignant tumors, including superficial bladder cancer, hairy cell leukemia, newly diagnosed chronic myelogenous leukemia, and Kaposi’s sarcoma. For superficial bladder cancer, the creation of a nonspecific inflammatory effect following transurethral resection using an intravesical injection of bacille Calmette-Gue´rin reduces the recurrence rate in 50% of patients [29]. There is also evidence that immune control of tumors may be local, as well as systemic. For example, tumors that are apparently dormant may selectively metastasize to areas that have been irradiated. The above lines of evidence are not as definitive as one would like in demonstrating the importance of the immune system in the control of tumors; however, as can be seen from Box 2, the immune reaction to tumors based on human patterns of neoplasia and animal models is quite diverse. Thus, again we must rely on animal models to more clearly demonstrate effects of the immune system on tumors and tumor derived factors effects on immune system as listed in Box 3.

Study of aging, tumor susceptibility, and immunity in an animal model Rate of thymic evolution with age correlated with tumor susceptibility The factors that contribute to a functional decline in T cells with age that lead to increased tumor susceptibility may include: (1) the rate of thymic involution with age co-related with tumor susceptibility, (2) decreased activity of the cytotoxic T cell associated with the tumor susceptibility, and (3) attenuated activation pathways with age in immune cells (antigen stimulation by way of the T-cell receptor, costimulation, control of apoptosis). The thymus is the central lymphoid organ that provides a specialized microenvironment for maturation and selection for most T-lymphocytes. T-cell generation through this thymic-dependent pathway is essential because it establishes and helps to maintain the pool of peripheral T cells. This process also provides a repertoire of T-cell diversity that is necessary for potential immune responses to a vast number of antigens. With increasing age, the thymic lymphatic mass decreases, and thymocyte production correspondingly declines (thymic involution). There is a considerable body of evidence to suggest that the microenvironment of the thymus influences thymic involution and the function of the thymic T-cell emigrants with age. Bar-Dayan et al [30] reported that the thymus of Institute for Cancer Research (ICR) mice reaches its maximal size at the age of 1 month and thereafter, the size of the thymic cortex undergoes an exponential decline. In parallel with the decline in thymic size, the proliferative index of the peripheral cortex of 7-month-old mice was reduced significantly by

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45% compared with the 1-month-old mice. This study suggested that a reduction of the proliferative index of the thymic cortex in adult mice compared with young mice might account for the reduction of thymic cortical cellularity during thymic involution. Hameed et al [31] similarly observed that the proliferative capacity of thymocytes from C3H/HeJ mice decreased as the animals attained maturity; the decreased proliferative capacity of thymocytes in response to concanavalin A (Con A) stimulation observed between 4 and 24 weeks of age closely correlated to the drop in thymic weight and cortical cellularity that was observed during this period. Using a genetic approach, Hsu et al [32] analyzed the Con A-induced proliferative response of thymocytes in 22-month-old BXD RI strains of mice; they observed a positive correlation of the Con A-induced thymocyte proliferative response with the capability of thymocytes to mature to the CD4 + CD8 + stage. There are fewer studies on the contribution of aging factors to tumor susceptibility as the thymus involutes; however, our recent studies in recombinant inbred BXD mice demonstrated that the more rapidly the thymus involutes with age, the higher the susceptibility to the growth of transplanted breast cancer. Specifically, different BXD RI strains exhibited a markedly different host response to identical challenges with the TS/A breast cancer cell line. This is related to the rate of thymic involution because strains with late onset of thymic involution have a low susceptibility to growth and progression of TS/A cancers, whereas strains with rapid onset of thymic involution are highly susceptible to progression of the TS/A cancers. In these studies, BXD RI strains from each type of thymic involution were inoculated at 12 months of age with a histocompatible (H-2d) TS/A tumor. In each case, the BXD strain was of the H-2d haplotype. The tumor growth was graded after 30 days as follows: pattern 1, tumor cells failed to develop; pattern 2, implanted tumor cells initially grew but began a slow decline in size so that by 4 weeks of age no tumor was detected; pattern 3, implanted tumors grew slowly and attained a large size at day 50; and pattern 4, implanted tumors grew rapidly for 2 weeks with metastases to lymph nodes and pancreas. Strains BXD11, 28, 32, and D2 were the strains of BXD mice that were characterized as having an initially small thymus, as well as an early onset of thymic involution; all these strains had patterns 3 or 4 of tumor development by 30 days (Fig. 4). Similarly, BXD strains 24 and 31, which exhibited a thymus phenotype characteristic of an initially large thymus and an early onset of thymic involution also exhibited pattern 4 tumor growth after 30 days. In contrast, BXD strains characterized as having an initially small thymus and late onset involution, (eg, BXD9) exhibited pattern 2 tumor growth and completely rejected the tumor by 4 weeks of age. BXD1, BXD27, and parental B6 (H-2d) mice with initially large thymuses and with late onset thymic involution, exhibited either no tumor growth (pattern 1) or pattern 2 growth. In contrast, all mice with early onset thymic involution developed pattern 3 tumors that resulted in sacrifice of the mice or pattern 4 tumors that killed the mice [33]. These results indicated that late-onset thymic involution is correlated with the susceptibility of trans-

