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The Journal of Immunology, 2007, 178: 6115-6122.
Copyright © 2007 by The American Association of Immunologists, Inc.
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CD8+ T Cells Circumvent Immune Privilege in the Eye and Mediate Intraocular Tumor Rejection by a TNF-{alpha}-Dependent Mechanism1

Dru S. Dace, Peter W. Chen and Jerry Y. Niederkorn2

Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX 75390


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although intraocular tumors reside in an immune-privileged environment, T cells can circumvent immune privilege and mediate tumor rejection without inducing damage to normal ocular tissue. In this study, we used a well-characterized tumor, Ad5E1 (adenovirus type 5 early region 1), to analyze the role of CD8+ T cells in the pristine rejection of intraocular tumors. It has been previously documented that Ad5E1 tumor rejection can occur in the absence of CD8+ T cells. However, here we find that CD8+ T cells infiltrated intraocular Ad5E1 tumors in C57BL/6 mice. Surprisingly, CD8+ T cells from tumor-rejector mice could mediate intraocular tumor rejection following adoptive transfer to SCID mice. In determining the mechanisms behind CD8+ T cell-mediated tumor rejection, we discovered that antitumor CTL activity was neither observed nor necessary for rejection of the intraocular tumors. CD8+ T cells from rejector mice did not produce IFN-{gamma} in response to Ad5E1 tumor Ags or use FasL to mediate intraocular tumor rejection. Also, CD8+ T cells did not use perforin or TRAIL, as CD8+ T cells from perforin knockout (KO) and TRAIL KO mice conferred protection to SCID recipient mice following adoptive transfer. We discovered that CD8+ T cells used TNF-{alpha} to mediate tumor rejection, because Ad5E1 tumor cells were highly sensitive to TNF-{alpha}-induced apoptosis and CD8+ T cells from TNF-{alpha} KO mice did not protect SCID mice from progressive Ad5E1 tumor growth. The results indicate that CD8+ T cells circumvent immune privilege and mediate intraocular tumor rejection by a TNF-{alpha}-dependent manner while leaving the eye intact and vision preserved.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ocular immune privilege is necessary for maintaining vision. Immune-mediated inflammation can have devastating consequences on normal ocular cells and the architecture of the eye. However, the eye invokes several mechanisms for maintaining immune privilege and reducing the risk of immune-mediated injury. Immune privilege of the anterior chamber (AC)3 of the eye is arguably the most intensely studied immune-privileged site and employs numerous mechanisms to restrain immune-mediated inflammation. The reduced expression or absence of MHC class I Ags on the corneal endothelium lessens the likelihood of injury due to infiltrating CD8+ CTLs (1). The aqueous humor contains a number of anti-inflammatory and immunosuppressive factors that serve to quench inflammation (2, 3, 4). Also, Ags introduced into the AC induce an Ag-specific down-regulation of Th1 and Th2 immune responses, a phenomenon known as AC-associated immune deviation (4). Immune privilege allows the prolonged and sometimes permanent existence of foreign tissues and tumors (5, 6). However, ocular immune privilege can be circumvented. Ocular tumors can induce a robust systemic immune response that culminates in tumor rejection (7, 8, 9, 10, 11). Immune rejection of intraocular tumors follows two mutually divergent patterns. One pattern of rejection occurs by a CD4+ T cell-mediated process that coincides with the development of delayed-type hypersensitivity responses leading to ischemic necrosis and damage to both the tumor and innocent bystander ocular cells and culminates in phthisis of the eye, whereas 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 (6, 7, 12, 13, 14, 15, 16, 17). 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.

The adenovirus-induced tumor Ad5E1 (adenovirus type 5 early region 1) possesses many qualities that make it amenable for analyzing the immunoregulatory processes that circumvent immune privilege and promote tumor rejection without inflicting irreparable injury to the eye and jeopardizing vision. This tumor undergoes spontaneous rejection when injected into the AC of syngeneic C57BL/6 mice. Tumor rejection requires CD4+ T cells, IFN-{gamma}, and macrophages, but does not require TNF-{alpha}, FasL, TRAIL, perforin, B cells, NK cells, or CD8+ T cells (9, 10, 11, 18). Importantly, tumor rejection does not culminate in phthisis and instead the anatomical integrity of the eye is left intact (9).

Many tumor models describe the role of CD8+ T cells as being the primary mediators of tumor rejection (19, 20, 21), which occurs through the direct recognition of MHC class I and the subsequent cytolysis by CTLs. In some models of tumor rejection, CD4+ T cells function as helper cells that are needed only for the induction of CTLs (22, 23, 24, 25, 26). Once CD8+ T cells are primed, the depletion of CD4+ T cells does not affect the effector function of CD8+ T cells (26). The rejection of intraocular Ad5E1 tumors is unique, as it appears to be primarily mediated by CD4+ T cells and not CD8+ T cells (9). Although intraocular Ad5E1 tumors undergo rejection in mice depleted of CD8+ T cells (9), there is evidence that CD8+ T cells might play an ancillary role in Ad5E1 tumor rejection. Schurmans et al. indicated that CD8+ T cells were present in intraocular Ad5E1 tumors (9). Moreover, they demonstrated that a CD8+ CTL clone generated by s.c. immunization with adenovirus+ Ras-transformed Ad5E1 tumor cells was capable of mediating the rejection of established intraocular Ad5E1 tumors when adoptively transferred into syngeneic C57BL/6 mice (8). These observations prompted us to consider the hypothesis that intraocular Ad5E1 tumors elicited the generation of tumor-specific CD8+ T cells that were capable of circumventing immune privilege and mediating tumor rejection by a mechanism that preserved the anatomical integrity of the eye.

