Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0806489 on October 31, 2006

Published online before print October 31, 2006
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(Journal of Leukocyte Biology. 2007;81:421-429.)
© 2007 by Society for Leukocyte Biology

Ocular immune privilege is circumvented by CD4+ T cells, leading to the rejection of intraocular tumors in an IFN-{gamma}-dependent manner

Dru S. Dace, Peter W. Chen, Hassan Alizadeh and Jerry Y. Niederkorn1

Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

1 Correspondence: Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9057, USA. E-mail: jerry.niederkorn{at}utsouthwestern.edu

ABSTRACT

Although intraocular tumors reside in an immune-privileged site, they can circumvent immune privilege and undergo rejection, which typically follows one of two pathways. One pathway involves CD4+ T cells, delayed-type hypersensitivity (DTH), and the culmination in ischemic necrosis of the tumor and phthisis (atrophy) of the eye. The second pathway is DTH-independent and does not inflict collateral injury to ocular tissues, and the eye is preserved. In this study, we used a well-characterized tumor, Ad5E1, to analyze the role of IFN-{gamma} in the nonphthisical form of intraocular tumor rejection. The results showed that IFN-{gamma} induced tumor cell apoptosis, inhibited tumor cell proliferation, and promoted rejection by inhibiting angiogenesis. Microarray analysis revealed that IFN-{gamma} induced up-regulation of five antiangiogenic genes and down-regulation of four proangiogenic genes in Ad5E1 tumor cells. Although IFN-{gamma} knockout (KO) mice have progressively growing intraocular tumors, IFN-{gamma} was not needed for the elimination of extraocular tumors, as all IFN-{gamma} KO mice rejected s.c. tumor inocula. This represents a heretofore unrecognized role for IFN-{gamma} in circumventing ocular immune privilege and eliminating intraocular tumors. The findings also reveal that some IFN-{gamma}-independent tumor rejection processes are excluded from the eye and may represent a new facet of ocular immune privilege.

Key Words: angiogenesis • anterior chamber

INTRODUCTION

One of the earliest observations of ocular immune privilege was made by the Dutch ophthalmologist van Dooremaal [1], who noted the prolonged survival of mouse skin grafts placed in the anterior chamber (AC) of the dog eye. It was another 75 years until Medawar [2] recognized the significance of the extended survival of foreign tissues placed in the AC of the eye or in the brain and coined the term "immune privilege." Medawar concluded that the apparent absence of patent lymphatic vessels draining the brain and eye resulted in the sequestration of antigens and thus created a condition akin to what we now know as "immunological ignorance." However, seminal studies performed over the past 30 years by Streilein [3 ] have provided important insights and have demonstrated that immune privilege is the product of multiple anatomical, physiological, and immunoregulatory processes [4 ].

The AC possesses a variety of mechanisms that dampen immune inflammation in the eye. Aqueous humor contains numerous anti-inflammatory and immunosuppressive factors, which serve to quench inflammation [3 4 5 ]. Also, antigens introduced into the AC induce antigen-specific down-regulation of Th1 immune responses, a phenomenon known as AC-associated immune deviation (ACAID) [5 ], which permits the sustained growth of many tissue allografts and tumors [6 ]. However, ACAID is not always induced, and some tumors elicit a robust, systemic immune response, which culminates in tumor rejection [7 ]. Immune rejection of intraocular tumors follows two mutually divergent patterns. One pattern of rejection occurs by a CD4+ T cell-mediated process, which coincides with the acquisition of delayed type hypersensitivity (DTH) responses, leading to ischemic necrosis and damage to the tumor and innocent bystander ocular cells and culminates in phthisis of the eye [7 ]. The second pattern of T cell-mediated intraocular tumor rejection is characterized by an intraocular T cell infiltrate, piecemeal necrosis of intraocular tumor cells, preservation of normal ocular cells, and the absence of phthisis [7 , 8 ]. The immunoregulatory process that determines which of these two pathways is invoked has an enormous impact on the fate of the eye and the preservation of vision.

Ad5E1 is an adenoviral- gene transformed embryonic cell tumor that has been used to characterize the circumvention of ocular immune privilege and to analyze the immune mechanisms leading to the nonphthisical form of intraocular tumor rejection [9 10 11 ]. Ad5E1 tumor rejection is dependent on CD4+ T cells and IFN-{gamma} but does not require CD8+ CTLs, perforin, TNF-{alpha}, NK cells, B cells, or Fas ligand (FasL) [10 , 11 ]. Although IFN-{gamma} is necessary for intraocular Ad5E1 tumor rejection, its exact role remains unknown. Therefore, we investigated the mechanisms by which IFN-{gamma} contributes to tumor rejection in an immune-privileged site.