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Fig. 4. Syngeneic murine breast cancer cells TS/A were injected subcutaneously into 14 BXD RI strains and the parental strains, DBA/2 and B6 (H-2d) mice (1.2  105 cells/flank at three sites in 50 ml of phosphate buffer saline (PBS) for each site, 5 mice per BXD RI strain). Tumor volumes were measured every 5 days. Each point represents the mean volume ± SD of the three tumors of the five mice from each group. (From Grizzle et al. BXD recombinant inbred mice represent a novel T cell-mediated immune response tumor model. Int J Cancer 2002;101:270 – 9. D 2002, John Wiley & Sons.)

planted tumors to develop and progress. This novel finding opens up a new avenue for re-evaluation of the role of the thymus in tumor susceptibility.

Tumor susceptibility and T-cell function in the BXD RI animal model Several mechanisms have been proposed for the induction of tumor-specific, T-cell immunity. In one model, tumor-specific T-cell immunity is induced as a result of direct priming of naive CD8 + T cells by tumor cells that have migrated

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from the site of the tumor to lymphoid tissues/aggregates. Alternatively, tumorspecific, T-cell responses may result from cross-presentation of tumor cellderived antigens (Ags) by professional antigen presenting cells (APCs), such as bone marrow-derived dendritic cells. Professional APCs not only present Ags derived from endogenously-produced Ags in the context of MHC class I molecules, but also have the capacity to present Ags ingested from exogenous sources. This alternative pathway for priming of CD8 + T cells (also referred to as cross-priming) could play a role in the induction of tumor-specific, T-cell responses, either by capture of tumor Ags in the periphery or by representation of Ags from tumor cells that have migrated to the lymph node or proteins from tumor cells that have drained by way of lymphatics into lymph nodes. As discussed earlier, cytotoxic T lymphocytes (CTLs) contain cytoplasmic granules that contain perforin (perforating protein) and associated granule proteases (granzymes) that lead to the equilibration of ionic gradients, osmotic lysis, and nuclear disintegration. The formation of membrane pores by perforin also is a prerequisite for apoptosis of target cells. Granzymes, which also are located in the secretory granules of cytotoxic lymphocytes, gain entry into the target cell through perforin pores and mediate target cell DNA degradation through the activation of caspase 3, which is the protease responsible for cleavage of poly (adenine diphosphate ribose) polymerase. The pathophysiologic significance of perforin-mediated cytotoxicity was confirmed in experiments with perforin-deficient animals, which demonstrated its role in protection against tumors [34,35]. The age-related decline in the functional activity of CTL has been recognized for many years. In mice, the deterioration at the molecular level (perforin and granzymes) in the lytic mechanisms may be responsible, in part, for this decline in CTL activity. A consistent decrease in the number of perforinpositive granules, their size, and intensity of staining was reported in aged mice. Furthermore, when stimulated in mixed lymphocyte culture, the cells from young mice showed much higher lytic activity than those from aged mice [34,35]. Tumors may escape control by the immune system by a decline in CTL function with age that leads to decreased tumor susceptibility, bypass of immune surveillance secondary to accumulation of mutated cells in the tumor with aging, by both, or by other mechanisms. We have used recombinant inbred BXD mice to dissect out the above possibilities [33]. H-2d recombinant inbred (RI) strains of BXD mice were injected with syngeneic breast cancer cells (TS/A), and the growth of the tumor was monitored over time. There was a dramatic difference in the growth of the implanted breast cancer cells among the 14 BXD RI strains. As discussed earlier there were four patterns of tumor development (see Fig. 4). To determine if the T-cell immune responses affected tumor development, the infiltrated T cells in the tumor and the tumor-specific CTL were analyzed. The immunohistochemistry indicated that many of the CD3 + T cells infiltrated into the tumor; the extent of the infiltration by CD3 + cells was associated with the ability to eliminate the implanted tumor

