splenectomy promotes indirect elimination of intraocular ...characterized the immune mechanisms...

Research Article

Splenectomy Promotes Indirect Elimination of IntraocularTumors by CD8þ T Cells That Is Associated with IFNg- andFas/FasL-Dependent Activation of IntratumoralMacrophages

Maxine R. Miller1, Jonathan B. Mandell1, Kelly M. Beatty1, Stephen A.K. Harvey1, Michael J. Rizzo1,2,Dana M. Previte1,2, Stephen H. Thorne3,4,5, and Kyle C. McKenna1,3,5

AbstractOcular immune privilege (IP) limits the immune surveillance of intraocular tumors as certain immuno-

genic tumor cell lines (P815, E.G7-OVA) that are rejected when transplanted in the skin grow progressivelywhen placed in the anterior chamber of the eye. As splenectomy (SPLNX) is known to terminate ocular IP, wecharacterized the immune mechanisms responsible for rejection of intraocular tumors in SPLNX mice as afirst step toward identifying how to restore tumoricidal activity within the eye. CD8þ T cells, IFNg , and FasL,but not perforin, or TNFa were required for the elimination of intraocular E.G7-OVA tumors that culminatedin destruction of the eye (ocular phthisis). IFNg and FasL did not target tumor cells directly as the majority ofSPLNX IFNgR1�/� mice and Fas-defective lpr mice failed to eliminate intraocular E.G7-OVA tumors thatexpressed Fas and IFNgR1. Bone marrow chimeras revealed that IFNgR1 and Fas expression on immune cellswas most critical for rejection, and SPLNX increased the frequency of activated macrophages (Mf) withinintraocular tumors in an IFNg- and Fas/FasL-dependent manner, suggesting an immune cell target of IFNgand Fas. As depletion of Mfs limited CD8 T cell–mediated rejection of intraocular tumors in SPLNX mice, ourdata support a model in which IFNg- and Fas/FasL-dependent activation of intratumoral Mfs by CD8þ T cellspromotes severe intraocular inflammation that indirectly eliminates intraocular tumors by inducing phthisis,and suggests that immunosuppressive mechanisms that maintain ocular IP interfere with the interactionbetween CD8þ T cells and Mfs to limit the immunosurveillance of intraocular tumors. Cancer Immunol Res;2(12); 1175–85. �2014 AACR.

IntroductionOcular "immune privilege" (IP) is exemplified by the obser-

vations that certain immunogenic tumor cell lines (P815 andE.G7-OVA), which are rejected by host immune responseswhen transplanted in the skin, grow progressively when placedinto the anterior chamber (a.c.) of the eye (1). IP is not immuneignorance, as several studies have shown that intraoculartumor growth primes systemic immune responses to tumorantigens (2–4). Rather, ocular immune responses are very

tightly regulated to limit inflammation during pathogenremoval so that certain intraocular tissues, which do notregenerate and are essential for vision, are not damaged (5, 6).

Ocular IP is maintained by anatomic and biochemicalbarriers to host immune responses along with the generationof systemic tolerance to antigens encountered within the eye(reviewed in ref. 7). Splenectomy (SPLNX) terminates ocular IPand promotes rejection of immunogenic tumors transplantedin the a.c. of the eye (8). However, the mechanism of tumorelimination has not been defined. Herein, we characterize therequirements for elimination of intraocular tumors in SPLNXmice as a first step toward identifying how to overcome IP andrestore tumoricidal activity within intraocular tumors. Wedemonstrate that CD8þ T cells, macrophages (Mf), IFNg , andFasL, but not perforin, or TNFawere necessary for intraoculartumor elimination. Although tumors expressed IFNgR1 andFas, IFNg and FasL did not directly target tumors. Rather, IFNgand Fas/FasL interactions were required for intratumoral Mfactivation that was associated with severe ocular inflamma-tion, which indirectly eliminated intraocular tumors by induc-ing complete destruction of the eye (ocular phthisis). Our datasuggest that immunosuppressive mechanisms that preserveocular IP interfere with the complex interplay between CD8þ

1Department ofOphthalmology, University of Pittsburgh, Pittsburgh, Penn-sylvania. 2Graduate Program in Immunology, University of Pittsburgh,Pittsburgh, Pennsylvania. 3Department of Immunology, University of Pitts-burgh, Pittsburgh, Pennsylvania. 4Department of Surgery, University ofPittsburgh, Pittsburgh, Pennsylvania. 5University of Pittsburgh CancerInstitute, Pittsburgh, Pennsylvania.