The pleiotropic nature of IFN-{gamma} enables it to participate in multiple tumor rejection mechanisms. Upon binding to its receptor, IFN-{gamma} can exert direct, antiproliferative [12 ], proapoptotic [13 ], and antiangiogenic [14 ] effects on tumor cells. IFN-{gamma} also exerts numerous effects on host cells that contribute to antitumor responses, including activation of innate and adaptive immune responses [15 ] and up-regulation of tumor necrosis family cytotoxic ligands, such as TNF-{alpha}, FasL, and TRAIL [16 , 17 ].

Given the numerous effects that IFN-{gamma} exerts on tumor cells and host cells, we sought to determine which cell populations responded to IFN-{gamma} and contributed to rejection of intraocular Ad5E1 tumors. The results indicated that IFN-{gamma} acted directly on tumor cells, leading to an inhibition of tumor cell proliferation, increased tumor cell apoptosis, and down-regulation of angiogenesis in the tumor-containing eye. Also, Ad5E1 tumors grew progressively in the eyes of ß2-microglobulin knockout (ß2M KO) mice, which was a result of their inability to produce IFN-{gamma} in response to tumor antigens. However, ß2M KO mice and IFN-{gamma} KO mice rejected s.c. Ad5E1 tumors. This suggests an unrecognized facet of immune deviation in which IFN-{gamma}-independent immune processes can mediate tumor rejection at s.c. sites but are excluded from the eye.

MATERIALS AND METHODS

Animals
C57BL/6 mice, IFN-{gamma} KO mice, ß2M KO mice, CD8 KO mice, and IFN-{gamma} receptor (IFN-{gamma}R) KO mice were obtained from The Jackson Laboratory (Bar Harbor, ME). TRAIL KO breeding pairs were kindly provided by Dr. Thomas Griffith (University of Iowa, Iowa City) and were bred at the University of Texas Southwestern Medical Center Animal Resource Center (Dallas). All animals were housed and cared for in accordance with the National Institutes of Health Guidelines on Laboratory Animal Welfare and the Association for Research in Vision and Ophthalmology, Use of Animals in Ophthalmic and Vision Research.

Tumor cells
Dr. Rene Toes (Leiden University Medical Center, The Netherlands) kindly provided Ad5E1 tumor cells [18 ], which were cultured in complete DMEM (Gibco-BRL, Grand Island, NY).

AC and s.c. tumor injections
Tumor cell suspensions (3x105 cells/5 µl) were injected into the AC as described previously [11 ], and tumor volume was recorded three times per week as the percentage of AC occupied with tumor. In other experiments, Ad5E1 tumor cells (3x105 cells/5 µl) were injected s.c., and inoculation sites were palpated three times per week to assess s.c. tumor growth.

In vitro stimulation of CD4+ T cells and IFN-{gamma} ELISA
Tumor-bearing animals (Day 17 post-tumor injection) were killed, and spleens were obtained, homogenized, and dissociated through nylon mesh. CD4+ T cells were isolated from spleens using mouse CD4 MicroBeads and magnetic cell sorting (Miltenyi Biotec, Auburn, CA).

To obtain APCs, naïve mouse spleens were homogenized and dissociated through nylon mesh. Spleen cells were incubated at 37°C for 30 min with 1 mg/ml collagenase D (Roche, Indianapolis, IN), then plated on PrimariaTM tissue-culture dishes (BD Biosciences, San Jose, CA), and incubated for 4 h at 37°C. Nonadherent cells were aspirated, leaving adherent macrophages and dendritic cells to serve as APCs.

CD4+ T cells (1x106) were incubated 72 h at 37°C in 35 x 10 mm tissue-culture dishes (BD Biosciences) in triplicate under the following conditions: alone (negative control), with 5 µg anti-CD3 (positive control), with APCs (1x106), or with APCs and mitomycin C-treated Ad5E1 stimulator cells (1x106). Levels of IFN-{gamma} in cell supernatants were determined using a mouse IFN-{gamma} Quantikine ELISA kit (R&D Systems, Minneapolis, MN).

Bone marrow (BM) chimeras
BM cells were collected from the femur and tibia of killed donor mice. Recipient mice were lethally irradiated (800 cGy) and injected i.v. at a 1:1 donor:recipient ratio (~4x107 cells/mouse) of C57BL/6 or IFN-{gamma}R KO donor BM cells. One month later, mice were challenged in the AC with Ad5E1 tumor cells as described above.

