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Maintenance of Immune Tolerance Depends on Normal Tissue Zita F. H. M. Boonman, Geertje J. D. van Mierlo, Marieke F. Fransen, Rob J. W. de Keizer, Martine J. Jager, Cornelis J. This information is current as M. Melief and René E. M. Toes of September 27, 2021. J Immunol 2005; 175:4247-4254; ; doi: 10.4049/jimmunol.175.7.4247 http://www.jimmunol.org/content/175/7/4247 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Maintenance of Immune Tolerance Depends on Normal Tissue Homeostasis

Zita F. H. M. Boonman,* Geertje J. D. van Mierlo,† Marieke F. Fransen,† Rob J. W. de Keizer,* Martine J. Jager,* Cornelis J. M. Melief,† and Rene´E. M. Toes1†‡

Ags expressed at immune privileged sites and other peripheral tissues are able to induce T tolerance. In this study, we analyzed whether tolerance toward an intraocular tumor expressing a highly immunogenic CTL is maintained, broken, or reverted into 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 node, and systemic tolerance as evidenced by the inability of these mice to reject an otherwise 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 Downloaded from cell maturation and preceded the induction of systemic tumor-specific CTL immunity. Our data show that normal tissue ho- meostasis 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 can act as a switch for the induction of CTL immunity. The Journal of Immunology, 2005, 175: 4247–4254.

entral tolerance, induced in the , plays a pivotal tomical location where transplanted foreign tissue survives for an http://www.jimmunol.org/ role in the prevention of undesired immune attack against extended period of time in an immunocompetent recipient. Evo- C the own tissues of the body. However, next to central lutionary, immune privilege of the eye is important because the tolerance, tolerance induced in the periphery is also important in few irregularities in the delicate of the eye can already maintaining the homeostasis and integrity of the body (1). Recog- severely affect vision and thus survival. Therefore, ocular immune nition of Ags in a noninflammatory environment has been postu- privilege is likely to play an important role in the protection of the lated to induce tolerance, whereas Ag recognition in a proinflam- eye against hazardous immune responses. matory context will lead to immunity. For example, activation of Originally it was believed that immune privilege in the eye was Ag-presenting dendritic cells (DC),2 either through inflammatory

due to lack of lymphatic drainage and the presence of -ocular by guest on September 27, 2021 signals delivered via the innate , as occurs during barriers (8). Consequently, ocular Ag would be hidden from the microbial or viral infection, or through interaction with CD4ϩ T immune system while immune cells could not enter the eye, cells or anti-CD40 Abs results in the development of strong CD8ϩ thereby sparing the from the destructive effects of inflam- CTL responses (2–5). In contrast, little information is available on matory cells (9). Currently, it is acknowledged that ocular immune the magnitude of the against self-Ags or tumor Ags (6), particularly if alarm signals are derived from injured cells privilege is much more multifaceted. It is an active process using that have not been exposed to pathogens or but instead are numerous mechanisms to maintain organ . These mecha- released during “spontaneous” tissue damage. For instance, it is nisms include, among others, the production and activity of im- not known whether tolerance against a defined Ag could be broken munosuppressive and the expression of and once the tissue (bearing this Ag) is damaged. TNF-related apoptosis-inducing ligand (10–12). Another aspect of A classical example of an organ involved in preventing and ocular immune privilege is an experimental phenomenon called dampening undesired immune attack is the eye. The eye being an anterior chamber acquired immune deviation (ACAID). This fea- “immune privileged site” is very efficient at preventing immune- ture describes how the inoculation of foreign Ag into the anterior mediated elimination of cells present in the eye that express for- chamber (AC) of the eye can generate systemic tolerance resulting eign Ag (7). Originally, an immune privileged site was an ana- in an inhibition of the delayed-type (DTH) against the inoculated foreign Ag (13). Although ocular immune privilege and ACAID apply to both *Department of Ophthalmology, †Department of Immunohematology and Blood MHC class II- and class I-restricted responses, most studies Transfusion, and ‡Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands on ocular immune privilege and ACAID have focused on MHC ϩ Received for publication February 22, 2005. Accepted for publication July 14, 2005. class II-restricted CD4 T cell-mediated immunity (7, 14). These The costs of publication of this article were defrayed in part by the payment of page studies showed an important role for eye-derived APCs that acti- charges. This article must therefore be hereby marked advertisement in accordance vate NKT cells in the that are required for the generation of with 18 U.S.C. Section 1734 solely to indicate this fact. CD8ϩ T regulatory cells capable of suppressing MHC class II- 1 Address correspondence and reprint requests to Dr. Rene´E. M. Toes, Department restricted DTH-responses (15). However, MHC class I-restricted of Rheumatology C4-R, Leiden University Medical Center, Albinusdreef 2, 2300 RC, ϩ Leiden, The Netherlands. E-mail address: [email protected] CD8 T cell responses are also affected by the presence of Ag in 2 Abbreviations used in this paper: DC, ; AC, anterior chamber; ACAID, the eye. For example, CTL immunity toward OVA is inhibited AC acquired immune deviation; Ad5E1A, adenovirus type 5 early region 1A; DLN, when OVA is injected into the AC (16). The inhibition of CTL draining ; NDLN, nondraining lymph node; CB, ciliary body; DTH, de- layed-type hypersensitivity. responses was the result of functional unresponsiveness rather than

