An MDM2 inhibitor achieves synergistic cytotoxic effects with adenoviruses lacking E1B55kDa gene on mesothelioma with the wild-type p53 through augmenting NFI expression.

A majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.

Characteristic of slow growth in cell culture of adenovirus type 54 causing nationwide outbreak epidemic keratoconjunctivitis in Japan.

PURPOSE: To characterize the virological features of adenovirus type 54 (Ad54) causing nationwide outbreak of severe epidemic keratoconjunctivitis (EKC) in Japan, we comparatively analysed the viral propagation phenotype of Ad54 and other Ads: type 37 (Ad37), 64 (Ad64), and 5 (Ad5), in A549 cells quantitatively. STUDY DESIGN: Laboratory investigation. METHODS: We compared the growth rate of Ads using copy numbers and cytopathic effect observation during propagation in A549 cell lines. Expressions of mRNA of E1 gene were also calculated and compared. Phylogenetic analysis of the region, including putative promoter of E1 gene and E1 open reading frame (ORF), were performed. RESULTS: Increases in viral loads, growth rate, and viral propagation were slower for Ad54 than for other Ads. The expression level of the E1 gene per infected cell was lower for Ad54 than for other Ad types on post-infection day 1. Phylogenetic analysis of the E1 gene putative promoter and ORF revealed Ad54 was the closest to Ad type 8. CONCLUSION: The propagation of Ad54 in A549 is slow compared with Ad37, Ad64 and Ad5. This slow propagation could have been caused by slow genomic replication resulting from delayed viral entry or E1 transcription initiation. The EKC caused by Ad54 needs more attention because the slow propagation of Ad54 may contribute to prolonged disease duration.

Cell transformation by the adenovirus oncogenes E1 and E4.

Extensive studies on viral-mediated oncogenic transformation by human adenoviruses have revealed much of our current understanding on the molecular mechanisms that are involved in the process. To date, these studies have shown that cell transformation is a multistep process regulated by the cooperation of several adenoviral gene products encoded in the early regions 1 (E1) and 4 (E4). Early region 1A immortalizes primary rodent cells, whereas co-expression of early region protein 1B induces full manifestation of the transformed phenotype. Beside E1 proteins, also some E4 proteins have partial transforming activities through regulating many cellular pathways. Here, we summarize recent data of how adenoviral oncoproteins may contribute to viral transformation and discuss the challenge of pinpointing the underlying mechanisms.

Temporal dynamics of adenovirus 5 gene expression in normal human cells.

Adenovirus executes a finely tuned transcriptional program upon infection of a cell. To better understand the temporal dynamics of the viral transcriptional program we performed highly sensitive digital PCR on samples extracted from arrested human lung fibroblasts infected with human adenovirus 5 strain dl309. We show that the first transcript made from viral genomes is the virus associated non-coding RNA, in particular we detected abundant levels of virus associated RNA II four hours after infection. Activation of E1 and E4 occurred nearly simultaneously later in infection, followed by other early genes as well as late genes. Our study determined that genomes begin to replicate between 29 and 30 hours after infection. This study provides a comprehensive view of viral mRNA steady-state kinetics in arrested human cells using digital PCR.

Early detection of virus infection in live human cells using Raman spectroscopy.

Virus infection of a human cell was determined only 3 h after invagination. We used viral vector Ad-CMV-control (AdC), which lacks the E1 gene coding for early polypeptide 1 (E1). AdC can replicate in human embryonic kidney 293 (HEK293) cells into which the E1 gene has been transfected. According to partial least-square regression discriminant analysis, it was assumed that two kinds of reaction take place in the cell during viral invasion. The first response of the cell was determined 3 h after the virus invasion, and the second one was determined ∼9 h later. The first one seems to be due to compositional changes in DNA. Analysis of large-scale datasets strongly indicated that the second reaction can be attributed to a reduction in protein concentration or uptake of phenylalanine into the nucleus.

Pancreatic carcinoma-specific immunotherapy using novel tumor specific cytotoxic T cells.

