although tumor immunosurveillance was first hypothesized more than four decades ago , the observed absence of spontaneous tumors in several immune - compromised mouse strains initially dampened enthusiasm for this idea .
mabs specific for immunoregulators have sometimes proven useful in establishing the importance of such molecules in immunity against experimental tumors ( 13 ) .
production of gene - targeted mice for specific effector molecules has greatly aided this endeavor ( 414 ) . nevertheless , experimental tumor models examined to date have provided limited information on tumor initiation and development .
ifn- ( 15 ) and perforin ( pfp ; references 5 , 14 , and 16 ) , key molecules of both the innate and adaptive immune systems , contribute to tumor prevention in mice treated with the chemical carcinogen , methylcholanthrene ( mca ) , or in mice deficient for p53 expression . in mca- ( 10 ) and
other carcinogen - induced cutaneous malignancy models ( 17 ) , the importance of antitumor immunity mediated by nk cells , nk t cells , and  t cells has also been illustrated .
however , in most of these models tumor development involves the transformation of many cells at one time and only a defined spectrum of tumors ( lymphomas and cutaneous malignancies ) is observed .
an alternative model of tumor induction is that spontaneous tumors can arise from a single cell of any tissue that has acquired the appropriate genetic mutations with age in any given environment .
herein , that environment has been controlled , and spontaneous tumor initiation compared in wt and gene - targeted mice of the same genetic background to illustrate the key role pfp and ifn- play in tumor immunosurveillance of lymphoma and lung adenocarcinoma .
inbred c57bl/6j and balb / c wt mice were purchased from the walter and eliza hall institute of medical research , melbourne , australia .
the following gene - targeted mice were bred and maintained at the austin research institute biological research laboratories , heidelberg , australia and the peter maccallum cancer institute , east melbourne , australia : c57bl/6j pfp - deficient ( b6 pfp ) ( targeted in c57bl/6j es cells and provided by d. kagi [ amgen institute , toronto , canada ] ; reference 18 ) ; c57bl/6j tnf - deficient ( b6 tnf ) ( targeted in c57bl/6j es cells [ reference 19 ] and provided by dr .
sedgwick and originally from the centenary institute of cancer medicine and cell biology , sydney , australia ) ; c57bl/6 ifn--deficient ( b6 ifn-)(backcrossed to c57bl/6j for 10 generations from genentech , inc . ; reference 20 ) ; c57bl/6 il-12p40-deficient ( b6 il-12)(backcrossed to c57bl/6j for 10 generations from hoffmann - la roche ; reference 21 ) ; c57bl/6 il-18deficient ( b6 il-18 ) ( backcrossed to c57bl/6j for eight generations and provided by dr .
s. akira , osaka university , japan ; reference 22 ) ; balb / c pfp ( backcrossed to balb / c for eight generations using microsatellite mapping ) , balb / c ifn- ( backcrossed to balb / c for 10 generations from genentech , inc . , reference 20 ) ; and balb / c pfp ifn-. f1 heterozygote progeny of some gene - targeted mice and their corresponding wt were bred as follows : ( b6  b6 ifn-)f1 ; ( balb / c  balb / c ifn-)f1 ; and ( balb / c  balb / c pfp)f1 .
all aging mice were routinely screened for viruses , parasites , and other microbes and tested negative over the entire course of the experiment .
any mouse with an abnormality ( palpable mass , abdominal distension , weight loss > 10% , ruffled fur ) was killed , its age recorded , and a postmortem performed .
mean lifespan  sem was calculated and probability of significance determined using a mann - whitney rank sum u - test .
the significance of proportions of tumors and , in particular , disseminated lymphomas , was determined by a fisher 's exact test .
a full autopsy was performed at sacrifice and tumor ( macroscopically detected ) , spleen , liver , thymus , and lymph nodes were routinely examined by histology after fixing these tissues in formalin and on occasions also fresh frozen .
the preparation and staining of sections for histology were performed by the department of anatomical pathology , austin and repatriation medical centre , heidelberg , australia .