In this study, we confirm the presence of CD8+ T cells in intraocular Ad5E1 tumors. Interestingly, CD8+ T cells that were isolated from tumor-rejector C57BL/6 mice were able to confer protection when adoptively transferred to SCID mice. We then attempted to determine the mechanism behind CD8+ T cell-mediated tumor rejection. We found that CD8+ T cells did not use CTLs, IFN-{gamma}, FasL, perforin, or TRAIL, but did use TNF-{alpha} to mediate intraocular Ad5E1 tumor rejection. This study is unique because it demonstrates that: 1) CD8+ T cells circumvent immune privilege; 2) CD8+ T cells mediate intraocular tumor rejection in a TNF-{alpha}-dependent manner without the induction of ocular tissue damage; 3) CD8+ effector T cells are not conventional "CTLs"; and 4) CD8+ T cell-mediated rejection of intraocular tumors is an ancillary pathway, separate from a CD4+ T cell-derived IFN-{gamma}-dependent-pathway of intraocular tumor rejection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

C57BL/6 (H-2b) mice were obtained from either The Jackson Laboratory or the National Cancer Institute (Frederick, MD). CD8 knockout (KO) mice (B6.129S2-Cd8atm1Mak/J), IFN-{gamma} KO mice (B6.129S7-Ifngtm1Ts/J), perforin KO mice (C57BL/6-Prf1tm1Sdz/J), TNF-{alpha} KO mice (B6;129S6-Tnftm1Gkl/J), and SCID mice (B6.CB17-Prkdcscid/SzJ) were obtained from The Jackson Laboratory. TRAIL KO breeding pairs were kindly provided by Dr. T. Griffith (University of Iowa, Iowa City, IA) and were bred at the University of Texas Southwestern Medical Center Animal Resource Center (Dallas, TX). All animals were housed and cared for in accordance with the guidelines of the University Committee for the Humane Care of Laboratory Animals, National Institutes of Health Guidelines on Laboratory Animal Welfare, and the Association for Research in Vision and Ophthalmology statement about the Use of Animals in Ophthalmic and Vision Research.

Tumor cells

Ad5E1 tumor cells were kindly provided by Dr. R. E. M. Toes (Leiden University Medical Center, Leiden, The Netherlands). The tumor cells were generated by the transformation of C57BL/6 mouse embryo cells with a plasmid encoding the human Ad5E1 and propagated as previously described (20, 27). Single-cell suspensions of Ad5E1 tumor cells were washed in HBSS (Cambrex) and suspended in HBSS for AC injections. Tumor cells were cultured in DMEM (Invitrogen Life Technologies) containing 10% heat-inactivated FCS, 1% L-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, 1% HEPES buffer, and 1% antibiotic-antimycotic solution.

P815 murine mastocytoma cells (DBA/2 origin) were obtained from the American Type Culture Collection and were cultured in complete DMEM as mentioned above.

AC injections

Tumor cell suspensions were injected into the AC as previously described (28). Mice were anesthetized with 0.66 mg/kg ketamine hydrochloride (Vetalar; Parke-Davis) given i.p. The eye was viewed by low power (x8) under a dissecting microscope and a sterile 30-gauge needle was used to puncture the cornea at the corneoscleral junction, parallel and anterior to the iris. A glass micropipette (diameter ~80 µm) was fitted onto a sterile infant feeding tube (size 5 French; Tyco Healthcare Group) and mounted onto a 0.1-ml Hamilton syringe. A Hamilton automatic dispensing apparatus was used to inject 5 µl of a monocellular suspension of Ad5E1 tumor cells (3 x 105 cells/5 µl). Eyes were examined three times per week, and the tumor volume was recorded as the percentage of AC occupied with tumor (28).

Flow cytometry of tumor-bearing eyes

C57BL/6 mice bearing intraocular Ad5E1 tumors were sacrificed between days 11–13 after tumor injection and tumor-bearing eyes, naive eyes, and spleens were collected. Tissues were homogenized, and cell suspensions were passed through a 70-µm nylon cell strainer. Erythrocytes were lysed and tissues were resuspended in HBSS containing 0.3% BSA. Cells were then double-stained with fluorochrome-conjugated Abs (1 µg/ml), either IgG-FITC and IgG-PE or anti-CD8-FITC and anti-CD3-PE (BD Pharmingen). Cells were incubated with Abs for 30 min at 4°C, washed three times with HBSS, and resuspended in PBS containing 0.3% BSA. Cells were then assessed for fluorescence in a FACScan flow cytometer (BD Biosciences) and the results were analyzed using CellQuest version 3.1f software (BD Biosciences).

