Deviant Immune Response After Intracameral Inoculation

8/6/2019 Deviant Immune Response After Intracameral Inoculation

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Deviant immune responses to allogeneictumors injected intracamerally and

subcutaneously in miceJerry Niederkorn, J. Wayne Streilein, and John A. Shadduck

The ability to introduce carefully controlled numbers of viable cells into the anterior chamberof mouse eyes made it possible to examine the interrelationship between presentation of anti-gens intracamerally and into conventional body sites and their synergistic/antagonistic effectson the immune system. P815 mastocytoma (DBA/2; H-2A) cells are syngeneic w ith BALBIchosts at the major histocompatibility complex but differ at numerou s m inor histocompatibilityloci. When these cells are injected intracamera lly into BAL BIc mice, they subvert the host'simmune response; that is, twnor cells injected subcutaneously developed into tumors. Thedynamics of this anterior chamber-ass ociated immun e deviation was manipulable. When sub-cutaneous (SC ) inoculations preceded intracameral (IC ) inoculations by 5 days or more, sys-temic anti-DBA 12 immunity elicited by SC inoculation prevented successful engraftment ofP815 tumors in the anterior chamber. A s the time interval between SC and IC inoculations ofP815 cells decreased, the balance between destruction or survival of intraocular tumor s wastipped in favor of tumor growth. Intrao cular tum or growth increased when IC inoculationspreceded SC inoculations and was most impressive w hen this interval was 7 days. In these m icethe tumors grew briskly and a ggressively in a fashion compara ble to that seen in hosts notreceiving prior SC inoculations. The apparent ca pacity of the immune system to prevent orenhance the growth of tumors can be successfully m anipulated in ways that suggest the possi-bility of therapeutic benefit in ophthahnologic disease.

Key words: anterior chamber, immune response, allogeneic tumors,keratoconjunctivitis, minor histocompatibility antigens

T:ansplantation antigens placed into theanterior chamber of the eye impact upon theimmune system in a manner quite differentfrom that after their introduction into otherFrom the Depar tments of Ophthalmology (Dr . Nieder-korn), Cell Biology and Internal Medicine, (Dr. Strei-lein), and Pathology (Dr. Shadduck), SouthwesternMedical School, Th e Unive rsity of Texas Health Sci-ence Center at Dal las.Supported in par t by U.S.P.H.S. grants EY-01330, AI-10678, and CA-09082.Submitted for publication May 27, 1980.Repr int requests: Jerry Niederkorn, Ph.D ., De par tm entof Cell Biology, The University of Texas Health Sci-ence Center , 5323 Harry Hines Blvd., Dal las, Texas

75235.

body sites. The time-h onored example of thisdifference is the observation that allogeneictissues grafted into the anterior chamber en-joy prolonged, sometimes indefinite survivalwhereas comparable tissues grafted to otherbody sites are rejected with dispatch.*~ 3 Thelabel often ap plied to the ante rior chamber inthis context is "immunologically privilegedsite." Until recently, the "privilege" affordedto allogeneic tissues in the anterior chamberwas thought to result from the anatomical ob-servation that the anterior chamber lacks alymphatic drainage route by which antigencan escape into the systemic circulation. Theresultant afferent blockade was thought toprevent alloantigens placed in the anterior0146-0404/81/030355+0 9$00.90/0 1981 Assoc. for Res. in Vis. and Oph thal. , Inc. 355


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35 6 Niederkorn et at. Invest. Ophthalmol. Vis. Sci.March 1981

10 15 20 25 5 10 15 20 25Days After Injection

Fig. 1. Growth patterns of anterior chambertumors following IC inoculation of 105 P815 mas-tocytoma cells. No keratoconjunctivitis was ob-served. A, DBA/2 mice. B, BALB/c mice. As-terisk, Ocular phthisis; parentheses, number ofeyes.

