Characterization of the Mouse IFN-L Ligand-Receptor System: IFN-Ls Exhibit Antitumor Activity Against B16 Melanoma

Research Article

Characterization of the Mouse IFN-L Ligand-Receptor System: IFN-Ls Exhibit Antitumor Activity against B16 Melanoma

Ahmed Lasfar,1 Anita Lewis-Antes,1 Sergey V. Smirnov,1 Shubha Anantha,1 Walid Abushahba,1 Bin Tian,1 Kenneth Reuhl,3 Harold Dickensheets,4 Faruk Sheikh,4 Raymond P. Donnelly,4 Elizabeth Raveche,2 and Sergei V. Kotenko1

Departments of 1Biochemistry and Molecular Biology and 2Pathology and Laboratory Medicine, University of Medicine and Dentistry-New Jersey Medical School, Newark, New Jersey; 3Department of Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey; and 4Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland

Abstract (2, 3). In contrast, studies with IFN-g and IFN-g receptor knockout mice (4–6) and in people bearing inactive variant forms of Recently discovered type III IFNs (IFN-L) exert their antiviral g and immunomodulatory activities through a unique receptor the IFN- receptor (7, 8) revealed that the antiviral activity of g g complex composed of IFN-LR1 and interleukin-10 receptor 2. IFN- is not its primary physiologic function. IFN- is a Th1 To further study type III IFNs, we cloned and characterized cytokine that stimulates cell-mediated immune responses that are mouse IFN-L ligand-receptor system. We showed that, similar critical for the development of host protection against intracel- to their human orthologues, mIFN-L2and mIFN- L3 signal lular parasites and is a part of antiviral and antitumor defenses through the IFN-L receptor complex, activate IFN stimulated (9–11). Therefore, both type I and type II IFNs stimulate a variety gene factor 3, and are capable of inducing antiviral protection of innate and adaptive immune mechanisms that contribute to and MHC class I antigen expression in several cell types eliminating viral infections (1, 9–13). including B16 melanoma cells. We then used the murine B16 IFNs are part of a larger family of proteins that also includes six interleukin-10 (IL-10)-related cytokines: IL-10, IL-19, IL-20, IL-22, melanoma model to investigate the potential antitumor IL-24, and IL-26. These diverse cytokines were first grouped into activities of IFN-Ls. We developed B16 cells constitutively the same family because they all use receptors that share expressing murine IFN-L2(B16.IFN-L 2cells) and evaluated common motifs in their extracellular domains and, therefore, their tumorigenicity in syngeneic C57BL/6 mice. Although form the class II cytokine receptor family (CRF2). Subsequently, constitutive expression of mIFN-L2in melanoma cells did not IFNs and IL-10-related cytokines are referred to as CRF2 affect their proliferation in vitro, the growth of B16.IFN-L2 cytokines. The most recent addition to the CRF2 family, type III cells, when injected s.c. into mice, was either retarded or IFNs or IFN-Es (also known as IL-28/29), show structural features completely prevented. We found that rejection of the modified of the family of IL-10-related cytokines but possess antiviral tumor cells correlated with their level of IFN-L2expression. activity, which defines them as a new type of IFNs (1, 14–16). We then developed IFN-L-resistant B16.IFN-L2cells (B16.IFN- Antiviral activity of IFN-Es was shown for several viruses in L2Res cells) and showed that their tumorigenicity was also in vitro experiments and also in a mouse model of vaccinia virus highly impaired or completely abolished similar to B16.IFN-L2 infection (1, 14, 15, 17). cells, suggesting that IFN-Ls engage host mechanisms to Three distinct human IFN-E proteins, IFN-E1, IFN-E2, and IFN- inhibit melanoma growth. These in vivo experiments show the E3, have been characterized and found to bind and signal through antitumor activities of IFN-Lsandsuggesttheirstrong a receptor complex composed of the unique IFN-ER1 chain and the therapeutic potential. (Cancer Res 2006; 66(8): 4468-77) IL-10 receptor 2 (IL-10R2) chain, which is shared with the IL-10, IL- 22, and IL-26 receptor complexes (14, 15, 18–22). In contrast, all Introduction type I IFNs exert their biological activities through a heterodimeric IFNs are the key cytokines in the establishment of a receptor complex composed of IFN-aR1 and IFN-aR2 chains, and multifaceted antiviral response. Three distinct types of IFNs (type IFN-g engages IFN-gR1 and IFN-gR2 chains to assemble a I, II, and III) are recognized based on their structural features, functional receptor complex. IFNs activate primarily the Jak-signal receptor usage, and biological activities. Although all IFNs are transducers and activators of transcription (STAT) signal trans- important mediators of antiviral protection, their roles in antiviral duction pathway. IFN-g causes the phosphorylation of Jak1 and defense vary. Type I IFNs (IFN-a/h/N/q/n in humans) possess Jak2, leading primarily to the mobilization of STAT1. Type I and strong intrinsic antiviral activity and are able to activate a potent type III IFNs, acting through distinct receptors, stimulate similar antiviral state in a wide variety of cells (1). The essential role of signaling events that include activation of Jak1 and Tyk2 kinases the functional type I IFN system in the induction of antiviral and several latent transcriptional factors of the STAT family resistance is clearly shown in type I IFN receptor knockout mice including STAT1, STAT2, STAT3, STAT4, and STAT5 (15, 23). because such animals are highly susceptible to viral infections Activated STAT1, STAT3, and STAT5 form homodimers and heterodimers and bind to an IFN-g activation site (GAS) in the promoters of IFN-inducible genes. IFN receptor engagement also leads to the activation of the IFN-stimulated gene factor 3 (ISGF3) Requests for reprints: Sergei V. Kotenko, Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry-NewJersey Medical School, transcription complex. ISGF3 is composed of STAT1 and STAT2 Newark, NJ 07103. Phone: 973-972-3134; Fax: 973-972-5594; E-mail: kotenkse@ and IFN regulatory factor 9 (ISGF3 or p48). ISGF3 regulates gene umdnj.edu. I2006 American Association for Cancer Research. transcription by binding to an IFN-stimulated response element doi:10.1158/0008-5472.CAN-05-3653 (ISRE). Consequently, biological activities induced by either type I