the following reagents were used : anti tcr - apc ( h57597 ; bd pharmingen ) ; nk1.1-pe ( pk136 ; bd pharmingen ) ; cd4-fitc ( ct4 ; caltag laboratories ) ; cd8-apc ( 53.6.7 ; bd pharmingen ) ; cd8-biotin ( 53.5.8 ; bd pharmingen ) ; cd11c - fitc ( hl3 ; bd pharmingen ) ; b220-pe ( ra36b2 ; caltag laboratories ) ; thy-1-pe ( 30-h12 ; bd pharmingen ) ; cd45.2-fitc ( 104 ; bd pharmingen ) ;  tcr - biotin ( clone gl3 ; bd pharmingen ) ; mac-1-biotin ( m170 ; caltag laboratories ) ; goat anti  mouse ig - fitc ( silenus laboratories ) ; and streptavidin - percp ( bd pharmingen ) .
anti - fc receptor ( 2.4g2 ) was used to prevent nonspecific binding by mab .
fresh splenocytes from 6-wk - old b6 or balb / c mice were always used as labeling controls .
a number of disseminated lymphomas were transplanted directly from b6.pfp , balb / c pfp , or b6
two representative experiments are shown using a b cell lymphoma from b6.pfp mice , pnk7 ( b220igtcr- ) , and a t cell lymphoma from b6 ifn- mice , bg18 ( cd8-cd4 tcr-ig ) .
groups of five wt or gene - targeted mice were injected intraperitoneally with increasing numbers of lymphoma cells and observed daily for tumor growth for > 100 d. the supplemental figures are a ( fig .
s2 ) flow cytometric analysis of lymphomas arising in balb pfp and b6 pfp mice .
inbred c57bl/6j and balb / c wt mice were purchased from the walter and eliza hall institute of medical research , melbourne , australia .
the following gene - targeted mice were bred and maintained at the austin research institute biological research laboratories , heidelberg , australia and the peter maccallum cancer institute , east melbourne , australia : c57bl/6j pfp - deficient ( b6 pfp ) ( targeted in c57bl/6j es cells and provided by d. kagi [ amgen institute , toronto , canada ] ; reference 18 ) ; c57bl/6j tnf - deficient ( b6 tnf ) ( targeted in c57bl/6j es cells [ reference 19 ] and provided by dr .
sedgwick and originally from the centenary institute of cancer medicine and cell biology , sydney , australia ) ; c57bl/6 ifn--deficient ( b6 ifn-)(backcrossed to c57bl/6j for 10 generations from genentech , inc . ; reference 20 ) ; c57bl/6 il-12p40-deficient ( b6 il-12)(backcrossed to c57bl/6j for 10 generations from hoffmann - la roche ; reference 21 ) ; c57bl/6 il-18deficient ( b6 il-18 ) ( backcrossed to c57bl/6j for eight generations and provided by dr .
s. akira , osaka university , japan ; reference 22 ) ; balb / c pfp ( backcrossed to balb / c for eight generations using microsatellite mapping ) , balb / c ifn- ( backcrossed to balb / c for 10 generations from genentech , inc . , reference 20 ) ; and balb / c pfp ifn-. f1 heterozygote progeny of some gene - targeted mice and their corresponding wt were bred as follows : ( b6  b6 ifn-)f1 ; ( balb / c  balb / c ifn-)f1 ; and ( balb / c  balb / c pfp)f1 .
all aging mice were routinely screened for viruses , parasites , and other microbes and tested negative over the entire course of the experiment .
any mouse with an abnormality ( palpable mass , abdominal distension , weight loss > 10% , ruffled fur ) was killed , its age recorded , and a postmortem performed .
mean lifespan  sem was calculated and probability of significance determined using a mann - whitney rank sum u - test .
the significance of proportions of tumors and , in particular , disseminated lymphomas , was determined by a fisher 's exact test .
a full autopsy was performed at sacrifice and tumor ( macroscopically detected ) , spleen , liver , thymus , and lymph nodes were routinely examined by histology after fixing these tissues in formalin and on occasions also fresh frozen .
