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The adaptive immune response tosporadic cancer

Summary: Most of the current experimental cancer models do not reflectthe pathophysiology of real-life cancer. Cancer usually occurs sporadicallyand is clonal in origin. Between tumor initiation and progression,clinically unapparent pre-malignant cells may persist for years or decadesin humans. Recently, mouse models of sporadic cancer have beendeveloped. The mouse germ-line can be engineered with high precisionso that defined genes can be switched on and off in the adult organism in atargeted manner. Analysis of the immune response against sporadic tumorsrequires the knowledge of a tumor antigen. Ideally, a silent oncogene, forwhich the mice are not tolerant, is stochastically activated in individualcells. This approach offers the opportunity to analyze the adaptive immuneresponse throughout the long process of malignant transformation andmost closely resembles cancer in humans. In such a model with the highlyimmunogenic SV40 large Tantigen as a dormant oncogene, we discoveredthat sporadic cancer is recognized by the adaptive immune system at thepre-malignant stage, concomitant with the induction of tumor antigen-specific tolerance. These results demonstrated that even highly immuno-genic sporadic tumors are unable to induce functional cytotoxic Tlymphocytes. Based on this model, we conclude that immunosurveillanceplays little or no role against sporadic cancer and that tumors must notescape immune recognition or destruction.

Keywords: sporadic cancer, immunosurveillance, tolerance, rejection antigen, cytotoxicT lymphocytes, antibodies

Introduction

Spontaneous tumor-induced immune responses have been

analyzed for decades. As cancer is a heterogeneous disease and

experimental models are different, virtually all possibilities of

tumor–immune cell interactions have been demonstrated. It

has been postulated that immune cells recognize and eliminate

tumor cells, whereas other models suggest that tumor cells

recruit immune cells that they require for growth. While the

former has been firmly established for a variety of virus-

induced tumors in mice and humans (1, 2), the latter was first

observed by Virchow almost 150 years ago and has been

confirmed countless times in experimental carcinoma models,

commonly referred to as inflammation-induced cancer (3–6).

Because the immune response to virus-induced tumors is

fundamentally different from that against spontaneous tumors

(1), it is not reviewed here.

Gerald Willimsky

Thomas Blankenstein

Immunological Reviews 2007

Vol. 220: 102–112

Printed in Singapore. All rights reserved

r 2007 The Authors

Journal compilation r 2007 Blackwell Munksgaard

Immunological Reviews0105-2896

Authors’ addresses

Gerald Willimsky1, Thomas Blankenstein1,2

1Institute of Immunology, Charite Campus Benjamin

Franklin, Berlin, Germany.2Max-Delbruck-Center for Molecular Medicine, Berlin,

Germany.

Correspondence to:

Thomas Blankenstein

Max-Delbruck-Center for Molecular Medicine,

Robert-Rossle-Str. 10, 13092 Berlin, Germany

Tel.: 1149 30 9406 2816

Fax: 1149 30 9406 2453

e-mail: [email protected]

Acknowledgements

The work described here was supported by grants from the

Deutsche Forschungsgemeinschaft (SFB633 and TR36)

and the European Community (FP6 program ‘ATTACK’).

102

Most experiments with non-viral tumors have been

performed by tumor transplantation. Through these studies,

important discoveries have been made. (i) Tumor cells differ

greatly in inherent immunogenicity (7, 8). (ii) If transplanted

tumor cells are rejected, it requires T cells, CD81 cytotoxic

and/or CD41 helper T cells. In most cases, tumor transplant

rejection requires antigen cross-presentation by activated

dendritic cells for T-cell activation and interferon-g (IFN-g)

and perforin as effector molecules (9, 10). (iii) Tumor

transplantation rejection antigens are tumor specific, the result

of somatic mutation, and are usually not cross-protective

between different tumor lines (11). (iv) Under

immunological pressure, for example in response to effective

immunotherapy, tumor cells can escape immune recognition

or destruction by various mechanisms (12). (v) Under

rigorous experimental conditions, e.g. with established

tumors, immunotherapy approaches can be informative and

form a solid basis for clinical studies (13). (vi) Last but not

least, mouse tumor transplantation studies have led to the

discovery of the major histocompatibility locus, H-2 in the

mouse, and the development of inbred strains.

There are many more important discoveries that resulted

from tumor transplantation studies. However, the spontaneous

immune response against transplanted tumor cells, usually

injected as a single-cell suspension, is highly artificial.

Transplanted tumors may be more accessible for infiltrating

immune cells. Their growth is often analyzed at a site different

from its origin, and it is difficult to exclude phenotypic

changes occurring during in vitro cell culture. Transplanted

tumor cells usually grow very fast, because they have acquired

malignancy in their primary host. Successful tumor

transplantation requires a large number of cells to be injected.

Many cells rapidly die after injection, which can lead to

artificial T-cell recognition, and, in general, the host is

exposed to a large number of tumor cells at a single time-

point that does not reflect cancer development as it occurs in

humans. Importantly, the immune response to primary tumors

cannot be deduced from tumor transplantation experiments,

because tumors that progressively grew in an immune-

competent primary host were promptly rejected following

transplantation to a naive immune-competent recipient (14).

