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The Journal of Immunology, 2005, 175: 4247-4254.
Copyright © 2005 by The American Association of Immunologists

Maintenance of Immune Tolerance Depends on Normal Tissue Homeostasis

Zita F. H. M. Boonman*, Geertje J. D. van Mierlo{dagger}, Marieke F. Fransen{dagger}, Rob J. W. de Keizer*, Martine J. Jager*, Cornelis J. M. Melief{dagger} and René E. M. Toes1,{dagger},{ddagger}

* Department of Ophthalmology, {dagger} Department of Immunohematology and Blood Transfusion, and {ddagger} Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ags expressed at immune privileged sites and other peripheral tissues are able to induce T cell tolerance. In this study, we analyzed whether tolerance toward an intraocular tumor expressing a highly immunogenic CTL epitope is maintained, broken, or reverted into immunity in the event the anatomical integrity of the eye is lost. Inoculation of tumor cells into the anterior chamber of the eye of naive B6 mice leads to progressive intraocular tumor growth, an abortive form of CTL activation in the tumor-draining submandibular lymph node, and systemic tolerance as evidenced by the inability of these mice to reject an otherwise benign tumor cell inoculum. Loss of anatomical integrity of the eye as a consequence of phthisis resulted in loss of systemic tolerance and the emergence of effective antitumor immunity against an otherwise lethal tumor challenge. Phthisis was accompanied by dendritic cell maturation and preceded the induction of systemic tumor-specific CTL immunity. Our data show that normal tissue homeostasis and anatomical integrity is required for the maintenance of ocular tolerance and prevention of CTL-mediated immunity. These data also indicate that tissue injury in the absence of viral or microbial infection can act as a switch for the induction of CTL immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Central tolerance, induced in the thymus, plays a pivotal role in the prevention of undesired immune attack against the own tissues of the body. However, next to central tolerance, tolerance induced in the periphery is also important in maintaining the homeostasis and integrity of the body (1). Recognition of Ags in a noninflammatory environment has been postulated to induce tolerance, whereas Ag recognition in a proinflammatory context will lead to immunity. For example, activation of Ag-presenting dendritic cells (DC),2 either through inflammatory signals delivered via the innate immune system, as occurs during microbial or viral infection, or through interaction with CD4+ T cells or anti-CD40 Abs results in the development of strong CD8+ CTL responses (2, 3, 4, 5). In contrast, little information is available on the magnitude of the immune response against self-Ags or tumor Ags (6), particularly if alarm signals are derived from injured cells that have not been exposed to pathogens or toxins but instead are released during "spontaneous" tissue damage. For instance, it is not known whether tolerance against a defined Ag could be broken once the tissue (bearing this Ag) is damaged.

A classical example of an organ involved in preventing and dampening undesired immune attack is the eye. The eye being an "immune privileged site" is very efficient at preventing immune-mediated elimination of cells present in the eye that express foreign Ag (7). Originally, an immune privileged site was an anatomical location where transplanted foreign tissue survives for an extended period of time in an immunocompetent recipient. Evolutionary, immune privilege of the eye is important because the few irregularities in the delicate anatomy of the eye can already severely affect vision and thus survival. Therefore, ocular immune privilege is likely to play an important role in the protection of the eye against hazardous immune responses.

Originally it was believed that immune privilege in the eye was due to lack of lymphatic drainage and the presence of blood-ocular barriers (8). Consequently, ocular Ag would be hidden from the immune system while immune cells could not enter the eye, thereby sparing the organ from the destructive effects of inflammatory cells (9). Currently, it is acknowledged that ocular immune privilege is much more multifaceted. It is an active process using numerous mechanisms to maintain organ function. These mechanisms include, among others, the production and activity of immunosuppressive cytokines and the expression of Fas ligand and TNF-related apoptosis-inducing ligand (10, 11, 12). Another aspect of ocular immune privilege is an experimental phenomenon called anterior chamber acquired immune deviation (ACAID). This feature describes how the inoculation of foreign Ag into the anterior chamber (AC) of the eye can generate systemic tolerance resulting in an inhibition of the delayed-type hypersensitivity (DTH) against the inoculated foreign Ag (13).