the preparation and staining of sections for histology were performed by the department of anatomical pathology , austin and repatriation medical centre , heidelberg , australia .
the following reagents were used : anti tcr - apc ( h57597 ; bd pharmingen ) ; nk1.1-pe ( pk136 ; bd pharmingen ) ; cd4-fitc ( ct4 ; caltag laboratories ) ; cd8-apc ( 53.6.7 ; bd pharmingen ) ; cd8-biotin ( 53.5.8 ; bd pharmingen ) ; cd11c - fitc ( hl3 ; bd pharmingen ) ; b220-pe ( ra36b2 ; caltag laboratories ) ; thy-1-pe ( 30-h12 ; bd pharmingen ) ; cd45.2-fitc ( 104 ; bd pharmingen ) ;  tcr - biotin ( clone gl3 ; bd pharmingen ) ; mac-1-biotin ( m170 ; caltag laboratories ) ; goat anti  mouse ig - fitc ( silenus laboratories ) ; and streptavidin - percp ( bd pharmingen ) . anti - fc receptor ( 2.4g2 ) was used to prevent nonspecific binding by mab .
fresh splenocytes from 6-wk - old b6 or balb / c mice were always used as labeling controls .
a number of disseminated lymphomas were transplanted directly from b6.pfp , balb / c pfp , or b6
two representative experiments are shown using a b cell lymphoma from b6.pfp mice , pnk7 ( b220igtcr- ) , and a t cell lymphoma from b6 ifn- mice , bg18 ( cd8-cd4 tcr-ig ) .
groups of five wt or gene - targeted mice were injected intraperitoneally with increasing numbers of lymphoma cells and observed daily for tumor growth for > 100 d.
s2 ) flow cytometric analysis of lymphomas arising in balb pfp and b6 pfp mice .
we undertook to monitor spontaneous tumor development in wt c57bl/6 ( b6 ) and balb / c mice or those that were deficient in ifn- and/or pfp .
b6 mice deficient in il-12 , il-18 , or tnf were also examined , as all of these cytokines have been shown to regulate the expression of ifn- and other effector molecules in lymphocytes ( 2327 ) .
b6 ifn- mice developed disseminated lymphomas ( 16/32 ( 50% ) and mean lifespan 491  104 d ) ( fig .
1 a ) . a few mice developed thymic lymphomas ( n = 2 ) or sarcoma ( n = 1 ) . like wt b6 mice ( 0/39 mice ) , b6 mice that were deficient in tnf ( 0/36 ) , il-12 ( 0/29 ) , or il-18 ( 0/27 ) did not develop any tumors over the same observation period ( fig .
1 a ) . these data illustrate spontaneous lymphoma formation in ifn-deficient mice and suggest that more than one of the upstream mediators of ifn- or an alternative cytokine must be required to regulate the protective effects of ifn-. consistent with our previous study ( 16 ) , b6 pfp mice died from aggressive disseminated lymphomas affecting the spleen , liver , and lymph nodes from 300 d onwards , with 57% ( 12/21 ) succumbing by the end of the experiment ( mean lifespan = 510  119 d ; fig .
the appearance of tumors was recorded in mice of ( a ) c57bl/6 and ( b ) balb / c backgrounds as indicated .
groups of mice ( number in parentheses ) were evaluated on a weekly basis and , when moribund , tumor type ( white squares , thymic lymphoma ; black squares , disseminated lymphoma ; black triangles , sarcoma ; white triangles , lung adenocarcinoma ; and white circles , other tumors ) recorded against the age at the time of death / autopsy ( in days ) .
no tumors were observed in heterozygote control groups of ( b6  b6 ifn-)f1(n = 17 ) , ( balb / c  balb / c ifn-)f1 ( n = 12 ) , and ( balb / c  balb / c pfp)f1 ( n = 18 ) mice over a 750-d period .
a small number of the oldest surviving balb / c ifn- mice ( 700750 d ) also developed nonmalignant mucosal hamartomas in the stomach .
of great interest was a considerably distinct pattern of tumor development observed in similar gene - targeted mice on a balb / c background ( fig .
c ifn- mice did not develop disseminated lymphoma ( 0/19 , p = 0.0001 ) . the finding that ifn- was not essential for host protection from this type of tumor in balb / c mice illustrated that strain - specific genetic factors can influence the nature of the immune response to these spontaneous lymphomas . by contrast , pfp was critical in host protection from disseminated lymphoma in both strains ( fig .