Various non-transgenic primary tumor models have been

used that better resemble typical tumor development. Most

often, chemical or physical carcinogens have been used to

induce autochthonous tumors. Examples are methyl-

cholanthrene (MCA) (15), a combination of DMBA (7,12-

dimethylbenzanthracene) and TPA (12-O-tetradecanoylphorbol

-13-acetate) (16, 17), or ultraviolet (UV) irradiation (7).

Although these carcinogens often contribute to cancer in

humans and therefore represent important experimental

models, the problem is that the potential rejection antigens

(18) are not known and are unique for each individual tumor

(11). Thus, the tumor-specific T-cell response cannot be

followed in the primary tumor-bearing host. This circumstance

makes it difficult, if not impossible, to judge the influence of

antigen-specific T cells on primary tumor development.

Recombination-activating gene-1 (Rag-1) knockout mice that

have no T cells, natural killer T (NKT) cells, and B cells do not

develop MCA-induced tumors significantly more frequently or

with shorter latency compared with control littermates (19).

Rag-2 knockout mice that have an identical phenotype as Rag-1

knockout mice also do not spontaneously develop tumors more

frequently compared with wildtype control mice (20). This

finding is not surprising, because nude mice that lack the

thymus and all thymus-dependent T cells also do not develop

MCA-induced or spontaneous tumors more frequently than

control mice (21, 22). Some investigators obtained different

results in the immune-deficient mice. Possible explanations for

the contradictory results have been reviewed elsewhere (23, 24).

In any case, tumor models that do not allow the analysis of

tumor-specific T-cell responses in the primary tumor-bearing

host have been widely misinterpreted with regard to a

spontaneous protective anti-tumor T-cell response (24).

Sporadic cancer models

Cancer in most cases occurs sporadically, is clonal in origin,

and arises through sequential accumulation of somatic muta-

tions and/or epigenetic changes in genes, whose gain or loss of

function is associated with malignant transformation. It is

thought that in a Darwinian selection process, continuously

more malignant clones emerge. It is the combined and

occasionally synergistic action between activated oncogenes

and inactivated tumor suppressor genes that cause malignancy.

This knowledge and techniques to modulate the mouse germ-

line with high precision and at the single-gene level allow

the construction of cancer models that better mimic the

pathophysiology of real-life cancer. Several excellent reviews

on the development of mouse cancer models toward sporadic

cancer have been published (25–27). Only developments as

they are relevant for tumor immunology are briefly recapitu-

lated here.

In first-generation mouse models, transgenic mice were

generated, in which an oncogene (e.g. SV40 Tag, Myc)

was expressed in a cell type-specific fashion. In these

transgenic mice, the oncogene was expressed constitutively in

Immunological Reviews 220/2007 103

Willimsky & Blankenstein � The immune response to sporadic cancer

a specific tissue (28–32). Alternatively, with the possibility of

gene-targeting by homologous recombination, tumor

suppressor genes have been inactivated in the germ-line by

use of genetically modified embryonic stem cells. By Cre–LoxP-

mediated recombination, these genes can be inactivated and

tumors can be induced in a cell type-specific fashion, e.g. by

application of Cre recombinase using adenoviruses (33), but

typically by crosses to a second transgenic mouse that carries

the recombinase gene (34–36). For immunological analysis,

these models bear several disadvantages. Large numbers of

cells, almost a whole organ, are simultaneously transformed,

resulting in an untypical short tumor latency period and fast

non-clonal tumor growth (Fig. 1). The polyclonality probably

alters the microenvironment that is known to be able to either

stimulate or inhibit tumor growth. Furthermore, transgenic

oncogenes expressed by tissue-specific promoters are self-

antigens, so that it is very difficult to exclude tolerance, even

though occasionally such mice have retained immune

competence for the oncogene (37–40). Tumor-specific

antigens that likely occur during multi-step carcinogenesis in

oncogene-transgenic or tumor suppressor gene-deficient mice

are not known.

To avoid tolerance, the oncogene can be activated in a cell

type-specific manner in the adult animal by temporally

controlled transgene expression (41). In this case, the

oncogene is regulated by a minimally active promoter that

includes a tetracycline-responsive element (TRE). Gene

expression is obtained by a second transgene, which encodes

a transactivator under the control of a cell type-specific

promoter, usually by intercrossing the respective single-

transgenic mice. Feeding double-transgenic mice with

doxycycline, a derivative of tetracycline, will then induce or

repress transgene expression, depending on the use of Tet-on

or Tet-off systems (42–44). In addition to controlling

oncogene expression in a time-dependent manner, these

models also allow examination of whether malignancy

requires persistent oncogene expression (oncogene

addiction) (42–45) or whether the initiating oncogene

becomes dispensable with progressive malignancy. Another

strategy to regulate gene expression conditionally is the

use of fusion proteins with the estrogen receptor hormone-

binding domain. The estrogen receptor is engineered in

such a way that exogenous administration of the synthetic

estrogen antagonist tamoxifen results in the transgene

product relocating into the nucleus. This approach has

been used successfully by directly fusing the c-Myc oncogene

to the estrogen receptor (46). Additionally, it can be used

to regulate the recombination activity of the recombinases

Cre and FLP.