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 4248 IMMUNE TOLERANCE IN NORMAL TISSUE HOMEOSTASIS

ϩ of Ag-specific CD8 T cells. This result was de- at the corneoscleral junction, parallel and anterior to the iris. A glass mi- termined by analyzing the response of Ag-specific T cells from cropipette (80 ␮m in diameter) was fitted into a sterile infant feeding tube, TCR-transgenic mice. Likewise, it has been postulated that the which was mounted onto a sterile 0.1-ml Hamilton syringe. The pipette, loaded with Ad5E1A-transformed tumor cells (0.75 ϫ 105 cells/4 ␮l) was emergence of T regulatory cells inhibits CTL immunity. ACAID introduced through the puncture site of the cornea, and 4 ␮l of the tumor can be induced in both naive mice and presensitized (17), cell suspension was delivered into the AC. The eyes were examined three and once induced, it is very long lasting and highly resistant to times a week with a dissecting to observe and document tumor termination (7). growth and the anatomical integrity of the eye. 7 The disadvantage of ocular immune privilege is that intraocular Subcutaneous inoculations. E1A-expressing tumor cells (1 ϫ 10 ) were injected s.c. into 7- to 10-wk-old male mice in 200 ␮l of PBS. Tumor size tumors, although rare, can grow unhampered leading to severe was measured twice weekly with calipers in three dimensions. Mice were mortality and morbidity. Typically, an intraocular tumor, such as a sacrificed when tumor size exceeded 1 cm3 to avoid unnecessary suffering. melanoma, grows quietly and insidiously without the patient no- ticing anything until vision becomes obscured by the enlarging tumor. At such time, distant metastases may have already occurred Serial paraffin-embedded 4-␮m sections of a murine tumor-bearing eye from which the patient will eventually die. In some cases, progres- were stained with H&E stain. sive intraocular tumors present as phthisis (i.e., disintegrate) in CFSE labeling and adoptive transfer which the tumor-containing eye has shrunken (18, 19) because of extensive infiltration of tumor or inflammatory cells into the ciliary Single cell suspensions were generated from spleen and peripheral lymph nodes of strain 42 mice. Erythrocytes were depleted by ammonium chlo- body (CB). The normal functioning CB produces aqueous humor ride treatment (2 min on ice). Cells were washed once in cold medium and to maintain the regular intraocular pressure of the eye. Once 90% once in cold PBS, after which they were resuspended in PBS at 1 ϫ 107 Downloaded from or more of the CB is infiltrated or damaged by tumor or inflam- cells/ml and incubated with 0.5 ␮M CFSE (Molecular Probes) for 30 min matory cells, it cannot produce aqueous humor any longer result- at 37°C. FCS was added to a concentration of 5% FCS, and the cells were washed in PBS. TCR-transgenic CD8ϩ T cells (3 ϫ 106) were injected into ing in hypotonia of the eye followed by phthisis, or literally shrink- the tail vein of intraocular tumor-bearing mice in 200 ␮l of PBS. age of the eye . Previously, we have shown that, although intraocular tumor Ags ϩ are constitutively presented to CD8 T cells in the local draining Single cell suspensions of , lymph nodes, and tumor-containing http://www.jimmunol.org/ lymph nodes (DLN), it did not lead to tumor eradication (20). eyes were prepared by mechanical disruption. Blood samples and cell sus- Moreover, Ag presentation did not lead to proper systemic CTL pensions of spleens were depleted of erythrocytes by ammonium chloride immunity, as tumor-specific CTLs were not found outside the tu- treatment for 5 min at room temperature. Cells were stained with directly allophycocyanin-conjugated mAb against CD8 (clone 53-6.7; BD Pharm- mor DLN. We studied the consequences for ocular tolerance and b ingen) combined with PE-conjugated E1A234–243-loaded H-2D tetramers immunity in case “spontaneous” phthisis occurs resulting from (E1A-TM) or PE-conjugated HPV16-loaded H-2Db tetramers as a control. progressive tumor growth. Our data show that intraocular tumor After CD11c enrichment, cells were stained with allophycocyanin-conju- growth