Although ocular immune privilege and ACAID apply to both MHC class II- and class I-restricted T cell responses, most studies on ocular immune privilege and ACAID have focused on MHC class II-restricted CD4+ T cell-mediated immunity (7, 14). These studies showed an important role for eye-derived APCs that activate NKT cells in the spleen that are required for the generation of CD8+ T regulatory cells capable of suppressing MHC class II-restricted DTH-responses (15). However, MHC class I-restricted CD8+ T cell responses are also affected by the presence of Ag in the eye. For example, CTL immunity toward OVA is inhibited when OVA is injected into the AC (16). The inhibition of CTL responses was the result of functional unresponsiveness rather than clonal deletion of Ag-specific CD8+ T cells. This result was determined by analyzing the response of Ag-specific T cells from TCR-transgenic mice. Likewise, it has been postulated that the emergence of T regulatory cells inhibits CTL immunity. ACAID can be induced in both naive mice and presensitized animals (17), and once induced, it is very long lasting and highly resistant to termination (7).

The disadvantage of ocular immune privilege is that intraocular tumors, although rare, can grow unhampered leading to severe mortality and morbidity. Typically, an intraocular tumor, such as a melanoma, grows quietly and insidiously without the patient noticing anything until vision becomes obscured by the enlarging tumor. At such time, distant metastases may have already occurred from which the patient will eventually die. In some cases, progressive intraocular tumors present as phthisis (i.e., disintegrate) in which the tumor-containing eye has shrunken (18, 19) because of extensive infiltration of tumor or inflammatory cells into the ciliary body (CB). The normal functioning CB produces aqueous humor to maintain the regular intraocular pressure of the eye. Once 90% or more of the CB is infiltrated or damaged by tumor or inflammatory cells, it cannot produce aqueous humor any longer resulting in hypotonia of the eye followed by phthisis, or literally shrinkage of the eye bulb.

Previously, we have shown that, although intraocular tumor Ags are constitutively presented to CD8+ T cells in the local draining lymph nodes (DLN), it did not lead to tumor eradication (20). Moreover, Ag presentation did not lead to proper systemic CTL immunity, as tumor-specific CTLs were not found outside the tumor DLN. We studied the consequences for ocular tolerance and immunity in case "spontaneous" phthisis occurs resulting from progressive tumor growth. Our data show that intraocular tumor growth leads to the inability of the AC tumor-bearing mice to control an otherwise unharmful tumor cell inoculum placed at a distant site in the body. Tissue damage in the absence of microbial or viral infection can break this established tolerance against tumor Ags, which suggests that normal tissue homeostasis is required for the maintenance of ocular tolerance.


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

C57BL/6 mice were purchased from Charles River Breeding Laboratories. TAP–/– mice and CD40–/– mice (both on C57BL/6 background) were purchased from The Jackson Laboratory. Strain 42 mice, bred at the Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek-Preventie en Gezondheid (TNO-PG) are TCR transgenic mice expressing the TCR {alpha} and {beta} chains derived from the H-2b-restricted early region 1A of human adenovirus type 5 (Ad5E1A)234–243-specific CTL clone 5 (21). Mice were kept at the Leiden University Medical Center Animal Facility (Leiden, the Netherlands) and used at 5–13 wk of age in accordance with national legislation and approval of the Animal Experimental Committee of the Leiden University.

Tumor cells

Mouse embryo cells transformed by the Ad5E1A plus EJ-ras were cultured in IMDM (Invitrogen Life Technologies) supplemented with 8% (v/v) FCS, 50 µM 2-ME, glutamine, and penicillin, as described (21, 22). Different kinds of Ad5E1A plus ras-transformed tumor cell lines were used. Some lines were progressively growing s.c. and therefore lethal in naive mice and others were nonlethal because they are rejected after a short period of growth following s.c. injection into naive mice (21, 23).

Tumor experiments

Intracameral inoculations. A previously described technique for deposition of a definite number of tumor cells into the AC of the mouse has been used (24). Mice were anesthetized with a mixture (ratio 1:1) of xylozine (2% Rompun; Bayer) and ketamine hydrochloride (Aescoket; Aesculaap) 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 (80 µm in diameter) was fitted into a sterile infant feeding tube, which was mounted onto a sterile 0.1-ml Hamilton syringe. The pipette, loaded with Ad5E1A-transformed tumor cells (0.75 x 105 cells/4 µl) was introduced through the puncture site of the cornea, and 4 µl of the tumor cell suspension was delivered into the AC. The eyes were examined three times a week with a dissecting microscope to observe and document tumor growth and the anatomical integrity of the eye.