Oncogene expression can also be induced by controlled

activation using a Cre- (or FLP-) mediated recombination

strategy. In these mice, an attenuator separates the promoter

from the oncogene. The attenuator can be any gene whose

expression prevents expression of the 30-located oncogene and,

therefore, serves as a stop-cassette. It is flanked by LoxP

recognition sites for Cre site-specific recombinase. The

oncogene can be activated by Cre recombinase-mediated

deletion of the stop-cassette. A large number of Cre

recombinase gene transgenic mice are available that express the

recombinase under different promoters. In mice double

transgenic for the conditional oncogene and recombinase

genes, the oncogene can be expressed in a tissue-specific

fashion, which likely will result in tolerance for the oncogene.

Alternatively, Cre recombinase expression may be achieved

by transcriptional activation (tetracycline, IFN, polyinosinic

–polycytidylic acid) or post-translational activation (4-hydro-

xytamoxifen, mifepristone) in the adult organism. The inducible

expression/activation models better mimic de novo expression of

the oncogene comparable to somatic mutations that typically

activate oncogenes (47). In principle, the mice should not have

developed tolerance for the oncogene at the time of Cre

recombinase expression and oncogene activation. However,

Fig. 1. Growth kinetics of sporadic versus non-sporadic cancer.A schematic drawing is shown of the growth kinetics of transplantedtumor cells, primary tumors in mice, in which an oncogene is expressedin a cell type-specific fashion (whole organ tumor), and primary tumorsin mice, in which an oncogene is activated in individual cells (sporadictumor). Sporadic tumors, as observed in LoxP–Tag mice, are characterizedby pre-malignant lesions at around 6–9 months of age and a variable butusually very long latency period, until tumors progress. The tissue-specific expression of SV40 T antigen in LoxP–Tag�Alb–Cre mice resultsin transformation of a large number of cells and tumor progression aftera short latency period. As sporadic tumors are induced in a stochasticfashion, tumor initiation and progression can be variable from mouse tomouse. Therefore, each red line represents an individual LoxP–Tagmouse.

104 Immunological Reviews 220/2007


tumor development is not sporadic, because the oncogene is

activated in many somatic cells.

It is difficult to assess how tightly Cre recombinase

expression can be controlled or whether attenuated oncogene

expression can be prevented. Transgenic models with

temporally controlled and reversible systems could be leaky.

The leakiness could be due to spontaneous (tamoxifen-

independent) Cre recombinase expression in double-

transgenic mice (authors’ unpublished observations),

oncogene expression in the non-induced situation (e.g. in the

absence of tetracycline), or incomplete function of the stop-

cassette. Potentially, leakiness may lead to partial tolerance for

the oncogene before tumor development starts. If leakiness of

conditional Cre recombinase expression is a rare phenomenon

or attenuated oncogene expression is low, immune

competence for the oncogene might be comparable to that of

non-transgenic mice. Importantly, in this situation leakiness is

stochastic and occurs in single cells, which faithfully mimics

sporadic tumor development. A comparable situation is

obtained by the ‘hit and run’ strategy, the random activation

of an oncogene in individual cells by spontaneous

chromosomal recombination (48, 49). This approach has

been pioneered with a mutant yet incomplete Ras oncogene

that is activated by homologous recombination with the

wildtype allele. The above models of stochastic oncogene

activation have a great advantage, in that no additional

manipulation of the transgenic mice is required. The

disadvantage is that the random oncogene activation is

difficult to predict and that tumor development and latency

period may be variable from mouse to mouse, even in an

inbred strain.

As an alternative to accidental oncogene activation, models

of Cre recombinase-mediated stop-cassette deletion offer the

possibility for administering the Cre recombinase exogenously.

This model uses Cre recombinase gene encoding vectors such

as adenoviruses (14, 50–53) or retroviruses (54). Careful

vector titration offers the possibility to hit only a few cells for

tumor onset, which resembles sporadic cancer. However, the

vectors are immunogenic, so that an immune response is

induced against the virus-infected cells (50). Therefore, viral

vector-induced oncogene activation does not allow for the

analysis of the immune response against spontaneous tumors

but is potentially an interesting model of analyzing the

immune response against virus-induced tumors. The

limitation is the tropism of many viral vectors. Adenoviruses

that have most often been used for Cre recombinase gene

delivery have a high tropism for the liver. The route of

administration can compensate at least partially for the

tropism. Intranasal application of Cre recombinase-encoding

adenoviruses has been shown to specifically induce

recombination in the lung epithelium (51–53). Recombinant

Cre recombinase protein has also been used for LoxP-mediated

recombination, because it can be taken up by cells in a

biologically active form (55, 56). The efficacy and cellular

tropism of Cre protein in vivo is currently unknown. Local gene

activation in the adult mouse is still difficult for many organs.