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Fig. 5. Growth of the implanted breast cancer cells is associated with T-cell infiltration in the tumor sites. Twelve-month-old BXD RI mice were implanted with TS/A tumor cells. At the indicated time points, the tumors were fixed in formalin and sections of the paraffin-embedded specimens were examined for T-cell infiltration based on staining with anti-CD3 and H&E. (From Grizzle et al. BXD recombinant inbred mice represent a novel T cell-mediated immune response tumor model. Int J Cancer 2002;101:270 – 9. D 2002, John Wiley & Sons.)

cells. Furthermore, CTLs of the lymph nodes also were co-related with eliminated tumor development in type 3 and 4 pattern mice (Fig. 5).

Disrupted activation pathways in T cells with age (stimulation by way of the T-cell receptor for antigen, costimulation, apoptosis control) The increased tumor growth in elderly mice seemed to depend also on the increased occurrence of T lymphocytes that were devoid of the co-stimulatory molecule CD28 [36 – 41]. The cells that are devoid of CD28 are unable to expand clonally. CD8 T cells contain a distinct subset of CD8 + CD28 cells. Stimulation of a T cell by way of the T-cell antigen receptor (TCR) and CD28 results in T-cell responses such as proliferation, IL-2 secretion, and activation of cytolytic effector function from memory precursor cytotoxic T lymphocytes (pCTL). By contrast, stimulation of the TCR without CD28 stimulation, for instance by antigenpresenting cells that lack CD80/86, can lead to anergy [42]. The loss of CD28 expression because of aging is more prominent in CD8 + than CD4 + T cells. Recent studies demonstrate that such modulation or loss of CD28 expression on

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T cells are attributed to two regulatory sequences, sites a and b, in the gene promoter [43]. Changes in the levels of cell surface expression of CD28 are associated with the coordinate modulation of the protein-binding activities of sites a and b. Although the binding activities of a and b are independent of each other, they constitute a functionally singular transcriptional initiator (INR) element. Among CD28null T cells, this ab-INR is inoperative because of the lack of a- and b-binding complexes. This might be the cause of the higher decline in the CD28 expression in CD8 + than in CD4 + T cells [43]. The increase in the presence of CD8 + CD28 T cells leads to a scenario where less CD8 + T cells are available for encountering future antigenic challenges, such as tumor immune surveillance. CD4 + CD28 T cells can react with autoantigens, undergo clonal expansion, and produce high concentrations of IL-2 and IFN-g. A concomitant loss of CD40 expression also has been observed which in turn can make CD4 + T cells incapable of promoting B-cell differentiation and immunoglobulin secretion. Hence, the age-related accumulation of CD4 + CD28 cells can direct the immune response toward reacting with autoantigens and away from generating B-cell responses against foreign antigens; thus, age-related accumulation of CD8 + CD28 cells lead to attenuated host immune surveillance and increased tumor development. Although CD28 is constitutively expressed on T cells, the levels of its expression on the cell surface are constantly modulated. In humans, CD28 is downregulated following activation, and levels of expression decline progressively with frequencies of CD-28 negative cells of up to 50% of the total CD4 compartment found among some individuals older than 65 years. It is not clear whether all CD8 + CD28 cells are cytotoxic effectors, whether they are functionally homogeneous or if they include a group of distinct maturational stages in differentiation of CTLs. As cytotoxic effector CD8 + cells mature they may lose CD28 expression. Although they may acquire intracellular perforin, these CD8 + CD28 T cells become anergic and relatively resistant to apoptosis and become auto-reactive T cells with age. As a result, they cannot be activated and will not kill mutated cells; this may lead to development of tumors.

Transcription factors in immunosenescence T cells In mice and humans, aging leads to a decline in the ability to mount strong T-cell responses to new antigens and to previously encountered recall antigens. The research at several laboratories has shown that T cells from healthy, old mice exhibit multiple defects early in signal transduction cascades. These include declines in tyrosine-specific protein phosphorylation, including phosphorylation of the TCR-associated CD3z chain, calcium signal generation, and phosphorylation of multiple substrates in responses initiated by the protein kinase C (PKC) activator phorbol myristoyl acetate (PMA) and by the calcium ionophore ionomycin. Also, these were decreased in Shc-tyrosine phosphorylation, and activation of the extracellular signal-related kinase, Ras-associated factor (RAF)