leads to the inability of the AC tumor-bearing mice to gated mAb against CD11c (clone HL3; BD Pharmingen) combined with stainings for CD80 (clone 16-10A1; BD Pharmingen), CD86 (clone GL1, control an otherwise unharmful tumor cell inoculum placed at a BD Pharmingen), CD40 (clone 3/23; BD Pharmingen), I-A/I-E (clone M5/ by guest on September 27, 2021 distant site in the body. Tissue damage in the absence of microbial 114.15.2; BD Pharmingen), or H-2Kb (clone AF6-88.5; BD Pharmingen). or viral infection can break this established tolerance against tumor Data acquisition and analysis were done on a BD Biosciences FACScan Ags, which suggests that normal tissue homeostasis is required for with CellQuest software. the maintenance of ocular tolerance. Separation of CD11cϩ and CD11cϪ Materials and Methods Peripheral lymph nodes of tumor-bearing mice were treated with collage- nase (250 U/ml; Sigma-Aldrich) and DNase (50 ␮g/ml; Sigma-Aldrich) for Mice 30 min at 37°C. CD11cϩ cells were positively selected using magnetized ϳ ϩ C57BL/6 mice were purchased from Charles River Breeding Laboratories. Ab for CD11c (N418; Miltenyi Biotec). A purity of 90% of the CD11c TAPϪ/Ϫ mice and CD40Ϫ/Ϫ mice (both on C57BL/6 background) were cell was obtained as determined by FACS analysis. purchased from The Jackson Laboratory. Strain 42 mice, bred at the Ned- erlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek- Results Preventie en Gezondheid (TNO-PG) are TCR transgenic mice expressing Collapse of the anatomical integrity of a tumor-containing eye the TCR ␣ and ␤ chains derived from the H-2b-restricted early region 1A leads to systemic distribution of endogenous tumor-specific CTL of human adenovirus type 5 (Ad5E1A)234–243-specific CTL clone 5 (21). Mice were kept at the Leiden University Medical Center Facility Recently, we described that intraocular tumor Ags are presented to (Leiden, the Netherlands) and used at 5–13 wk of age in accordance with tumor-specific CTL in tumor DLN (20). The Ag is likely to be national legislation and approval of the Animal Experimental Committee derived from the ocular growing tumor, as previous control exper- of the Leiden University. iments have shown that leakage of tumor cell suspension into the Tumor cells conjunctival sac during the inoculation into the AC does not lead Mouse cells transformed by the Ad5E1A plus EJ-ras were cultured to T cell priming in the DLN (20). However, no effective antitumor in IMDM (Invitrogen Technologies) supplemented with 8% (v/v) immune response could be found in the tumor-bearing animals ϩ FCS, 50 ␮M 2-ME, glutamine, and penicillin, as described (21, 22). Dif- because the endogenous tumor-specific CD8 T cells do not dis- ferent kinds of Ad5E1A plus ras-transformed tumor cell lines were used. tribute systemically. For these studies we used a tumor model of Some lines were progressively growing s.c. and therefore lethal in naive tumor cells transformed by Ad5E1A that do not metastasize to mice and others were nonlethal because they are rejected after a short period of growth following s.c. injection into naive mice (21, 23). other parts of the body. These tumor cells harbor a highly immu- nogenic CTL epitope that is recognized by E1A-specific CTL. As Tumor experiments no effective tumor-specific CTL responses are generated, E1A- Intracameral inoculations. A previously described technique for deposi- transformed tumors are not cleared and grow progressively in the tion of a definite number of tumor cells into the AC of the mouse has been AC of the eye of syngeneic C57BL/6 mice. However, upon further used (24). Mice were anesthetized with a mixture (ratio 1:1) of xylozine (2% Rompun; Bayer) and ketamine hydrochloride (Aescoket; Aesculaap) experimentation it was found that in some cases, several weeks given i.p. The eye was viewed by low power (ϫ8) under a dissecting after tumor inoculation, systemic E1A-specific CTL responses microscope, and a sterile 30-gauge needle was used to puncture the cornea were present. The presence or absence of E1A-specific CTLs in the The Journal of Immunology 4249