Fig. 2. A nterior chamber of BALB/c mouse filledwith growing mastocytoma 10 days after IC inocu-lation of 105 P815 cells,chamber from reaching the immune systemand thereby preclude the elicitation of animmune response.Studies recently reported from this labora-tory"~G and corroborated by others7 havedemonstrated conclusively that antigen in-troduced into the anterior chamber of rateyes does, in fact, make itself known to thesystemic immune apparatus. Semiallogeneic(Fj hybrid) lymphoid cells placed in the an-terior chambers of parental strain rats, dispa-rate at the rat major histocompatibility com-plex (MHC) Rt-1, evoke the appearance inthe host's serum of specific anti-Rt-1 alloan-tibodies. Moreover, the allograft immune re-sponse of intracamerally inoculated rats istransiently suppressed, so that orthotopicallografts of skin survive on these animals

for extended intervals. Experimental dissec-tion of this phenomenon, termed F t lympho-cyte-induced immune deviation (FiLI-ID),proved to be difficult in rats because of thelack of appropriate immunologic reagentsable to identify functionally distinct lympho-cyte subsets and because of the lack of MHCcongenic and recombinant rat strains compa-rable to those available in mice.The experiments reported in this paperdescribe the adaptation of the technique ofintracameral (IC) inoculation of cellular sus-pensions to adult mice. The results revealthat the phenomenon of FjLI-ID first dem-onstrated in rats apparently applies as well tomice and may in fact be a general phe-nomenon.Materials and methods

Experimental animals. Adult female BALB/c(H-2a), DBA/2 (H-2) and C57BL/6 (H-2b) micewere purchased from Jackson Laboratories, BarHarbor, Maine, and used as experimental subjectswhen they were between 3 and 5 months of age.BALB/c and DBA/2 mice share similar H-2 hap-lotypes but differ at multiple minor histocompati-bility loci.Tumor cells. P815 mastocytoma (DBA/2) cellswere cultivated in suspension cultures in Falcon75 cm2 tissue culture flasks (Falcon Plastics, Ox-nard, Calif.) with Dulbecco's modified Eagle'sminimal essential medium supplemented with10% heat-inactivated fetal calf serum and gen-tamicin (0.05 mg/ml; Schering Corp ., Kenilworth,N.J.). EL-4 lymphoma (C57BL/6) was maintainedby serial passage as ascites in C57BL/6 mice.Monocellular suspensions of P815 cells and EL-4cells were washed in Hanks' balanced salt solution(HBSS) and resuspended in HBSS for subcutane-ous (SC) and IC inoculations.Anterior cJiamber inoculations. A modifiedquantitative technique for depositing a definitenumber of tumor cells into the anterior cham berofthe mouse eye was employe d.8The technique is asfollows. Mice were deeply anesthetized with 0.66mg of ketamine hydrochloride (Vetalar; Parke,Davis & Co,, Detroit, Mich.) given intramuscular-ly. The eye was viewed und er the low power (8x)of a dissecting microscope, and a sterile 30-gaugeneedle was used to puncture the cornea at thecorneoscleral junction, parallel and anterior to theiris. The aqueous humor was expressed by com-pressing the cornea with the back of a scalpel


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Volume 20Number 3 Immune responses to intracameral alloantigens 357

Table I. Ability of P815 tumor cells to immunize BALB/c mice to DBA/2 alloantigensPrimary exposure No . of mice Secondary exposure ResultsDB A/2 skin graft 19 No ne (first set)DB A/2 skin graft 8 DBA /2 skin graft (second set)P815 injected SC 8 DBA /2 skin graftP815 injected IC 11 DBA /2 skin graftDBA /2 skin graft 10 P815 injected ICNon e 10 P815 injected IC

MST = 10.8 days (10.2 - 11.8)*MST = 7.0 0.0 daysMST = 7.0 0.0 daysMST = > 20 days0/10 Tumorsdeveloped10/10 TumorsdevelopedBALB/c mice were exposed to DBA/2 alloantigens as indicated as a primary exposure (day 0) and secondary exposure (day 14).*95% confidence limits.