Cancer Res 2006; 66: (8). April 15, 2006 4468 www.aacrjournals.org

V V V or type III IFNs are also similar and include induction of antiviral 5 -GCCGCCCTTGTCACCTGCCGC-3 (mR12-1F), 5 -GGGATGTACATGT- V V protection and up-regulation of MHC class I antigen expression in GGCGGGCCGACCGGTGG-3 (mR12-2F), 5 -GCTATCAGTAGAAGCAA- V V several cell types (15). GAGTGAG-3 (mR12-5R), and 5 -GCCTCTAGAGCTCTTTTGTCCCCTG- V Sequences of the murine IFN-Es and their receptor were GAGCCTC-3 (mR12-6R) and the same pool of cDNAs were used for nested PCR (mR12-1 and mR12-5 primers for the first round and mR12-2 previously reported by us (15, 17). Here we present detailed F R F E and mR12-6R primers for the second round) to amplify a cDNA fragment analysis of the mouse IFN- antiviral system and its comparison encoding the mIFN-ER1 extracellular domain. The resulting PCR product E with the human IFN- antiviral system. We showthat similar to was digested with BsiWI and XbaI restriction endonucleases and cloned E their human orthologues, mouse IFN- s possess strong antiviral into KpnI and NheI restriction sites of either plasmid pEF-IL-10R1/mIFN- and immunomodulatory activities. Importantly, for the first time, ER1 or plasmid pEF3-IL-10R1/gR1, resulting in plasmids pEF-mIFN-ER1/ we show antitumor activity of IFN-Es against B16 melanoma, ER1 and pEF-mIFN-ER1/gR1, respectively. suggesting that type III IFNs may have therapeutic potential in The nucleotide sequences of the modified regions of all constructs were cancer treatment. verified in their entirety by DNA sequencing. The assigned GenBank accession numbers were as follows: mouse IFN-E2 and IFN-E3 cDNAs from 129/Sv strain, AY869695 and AY869696, respectively (17); mouse IFN-ER1 Materials and Methods cDNA, AY184375 (15); and mouse IFN-k1 gene from 129/Sv and FVB strains, Plasmid construction. Based on the sequence of the mouse genome, DQ340653 and DQ340654, respectively. primers were designed to amplify and clone the appropriate regions of the Cells, transfection, flow cytometry, and electrophoretic mobility cDNAs encoding mouse ligands and their receptor into corresponding shift assay. The 16-9 hamster-human somatic cell hybrid line and its vectors. These primers contained sequences homologous to coding regions derivative 16-9 cell line expressing the human chimeric IFN-E receptor of the corresponding genes and sequences recognized by restriction complex (hIFN-ER1/gR1 + hIL-10R2; ref. 15) were maintained in Ham’s F12 endonucleases to facilitate cloning of these genes. Mouse IFN-k2 and medium with 10% fetal bovine serum (FBS). Colorectal adenocarcinoma IFN-k3 genes were amplified by PCR with primers 5V-CCGGTAC- HT29 cells were maintained in RPMI medium with 10% FBS. COS-1 cells, a V V CATGCTCCTCCTGCTGTTGCCTCTGC-3 (mifnl-2F) and 5 -GAGAATTC- SV40-transformed fibroblast-like simian CV-1 cell line, mouse L929 (s.c. CAGGTCAGACACACTGGTCTCC-3V (mifnl-4R) and mouse genomic DNA connective tissue) cells, NIH 3T3 fibroblast-like cells, B16 melanoma cells, from 129/Sv strain, and cloned into the pcDEF3 (pEF) vector (24) with the and derivatives were grown in DMEM with 10% FBS. Primary keratinocytes use of KpnI and EcoRI restriction endonucleases, generating plasmids and bone marrow–derived macrophages were generated from C57BL/6 pEF-mIFN-E2gene and pEF-mIFN-E3gene. After transfection of these mice as described (26, 27). Splenocytes were isolated as follows: spleen was plasmids into COS-1 cells, total RNA was isolated from the transfectants, extracted, placed into RPMI medium with 5% FBS, and minced. After converted to cDNA, and amplified with mifnl-4R and either mifnl-2F or gravitational sedimentation of large tissue clumps, the remaining cells in V V 5 -CCGGATCCTGTCCCCAGGGCCACCAGGC-3 (mifnl-6F) primers to ob- suspension were harvested, pelleted by centrifugation, and used for tain mIFN-E2 and mIFN-E3 cDNA fragments, which were cloned into experiments. either the KpnI and EcoRI restriction sites of the pEF vector or the BamHI 16-9 and COS-1 cells were transfected as described (15). COS cell and EcoRI restriction sites of the pEF-SPFL vector (24), respectively, supernatants were collected at 72 hours and used as a source of the resulting in plasmids pEF-mIFN-E2, pEF-mIFN-E3, pEF-FL-mIFN-E2, and expressed proteins. pEF-mIFN-E2 or pEF-mIFN-E3 plasmid was transfected pEF-FL-mIFN-E3. Because plasmid pEF-SPFL encodes the IFN-gR2 signal into 0.5 Â 106 to 1 Â 106 B16 cells with the use of transIT-LT1 reagent peptide followed by the FLAG epitope (24), this abutted the reading (Mirus, Madison, WI) and G418-resistant colonies (800 Ag/mL) were frames of the IFN-Es to the frame of the FLAG epitope. Therefore, these harvested and tested for the production of mIFN-Es. plasmids encode mIFN-Es tagged at their NH2 terminus with the FLAG To assess the growth (proliferation rate) of parental and transfected B16 epitope (FL-mIFN-E2/3). The pEF-mIFN-E plasmids encode intact mIFN- cells, the cells were seeded at 0.5 Â 105 per well in six-well plates in Es with their own signal peptides. triplicates, grown at 37jC, and counted after 24, 48, 72, and 120 hours. Genomic DNA from a feral mouse and various mouse strains (129/Sv, To detect changes in MHC class I antigen (H-2Kb) expression, cells were C57BL/6, FVB, and CD1) and primers 5V-CCGGTACCATGGCTACAGTGT- treated for 72 hours with cytokines or cell-conditioned medium, harvested, b GCCTGCTGGGT-3V (mifnl-1F)and5V-CCGAATTCAGACACACTGGTCTT- and incubated with mouse monoclonal antibody against H-2K (eBioscien- V k CACTGGCC-3 (mifnl-3R) were used for PCR to amplify mouse IFN- 1 ces, San Diego CA), followed by incubation with FITC-goat antimouse gene fragment. The resulting PCR products were either cloned into pCR2.1 immunoglobulin (Sigma, St. Louis, MO). Cell-surface staining was analyzed vector (Invitrogen, Carlsbad, CA) and sequenced or subjected to direct by flowcytometry. sequencing with primers 5V-CCGGATCCTCCTTCCAAGCCCACCCCAACC-3V To evaluate STAT activation, cells were treated for 15 minutes at 37jC V V (mifnl-5F) and 5 -CCAGAATTCTCCACTGATGAG-3 (mifnl-10R). Feral mouse with various cytokines and used for electrophoretic mobility shift assay k IFN- 1 gene fragment was also amplified by PCR with primers mifnl-1F and (EMSA) experiments with GAS probe as described (15, 19). Human and E 5V-TTCGCTAGCGACACACTGGTCTTCACTGGC-3V (mifnl-15R) and cloned murine mIFN- s were from PeproTech, Inc. (Rocky Hill, NJ); mIFN-aAwas into the pEF2-X-FL vector (25) with the use of KpnI and NheI restriction from R&D Systems (Minneapolis, MN). endonucleases, generating plasmids pEF-mIFN-E1gene-FL. In this plasmid, Virus infection and antiviral protection. Vesicular stomatitis virus the stop codon of the mIFN-k1 gene encoded in the last exon (exon 5) was (VSV; an RNA rhabdovirus, Indiana strain) infected or uninfected B16 cells deleted and replaced with the FLAG epitope (mIFN-E1gene-FL). were disrupted and the RNA was isolated as described (15) and used for Mouse IFN-ER1 cDNA was cloned with the use of nested PCR as follows. nested reverse transcription-PCR (RT-PCR) with mIFN-E-specific primers A first round of PCR was done with primers 5V-GCTGCATCTTCCTA- with the use of the GeneAmp RT-PCR kit (Perkin-Elmer, Boston, MA). The