FIGURE 1. Two types of progres- sive intraocular Ad5E1A-expressing tumor growth in C57BL/6 mice. Sag- ittal view of a naive (tumor-free) eye (A) and two types of progressive in- traocular tumor growth: nonphthisis (B) vs phthisis (C). Both types show anterior and posterior tumor expan- sion. There is no tumor growth out- side the eye bulb. Note the decrease in size of both intraocular tumor and eye in the phthisis intraocular tumor (C). Box (inset)inA–C are enlarged in D and E, which show the CB in detail of the naive eye (D), the non- phthisis (E), and phthisis intraocular tumor in which the CB has been com- pletely destructed. C, cornea; L, lens; Downloaded from VB, vitreous body.

periphery appeared to be correlated with two distinct patterns of served until 6 wk after intracameral tumor injection, they would http://www.jimmunol.org/ progressive intraocular tumor growth. The first pattern was char- not occur later during progressive intraocular tumor growth (non- acterized by a developing intraocular tumor that grows on the iris phthisis) (Fig. 3A). Clinically phthisis looks somewhat similar to into the AC and progressively expands into the posterior segment prephthisis but the clinical signs are more severe: the eye bulb has of the eye. Eventually the whole eye is filled with tumor cells become at least half its original size, the AC is completely oblit- without damaging the barriers of ocular tissue or orbit, leaving the erated, and the cornea is opaque, edematous, and thickened. The intraocular and surrounding eye bulb intact (Fig. 1B). tumor, although smaller in size, is still present in the phthisis eye However, in another 60% of mice, the eye bulb shrinks (phthisis). (Fig. 3D). In these eyes, extensive tumor infiltration of the CB occurred. This As depicted in Fig. 2, the presence of E1A-specific CTL in is likely to disrupt the aqueous humor production of the CB re- DLN, NDLN, spleen, and tumor was determined by E1A-Db tet- by guest on September 27, 2021 sulting in hypotonia of the tumor-bearing eye leading to collapse ramer-. E1A-specific CTLs were readily detectable sys- of the anatomical intraocular integrity and shrinkage of the eye temically in mice undergoing complete phthisis of the eye; how- bulb (Fig. 1C, phthisis). To examine in depth whether these two ever not in mice with nonphthisis. In the latter case, E1A-specific patterns of intraocular tumor growth are associated with systemic CTLs were only found in the tumor DLN. Although, a relatively antitumor responses, we investigated DLN, nondraining lymph small number of E1A-specific CTLs were detected in some mice nodes (NDLN), and spleen for the presence of endogenous E1A- with prephthisis (20%), probably as a consequence of the ongoing specific CTLs using E1A/Db tetramers. As shown in Fig. 2A, tu- phthisis process, no E1A-specific CTLs were detected systemically mor-specific CD8ϩ T cells were observed in the tumor DLN, in the majority of mice (80%) with prephthisis intraocular tumors. NDLN, and spleen of mice with phthisis intraocular tumor growth Together these findings indicate that the occurrence of phthisis 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 intraoc- ular phthisis tumors but not in the tumor-bearing eyes of the non- phthisis 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-contain- ing phthisis eye.