Adoptive transfer experiments

C57BL/6, perforin KO, TRAIL KO, and TNF-{alpha} KO mice were injected with Ad5E1 tumors as described above. The tumors were spontaneously rejected in these animals within 2–3 wk after tumor injection. Following rejection, these animals were sacrificed, splenocytes were collected, and erythrocytes were lysed. Splenocytes were then incubated with CD8-specific microbeads (10-µl beads per 107 cells; Miltenyi Biotec) in PBS with 0.5% BSA for 15 min at 4°C. The cells were washed with 0.5% BSA in PBS followed by magnetic separation using LS+ columns as described by the manufacturer (Miltenyi Biotec). The retained cells were eluted from the column. To ensure that no contaminating CD4+ T cells were present, the cells were subsequently treated with 1 µg/ml anti-mouse CD4 (BD Pharmingen) followed by incubation with Low-Tox rabbit complement (Cedarlane Laboratories). Cells were washed three times, resuspended in HBSS, and injected i.v. into SCID or IFN-{gamma} KO mice at a 1:1 donor to recipient ratio (~1 x 107 cells/mouse). Following adoptive transfer, recipient mice were injected with Ad5E1 tumors as described above.

CTL assay

A standard 4-h 51Cr release assay, as previously described (29), was used to measure CTL activity in vitro. Briefly, single-cell suspensions of lymphocytes in complete RPMI 1640 medium (BioWhittaker) containing 10% heat-inactivated FBS (HyClone), 2 mM L-glutamine (BioWhittaker), 1 mM sodium pyruvate (BioWhittaker), 1% penicillin-streptomycin-Fungizone (BioWhittaker), 1% nonessential amino acids (BioWhittaker), 1% HEPES buffer (BioWhittaker), and 5 x 10–5 M 2-ME (Sigma-Aldrich) were prepared from various spleens and used as effector cells. Experimental and control effector lymphocytes were boosted in vitro for 96 h at 37°C with mitomycin C-treated Ad5E1 tumor cells or mitomycin C-treated P815 tumor cells. The in vitro boosted effector cells were washed and resuspended in complete RPMI medium. Effector cells were dispensed along with 2 x 104 51Cr-labeled Ad5E1 or P815 cells/well in triplicate at several E:T ratios (100:1, 50:1, 25:1, and 12.5:1) in a 96-well U-bottom microtiter plate (Corning), in a total volume of 200 µl/well. Tumor cells were also incubated alone (spontaneous release) or with 50 µl of Zapoglobin (Beckman Coulter) lytic reagent (total release). The plate was incubated at 37°C for 4 h. The plate was then centrifuged at 800 rpm for 6 min before harvesting 100 µl of the supernatant from each well and counting on a gamma counter. Cytotoxicity was determined by the amount of 51Cr released by the target cells, and the specific lysis was calculated as follows: [(experimental cpm) – (spontaneous release cpm)] ÷ [(maximum release cpm) – (spontaneous release cpm)] x 100%.

In vitro stimulation of T cells and IFN-{gamma} ELISA

Tumor-bearing animals (day 17 after tumor injection) were killed and spleens were obtained. CD4+ and CD8+ T cells were isolated from spleens using mouse CD4 and CD8 microbeads and magnetic cell sorting as described above.

APCs were obtained by mincing the spleens from naive animals and incubating them with 1 mg/ml collagenase D (Roche) at 37°C for 30 min. Cells were plated on Primaria tissue culture dishes (BD Biosciences) and incubated for 4 h at 37°C. Nonadherent cells were aspirated, leaving adherent macrophages and dendritic cells, which were collected and used as APCs.

T cells (1 x 106) were incubated alone (negative control), with 5 µg of anti-CD3 (positive control), with mitomycin C-treated Ad5E1 stimulator cells (1 x 106), or with APCs and mitomycin C-treated Ad5E1 stimulator cells (1 x 106) in 35 x 10 mm tissue culture dishes (BD Biosciences) in triplicate. Cells were incubated for 72 h at 37°C. Supernatants were harvested and levels of IFN-{gamma} in cell supernatants were determined using a mouse IFN-{gamma} Quantikine ELISA kit (R&D Systems).

Detection of Fas expression by flow cytometry

Ad5E1 tumor cells were cultured in medium alone or in medium containing 20 U/ml recombinant murine IFN-{gamma} for 72 h. Cells were washed and then stained with either hamster IgG or hamster anti-mouse Fas (clone Jo-2; BD Pharmingen) followed by incubation with biotinylated anti-hamster mixture and streptavidin-PE (BD Pharmingen). After washing, cells were resuspended in 0.5 ml of 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 Fas expression.

Annexin V apoptosis assay

Tumor cell apoptosis was evaluated using an annexin V apoptosis assay (30). Single-cell suspensions of Ad5E1 tumor cells (1 x 105 cells/ml) were added to 24-well plates (catalog no. 3526; Corning). Cells were suspended in either medium alone or medium containing various concentrations of murine TNF-{alpha} (1, 10, or 100 ng/ml). Staurosporine (3 µg/ml; Sigma-Aldrich) was used as a positive control for inducing apoptosis (31). Cultures were incubated for 24, 48, and 72 h at 37°C. Following incubation, cells were removed by trypsinization and resuspended in PBS. Tumor cells were stained with a TACS annexin V-FITC kit (R&D Systems). Cell suspensions were evaluated using a FACScan flow cytometer (BD Biosciences), and the results were analyzed using CellQuest version 3.1f software (BD Biosciences). Propidium iodide-negative cells that stained positively with annexin V were considered to be apoptotic.