Pancreatic cancer represents one of the most lethal human cancers. Investigation of the effective targeting to the tumor cells is essential for both primary tumors and metastases. Tumor specific cytotoxic T lymphocytes (CTLs) have recently been considered to be the attractive vehicles for delivering therapeutic agents toward various tumor diseases. This study was to explore the distribution pattern of CTL carrying the lentiviral vectors with the characteristic of adenoviral E1 gene under the control of the cell activation-dependent CD40 ligand promoter (Lenti/hCD40L/E1AB). Following transduction with adenoviral particles containing chimeric type 5 and type 35 fiber proteins (Ad5/35-TRAIL), these CTLs produced infectious virus when exposed to SW1990 cells. We found that the novel CTL harboring Lenti/hCD40L/E1AB and Ad5/35-TRAIL inhibited pancreatic cancer cell growth and angiogenesis in vitro and in vivo. Furthermore, Ad5/35-TRAIL transduced CTL induced significant apoptosis in pancreatic carcinoma cell lines and upregulated IFN-gamma (IFN-γ) secretion of CTLs. Importantly, Ad5/35-TRAIL transduced CTLs had no inhibitory effect on normal cells. Thus, the novel CTLs may be safe and feasible for the development of gene therapy approaches to pancreatic carcinoma.

The generation and analyses of a novel combination of recombinant adenovirus vaccines targeting three tumor antigens as an immunotherapeutic.

Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4+ and CD8+ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no "antigenic competition" in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies.

Gene therapy targeting hepatocellular carcinoma by a dual-regulated oncolytic adenovirus harboring the focal adhesion kinase shRNA.

Cancer targeting gene-viro-therapy (CTGVT) approach has become a hotspot and a trend in the field of cancer biotherapy and oncolytic adenovirus is an ideal vector to carry the targeting genes. In this study, we used human telomerase reverse transcriptase (hTERT) promoter to control the adenovirus early region 1a (E1A) and the human α-fetoprotein (AFP) promoter integrated with hypoxia response element (HRE) to control the adenovirus early region 1b (E1B). Then the novel double-regulated adenovirus Ad-hTERT-HREAF (named SG505) was engineered. The short-hairpin RNA against focal adhesion kinase (FAK) was inserted into SG505 and thus forming Ad-hTERT-HREAF-shRNA (called SG505­siFAK). Then various oncolytic adenoviruses were examined to verify whether they could suppress liver cancer cells selectively and efficiently both in vitro and in vivo. Both replicative and replication-defective adenoviruses carrying FAK-shRNA significantly inhibited the expression of FAK in Hep3B and SMMC-7721 cell lines and efficiently suppressed the growth of liver cancer cell lines with minor effect to normal cells. Furthermore, the recombined oncolytic adenoviruses, SG505-siFAK, SG505-EGFP and SG505 were able to selectively propagate in AFP-positive liver cancer cells in vitro and the SG505-siFAK efficiently suppressed the expression of FAK. SG505-siFAK showed the most potent tumor inhibition capability among the three recombined adenovirus with IC50 levels of 0.092±0.009 and 0.424±0.414 pfu/cell in the Hep3B and HepG2 cell line, respectively. Animal experiment further confirmed that SG505-siFAK achieved the most significant tumor inhibition of Hep3B liver cancer xenografted growth by intratumoral injection comparing to the intravenous injection among the three recombined viruses. Immunohistochemical results indicated that FAK expression was downregulated significantly in the tumors treated with SG505-siFAK. The dual-regulated oncolytic adenovirus SG505-siFAK was proven to inhibit the growth of liver cancer cells selectively and efficiently, therefore SG505-siFAK could be a potential agent for future clinical trials of hepatocellular carcinoma.

Extended evaluation of a phase 1/2 trial on dosing, safety, immunogenicity, and overall survival after immunizations with an advanced-generation Ad5 [E1-, E2b-]-CEA(6D) vaccine in late-stage colorectal cancer.

A phase 1/2 clinical trial evaluating dosing, safety, immunogenicity, and overall survival on metastatic colorectal cancer (mCRC) patients after immunotherapy with an advanced-generation Ad5 [E1-, E2b-]-CEA(6D) vaccine was performed. We report our extended observations on long-term overall survival and further immune analyses on a subset of treated patients including assessment of cytolytic T cell responses, T regulatory (Treg) to T effector (Teff) cell ratios, flow cytometry on peripheral blood mononuclear cells (PBMCs), and determination of HLA-A2 status. An overall survival of 20 % (median survival 11 months) was observed during long-term follow-up, and no long-term adverse effects were reported. Cytolytic T cell responses increased after immunizations, and cell-mediated immune (CMI) responses were induced whether or not patients were HLA-A2 positive or Ad5 immune. PBMC samples from a small subset of patients were available for follow-up immune analyses. It was observed that the levels of carcinoembryonic antigen (CEA)-specific CMI activity decreased from their peak values during follow-up in five patients analyzed. Preliminary results revealed that activated CD4+ and CD8+ T cells were detected in a post-immunization sample exhibiting high CMI activity. Treg to Teff cell ratios were assessed, and samples from three of five patients exhibited a decrease in Treg to Teff cell ratio during the treatment protocol. Based upon the favorable safety and immunogenicity data obtained, we plan to perform an extensive immunologic and survival analysis on mCRC patients to be enrolled in a randomized/controlled clinical trial that investigates Ad5 [E1-, E2b-]-CEA(6D) as a single agent with booster immunizations.