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and c-Jun N-terminal kinase/stress-activated protein kinase pathway in response to stimulation by polyclonal stimuli. The age-dependent loss of T-cell functions also may depend on the defects in the generation of second messengers following the receptor activation in the T cells. The initial event of the signal transduction following TCR activation involves the activation of early genes such as c-fos and c-myc, the products of which are of great importance in the differentiation of T cells and in their effector functions. The transcription factors are molecules of crucial importance in the mechanisms of transmission of extracellular signals from the cytoplasm to the cell nucleus. Recent studies revealed a decrease in the availability of transcription factors as a result of the age-dependent decline of the signals of cell proliferation. The activation of T cells depends on the transcription factors, such as the activator protein-1 (AP-1), nuclear factor kappa-B (NF-kB), and the nuclear factor of activated T cells (NFAT). These proteins play a decisive role in the transcription of certain genes that are involved in the induction of the immune response. The NF-kB regulates the expression of a great number of cytokines, like IL-1, IL-2, IL-6, IL-8, IFN-b, TNF-a, the granulocyte-macrophage colony stimulating factor, and the molecules of adhesion (e.g., ICAM). The induction of NF-kB was decreased in the activated T cells of elderly subjects. This latter fact did not seem to depend on the type of stimulus, the altered composition of the p5O – p65 subunits, or the NF-kB levels that were found in the cytosol of inactivated T cells, but it may be attributed to an altered regulation of the inhibitory subunit I-kB or to a decreased proteasomal activity. Studies on the other transcription factors in immune cells demonstrated a decrease of the NFAT in activated T cells of the elderly. NFAT is a specific activator of transcription of the antigen receptors and is responsible for the specific inducibility of T cells by IL-2. The binding sites of NFAT have been observed in the regulatory regions of the genes of the cytokines IL-3, IL-4, GM-CSF, and TNF-a. The induction of AP-1 also is defective after the activation of T cells. Therefore, it seems to be evident that the immunosenescence involves also the transcription factors (ie, even the latter may contribute to the functional changes of the cells in the elderly). Multiple transcriptional defects could lead to T-cell replicative senescence. How T-cell replicative senescence is co-related to decreased tumor susceptibility is still elusive, and needs to be further studied. In particular, a T-cell dependent animal model that develops tumors with age needs to be developed. Similarly, our observations on how the growth of transplanted breast cancers are modulated in DBA/2 strains of mice need to be confirmed with respect to tumors developing from B6 animals and with respect to the development of tumors in D and B strains.

The role of tumor-associated macrophages Tumor-associated macrophages that develop from monocytes that are attracted into tumors by chemotactic agents such as macrophage colony-stimulating factor

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receptor (M-CSF), TGF-b, and monocyte chemoattractant protein (MCP)-1, play an important role in the body’s immune reaction to tumors [44]. In contrast to lymphocytes, which in general aid in minimizing tumor growth and progression, TAMs actually may promote growth of some tumors by way of induction of angiogenesis, formation of stroma, and expression of growth factors; however, the progression of tumors that are susceptible to antitumor effects of TAMs may be inhibited by the cytotoxic action of TAMs. In general, without direct action of the tumor on TAMs, TAMs will inhibit tumor development and progression. If the tumor produces a pattern of products that affect TAMs in a specific manner, then the balance of the action of TAMs may be to inhibit the action of the immune system and to shift the expression of TAM cytokines so that the tumor is stimulated to progress. This pattern is demonstrated in Fig. 6 where the larger arrows indicate the predominant pathway. In conclusion, the study of aging, susceptibility of tumor, and immunity is at an initial stage, and numerous excellent questions challenge immunologists and tumor biologists. Because unpredicted factors contribute to tumor susceptibility in the human population, we feel that the development of an animal model is essential to dissect out these complicated issues.

Fig. 6. Cytotoxic lymph node T-cell response at day 30 after injection of the TS/A cell line. The lymph node T cells were isolated from all TS/A tumor cell-challenged BXD strains and stimulated for 5 days at 370C, then co-cultured with 51Cr labeled target cell TS/A at different ratios (E:T = 10:1, 50:1, and 100:1) for 6 hours. Specific release was calculated and results are expressed as means ± standard deviations for at least five animals. E, effector; T, target. (From Grizzle et al. BXD recombinant inbred mice represent a novel T cell-mediated immune response tumor model. Int J Cancer 2002;101:270 – 9. D 2002, John Wiley & Sons.)

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