Detection of membrane TNF-{alpha} expression by flow cytometry

Spleen-derived CD8+ T cells from tumor-rejector mice were isolated by CD8 microbead separation as described above. CD8+ T cells were incubated with either medium alone (negative control), PMA plus ionomycin (50 ng/ml and 1 µg/ml, respectively; positive control), or mitomycin C-treated Ad5E1 tumor cells for 24 h at 37°C. Cells were then harvested, washed, and stained with 1 µg/ml rat anti-mouse TNF-{alpha} Ab (R&D Systems) or 1 µg/ml rat IgG isotype control Ab for 30 min at 4°C. Cells were stained with a FITC-conjugated anti-rat IgG Ab (BD Pharmingen) for 30 min at 4°C. Fluorescence was detected with a FACScan flow cytometer (BD Biosciences). Results were analyzed using CellQuest v.3.1f software (BD Biosciences).

Statistics

The Mann-Whitney U test was performed for in vivo Ad5E1 tumor growth experiments. Differences between groups were considered statistically significant once p values fell below 0.05. For all other experiments, a Student’s t test was used to assess the statistical significance of the differences between experimental and control groups. p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD8+ T cells infiltrate intraocular Ad5E1 tumors

Although CD8+ T cells are not required for the rejection of intraocular Ad5E1 tumors, CD8+ T cells may still play a role in their rejection. Schurmans et al. indicated that CD8+ T cells were present in intraocular Ad5E1 tumors as detected by immunohistochemistry (9). To verify this observation and quantitate the numbers of CD8+ T cells infiltrating Ad5E1 tumors, tumor-bearing and naive eyes were obtained from C57BL/6 mice at a time point that roughly coincided with the onset of tumor rejection (day 13 after tumor injection). Ocular tissue was homogenized and stained with Abs to detect CD8+CD3+ T cells. Fluorescence was detected by flow cytometry, and splenocytes were used as a positive control for staining CD8+ T cells. Isotype control staining of tissues was negative (data not shown), and the location of quadrants was based on the fluorescence of isotype controls. A significant number of CD8+CD3+ T cells infiltrated Ad5E1 tumor-bearing eyes (Fig. 1). Naive eyes contained a very low number (~1.0%) of CD8+CD3+ T cells, which may be due to T cells circulating within the blood vasculature of the eye at the time of tissue harvest. The increased number of CD8+CD3+ T cells in tumor-bearing eyes suggests that CD8+ cells migrate to the eye in a tumor-dependent manner.


Figure 1
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  		FIGURE 1. CD8+ T cells are present in Ad5E1 tumor-bearing eyes. C57BL/6 mice bearing Ad5E1 tumors were sacrificed on day 13 after tumor injection and tumor-bearing eyes, naive eyes, and spleens were harvested. Cells from tissues were stained for the presence of CD8+CD3+ cells using anti-CD8-PE and anti-CD3-FITC Abs. Cells were also stained with IgG-PE and IgG-FITC as isotype controls. Staining was then assessed by flow cytometry. CD8+CD3+ cells were present in splenocyte suspensions (positive control) and Ad5E1 tumor-bearing eyes. Very low numbers of CD8+CD3+ cells were observed in naive eyes, indicating these cells migrate to tumor-bearing eyes.

 
CD8+ T cells from rejector mice can be adoptively transferred to SCID mice and produce tumor rejection

Although CD8+ T cells migrate to intraocular tumors, it was important to confirm that CD8+ T cells were tumor-specific and could mediate tumor rejection. Accordingly, Ad5E1 tumor cells were injected into the eyes of C57BL/6 mice. Following tumor rejection, CD8+ T cells were harvested from the spleens of rejector mice and purified using magnetic bead sorting. CD8+ T cell suspensions were purged of contaminating CD4+ T cells by using anti-CD4 Ab plus complement. Purified CD8+ T cells were adoptively transferred to SCID mice by i.v. injection (~1 x 107 cells/mouse) and the mice were immediately challenged with an AC injection of Ad5E1 tumor cells. Surprisingly, the adoptive transfer of CD8+ T cells from rejector mice led to Ad5E1 tumor rejection in SCID mice, whereas naive SCID mice exhibited progressive tumor growth (Fig. 2). Thus, CD8+ T cells can function as antitumor effector cells and can mediate tumor rejection independently of CD4+ T cells once they are primed.


Figure 2
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  		FIGURE 2. Adoptive transfer of CD8+ T cells from C57BL/6 mice that previously rejected Ad5E1 tumors protects SCID recipients from Ad5E1 tumor outgrowth. Ad5E1 tumor cells were injected into the AC of C57BL/6 mice. Splenocytes were collected after intraocular tumor rejection (between days 21 and 28 after tumor injection). CD8+ T cells were isolated from splenocytes by magnetic bead sorting and further purified by treatment with an anti-CD4 Ab plus complement. CD8+ T cells (~1 x 107 cells/mouse) were injected i.v. into SCID recipients. SCID mice were then challenged in the AC with Ad5E1 tumor cells on the same day as the adoptive transfer. Tumor growth was monitored 2–3 times per week and scored as the percentage of an AC occupied by a tumor. Each data point represents the average percentage of ACs occupied with tumors in five mice from each treatment group. Tumors grew progressively in naive SCID mice (n = 5), but were rejected in SCID mice that received rejector CD8+ T cells (n = 5). Significant differences (p = 0.025) were observed at day 15 after tumor injection and at all time points thereafter. This experiment was repeated two additional times with similar results.