Cytokines, antibodies, and reagents
Recombinant murine (rm)IFN-{gamma} was purchased from R&D Systems. Purified rat antimouse CD16/CD32 (Fc{gamma}RIII/II) mouse BD Fc BlockTM, hamster antimouse IFN-{gamma}R ß-chain, biotin-conjugated mouse anti-Armenian and Syrian hamster IgG (cocktail) mAb, streptavidin-PE (SAv-PE) conjugate, rat antimouse CD31, and rat IgG were purchased from BD PharMingen (San Diego, CA).

Flow cytometric analysis
Surface expression of IFN-{gamma}R was assessed by flow cytometry. Nonspecific FcRs were blocked with rat antimouse CD16/CD32. Cells were washed and then stained with hamster IgG or hamster antimouse IFN-{gamma}R, followed by incubation with biotinylated antihamster cocktail and SAv-PE. After washing, cells were resuspended in 0.5 ml 2% formalin in PBS and assessed for fluorescence in a FACScan flow cytometer (BD Biosciences). Results were analyzed using CellQuest v.3.1f software (BD Biosciences). Splenocytes were used as a positive control for IFN-{gamma}R expression.

Annexin V apoptosis assay
Tumor cell apoptosis was evaluated using the Annexin V apoptosis assay [19 ]. Single-cell suspensions of Ad5E1 tumor cells (1x105 cells/ml) were added to 24-well plates (Corning Inc., Corning, NY). Cells were cultured in medium alone or medium containing various concentrations of mIFN-{gamma}. Staurosporine (Sigma-Aldrich, St. Louis, MO) was used as a positive control for inducing apoptosis [20 ]. Following incubation, cells were stained with a TACSTM Annexin V FITC kit (R&D Systems), and fluorescence was detected with a FACScan flow cytometer as described above. Annexin V-positive, propidium iodide-negative cells were considered to be apoptotic.

Proliferation assay
Single-cell suspensions of Ad5E1 tumor cells (1x105 cells/ml) were added to 24-well plates. Cells were cultured in medium alone or medium containing mIFN-{gamma}. Cells were incubated for 48 h at 37°C, then pulsed with 100 µCi 3H-thymidine, and incubated for an additional 24 h. Wells were washed with PBS, cells lysed with 10% SDS, and radioactivity counted in a liquid scintillation counter.

Tumor vessel density
The density of intraocular tumor blood vessels was assessed using the plasma marker FITC-dextran as described previously [21 ]. FITC-dextran (200 µL; 2x106 Da, 25 mg/ml, Sigma-Aldrich) was injected i.v. into tumor-bearing mice. Thirty minutes later, mice were anesthetized with ketamine hydrochloride, and the major posterior ocular artery was ligated with a 2-0 silk suture (Vanguard Surgical Systems, Houston, TX). Mice were killed, and intact tumor-bearing or naïve eyes were enucleated and placed in ice-cold PBS. Eyes were homogenized and centrifuged, and supernatant was collected. Blood samples were collected by cardiac puncture and heparinized. Plasma was separated and stored in the dark at 4°C. Eye supernatants or plasma were placed in wells of a flat-bottom, 96-well plate (50 µl), and fluorescence was measured at 490 nm (DTX 880 multimode detector, Beckman Coulter, Fullerton, CA). The tumor/plasma fluorescence ratio was determined and used as a measure of the tumor blood vessel density [21 ].

Histology
Tumor-containing eyes were removed from killed mice, fixed in formalin, embedded in paraffin, and cut into 5-µm sections, which were stained with H&E to examine tumor pathology. Other sections were incubated with anti-CD31 (PECAM-1) or rat IgG1 isotype control using the Vectastain Elite ABC system (Vector Laboratories, Burlingame, CA).

Microarray analysis
Ad5E1 tumor cells were incubated for 72 h in medium alone or in medium containing 20 U/ml rmIFN-{gamma}. Cells were submitted to the University of Texas Southwestern Medical Center Microarray Core Facility for total RNA isolation, cDNA synthesis, sample hybridization to the Mouse Genome 430 2.0 array (Affymetrix, Santa Clara, CA), and array scanning. Array data were globally normalized and analyzed using GeneSpring 7.2 software (Agilent Technologies, Palo Alto, CA) and are viewable at http://microarray.swmed.edu/smcdb/swarray_db.html.

Statistics
Comparison among experiments was performed by one-way ANOVA. The differences were considered significant at P< 0.05.