Note: Supplementary data for this article are available at Cancer Immu-nology Research Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Kyle C. McKenna, Department of Biology, Fran-ciscan University of Steubenville, Sts. Cosmas and Damian Hall, 1235University Boulevard, Steubenville, OH 43952. Phone: 740-283-6765; Fax:740-283-6363; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-14-0093-T

�2014 American Association for Cancer Research.

CancerImmunology

Research

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Published OnlineFirst September 23, 2014; DOI: 10.1158/2326-6066.CIR-14-0093-T

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T cells and intratumoral Mfs necessary to eliminate intraoc-ular tumors.

Materials and MethodsTumor cell lines

P815, provided by Dr. Judith A. Kapp (University of Alabama,Birmingham, AL) in 2006, and E.G7-OVA (ATCC CRL-2113),obtained from the ATCC in 2008, were used. E.G7-OVA weretransduced to express firefly luciferase (Luc-E.G7) using Vira-Power (Invitrogen). Luc-E.G7 cultures initiated from frozenstocks were cultured monthly with 1.0 mg/mL G418 sulfate tomaintain the expression of OVA and used in experimentationfor no longer than 3 months, at which time a new Luc.E.G7culture was initiated. Luc-E.G7 cultures were authenticatedevery 3 months, in comparison with ATCC CRL-2113 byevaluating bioluminescence and sensitivity to lysis by OVA-specific CD8þ OT-I CTL. P815 was authenticated by IDEXXRADIL in 2014 by direct comparison with P815 obtained fromthe ATCC (ATCC TIB-64) in 2014 (Supplementary Table SI).

MiceMale and female mouse strains older than 7 weeks from The

Jackson Laboratory, detailed in Supplementary Table SII, wereused. OT-Imice (9)were crossedwith gld or IFNgR1�/�mice togenerate OT-I mice deficient in FasL or IFNgR1. All experi-mentation was approved by the IACUC at the University ofPittsburgh.

SplenectomyMice were anesthetized with a solution of ketamine (100

mg/kg) and xylazine (10mg/kg). The skin above the spleen wasprepared for surgery by depilation and contact sterilization.Sterile scissors were used to open the skin and fascia tovisualize the spleen. The spleen was gently held with sterileforceps, splenic vessels cauterized, and the spleen removed.The incision site was closed with sterile surgical staples(Roboz) that were removed 10 to 14 days after surgery.

Ocular injections and intraocular tumor growthmeasurements

Tumor cell lines (104 cells) were injected into the a.c. of theeye as previously described (4). P815 tumor growth was mon-itored by visual inspection (Supplementary Fig. S1), and scoredas either progressive or phthisical at least 17 days after tumorchallenge. A very significant correlation was observed betweenbioluminescence measurements and Luc-E.G7 numbers invitro (Supplementary Fig. S1C). Hence, the sequential growthof Luc-E.G7 in the a.c. of the eye of live mice was monitored bybioluminescent imaging (BLI) using an IVIS imager (CaliperLife Sciences) following sedation of mice with isoflurane andwithin 15 minutes after intraperitoneal injection of 6 mg D-luciferin salt (Gold Biotechnology; Supplementary Fig. S1D).Background bioluminescence was defined at 104 photons/second (Supplementary Fig. S1E). Rejection of Luc-E.G7tumors was scored in individual mice as a two-log decreasein tumor bioluminescence that wasmaintained for at least twosuccessive measurements.

In vivo depletion of immune cell subsets and Fas/FasLneutralization

To selectively removeCD4þ orCD8þT cells,micewere givenintraperitoneal injections of anti-CD4 (clone GK1.4) or anti-CD8 (clone 2.43) antibodies fromBioXCell. Antibody treatment(0.2–0.4 mg) began 3 days before or 7 days after ocular tumorinjections and continued every 3 to 4 days thereafter (0.1 mginjections). Depletion was greater than 96%, as determined bythe flow cytometric analysis of peripheral blood (data notshown). Macrophages were depleted by subconjunctival(scon.) injections (10 mL) or, scon. and intravenous (i.v.)injections (100–200 mL) as indicated. Neutralization of Fas/FasL interactions was accomplished by 0.1 mg intraperitoneal(i.p.) injections of Ultra-LEAF anti-mouse CD178(FasL) anti-bodies (BioLegend) that were given before tumor challengeand every 3 to 4 days thereafter. Equivalent injections ofHamster IgG (BioXCell) were given to control for antibodyinjection.