GAGGCTCCAG-3V(mR12-3F) and 5V-GAGAATGTGTAGATGGACCACCAG-3V first round was done with primers mifnl-2 and mifnl-4 and the second (mR12-8R) using cDNAs obtained from mouse bone marrowcells as round with mifnl-6 and mifnl-4. RNA samples were also used for RT-PCR template. The first-round PCR product was used as a template for a second with murine h-actin-specific primers. Antiviral assays were done essentially round of PCR amplification, with primers 5V-AGCGCTAGCGGCAATGCCCT- as described (28). After incubation with test IFNs for 24 hours, HT-29 or B16

CACTCTTGCTTC-3V (mR12-4F)and5V-GCGAATTCACCTGACCAAGTA- cells were challenged with VSV and then further incubated until controls ATCTCC-3V(mR12-7R), to generate the extracellular domain of mIFN-ER1, showed full killing by virus (1-2 days). Cells not killed were visualized by which was subsequently cloned into plasmid pEF3-IL-10R1/gR1 (19), using staining with crystal violet. NheI and EcoRI restriction endonucleases to excise a cDNA fragment Mice, tumor transplantation, and histologic analysis of tumor encoding the IFN-gR1 intracellular domain and replace it with that tissue. Female C57BL/6 mice, 6 to 8 weeks old, were obtained from Charles for mIFN-ER1, resulting in plasmid pEF-IL-10R1/mIFN-ER1. Primers River Laboratories, Inc. (Wilmington, MA). Mice were injected s.c. on the www.aacrjournals.org 4469 Cancer Res 2006; 66: (8). April 15, 2006

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2006 American Association for Cancer Research. Cancer Research flank with 0.5 Â 106 or 1 Â 106 cells in 0.1 mL of PBS and tumors grewin the The log-rank test was used to test whether survival curves were identical. All site of injection. Mice were checked for tumor growth by palpation of the statistical analyses were done with the use of the Survival package in injection site every 1 to 2 days. Mice were assigned tumor positive after first program R (29). detection of tumors; animals were marked and kept for further analyses of tumors. Mice that rejected the primary tumor cells were challenged s.c. with parental B16 cells on the opposite flank. Tissue extraction for histology was Results done in mice with similar tumor size. Animals with massive tumor burden Cloning and characterization of mouse IFN-Ls and their were euthanized. Extracted tumors were immediately fixed in 10% E phosphate-buffered formalin. Paraffin-embedded sections were stained receptor. There are three IFN- genes clustered on human E E with H&E and analyzed by an expert pathologist blinded to treatment chromosome 19 encoding highly homologous IFN- 1, IFN- 2, assignment. and IFN-E3 proteins (ref. 15; Fig. 1). Further analysis of this The Kaplan-Meier estimator was used to calculate the median survival genomic region suggested that, after the divergence of the IFN-k1 (tumor appearance) time and to derive tumor appearance (survival) curves. and IFN-k2 genes, a more recent duplication event occurred in