Subcutaneous inoculations. E1A-expressing tumor cells (1 x 107) were injected s.c. into 7- to 10-wk-old male mice in 200 µl of PBS. Tumor size was measured twice weekly with calipers in three dimensions. Mice were sacrificed when tumor size exceeded 1 cm3 to avoid unnecessary suffering.

Histology

Serial paraffin-embedded 4-µm sections of a murine tumor-bearing eye were stained with H&E stain.

CFSE labeling and adoptive transfer

Single cell suspensions were generated from spleen and peripheral lymph nodes of strain 42 mice. Erythrocytes were depleted by ammonium chloride treatment (2 min on ice). Cells were washed once in cold medium and once in cold PBS, after which they were resuspended in PBS at 1 x 107 cells/ml and incubated with 0.5 µM CFSE (Molecular Probes) for 30 min at 37°C. FCS was added to a concentration of 5% FCS, and the cells were washed in PBS. TCR-transgenic CD8+ T cells (3 x 106) were injected into the tail vein of intraocular tumor-bearing mice in 200 µl of PBS.

Flow cytometry

Single cell suspensions of spleens, lymph nodes, and tumor-containing eyes were prepared by mechanical disruption. Blood samples and cell suspensions of spleens were depleted of erythrocytes by ammonium chloride treatment for 5 min at room temperature. Cells were stained with directly allophycocyanin-conjugated mAb against CD8 (clone 53-6.7; BD Pharmingen) combined with PE-conjugated E1A234–243-loaded H-2Db tetramers (E1A-TM) or PE-conjugated HPV16-loaded H-2Db tetramers as a control. After CD11c enrichment, cells were stained with allophycocyanin-conjugated mAb against CD11c (clone HL3; BD Pharmingen) combined with stainings for CD80 (clone 16-10A1; BD Pharmingen), CD86 (clone GL1, BD Pharmingen), CD40 (clone 3/23; BD Pharmingen), I-A/I-E (clone M5/114.15.2; BD Pharmingen), or H-2Kb (clone AF6-88.5; BD Pharmingen). Data acquisition and analysis were done on a BD Biosciences FACScan with CellQuest software.

Separation of CD11c+ and CD11c– populations

Peripheral lymph nodes of tumor-bearing mice were treated with collagenase (250 U/ml; Sigma-Aldrich) and DNase (50 µg/ml; Sigma-Aldrich) for 30 min at 37°C. CD11c+ cells were positively selected using magnetized Ab for CD11c (N418; Miltenyi Biotec). A purity of ~90% of the CD11c+ cell population was obtained as determined by FACS analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Collapse of the anatomical integrity of a tumor-containing eye leads to systemic distribution of endogenous tumor-specific CTL

