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Immunology |
Department of Molecular Immunology, German Cancer Research Center [R. G., B. A., G. J. H.], and Department of Surgery, University of Heidelberg [E. R., E. K.], 69120 Heidelberg, Germany
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ABSTRACT |
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(Mig) and IFN-inducible protein 10 (IP10). This remodeling of the vasculature in a proinflammatory environment may directly affect lymphocyte extravasation and effector function. Therefore, irradiation/adoptive transfer therapy combines antigen-driven tumor cell eradication with antiangiogenic effects on tumor endothelium, a powerful synergy that has not been previously appreciated. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Once successfully activated, effector T cells must be able to recirculate, enter tumor sites, and function within the tumor microenvironment to trigger the rejection process. Although tumor infiltration by effector T cells is a prerequisite for rejection (7) , it is no guarantee for an immunologically specific interaction (8) . There is accumulating evidence that local factors such as tumor-derived stroma (9 , 10) and the cytokine micromilieu (11) play a crucial role in tumor immunity. Thus, for immune-mediated destruction of solid tumors, the effector phase is as important as the initial priming of T cells. There is also a clear correlation between tumor size and therapeutic success (6) . Immunotherapy in classical transplantation models and experimentally induced metastasis is usually started simultaneously with tumor challenge or at a low tumor burden and is, therefore, not suited to destroy solid tumors. It is not surprising then, that the translation of tumor immune therapy to the clinic has been proven ineffective, and the numbers of responders remain small (5) . Transgenic mice developing autochthonous tumors might better reflect the clinical situation in terms of tissue tropism and growth kinetics. As is apparent in human cancers, however, these mouse models of de novo tumorigenesis confirm that currently available immunotherapeutic strategies are most effective either in a preventive setting (12 , 13) or in a setting in which the growth of solid tumors is merely delayed (14 , 15) .
The RIP1-Tag5 transgenic mouse is created by expressing Tag3 under the control of the RIP (16) . Onset of oncogene expression in pancreatic islets of Langerhans in adult mice causes transformation of ß-cells and progressive tumor development, although an immune response against Tag is evident (17) . During multistage carcinogenesis, normal and hyperproliferative islets are infiltrated by lymphocytes. However, the lymphocytic infiltration is lost during the transition from angiogenic islets to highly vascularized, solid tumors. We have previously shown that increasing the frequency of Tag-specific lymphocytes and providing costimulation on tumor cells failed to provoke immune-mediated tumor destruction (18 , 19) . Thus, it became apparent that a potent tumor antigen, together with highly activated, self-reactive lymphocytes, is not sufficient for tumor rejection, although these factors are nonetheless considered to be essential for T-cell based immunotherapy (20) . We, therefore, postulated that a permissive tumor microenvironment is required for effector function and subsequently demonstrated that irradiation of transgenic mice renders solid tumors accessible for lymphocytic infiltration (19 , 21) . Until now, however, the potential therapeutic efficacy of this treatment was not further investigated.
In normal tissue, radiation causes an inflammatory response that includes cytokine release, up-regulation of adhesion molecules, and a subsequent increase in lymphocyte adhesion (22 , 23) . Therefore, if irradiation is an inflammatory stimulus, is it able to promote inflammation in a highly angiogenic tumor? We evaluated the impact of irradiation on established tumors in transgenic mice and its efficacy for immunotherapy. We demonstrate that even persistently high levels of tumor-specific T cells, used alone, failed to eradicate solid tumors, but a combination of irradiation and multiple T cell transfers led to complete tumor regression. This antitumor effect was mediated by a proinflammatory environment and a dramatic lymphocytic influx. In addition, the combination of irradiation and adoptive transfers reestablished an almost normal vasculature within tumors early after the initiation of therapy, thus breaking the vicious cycle of tumor-induced angiogenesis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30 weeks. TagTCR1 mice express the MHC class II-restricted T-cell receptor specific for Tag; peripheral lymphocytes represent 10% transgenic CD4+ T cells with a normal CD4+:CD8+ T-cell ratio. TagTCR1 mice, kindly provided by Irmgard Förster (24)
, and Rag-1-/- mice were backcrossed to C3HebFe (Jackson Laboratory, Bar Harbor, ME) and kept under pathogen-free conditions at the German Cancer Research Center.
Adoptive Transfers.
RIP1-Tag5 mice were lethally irradiated with 10 Gy (X-ray source, Buchler, Braunschweig, Germany, 0.701 Gy/min). One day after irradiation, 5 x 106 bone marrow cells from Rag-1-/- or C3HebFe mice were injected i.v. Three weeks later, 5.0 x 106 or 2.5 x 106 in vitro activated TagTCR1 cells were injected i.v. and i.p. TagTCR1 cells were isolated from lymph nodes and 1.5 x 107 cells/six-well plate were cultured in RPMI medium supplemented with 10% FCS, 2 mM glutamine, 100 units/ml penicillin/100 µg/ml streptomycin, 0.05 mM 2-mercaptoethanol, 10 units recombinant IL-2/ml and 25 nM Tag peptide 362–384. C3H-derived lymphocytes were cultured with 300 ng/ml ConA. Cells were harvested after 72 h.