Flow cytometric analysisFifteen to 16 days after tumor challenge, single-cell suspen-

sions ofwhole tumor–bearing eyeswere prepared as previouslydescribed (10); Fc receptors were blocked and then stainedwith combinations of fluorescently conjugated antibodiesfrom BD Pharmingen to the following cell surface molecules:CD45, CD11b, Thy 1.2, GR-1, and/or F4/80 in FACS buffer (PBSþ 2% fetal bovine serum). Cells were then washed and fixed inCytofix/Cytoperm (BD Pharmingen) and in some experimentsincubated with PE-conjugated anti-CD68, or polyclonal rabbitanti-mouse NOS2 antibody (BD Pharmingen) in Perm/Washbuffer (BD Pharmingen). Cells treated with polyclonal anti-mouse NOS2were then stainedwith AlexaFluor 546 anti-rabbitIgG (R&D Systems). Events were collected using a FACSDivaFlow Cytometer (Becton Dickinson) and analyzed using FACS-Diva (Becton Dickinson) and FlowJo (TreeStar) software.

Generation of bone marrow chimeric miceMice were irradiated (10 Gy) in a Cs source irradiator

(Nordion) and then injected i.v. with bone marrow (4.5 �106 cells) isolated from femurs, and tibias of B6, lpr, orIFNgR1�/� mice. Experimentation was performed 8 weekslater after reconstitution of the immune system.

Gene array analysis and RT-PCRFifteen to 16 days after ocular tumor challenge, eyes were

removed, homogenized in RLT buffer (from the RNeasy Kit;Qiagen) in a Tenbroeck frosted glass tissue grinder, then storedat �80�C until isolation of total RNA using Qiashredder andRNeasy kits (Qiagen). cDNA was synthesized using a HighCapacity cDNAReverse Transcription Kit and quantitative RT-PCR was performed using a StepOne Plus instrument withcommercially available TaqMan primer probe sets (AppliedBiosystems). Pyruvate decarboxylase (Pcx) was used as thenormalizing (housekeeping) gene. Extracted RNA (~500 ng)was also processed using a 30 IVT Express Kit to yield amplifiedRNA (~20 mg), which was hybridized to M430 2.0 microarrays;the microarrays were scanned using a GeneChip 3000 Arrayscanner (Affymetrix Inc.). Raw data were processed using

Miller et al.

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Affymetrix GCOS v.1.4 software with default settings, thenexported to Microsoft Excel. The ratio (mean SPLNX:meancontrol) was calculated for each microarray panel, with can-didate panels having ratios �1.5 or �0.67. Valid candidatesshowed detectable (i.e., called Present by GCOS) transcript inboth samples of the higher-expressing group, and no overlap ofvalueswas found between groups. The dataset is available fromthe NCBI GEO database (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE57101).

Adoptive transfer of OT-I T cellsIn some experiments, na€�ve CD8þ T cells were isolated from

spleens of OT-I, OT-I X gld, or OT-I X IFNgR1�/� mice usingmouseCD8T-cell enrichment kits (Stemcell Technologies) andthen injected i.v. via tail-vein injection (3.0 � 106 cells). Togenerate OT-I T-cell effectors, splenocytes from OT-I micewere stimulated in vitro for 3 days with 0.1 mg/mL SIINFEKLpeptide, and dead cells were removed by centrifugation overFico/Lite LM (mouse; Atlanta Biologicals). OT-I T-cell effectors(1.0–3.0 � 106 cells) were injected i.v.

Statistical analysisPrism (GraphPad) software was used for statistical analysis.