blade, and the evacuated fluid was blotted withsterile 4 by 4 gauze pads. A glass micropipet (ap-proximately 80 (xm in diameter) was fitted into asterile infant feeding tube (No. 5 French; CutterLaboratories, Inc., Berkeley, Calif.), which wasmounted onto a sterile 0.1 ml Hamilton syringe(Hamilton Co., Inc., Whittier, Calif.). A Hamiltonautomatic dispensing apparatus was fitted onto theloaded syringe and was used to dispense 5 fAquantities of P815 cell suspensions. The pipetloaded with P815 cell suspension (2 x 107 cells/ml = 1 X 10

5cells/5 /xl) was introduced throughthe puncture site of the cornea, and 5 fi\ of theP815 cell suspension were delivered into the an-terior chamber. As the needle was withdrawn, th eiris prolapsed and plugged the perforated cornea,thereby minimizing leakage of the inoculum.

SC inoculations. Tumor cells, 1 x 105, (P815or EL-4) suspended in 0.1 ml of HBSS were inocu-lated subcutaneously into the right rear flanks atvarious times relative to anterior chamber inocu-lations.Skin grafting. Full thickness skin grafts wereprepared as described elsewhere.9 Grafts wereapplied orthotopically and wrapped in plaster ofparis bandages. Casts were removed 7 days later,and the grafts were inspected for evidence of re-jection. Destruction was judged complete whenall remnants of surface epidermis were gone. Me-dian survival times (MSTs) were calculated.Experiments and results

Our experimental approach was, first, toestablish that successful inoculation of repro-ducible numbers of viable cells into the an-terior chamber of murine eyes was feasibleand, second, to determine whether alloge-neic cells injected intracamerally could make

an impact on the systemic immune apparatusof recipient allogeneic mice. The mas-tocytoma, P815, which originated in DBA/2mice, was selected because of its ease ofmaintenance in vitro and its faithful inductionof tumors when inoculated into syngeneicDBA/2 adult mice and because this particulartumor cell line has been used extensively bycellular immunologists as a target of in vitroassays of T cell-media ted immunity.10Growth patterns ofP815 tumor cells inocu-lated into the anterior chamber of syngeneican d allogeneic eyes. P815 cells, at a concen-tration of 105/5 fi\, were inoculated into theanterior chambers of eyes of two panels ofadult animals: DBA/2 and BALB/c mice. Thepattern of tumor growth in both panels wassimilar (Fig. 1). During the first 4 days afterinoculation, no evidence of tumor could beseen with aid of a dissecting microscope.However, between 5 and 6 days white tufts oftumor cells were detected anterior to the iris.These masses grew rapidly, and by 8 to 12

days after inoculation they completely filledthe anterior chamber (Fig. 2). Characteristi-cally, at this point a cone-shaped wedge oftumor tissue extended toward the cornea, re-sulting in corneal perforation between days12 and 14. Within the next 2 to 4 days, theaffected eyes rapidly regressed in size andwere replaced by organizing fibrous tissue.Thus, despite the fact that P815 cells ex-press minor histocompatibility antigens aliento the BALB/c strain of mice,11 no evidenceof transplantation immunity was observed;

the growth of P815 cells in the eyes of both


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358 Niederkorn et al. Invest. Ophthalmol. Vis. Sri.March 1981

5 10 15 20 25 5 10 15 20 25 5 10 15 20 25Days After Injection

=duration)

5 10 15 20 25

( ) = Naof EyesKC = Keratoconjunctivitis (* = Ocular PhthisisFig. 3. Influence of P815 mastocytoma cells inoculated subcutaneously on growth patterns ofintracamerally inoculated P815 cells. Label in upper left corner of each panel represents timeinterval b etween SC and IC inoculations of P815 cells. Minus sign indicates that SC inocula-tion preceded IC inoculation by (N) days.