Flow Cytometry.
Lymph node or tumor cell suspensions were stained with biotinylated anti-clonotype (TagTCR) antibody 9H5 [10 µg/ml; from I. Förster (24)
], followed by streptavidin-Red670 (1.7 µg/ml, Life Technologies, Inc., Eggenstein, Germany), and PE-conjugated anti-CD4 (GK1.5, 2.0 µg/ml; BD PharMingen, Heidelberg, Germany) for two-color analysis. Cells were analyzed on a FACScan (Becton Dickinson).
Assessment of Tumor Size.
Solid tumors were dissected from pancreatic tissue after perfusion of 1x HBSS/5 mM glucose through the common bile duct into the pancreas. Tumor size was measured with a caliper and the formula [volume = 0.52 x (width)2
x length] was applied to determine the tumor burden (mm3) per mouse.
Western Blotting.
Pancreases were disrupted in 50 mM Tris (pH 8.0), 1% 2-mercaptoethanol, 0.3% SDS, and 1 tablet of protease inhibitors/10 ml (Roche, Mannheim, Germany). Extracts were heated for 10 min at 95°C and clarified by centrifugation. Fifteen µg of total protein extract were separated on 10% SDS/PAGE. The membrane was blocked in 1x PBS, 0.1% Tween 20, and 5% milk powder and probed with 1 µg/ml SV40T-Ag polyclonal mouse antibody (416; Dianova, Hamburg, Germany), antimouse IgG-horseradish peroxidase (10 ng/ml; Vector/Alexis Biochemicals, Grünberg, Germany) and SuperSignal chemiluminescent substrate (Pierce, Rockford, IL).
Immunohistochemistry and in Situ Hybridization.
Staining was performed as described (19)
with the following reagents: anti-Tag (rabbit polyclonal, 1:1000; from D. Hanahan); anti-CD4 (GK1.5, 10 µg/ml; BD PharMingen); anti-CD8 (Ly-2, 10 µg/ml; BD PharMingen), and anti-CD11b (Mac-1, 10 µg/ml; BD PharMingen). For in situ hybridization the following PCR fragments were subcloned into Bluescript vector: 639 bp Mig: forward primer (FP): 5'-CATCATCTTCCTGGAGCAGTGT-3', reverse primer (RP): 5'-GACACTTGCCCAGATCTGATGT-3'; 341 bp IP10: FP: 5'-CAAGGGATCCCTCTCGCAAGGA-3', RP: 5'-AGGGCAATTAGGACTAGCCATC-3'. Digoxigenin-labeled (dig-11-UTP; Roche) sense and antisense probes were synthesized from XbaI or BamHI linearized plasmids, using T3 and T7 polymerase (Roche), respectively. Ten-µm cryosections were treated with 4% paraformaldehyde and 0.2 M HCl, acetylated with acetic anhydride for 10 min, and hybridized with 250 ng of RNA probe/section in 100 µl of hybridization buffer [1% blocking reagent (Roche; 50% formamide, 5x SSC, 1 mg/ml yeast RNA, 0.5% Tween 20, 0.1 mg/ml heparin, 0.1% CHAPS, and 5 mM EDTA) at 55°C overnight. Sections were washed in 2x SSPE/0.3% CHAPS, treated with RNase A (20 µg/ml at 37°C for 30 min), followed by a wash in 50% formamide and 2x SSPE for 1.5 h at 50°C. RNA hybrids were detected with antidigoxigenin, alkaline phosphatase Fab-fragments (7.5 units/ml; Roche), followed by incubation with chromogenic substrate (0.45 mg/ml 4-nitroblue tetrazolium chloride and 0.18 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate; Roche) in a buffer containing 0.1 M Tris (pH 9.5), 1 M NaCl, 0.05 M MgCl2, 0.1% Tween 20, and 0.24 mg levamisole (Sigma Chemical Co., Taufkirchen, Germany). Sections were counterstained in 1% methyl green.
TUNEL Assay/Vessel Staining.
Staining for apoptotic cells was performed on 5-µm frozen sections using in situ cell-death detection kit-fluorescein (Roche). Subsequently, endothelial cells were stained with MECA32 (5 µg/ml; BD PharMingen) and Cy3-conjugated antirat IgG Fab-fragments (2 µg/ml; Dianova).
Intravital Microscopy.
Intravital microscopy was performed as described previously for the rat (25)
. The pancreatic head was immobilized in a 37°C immersion chamber with Ringer’s solution. Animals received i.v. injections of 1 µg/g FITC-labeled, autologous mouse erythrocytes to measure erythrocyte velocity, followed by 50 µg/g FITC-labeled albumin (Sigma Chemical Co.) to obtain maximal vessel contrast for video recording of morphology and to evaluate vessel diameters.