Figure 1. Mouse and human IFN-Es and IFN-ER1. The alignment of the amino acid sequences of human and mouse IFN-Es (accession numbers for hIFN-E1, hIFN-E2, hIFN-E3, mIFN-E2, and mIFN-E3 are AY184372, AY184373, AY184374, AY869695, and AY869696, respectively; A) and IFN-ER1 receptor chains (accession numbers for hIFN-ER1 and mIFN-ER1 are AF439325 and AY184376, respectively; D). The consensus sequence is shown on the bottom. Identical amino acids corresponding to the consensus sequence are shown in black outline with white lettering. Similar amino acids are shown in gray outline with white lettering. Amino acid residues are numbered starting from first Met residue (signal peptide amino acids are included). Predicted signal peptides and IFN-ER1 transmembrane domains are boxed. Potential glycosylation sites are underlined as well as intracellular Tyr-based motifs which are conserved between mIFN-ER1 and hIFN-ER1. B, schematic of the regions of human chromosome 19 and mouse chromosome 7 containing IFN-E gene clusters. Although mouse and human IFN-E loci are colinear, there are three functional IFN-E genes in humans whereas only two genes in the mouse genome encode functional proteins, mIFN-E2 and mIFN-E3. Sequence variations within the mIFN-k1W pseudogene are shown by PCR with primers corresponding to exons 1 and 3 of the gene and genomic DNAobtained from different mouse strains: CD1, FVB, C57BL/6, 129/Sv, and wild-type feral mouse (w.t.). C, conditioned media (10 AL) from COS-1 cells transfected with plasmids pEF-SPFL (mock), pEF-FL-hIFN-E1(FL-hIFN-k1), pEF-FL-IFN-E2(FL-hIFN-k2), pEF-FL-IFN-E3(FL-hIFN-k3), pEF-FL-mIFN-E2(FL-mIFN-k2), pEF-FL-mIFN-E3(FL-IFN-k3), and pEF-mIFN-E1gene-FL (mIFN-k1-FL) were evaluated by Western blotting with FLAG monoclonal antibody. The molecular weight markers are shown on the right.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2006 American Association for Cancer Research. Antitumor Activities of IFN-Es which a fragment containing the IFN-k1 and IFN-k2 genes was receptor is significantly altered by a short insertion and substi- copied and integrated back into the genome in a head-to-head tutions of several amino acid residues, resulting in a longer and orientation with the IFN-E1-IFN-E2 segment (Fig. 1B). Divergence more negatively charged region in the mouse receptor (18 of 20 within this region created the IFN-k3 gene, which is almost amino acids are negatively charged; Fig. 1D). Two tyrosines, Tyr343 identical to the IFN-k2 gene. However, in the duplicated fragment, and Tyr517, of human IFN-ER1 (hIFN-ER1) can independently the part which contained the IFN-k1 gene was extensively mutated mediate STAT2 activation by IFN-Es (23). Interestingly, the Tyr341- so that only separate pieces which do not encode a functional gene based motif of mIFN-ER1 (YLERP) shows similarities with that (IFN-k4W;Fig.1B) can be found in this region. surrounding Tyr343 of hIFN-ER1 (YIEPP). In addition, the COOH- Analysis of the mouse genome revealed that the region colinear terminal amino acid sequence of mIFN-ER1 containing Tyr533 with the human IFN-k (hIFN-k) gene cluster is located on (YLVRstop) is very similar to the COOH-terminal amino acid chromosome 7A3 and has a similar organization with the hIFN-k sequence of hIFN-ER1 containing Tyr517 (YMARstop). Therefore, locus (Fig. 1B). Two genes colinear with the hIFN-k2 and hIFN-k3 both the mouse and human IFN-ER1 chains contain similar genes seemed to be intact and were predicted to encode functional docking sites for STAT2 recruitment and activation, YAEXP and proteins, which were designated mouse IFN-E2 (mIFN-E2) and YAXRstop (where A is hydrophobic). Thus, Tyr341- and Tyr533- mIFN-E3 in accordance with the corresponding human genes. based motifs on mIFN-ER1 are also likely to mediate STAT2 Mouse IFN-E2 and IFN-E3 also showhigher sequence identity to recruitment and, therefore, mediate ISGF3 activation, which is human IFN-E2 and IFN-E3 than to hIFN-E1(Fig.1A). In contrast to responsible for most of the IFN-E-induced biological activities (23). the IFN-k2 and IFN-k3 genes, the mIFN-k1 gene seemed to have lost Cloning and sequence analysis of murine IFN-Es and IFN-ER1 the entire exon 2 and acquired a stop codon within exon 1; however, revealed that mIFN-E2 and mIFN-E3 are very similar to their exons 3, 4, and 5 are intact. To investigate whether these mutations human orthologues and that mouse genome does not encode are strain specific, the IFN-E1 genomic fragments from several functional IFN-E1 protein. Because there were variations in the mouse strains were cloned and sequenced. Although substantial intracellular domain of the mIFN-ER1 in comparison with hIFN- sequence variations have been revealed in the region between exons ER1, which could lead to differences in signaling and biological 1 and 3 (Fig. 1B), the stop codon within exon 1 was present in all the activities, we studied the mouse IFN-E ligand-receptor system. strains and exon 2 could not be predicted in any of the strains. Signaling by mouse IFN-Ls. We first used a previously The mouse IFN-k2 and IFN-k3 genes were cloned into the pEF described hamster cell line that expresses a modified human mammalian expression vector and transfected into COS-1 cells. IFN-E receptor complex (15) to showthat mIFN- Es can signal The individual mIFN-E cDNAs were synthesized from RNA isolated through human IFN-E receptor complex. Hamster cells transfected from the transfected COS cells and subsequently cloned into the with chimeric human IFN-ER1/gR1 and IL-10R2 were responsive to pEF-SPFL plasmid in-frame with the FLAG epitope (referred to both human and mouse IFN-Es as measured by STAT1 activation hereafter as FL-mIFN-Es). The immunoblots of conditioned media in EMSA and up-regulation of MHC class I antigen expression from transfected COS-1 cells revealed that secreted FL-mIFN-E2 (Fig. 2A, middle, and data not shown). Parental hamster cells were migrated on an SDS-PAGE gel as a broad band of f30 to 36 kDa unresponsive to either human or mouse IFN-Es (ref. 15; Fig. 2A, whereas FL-mIFN-E3 appeared as two distinct bands of f22 to 25 left). Interestingly, expression of murine IFN-ER1/gR1 alone and 35 kDa, respectively (Fig. 1C), suggesting possible glycosylation rendered hamster cells responsive to both human and mouse of mIFN-Es. Both mIFN-E2 and mIFN-E3 possess a potential site IFN-Es as determined by EMSA with a GAS DNA probe (Fig. 2A, for N-linked glycosylation (Asn105-Met-Thr in mIFN-E2and right), indicating that hamster IL-10R2 can dimerize with murine Asn107-Asp-Ser in mIFN-E3; Fig. 1A). Treatment with peptide IFN-ER1 to mediate signaling in response to either human or N-glycosidase F (PNGase F) reduced the apparent molecular mouse IFN-Es. These experiments showthat mouse and human weights of these proteins, confirming that they are glycosylated IFN-Es are not species specific. (ref. 17 and data not shown). In a search for mouse cell lines responsive to IFN-Es, we Because there were several open reading frames in the region screened several mouse cell lines by measuring up-regulation of between exons 1 and 3 of the mIFN-k1 gene, we investigated MHC class I antigen expression and STAT activation in response whether these frames could lead to the production of the modified to mIFN-Es. Several mouse fibroblast-like cell lines, such as L929 IFN-E1 protein. The entire mIFN-k1 gene was cloned from the wild- and NIH 3T3, did not respond to mIFN-Es but responded well to type strain into the pEF-X-FL mammalian expression vector in the mIFN-a (Figs. 2C and 3A, and data not shown). This correlated way that a potential IFN-E1 protein would be tagged on its COOH with earlier findings that several human cell types, such as primary terminus with the FLAG epitope. The plasmid was then transfected fibroblasts and human umbilical vein endothelial cells, do not into COS-1 cells and the conditioned medium was analyzed by express IFN-ER1 and therefore are not responsive to IFN-Es.5 In Western blotting for the presence of a FLAG-tagged protein. We contrast, mouse B16 melanoma cells strongly responded to both failed to detect the presence of a FLAG-tagged protein (Fig. 1C), mouse IFN-E and IFN-a (Figs. 2B and 3A). Using EMSA with either showing that the mouse IFN-k1 gene is a pseudogene. GAS or ISRE DNA probes, we showed that both type I and type III The mouse IFN-ER1 (mIFN-ER1) chain was also cloned and it is mIFNs induced formation of similar STAT-DNA-binding complexes f67% similar to its human counterpart (Fig. 1D). The receptor is in intact B16 cells (Fig. 2B). Treatment of B16 cells with either encoded on mouse chromosome 4D3. Although the mouse and mouse IFN-E or IFN-a stimulated formation of the ISGF3 complex, human IFN-ER1 sequences are very similar, only two of three which binds to ISRE probe and is specifically induced only by type I tyrosine residues of the human receptor intracellular domain are and type III IFNs (Fig. 2B, right). EMSA with the GAS probe showed conserved in the mouse orthologue. In addition, the mouse receptor contains three additional tyrosine residues. There is also a stretch of negatively charged residues close to the end of the human receptor intracellular domain. This region in the mouse 5 Unpublished observations. www.aacrjournals.org 4471 Cancer Res 2006; 66: (8). April 15, 2006