panels of animals was indistinguishable.Corneal perforation, regression of the tu-mor, and, finally, resorption of the eyes insyngeneic recipients were probably a conse-quence of the uncontrolled local growth ofthe tumor. It is presumed, although there isno direct proof, that the tumor brings aboutits own destruction by progressively com-promising the blood supply to the orbit untilinfarction takes place. As a consequence, thetumor and the eye are both destroyed andresorbed, i.e., ocular phthisis.Growth patterns ofP815 tumor cells inocu-lated subcutaneously into syngeneic and al-logeneic mice. Inoculations of 105 P815 cellswere placed subcutaneously into panels ofadult DBA/2 and BALB/c mice. In DBA/2recipients palpable tumor nodules appearedat inoculation sites within 5 days. Thesemasses grew rapidly, reaching approximately4 cm in diameter within 19 days after inocu-lation. Unrestrained growth of these tumorseventually proved fatal for the recipients,death occurring within 3 weeks of originalimplantation. By contrast, no evidence of SCtumors was seen in BALB/c recipients. The

failure of subcutaneously injected P815 cells

to grow in BALB/c hosts was found, as ex-pected, to be due to the ability of these cellsto elicit allograft immunity directed at theminor histocompatibility antigens expressedon the tumor cells. This was proved to be thecase as follows. Panels of BALB/c mice thatreceived 105 P815 tumor cells subcutane-ously on day 0 were grafted with DBA/2 skinorthotopically on day 14. Accelerated rejec-tion of these grafts (MST = 7.0 0.0 days)compared to the speed of rejection of DBA/2skin grafts placed on immunologically virginBALB/c recipients (MST = 10.8, 95% confi-dence limits of 10.2 to 11.8) indicates that theinitial SC encounter with P815 cells elicitedallograft immunity in BALB/c hosts (seeTable I). Taken together with the P815 tumorcell growth patterns in the anterior chambersdescribed above, these data support the hy-pothesis that a histoincompatible tumor graftplaced subcutaneously elicits a specific im-mune response that prevents malignant cellsfrom establishing a viable tumor in situ;by contrast, the immunologically privilegedstatus of the anterior chamber of the eye"protects" locally injected allogeneic tumorcells from immune recognition and/or de-


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struction, and as a consequence the malig-nant cells are able to develop into a progres-sively growing tumor that leads to oculardestruction with a tempo and vigor indistin-guishable from those seen in syngeneic hosts.A considerable body of evidence indicatesthat the P815 tumor cell line expresses bothtransplantation alloantigens and tumor-spe-cific antigens.10* n In order to determinewhether the failure of P815 cells to grow inthe SC tissues of BALB/c mice was due to animmune response directed at minor his-tocompatibility differences between DBA/2and BALB/c strains, BALB/c recipients ofsubcutaneously injected P815 cells weregrafted 2 weeks thereafter with DBA/2 skin.In a related experiment, BALB/c mice weregrafted orthotopically with DBA/2 skin. Twoweeks later, they received 105 P815 cells in-jected into the anterior chamber. This latterexperiment examined formally the hypothe-sis that pre-existent systemic anti-DBA/2 al-loimmunity can prevent the development ofDBA/2 tumor in the anterior chamber. Theresults of these experiments are presented inTable I. For the sake of comparison conven-tional first- and second-set graft survivaltimes are included in the table. It can be seenthat an SC inoculation of P815 tumor cellseffectively immunized BALB/c mice to DBA/2alloantigens; these animals rejected sub-sequent DBA/2 skin grafts in an acceler-ated fashion, i.e., 7.0 0.0 days (comparewith MSTs of first- and second-set grafts).Moreover, active immunization of BALB/cmice with DBA/2 skin prevented P815 cellsfrom establishing a successful graft in the an-terior chamber. These results offer strongcircumstantial evidence in support of the hy-pothesis that subcutaneously injected P815cells immunize BALB/c mice to DBA/2minor histocompatibility antigens and thatthis immunization can express itself in theanterior chamber.The data do not address directly, however,the question of whe ther P815 tumor cells alsocan immunize BALB/c mice to the tumor-specific antigen(s). Experiments are underway to test this possibility. At present w e arewilling to conclude that, at the very least, the