RNA, cDNA, Quantitative PCR.
RNA was prepared from snap-frozen tumors using RNeasy mini kit (Qiagen, Hilden, Germany). Three µg of RNA were treated with DNaseI (Promega, Mannheim, Germany), and cDNA synthesis was primed with random hexamers (Amersham Pharmacia, Freiburg, Germany), followed by reverse transcription using 1 unit of M-MLV (Life Technologies, Inc.)/15 µl. Reaction was stopped with 160 µl of H2O. cDNA (2.5 µl) was analyzed using real-time PCR TaqMan technology (Applied Biosystems, Weiterstadt, Germany). The mouse hypoxanthine phosphoribosyltransferase (HPRT) gene served as the internal standard. The following primers and probes were used: HPRT (amplicon length: 93 bp): FP, 5'-ACACCTGCTAATTTTACTGGCAACA-3'; RP, 5'-TGGAAAAGCCAAATACAAAGCCTA-3'; probe, 5'-CTCGTATTTGCAGATTCAACTTGCGCTCATC-3'; IFN- (95-bp): FP, 5'-ATCTGGCTCTGCAGGATTTTCA-3'; RP, 5'-TCAAGTGGCATAGATGTGGAAGAA-3'; probe, 5'-CCTTTTGCCAGTTCCTCCAGATATCCAAGAAG-3'; TNF-
(94 bp): FP, 5'-ACAAGGCTGCCCCGACTA-3'; RP, 5'-CTTGACGGCAGAAGGAGGTT-3'; probe: 5'-CCCACACCGTCAGCCGATTTGC-3'; Mig (144 bp): FP, 5'-GAGGAACCCTAGTGATAAGGAATGC-3'; RP, 5'-TCTTCAGTGTAGCAATGATTTCAGTTT-3'; probe: 5'-ATCAGCACCAGCCGAGGCACG-3'; IP10 (108 bp): FP, 5'-CCTTCACCATGTGCCATGC-3'; RP, 5'-TCTTACATCTGAAATAAAAGAGCTCAGGT-3'; probe, 5'-CCCCACACCCTCCTTGTCCTCCC-3'.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, which was not significantly increased when compared with untreated tumors. For adoptive transfers, lymph node cells from transgenic mice expressing a T-cell receptor specific for Tag (TagTCR1) were activated in vitro. Transferred cells represent a pool of lymphocytes with 10% transgenic CD4+ T cells, but otherwise there is a normal CD4+:CD8+ T-cell ratio (24)
. Adoptive transfer in nonirradiated RIP1-Tag5 mice resulted in an accumulation of T cells at the periphery of solid tumors as early as 2 days after transfer. These cells, however, failed to efficiently penetrate into tumor tissue (Fig. 1
, C and D). In contrast, adoptively transferred T cells in irradiated mice migrated into solid tumors and accumulated over time (Fig. 1, E and F)
. This experiment demonstrates that accessibility of tumor-specific lymphocytes to solid tumors requires "conditioning" of the organ by irradiation.
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Tag expression in RIP1-Tag5 mice starts at 10 weeks of age and results in the stepwise development of insulinomas (19)
. The incidence of multifocal islet tumors increased with age and correlated with the amount of Tag present in a given pancreas (Fig. 2A)
. To examine whether enhanced T-cell extravasation correlates with the rejection of already established tumors in RIP1-Tag5 mice, 22-week-old mice were irradiated and received a single adoptive transfer of 5 x 106 TagTCR lymphocytes 3 weeks later. Treated animals showed no survival advantage over the untreated control group (data not shown). All of the transgenic mice developed insulinomas and died of hypoglycemia between week 27 and 34. Clearly, infiltrating activated antitumor lymphocytes are either poor effectors or they do not survive long enough to impact tumor growth. To monitor the fate of adoptively transferred T cells, we analyzed migration and frequency of TagTCR and CD4+ double-positive cells for up to 20 days after transfer by using flow cytometry. Tag-specific CD4+ T cells preferentially expanded in the draining lymph nodes of the pancreas and accumulated in tumor tissue reaching maximum numbers at day 7 (Fig. 2B)
. Although no tumor cells were found within draining lymph nodes (data not shown), Tag is efficiently presented in pancreatic lymph nodes of RIP1-Tag5 mice and naïve 5,6-carboxy-succinimidyl-fluorescein-ester (CFSE)-labeled TagTCR lymphocytes are primed to proliferate.4
In addition, up-regulation of T-cell receptor expression was exclusively observed on T cells populating the pancreatic lymph nodes and solid tumors at day 7 after adoptive transfer (Fig. 2C)
. In vivo expansion, however, was short lived and rapidly declined within 20 days after transfer. These data demonstrate that, after irradiation and adoptive transfer, highly activated antitumor lymphocytes are initially found at tumor and tumor-draining sites. It is likely that these cells disappear by activation-induced cell death (26
, 27)
. The small remaining T-cell population infiltrating tumors at day 20 is unable to prevent progressive tumor growth.