In a model of tamoxifen-inducible Cre recombinase activation,

the magical touch mouse, it has been shown that topical

application of tamoxifen onto the skin induced local gene

activation due to LoxP-mediated recombination (57).

In a model that most clearly should reveal the ability of the

immune system to recognize and eliminate spontaneous

tumors, immune-competent mice should be prone to tumor

development with high incidence without experimental

interference, and these tumors, ideally one per mouse, should

originate from single cells that express a tumor-specific

transplantation rejection antigen already in the earliest phase

of malignant transformation. This outcome is the case, if the

transforming oncogene is present at the same time as

the tumor-specific transplantation rejection antigen. If the

oncogene is silent and only in rare cases spontaneously

activated due to genetic or epigenetic mechanisms, the mice

should not have developed tolerance to the oncogene. We have

established a transgenic mouse model, termed LoxP–Tag, that

essentially fulfills these criteria (14). The transgene consists of a

ubiquitously active promoter (chimeric chicken b-actin-b-

globin) and the gene encoding the SV40 small and large T

antigen (Tag). The promoter and Tag are separated by the gene

for chloramphenicol acetyl transferase (CAT) that acts as a stop-

cassette by transcriptional termination. The CAT gene is flanked

by LoxP recognition sites for the Cre recombinase. Surprisingly,

the mice spontaneously develop sporadic cancer without any

experimental manipulation. Tumors develop from single cells

that express Tag as the initiating oncogene and as the tumor

antigen. Tumor progression occurs after a variable but usually

very long latency period, compatible with the multistage

process of malignancy (Fig. 1). The tumor growth kinetics of

these sporadic tumors is fundamentally different from those

tumors that result from oncogene expression in the whole

organ. While in the latter case tumors grow with no or very

short latency, the sporadic tumors in LoxP–Tag mice can be

detected at the microscopic level at around 6–9 months, by

immunohistology with anti-Tag antibodies. These tumors

remain unapparent in most mice for a very long time, up to

2.5 years of age, at which time they progress and then usually

grow very quickly. This general growth kinetics is comparable



to that observed often in humans. Following the tumor-

initiating carcinogen exposure, for example the UV light-

induced sunburn of the skin or tobacco smoke in the lung, a

pre-malignant clinically unapparent state can last for years or

decades. The tumors in LoxP–Tag mice occur at different sites;

renal cell carcinomas and osteosarcomas are relatively frequent.

The tumor-specific immune response can be followed

throughout this long process of tumor development. Analysis

of tumors by reverse-transcriptase polymerase chain

reaction (RT-PCR) revealed at least two mechanisms associated

with Tag expression. In some tumors, Tag mRNA was

detected on differentially spliced bicistronic messages as a

result of a read-through mechanism. In others, spontaneous

(Cre/LoxP-independent) deletion of the attenuator including

the small-T region was detected, probably placing Tag in

closer proximity to the promoter. Tumor cells with bicistronic

mRNA expressed CAT, the attenuator gene, whereas tumors

with spontaneous attenuator deletion did not express the

CAT gene.

The B-cell response to sporadic cancer

It has been known for a long time that antibodies directed

against tumor cell-associated antigens occur frequently in

cancer patients (58). By expression cloning from cDNA

libraries of patients’ tumor cells screened with serum anti-

bodies of the cancer patients, almost 2000 antigens were

identified (59, 60). Nearly all of the recognized antigens are

self-antigens. Somatically mutated antigens were identified

rarely, e.g. p53 containing a point mutation leading to a single

amino acid exchange was identified in one case (61). Even

though tumor cell-derived self-antigens are often classified as

tumor antigens, self-antigens are usually poor tumor trans-

plantation rejection antigens. Most of the antigens appear to

have intracellular localization, indicating that the self-reactive B

cells have no access to the antigens, except during cell death

and release of the self-antigens. Therefore, it does not seem to

be necessary to delete such self-reactive B cells clonally. This

assumption is compatible with the finding that the tumor-

reactive antibodies occur in cancer patients with progressive

disease without indication of tumoricidal activity. In some

studies, the antibodies were associated with a poor prognosis,

e.g. in patients with colorectal cancer (62). However, this

correlation may simply reflect tumor burden, because the

presence of tumor-reactive antibodies has been correlated with

tumor burden (63). The tumor-derived self-antigens were

identified by immunoglobulin G (IgG) antibodies in cancer

patients’ sera. Because T-cell help is usually necessary for Ig

class switch, it is often assumed that the tumors in humans

induce functional T cells.

In mouse tumor models, B cells or their products inhibited

tumor rejection or supported tumor development (64–66).

Different mechanisms have been suggested to be responsible.

Qin et al. (65) demonstrated in a tumor transplantation model that

B cells inhibited T-cell activation in the priming phase, perhaps by

competing with other antigen-presenting cells, such as dendritic

cells, for the tumor antigens. An interesting mechanism for how B

cells promote tumor growth was suggested by de Visser et al. (66).