ResultsSPLNX promotes CD8 T cell–mediated rejection ofintraocular tumorsPreliminary studies showed that P815 tumors that normally

grewprogressively when transplanted in the a.c. of the eyewere

rejected in SPLNXmice by a process that culminated in ocularphthisis (8). To characterize the requirements for eliminationof intraocular tumors, wemonitored intraocular tumor growthafter injection of 104 P815 tumor cells (H-2d) into the a.c. ofSPLNX Balb/C J mice or Balb/C J mice that were not surgicallymanipulated (hereafter referred to as controls). Themajority ofSPLNX mice rejected P815 tumors with phthisis beginning 10to 14 days after tumor challenge, whereas progressive intra-ocular tumor growth was observed in controls (Table 1),confirming the previous observations (8). This delayedimmune response suggested the contribution of adaptiveimmunity. Therefore, intraocular tumor growth was moni-tored in SPLNX mice that received depleting anti-CD4 oranti-CD8 antibodies. Removal of CD4þ T cells before (Table 1,Exp. 2) or at 7 days after tumor challenge (Table 1, Exp. 4) didnot influence the rejection of intraocular tumors in SPLNXmice. In contrast, SPLNX mice depleted of CD8þ T cells(Table 1, Exp. 3 and Exp. 4) did not reject intraocular tumors.

Intraocular growth of transplanted bioluminescentLuc-E.G7 tumors (H-2b) that expressedOVAas a defined tumorantigen (11) was also monitored by BLI in SPLNX and controlC57Bl/6 J (B6) mice that received a mock surgery (Fig. 1A–C).During the first 14 days after tumor challenge, equivalenttumor growth was observed (Fig. 1A and B). However, by day21, the majority of SPLNX mice eliminated these establishedintraocular tumors (Fig. 1B and C) resulting in ocular phthisis.The rejection of intraocular Luc-E.G7 tumors required CD8þ Tcells as progressive intraocular tumor growth was observed inSPLNX mice deficient in CD8þ T cells (CD8�/� mice) and inSPLNX B6 mice given depleting anti-CD8 antibodies (Fig. 1D)

Table 1. Splenectomy promotes CD8 T cell–mediated rejection of intraocular P815 mastocytomas inBalb/C mice

Experiment Surgery Antibody treatmentNumber of mice with tumor/total,number of mice (percentage with tumor)

1 None None 5/8 (63%)Splenectomy None 0/7 (0%) P ¼ 0.0256

2 Splenectomy Rat IgG 0/4 (0%)Splenectomy Anti-CD4 1/4 (25%) ns

3 None None 6/7 (86%)Splenectomy Rat IgG 0/8 (0%) P ¼ 0.0014Splenectomy Anti-CD8 8/8 (100%) ns

4 None Rat IgG 6/6 (100%)Splenectomy Rat IgG 0/6 (0%) P ¼ 0.0022None Anti-CD4 5/5 (100%)Splenectomy Anti-CD4 0/5 (0%) P ¼ 0.0079None Anti-CD8 5/5 (100%)Splenectomy Anti-CD8 4/5 (80%) ns

NOTE:Balb/C Jmicewere used in these experiments and104 P815 tumor cellswere injected in the a.c. of the eye. In experiments 2 and3, antibody treatments began 1 day before tumor challenge and continued every 3 to 4 days thereafter. In experiment 4, antibodytreatments began 7 days after tumor challenge. Splenectomized mice were compared with control mice by the Fisher exact test.P values are shown.Abbreviation: ns, not statistically significant.

Mechanisms of Intraocular Tumor Elimination

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but not in SPLNX B6 mice given Rat IgG as a control forantibody administration. Combinedwith our equivalent obser-vations in theP815/Balb/C J intraocular tumormodel (Table 1),these data identified a common requirement for CD8þ T cellsin phthisical rejection of intraocular tumors in SPLNX mice.

Rejection of intraocular tumors requires FasLand IFNg

CD8þ T cells can directly eliminate tumors by perforin-dependent cytolytic activity, FasL-induced apoptosis, or viathe production of tumoricidal cytokines, including TNFa andIFNg (12). Therefore, to determine the contribution of thesemolecules in the control of intraocular tumors, Luc-E.G7intraocular tumor growth was monitored in SPLNX andcontrol mice with genetic deficiencies in perforin (pfp�/�),FasL (gld), TNFa (TNFa�/�), or IFNg (IFNg�/�). Themajorityof controls in these immunodeficient strains showed pro-gressive intraocular tumor growth (Fig. 2). SPLNX pfp�/�

mice rejected established intraocular Luc-E.G7, which indi-cated that lytic activity of CD8þ T cells was not required toeliminate these intraocular tumors. Similarly, P815 tumorsthat grew progressively in pfp�/� control mice on a Balb/CByJ background were rejected when pfp�/�mice were SPLNXbefore tumor challenge (Supplementary Table SIII). Hence,perforin was dispensable for rejection of intraocular tumors.TNFa was also not required for rejection of Luc-E.G7 tumorsin SPLNX mice (Fig. 2C). However, FasL and IFNg werenecessary as intraocular Luc-E.G7 tumors grew progressivelyin SPLNX gld and IFNg�/� mice (Fig. 2B and D). A greater

frequency of SPLNX Balb/C ByJ mice given anti-FasL anti-bodies showed progressive intraocular P815 tumor growth incomparison with SPLNX mice given control antibodies (Sup-plementary Table SIII, Exp. 3), which indicated that Fas/FasLinteractions also contributed to elimination of P815 tumorsin SPLNX Balb/C J mice.