contrast, murine and human type I IFNs do not have substantial activity on each other’s cells (30). Consistent with our initial findings (15), we also found that mIFN-Es induced antiviral protection in mouse B16 cells or human HT29 cells infected with VSV, an RNA rhabdovirus (Fig. 3B and data not shown). Antiviral potency of mIFN-E2 on B16 cells against VSV was comparable with that of mIFN-E (f107 IFN-a-relevant units/mg). We also showed that, similar to their human orthologues, expression of mIFN-k genes was induced in response to viral infections. Consistent with their role in antiviral protection, the expression of mIFN-E2/IFN-E3 mRNAs was detected in B16 cells infected with VSV (Fig. 3C). Antitumor activities of mIFN-Ls. Type I IFNs are approved for the treatment of various malignancies including melanomas. Therefore, we used a gene therapy approach to investigate whether type III IFNs may also possess antitumor activities. Cytokine gene therapy has shown many advantages in comparison with systemic administration, which requires the injection of high doses of a cytokine, often leading to adverse side effects (31–33). Modified tumor cells constitutively producing a cytokine at the tumor site have been shown to be highly effective in treating solid cancers (34, 35). B16 cells were transfected with plasmid pEF-mIFN-E2, and G418- resistant cells were selected, pooled together, and designated E Figure 2. STAT activation. Parental hamster cells (A, left), hamster cells B16.IFN- 2 cells. The plasmid contains a strong elongation factor expressing either hIFN-ER1/gR1 and hIL-10R2 (A, middle) or mIFN-ER1/gR1 1a promoter that provides constitutive production and secretion of (A, right), mouse B16 melanoma cells (B), and L929 cells, primary mouse mIFN-E2 from B16.IFN-E2 cells. To assess IFN-E production and keratinocytes, macrophages, and splenocytes (C) were left untreated or treated with either recombinant hIFN-E1, mIFN-E2, or mIFN-a (10 ng/mL), or with activity in B16.IFN-E2 cells, we tested expression of MHC class I COS cell-produced mIFN-Es (100 AL COS cell conditioned media; E2 and E3 antigen, which is induced by IFN-Es in B16 cells (Fig. 3A). As shown indicate whether mIFN-E2 or mIFN-E3 were used). Cellular lysates were prepared and evaluated for STAT activation by EMSAs with either ISRE or GAS in Fig. 4A, a significant increase of MHC class I antigen expression DNA probes as indicated. STAT1 and STAT3 antibodies were used to show was observed in B16 cells transfected with pEF-IFN-E2 plasmid identity of STAT DNA-binding complexes (B). Arrows, positions of STAT (B16.IFN-E2 cells) in comparison with B16 cells transfected with DNA-binding and ISGF3 complexes in EMSAs.

IFN-induced activation of STAT1 and STAT3 DNA binding complexes (Fig. 2B, left). STAT identity was defined by supershifting the complexes with STAT1 or STAT3 antibodies (Fig. 2B, left). In addition to B16 cells, mouse keratinocyte-like PAM212 cells and mouse primary keratinocytes were also responsive to IFN-Esas shown by EMSA (Fig. 2C; ref. 17 and data not shown). In contrast, IFN-a, but not IFN-E, induced STAT activation in mouse bone marrow–derived macrophages and mouse primary splenocytes (Fig. 2C). Therefore, we concluded that similar to their human orthologues, mouse type I and type III IFNs use common signaling pathways to activate gene transcription. However, the target cells for IFN-E and IFN-a only partially overlap. Immunomodulatory and antiviral activities of mouse IFN-Ls. We next determined whether the mouse IFN-E system is capable of mediating biological activities similar to those induced by human IFN-Es, including antiviral protection and up-regulation of MHC class I antigen expression. We first showed that MHC class I antigen expression was strongly up-regulated in mouse B16 cells and PAM212 cells, and not in mouse fibroblast-like cell lines such Figure 3. Biological activities. A, mouse B16 cells, mouse NIH 3T3 cells, and as L929 and NIH 3T3 cells, in response to treatment with either human HT29 cells were left untreated (thin lines) or treated with recombinant E E mouse or human IFN-Es(Fig.3A and data not shown). In addition, mIFN-a, mIFN- 2, or hIFN- 1 (10 ng/mL; thick lines) and the level of MHC class I antigen expression was shown by flow cytometry. B, antiviral protection in both mouse IFN-E2 and IFN-E3 were capable of up-regulating response to either mIFN-E2 or mIFN-a was evaluated on B16 cells infected with MHC class I antigen expression in several human cell lines such as VSV using cytopathic effect reduction assay. An equal number of cells was plated in all wells and treated with 2-fold serial dilutions of indicated cytokines. epitheloid carcinoma HeLa S3 cells, lung carcinoma A549 cells, Twenty-four hours later, the virus was added. Antiviral activity is shown as the keratinocyte HaCaT cells, hepatoma HuH7 cells, and colorectal amount of mIFN-a or mIFN-E2 for 50% protection of the cells from cytopathic carcinoma HT-29 cells, which are responsive to hIFN-Es (ref. 15; effect. C, expression of mIFN-Es mRNAwas evaluated by RT-PCR in virus-infected cells. B16 cells were left untreated (À) or infected with VSV and Fig. 3A and data not shown). These experiments again showed that the cells were collected at postinfection times (4, 8, and 24 hours), RNAwas mouse and human IFN-Es are active between these species. In isolated, and RT-PCR was done.