They detected antibody deposits within the tumor lesion in a

transgenic model of inflammatory skin cancer with the human

papilloma virus 16 oncogene. These antibodies induced the

infiltration of innate immune cells, probably neutrophilic

granulocytes, that were suggested to be responsible for the

tumor-promoting inflammatory response (66). In another

model of inflammation-induced skin tumors, antibodies against

mutant oncogenic Ras correlated with tumor burden (67).

Spiotto et al. (68) induced the expression of a tumor-specific

antigen [green fluorescence protein (GFP)] by Cre–LoxP-

mediated recombination in established tumors. Within 3 weeks,

large amounts of anti-GFP IgG1 antibodies were detected in the

sera of the tumor-bearing mice. Pre-existing CD41 T-cell

responses to other tumor antigens likely potentiated the anti-GFP

IgG antibody response, because no anti-GFP CD41 T-cell response

was detected (68). Thus, the detection of anti-IgG antibodies

against tumor-derived antigens does not necessarily indicate a

functional CD41 T-cell response against these antigens. In another

tumor transplantation model, Preiss et al. (69) showed that

immunization of mice with a mammary adenocarcinoma cell

line induced antibodies to similar self-antigens, as they were also

detected in cancer patients. Eight tumor-associated antigens were

cloned by serum IgG antibodies that were induced by tumor cell

immunization. None of the antigens served as a rejection antigen,

even though all induced IgG antibodies. However, for one of the

identified antigens, vimentin, it was shown that immunization

with plasmid DNA encoding vimentin induced tumor immunity

in vimentin gene-deficient but not vimentin gene-competent

mice. These data demonstrated that IgG antibodies do not

indicate tumor immunity. Remarkably, tissue damage that was

induced by injection of adenoviruses induced antibodies against

the same antigens that were also induced by tumor cell

immunization (69). These data indicated that the humoral

response against tumors is primarily the result of cell death and

is not a specific anti-tumor response. However, tumor-specific

antibodies have also been described (70). If directed against cell

surface receptors, they can inhibit tumor growth, if induced by

vaccination (70, 71).



In transgenic mice expressing Tag under the control of the

insulin gene regulatory region, antibody responses to Tag have

been detected in some but not other lines, correlating with the

time point of oncogene expression (37). Anti-Tag antibodies

were detected in mice in which Tag expression was not

detected before 6 weeks after birth and, therefore, were not

tolerant for Tag. Sporadic tumors in LoxP–Tag mice induced

anti-Tag antibodies with a high reproducibility, despite the

stochastic nature of the model (14). Obviously, the mice were

not tolerant for Tag. When the LoxP–Tag mice were crossed to

Alb–Cre transgenic mice that express the Cre recombinase by

the albumin promoter, the stop cassette that inhibited Tag

expression was deleted early in the life of the mice. These

double-transgenic mice were completely tolerant for Tag, and

the liver carcinomas that occurred at a relatively young age

were unable to induce Tag-specific antibodies. In the single

transgenic LoxP–Tag mice with sporadic tumors, the Tag-

specific antibodies served as surrogate markers for tumor

growth and recognition of the tumor by the adaptive immune

system. Usually, the anti-Tag antibodies in LoxP–Tag mice were

detectable many months before obvious tumor burden,

indicating a long latency period between the initiating

oncogenic event of Tag expression and tumor progression.

Analysis of serum antibodies over time revealed that the Tag-

specific antibodies increased over time to very high levels, until

the mice became moribund. Anti–Tag antibody concentrations

in tumor-bearing LoxP–Tag mice were even higher than in

young tumor-free mice after Tag-specific immunization. In

most LoxP–Tag mice, no macroscopically visible tumor was

detectable at the time of the first detection of the anti-Tag

antibodies. This finding indicated that B cells recognized the

tumor at early stages of tumor development. Because the half-

life of antibodies in the serum is relatively short and high

antibody titers are dependent on the presence of the antigen,

the persistent anti-Tag antibodies likely reflected persistent yet

latent tumor cells. We therefore assume that B cells recognized

small pre-malignant lesions that expressed Tag. Because Tag is a

nuclear antigen, not accessible to antibodies, and because anti-

Tag antibodies occurred long before tumor burden, it is likely

that antigen release caused by dying cells and generated during

the slow process of malignant transformation sustained the

humoral anti-Tag response. The anti-Tag antibodies were of the

IgG subtype, indicating CD41 T-cell help, even though so far

we have no direct evidence for a Tag-specific CD41 T-cell

contribution. When we analyzed the anti-Tag antibodies for

their Ig sub-classes, we found in virtually all mice Tag-specific

IgG2a, IgG2b, and occasionally IgA but no IgG1 or IgG3. IgG2a

indicates IFN-g involvement, and IgG2b and IgA indicate

transforming growth factor-b1 (TGF-b1) involvement in the

anti-tumor response. Paradoxically, these cytokines reciprocally

inhibit each other and their possible role in sporadic tumor

development is discussed below.