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. A group of 25-week-old tumor-bearing mice was again irradiated, followed by repeated injections of 2.5 x 106 activated antitumor TagTCR1 cells. Control groups consisted of transgenic mice that received: (a) repetitive adoptive transfers without prior irradiation; (b) irradiation alone; or (c) irradiation and repeated injections of ConA-activated lymphocytes. Blood glucose levels and body weight were monitored every 10 days. None of the mice developed diabetes or dramatic weight loss during therapy (data not shown). All of the control animals became hypoglycemic because of the growth of insulin-secreting tumors, and mice in groups (b) and (c) died between weeks 26 and 34 (Fig. 3B)
, similar to untreated RIP1-Tag5 mice. Mice receiving adoptive transfers without irradiation (group a) had a minor survival advantage compared with other control mice (Fig. 3B
; P = 0.0129); a similar survival advantage is observed in RIP1-Tag5 x TagTCR1 double transgenic mice.4
Notably, ConA-activated T cells infiltrated tumor tissue but were unable to prevent tumor growth (data not shown). In contrast, the life span of transgenic mice was dramatically increased when irradiation was combined with repeated transfers of Tag-specific lymphocytes. At the age of 46 weeks, 27 of 32 treated mice were still alive. Therapy with Tag-primed lymphocytes derived from C3H mice instead of TagTCR cells was equally efficient (data not shown). The same therapeutic outcome was observed in T-cell-deficient as well as immunocompetent mice, which indicated that potential intrinsic regulatory mechanisms are negligible during the effector phase in these studies (Fig. 3B)
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. Again, this result shows that the efficacy of adoptive transfers depends strictly on an initial radiation stimulus and, in addition, leads to complete macroscopic tumor rejection.
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. Only in rare cases, did irradiation treatment alone cause some discoloring that resulted in tumors with a "patchy" red/white appearance. The change in color evolved gradually and was most prominent after the second adoptive transfer, which suggests a potential role of infiltrating lymphocytes. Histological analysis of tumors, recovered 6 weeks after irradiation alone, showed a mild T-cell infiltration (Fig. 4, B and C)
and a Tag expression pattern similar to untreated tumors (Fig. 4D)
. In contrast, white tumor nodules displayed an extremely high degree of lymphocytic infiltration with colocalization of CD4+ and CD8+ T cells (Fig. 4, E and F)
. Lymphocyte penetration into tumor tissue correlated with a decreased number of Tag-expressing tumor cells (Fig. 4G)
, most likely because of immune-mediated tumor cell destruction. After the second adoptive transfer, the number of intrapancreatic lymph nodes was increased by a factor of 2. Thus, de novo formation of lymph node tissue in close vicinity to tumors may contribute to therapeutic efficacy.
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. Because tumor-cell apoptosis has been reported to be an integral part of the RIP-Tag tumorigenesis (30)
, we infer that the TUNEL assay detects apoptotic tumor cells (Fig. 5A)
. A moderate increase in the apoptotic index was observed in irradiated tumors (data not shown) and in tumors after irradiation and two adoptive transfers (Fig. 5C)
. Surprisingly, combined irradiation/transfer therapy did not induce endothelial cell death, but enhanced vessel density in the tumors of treated mice (Fig. 5C)
. Neither irradiation nor adoptive transfers alone induced vessel death or any morphological changes to the vasculature (data not shown). We then used intravital microscopy to analyze tumor vessel morphology and hemodynamic parameters in vivo. Untreated RIP1-Tag5 tumors displayed an irregular vascular network with large vessels juxtaposed to small vessels (Fig. 5B)
, loss of vessel hierarchy, and zones of neovascularization next to large hemorrhagic areas. However, tumors of white appearance obtained after therapy (n = 3), have a uniform vascularity (Fig. 5D)
. The majority of vessels (83%) displays diameters of 10 µm, thus resembling capillaries of the normal circulation. In addition, structurally defined arterioles, capillaries, and venules become apparent, whereas vascular sinusoids and lacunas, typical markers of high angiogenic activity, were absent (data not shown). Erythrocyte velocity was not affected by alteration in vessel morphology (data not shown). Collectively, these data show that irradiation and subsequent massive lymphocytic infiltration transform the aberrant, highly angiogenic vasculature into a network of small capillaries of normal appearance, most likely because of the inhibition of ongoing angiogenesis.