Construction and characterization of E1- and E3-deleted adenovirus vectors expressing two antigens from two separate expression cassettes.

Here we describe a series of replication-defective adenovirus vectors designed to express transgene products from two expression cassettes placed into the deleted E1 and E3 domains. Vectors that contained an E1 cassette with a cytomegalovirus promoter in the forward orientation and an E3 cassette with the chicken ß-actin promoter in the reverse orientation grew to acceptable yields and expressed both transgenes. Additionally, they elicited immune responses to both transgene products. Levels of expression and the vectors' immunogenicity were influenced by the presence of regulatory elements shared between the two expression cassettes. Specifically, vectors that carried the same intron and enhancer in both expression cassettes could be rescued and expanded, but they were poorly immunogenic. Deletion of the enhancer or both the enhancer and the intron from the E3 cassette increased T- and B-cell responses to both transgene products.

Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication.

First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

Antitumor effects of telomerase-specific replication-selective oncolytic viruses for adenoid cystic carcinoma cell lines.

We evaluated the antitumor effect of a telomerase-specific replication-selective adenovirus (Telomelysin, OBP-301) for adenoid cystic carcinoma (ACC) in vitro and in vivo. Adenovirus E1 gene expression was controlled by human telomerase reverse transcription (hTERT). Infection of ACC cells by OBP-301 induced high E1A mRNA expression and subsequent oncolytic cell death in a dose-dependent manner. Using OBP-401 (TelomeScan), a genetically engineered adenovirus that carries the GFP gene under the control of the cytomegalovirus (CMV) promoter at the deleted E3 region of OBP-301, ACC cells expressed bright GFP fluorescence as early as 12 h after OBP-401 infection. The fluorescence intensity gradually increased in a time-dependent manner, followed by rapid cell death due to the cytopathic effect of OBP-401, as evidenced by the floating, highly light-refractive cells using phase-contrast microscopy. Effects of intratumorally injected OBP-401 against established Acc2 xenograft tumors were seen in BALB/c nu/nu mice. The levels of GFP expression following ex vivo infection of OBP-401 may be of value as a positive predictive marker for the outcome of telomerase-specific virotherapy. Our data clearly indicated that telomerase-specific oncolytic adenoviruses have significant therapeutic potential against human ACC in vitro and in vivo. These results suggest that treatment with OBP-301 and OBP-401 may improve the quality of life of oral cancer patients.

Impact of E1 and Cre on adenovirus vector amplification: developing MDCK CAV-2-E1 and E1-Cre transcomplementing cell lines.

Adenovirus vectors have been extensively studied through the manipulation of viral genome. However, little attention is being paid to their producer cell-lines; cells are selected according to virus yields, neglecting the expression profile of transcomplementing gene products underlying cell performance. This work evaluates the impact of E1 (E1A and E1B) and Cre recombinase levels in the production of E1-deleted and helper-dependent canine adenovirus type 2 (CAV-2) vectors using MDCK cells. E1A and E1B gene expression and Cre activity were evaluated in different cell clones and compared with the corresponding cell productivity and susceptibility to oxidative stress injury. CAV-2 production was proportional to E1A expression (the highest levels of E1A corresponding to productivities of 3000-5000 I.P./cell), while E1B prolonged host cell viability after infection, conferring protection against apoptosis. Cre recombinase counteracted E1B anti-apoptotic properties, however viral production was maintained under high levels of Cre. Yet, Cre recombinase side effects can be reduced using cell lines with lower Cre-activities, without compromising the excision efficiency of helper vector packaging signal. These results highlight the influence of transcomplementing gene products on CAV-2 producer cell line performance, and the ability to express high levels of E1A and E1B as an important feature for cell line establishment and high adenovirus titers.

Adenoviral vectors stimulate glucagon transcription in human mesenchymal stem cells expressing pancreatic transcription factors.