The T-cell response to sporadic cancer

Because the analysis of tumor-specific endogenous T-cell

responses against autochthonous tumors requires the knowl-

edge of a tumor antigen and immune competence to that

tumor antigen before tumor development, only a few studies

analyzed spontaneous tumor-specific T-cell responses in the

primary tumor-bearing host (38, 40, 72). In these mice, the

response against Tag expressed by the rat insulin promoter was

analyzed. The T-cell response to sporadic cancer and particu-

larly to sporadic cancer in the pre-malignant phase has not

been analyzed before. This was most important, however,

because as we have seen above, a tumor antigen-specific B-cell

response and IgG antibodies occurred very early in the process

of tumor development. This finding made it likely that T cells

also recognized the tumor antigen in an early phase. We mainly

have analyzed the CD81 cytotoxic T lymphocyte (CTL)

response in LoxP–Tag mice. Because we could not exclude that

leakiness of the attenuator resulted in Tag expression in

addition to that found in the tumor that could result in CTL

tolerance, we analyzed the Tag-specific immune competence in

young tumor-free transgenic mice. Therefore, mice were

immunized with tumor cells genetically modified to express

the immunostimulatory molecules interleukin-7 (IL-7) and

B7.1 and Tag. Previously, it was shown in tumor transplantation

models that these molecules increased the immunogenicity of

the tumor cells, leading to effective tumor immunity (73, 74).

The mice were challenged with a different tumor cell line that

expressed Tag as a shared antigen. Most of the immunized

LoxP–Tag mice rejected the Tag-positive but not the Tag-

negative challenge tumor, demonstrating Tag-specific tumor

immunity. This result also indicated that leakiness of the

attenuator that inhibited Tag expression was a too rare phe-

nomenon on a per cell basis or a too low-abundance phenom-

enon on a per molecule basis to induce tolerance. This

assumption was confirmed by T-cell analysis with H-2Kb/Tag

peptide IV tetramers. Several CTL epitopes of Tag have been

identified, and epitope IV is a dominant peptide antigen (75,

76). Tag-immunized LoxP–Tag mice showed an increase of Kb/

peptide IV tetramer-positive cells that was comparable to that

seen in wildtype mice. Thus, LoxP–Tag mice have retained

normal immune competence against the immunodominant

Tag epitope. Interestingly, when LoxP–Tag mice were



immunized in a Tag-specific fashion and had rejected a lethal

challenge of transplanted Tag-positive tumor cells, they also

efficiently controlled primary tumor development. None of the

immunized mice developed tumors during an observation

time of 2 years, whereas all naive mice developed a tumor.

Thus, the failure of T cells to control primary tumors does not

reflect their inherent inability to control Tag-induced tumors.

Comparable data have been obtained in RIP-Tag mice, which if

vaccinated before tumor development, efficiently generated

effector T cells that prevented tumors long term (38, 40, 72).

Therapeutic vaccination at the time of established small tumors

failed in most of the mice (40). This outcome was not due to

the failure of T-cell activation but probably the inability of T

cells to infiltrate the established tumor. A similar observation

has been made in an autochthonous model with chemically

induced tumors (74). Because LoxP–Tag mice could but never

spontaneously did control the primary tumor, we analyzed

whether the primary tumor was specifically recognized and

induced a CTL response. Kb/peptide IV tetramer-positive cells

comprising between 3% and 18% of the CD81 T cells

spontaneously expanded in tumor-bearing LoxP–Tag mice.

This expansion of tumor antigen-specific CD81 T cells

was comparable to that seen in Tag-immunized tumor-free

LoxP–Tag mice. Thus, the primary tumor induced a strong

spontaneous B-cell and an apparently strong CTL response.

The progressive growth of tumors in the presence of a

tumor-specific CTL response raised the question of whether

the tumors had to evade their destruction and, thus, became

‘low’ immunogenic. The immunogenicity of tumor cells is

usually analyzed by tumor transplantation into T-cell-

competent mice. Low- or non-immunogenic tumor cells are

not rejected, even after immunization. Some spontaneous

tumor cell lines fall into this category. Intermediate

immunogenic tumor cells are often rejected in immunized

mice but grow in naive mice. Many chemical carcinogen-

induced, e.g. via MCA, tumor cells fall into this category (11).

Highly immunogenic tumor cells are rejected in naive mice. A

variety of UV irradiation-induced tumor cells fall into this

category (7). Because UV irradiation induces immune

suppression, it has been assumed that it was this fact that

allowed the immunogenic tumors to grow. In general, it is

often believed that the lack of immunogenicity of tumor cells

reflects a selection process to avoid T-cell recognition.