Phthisis precedes the emergence of tumor-specific CTL To determine which of the two features, intraocular CTLs or phthi- sis occurred first, we set out to analyze the presence of endogenous E1A-specific CTLs in different stages of tumor growth: nonphthi- FIGURE 2. Collapse of the anatomical integrity of a tumor-containing sis, prephthisis, and phthisis intraocular tumors (Fig. 3). These eye results in systemic distribution of endogenous tumor-specific CTLs. Endogenous E1A-specific CTLs are only observed in the tumor DLN of three different phases of intraocular tumor growth were defined mice with nonphthisis intraocular tumor growth and have distributed sys- macroscopically and confirmed microscopically. The clinical temically in mice with phthisis intraocular tumor growth. Mice bearing an prephthisis signs are commencing microphthalmos (i.e., small eye) Ad5E1A-expressing tumor were sacrificed 63 days after tumor inoculation. and cornea-cloudiness (Fig. 3, B and C). When these prephthisis FACS analysis was performed in DLN, NDLN, spleen, and intraocular signs are present, phthisis will occur 2–3 wk from this time point. tumors using Db/E1A tetramers. One representative experiment of 10 is However, when these clinical signs of prephthisis were not ob- shown. 4250 IMMUNE TOLERANCE IN NORMAL TISSUE HOMEOSTASIS

Intraocular tumor Ags are cross-presented by CD11cϩ cells to naive CTL in local DLN The results we describe illustrate systemic distribution of tumor- specific CD8ϩ T cells after collapse of the anatomical integrity of the tumor-containing eye (phthisis). As no such systemic distribu- tion 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 ac- tivated. 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 intracam- eral injection of E1A-expressing tumor cells, secondary lymphoid Downloaded from organs were examined. Selective in vitro outgrowth of lymph node and spleen cultures followed by PCR showed the presence of tu- mor 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-express- http://www.jimmunol.org/ ing tumor cells into wild-type and TAPϪ/Ϫ mice. TAPϪ/Ϫ 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 de- ficient from TAP. Nineteen days after tumor inoculation, at a time intraocular tumors had developed, CFSE-labeled E1A-specific CD8ϩ T cells derived from TCR-transgenic mice were injected. by guest on September 27, 2021 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 TAPϪ/Ϫ mice, indicating that host-derived APC presented the ocular tumor Ag in a TAP-dependent fashion.

FIGURE 3. Phthisis precedes the emergence of tumor-specific CTLs. Endogenous E1A-specific CTLs are never observed in a nonphthisis in- traocular tumor (A) or in the very early stages of preprephthisis when the tumor-bearing eye starts to show some signs of collapse (B). Tumor-spe- cific 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 inoc- ulation either for histology of the intraocular tumors or for FACS analysis performed in tumor-bearing eyes using Db/E1A tetramers. One represen- tative experiment of three is shown. FIGURE 4. Host-derived APCs are required for presentation of tumor Ags. CFSE-labeled E1A-specific CD8ϩ T cells were adoptively transferred into wild-type mice or TAPϪ/Ϫ mice bearing 19-day-old tumors in the AC precedes the emergence of systemically distributed tumor-specific of the eye. Three days posttransfer proliferation of adoptively transferred CTL, suggesting that during phthisis signals are released that trig- E1A-specific T cells in DLN, NDLN, and spleen was analyzed by FACS. ger proper CTL responses. One representative experiment of three is shown. The Journal of Immunology 4251