Recently, we described that intraocular tumor Ags are presented to tumor-specific CTL in tumor DLN (20). The Ag is likely to be derived from the ocular growing tumor, as previous control experiments have shown that leakage of tumor cell suspension into the conjunctival sac during the inoculation into the AC does not lead to T cell priming in the DLN (20). However, no effective antitumor immune response could be found in the tumor-bearing animals because the endogenous tumor-specific CD8+ T cells do not distribute systemically. For these studies we used a tumor model of tumor cells transformed by Ad5E1A that do not metastasize to other parts of the body. These tumor cells harbor a highly immunogenic CTL epitope that is recognized by E1A-specific CTL. As no effective tumor-specific CTL responses are generated, E1A-transformed tumors are not cleared and grow progressively in the AC of the eye of syngeneic C57BL/6 mice. However, upon further experimentation it was found that in some cases, several weeks after tumor inoculation, systemic E1A-specific CTL responses were present. The presence or absence of E1A-specific CTLs in the periphery appeared to be correlated with two distinct patterns of progressive intraocular tumor growth. The first pattern was characterized by a developing intraocular tumor that grows on the iris into the AC and progressively expands into the posterior segment of the eye. Eventually the whole eye is filled with tumor cells without damaging the barriers of ocular tissue or orbit, leaving the intraocular structures and surrounding eye bulb intact (Fig. 1B). However, in another 60% of mice, the eye bulb shrinks (phthisis). In these eyes, extensive tumor infiltration of the CB occurred. This is likely to disrupt the aqueous humor production of the CB resulting in hypotonia of the tumor-bearing eye leading to collapse of the anatomical intraocular integrity and shrinkage of the eye bulb (Fig. 1C, phthisis). To examine in depth whether these two patterns of intraocular tumor growth are associated with systemic antitumor responses, we investigated DLN, nondraining lymph nodes (NDLN), and spleen for the presence of endogenous E1A-specific CTLs using E1A/Db tetramers. As shown in Fig. 2A, tumor-specific CD8+ T cells were observed in the tumor DLN, NDLN, and spleen of mice with phthisis intraocular tumor growth in contrast to the nonphthisis eye tumor group in which tumor-specific CTLs were only found in the DLN and not in NDLN and spleen. In addition, tumor-specific CTLs were found in the intraocular phthisis tumors but not in the tumor-bearing eyes of the nonphthisis group. These findings indicate that in mice with phthisis intraocular tumor growth, endogenously triggered tumor-specific CTLs can leave their primary priming site, the tumor DLN, and are able to spread systemically eventually invading the tumor-containing phthisis eye.



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  		FIGURE 1. Two types of progressive intraocular Ad5E1A-expressing tumor growth in C57BL/6 mice. Sagittal view of a naive (tumor-free) eye (A) and two types of progressive intraocular tumor growth: nonphthisis (B) vs phthisis (C). Both types show anterior and posterior tumor expansion. There is no tumor growth outside the eye bulb. Note the decrease in size of both intraocular tumor and eye in the phthisis intraocular tumor (C). Box (inset) in A–C are enlarged in D and E, which show the CB in detail of the naive eye (D), the nonphthisis (E), and phthisis intraocular tumor in which the CB has been completely destructed. C, cornea; L, lens; VB, vitreous body.

 


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  		FIGURE 2. Collapse of the anatomical integrity of a tumor-containing eye results in systemic distribution of endogenous tumor-specific CTLs. Endogenous E1A-specific CTLs are only observed in the tumor DLN of mice with nonphthisis intraocular tumor growth and have distributed systemically in mice with phthisis intraocular tumor growth. Mice bearing an Ad5E1A-expressing tumor were sacrificed 63 days after tumor inoculation. FACS analysis was performed in DLN, NDLN, spleen, and intraocular tumors using Db/E1A tetramers. One representative experiment of 10 is shown.

 
Phthisis precedes the emergence of tumor-specific CTL

To determine which of the two features, intraocular CTLs or phthisis occurred first, we set out to analyze the presence of endogenous E1A-specific CTLs in different stages of tumor growth: nonphthisis, prephthisis, and phthisis intraocular tumors (Fig. 3). These three different phases of intraocular tumor growth were defined macroscopically and confirmed microscopically. The clinical prephthisis signs are commencing microphthalmos (i.e., small eye) and cornea-cloudiness (Fig. 3, B and C). When these prephthisis signs are present, phthisis will occur 2–3 wk from this time point. However, when these clinical signs of prephthisis were not observed until 6 wk after intracameral tumor injection, they would not occur later during progressive intraocular tumor growth (nonphthisis) (Fig. 3A). Clinically phthisis looks somewhat similar to prephthisis but the clinical signs are more severe: the eye bulb has become at least half its original size, the AC is completely obliterated, and the cornea is opaque, edematous, and thickened. The tumor, although smaller in size, is still present in the phthisis eye (Fig. 3D).



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  		FIGURE 3. Phthisis precedes the emergence of tumor-specific CTLs. Endogenous E1A-specific CTLs are never observed in a nonphthisis intraocular tumor (A) or in the very early stages of preprephthisis when the tumor-bearing eye starts to show some signs of collapse (B). Tumor-specific CTLs are found in small numbers in prephthisis tumors (C) and are found in large amounts in phthisis intraocular tumors (D). In the prephthisis intraocular tumor (B and C), the eye has started to deteriorate, increasingly showing reduced eye bulb size, opacified and thick cornea, and increasing collapse of the AC and vitreous body (VB). Mice bearing an Ad5E1A-expressing tumor were sacrificed at several time points after tumor inoculation either for histology of the intraocular tumors or for FACS analysis performed in tumor-bearing eyes using Db/E1A tetramers. One representative experiment of three is shown.