To analyze the immunogenicity of sporadic tumors that had

grown in LoxP–Tag mice, a number of tumor cell lines were

established. Not surprisingly, they all expressed Tag. Most of

these cell lines grew progressively as a tumor in T-cell-deficient

Rag knockout or severe combined immunodeficient (SCID)

mice. All of these tumors were rejected in naive wildtype

(C57BL/6) mice. Several of the tumor cell lines were also

analyzed in naive young LoxP–Tag mice, which showed that

they were all rejected. These data demonstrated that the

sporadic tumors that had grown in immune-competent mice

were highly immunogenic. One can conclude, therefore, that

sporadic tumors do not escape recognition by T cells in their

primary host. Immune suppression such as that induced by UV

irradiation is not necessary for the growth of highly

immunogenic tumors. Two of the tumor cell lines of

LoxP–Tag mice were also injected in LoxP–Tag�Alb–Cre

transgenic mice. In these double-transgenic mice, the Cre

recombinase is expressed by the liver-specific albumin

promoter. This expression leads to deletion of the stop-

cassette and Tag expression early in life, so that the mice are

tolerant for Tag. In these double-transgenic mice, the

transplanted tumor cells grew with kinetics comparable to that

in T-cell-deficient SCID mice. This finding suggested that Tag

was the dominant rejection antigen responsible for the high

immunogenicity. Because young LoxP–Tag mice rejected these

tumors, additional confirmation was given that LoxP–Tag mice

have retained normal immune competence against Tag.

Furthermore, these experiments demonstrated that highly

immunogenic tumors do not have to escape immune

recognition during progression in their primary immune-

competent host.

This paradoxical situation, where the tumors progressively

grew despite strongly expanded tumor-specific CD81 T cells,

raised the question of whether the CD81 T cells were

functional. To analyze these cells, the mice were subjected to

an in vivo killing assay, in which Tag peptide IV-loaded and non-

loaded spleen cells, labeled with different amounts of

fluorescent dye, were injected into mice, and the relative

disappearance of the two spleen cell populations was analyzed

1 day later. Tag-immunized but not naive young tumor-free

LoxP–Tag mice specifically killed the Tag peptide IV-loaded

spleen cells. Old tumor-bearing LoxP–Tag mice were unable to

kill the Tag peptide IV-loaded spleen cells, despite the large

number of Kb/peptide IV tetramer-positive cells. This finding

indicated that the Tag-specific CD81 T cells were non-

functional, e.g. anergic, and that the tumor had induced

tolerance. To confirm this assumption, we asked whether

LoxP–Tag mice with a primary sporadic tumor were still able

to reject LoxP–Tag-derived tumor cells after transplantation.

While wildtype mice and young tumor-free LoxP–Tag mice

rejected the Tag-positive tumor graft, old LoxP–Tag mice with

primary tumors failed to reject the transplanted tumor cells.

This experiment firmly established that sporadic tumors,



instead of escaping immune recognition, induced tolerance.

Because most LoxP–Tag mice were old at the time of tumor

burden, one could not exclude that a general age-dependent

decline of immune competence was responsible for the

inability to reject transplanted Tag-positive tumor cells.

However, old wildtype mice rejected the Tag-positive tumor

cells as efficiently as young mice. Therefore, tolerance is

tumor-induced and not due to the decline of immune

responses in aged mice.

The data reviewed so far are in sharp contrast to the

hypothesis that spontaneous tumors are recognized and

eliminated by T cells, termed cancer immunosurveillance

(77–79). Recently, attempts have been made to revive this

hypothesis (80). The proponents based their theory on

experiments showing that immune-deficient mice, such as T-

cell-deficient Rag-2 knockout mice, developed MCA-induced

or spontaneous tumors more frequently or with shorter latency

in comparison with wildtype control mice. However, these

data could not be reproduced (19). The possible reasons for

this discrepancy have been reviewed elsewhere (23, 24).

Altered inflammatory responses in the life-long immune-

deficient mice (81) and the use of non-littermates as controls

are likely reasons. IFN-g-receptor knockout mice were more

susceptible to MCA-induced tumors in comparison with

control littermates (82). The mechanism appeared to be a

reduced tissue repair and MCA-encapsulation response, with

no indication of tumor-specific T-cell involvement (19, 23). In

any case, these tumor models did not allow for monitoring

tumor-specific T-cell responses, because no tumor antigens

were known. The potential pitfalls of antigenically non-defined

tumor models with regard to T-cell-mediated anti-tumor

responses have been reviewed recently (24). The fact that

highly immunogenic sporadic tumors in immune-competent

LoxP–Tag mice induced tolerance and progressively grew

revealed the absence of immunosurveillance. One could argue

that occasionally LoxP–Tag mice rejected a sporadic tumor,

unrecognized by us. However, those LoxP–Tag mice that

rejected a tumor once, the transplanted tumor, remained

tumor-free throughout their life.

Why were the sporadic tumors in LoxP–Tag mice not

rejected, despite their immunogenicity? Two alternatives exist.