Fig. 4. Intense ocular inflammation of BALB/cmouse inoculated first intracamerally on day 0 andthen subcutaneously with 105 cells on day 4.

tumor cells express DBA/2 minor antigensand elicit allograft immunity in BALB/cmice. With this in mind and with the obser-vations that there is a marked disparity ingrowth patterns of P815 cells injected intothe SC tissue compared to that followinginoculation into the anterior chamber ofBALB/c mice, we recognized an opportunityto examine the putative relationship betweenalloantigens implanted as tumor cells at bothsites.Influence ofSC inoculation ofP8l5 cells ongrowth pattern of intracamerally inoculatedP815 cells. The next series of experimentswas designed to determ ine w hether flank in-oculation of DBA/2 alloantigens (expressedon P815 cells) before, after, or simulta-neously with IC inoculation of P815 cellsmight influence the growth of the latter inthe anterior chamber. These data are sum-marized in Fig. 3 and Table I. Evidence sup-porting the conclusion that SC injections ofP815 cells induced anti-DBA/2 transplanta-tion immunity was found in panels where SCinjection of 105 P815 cells preceded IC injec-tion by 7 or 5 days. In neither group ofBALB/c mice were SC or anterior chambertumors observed. This finding corroboratespreviously published reports that a state ofpre-existing specific immunity can rob theanterior chamber of its ability to confer im-munologic privilege.12

When SC injections of P815 cells pre cededIC injections by only 4 days, tumors devel-


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360 Niederkorn et al. Invest. Ophthalmol. Vis. Sci.March 1981

Table II. Temporal relationships of IC and SC injections of P815: effects on tumor growthDay*ofSC inocu-lation ofP815 cells

- 7- 5- 4- 2

0+2+4+5+7

No. ofeyes injected

202010101810

101010

Eyes withtumors (%)00

(0)10(100)9(90)17(94)8(80)10(100)10(100)10(100)

Eyes with kerato-conjunctivitis (%)00(0)6(60)0

(0)2(11)8(80)

4(40)6(60)10(100)

Eyesdestroyed (%)00(0)6(60)0(0)0(0)6(60)4(40)6(60)10(100)

Mice developingSC tumors (%)0/100/10(0)0/5(0)(5/5)(100)9/ 9(100)0/5(0)0/5(0)5/ 5(100)0/5(0)

*Day = day of bilateral IC inoculation of P815 cells.

oped in the anterior chambers. These neo-plasms appeared asfloccularaggregates 5 to 7days after inoculation and rapidly increasedin size thereafter. Their growth was accom-panied by intense signs of inflammation; thatis, the conjunctivae became injected andedematous, the cornea developed severekeratitis, the globes swelled to enormoussize, and ultimately these eyes were de-stroyed, becoming desiccated between 21and 24 days after inoculation.

Tumor also developed in eyes of BALB/cmice that received SC inoculations of P815 2days prior to IC injections. In these animals,evidence of tumor appeared earlier (within 4to 5 days of IC inoculation), which grew tofill the anterior chamber by 9 days thenrapidly defervesced. Between days 14 and 25after inoculation gross ocular morphology re-turned to normal. At no time was keratocon-junctivitis resembling that described aboveobserved in the eyes of these animals. Simi-larly, if P815 cells were simultaneously in-oculated subcutaneously and intracamerally,tumors appeared in the anterior chamber by5 days. These neoplastic collections grewrapidly, filling the anterior chamber betweendays 9 and 14; a minority of eyes at this timehad an accompanying keratoconjunctivitis.However, in every instance the inflamma-tion receded, tumor regression intervened,