 
As depicted in Fig. 2, the presence of E1A-specific CTL in DLN, NDLN, spleen, and tumor was determined by E1A-Db tetramer-staining. E1A-specific CTLs were readily detectable systemically in mice undergoing complete phthisis of the eye; however not in mice with nonphthisis. In the latter case, E1A-specific CTLs were only found in the tumor DLN. Although, a relatively small number of E1A-specific CTLs were detected in some mice with prephthisis (20%), probably as a consequence of the ongoing phthisis process, no E1A-specific CTLs were detected systemically in the majority of mice (80%) with prephthisis intraocular tumors. Together these findings indicate that the occurrence of phthisis precedes the emergence of systemically distributed tumor-specific CTL, suggesting that during phthisis signals are released that trigger proper CTL responses.

Intraocular tumor Ags are cross-presented by CD11c+ cells to naive CTL in local DLN

The results we describe illustrate systemic distribution of tumor-specific CD8+ T cells after collapse of the anatomical integrity of the tumor-containing eye (phthisis). As no such systemic distribution was observed in tumor-bearing mice with intact intraocular structures, these findings indicate that signals are released during phthisis development, which is beneficial to the induction of CTL response. These signals could relate to Ag presentation, as well as to the context in which Ag presentation occurs. Activation of CTL is generally accepted to result from cross-presentation of Ag by DCs that have acquired Ag from tumor cells. However in some instances, direct presentation of Ag by tumor cells have also been implicated as the predominant mechanism by which CTLs are activated. To study whether E1A-specific CTL are primed directly by tumor cells in tumor DLN or by host APC, we analyzed whether intraocular tumor cells can be found in the secondary lymphoid organs of tumor-bearing animals. Twenty-one days after intracameral injection of E1A-expressing tumor cells, secondary lymphoid organs were examined. Selective in vitro outgrowth of lymph node and spleen cultures followed by PCR showed the presence of tumor cells in tumor DLN, but not in NDLN or spleen (data not shown). Subsequently we investigated whether these tumor cells, which are able to reach the lymphoid organs, were capable of presenting tumor Ag in vivo. Therefore, we injected E1A-expressing tumor cells into wild-type and TAP–/– mice. TAP–/– mice cannot cross-present the E1A-Ag because presentation of this epitope is strictly TAP-dependent (25, 26). In case the E1A epitope is presented directly by tumor cells to E1A-specific CTL, it is expected that the E1A-epitope will also be presented in mice deficient from TAP. Nineteen days after tumor inoculation, at a time intraocular tumors had developed, CFSE-labeled E1A-specific CD8+ T cells derived from TCR-transgenic mice were injected. Three days later, division of tumor-specific T cells in different lymphatic organs was analyzed. As shown in Fig. 4, only division of CFSE-labeled cells was observed in tumor-bearing wild-type mice but not in TAP–/– mice, indicating that host-derived APC presented the ocular tumor Ag in a TAP-dependent fashion.



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  		FIGURE 4. Host-derived APCs are required for presentation of tumor Ags. CFSE-labeled E1A-specific CD8+ T cells were adoptively transferred into wild-type mice or TAP–/– mice bearing 19-day-old tumors in the AC of the eye. Three days posttransfer proliferation of adoptively transferred E1A-specific T cells in DLN, NDLN, and spleen was analyzed by FACS. One representative experiment of three is shown.