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mRNA in RIP1-Tag5 tumors, and a 2-fold up-regulation of TNF- mRNA, when compared with untreated mice or mice receiving adoptive transfer alone (Fig. 6A)
. Elevated IFN- expression correlated with enhanced expression of two distinct members of the CXC-chemokine family, namely Mig and IP10 (Fig. 6A)
. These findings imply that irradiation alone has a moderate, but distinct effect on the tumor microenvironment that is not sufficient to prevent tumor growth but that facilitates lymphocyte extravasation. Subsequently, migration of adoptively transferred lymphocytes into tumor tissue led to a dramatic increase in IL-12 (data not shown), IFN-, TNF-, Mig, and IP10 expression (Fig. 6A)
. Although IFN- and TNF- in themselves enhance immunological recognition of tumor cells (31
, 32)
, they are potent inducers of Mig and IP10 and can also act synergistically (33)
. The antitumor effects of IL-12 have been attributed to an up-regulation of IFN-, which in turn induces Mig and IP10 expression (34)
. In addition, Mig and IP10 are shown to have antiangiogenic effects in in vivo Matrigel assays (35)
. Because reduced angiogenesis is a feature of regressing RIP1-Tag5 tumors (Figs. 4A
and 5, C and D
), we analyzed chemokine expression and localization in tumor tissue by in situ hybridization. A few Mig- or IP10-positive cells were present in irradiated tumors (Fig. 6, B and C)
, but a dramatic increase became apparent after combined irradiation/transfer therapy (Fig. 6, E and F)
. Tumor cells did not secrete Mig or IP10. On the basis of the expression patterns shown by in situ hybridization and immunohistochemistry (Fig. 6, D and G)
, we infer that a subset of infiltrating CD11b+ monocytes/macrophages display a high chemokine expression. The strong correlation between RIP1-Tag5 tumor regression and high levels of Mig and IP10, together with their documented antiangiogenic and chemotactic activities (35
, 36)
, implies a crucial role for these factors in reestablishing a uniform vascularization and in recruiting activated effector cells.
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DISCUSSION |
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In humans and mice, immunogenic tumors can coexist with antitumor lymphocytes without any impact on tumor progression (1 , 21) . Furthermore, there is evidence for tolerization of self-specific T cells in the presence of a growing tumor (1 , 37) . Indeed, in RIP1-Tag5 mice, we cannot rule out the presence of regulatory mechanisms that interfere with the initial priming of the endogenous immune response. In the present study, we focused on the effector arm of antitumor immunity by performing adoptive transfers with a mixture of activated lymphocytes, including MHC class II-restricted T cells with anti-Tag specificity and CD8+ T cells of the normal repertoire. It is well accepted that CD4+ T cells have the capacity for tumor rejection, but tumor immunity may require a collaborative effort of multiple immunological effectors (38) . Although transferred anti-Tag TCR cells proliferate in vivo after encountering the antigen, their life span is short. In the autochthonous RIP1-Tag5 tumor model, we show that the elimination of established tumors by immune cells occurs over a period of weeks, not days, which emphasizes the need for effector cells to persist in the host. Repetitive infusions are required to maintain a consistently high level of antitumor effector cells. In a clinical setting, vaccination strategies may provide a more efficient way to generate and restimulate sufficient numbers of antitumor lymphocytes.
Because we had previously hypothesized a crucial role for the microenvironment in tumor escape from immune destruction (21) , the failure of adoptive transfers alone to prolong the life of transgenic mice was not unexpected. Indeed, there is precedence for progressive tumor growth even in the presence of highly activated T cells specific for a tumor antigen (4 , 14 , 19) . Viral infections, for instance, have been shown to slightly increase the efficacy of antitumor responses, most likely because of bystander effects (4 , 14) . Similarly, a locally applied infectious agent, such as bacillus Calmette-Guérin (BCG), triggers mononuclear cell infiltration into the superficial layers of the bladder and is used in the treatment of bladder cancer (39) . Moreover, genetically engineered tumor cells, which locally increase cytokine concentrations, interfere with tumor growth in mouse transplantation models (11 , 31 , 32) . Thus, the concept that inflammatory mediators enhance tumor regression is not new, but has been attributed mainly to a general boost in effector function. Similarly, irradiation of the tumor-bearing host before the administration of ex vivo activated tumor-infiltrating lymphocytes was shown to be more effective than adoptive transfers alone (6 , 40) . However, in those experiments, the effect of irradiation was mainly attributed to a survival advantage of adoptively transferred cells and the elimination of a postulated, intrinsic, cellular suppressor activity (41) .
In this study, we show that irradiation of tumor-bearing RIP1-Tag5 mice with a dose that does not affect tumor growth, "conditions" solid tumors and renders them susceptible to lymphocyte infiltration. Subsequent tumor rejection is observed only when irradiation is combined with the action of tumor-antigen-specific T cells. Infiltration of nonspecifically primed T cells has no impact on tumor growth. Strikingly similar observations were made in a transgenic mouse model for liver autoimmunity, in which "conditioning" of the organ by irradiation or bacterial infection, is a prerequisite for tissue infiltration, and only self-reactive lymphocytes cause liver damage (42) . Thus, our data present evidence for the hypothesis that, in a noninflammatory environment, effector and target cells can coexist without initiating autoimmunity or tumor rejection (43) .