 
Absence of CTL activity in hosts that have rejected intraocular Ad5E1 tumors

CD8+ T cells are classically defined as CTLs, and the ability of CD8+ T cells to mediate tumor rejection is often reflected by their ability to lyse tumor targets in vitro in a standard CTL assay. Accordingly, we performed a CTL assay with a variety of immunologically impaired mouse strains that were injected with Ad5E1 tumors. Surprisingly, no CTL activity was found against Ad5E1 tumor cells in C57BL/6 mice that rejected either intraocular or s.c. Ad5E1 tumors (Table I). As expected, IFN-{gamma} KO and CD4 KO mice that were susceptible to progressive intraocular Ad5E1 tumor growth did not develop tumor-specific CTLs. C57BL/6 mice injected s.c. with the DBA/2 mastocytoma P815 developed tumor-specific CTLs against P815 target cells but failed to lyse the negative control B16LS9 tumor cells, indicating that C57BL/6 mice develop tumor-specific allogeneic CTLs. BALB/c mice injected s.c. with Ad5E1 tumor cells developed CTLs that effectively lysed Ad5E1 tumor cells targets, indicating that Ad5E1 tumor cells were susceptible to lysis by allogeneic CTLs. Because no CTL activity was detected against Ad5E1 tumors in C57BL/6 mice, we referred to these CD8+ T cells as "CD8+ antitumor effector cells" rather than "CTLs".


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  		Table I. Absence of CTL activity in hosts that have rejected intraocular Ad5E1 tumors

 
CD8+ T cells do not use IFN-{gamma}, FasL, perforin, or TRAIL to mediate Ad5E1 tumor rejection

Because CD8+ T cells could independently produce intraocular tumor rejection, we determined the mechanisms that CD8+ T cells used to eliminate intraocular Ad5E1 tumors. CD8+ T cells can use multiple mechanisms to produce tumor rejection, including IFN-{gamma}, FasL, perforin/granzyme, TRAIL, and TNF (32, 33, 34, 35). We examined each of these mechanisms. The first potential mechanism of tumor rejection explored was CD8+ T cell-derived IFN-{gamma}. Ad5E1 tumor cells grow progressively in the eyes of IFN-{gamma} KO mice (10), and IFN-{gamma} invokes several antitumor properties upon binding to Ad5E1 tumor cells, including the induction of apoptosis, the inhibition of proliferation, and the prevention of tumor angiogenesis (18). Therefore, we examined whether CD8+ T cells from C57BL/6 rejector mice produced IFN-{gamma} in response to Ad5E1 tumor Ags. CD8+ T cells did not produce IFN-{gamma}, either in response to Ad5E1 tumors alone or when cocultured with APCs and Ad5E1 tumor Ags (Fig. 3A). By contrast, CD4+ T cells from C57BL/6 rejector mice produced significant amounts of IFN-{gamma} in response to Ad5E1 tumor Ags, suggesting that CD8+ T cells do not use IFN-{gamma} to reject intraocular Ad5E1 tumors.


Figure 3
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  		FIGURE 3. CD8+ T cells do not use IFN-{gamma} or FasL to mediate Ad5E1 tumor rejection. A, CD8+ T cells do not produce IFN-{gamma} in response to Ad5E1 tumor Ags. CD4+ and CD8+ T cells from tumor-bearing (day 25–30 after tumor inoculation) C57BL/6 mice were assessed for their ability to secrete IFN-{gamma}. Cells were incubated for 72 h with the conditions indicated, after which cell supernatants were harvested and IFN-{gamma} secretion was determined by ELISA. CD4+ T cells produced significant (p < 0.001) levels of IFN-{gamma} in response to Ad5E1 tumor Ags, whereas CD8+ T cells did not. This experiment was repeated an additional time with similar results. B, Ad5E1 tumor cells do not express Fas. Ad5E1 tumor cells were incubated either with medium alone or medium containing 20 U/ml recombinant murine IFN-{gamma} for 72 h. Cells were stained with either anti-Fas Ab or an isotype control Ab and evaluated by flow cytometry. Freshly isolated splenocytes were used as a positive control. Ad5E1 tumor cells were negative for Fas expression even after stimulation with IFN-{gamma}.

 
Next, we determined whether CD8+ T cells use FasL to mediate Ad5E1 tumor rejection. Because FasL is abundantly expressed on activated CD8+ T cells (36), we examined the expression of a Fas receptor on Ad5E1 tumor cells by flow cytometry. We found that Ad5E1 tumor cells did not express Fas (Fig. 3B). Also, the stimulation of Ad5E1 tumor cells with IFN-{gamma}, which is known to up-regulate Fas expression on cells (37), did not induce Fas expression. Thus, Ad5E1 tumor cells do not express a Fas receptor and CD8+ T cells, therefore, cannot use FasL to mediate Ad5E1 tumor rejection.

We next examined CD8+ T cells for their capacity to use perforin or TRAIL to mediate Ad5E1 tumor rejection. Perforin is a well-defined molecule used by CD8+ T cells to lyse tumor cells, and TRAIL induces the apoptosis of Ad5E1 tumor cells (10). To explore the roles of perforin and TRAIL, perforin KO and TRAIL KO mice were challenged with an intraocular injection of Ad5E1 tumor cells. As previously reported, these mice rejected an intraocular challenge of Ad5E1 (9, 18). CD8+ T cells were isolated from tumor-rejector mice by magnetic bead sorting and were treated with an anti-CD4 Ab and complement as described earlier. The purified CD8+ T cells were adoptively transferred to SCID mice before challenging them with AC injections of Ad5E1 tumor cells. The results showed that SCID mice were able to reject Ad5E1 tumors following the adoptive transfer of perforin KO CD8+ T cells (Fig. 4A) or TRAIL KO CD8+ T cells (Fig. 4B). By contrast, tumors grew progressively in naive SCID mice. Thus, CD8+ T cells do not use either perforin or TRAIL to reject intraocular Ad5E1 tumors.