The first is that the tumor was recognized too late and sneaked

through (83). It has been demonstrated that even if functional

CTLs were induced, they were unable to reject the already-

established tumor (24, 38, 72). The second alternative is that

sporadic tumors were unable to induce functional CTLs. In the

first alternative, it would have been possible to detect a window

of functional Tag-specific CTLs, while in the second alternative,

tolerance for Tag would have been manifested at the time when

the tumor was initially recognized. To distinguish between

these two possibilities, we recently conducted a time course

analysis of anti-Tag IgG antibodies versus Tag-specific

tolerance. As mentioned above, anti-Tag IgG antibodies in the

serum of LoxP–Tag mice were the best surrogate marker for

tumor development and indicated the time point when an

adaptive immune response was initiated. At the time when the

anti-Tag IgG antibodies were detected for the first time, no

macroscopically visible tumor was present in most LoxP–Tag

mice. Additionally, the anti-Tag IgG antibodies persisted, up to

a year or even longer, before obvious tumor burden. Thus, an

adaptive immune response was detected against pre-malignant

lesions. Analysis of Tag-specific CTLs by the in vivo killing assay

mentioned above revealed that LoxP–Tag had developed Tag-

specific tolerance simultaneously with the first detection of

anti-Tag antibodies (Fig. 2). Furthermore, there was an almost

perfect correlation between the initial detection of anti-Tag

antibodies in the serum and the failure to reject transplanted

Tag-positive tumor cells at that time point. These data

demonstrate that pre-malignant lesions induced Tag-specific

tolerance and that we failed to detect a window of functional

CTL activation. We therefore concluded that sporadic

immunogenic tumors were unable to activate functional CTLs

(authors’ unpublished observations). It follows that there is no

need to escape recognition or destruction by CTLs. A critical

review of the literature indicates that direct evidence of escape

Fig. 2. Tumor-specific tolerance occurs concomitant with recogni-tion of pre-malignant sporadic lesions by the adaptive immune

system. Anti-SV40 T antigen (Tag) IgG antibodies in the serum ofLoxP–Tag mice marked the time-point when the mice had developed Tag-specific CTL tolerance. The scheme illustrates two important points: (i)tumor latency is not caused by T-cell control and (ii) paradoxically, theantibodies indicate CTL tolerance (split tolerance). Small tumor indicatesthat no macroscopically visible tumor was detected.



from spontaneous CTL attack by non-viral primary tumors is

lacking (24).

TGF-b1 and IFN-g

TGF-b1 is a prototypical member of a family of conserved

proteins (TGF-b1, TGF-b2, and TGF-b3) with similar yet

highly pleiotropic biological activities. TGF-b1 can be pro-

duced and secreted by most cell types, usually in a latent form.

The latent form requires activation before TGF-b1 can exert

biological activity. TGF-b1 plays a crucial role in the develop-

ment, growth, regulation, and differentiation of many tissues.

It inhibits the proliferation of most cells but can stimulate

the growth of mesenchymal cells. It exerts immunosuppressive

effects and enhances the formation of extracellular matrix.

TGF-b1 is involved in wound repair processes and in the

initiation of inflammatory reaction, while it effectively in-

hibits T-cell function (84–87). Interestingly, it has recently

been shown that TGF-b1 is also required for the generation

of an inflammatory T-cell subset in vivo, termed Th17 cells

(88, 89).

The tumor-induced tolerance in LoxP–Tag mice was

associated with tumor-specific IgG antibodies mainly of IgG2a

and IgG2b subtypes. The class switch to IgG2a is induced by

IFN-g and to IgG2b by TGF-b1 (90). Therefore, the isotype

composition indicated an involvement of IFN-g and TGF-b1

during the tumor-induced immune response. To analyze a

possible role of IFN-g during the anti-tumor response,

sporadic tumor development was observed in LoxP–Tag/IFN-

g-deficient and LoxP–Tag/IFN-g-competent mice. The absence

of IFN-g slightly reduced the tumor latency in about half of the

mice, but all mice from both groups developed tumors that did

not differ in spectrum or number. IFN-g-deficient Tag-negative

mice remained tumor-free during 20 months of observation.

As we have seen that the sporadic tumors are unable to induce

functional CTLs, the shortened latency in some IFN-g-deficient

compared with IFN-g-competent mice was not due to a tumor-

specific response but likely could be explained by altered

inflammatory responses between both types of mice. It is well

established that inflammatory responses critically influence

tumor development (3–6). To explain the IgG2b anti-Tag

antibodies, we determined serum TGF-b1 levels. In tumor-

bearing mice, substantially elevated TGF-b1 concentrations

were detected in comparison with young LoxP–Tag and old

wildtype mice. The tumor cells in most cases did not produce

TGF-b1. Which host cells produce the excess amounts of TGF-

b1 is currently not known.

Compatible with the fact that TGF-b1 negatively regulates

IFN-g, tumor-bearing LoxP–Tag mice showed a generalized

and almost complete IFN-g deficiency as detected by a sensitive

in vivo IFN-g capture assay. Whereas young LoxP–Tag and old

wildtype mice showed substantial amounts of IFN-g in serum

(500 pg/ml or more), virtually no IFN-g could be detected in

the sera of tumor-bearing mice. It therefore seemed that

TGF-b1 dominated over IFN-g. It is currently not known

when, relative to each other, the cytokines are expressed.

TGF-b1 was already elevated in the serum of mice with

pre-malignant lesions. The fact that in most cases TGF-b1 was

not produced by the tumor cells raises the question of whether

it is directly responsible for the Tag-specific tolerance.

Alternatively, elevated TGF-b1 could be the result of the

default immune response against the persistent immunogenic

tumor antigen.

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