and the eyes returned toward a normal ap-pearance.Thus, if SC injections of P815 tumor cellspreceded or coincided with IC injections ofP815 cells, a host response was elicited (wepresume the response to be immunologic innature) which was capable of controlling ICtumor growth. If the SC injection precededthe IC injection by 5 or more days, the hostresponse absolutely prevented IC tumor de-velopment, and there was negligible inflam-mation. However, as the interval betweenthe two tumor cell injections became pro-gressively shorter, a less efficient host re-sponse took charge, a response in which in-tense inflammation was a significant compo-nent. When the two injections were placed atvirtually the same time, the inflammationwas not sufficient to cause permanent oculardestruction but did rid the eye of the tumor-ous tissue.

SC injections of P815 cells also were madeat timed intervals after IC injection of tumorcells: at 2, 4, 5, and 7 days. In every instanceinjected eyes in every panel of animals de-veloped evidence of anterior chamber tumorwithin 5 days of injection. These tumors grewrapidly, filled the anterior chamber within 10days, and were accompanied by intenseinflammatory changes especially prominentin the conjunctiva and cornea (Fig. 4). In


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animals whose SC injection of P815 cells fol-lowed IC injection by 2 days, a minority ofeyes resolved both the inflammation andtumor infiltration, returning to normal within25 days. In the remainder of subjects theeyes were destroyed by the reaction withinthe globe; two eyes developed corneal perfo-ration. In panels of BALB/c mice whose SCinjections of P815 followed the IC injectionby 4, 5, and 7 days, phthisis of all injectedeyes occurred, and corneal perforation wascommon; in th e last panel ( + 7-day interval),every eye underwent corneal perforation. Itwould appear therefore that SC injections oftumor cells that follow IC injection of tumorcells cause progressively less host responseable to control the growth of the oculartumor. That these eyes developed intensekeratoconjunctivitis suggests that a host im-mune response had been initiated; however,the response was inadequate to the task, andthe tum ors destroyed the eyes as successfullyas though noflank njections had been made.

Influence ofIC inoculation ofPSlS cells ongrowth pattern of subcutaneously injectedtumor cells. The preceding description fo-cused on the capacity of tumor cells placedsubcutaneously in BALB/c mice to influencethe growth of tumor cells injected into theanterior chamber of the eye. On the basis ofour previous studies,6 we suspected thattumor cells placed in the anterior chambermight alter the capacity of tumor cells togrow in the SC inoculation site. All the ani-mals described above were examined at regu-lar intervals for the appearance of SC tumors.The summary of these observations are pre-sented in Table II. It should be restatedfor the sake of comparison that under nocircumstance did local tumors appear inBALB/c mice if 105 P815 cells were injectedsubcutaneously without other experimentalmanipulation; w e took this as evidence of thevigor of allograft immunity elicited by theseallogeneic tumor cells placed at this site. Toour surprise, SC tumors did appear in certainpanels of animals that also received IC injec-tions of P815 cells (Fig. 5). Specifically, SCrumors developed in mice whose IC injec-tions (1) preceded the SC injection by 5 days,