Figure 4
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  		FIGURE 4. CD8+ T cells do not use perforin or TRAIL to reject intraocular Ad5E1 tumors. Following the rejection of an AC challenge with Ad5E1 tumor cells, perforin KO and TRAIL KO mice were sacrificed and their splenocytes harvested. CD8+ T cells were isolated from splenocytes by magnetic bead sorting and further purified by treatment with anti-CD4 Ab plus complement. CD8+ T cells (~1 x 107 cells/mouse) were injected i.v. into SCID recipients. SCID mice were then challenged in the AC with Ad5E1 tumor cells on the same day as the adoptive transfer and tumor growth was monitored 2–3 times per week. Tumor growth was scored as the percentage of an AC occupied by a tumor. Each data point represents the average percentage of ACs occupied with tumors in five mice from each treatment group. A, Tumors grew progressively in naive SCID mice (n = 5) but were rejected in SCID mice that received perforin KO CD8+ T cells (n = 5). Significant differences (p = 0.04) were observed between groups on day 10 after tumor injection and at all time points thereafter. B, Tumors grew progressively in naive SCID mice (n = 5) but were rejected in SCID mice that received TRAIL KO CD8+ T cells (n = 5). Significant differences (p = 0.03) were observed between groups on day 12 after tumor injection and at all time points thereafter.

 
CD8+ T cells use TNF-{alpha} to mediate Ad5E1 tumor rejection

We next examined whether CD8+ T cells use TNF-{alpha} to mediate Ad5E1 tumor rejection. Ad5E1 tumor cells cultured with various doses of TNF-{alpha} were highly sensitive to TNF-{alpha}-mediated apoptosis (Fig. 5A). TNF-{alpha} KO mice rejected an intraocular challenge of Ad5E1 tumors (9), but when CD8+ T cells from tumor-rejector TNF-{alpha} KO mice were adoptively transferred to SCID mice, TNF-{alpha} KO CD8+ T cells did not protect the mice against tumor challenge and Ad5E1 tumor cells grew progressively (Fig. 5B). Ad5E1 tumors grew at roughly the same tempo as Ad5E1 tumors grew in naive SCID mice. Thus, CD8+ T cells require TNF-{alpha} to mediate intraocular Ad5E1 tumor rejection. Also, cell membrane expression of TNF-{alpha} on CD8+ T cells was up-regulated in response to Ad5E1 tumor cell Ag stimulation (Fig. 5C), further suggesting a role for TNF-{alpha} in CD8+ T cell rejection of intraocular Ad5E1 tumors. Soluble TNF-{alpha} was not released from CD8+ T cells in response to Ad5E1 tumor Ags as detected by ELISA (data not shown), demonstrating that membrane TNF-{alpha} is responsible for Ad5E1 tumor rejection.


Figure 5
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  		FIGURE 5. CD8+ T cells use TNF-{alpha} to mediate Ad5E1 tumor rejection. A, Ad5E1 tumor cells are highly sensitive to TNF-{alpha}-mediated apoptosis. Ad5E1 tumor cells were incubated with medium alone (negative control), staurosporine (positive control), or medium containing various doses of TNF-{alpha} for 24, 48, and 72 h. Apoptosis was determined by flow cytometry using the TACS annexin V FITC kit. TNF-{alpha} induced significant (p < 0.001) apoptosis compared with medium alone at all doses and time points examined. B, CD8+ T cells require TNF-{alpha} to mediate Ad5E1 tumor rejection. Following the rejection of an AC challenge of Ad5E1, TRAIL KO and TNF KO mice were sacrificed and their splenocytes harvested. CD8+ T cells were isolated from splenocytes by magnetic bead sorting and further purified by treatment with an anti-CD4 Ab plus complement. CD8+ T cells (~1 x 107 cells/mouse) were injected i.v. into SCID recipients. SCID mice were then challenged AC with Ad5E1 tumors on the same day as the adoptive transfer. Tumor growth was monitored 2–3 times per week and scored as the percentage of the AC occupied by a tumor. Each data point represents the average percentage of ACs occupied with tumors in five mice from each treatment group. Tumors grew progressively in naive SCID mice (n = 5) and in SCID mice that received TNF-{alpha} KO CD8+ T cells (n = 5). No significant difference was observed between groups throughout the duration of the experiment (p > 0.05 at all time points). C, CD8+ T cells up-regulate membrane TNF-{alpha} in response to Ad5E1 tumor Ags. Following the rejection of an AC challenge of Ad5E1, C57BL/6 mice were sacrificed and their splenocytes harvested. CD8+ T cells were isolated from splenocytes by magnetic bead sorting. CD8+ T cells were treated with either medium alone, PMA plus ionomycin, or mitomycin C-treated Ad5E1 tumor cells for 24 h. Cells were then stained for the presence of membrane-bound TNF-{alpha}. Histogram analysis shows membrane TNF-{alpha} expression of CD8+ T cells following exposure to medium only (shaded histogram), PMA plus ionomycin (broken histogram), and Ad5E1 tumor Ags (bold histogram).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Unlike most tumors and tissue allografts, Ad5E1 tumors undergo immune rejection following transplantation into the AC of the eye in wild-type syngeneic mice. Interestingly, the rejection of Ad5E1 tumors in the AC is IFN-{gamma}-dependent, as the tumor grows progressively in the eyes of IFN-{gamma} KO mice (10). By contrast, Ad5E1 tumor cells transplanted to nonimmune-privileged body sites (e.g., subcutaneously) are rejected, both in wild-type and IFN-{gamma} KO mice (18). Thus, an IFN-{gamma}-independent tumor rejection process functions at extraocular locations but is excluded from the AC of the eye. However, intraocular Ad5E1 tumors succumb to an IFN-{gamma}-dependent immune effector mechanism that is functional in the AC and produces tumor rejection that not only rids the eye of a potentially life-threatening neoplasm but also preserves the anatomical integrity of the eye.