RESULTS

TRAIL is not necessary for intraocular rejection of the Ad5E1 tumor
We hypothesized previously that TRAIL might be the primary mediator of intraocular rejection of Ad5E1 tumors [11 ]. This was supported by several observations. First, TRAIL is expressed constitutively in human and murine eyes and participates in tumor surveillance [11 , 22 ]; second, Ad5E1 tumor cells express TRAIL receptors and are sensitive to TRAIL-induced apoptosis in vitro [11 ]; and third, IFN-{gamma} KO mice have diminished TRAIL expression in the eye and fail to reject intraocular Ad5E1 tumors [11 ]. To determine if TRAIL was necessary for intraocular rejection of Ad5E1 tumors, TRAIL KO mice were examined for their ability to reject intraocular Ad5E1 tumors. It is surprising that TRAIL KO mice rejected Ad5E1 tumor cells at the same tempo as wild-type C57BL/6 mice (Fig. 1A ). The results indicate that although TRAIL can induce apoptosis of Ad5E1 tumor cells in the eye [11 ], it is not necessary for rejection of intraocular Ad5E1 tumors.


Figure 1
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  		Figure 1. Intraocular tumor growth in C57BL/6, TRAIL KO, CD8 KO, and ß2M KO mice. Ad5E1 tumor cells were injected into the AC on Day 0, and tumor growth was scored as the percentage of AC occupied by tumor in (A) TRAIL KO mice (n=5), (B) CD8 KO mice (n=5), and (C) ß2M KO mice (n=5).

 
ß2M is necessary for intraocular rejection of the Ad5E1 tumor
A previous report indicated that intraocular Ad5E1 tumors underwent rejection in CD8+ T cell-depleted mice [10 ]. However, it was also shown that adoptively transferred Ad5E1-specific CD8+ CTLs mediated rejection of intraocular Ad5E1 tumors [9 ]. Therefore, the role of CD8+ CTLs in the rejection of intraocular Ad5E1 tumors is unclear. To address this discrepancy, we performed experiments using CD8 KO and ß2M KO mice, which are defective in CTLs and MHC Class I expression, respectively [23 ]. CD8 KO mice and wild-type C57BL/6 mice rejected intraocular Ad5E1 tumors (Fig. 1B) . However, Ad5E1 tumor cells grew progressively in the eyes of ß2M KO mice (Fig. 1C) .

The discrepancy between CD8 KO and ß2M KO mice may be explained by the diminished IFN-{gamma} responses that have been reported in ß2M KO mice [24 , 25 ]. As IFN-{gamma} KO mice and CD4 KO mice fail to reject intraocular Ad5E1 tumors [10 , 11 ], we examined the ability of CD4+ T cells from ß2M KO mice to produce IFN-{gamma} in response to Ad5E1 tumors. C57BL/6 splenic APCs were pulsed with mitomycin C-treated Ad5E1 tumor cells and used to stimulate CD4+ T cells from wild-type C57BL/6 mice, ß2M KO mice, and CD8 KO mice. CD4+ T cell production of IFN-{gamma} was assessed by ELISA 72 h later. The results shown in Figure 2 demonstrate that CD4+ T cells from wild-type and CD8 KO mice produced significant levels of IFN-{gamma} in response to Ad5E1 tumors, yet CD4+ T cells from ß2M KO mice produced no detectable IFN-{gamma} in response to Ad5E1 tumor antigens. It is important that ß2M KO mice were able to produce IFN-{gamma} in response to anti-CD3 stimulation.


Figure 2
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  		Figure 2. Deficient IFN-{gamma} production by ß2M KO mice in response to Ad5E1 tumor stimulation. CD4+ cells from tumor-bearing (Day 25 post-tumor inoculation) C57BL/6, ß2M KO, and CD8 KO mice were incubated for 72 h with the conditions indicated, after which cell supernatants were harvested, and IFN-{gamma} secretion was determined by ELISA. ß2M KO mice produced normal levels of IFN-{gamma} in response to anti-CD3 stimulation yet were deficient in secretion of IFN-{gamma} in response to Ad5E1 tumor stimulation (**P<0.001).

 
IFN-{gamma}-independent tumor rejection occurs at extraocular sites but is excluded from the eye
The requirement of IFN-{gamma} for rejection of intraocular Ad5E1 tumors raised the question as to whether IFN-{gamma} was needed for the rejection of Ad5E1 tumors in general or if it was a unique property of the intraocular tumors. Accordingly, Ad5E1 tumor cells (3x105 cells/5 µl) were injected s.c. in panels of ß2M KO mice, IFN-{gamma} KO mice, and wild-type C57BL/6 mice, and s.c. tumor growth was assessed three times per week. Although previous results indicated that Ad5E1 tumor cells grew progressively when transplanted into the eyes of ß2M KO mice and IFN-{gamma} KO mice, no tumors developed in any of the naïve mice injected s.c. with Ad5E1 tumor cells, indicating that an IFN-{gamma}-independent form of tumor rejection occurred at s.c. sites but was excluded from the eye (Table 1 ).