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To examine whether the constitutive secretion of IFN-E inhibited tumor growth in vivo,0.5Â 106 B16.IFN-E2 cells, parental B16 cells, or B16.vector cells were injected s.c. into immunocompetent syngeneic C57BL/6 mice. All mice injected with parental B16 cells or B16 cells transfected with an empty vector (B16.vector) developed tumors in <20 days whereas the tumorigenicity of IFN- E-producing B16.IFN-E2 cells was highly impaired or completely abolished (Fig. 4C). Although some mice developed B16.IFN-E2 tumors, the tumors appeared later and grewabout thrice slower than parental B16 tumors. To examine if the presence of B16 cells producing IFN-E2 in the same anatomic site used for the tumor implantation could affect the growth of parental B16 cells, we mixed both types of cells at equal amounts and injected them into the mice. The tumor onset was delayed in 100% of the mice injected with a mixture of 0.5 Â 106 B16 cells and 0.5 Â 106 B16.IFN-E2. Furthermore, 30% of the mice remained tumor-free over a 3-month observation period (Fig. 4D). The level of IFN-E production correlates with the level of MHC class I antigen expression in B16.IFN-E2. To study the correlation between the level of IFN-E secretion from B16.IFN-E2 cells and the tumorigenicity, we selected three different clones of B16.IFN-E2 cells based on the level of MHC class I antigen expression in these E E E clones. The clones were respectively designated B16.IFN- 2L (low; Figure 4. mIFN- 2-producing B16.IFN- 2 cells and their tumorigenicity. A, the E E basal level of MHC class I antigen expression was evaluated by flow cytometry in Fig. 5A), B16.IFN- 2M (intermediate; Fig. 5B), and B16.IFN- 2H B16 cells transfected with an empty vector (B16.vector cells, filled histogram) cells (high; Fig. 5C). The level of MHC class I antigen expression in and in B16 cells transfected with plasmid pEF-mIFN-E2 (B16.IFN-E2 cells; E unfilled histogram with dashed line). B, B16 cells were left untreated these clones correlated with the level of IFN- production, which (filled histogram) or treated with the conditioned medium of B16.IFN-E2 cells was evaluated by comparing the ability of conditioned medium (unfilled histogram with dashed line) and the level of MHC class I antigen from the clonal cells to induce MHC class I antigen expression in expression was evaluated by flow cytometry. Inset, dose-response curve of E MHC class I antigen induction on B16 cells after treatment with serial dilutions B16 cells relative to the standard recombinant IFN- 2. The level of of conditioned medium [supernatant (SN)] from B16.IFN-E2 cells (.)or secreted IFN-E2 was estimated to be 1 to 5 ng for B16.IFN-E2L B16.vector cells (o). Ordinate, mean of fluorescence (MF); abscissa, dilution E E cells, 20 to 50 ng for B16.IFN- 2M cells, and 100 to 150 ng for factor. C, tumorigenicity of B16.IFN- 2 cells was assessed in syngeneic E 6 C57BL/6 mice in comparison with parental B16 cells and B16.vector cells. B16.IFN- 2H cells produced by 10 cells every 24 hours. Each mouse was injected s.c. in the flank with either 0.5 Â 106 B16 parental cells We next assessed the tumorigenicity of the clones in vivo.As (14 mice), B16.vector cells (5 mice), or B16.IFN-E2 cells (12 mice). Tumor development was monitored five times a week by palpation of the injection site. shown on the Fig. 5 (right), a close correlation existed between the Whereas median tumor appearance in mice injected with either B16 cells or level of IFN-E production and the inhibition of tumor growth B16.vector cells was similar (18 and 16 days, respectively), tumor appearance in in vivo. B16.IFN-E2H cells producing the highest amount of mIFN- mice injected with B16.IFN-E2 cells was significantly delayed (median tumor E E appearance, 39.5 days; P =1.7Â 10-6, B16.IFN-E2 compared with B16 tumors; 2 were rejected at a higher rate than B16.IFN- 2M and B16.IFN- P =1.3Â 10-5; B16.IFN-E2 compared with B16.vector tumors). D, C57BL/6 E2L cells secreting lower amount of mIFN-E2. These experiments mice were injected with either 106 parental B16 cells alone (6 mice) or a mixture E Â 6 Â 6 E showed that tumorigenicity of B16.IFN- 2 cells negatively corre- of 0.5 10 parental B16 cells with 0.5 10 of either B16.IFN- 2 cells (6 mice) E or B16.vector cells (6 mice). Tumor appearance in mice injected with lated with the increase of mIFN- 2 production. We again observed B16/B16.IFN-E2 was significantly delayed [median tumor appearance, 43 days that in animals that developed B16.IFN-E2 tumors, the tumors versus 13 days for B16 tumors (P =3.8Â 10-4) or 13 days for B16/B16.vector tumors (P =5.0Â 10-4)]. appeared later and grewslower than parental B16 tumors. Importantly, B16.IFN-E2 tumor cells extracted from animals that succumbed to the tumors still preserved their in vitro preimplan- control pEF plasmid (B16.vector), suggesting a secretion of IFN-E tation characteristics: the cells maintained mIFN-E2 production from B16.IFN-E2 cells. Production of IFN-E2 in the culture medium and retained up-regulation of MHC class I antigen expression (data was confirmed by the treatment of parental B16 cells with not shown). conditioned medium obtained from B16.IFN-E2 cells after 3 days Our results showed that IFN-E played an important role against of cell culture. The expression level of MHC class I antigen was the establishment of B16 melanoma tumors. IFN-E might inhibit markedly up-regulated in B16 cells after treatment with the tumor formation by acting directly on B16 melanoma cells or B16.IFN-E2 cell–conditioned medium in a dose-dependent manner through indirect mechanisms such as stimulation of antitumor (Fig. 4B). These results showthat B16.IFN- E2 stable transfectants immune responses or inhibition of angiogenesis. To separate direct constitutively secrete and respond to autocrine mIFN-E2. However, and indirect effects of IFN-E on tumor growth, we generated we did not observe any difference in the in vitro proliferation rate B16.IFN-E2 cells which were unresponsive to IFN-E. Clones of by counting the cells over a 5-day period between B16.IFN-E2 cells B16.IFN-E2 cells were selected for their low level of MHC class I and parental B16 cells or B16.vector cells transfected with an empty antigen expression comparable to that of parental B16 cells. The vector (data not shown). clones were further screened for sustained IFN-E2 production. A Because IFN-Es signal in a similar manner as type I IFNs and clonal cell population, designated B16.IFN-E2Res, was selected possess overlapping biological activities (15), we hypothesized that which maintained IFN-E2 production but the cells lost respon- the constitutive expression of IFN-E at the tumor site may affect siveness to IFN-E. As shown in Fig. 6A, treatment of parental B16 the in vivo tumorigenicity of B16 cells, as described for IFN-a (34). cells with the supernatant from B16.IFN-E2Res cells resulted in the www.aacrjournals.org 4473 Cancer Res 2006; 66: (8). April 15, 2006

tumors to develop was observed (Fig. 6B), suggesting that IFN-E can act through indirect mechanisms mediated by the host to suppress tumor growth. In the mixed transplantation assay, when the mixture of 0.5 Â 106 B16 cells and 0.5 Â 106 B16.IFN-E2Res cells was injected into mice, tumors were delayed or completely rejected (Fig. 6C), similar to results obtained with the mixture of B16 and B16.IFN-E2 cells (Fig. 4D). These experiments indicated that IFN-E produced by tumor cells induced indirect antitumor effects mediated by the host. To study whether B16.IFN-E2 cells, sensitive or resistant to IFN-E treatment, generate a long-lasting memory antitumor response, mice that had rejected B16.IFN-E2 or B16.IFN-E2Res tumors were challenged by injection of 0.5 Â 106 parental B16 cells in the opposite flank. Whereas 100% of naive mice developed tumors, f90% of mice that had previously rejected transfected modified B16 melanomas succumbed to parental B16 tumors (Fig. 6D), implying that mice in which B16.IFN-E2 tumors were eliminated did not develop a significant sustained antitumor immunity. When B16.IFN-E2 cells, sensitive or resistant to IFN-E, were injected into mice, some animals developed tumors albeit with significant delay compared with tumors from parental B16 cells. In