The rejection of intraocular Ad5E1 tumors does not require CD8+ T cells, as these tumors undergo rejection in CD8+ T cell-depleted and CD8 KO mice (9, 18). However, the presence of CD8+ T cells in intraocular Ad5E1 tumors led us to investigate the role of CD8+ T cells in tumor rejection. Interestingly, Ad5E1 tumor cells do not undergo tumor-specific CTL-mediated lysis as determined by a 4-h 51Cr release assay, and we were consistently unable to detect CTL activity against syngeneic Ad5E1 tumor cells in C57BL/6 mice. There is evidence that ocular immune privilege involves the functional silencing of CD8+ CTLs. For example, the injection of a soluble Ag into the AC elicits the expansion of Ag-specific CD8+ T cells in regional lymph nodes; however, the cytolytic activity of the CD8+ T cells is severely reduced (38). Other studies showed that even though CD8+ T cells infiltrate allogeneic intraocular tumors, they fail to differentiate into fully functional cytotoxic effector cells (39). However, termination of ocular immune privilege results in the acquisition of terminally differentiated, tumor-specific CD8+ CTLs (40). Thus, there is evidence that immune privilege in the eye includes the silencing of CD8+ CTLs. We demonstrated that Ad5E1 tumor cells were susceptible to Ag-specific allogeneic CTLs, because CTLs from Ad5E1-immunized BABL/c mice readily lysed Ad5E1 tumor cells. Although Ad5E1 tumor cells were susceptible to allospecific CTL-mediated killing, they were poor immunogens for eliciting tumor-specific CTL responses in the syngeneic host. The lack of CTL killing in C57BL/6 mice is intriguing because CD8+ T cells can mediate tumor rejection as shown by the adoptive transfer of rejector CD8+ T cells to susceptible SCID mice, yet the same CD8+ T cells do not exhibit CTL activity. This underscores the functional plasticity of CD8+ effector cells and demonstrates that CD8+ T cells are not limited to perforin-mediated processes to rid the host of a tumor. A previous study has shown that tumor rejection can occur in wild-type mice without the presence of any detectable CTL activity (41). Yamaguchi et al. showed that Meth A cells, exhibiting viral Ags, were resistant to CTL mediated killing yet underwent rejection in wild-type mice (41). When compared with another tumor cell line that induced significant CTL activity, CTL resistant Meth A cells were rejected in wild-type mice at a similar time course (41). This indicates that in vitro CTL activity is not necessarily a true measure of CD8+ T cell-mediated rejection in vivo.

We have been unable to detect specific CTL activity against Ad5E1 tumor cell targets. By contrast, Schurmans et al. demonstrated the lysis of Ad5E1 tumor cells by splenic effector cells from C57BL/6 mice isolated 12 days after s.c. Ad5E1 tumor injection and 6 wk after AC Ad5E1 tumor injection (8). There are several possible explanations for this discrepancy. First, the difference observed between their s.c. injected mice and ours may be due simply to tumor burden. Schurmans et al. injected 1 x 107 Ad5E1 tumor cells s.c. (8), whereas we injected 3 x 105 Ad5E1 tumor cells s.c. In support of this hypothesis, McKenna and Kapp have noted that tumor burden affects CTL generation, as CTL activity was detected in the spleen when 1 x 106 but not 1 x 104 E.G7-OVA tumors were injected s.c. (42). Therefore, we may observe an Ad5E1-specific CTL response if we increased our s.c. tumor burden. Second, the time at which the splenic effector cell isolation takes place may be crucial for the detection of CTL activity. Schurmans et al. collected splenic effector cells 6 wk after tumor injection (8), whereas we collected splenic effector cells 3 wk after tumor injection. More importantly, Schurmans et al. noted no CTL activity in splenic effector cells isolated from mice 12 days after AC Ad5E1 tumor injection (8), which is consistent with our observations. Therefore, the absence of splenic effector cell lytic activity at the 3-wk time point does not rule out that CD8+ CTL activity may be occurring in the eye at these time points of intraocular tumor rejection. We hypothesize that splenic CTL activity may increase as splenic effector cells are isolated between 3 and 6 wk after tumor injection.