IFNgR1 and Fas expression on hematopoietic cells iscritical for intraocular tumor rejection

Flow cytometric analysis indicated that Luc-E.G7 tumorsexpressed both IFNgR1 (Fig. 3A) and Fas (Fig. 3D), suggestingthat IFNg and FasL expressed by CD8þ T cells could targettumor cells directly. However, 3-day culture of Luc-E.G7 orP815 tumors with IFNg at concentrations ranging from 12.5 to5,000 U/mL did not affect tumor viability and had only amodest effect on growth (Supplementary Fig. S2). In addition,EL-4 cells, the parental cell line of Luc-E.G7 are resistant toFasL-induced apoptosis due to overexpression of cellularFLICE inhibitory protein (c-FLIP; ref. 13). Hence, we testedthe hypothesis that IFNg and FasL acted on nontumor (stro-mal) cells within the tumor microenvironment to eliminateintraocular tumors bymonitoring intraocular Luc-E.G7 growthin SPLNX and control IFNgR1�/� and Fas-defective lpr mice.Luc-E.G7 tumors grew progressively in themajority of controlsof both the strains. Despite tumor expression of IFNgR1 andFas, the majority of SPLNX IFNgR1�/� (Fig. 3B) and SPLNX lpr(Fig. 3E) failed to eliminate intraocular Luc-E.G7 tumors.Similarly, progressive intraocular P815 tumor growth wasobserved in SPLNX and control IFNgR1�/� on a Balb/C ByJ

Figure 1. Splenectomy promotesCD8 T cell–mediated rejection ofintraocular Luc-E.G7 tumors.Intraocular Luc-E.G7 (H-2b)tumor growth monitored by BLIin C57Bl/6 (B6) mice that weresubjected to a mock surgicalprocedure (A) or weresplenectomized (SPLNX, B). Eachline represents measurementsfrom an individual mouse withineach treatment group that wasused to generate survival curvesshown in C. The P value indicatescomparison of groups by a log-rank test. Percentage of SPLNXCD8�/� H-2b mice, SPLNX B6, orSPLNX B6 mice that were givendepleting anti-CD8 antibodies orrat IgG with intraocular Luc-E.G7tumors (D). A to C, singleexperiment reproduced insubsequent figures. Data in D arecombined from five independentexperiments. P values in D indicatecomparison with the SPLNX ratIgG–treated group by a log-ranktest. n, number of mice per group.

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background (Supplementary Table SIII, Exp. 2). These dataindicated that IFNg and FasL indirectly eliminated intraoculartumors by targeting stromal cells within the tumormicroenvironment.

Tumor stroma comprised hematopoietic and nonhemato-poietic cells (14). To determine the critical cell populations thatexpressed IFNgR1 and FasL, bone marrow chimeras weregenerated with selective deficiencies in IFNgR1 or Fas inhematopoietic cells, nonhematopoietic cells, or both cell popu-lations. IFNgR1 expression on hematopoietic cells was criticalfor intraocular tumor rejection in SPLNX mice as a lesserfrequency of B6 recipients of IFNgR1�/� bonemarrow rejectedLuc-E.G7 intraocular tumors in comparison with B6 recipientsof B6 bone marrow (Fig. 3C). Indeed, the percentage of B6recipients of IFNgR1�/� bone marrow was statistically com-parable with IFNgR1�/� recipients of IFNgR1�/� bone mar-row. SPLNX IFNgR1�/� recipients of B6 bone marrow rejectedintraocular Luc-E.G7 tumors confirming the necessity forIFNgR1 on hematopoietic cells. Fas expression on hematopoi-etic cells was also required for intraocular tumor elimination asall SPLNX B6 recipients of lpr bone marrow demonstratedprogressive intraocular Luc-E.G7 tumor growth (Fig. 3F),whereas SPLNX lpr recipients of B6 bone marrow rejectedintraocular Luc-E.G7 tumors (Fig. 3F).