We also demonstrate that irradiation changes the tumor microenvironment by increasing the expression of adhesion molecules, cytokines, and chemokines. The effect is pleiotropic and surprisingly sustained, such that even 3 weeks after irradiation, an infusion of activated, self-specific T cells causes tissue damage. We have evidence that tumor escape in RIP1-Tag5 mice is linked to the sequential transformation of the vasculature during angiogenesis.5
Solid tumors display an aberrant vasculature with a loss of vessel hierarchy and leukocyte adhesion. Reduced leukocyte adherence to tumor endothelium has also been demonstrated in other animal models (25
, 44)
, and angiogenic stimuli are known to directly suppress expression of multiple adhesion molecules on tumor-associated vessels (45)
. It is, therefore, reasonable to speculate that radiation-induced changes to the endothelium reverse the nonadhesive phenotype of tumor endothelium and, thus, directly facilitate leukocyte-endothelium interactions. Although radiation therapy kills normal endothelial cells (22)
, we did not observe apoptosis in tumor vessels of irradiated RIP1-Tag5 mice. It is possible that a proangiogenic environment protects tumor endothelium from apoptosis. For instance, in vitro, high levels of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) confer radiation-resistance on normal endothelial cells (28
, 29)
. Nevertheless, combined irradiation/adoptive transfer therapy showed profound effects on RIP1-Tag5 tumors such as a dramatic transformation of the vasculature into a uniform network of capillaries with almost normal appearance. Again, no endothelial cell death or necrosis was obvious, but we observed a reduction in Tag+ cells and dramatically enhanced expression of IFN-, TNF-, and the angiostatic chemokines Mig and IP10. In immunocompromised mice, intratumoral Mig treatment reduces the tumor burden either by decreasing vessel density (46)
or by inducing endothelial cell death and necrosis (47)
. Our data imply that elevated expression of endogenous Mig and IP10 in RIP1-Tag5 tumors plays a crucial role in reestablishing a more normal, functional vasculature, which clearly argues for an angiogstatic effect of Mig and IP10 under physiological conditions. Mig and IP10 also display chemotactic properties and enhance leukocyte adhesion in vitro (33
, 36)
. Because we observed not only decreased angiogenic activity in RIP1-Tag5 tumors but also massive leukocytic infiltration, we propose a dual role for Mig and IP10 in irradiation-triggered tumor rejection, namely, inhibiting angiogenesis and promoting lymphocyte chemotaxis. Although it is not clear which cell types secrete high amounts of Mig and IP10 during antitumor therapy, we show that a subset of infiltrating CD11b+ cells, but not tumor cells, are likely to express high levels of both chemokines. Contribution by other cell types, e.g., endothelial cells, cannot be excluded.
As a potential sequence of events, we postulate that a proinflammatory stimulus activates the host immune system and enables monocytes/macrophages to gain access to tumors. The permissive environment, including an altered vessel morphology, provides all of the necessary components for a localized, antigen-driven immune response, in which CD4+ T cells mediate specificity and recruit CD8+ T cells and other effector cells, like monocytes/macrophages. Then, convergence and collaboration of leukocytes within the tumor ultimately result in its destruction. In the present study, inflammation was induced by irradiation, but more specific mediators such as immunostimulatory bacterial DNA should also be effective. Irradiation alone induces proinflammatory events, which do not reach a critical threshold to activate the endogenous immune system derived from bone marrow reconstitution. It is only by subsequently infusing activated effector cells that tumor rejection occurs. However, the infused cells have a short life span, necessitating repeated transfers for a sustained antitumor response.
Although we have increasing knowledge about molecules such as Mig and IP10, and their functions during an antitumor response, no single agent has thus far been identified that is able to trigger the entire complex cascade of events needed to eradicate solid tumors. In human tumors, the degree of infiltration depends on the tumor type and is variable and unpredictable (21) . We envision that more promising immunotherapeutic strategies against cancer will use a combination of nonspecific inflammatory stimuli to optimize T-cell infiltration, with continuous effector priming at the tumor site to elicit a sustained antitumor inflammatory response.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 405), the European Community (QLG1-CT-1999-00202) and a postdoctoral grant of the Infektionsforschung-Stipendienprogramm. 
2 To whom requests for reprints should be addressed, at Department of Molecular Immunology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Phone: 49-6221-423754; Fax: 49-6221-401629; E-mail: r.ganss{at}dkfz.de.
3 The abbreviations used are: Tag, SV40 T antigen; RIP, rat insulin gene promoter; VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; ConA, concanavalin A; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-fluorescein nick-end labeling; Mig, monokine induced by IFN-; IP10, interferon-inducible protein 10; IL, interleukin; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TNF, tumor necrosis factor.