CD8+ T cells mediate tumor and allograft rejection by several mechanisms. These include the expression of IFN-{gamma}, FasL, perforin, TRAIL, and TNF-{alpha} (32, 33, 34, 35). To determine which one of these was used to mediate Ad5E1 tumor rejection, we systematically tested each of these components. IFN-{gamma} was the first candidate, because previous work has shown the importance of IFN-{gamma} in Ad5E1 tumor rejection (10, 18). However, unlike CD4+ T cells, CD8+ T cells did not secrete IFN-{gamma} in response to Ad5E1 tumor Ags. Thus, it is unlikely that CD8+ T cells use IFN-{gamma} to mediate Ad5E1 tumor rejection in the eye. We also systematically ruled out CD8+ T cell expression of FasL, perforin, and TRAIL as mechanisms of tumor rejection. Ad5E1 tumor cells are Fas-negative, thus ruling out Fas-induced apoptosis as a mechanism used by CD8+ T cells for the rejection of intraocular Ad5E1 tumors. Perforin and TRAIL-induced apoptosis are unlikely candidates, because effector mechanisms such as CD8+ T cells from perforin KO and TRAIL KO mice produced Ad5E1 tumor rejection when adoptively transferred to SCID mice. Surprisingly, Ad5E1 tumor cells were highly sensitive to TNF-{alpha}. Even 1 ng/ml TNF-{alpha} mediated 70% apoptosis in just 24 h after being mixed with Ad5E1 tumor cells. SCID mice adoptively transferred with CD8+ T cells from tumor-rejector TNF-{alpha} KO mice were no longer protected from Ad5E1 tumor outgrowth. The most likely explanation for this finding is that CD8+ T cells require TNF-{alpha} for the rejection of intraocular Ad5E1 tumors. However, it is possible that T cells from TNF-{alpha} KO mice have a defect in their capacity to migrate to and enter the Ad5E1 tumor-containing eye. This explanation is unlikely because Ad5E1 tumors undergo rejection in the eyes of TNF-{alpha} KO mice, indicating that lymphocytes from TNF-{alpha} KO mice are capable of migrating to and entering Ad5E1 tumor-containing eyes (9). Also, we show that CD8+ T cells from tumor-rejector mice up-regulate membrane-bound TNF-{alpha} in response to Ad5E1 tumor Ags, but not the soluble form of TNF-{alpha}. Membrane-bound TNF-{alpha} has been implicated as a mediator of CD8+ T cell-mediated tumor lysis (34). Ratner and Clark demonstrate that cloned CTLs expressed a membrane form of TNF-{alpha} that killed TNF-{alpha}-sensitive target cells (34). This killing was a "slow" (18 h) lytic reaction using this surface-associated TNF-{alpha}. This observation may demonstrate why no killing was observed in our study, as no Ad5E1 tumor lysis was detected after 4 h of effector and target interaction. The importance of CD8+ T cell-derived TNF-{alpha} has been demonstrated in mediating tumor rejection at nonimmune-privileged sites (34, 43, 44). Moreover, our study demonstrates that CD8+ T cells use TNF-{alpha} to mediate tumor rejection in an immune-privileged environment without inflicting significant damage to bystander ocular tissue.

In our adoptive transfer of primed CD8+ T cells to SCID recipient mice, Ad5E1 tumor rejection occurred independently of CD4+ T cell help. Although it is possible that a small number of CD4+ T cells were present in the adoptively transferred cells, this seems unlikely because the CD8+ T cell suspensions were positively selected using immunomagnetic beads that are highly specific for CD8+ cells, and the CD8+ T cell-enriched cell suspensions were purged of any residual CD4+ T cells by in vitro treatment with an anti-CD4 Ab and complement. On the surface, our observations are similar to the findings of Valujskikh et al., who showed that primed CD8+ T cells from female donors rejected male skin grafts independently of CD4+ T cells in a manner that did not inflict damage to the host’s innocent bystander cells (45). However, in the study by Valujskikh et al. the effector function of the CD8+ T cells was IFN-{gamma} dependent, which is in sharp contrast to the findings in our study where the CD8+ T cells did not produce IFN-{gamma}, suggesting that IFN-{gamma} is not required for the rejection of the syngeneic tumor (45). CTL activity in both studies was not detected toward the respective target cells even though a mononuclear cell infiltrate was observed in rejecting tissue, and the processing and presentation of relevant peptides by host APCs were necessary to generate CD8+ T effector cells (45). It bears noting that F4/80+ macrophages are involved in rejecting Ad5E1 intraocular tumors (data not shown) and that the depletion of ocular macrophages with clodronate liposome injection results in progressive Ad5E1 tumor growth (11). This suggests that ocular APC function may be necessary for tumor Ag presentation to CD8+ T cells and eventual tumor rejection in the eye.

We propose that, following intraocular injection of Ad5E1 tumor cells, T cells circumvent immune privilege, infiltrate intraocular Ad5E1 tumors, and mediate tumor rejection. This T cell-mediated rejection involves two separate pathways: 1) a primary pathway involving CD4+ T cells expressing IFN-{gamma} that binds directly to Ad5E1 tumors resulting in decreased proliferation, increased apoptosis, and the inhibition of tumor cell angiogenesis; and 2) an ancillary pathway involving TNF-{alpha}-expressing CD8+ T cells that mediates tumor cell apoptosis without inflicting injury to normal ocular tissues. Also, the CD8+ effector T cells are not conventional "CTLs" but mediate tumor rejection via TNF-{alpha}-induced apoptosis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

2 Address correspondence and reprint requests to Dr. Jerry Y. Niederkorn, Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390. E-mail address: jerry.niederkorn{at}utsouthwestern.edu Back

3 Abbreviations used in this paper: AC, anterior chamber; Ad5E1, adenovirus type 5 early region 1; KO, knockout. Back

Received for publication December 8, 2006. Accepted for publication March 7, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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