 
Because CD11c+ cells are most likely the host-derived APCs responsible for cross-presentation of tumor Ags to CTL (27), and because we have shown recently that the E1A-Ag is presented by CD11c+ cells (but not by CD11c– cells) from (s.c.) growing tumors, we subsequently studied whether phthisis would lead to maturation of these cells as APC activation is considered crucial for induction of CTL immunity (2, 28). For this purpose, CD11c+ cells in tumor DLN of mice with phthisis eye tumors and nonphthisis intraocular tumors were analyzed. In Fig. 5 it is shown that CD11c+ cells isolated out of the DLN from mice with phthisis intraocular tumor growth had up-regulated several surface markers such as CD80, CD86, CD40, and MHC class I and class II in comparison with the CD11c+ cells isolated from nonphthisis eye tumor-bearing mice. CD11c+ cells isolated out of the phthisis intraocular tumors also displayed an up-regulation of these activation markers contrary to the CD11c+ cells out of the nonphthisis eye tumors (data not shown). Together these findings indicate that collapse of the anatomical integrity of a tumor-containing eye led to an activated phenotype of the CD11c+ cells present in tumor DLN and the phthisis intraocular tumor itself. As presentation of the E1A epitope depends on host-derived APCs, our results indicate that the emergence of systemic CTL immunity results from the presentation of the E1A epitope by DCs that are endowed with the capacity to activate CTLs allowing their systemic distribution as a consequence of phthisis.



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  		FIGURE 5. Collapse of the anatomical integrity of the tumor-containing eye leads to activated CD11c+ cells. C57BL/6 mice were injected intracamerally with 0.75 x 105 E1A-expressing tumor cells. When phthisis (broken histogram) or nonphthisis (shaded histogram) intraocular tumor growth had developed, mice were sacrificed and CD11c+ cells were isolated out of the lymph nodes and intraocular tumors for analysis of CD80, CD86, CD40, MHC class I and class II expression. Naive tumor-free mice were included in the experiment (thick histogram). One representative experiment of three is shown.

 
Collapse or preservation of intraocular tumor-containing structures determines the outcome of sequential immune responses; immunity vs tolerance

As outlined, an intraocular tumor in a deteriorating eye leads to systemic spread of tumor-specific CTLs. To analyze whether these systemic CTLs are effective in vivo, mice with primary intraocular tumors with or without phthisis were challenged s.c. with Ad5E1A plus ras-transformed tumor cell lines. For these experiments, we took advantage of two tumor cell lines that have different s.c. growth patterns in naive mice. One tumor cell line grows progressively and is lethal in all naive C57BL/6 mice (22). The other tumor cell line is rapidly eradicated after s.c. injection in naive C57BL/6 mice (21). In a group of 30 mice with intraocular tumors, 17 mice developed phthisis 8 wk post tumor inoculation. The remaining 13 tumor-bearing mice maintained "normal" intraocular integrity. Injecting the lethal progressive E1A-expressing tumor s.c. in both groups resulted in a 100% tumor-take and death in mice without any phthisis signs in contrast to 0% tumor outgrowth in mice with phthisis (Fig. 6A). All naive mice developed lethal s.c. tumors (Fig. 6A). As rejection of these E1A-expressing tumors crucially depends on E1A-specific CD8+ CTL immunity (29), these results indicate that phthisis leads to the induction of strong tumor-specific CTL responses that protect against a secondary tumor challenge.



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  		FIGURE 6. Collapse or preservation of intraocular tumor-containing structures determine the outcome of sequential immune responses: immunity vs tolerance. Secondary "lethal" (A) or "nonlethal" (B) syngeneic s.c. tumors were inoculated (10 x 106 E1A-expressing tumor cells) in mice with progressive nonphthisis ({diamondsuit}) or phthisis ({blacktriangleup}) intraocular tumor growth and in naive mice (primary s.c. tumor ({blacksquare})). Secondary "lethal" tumors (A) develop s.c. in naive mice ({blacksquare}) and in mice with nonphthisis intraocular tumor growth ({diamondsuit}). Secondary s.c. tumor growth was inhibited in mice with phthisis intraocular tumor growth ({blacktriangleup}). Secondary "nonlethal" tumors (B) only develop in mice with nonphthisis intraocular tumor growth ({diamondsuit}) and was inhibited in mice with phthisis intraocular tumor growth ({blacktriangleup}) and in naive mice ({blacksquare}).