9 of 25 balb / c pfp mice developed disseminated lymphoma ( mean lifespan = 597  82 d ) , and a few mice developed thymic lymphomas ( n = 2 ) or sarcoma ( n = 1 ; fig . 1 b ) . the earlier onset ( mean lifespan = 368  120 d ) ( p < 0.0001 , compared with pfp mice ) and greater frequency ( 14/20 ) of disseminated lymphoma in balb / c mice deficient in both pfp and ifn- , revealed that in the absence of pfp , ifn- did play an important role in delaying the development of disseminated lymphoma ( fig .
these data supported a general role for ifn- in protection from spontaneous lymphoma , but suggested that the relative role of ifn- was strain specific .
lymphocyte - mediated immunosurveillance of epithelial malignancy has never been demonstrated , except where viral infection may contribute to oncogenesis ( e.g. , ebv , hpv transplant recipients ) or when carcinogens were also administered , and we therefore felt the development of spontaneous epithelial tumors in gene - targeted mice warranted particular attention .
while ifn- and pfp were especially important for the control of disseminated lymphoma , a significant incidence of lung adenocarcinoma was detected in balb / c ifn- mice ( 3/19 , p = 0.0269 ) and balb / c pfp mice ( 3/25 , p = 0.048 ) ( fig .
notably , it is relevant that the onset of lung adenocarcinomas was very late in balb / c ifn- ( 681  43 d ) and balb / c pfp ( 693  67 d ) mice .
indeed , the development of all tumors , including disseminated lymphomas was significantly delayed in balb / c mice compared with b6 mice .
far fewer balb / c pfp ifn- , b6 ifn- , and b6 pfp mice remained alive ( free of lymphoma ) over the first 600 d of life and therefore larger numbers of these will likely be required to observe adenocarcinomas .
f1 progeny of gene - null mice and wt mice lacking either one allele of pfp or ifn- did not develop any spontaneous tumors ( fig .
1 ) . determining whether the immune system controls other epithelial malignancies is an important issue for future long - term studies and only a similar study of far larger groups of mice ( 100 ) will reveal whether significant numbers of other epithelial malignancies emerge in ifn- and pfp mice .
6 of the 16 lymphomas arising in b6 ifn- mice were analyzed for phenotype and while five were tcr- , no unique lymphoma phenotype was apparent ( unpublished data ) .
several previous studies have indicated that t cell death after antigen - driven expansion may be regulated by ifn- ( 28 , 29 ) , and therefore possibly disrupted homeostasis and failed immunosurveillance by ifn- loss , may be the cause of t cell lymphoma in b6 ifn- mice .
all of the disseminated lymphomas in b6 pfp mice were of b cell origin ( b220 sig cd4cd8 tcr ) , or plasmacytomas ( b220 cd4 cd8 tcr sig ; also defined histologically , unpublished data ) .
our previous study of pfp mice with additional p53 loss indicated a majority of lymphomas of b cell origin , however some p53pfp mice also developed lymphomas of non - b cell origin ( 16 ) .
one explanation for the development of b cell lymphomas in pfp mice is the recognized role for pfp in controlling the survival of some apcs , like b cells ( 30 , 31 ) .
however , it remains unclear why only lymphomas of b cell origin developed in b6 pfp mice when enhanced expansions of cd8 t cells were observed in these mice , particularly after challenge with foreign antigens ( e.g. , viral or bacterial infection ; references 28 , and 3234 ) .