Fig. 5. SC tumor in BALB/c mouse inoculatedlocally witli 105 P815 cells and simultaneously withan IC inoculation of 105 P815 cells.(2) followed the SC injection by 2 days, or (3)were given simultaneously with SC injec-tions. No SC tumors were discovered atother time intervals tested. In all instancesthese unexpected SC tumors appeared ap-proximately 9 days after SC inoculation, irre-spective of the timing of the IC tumor cellinjection. SC tumors grew and then re-gressed over the subsequent 2 to 7 days. Noprogressively growing SC tumor became es-tablished or threatened the life of the hostThe power of DBA/2 allogeneic tumorcells placed in the anterior chamber to affectthe nature of the BALB/c hosts' response toDBA/2 antigens placed elsewhere was dra-matically seen in the following experiment.P815 cells (1 X I05) were inoculated into theanterior chamber of BALB/c animals on day0. Fourteen days later these animals weregrafted orthotopically with DBA/2 skins. Thegrafts enjoyed a surprisingly prolonged ten-ure on their allogeneic host (see Table I).Not only were they not rejected in second-setfashion; they survived longer than did D BA/2grafts on normal BALB/c hosts. Thus the ICpresentation of alloantigens subverted the al-lodestructive host immune response and in-stead allowed prolonged acceptance of theseallodisparate grafts.

These data clearly demonstrate that thepresence of tumor cells in the anteriorchamber can profoundly affect the capacity ofthe immune response to deal with tumorcells placed subcutaneously. Tumor growing


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362 Niederkorn et at. Invest. Ophthalmol. Vis. Sci.March 1981

in the anterior chamber seems to blunt, sup-press, and/or delay the development of aneffective and destructive alloimmune re-sponse; as a consequence, for a transient in-terval the tumor is able to establish itself andgrow. Ultimately, however, the host immuneresponse gains ascendance, and tumor re-gression is procured, presumably by the ac-tion of alloimmune T lymphocytes.Discussion

The ability to introduce carefully con-trolled numbers of viable cells into the an-terior chamber of mouse eyes is an im portanttechnologic achievement. Although theseexperiments are not the first to claim successin this regard (Gallie et al.13 and Boone andDuPree8 reported successful inoculation ofhuman melanoma cells into the anteriorchambers of the eyes of nude mice), the useof an allogeneic tumor model makes it possi-ble to examine the interrelationships be-tween presentations of antigens intracam-erally and into conventional body sites. Wehave been rewarded with an illuminating andin many ways surprising array offindings.Wefeel that the most significant observation isthat intraocular allogeneic DBA/2 tumorcells, syngeneic with BALB/c hosts at theMHC but differing at numerous minor his-tocompatibility loci, can subvert the host'simmune response in such a manner thattumor cells injected subcutaneously are ableto proliferate transiently. Since P815 tumorsnever develop in SC tissues of unmanipu-lated BALB/c hosts, the deviation seen in thehost's immune response in these experimentsis impressive.Our studies have not proceeded far enoughto have gained any meaningful insight intothe nature of the contributing immunologicfactors. In the antecedent rat model of thissame (or very similar) phenomenon,6 it wasconcluded that deviation of the systemic im-mune response achieved by intracamerallyinjected allogeneic cells required that (1) thetime interval between IC injection and testallograft be confined within narrow limits, (2)the eye containing the alloantigenic cell in-oculum must remain anatomically intact for

at least 48 hr, and (3) a normally functioningspleen must be present. Adaptation of themodel to the murine system will permit amore sophisticated analysis of the cellularbasis of immune deviation of this type. Theimmunologic interrelationship between SCand IC inoculation of the P815 tumor also wasrevealed in the capacity of flank tumor cellsto modulate growth of IC tumors. The mostobvious direct effect occurred when SC in-jections preceded IC injections by 5 or moredays: the systemic anti-DBA/2 immunityelicited by SC tumor cell injections pre-vented successful engraftment of P815 cellsin the anterior chamber. More interestingly,however, was the promotion of IC tumorgrowth by SC injections performed near to orsimultaneously with the time of IC injec-tions. The pattern of tumor growth in an-terior chambers under these circumstances,the severe keratoconjunctivitis, and the suc-cessful destruction of the tumor with preser-vation of the eye indicate that the two inde-pendent injections achieved a state of tran-sient balance in the anterior chamber. Bycarefully selecting the sequence and time in-terval between SC and IC injections of P815tumor cells, it was possible to predict the"winning side"; that is, given one protocol,the tumor would be destroyed and the eyesaved from destruction, whereas with an-other protocol the tumor gained ascendancyand proliferated mindlessly until both tumorand eye succumbed. These startling obser-vations suggest that experimental dissectionof the cellular and molecular basis of the im-mune response in this new model mightbring important new insights into the physio-logic process by which the immune responsecan be enlisted in a host's effort to containmalignant neoplasms in the anterior chamberof the eye.