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  		Table 1. IFN-{gamma}-Independent Tumor Rejection Occurs at Extraocular Sites but is Excluded from the Eye

 
Role of IFN-{gamma}Rs on host cells in the rejection of intraocular Ad5E1 tumors
There are two possible pathways that IFN-{gamma} could use to eliminate intraocular Ad5E1 tumors: directly, by inducing tumor cell death, or indirectly, by activating antitumor responses in the host. We generated BM chimeras to test these possibilities. C57BL/6 mice were irradiated and reconstituted with BM cells from IFN-{gamma}R KO donors and challenged AC with Ad5E1 tumor cells. Similar experiments were performed using BM chimeras, in which irradiated IFN-{gamma}R KO mice were reconstituted with BM cells from wild-type mice. The absence of IFN-{gamma}Rs on hematopoietic cells (Fig. 3A ) or nonhematopoietic cells (Fig. 3B) did not affect Ad5E1 tumor rejection, as intraocular Ad5E1 tumors were rejected in these animals similarly to wild-type C57BL/6 mice. Intraocular tumors were also rejected in IFN-{gamma}R KO hosts, which lacked IFN-{gamma}Rs on hematopoietic and nonhematopoietic cells (Fig. 3C) . Together, these results demonstrated that intraocular Ad5E1 tumor rejection did not require host cell IFN-{gamma}R expression, suggesting that IFN-{gamma} mediates Ad5E1 tumor rejection directly.


Figure 3
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  		Figure 3. IFN-{gamma}R is not needed on the host cells to reject the Ad5E1 tumor. Recipient mice were irradiated lethally (800 cGy) and injected i.v. with C57BL/6 or IFN-{gamma}R KO donor BM. One month later, tumor cells (3x105 cells/5 µl) were injected into the AC. Tumor growth was scored as the percentage of AC occupied by tumor. (A) C57BL/6 recipients (n=5) of IFN-{gamma}R KO BM, (B) IFN-{gamma}R KO recipients (n=5) of C57BL/6 BM, and (C) IFN-{gamma}R KO animals all rejected the Ad5E1 tumor, indicating that IFN-{gamma}R is not needed on any host tissue to reject the tumor. (D) IFN-{gamma}R expression on Ad5E1 tumor cells. Cells were stained with anti-IFN-{gamma}R (open histogram) or an isotype control antibody (shaded histogram) and evaluated by flow cytometry. Analysis revealed a 20% increase in positively staining cells compared with the IgG control.

 
Effect of IFN-{gamma} on Ad5E1 tumor cells
For IFN-{gamma} to exert its effects, it must bind to its high-affinity receptor [26 ]. As IFN-{gamma}R expression is not necessary on host cells for tumor rejection, we hypothesized that Ad5E1 tumors must express IFN-{gamma}R. We confirmed that Ad5E1 tumor cells express the IFN-{gamma}R by flow cytometric analysis (Fig. 3D) . Upon binding of IFN-{gamma} to its receptor on Ad5E1 tumor cells, there are several possible mechanisms that IFN-{gamma} could use to mediate Ad5E1 rejection. First, we examined whether IFN-{gamma} induced apoptosis in vitro. Ad5E1 tumor cells were exposed to 20–2000 U/ml IFN-{gamma} for 24–72 h, and apoptosis was determined using Annexin V staining. IFN-{gamma} induced modest, albeit statistically significant (P<0.0008), amounts of apoptosis of Ad5E1 tumor cells at all doses and time-points tested (Fig. 4A ).


Figure 4
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  		Figure 4. Effect of IFN-{gamma} on apoptosis and proliferation of Ad5E1 tumor cells in vitro. (A) Ad5E1 tumor cells were incubated with mIFN-{gamma}, and apoptosis was determined by Annexin V staining. IFN-{gamma} induced highly significant apoptosis compared with media alone at all doses for each time-point examined, as P values never exceeded 0.0046. (B) Proliferation of Ad5E1 tumor cells was determined by incubating tumor cells with various doses of rmIFN-{gamma}, and 3H-thymidine incorporation was examined. *P< 0.05 for all IFN-{gamma} groups compared with media control.

 
Second, we examined if IFN-{gamma} inhibited Ad5E1 tumor cell proliferation. Ad5E1 tumor cells were incubated with various doses of IFN-{gamma} and incubated for 72 h. 3H-thymidine incorporation assays indicated that IFN-{gamma} greatly inhibited tumor cell proliferation, and biologically relevant doses of IFN-{gamma} inhibited proliferation by as much as 50% (Fig. 4B) .

Studies have shown that IFN-{gamma} inhibits tumor angiogenesis [27 , 28 ]. We examined if IFN-{gamma} inhibited intraocular Ad5E1 tumor angiogenesis by histopathology and by assessing the quantity of FITC-dextran-labeled plasma contained in tumor-bearing eyes. Gross clinical observations revealed markedly greater intraocular tumor vascularization in IFN-{gamma} KO micecompared with wild-type hosts (Fig. 5A and 5B ), which was confirmed by histopathology (Fig. 5C 5D 5E 5F) .