 
In another group of 30 mice with intraocular tumors, 20 mice developed phthisis compared with 10 mice with nonphthisis intraocular tumor growth. Inoculation (s.c.) of the regressor E1A-expressing tumor cell clone led, as expected, to complete tumor rejection in naive mice and in mice with phthisis intraocular tumors (Fig. 6B). However, secondary s.c. tumors developed in all mice with nonphthisis intraocular tumor growth (Fig. 6B). Thus, because the ability of the mouse to control a challenge of this otherwise nonlethal (spontaneous regressing) tumor is lost in the case of intraocular tumor growth, these results show that intraocular tumor growth results in systemic tolerance that allows the outgrowth of an otherwise nonlethal tumor. More importantly, they also indicate that this tolerance is broken if anatomical integrity of the eye is lost. Together, these findings indicate that collapse or preservation of intraocular structures in a tumor-containing eye determines the systemic immunological immune responsiveness, respectively resulting in immunity or tolerance against secondary tumor challenges.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our results show that ocular immune privilege is broken when the anatomical integrity of the eye is disrupted. Mice with established tumors growing in the AC are incapable of rejecting an otherwise nonlethal tumor inoculated s.c., but are able to resist an aggressive, otherwise lethal tumor, when phthisis occurs spontaneously. These findings are strongly correlated with the systemic emergence of tumor-specific CTLs in case of phthisis, explaining the rejection of a s.c. tumor, as rejection of this tumor crucially depends on CTL-mediated immunity (21). In case no phthisis occurred, although tumor-specific CTL are present in tumor DLN, the animal could not mount a tumor-protective CTL response any longer, as reflected by the inability to reject a challenge with benign tumor cells. Because such CTL responses are crucial for the eradication of these s.c. tumors (21, 23, 30), these results indicate that ocular tumor growth in intact eyes leads to an "abortive" or "poised" CTL reaction.

Apparently, the tumor growing in the AC recruits effective suppressor mechanisms that do not only exert local, but also systemic effects that allow the s.c. growth of an otherwise nontumorigenic E1A-transformed tumor cell clone. The induction of such systemic effects appears to be specific of AC tumor growth because we did not observe these effects when the same tumor was injected s.c. (data not shown). The observation that the tolerance and suppressive mechanisms installed by the AC growing tumor are broken and converted into efficient antitumor immunity is intriguing as it indicates that disruption of anatomical (ocular) integrity can act as a switch determining whether immunity or tolerance prevails. This finding is in line with the clinical observation that trauma to one eye may cause destruction of the fellow eye, called sympathetic ophthalmia. Although the mechanisms underlying this condition are not fully understood, it is thought that the release of intraocular protein-Ags in the traumatized eye evokes a T cell-mediated (auto) immune reaction. These effector T cells will subsequently recognize similar Ag in the other eye leading to devastation of the nontraumatized eye (31). We consider it likely that trauma to the eye not only leads to the release of protein Ag, but also to the activation of DC as we observed that phthisis was strictly correlated with activation of CD11c+ cells in DLN. As Ag is present in both nonphthisis and phthisis eyes, the activation of DC as a consequence of trauma is conceivably an important parameter during the induction of T cell responses, and as in the case of sympathetic ophthalmia, the underlying mechanism responsible for destruction of the fellow eye.

It could be speculated that clonal expansion and systemic spread of tumor-specific CTL is a result of the ability of the tumor to metastasize systemically when the integrity of the eye is lost, allowing systemic CTL-induction. We, however, consider this very unlikely as systemic spread of the tumor used in this model cannot be detected when transplanted s.c. or in the AC. Tumor cells can only be detected by PCR in the tumor DLN and not in the NDLN, lungs, or spleen. Likewise, we have never observed micrometastases either macroscopically or microscopically. Moreover, no metastases are observed in T cell-deficient nude mice or in mice with phthisis. In addition, and more importantly, mice deficient for TAP are not able to activate adoptively transferred tumor-specific CTL derived from TCR-transgenic mice (32) (Fig. 5), showing that TAP-expressing APC of host origin are required for tumor Ag presentation. Thus, although tumor cells can be present in tumor DLN, they are apparently not able to present the tumor Ag directly, possibly as a result of sequestration or lack of costimulatory molecule expression.

As DCs are the predominant, if not the only, cells capable of generating CTL immunity (27, 33, 34), it is likely as outlined that the outcome of immune reactivity is a consequence of DC activation.