It is interesting that the histoincompatibleDBA/2 tumor grows so successfully in theanterior chamber of BALB/c eyes withoutevoking the inflammatory response seen intumor-injected eyes of animals that also wereinjected with tumor cells subcutaneously.The presumption is that intracamerally in-jected P185 cells make a significant impact


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upon the immune system. However, thenature of the resulting immune responseis qualitatively different from the responseelicited by SC tumors; tumor-bearing eyes inthese latter animals develop severe kerato-conjunctivitis. It is suspected, but t here is nodirect proof, that SC tumors elicit T cell-mediated immunity that in the absence ofantibody destroys SC tumor cells and tumorcells in the anterior chamber with dispatch.It is further possible that severe inflamma-tory reactions which take place in anteriorchambers of tumor-infested eyes of animalsinjected at nearly the same time in the flankwith P815 cells result from a dynamic in situinterplay of destructive T cells and protec tiveantibodies similar to the phenomenon of im-munologic enhancement.14 Alternatively, theinflammatory response seen in these eyesmay represent immunostimulation of thetumor, an idea first proposed by Prehn.15Studies to examine these and other possibili-ties are under way.Much remains to be investigated in thisnewly described ocular tumor model. Immu-nologic and other physiologic aspects of thebiology of intracamerally injected murinetumors are essentially unexplored. It mightbe anticipated that an entire storehouse ofinteresting and provocative new findings iswaiting. Although a weakly histoincompati-ble tumor has been employed in this firstversion of the model, there is good reasonto expect that some of the lessons learnedwill be applicable to tumor-specific trans-plantation antigens. In many ways, minorhistocompatibility antigens resemble tumor-specific antigens immunologically, especiallyin the types of effector immune responsesthey elicit. Moreover, faint glimpses of ther-apeutic implications can be seen in the dataalready collected. Certainly the possibilitycan be raised that inoculation of tumor cellssubcutaneously might be able to direct thehost's immune response such that the growthof similar tumor cells in the anterior chamb erwould be suppressed, the tumor eventuallyeradicated, and the architecture of the eyepreserved.

We gratefully acknowledge the expert technical assis-tance of Ms. Lanya Lonsberry, Rebecca Davila, andElizabeth Mayhew, and we appreciate careful prepara-tion of the manuscript by Ms. Helen Patterson.R E F E R E N C E S

1. Greene HSN: Heterologous transplantation of theBrown-Pearce tumor. Cancer Res 9:728, 1949.2. Medawar PB: Immunity to homologous grafted skin.III. The fate of skin homografts transplanted to thebrain, to subcutaneous tissue and to the anteriorchamber of the eye. Br J Exp Pathol 58:69, 1948.3. Kaplan HJ and Stevens TR: A reconsideration ofimmunological privilege within the anterior cham-ber of the eye. Transplantation 19:302, 1975.4. Kaplan HJ and Streilein JW: Immune response toimmunization via the anterior cham ber of the eye. I.F! lymphocyte-induced immune deviation. J Im-munol 118:809, 1977.5. Kaplan HJ and Streilein JW: Immune response toimmunization via the anterior chamber of the eye.II. An analysis of F, lymphocyte induced immunedeviation. J Immunol 120:689, 1978.6. Streilein JW and Kaplan HJ: Immunologic privilegein the Anterior Chamber. In Immunology and Im-munopathology of the Eye, Silverstein AM andO'Connor GR, editors. New York, 1979, Masson etCie., p. 174.

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