Viral gene carriers are being widely used as gene transfer systems in (trans)differentiation and reprogramming strategies. Forced expression of key regulators of pancreatic differentiation in stem cells, liver cells, pancreatic duct cells, or cells from the exocrine pancreas, can lead to the initiation of endocrine pancreatic differentiation. While several viral vector systems have been employed in such studies, the results reported with adenovirus vectors have been the most promising in vitro and in vivo. In this study, we examined whether the viral vector system itself could impact the differentiation capacity of human bone-marrow derived mesenchymal stem cells (hMSCs) toward the endocrine lineage. Lentivirus-mediated expression of Pdx-1, Ngn-3, and Maf-A alone or in combination does not lead to robust expression of any of the endocrine hormones (i.e. insulin, glucagon and somatostatin) in hMSCs. Remarkably, subsequent transduction of these genetically modified cells with an irrelevant early region 1 (E1)-deleted adenoviral vector potentiates the differentiation stimulus and promotes glucagon gene expression in hMSCs by affecting the chromatin structure. This adenovirus stimulation was observed upon infection with an E1-deleted adenovirus vector, but not after exposure to helper-dependent adenovirus vectors, pointing at the involvement of genes retained in the E1-deleted adenovirus vector in this phenomenon. Lentivirus mediated expression of the adenovirus E4-ORF3 mimics the adenovirus effect. From these data we conclude that E1-deleted adenoviral vectors are not inert gene-transfer vectors and contribute to the modulation of the cellular differentiation pathways.

Gene therapy using adenovirus against malignant mesothelioma.

BACKGROUND: Adenovirus vectors have been utilized for cancer gene therapies. The present study examined the oncolytic effects of adenovirus type 5 (Ad5) and fiber-substituted conditionally replicating adenovirus (CRAD) Ad5/F35 vectors on the human malignant mesothelioma cells MSTO-211H, NCI-H28, NCI-H2052, and NCI-H2452 cells. MATERIALS AND METHOD: For the adenovirus, the first mRNA/protein to be made (~1 h after infection) is E1A. Ad5F35 and Ad5 CRAD vectors containing the E1 gene controlled by the human midkine promoter (Ad5F35/MKp-E1 and Ad5/MKp-E1, respectively) were constructed. Western blotting and cell viability assays were carried out in cells transfected with Ad5/MKp-E1 and Ad5F35/MKp-E1. RESULTS: Coxsackie and adenovirus receptor (CAR), a cell surface target of Ad5, and CD46, a cell surface target of Ad35, were expressed in all the malignant mesothelioma cell lines examined here, as much as in HEK293 cells, with no significant differences in the expression levels among cells. Both Ad5/MKp-E1 and Ad5F35/MKp-E1 induced oncolysis of malignant mesothelioma cells in a viral particle-dependent manner, with similar efficacy. CONCLUSION: The results of the present study suggest that both Ad5/MKp-E1 and Ad5F35/MKp-E1 are useful for the gene therapy of human malignant mesothelioma.

Fiber-substituted conditionally replicating adenovirus for oncolysis of human renal carcinoma cells.

BACKGROUND: Adenovirus vectors have lately been highlighted in gene therapies. We investigated the oncolytic effects of a chimeric adenovirus type 5 (Ad5) with replacement of Ad5 fiber knob with adenovirus type 35 (Ad35) fiber knob (Ad5F35) on human renal cell carcinoma (RCC). MATERIALS AND METHODS: The conditionally replicating Ad5F35 vector was constructed and infected into RCC cell lines 786-O, ACHN, and RCC4-VHL. For these cells, reverse transcription-polymerase chain reaction and western blotting were carried out and the cell viability was assayed. RESULTS: In all RCC cell lines, it was found that CD46, a cell surface target of Ad35, was well-expressed, while coxsackie and adenovirus receptor (CAR), a cell surface target of Ad5, was considerably less expressed. The Ad5F35 vector induced oncolysis of RCC cells, with significantly higher efficacy as compared with that for the Ad5 vector. CONCLUSION: Ad5F35 vector could be a candidate for promising gene therapy of human RCC.

Preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors.