Figure 5
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  		Figure 5. Effect of IFN-{gamma} on tumor blood vessel density. (A) Clinical photograph of a tumor-bearing IFN-{gamma} KO mouse reveals extensive vascularization of the tumor by Day 13 post-tumor injection. (B) Clinical photograph of a tumor-bearing C57BL/6 on Day 13 post-tumor injection reveals a clear eye nearly devoid of tumor. (C) Histological examination confirms the presence of blood vessels in the tumor of IFN-{gamma} KO mice at 100x and at (E) 250x original magnifications (arrows). (D) Histological examination reveals a small tumor devoid of tumor blood vessels in C57BL/6 mice at 100x and at (F) 250x original magnifications. (G) To quantitate tumor vessel density, tumor-bearing animals were injected with the plasma marker FITC-dextran as described in Materials and Methods. Tumor/plasma fluorescence ratio is a measure of the tumor vessel density. Results compare tumor vessel density of C57BL/6 mice with ß2M KO mice and with (H) IFN-{gamma} KO mice.

 
We assessed tumor vascularity in mice by FITC-dextran quantitation. Tumor vessel density correlates with the amount of FITC-dextran that extravasates into the tumor and accumulates in the vessels [29 ]. Tumor-bearing eyes of ß2M KO (Fig. 5G ) and IFN-{gamma} KO mice (Fig. 5H) demonstrated significantly increased accumulation of FITC-dextran compared with tumor-bearing eyes of wild-type hosts.

We also examined blood vessel density by immunohistochemical staining of vascular endothelial cells with anti-CD31 (PECAM-1) antibody. Intraocular tumors (Day 13 postinjection) in IFN-{gamma} KO mice exhibited a greater number of CD31-positive cells (Fig. 6D and 6F ) than intraocular tumors in C57BL/6 mice (Fig. 6B) , supporting the hypothesis that IFN-{gamma} plays an antiangiogenic role in Ad5E1 tumor rejection.


Figure 6
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  		Figure 6. Ad5E1 tumor angiogenesis in wild-type C57BL/6 mice and IFN-{gamma} KO mice. Tumor-bearing eyes of C57BL/6 (A and B) or IFN-{gamma} KO mice (C–F) were harvested on Day 13 post-tumor injection. Paraffin sections were prepared and stained with an isotype control antibody (A, C, and E) or rat anti-mouse CD31 (B, D, and F). C57BL/6 mice are devoid of CD31 staining, whereas IFN-{gamma} KO mice display abundant staining of CD31 (arrows). Photographs were taken at 800x original magnification.

 
Our data show a correlation between the presence of IFN-{gamma} and the lack of Ad5E1 tumor angiogenesis. To see if IFN-{gamma} directly alters the expression of angiogenesis-related genes in Ad5E1 tumor cells, microarray analyses were performed. Ad5E1 cells were incubated in normal medium alone or with 20 U/ml IFN-{gamma} for 72 h. Angiogenesis-related genes were analyzed specifically for differential expression following IFN-{gamma} treatment. Table 2 shows nine candidate genes, which were altered in Ad5E1 cells by IFN-{gamma} treatment. Five antiangiogenesis-related genes were up-regulated following IFN-{gamma} treatment, including the chemokines CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC). Four proangiogenesis genes were down-regulated following exposure to IFN-{gamma}, including VEGF and PDGF-D. These results suggest that direct binding of IFN-{gamma} on Ad5E1 tumor cells may affect angiogenesis by altering expression of these genes, leading to tumor ischemia and resolution.


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  		Table 2. Differential Expression of Pro- and Antiangiogenic Genes by IFN-{gamma}-Treated Ad5E1 Tumor Cells, As Determined by Microarray Analysis

 
DISCUSSION

The rejection of intraocular tumors can occur by two processes, which have profoundly different histopathological patterns and consequences to the host. Phthisical rejection of intraocular tumors resembles a localized DTH reaction and is characterized by damage to microvascular endothelium of the tumor, ischemic bulk necrosis, innocent bystander destruction of normal host tissues, perivascular cuffing with mononuclear cells, and little or no evidence of cell-cell contact between immune effector cells and tumor cells and a conspicuous absence of piecemeal necrosis of intraocular tumor cells [6 7 8 , 30 31 32 33 ]. In sharp contrast, non-phthisical rejection of tumors is characterized by mononuclear infiltrates comprised of CD4+ T cells, CD8+ T cells, and F4/80+ macrophages; piecemeal destruction of tumor cells without innocent bystander damage to normal host tissue; and little or no evidence of ischemia [6 7 8 , 30 31 32 33 ]. The presence of infiltrating mononuclear cells is not sine qua non for either of these pathways, as both patterns of rejection have mononuclear infiltrates. The nonphthisical rejection of intraocular Ad5E1 tumors requires CD4+ T cells, IFN-{gamma}, and macrophages [34 ] but is also accompanied by an infiltration of CD8+ T cells (unpublished findings). Future studies will define the precise role of each of these immune elements in the nonphthisical form of intraocular tumor rejection.