IFNgR1 and FasL on CD8þ T cells are not required forintraocular tumor elimination

One explanation for impaired intraocular tumor rejection inSPLNX IFNgR1�/�, gld, and lpr mice is that priming of tumor-specific CD8þ T cells is impaired in these strains. Therefore, toaddress the functionality of tumor-specific CD8þ T cells in

Figure 2. FasL and IFNg are required for intraocular tumor regression.Percentage of mice with intraocular Luc-E.G7 tumors in SPLNX andcontrol pfp�/� (A), FasL-defective gld (B), TNFa�/� (C), and IFNg�/� (D) H-2b mice. n, number of mice per group. Data shown are pooled from threeindependent experiments in A, four experiments in B, and twoexperiments in D. C represents one experiment. P values indicatecomparison of groups within individual panels by a log-rank test.

Figure 3. IFNgR1 and Fas expression on hematopoietic cells is required for intraocular tumor regression. Cell surface expression of IFNgR1 (A) or Fas (D)on cultured Luc-E.G7 tumor cells. Black histograms, antibody-specific staining; gray histograms, background fluorescence after staining with isotypecontrol antibodies. Percentage of mice with intraocular Luc-E.G7 tumors in SPLNX and control IFNgR1�/� (B) or Fas-defective lpr (E) H-2b mice, andin indicated bone marrow chimeric H-2b mice (C and F). n, number of mice per group. Two or more independent experiments were combined in datapresented in B to F. P values in B and E are comparisons between treatment groups by log-rank tests. P values in C and F are comparisons with SPLNX B6recipients of B6 bone marrow by log-rank tests.






tumor-bearing mice, na€�ve OVA-specific CD8þ OT-I T cellswere transferred into CD8-deficient mice that were SPLNXor were not surgically manipulated before challenge withLuc-E.G7 tumors in the a.c. Intraocular Luc-E.G7 tumor burdenwas significantly lower in OT-I–transferred SPLNX CD8�/�

mice than in control CD8�/� mice that were not surgicallymanipulated although they received an equivalent number ofOT-I T cells (Fig. 4), which indicated that intraocular tumorgrowth in SPLNX but not control mice induced tumoricidalactivity in transferred OT-I T cells. To determine whetherIFNgR1 or FasL deficiency in CD8þ T cells abrogated tumor-icidal activity, OT-I mice were bred onto an IFNgR1�/� or gld(H-2b) background. SPLNX CD8-deficient mice that receivedna€�ve OT-I X IFNgR1�/� or OT-I X gld CD8þ T cells before Luc-E.G7 challenge also controlled intraocular tumor growth,indicating that deficiency in IFNgR1 or FasL on CD8þ T cellsdid not impair CD8 tumoricidal activity.

Intraocular tumor regression in SPLNX mice isassociated with IFNg- and Fas/FasL-dependent Mfactivation and markers of ocular inflammation

Previously, we and others demonstrated that rejection ofestablished E.G7 skin tumors required IFNg expression bytumor-specific CD8þ T cells to activate tumoricidal nitricoxide (NO) production by intratumoral Mfs (10, 15, 16). NOis synthesized in activated Mfs by the enzyme NO synthase 2(NOS2; ref. 17). Therefore, to determine whether a similarmechanism was involved in rejection of intraocular tumorsin SPLNX mice, we compared the frequency of NOS2þ Mfs byflow cytometry in day 16 Luc-E.G7 intraocular tumors ofSPLNX and control B6, gld, lpr, and IFNgR1�/� mice. Thistime point, immediately before Luc-E.G7 intraocular tumorregression in SPLNX B6 mice, was chosen as tumor burdenmeasured by BLI was statistically comparable in both thetreatment groups. Some intraocular tumors in SPLNX micedid, however, show lower tumor burden, indicating the begin-ning of tumor regression.