Figure 5. Correlation between the level of IFN-E production and tumorigenicity of B16.IFN-E2 cells. Three different clones of B16.IFN-E2 cells were selected based on their level of MHC class I antigen expression, which correlated with the concentration of IFN-E2 produced into conditioned media. The level of MHC class I antigen expression in these clones (unfilled histograms with dashed lines) and in parental B16 cells (filled histograms) was evaluated by flow cytometry. Clonal cells were designated as low (B16.IFN-E2L cells; A), intermediate (B16.IFN-E2M cells; B), and high (B16.IFN-E2H cells; C) IFN-E producers. Tumorigenicity of these clonal cell populations (right) was assessed in syngeneic C57BL/6 mice as described in Fig. 4 legend. Mice were injected with 106 of either parental B16 cells (4 mice; median tumor appearance, 18 days) or different clonal cell populations [8 mice for each population; low: median tumor appearance, 24 days (A); intermediate: median tumor appearance, 52 days (B); and high: median tumor appearance, 89 days (C)]. P values are shown in the figure. induction of MHC class I antigen expression, indicating that B16.IFN-E2Res cells secreted IFN-E2. However, B16.IFN-E2Res cells failed to up-regulate the expression of MHC class I antigen, Figure 6. IFN-E2-resistant B16.IFN-E2Res cells and their tumorigenicity. A, parental B16 cells or B16.IFN-E2Res cells were treated with either exogenous establish antiviral state, and activate STATs in the presence of recombinant mIFN-a (10 ng/mL), mIFN-E2 (10 ng/mL), or conditioned medium either their endogenous secreted mIFN-E2 or exogenous mIFN-E2 (supernatant) of either B16.IFN-E2Res cells or B16.vector cells in triplicate a and MHC class I antigen expression was evaluated by flow cytometry. Ordinate, (Fig. 6A and data not shown). In contrast, treatment with mIFN- mean of fluorescence. B and C, tumorigenicity of B16.IFN-E2Res cells was induced STAT activation, antiviral protection, and up-regulation of assessed in C57BL/6 mice as described in Fig. 4 legend. Mice were injected MHC class I antigen expression in B16.IFN-E2Res cells to the same with 106 of either parental B16 cells (5 mice) or B16.IFN-E2Res cells (10 mice; median tumor appearance, 17 days for B16 tumors and 41.5 days level as in parental B16 cells, indicating that the MHC class I for B16.IFN-E2Res tumors; P =2.0Â 10-4; B) or with a mixture of 0.5 Â 106 antigen expression and the Jak-STAT signaling pathway were still parental B16 cells with either B16.IFN-E2Res cells (8 mice) or B16.vector cells E (5 mice; median tumor appearance, 12.5 days for B16/B16.vector tumors and functional in B16.IFN- 2Res cells (Fig. 6A and data not shown). E Â -5 E 32 days for B16/B16.IFN- 2Res tumors; P =1.8 10 ; C). D, C57BL/6 Transfection of B16.IFN- 2Res cells with plasmid encoding mIFN- mice that rejected B16.IFN-E2 or B16.IFN-E2Res cells and stayed tumor-free for ER1 restored responsiveness of the cells to IFN-E (data not shown). at least 3 months were challenged with 0.5 Â 106 parental B16 cells, which E E were injected in the flank opposite to those used for first injection. Eight naive Therefore, B16.IFN- 2Res cells produced IFN- 2 but were resistant mice, 8 mice previously vaccinated with B16.IFN-E2Res cells, and 10 mice to IFN-Es due to mIFN-ER1 deficiency. vaccinated with B16.IFN-E2 cells were used in these experiments. Only one B16.IFN-E2Res cells (0.5 Â 106) were injected s.c. into mice and mouse in each previously vaccinated group did not develop tumors; all other f animals succumbed to tumors with similar median tumor appearance time tumor development was monitored. A significant delay ( 2-fold) (16 days in all groups; P = 0.42, naive versus B16.IFN-E2-vaccinated animals; in tumor appearance or a complete inability of B16.IFN-E2Res P = 0.36, naive versus B16.IFN-E2Res-vaccinated animals).

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2006 American Association for Cancer Research. Antitumor Activities of IFN-Es addition, B16.IFN-E2 cell–derived tumors seemed to growabout Discussion thrice slower than parental B16 tumors. To elucidate a mecha- Following the discovery of human IFN-E proteins (18), we nism(s) explaining the inhibition of tumor growth in vivo, tumors searched the mouse genome to identify murine IFN-E orthologues. removed from several mice in each treatment group were Although there are three genes encoding highly homologous but examined by light microscopy. Photomicrographs of B16 and distinct human IFN-E proteins (IFN-E1, IFN-E2, and IFN-E3), the E B16.IFN- 2 tumors are shown in Fig. 7. Histologically, tumors from search of the mouse genome revealed the existence of only two E mice injected with B16.IFN- 2 cells, which displayed delayed genes, representing mouse IFN-k2 and IFN-k3 gene orthologues, growth, differed in several aspects from the parental B16-derived encoding intact proteins (Fig. 1). The mouse IFN-k1 gene lesions. B16 tumors were large and composed of solid nests of orthologue contains a stop codon in the first exon and, therefore, neoplastic cells supplied by numerous small blood vessels. Focal it does not encode an intact protein. Mouse IFN-Es (mIFN-E2 and areas of the lesion showed prominent cellular degeneration and mIFN-E3) and IFN-E receptor (mIFN-ER1) orthologues were cloned areas of necrosis. In contrast, tumors derived from B16.IFN-E2 cells and found to be quite similar to their human counterparts (Fig. 1). were less vascular than B16 tumors, with nests of viable tumor cells Experiments showed that similar to their human counterparts, clustered around a thin-walled, centrally located vessel. Large mIFN-E2 and mIFN-E3 signal through the IFN-E receptor complex, confluent areas of necrosis were evident throughout the mass. The activate ISGF3, and are capable of inducing antiviral protection and mitotic rate of the B16 tumors was more than double those from MHC class I antigen expression in several cell types (Figs. 2 and 3). B16.IFN-E2 cells (14 versus 6 per 40Â field, respectively). No tumor- The results confirmed our previous observation that type III IFNs infiltrating immune cells were detected in tumors from any group (IFN-Es) engage a unique receptor complex, composed of IFN-ER1 of mice, including ones that received the tumor vaccines and were and IL-10R2 subunits, to induce signaling and biological activities challenged with parental B16 cells. similar to those of type I IFNs.

Figure 7. Tumor histology. H&E staining of parental B16 tumor (A and B) and B16.IFN-E2 tumor (C and D). A, low-magnification photomicrograph (Â10) of B16 tumor. Nests of tumor cells are surrounded by areas of necrosis. The tumor cells are oriented around thin-walled blood vessels (arrows). Ample melanin is present in many tumor cells. B, high-magnification micrograph (Â20) showing pleomorphic tumor cells surrounding blood vessels. Granules of melanin are present in numerous cells (arrows). No immune cells were in any region of the tumor. C, micrograph (Â10) of B16.IFN-E2 tumor. Tumor shows extensive areas of necrosis (N) with thin islands of viable tumor cells surrounding blood vessels (arrows). D, high-magnification micrograph (Â20) of B16.IFN-E2 tumor. Cells are round to polygonal and more uniform than those from B16 tumors. Necrotic debris surrounds the areas of viable cells. No immune cells were present in the tumor or adjacent to the necrotic fields. www.aacrjournals.org 4475 Cancer Res 2006; 66: (8). April 15, 2006