4 Ruth Ganss, unpublished observations.
5 E. Ryschich, J. Schmidt, E. Klar, G. J. Hämmerling, and R. Ganss. Transformation of the microvascular system during multistage tumorigenesis, submitted for publication.
Received 7/19/01. Accepted 12/27/01.
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S. Matsumura, B. Wang, N. Kawashima, S. Braunstein, M. Badura, T. O. Cameron, J. S. Babb, R. J. Schneider, S. C. Formenti, M. L. Dustin, et al. Radiation-Induced CXCL16 Release by Breast Cancer Cells Attracts Effector T Cells J. Immunol., September 1, 2008; 181(5): 3099 - 3107. [Abstract] [Full Text] [PDF]
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T. Iuchi, S. Teitz-Tennenbaum, J. Huang, B. G. Redman, S. D. Hughes, M. Li, G. Jiang, A. E. Chang, and Q. Li Interleukin-21 Augments the Efficacy of T-Cell Therapy by Eliciting Concurrent Cellular and Humoral Responses Cancer Res., June 1, 2008; 68(11): 4431 - 4441. [Abstract] [Full Text] [PDF]
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A. Jorritsma, A. D. Bins, T. N.M. Schumacher, and J. B.A.G. Haanen Skewing the T-Cell Repertoire by Combined DNA Vaccination, Host Conditioning, and Adoptive Transfer Cancer Res., April 1, 2008; 68(7): 2455 - 2462. [Abstract] [Full Text] [PDF]
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S. B. J. Wong, R. Bos, and L. A. Sherman Tumor-Specific CD4+ T Cells Render the Tumor Environment Permissive for Infiltration by Low-Avidity CD8+ T Cells J. Immunol., March 1, 2008; 180(5): 3122 - 3131. [Abstract] [Full Text] [PDF]
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P. Otahal, B. B. Knowles, S. S. Tevethia, and T. D. Schell Anti-CD40 Conditioning Enhances the TCD8 Response to a Highly Tolerogenic Epitope and Subsequent Immunotherapy of Simian Virus 40 T Antigen-Induced Pancreatic Tumors J. Immunol., November 15, 2007; 179(10): 6686 - 6695. [Abstract] [Full Text] [PDF]
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A. Perez-Diez, N. T. Joncker, K. Choi, W. F. N. Chan, C. C. Anderson, O. Lantz, and P. Matzinger CD4 cells can be more efficient at tumor rejection than CD8 cells Blood, June 15, 2007; 109(12): 5346 - 5354. [Abstract] [Full Text] [PDF]
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B. Zhang, N. A. Bowerman, J. K. Salama, H. Schmidt, M. T. Spiotto, A. Schietinger, P. Yu, Y.-X. Fu, R. R. Weichselbaum, D. A. Rowley, et al. Induced sensitization of tumor stroma leads to eradication of established cancer by T cells J. Exp. Med., January 22, 2007; 204(1): 49 - 55. [Abstract] [Full Text] [PDF]
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P. Otahal, T. D. Schell, S. C. Hutchinson, B. B. Knowles, and S. S. Tevethia Early Immunization Induces Persistent Tumor-Infiltrating CD8+ T Cells against an Immunodominant Epitope and Promotes Lifelong Control of Pancreatic Tumor Progression in SV40 Tumor Antigen Transgenic Mice. J. Immunol., September 1, 2006; 177(5): 3089 - 3099. [Abstract] [Full Text] [PDF]
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E. A. Reits, J. W. Hodge, C. A. Herberts, T. A. Groothuis, M. Chakraborty, E. K.Wansley, K. Camphausen, R. M. Luiten, A. H. de Ru, J. Neijssen, et al. Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy J. Exp. Med., May 15, 2006; 203(5): 1259 - 1271. [Abstract] [Full Text] [PDF]
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A. I. Garbe, B. Vermeer, J. Gamrekelashvili, R. v. Wasielewski, F. R. Greten, A. M. Westendorf, J. Buer, R. M. Schmid, M. P. Manns, F. Korangy, et al. Genetically Induced Pancreatic Adenocarcinoma Is Highly Immunogenic and Causes Spontaneous Tumor-Specific Immune Responses Cancer Res., January 1, 2006; 66(1): 508 - 516. [Abstract] [Full Text] [PDF]
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A. A. Lugade, J. P. Moran, S. A. Gerber, R. C. Rose, J. G. Frelinger, and E. M. Lord Local Radiation Therapy of B16 Melanoma Tumors Increases the Generation of Tumor Antigen-Specific Effector Cells That Traffic to the Tumor J. Immunol., June 15, 2005; 174(12): 7516 - 7523. [Abstract] [Full Text] [PDF]
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F. Avogadri, C. Martinoli, L. Petrovska, C. Chiodoni, P. Transidico, V. Bronte, R. Longhi, M. P. Colombo, G. Dougan, and M. Rescigno Cancer Immunotherapy Based on Killing of Salmonella-Infected Tumor Cells Cancer Res., May 1, 2005; 65(9): 3920 - 3927. [Abstract] [Full Text] [PDF]