This study examined the IFN-{gamma}-dependent mechanisms that culminate in the nonphthisical form of intraocular tumor rejection. Of the two primary pathways of intraocular tumor rejection, the nonphthisical form is the most desirable, as the tumor is eradicated, and the eye is preserved. We have shown previously that hosts deficient in IFN-{gamma} had a profound reduction of TRAIL expression on intraocular tissues and were unable to reject intraocular Ad5E1 tumors [11 ]. These findings, along with the observation that intraocular tumor rejection was accompanied by extensive tumor cell apoptosis, led us to suspect that the nonphthisical form of intraocular tumor rejection was mediated by TRAIL, which is normally expressed on multiple, intraocular tissues [11 , 22 ]. Other data supporting this hypothesis include: Ad5E1 cells express TRAIL-R2 and are susceptible to TRAIL-induced apoptosis; IFN-{gamma} enhances TRAIL expression on CD4+ T cells and ocular cells; IFN-{gamma} enhances tumor cell susceptibility to TRAIL-induced apoptosis; CD4+ T cells, corneal endothelial cells, and iris ciliary body cells express TRAIL and are capable of inducing apoptosis of tumor cells in vitro; and in vitro apoptosis induced by CD4+ T cells or corneal cells are partially blocked with anti-TRAIL antibody [11 ]. However, the role of TRAIL in mediating Ad5E1 rejection is questionable, as intraocular tumor rejection was unabated in TRAIL KO mice.

Immune-mediated tumor rejection has been thought to be mediated primarily by CD8+ CTLs. In many models, CD8+ T cells, in the absence of CD4+ T cells, are effective in eliminating tumors [35 , 36 ]. However, some studies have shown that CD4+ T cells, in the absence of CD8+ T cells, are effective in eliminating tumors [37 ]. CD4+ T cell-mediated rejection is often thought to occur by direct killing of MHC Class II+ tumors [38 ]. However, CD4+ T cell rejection of MHC Class II-negative tumors suggests that CD4+ T cells use additional effector mechanisms in promoting tumor rejection [37 ]. IFN-{gamma} is one of the immune elements that follows this pathway [28 , 37 ]. Rejection of intraocular Ad5E1 tumors is CD4+ T cell-dependent, as CD4 T cell-depleted mice cannot reject the tumor in the eye [10 ]. Rejection is IFN-{gamma}-dependent, as IFN-{gamma} KO mice fail to reject intraocular Ad5E1 tumors [11 ]. Therefore, it is likely that IFN-{gamma}-expressing CD4+ T cells are the primary mediators of intraocular Ad5E1 tumor rejection.

The study demonstrates that CD8+ T cells are not necessary for rejection of intraocular Ad5E1 tumors. Earlier reports offer conflicting conclusions. One study ruled out CD8+ T cell participation, as mice depleted of this population of cells were able to reject an intraocular Ad5E1 challenge [10 ]. By contrast, another report indicated that Ad5E1-specific CTLs could be generated in vitro and adoptively transferred to SCID recipients, resulting in intraocular tumor rejection [9 ]. We used two mouse models deficient in CD8+ T cells (i.e., ß2M KO mice and CD8 KO mice) to address this issue. The results were again conflicting. CD8 KO mice rejected intraocular tumors, and ß2M KO mice harbored progressively growing intraocular tumors. These results led to alternative hypotheses to explain the nonphthisical form of intraocular tumor rejection. The first hypothesis proposed that NKT cells mediated tumor rejection, as ß2M KO mice are deficient in NKT cells [39 ]. Tumor rejection by NKT cells has been documented previously [40 ], and their secretion of IFN-{gamma} is important for their antitumor activity [41 ]. However, intraocular Ad5E1 tumors are not rejected in MHC Class II-deficient mice [10 ] and transporter associated with antigen processing 1 (TAP-1) KO mice (data not shown), both of which have normal NKT cell repertoires [42 , 43 ], suggesting that the inability of ß2M KO mice to reject Ad5E1 tumors is not a result of their lack of NKT cells. Also, CD4+ T cells from TAP-1 KO mice cannot produce IFN-{gamma} in response to Ad5E1 tumor antigens (data not shown). This indicates that MHC Class I and not NKT cells is responsible for efficient production of CD4+ T cell-derived IFN-{gamma}.