Because CD11cϩ cells are most likely the host-derived APCs tion markers contrary to the CD11cϩ cells out of the nonphthisis responsible for cross-presentation of tumor Ags to CTL (27), and eye tumors (data not shown). Together these findings indicate that because we have shown recently that the E1A-Ag is presented by collapse of the anatomical integrity of a tumor-containing eye led CD11cϩ cells (but not by CD11cϪ cells) from (s.c.) growing tu- to an activated phenotype of the CD11cϩ cells present in tumor mors, we subsequently studied whether phthisis would lead to mat- DLN and the phthisis intraocular tumor itself. As presentation of uration of these cells as APC activation is considered crucial for the E1A epitope depends on host-derived APCs, our results indi- induction of CTL immunity (2, 28). For this purpose, CD11cϩ cate that the emergence of systemic CTL immunity results from cells in tumor DLN of mice with phthisis eye tumors and nonph- the presentation of the E1A epitope by DCs that are endowed with thisis intraocular tumors were analyzed. In Fig. 5 it is shown that the capacity to activate CTLs allowing their systemic distribution CD11cϩ cells isolated out of the DLN from mice with phthisis as a consequence of phthisis. intraocular tumor growth had up-regulated several surface markers such as CD80, CD86, CD40, and MHC class I and class II in Collapse or preservation of intraocular tumor-containing comparison with the CD11cϩ cells isolated from nonphthisis eye structures determines the outcome of sequential immune tumor-bearing mice. CD11cϩ cells isolated out of the phthisis in- responses; immunity vs tolerance traocular tumors also displayed an up-regulation of these activa- 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 Downloaded from took advantage of two tumor cell lines that have different s.c. growth patterns in naive mice. One tumor cell line grows progres- sively 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 re- http://www.jimmunol.org/ maining 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 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 CD8ϩ CTL immunity (29), these results indicate that phthisis leads to the induction of strong tumor-specific CTL responses that protect against a secondary tu- by guest on September 27, 2021 mor challenge. In another group of 30 mice with intraocular tumors, 20 mice developed phthisis compared with 10 mice with nonphthisis in- traocular 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 tu- mors (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 intraoc- ular 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 integ- rity of the eye is lost. Together, these findings indicate that col- lapse or preservation of intraocular structures in a tumor-contain- ing eye determines the systemic immunological immune responsiveness, respectively resulting in immunity or tolerance against secondary tumor challenges.

Discussion Our results show that ocular immune privilege is broken when the FIGURE 5. Collapse of the anatomical integrity of the tumor-contain- ϩ anatomical integrity of the eye is disrupted. Mice with established ing eye leads to activated CD11c cells. C57BL/6 mice were injected tumors growing in the AC are incapable of rejecting an otherwise intracamerally with 0.75 ϫ 105 E1A-expressing tumor cells. When phthisis nonlethal tumor inoculated s.c., but are able to resist an aggressive, (broken histogram) or nonphthisis (shaded histogram) intraocular tumor growth had developed, mice were sacrificed and CD11cϩ cells were iso- otherwise lethal tumor, when phthisis occurs spontaneously. These lated out of the lymph nodes and intraocular tumors for analysis of CD80, findings are strongly correlated with the systemic emergence of CD86, CD40, MHC class I and class II expression. Naive tumor-free mice tumor-specific CTLs in case of phthisis, explaining the rejection of were included in the experiment (thick histogram). One representative ex- a s.c. tumor, as rejection of this tumor crucially depends on CTL- periment of three is shown. mediated immunity (21). In case no phthisis occurred, although 4252 IMMUNE TOLERANCE IN NORMAL TISSUE HOMEOSTASIS