First-generation, E1/E3-deleted adenoviral vectors with diverse transgenes are produced routinely in laboratories worldwide for development of novel prophylactics and therapies for a variety of applications, including candidate vaccines against important infectious diseases, such as HIV/AIDS, tuberculosis, and malaria. Here, we show, for two different transgenes (both encoding malarial antigens) inserted at the E1 locus, that rare viruses containing a transgene-inactivating mutation exhibit a selective growth advantage during propagation in E1-complementing HEK293 cells, such that they rapidly become the major or sole species in the viral population. For one of these transgenes, we demonstrate that viral yield and cytopathic effect are enhanced by repression of transgene expression in the producer cell line, using the tetracycline repressor system. In addition to these transgene-inactivating mutations, one of which occurred during propagation of the pre-viral genomic clone in bacteria, and the other after viral reconstitution in HEK293 cells, we describe two other types of mutation, a small deletion and a gross rearranging duplication, in one of the transgenes studied. These were of uncertain origin, and the effects on transgene expression and viral growth were not fully characterized. We demonstrate that, together with minor protocol modifications, repression of transgene expression in HEK293 cells during viral propagation enables production of a genetically stable chimpanzee adenovirus vector expressing a malarial antigen which had previously been impossible to derive. These results have important implications for basic and pre-clinical studies using adenoviral vectors and for derivation of adenoviral vector products destined for large-scale amplification during biomanufacture.

Targeted expression of herpes simplex virus thymidine kinase in adenovirus-infected cells reduces virus titers upon treatment with ganciclovir in vitro.

BACKGROUND: Adenoviruses are a frequent cause of life-threatening infections in immunocompromised patients. Available therapeutics still cannot completely prevent fatal outcomes. By contrast, herpes viruses are well treatable with prodrugs such as ganciclovir (GCV), which are selectively activated in virus-infected cells by virus-encoded thymidine kinases. This effective group of prodrugs is not applicable to adenoviruses and other DNA viruses because they lack those kinases. METHODS: To render adenoviruses amenable to GCV treatment, we generated an adenoviral vector-based delivery system for targeted expression of herpes simplex virus thymidine kinase (HSV-TK) in wild-type adenovirus 5 (wt Ad5)-infected cells. HSV-TK expression was largely restricted to wt virus-infected cells by transcription of the gene from the Ad5 E4 promoter. Its activity is dependent on the adenoviral E1A gene product which is not produced by the vector but is only provided in cells infected with wt adenovirus. The anti-adenoviral effect of HSV-TK expression and concomitant treatment with GCV was assessed in vitro in four different cell lines or primary cells. RESULTS: E4 promoter-mediated HSV-TK background expression was sufficiently low to prevent cytotoxicity in the presence of low-levels GCV in cells not infected with wt Ad5. However, expression was several-fold increased in wt Ad5-infected cells and treatment with low levels of GCV efficiently inhibited wt Ad5 DNA replication. Genome copy numbers and output of infectious particles were reduced by up to > 99.99% and cell viability was greatly increased. CONCLUSIONS: We extended the concept of enzyme/prodrug therapy to adenovirus infections by selectively sensitizing adenovirus-infected cells to treatment with GCV.

Checkpoints in adenoviral production: cross-contamination and E1A.

Adenoviruses are widely used for overexpressing proteins in primary mammalian cells. Incorporation of the early viral gene, E1A, or viral cross-contamination can occur during amplification, and identification of these products is crucial as the transcription of unwanted genetic material can impact cell function and compromise data interpretation. Here we report methods for evaluation of contaminating adenovirus and E1 viral DNA.

Abrogating TNF-α expression prevents bystander destruction of normal tissues during iNOS-mediated elimination of intraocular tumors.

Although intraocular tumors reside in an immune privileged site, some tumors are rejected nonetheless. For example, intraocular adenovirus-induced (Ad5E1; adenovirus type 5 early region 1) tumors are rejected in syngeneic C57BL/6 mice by one of two pathways. One pathway leads to extensive necrosis of innocent bystander cells and culminates in destruction of the eye, a condition called phthisis. The second pathway is characterized by piecemeal tumor cell death that rids the eye of the tumor while preserving the architecture and function of the eye. To study the mechanisms of phthisical tumor rejection, we isolated a cell clone-designated clone 2.1 that consistently undergoes rejection in a phthisical manner. CD4(+) T cells and macrophages were required for phthisical rejection of intraocular clone 2.1 tumors and M1 macrophages were involved in mediating tumor rejection. In vitro and in vivo inhibition of iNOS (inducible nitric oxide synthase) abolished macrophage-mediated killing of tumor cells and rejection of intraocular tumors. A role for M1 macrophages was further supported by investigations showing that intraocular tumors grew progressively in IFN-γ KO (knockout) mice. Studies in mice deficient in TNF-α, TNF receptor-1, or TNF receptor-2 revealed that although TNF-α was not needed for tumor rejection, it was required for the development of necrotizing inflammation and phthisis of tumor-bearing eyes. Together, our findings suggest new strategies to successfully eliminate ocular tumors while preserving the integrity of the eye.