The second hypothesis as to why ß2M KO mice fail to reject the Ad5E1 tumor relates to insufficient IFN-{gamma} secretion by CD4+ T cells in these animals. Previous studies have shown that ß2M KO mice have a severely reduced capacity to produce IFN-{gamma} in response to bacterial or viral challenges [24 , 25 ]. Of particular interest was the decreased ability of ß2M KO CD4+ T cells to produce IFN-{gamma} in response to antigenic challenge [25 ]. Therefore, we tested the ability of CD4+ T cells from ß2M KO mice to produce IFN-{gamma} in response to APCs pulsed with Ad5E1 tumor antigens. This hypothesis was confirmed, as ß2M KO mice could not produce IFN-{gamma} in response to Ad5E1 tumor antigens. This inability was not a result of an intrinsic deficiency of ß2M KO mice to produce IFN-{gamma}, as they produced normal levels of IFN-{gamma} in response to CD3 stimulation. Rather, it appears that there was an impaired IFN-{gamma} response to Ad5E1 tumor antigens in the ß2M KO mice. The exact mechanism resulting in impaired IFN-{gamma} production in Class I-deficient mice is unknown. However, some data suggest that MHC Class I genes can influence the development of protective CD4+ Th1 responses [44 ]. Therefore, the absence of such genes could result in an impaired Th1 response and most notably, insufficient IFN-{gamma} expression. The role of MHC Class I and its effects on CD4+ T cells merit further investigation.

Some studies indicate that host cells, rather than tumor cells, are the targets of CD4+ T cell-derived IFN-{gamma} [27 , 28 , 37 ]. However, our results indicate that tumor cells are the target of IFN-{gamma}, which agrees with other tumor models [45 , 46 ]. The present findings demonstrate that IFN-{gamma} affects Ad5E1 tumor cells in multiple ways, including inhibition of proliferation, induction of apoptosis, and inhibition of angiogenesis. The effect of IFN-{gamma} on angiogenesis is noteworthy. The putative decrease in tumor vessel density was confirmed by CD31 immunohistochemistry and FITC-dextran fluorescence. Moreover, the microarray data demonstrate that IFN-{gamma} affected the expression of proangiogenic and antiangiogenic genes in Ad5E1 tumor cells. Of particular interest are IP-10, MIG, and I-TAC, three chemokines, which are IFN-inducible and antiangiogenic [14 ]. These chemokines bind in a highly selective manner to CXCR3, a chemokine receptor that is expressed on activated T cells and endothelial cells [47 ]. Therefore, expression of these two chemokines is important as they inhibit tumor angiogenesis and attract activated T cells to the tumor site to facilitate tumor rejection.

The results suggest that rejection of intraocular Ad5E1 tumors is IFN-{gamma}-dependent and acts directly on the tumor cells through multiple mechanisms, including the inhibition of proliferation and the induction of tumor cell apoptosis. IFN-{gamma} also up-regulates MHC Class I on the tumor cells (data not shown), which may increase their immunogenicity and facilitate rejection. However, the most important role IFN-{gamma} plays in intraocular Ad5E1 tumor rejection is in the inhibition of angiogenesis. In the presence of IFN-{gamma}, intraocular Ad5E1 tumors demonstrate significantly less vascular endothelial cells and tumor blood vessel density, which corresponds to up-regulation of five antiangiogenic-related genes and down-regulation of four proangiogenic-related genes. These mechanisms lead to the abrogation of ocular immune privilege and the rejection of Ad5E1 tumors in syngeneic C57BL/6 mice.

The present findings also reveal a heretofore unrecognized facet of immune deviation in which IFN-{gamma}-independent immune processes can mediate tumor rejection at s.c. sites but do not play a role in intraocular tumor rejection. Our results imply that this novel, IFN-{gamma}-independent immune mechanism is not induced by intraocular Ad5E1 tumors or is induced but abrogated in the eye. The exclusion of this IFN-{gamma}-independent mechanism of tumor rejection may represent yet a new facet of ocular immune privilege.

ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants EY05631 and EY016664 and an unrestricted grant from Research to Prevent Blindness, Inc. (New York, NY).

Received August 1, 2006; revised September 12, 2006; accepted September 29, 2006.

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D. S. Dace, P. W. Chen, and J. Y. Niederkorn
CD8+ T Cells Circumvent Immune Privilege in the Eye and Mediate Intraocular Tumor Rejection by a TNF-{alpha}-Dependent Mechanism
J. Immunol., May 15, 2007; 178(10): 6115 - 6122.
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