are not fully understood, it is thought that the release of intraocular -Ags in the traumatized eye evokes a T cell-mediated (auto) immune reaction. These effector T cells will subsequently recog- nize similar Ag in the other eye leading to devastation of the non- traumatized eye (31). We consider it likely that trauma to the eye not only leads to the release of protein Ag, but also to the activa- tion of DC as we observed that phthisis was strictly correlated with activation of CD11cϩ cells in DLN. As Ag is present in both nonphthisis and phthisis eyes, the activation of DC as a conse- quence 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, al- lowing 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 Downloaded from only be detected by PCR in the tumor DLN and not in the NDLN, , or spleen. Likewise, we have never observed micrometas- tases either macroscopically or microscopically. Moreover, no me- tastases 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 http://www.jimmunol.org/ 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 mol- ecule expression. FIGURE 6. Collapse or preservation of intraocular tumor-containing struc- tures determine the outcome of sequential immune responses: immunity vs As DCs are the predominant, if not the only, cells capable of tolerance. Secondary “lethal” (A) or “nonlethal” (B) syngeneic s.c. tumors generating CTL immunity (27, 33, 34), it is likely as outlined that by guest on September 27, 2021 were inoculated (10 ϫ 106 E1A-expressing tumor cells) in mice with progres- the outcome of immune reactivity is a consequence of DC sive nonphthisis (ࡗ) or phthisis (Œ) intraocular tumor growth and in naive activation. mice (primary s.c. tumor (f)). Secondary “lethal” tumors (A) develop s.c. in The data presented indicate that CD11cϩ cells display a rela- naive mice (f) and in mice with nonphthisis intraocular tumor growth (ࡗ). tively “resting” phenotype in mice with nonphthisis intraocular Secondary s.c. tumor growth was inhibited in mice with phthisis intraocular tumor growth, allowing Ag presentation in a nonimmunogenic or tumor growth (Œ). Secondary “nonlethal” tumors (B) only develop in mice even tolerogenic fashion (34, 35). It is conceivable that CTLs rec- with nonphthisis intraocular tumor growth (ࡗ) and was inhibited in mice with phthisis intraocular tumor growth (Œ) and in naive mice (f). ognizing Ag on such quiescent DCs go through an abortive/poised form of activation that ultimately leads to deletion as is also ob- served in other systems (32, 36, 37). This would explain our ob- tumor-specific CTL are present in tumor DLN, the animal could servation that we do not observe a systemic dispersal of tumor- not mount a tumor-protective CTL response any longer, as re- specific CTL that have been activated in the local DLN. However, flected by the inability to reject a challenge with benign tumor the inability of the animals to control the growth of otherwise cells. Because such CTL responses are crucial for the eradication nonlethal E1A-expressing tumors implanted s.c. could also result of these s.c. tumors (21, 23, 30), these results indicate that ocular from other mechanisms, such as the emergence of tumor-directed tumor growth in intact eyes leads to an “abortive” or “poised” CTL responses. Stimulation of the immune system reaction. under tolerogenic conditions as operative in the ocular environ- Apparently, the tumor growing in the AC recruits effective sup- ment might lead to the systemic emergence of such regulatory T ϩ pressor mechanisms that do not only exert local, but also systemic cells that inhibit the induction of tumor-specific CD8 T cells at effects that allow the s.c. growth of an otherwise nontumorigenic other sides. Thus, two levels of ocular tolerance might occur, one E1A-transformed tumor cell clone. The induction of such systemic is leading locally to an abortive form of T cell activation, whereas effects appears to be specific of AC tumor growth because we did the other results systemically in active inhibition of tumor-specific not observe these effects when the same tumor was injected s.c. immunity. Either way, our results indicate that phthisis can bypass (data not shown). The observation that the tolerance and suppres- ocular tolerance leading to the induction of proper systemic anti- sive mechanisms installed by the AC growing tumor are broken tumor immunity, conceivably as a result of local APC activation and converted into efficient antitumor immunity is intriguing as it (34). Although we cannot rule out the possibility that eyes under- indicates that disruption of anatomical (ocular) integrity can act as going phthisis are susceptible to some kind of low-grade infection a switch determining whether immunity or tolerance prevails. This that provides danger signals, the acquisition of the immunostimu- finding is in line with the clinical observation that trauma to one latory DC phenotype is acknowledged. The theory is in line with eye may cause destruction of the fellow eye, called sympathetic the concept that stressed cells release signals such as uric acid that ophthalmia. 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