Type I IFNs are recognized not only for their antiviral role but suggesting that the development of long-lasting immunity in this also for their antitumor activity. IFN-a is used clinically as a mouse tumor model was relatively weak. Therefore, the involve- treatment for several malignancies, including melanoma (1, 34). ment of antitumor mechanisms in response to IFN-E, which are Recently, the target cells for the antitumor action of both type I and likely to play an important role independent of adaptive immunity, type II IFNs were highlighted in animal experiments with trans- can be proposed. planted and carcinogen-induced tumors (36, 37). Using mice The immunosurveillance function of CTL, which can kill tumor deficient in IFN response, it was shown that whereas endogenously cells, is clearly important (36). Although neither lymphocyte produced type I and type II IFNs target immune cells to enhance recruitment in established B16.IFN-E tumors (Fig. 7) nor strong antitumor responses, only the direct action of type II IFN on cancer protective immunity in animals which rejected B16.IFN-E cells was cells seemed to be important to mobilize an effective antitumor detected (Fig. 6D), these results cannot be extrapolated to other response (37). Because cell signaling by IFN-Es is similar to type I tumors. For example, B16 cells expressing IFN-a also showed IFNs, which have shown antitumor effects, we investigated whether decreased tumorigenicity and failed to elicit protective immunity type III IFNs also possess antitumor activities using a gene therapy to subsequent challenge with B16 cells (39). In contrast, IFN-a- approach in the mouse B16 melanoma model. expressing TS/A and MC38 tumors were effective in the induction B16 cells respond to IFN-Es, as shown by up-regulation of MHC of protective immunity (40, 41). Therefore, the ability of IFN-E to class I antigen expression and antiviral activity (Figs. 2 and 3). By induce adaptive immune mechanisms in other tumor models gene transfer, we engineered B16 cells, which constitutively remains to be investigated. produced mIFN-E2(B16.IFN-E2 cells). In response to their Tumor rejection is a complex process which integrates both secretion of IFN-E, B16.IFN-E2 cells exhibited constitutively high immune and nonimmune mechanisms toward tumor destruction. level of MHC class I antigen expression (autocrine mIFN-E2 effect; In addition to adaptive immune mechanisms, tumor develop- Fig. 4A). ment and growth is also affected by tumor stromal cells, a B16 melanoma is an aggressive and highly malignant murine mixture of various cell types that reside at the tumor site and tumor. One hundred percent of immunocompetent C57BL/6 those recruited to the site, which are required to sustain tumor syngeneic mice, injected with parental B16 cells, developed tumors growth by providing growth factors, extracellular matrix support, within 3 weeks (Figs. 4–6). However, the constitutive production of blood supply, and waste removal (42). For example, under mIFN-E2 by B16.IFN-E2 cells markedly affected tumorigenicity of physiologic conditions, keratinocytes control melanocyte growth the cells (Figs. 4 and 5). B16.IFN-E2 cells were either rejected by the and behavior through cell interaction and paracrine mechanisms, host or grewat a slowerrate than control parental B16 cells. The whereas under melanoma conditions, normal communications antitumor effect of IFN-E was dose dependent (Fig. 5). B16.IFN-E2 are altered (43). We found that normal keratinocytes are highly cells also inhibited the growth of parental B16 cells when both cell responsive to IFN-E (Fig. 2C; ref. 15). Therefore, mIFN-E2 types were injected together (Fig. 4). B16 melanoma is a poorly produced by either IFN-E-resistant or IFN-E-sensitive B16.IFN- immunogenic tumor, characterized by inefficient MHC-restricted E2 melanomas could affect neighboring keratinocytes and, antigen presentation (38). The high level of constitutive MHC class perhaps, other tumor stromal cells, and could inhibit their I antigen expression in B16.IFN-E2 cells may render the cells more tumor-supportive function. Histologic analysis (Fig. 7) showed immunogenic and promote adaptive antitumor immune responses. large areas of tumor necrosis associated with reduced vascularity However, B16.IFN-E2 tumors did not display increased lymphocytic in B16.IFN-E2 tumors. IFN-Es may contribute to antiangiogenic infiltrates (Fig. 7) and failed to induce a strong memory response mechanisms by inhibiting the abilities of tumor or stroma to (Fig. 6D), suggesting that up-regulation of MHC class I antigen stimulate angiogenesis, which is required to maintain tumor expression on the surface of B16 cells was not sufficient to survival and proliferation in vivo (44). However, the precise significantly increase the immunogenicity of B16.IFN-E2 cells. mechanisms and mediators of IFN-E antitumor activities should To examine whether the antitumor effect of mIFN-E2 was due to be further investigated and compared with mechanisms induced a direct action on B16 cells or mediated by a host response, we by other types of IFNs for which the antiangiogenic effects have developed B16.IFN-E2Res cells, which produced mIFN-E2 but were been reported in several studies (1, 45). resistant to IFN-E treatment (Fig. 6A). B16.IFN-E2Res cells This study represents the first report of the use of IFN-Esina displayed reduced tumorigenicity and repressed the growth of model of cancer therapy. The results clearly showed that IFN-Es are parental B16 cells in vivo to a level comparable to IFN-E-sensitive effective in the mouse B16 melanoma model and engaged host B16.IFN-E2 cells (Fig. 6B and C). The impaired tumor growth of mechanisms to exert their antitumor functions. Although the IFN-E-resistant B16.IFN-E2Res cells, which still secreted mIFN-E2, precise mechanism(s) responsible for the antitumor activities of strongly suggested that host-defense mechanisms played a major type III IFNs remains to be elucidated, this study reveals the role in mediating IFN-E-induced antitumor activity. However, potential therapeutic antitumor properties of IFN-Es and suggests direct action of IFN-Es on the host is rather limited. Several cell their potential use in cancer therapy. types, including primary lymphocytes and macrophages, the major players in specific antitumor immunity, were found to be unresponsive to IFN-E (Fig. 2), suggesting that immune cells are Acknowledgments E not the primary targets of IFN- . Therefore, the function of Received 10/10/2005; revised 1/3/2006; accepted 2/14/2006. immune cells is unlikely to be directly altered by IFN-E. In contrast, Grant support: National Institute of Allergy and Infectious Diseases, USPHS grants virtually all cell types respond to type I IFNs, indicating that RO1 AI051139 and AI057468 and American Heart Association grant AHA #0245131N (S.V. Kotenko) and ES05022 grant support for the Molecular Pathology Core. mechanisms of antitumor activities of type I and type III IFNs are The costs of publication of this article were defrayed in part by the payment of page not identical. Our experiments also showed that only f10% of charges. This article must therefore be hereby marked advertisement in accordance E with 18 U.S.C. Section 1734 solely to indicate this fact. mice that rejected the B16.IFN- 2 cells, either sensitive or resistant We thank Jerry Langer and Martin Schwarz for the critical review of the text and to IFN-E, survived the parental tumor challenge (Fig. 6D), helpful suggestions.

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