most of the lymphomas in b6 pfp and b6 ifn- mice were diffuse large cell lymphomas ( see online supplemental materials ) .
flow cytometric analysis of disseminated lymphomas in balb / c pfp and balb / c pfpifn- mice revealed that all were also b220 sig cd4cd8 tcr ( unpublished data ) . importantly , these data supported a role for ifn- in also suppressing the earlier onset of b cell lymphomas .
interestingly , in the balb / c strain , a small number of the lymphomas arising in pfp and pfp ifn- mice showed an unusual histiocytic appearance ( fig . 1 , asterisk ) with a pale eosinophilic cytoplasm .
these tumors additionally expressed cd11c and mac-1 antigens ( see online supplemental materials ) , but did not all express other markers of b1 cells ( e.g. , cd5 , unpublished data ) .
of note , this type of histopathology occurs only rarely in humans , and interestingly the accumulations of activated histiocytes ( macrophages ) observed in pfp - deficient humans displaying familial hemophagocytic lymphohistiocytosis ( 35 ) are one example .
all the lung adenocarcinomas in both balb / c pfp and balb / c ifn- mice were well - differentiated papillary adenocarcinomas ( see online supplemental materials ) .
the malignancy of primary tumors arising was additionally confirmed by secondary transfer into mice of the same strain .
two representative experiments of > 10 using different disseminated lymphomas transferred from either b6 pfp or b6 ifn- mice are depicted in fig .
2 . the transfer of three different t cell lymphomas from b6 ifn- mice ( one shown ) demonstrated that these tumors grew at a similar rate in b6 ifn- and b6 wt mice ( fig .
2 ) . by contrast , and in concert with our previous data ( 16 ) ,
all b cell lymphomas from b6 pfp mice grew at low cell numbers in b6 pfp mice , but were avidly rejected when transferred into b6 wt mice ( fig .
thus far , all lymphomas of b cell origin arising in pfp mice , including five described herein , were rejected in wt mice by cd8 t cells ( reference 16 and unpublished data ) and in this study the donor tumor cells and recipient mice were genetically matched as closely as possible .
similar experiments with three different b cell lymphomas from balb / c pfp mice have also demonstrated rejection in balb / c wt mice and ( balb / c  balb / c . pfp)f1 heterozygous mice , but not balb / c pfp mice ( unpublished data ) . in concert with our previous lymphoma transfer studies ( 16 ) , it is likely that the lymphomas of b cell origin are generally being detected by cd8 t cells independently of any alloantigens potentially expressed by the tumor .
it is probable that the lack of detectable immunogenicity of lymphomas from ifn- mice indicates an important functional distinction between the activities of ifn- and pfp in eliminating potentially transformed cells .
consistent with the immune system functioning as a tumor - suppressor system , our results indicated that the immunoselection pressure of pfp on tumor cells was strong .
lymphomas from pfp - deficient mice have emerged in the absence of this significant immunoselection pressure , and they are avidly rejected in wt mice upon transfer .
immunoselection by ifn- appears far weaker from our spontaneous lymphoma transplant data , however it has been demonstrated in an experimental system using mca fibrosarcomas arising in ifn- receptor - deficient mice ( 36 ) .
primary lymphomas arising in b6 pfp ( top ) and b6.ifn- ( bottom ) were secondarily transplanted intraperitoneally ( 1010 cells in 0.2 ml pbs , as indicated ) into groups of five untreated b6 wt ( black circles or squares ) , b6 pfp ( white circles ) or b6 ifn- ( white squares ) mice .
the results are representative of seven primary lymphomas and three primary lymphomas transplanted from b6 pfp and b6 ifn- mice , respectively .
top , pnk-7 ( b cell lymphoma from b6 pfp mouse ) ; bottom , bg18 ( t cell lymphoma from b6 ifn- mouse ) .
previous studies have demonstrated that t cells ( 2 ) , nk t cells ( 37 ) , and nk cells ( 37 ) can all produce ifn- with antitumor activity .