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M. Berger, G. Bergers, B. Arnold, G. J. Hammerling, and R. Ganss Regulator of G-protein signaling-5 induction in pericytes coincides with active vessel remodeling during neovascularization Blood, February 1, 2005; 105(3): 1094 - 1101. [Abstract] [Full Text] [PDF]
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S. Demaria, N. Kawashima, A. M. Yang, M. L. Devitt, J. S. Babb, J. P. Allison, and S. C. Formenti Immune-Mediated Inhibition of Metastases after Treatment with Local Radiation and CTLA-4 Blockade in a Mouse Model of Breast Cancer Clin. Cancer Res., January 15, 2005; 11(2): 728 - 734. [Abstract] [Full Text] [PDF]
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G. M. Bendle, A. Holler, L.-K. Pang, S. Hsu, M. Krampera, E. Simpson, and H. J. Stauss Induction of Unresponsiveness Limits Tumor Protection by Adoptively Transferred MDM2-Specific Cytotoxic T Lymphocytes Cancer Res., November 1, 2004; 64(21): 8052 - 8056. [Abstract] [Full Text] [PDF]
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M. Chakraborty, S. I. Abrams, C. N. Coleman, K. Camphausen, J. Schlom, and J. W. Hodge External Beam Radiation of Tumors Alters Phenotype of Tumor Cells to Render Them Susceptible to Vaccine-Mediated T-Cell Killing Cancer Res., June 15, 2004; 64(12): 4328 - 4337. [Abstract] [Full Text] [PDF]
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N. Garbi, B. Arnold, S. Gordon, G. J. Hammerling, and R. Ganss CpG Motifs as Proinflammatory Factors Render Autochthonous Tumors Permissive for Infiltration and Destruction J. Immunol., May 15, 2004; 172(10): 5861 - 5869. [Abstract] [Full Text] [PDF]
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K. Chamoto, T. Tsuji, H. Funamoto, A. Kosaka, J. Matsuzaki, T. Sato, H. Abe, K. Fujio, K. Yamamoto, T. Kitamura, et al. Potentiation of Tumor Eradication by Adoptive Immunotherapy with T-cell Receptor Gene-Transduced T-Helper Type 1 Cells Cancer Res., January 1, 2004; 64(1): 386 - 390. [Abstract] [Full Text] [PDF]
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S. Teitz-Tennenbaum, Q. Li, S. Rynkiewicz, F. Ito, M. A. Davis, C. J. Mcginn, and A. E. Chang Radiotherapy Potentiates the Therapeutic Efficacy of Intratumoral Dendritic Cell Administration Cancer Res., December 1, 2003; 63(23): 8466 - 8475. [Abstract] [Full Text] [PDF]
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M. Chakraborty, S. I. Abrams, K. Camphausen, K. Liu, T. Scott, C. N. Coleman, and J. W. Hodge Irradiation of Tumor Cells Up-Regulates Fas and Enhances CTL Lytic Activity and CTL Adoptive Immunotherapy J. Immunol., June 15, 2003; 170(12): 6338 - 6347. [Abstract] [Full Text] [PDF]
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L. Peng, J. Kjaergaard, G. E. Plautz, M. Awad, J. A. Drazba, S. Shu, and P. A. Cohen Tumor-Induced L-Selectinhigh Suppressor T Cells Mediate Potent Effector T Cell Blockade and Cause Failure of Otherwise Curative Adoptive Immunotherapy J. Immunol., November 1, 2002; 169(9): 4811 - 4821. [Abstract] [Full Text] [PDF]
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)]; mesenteric LNs (
); pancreatic, tumor-draining LNs (
); ß-cell tumors (
); and spleen (
) at different time points after one adoptive transfer. Two mice per group and time point were analyzed. C, expression of T-cell receptor on TagTCR/CD4+ T cells; a representative staining of somatic (som), mesenteric (mes), and pancreatic (panc) LN cells and lymphocytes from ß-cell tumors, isolated 7 days after the first adoptive transfer. The histograms, the fluorescence intensity corresponding to the expression level of TagTCR in a total of 5,000 cells from somatic and mesenteric LNs and of 1,500 and 10,000, respectively, pancreatic and tumor-derived lymphocytes. The X axis, fluorescence intensity; the Y axis, relative cell number.
) Rag-1-/- bone marrow mice (n = 16) and immunocompetent (
) wild-type bone marrow mice (n = 16; P > 0.0001). Control mice received multiple adoptive transfers without irradiation (
; n = 12), irradiation treatment alone (
; n = 12), or irradiation treatment in combination with adoptively transferred ConA-stimulated lymphocytes (
), and mice after a combination of irradiation and two consecutive transfers (