The data presented indicate that CD11c+ cells display a relatively "resting" phenotype in mice with nonphthisis intraocular tumor growth, allowing Ag presentation in a nonimmunogenic or even tolerogenic fashion (34, 35). It is conceivable that CTLs recognizing Ag on such quiescent DCs go through an abortive/poised form of activation that ultimately leads to deletion as is also observed in other systems (32, 36, 37). This would explain our observation that we do not observe a systemic dispersal of tumor-specific CTL that have been activated in the local DLN. However, the inability of the animals to control the growth of otherwise nonlethal E1A-expressing tumors implanted s.c. could also result from other mechanisms, such as the emergence of tumor-directed regulatory T cell responses. Stimulation of the immune system under tolerogenic conditions as operative in the ocular environment might lead to the systemic emergence of such regulatory T cells that inhibit the induction of tumor-specific CD8+ T cells at other sides. Thus, two levels of ocular tolerance might occur, one is leading locally to an abortive form of T cell activation, whereas the other results systemically in active inhibition of tumor-specific immunity. Either way, our results indicate that phthisis can bypass ocular tolerance leading to the induction of proper systemic antitumor immunity, conceivably as a result of local APC activation (34). Although we cannot rule out the possibility that eyes undergoing phthisis are susceptible to some kind of low-grade infection that provides danger signals, the acquisition of the immunostimulatory DC phenotype is acknowledged. The theory is in line with the concept that stressed cells release signals such as uric acid that locally activate APC so that they present captured Ag along with proper costimulatory signals, allowing proper activation of passing T cells, even T cells with low avidity, in the DLN (38, 39, 40, 41).

Although the precise mechanism and pathways operative in ocular tolerance induction and maintenance are not completely elucidated, it is generally accepted that the eye contains various immune-modulating cytokines like vasoactive intestinal peptide, IL-10, and TGF-{beta}, which prevent and suppress the DTH response, possibly by modulating DC maturation (7). It is likely that upon loss of the anatomical integrity and destruction of the eye, fewer of these cytokines are produced resulting in a strong influence on the local immune response, especially when combined with the release of "danger" signals from the shrinking eye. This local environment could very effectively activate DCs that stimulate local immune responses that are inert to systemic tolerance mechanisms. In this way, the local mechanisms that normally drive, under steady-state conditions, an abortive or poised form of T cell activation are now converted into a machinery that results in the development of effective CD8+ CTL responses. These responses are not blunted by systemic regulatory circuits, which explains the capacity of mice with phthisis to persist against a challenge from a "malignant" Ad5E1A-transformed tumor cell line that normally kills an unimmunized mouse.

The incidence of natural occurring successful tumor immunity as a consequence of a secondary (autoimmune) neurological disease has been observed in humans (42, 43). Likewise, it has been described that melanoma patients can spontaneously develop a strong T cell response to melanocyte differentiation Ags. In some cases, these T cell responses can even cause vitiligo, an autoimmune disease characterized by depigmentation of the skin, indicating that potent antitumor immunity can result from cancer progression (44, 45, 46, 47). Thus also in humans, tumor growth can be associated with cessation of immune tolerance and induction of immunity (48). Although, the mechanisms underlying these paraneoplastic events are not known, it is tempting to speculate that they result from the generation of immunity following the release of danger or stress signals as a consequence of progressive tumor growth.

Altogether, our findings indicate that tolerance is rapidly installed once an Ag is presented in an immune privileged context. However, the tolerance is rapidly broken if the organ is damaged, indicating that the context of Ag presentation is not only the determining factor for initiation of immunity or tolerance, but also for the maintenance of tolerance.


   Disclosures
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 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 Address correspondence and reprint requests to Dr. René E. M. Toes, Department of Rheumatology C4-R, Leiden University Medical Center, Albinusdreef 2, 2300 RC, Leiden, The Netherlands. E-mail address: R.E.M.Toes{at}lumc.nl Back

2 Abbreviations used in this paper: DC, dendritic cell; AC, anterior chamber; ACAID, AC acquired immune deviation; Ad5E1A, adenovirus type 5 early region 1A; DLN, draining lymph node; NDLN, nondraining lymph node; CB, ciliary body; DTH, delayed-type hypersensitivity. Back

Received for publication February 22, 2005. Accepted for publication July 14, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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