it remains unclear which type of lymphocytes use ifn- to control spontaneous lymphomas or lung adenocarcinomas , and this issue is very difficult to address without mice conditionally deficient ( e.g. , nk cell , nk t cell , or t cell ) in ifn-. defining exactly how a pleiotropic cytokine like ifn- mediates host protection from spontaneous tumors will also be a challenging goal for future investigation given that the lymphomas arising in b6 ifn- mice appear nonimmunogenic when transferred into wt mice .
many potential direct and indirect activities of ifn- could suppress spontaneous tumor formation . although ifn- can suppress tumor angiogenesis ,
it is unlikely that this is the main mode of action in preventing the development of nonsolid tumors such as disseminated lymphomas .
it also remains feasible that loss of ifn- may result in tumor promotion by another factor normally suppressed by ifn-. gene - targeted mice have been extremely valuable when used in models where tumors are induced by viruses or carcinogens or simply when the mice were aged for spontaneous tumor formation .
the value of using several types of tumor induction models is no better illustrated by the lack of tumor development in tnf mice , but the reduced predisposition of these mice to carcinogen - induced skin tumors ( 38 ) .
although some other recent studies in aging mice have suggested that tumor immunosurveillance may exist ( 16 , 36 ) , our study directly demonstrated the specific importance of ifn- in the development of lymphomas and adenocarcinomas .
recently , a study of aging 129/sv mice doubly deficient for recombination activating gene ( rag)-2 and signal transducer and activator of transcription ( stat)-1 , the transcription factor mediating signaling by ifn- and ifn-/ receptors ( rag-2stat-1 ) , suggested that lymphocytes and ifn- may play an important role in host protection from breast and colon carcinomas ( 36 ) .
mice deficient in stat-1 alone displayed a low incidence of mammary carcinoma , however stat-1 regulates far more genes than ifns alone , including the mammary tumor suppressor , brca-1 ( 39 ) .
the lack of lymphoma reported in 129/sv stat-1 mice ( 32 ) may be because 129/sv behave like the balb / c strain .
nevertheless , we did not observe colon or breast adenocarcinomas in either balb / c or c57bl/6 strains of ifn--deficient mice .
the development of mammary and colon adenocarcinomas in 129/svev rag-2stat-1 mice may be a consequence of intestinal hyperplasia peculiar to 129 rag mice .
by contrast , the lung adenocarcinomas observed in balb / c pfp and balb / c ifn- mice occurred in strains of mice with no evidence of predisposing epithelial hyperplasia . the concept of whether lymphocytes play a key role in immunosurveillance beyond their own regulation ( i.e. , of nonlymphoid tissues ) has been debated for years ( 40 ) .
the adenocarcinomas observed in mice in our study suggested that future studies , monitoring spontaneous tumor development in gene - targeted mice alone and bred with other strains genetically prone to epithelial malignancies , will be most informative .
 the immunosurveillance of transformed cells by the immune system remains one of the most controversial and poorly understood areas of immunity . 
 gene - targeted mice have greatly aided our understanding of the key effector molecules in tumor immunity . herein , we describe spontaneous tumor development in gene - targeted mice lacking interferon ( ifn)- and/or perforin ( pfp ) , or the immunoregulatory cytokines , interleukin ( il)-12 , il-18 , and tumor necrosis factor ( tnf ) . both ifn- and pfp were critical for suppression of lymphomagenesis , however the level of protection afforded by ifn- was strain specific . 
 lymphomas arising in ifn--deficient mice were very nonimmunogenic compared with those derived from pfp - deficient mice , suggesting a comparatively weaker immunoselection pressure by ifn-. single loss of il-12 , il-18 , or tnf was not sufficient for spontaneous tumor development . 
 a significant incidence of late onset adenocarcinoma observed in both ifn- and pfp - deficient mice indicated that some epithelial tissues were also subject to immunosurveillance . 
Introduction
Materials and Methods
Mice.
Histopathology and Surface Phenotyping of Tumors.
Tumor Transplantation Experiments.
Online Supplemental Materials.
Results and Discussion