In all strains of mice tested and regardless of surgicaltreatment, intraocular Luc-E.G7 tumors were infiltrated byCD45þ CD11bþ myeloid cells composed of GR-1þ and GR-1�

cells (Supplementary Fig. S3). GR-1� cells also expressed F4/80

and CD68, indicating that they were Mfs (Supplementary Fig.S3). The number of GR-1þ cells was statistically equivalentbetween SPLNX and control mice of each strain although atrend of lower or higher GR-1þ cell numbers was observed inSPLNX B6 and SPLNX lpr mice, respectively (Fig. 5A). Thepercentage of NOS2þ GR-1þ cells in intraocular tumors wasvery low<5% (Supplementary Fig. S3B) and their numberswereequivalent between SPLNX and control mice in all the strainstested (Fig. 5B).Mfnumberswere comparable between SPLNXand control B6 or gld mice, whereas the number of Mfsincreased in SPLNX lpr and SPLNX IFNgR1�/� in comparisonwith their controls (Fig. 5C). A statistically significant 2.4-foldincrease in the number of NOS2þMfs was observed in SPLNXB6 mice in comparison with control B6 mice, whereas nodifferences in NOS2þ Mfs were observed between SPLNX andcontrol gld, lpr, or IFNgR1�/� mice (Fig. 5D). These dataindicated that both IFNg and Fas/FasL interactions wererequired for Mf activation and that increased Mf expressionof NOS2 correlated with intraocular tumor regression.

To determine whether NO production was required forelimination of intraocular tumors in SPLNX mice, Luc-E.G7tumor growth was monitored in SPLNX and control NOS2–deficient B6 mice. Luc-E.G7 grew progressively in all controlNOS2�/� mice, whereas the majority of SPLNX NOS2�/� micerejected intraocular Luc-E.G7 tumors (Fig. 5F). P815 tumorswere also rejected in SPLNX NOS2�/� mice on a Balb/Cbackground (Supplementary Table SIII, Exp. 4), which indicat-ed that NO production was dispensable for intraocular tumorelimination in SPLNX mice. Nevertheless, NOS2 expressionremained a very sensitive marker of Mf activation.

To evaluate additional genes associated with intraoculartumor rejection, oligonucleotide microarrays were used toidentify gene expression differences between whole tumor–bearing eyes (d15) from control or SPLNX mice (2 mice pergroup). Relative to controls, SPLNX mice showed increased(>1.5-fold) expression of 1,488 genes and decreased expressionof 523. Canonical pathways analysis (IPA) parsed 397 of thesegenes into 88 significantly enriched pathways (SupplementaryTable SIV), of which the most significant were inflammationpathways. As IFNg mRNA increased in intraocular Luc-E.G7tumors of SPLNX mice, a group of approximately 600 IFN-stimulated geneswasmanually assembled from theNCBIGenedatabase, the Interferome.org database, and Schoggins andcolleagues (18); themicroarrays surveyed 408 of these genes. Inwhole tumor–bearing eyes from SPLNX mice, 127 IFNg-stim-ulated genes (31.1%) showed increased expression, whereasonly nine (2.2%) showed decreased expression.

Only four of the 88 canonical pathways identified (Cell CycleControl of Chromosomal Replication, Estrogen-mediated S-phase entry, Role of CHK Proteins in Cell Cycle CheckpointControl, and Role of BRCA1 in DNA Damage Response) con-tained more genes showing decreased than increased expres-sion. A net decrease in cell-cycle activity and cell proliferationin the tumor-bearing eyes of SPLNX mice was confirmed byexamining 192 cell-cycle (19) genes present on themicroarrays:40 (20.8%) were downregulated, whereas only four (<1%) wereupregulated. Of note was Ki67, a marker of cell proliferation,with a valid decrease to 71% of control tumor–bearing eyes,

Figure 4. CD8 T-cell expression of IFNgR1 or FasL is dispensable fortumoricidal activity. Intraocular Luc-E.G7 tumor burden in SPLNX orcontrol CD8�/�H-2bmice that received naïveOT-I, OT-I X IFNgR1, orOT-I X gld CD8þ T cells before intraocular tumor challenge. This experimentwas performed twice with similar results.

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which was consistent with the lower tumor burden seen insome SPLNX mice at this time.The expression of inflammatory genes in tumor-bearing eyes

of SPLNX and control mice (day 16) was confirmed by RT-PCR(Supplementary Fig. S4). FasL, CXCL2, NOS2, and Tbet expres-sion was significantly greater in tumor-bearing eyes of SPLNXmice. IFNg expression was also greater in tumors of SPLNXmice, but the increase was not statistically significant. Fasexpression was comparable between SPLNX and controltumors. Combined, these data indicated that rejection ofintraocular tumors in SPLNX mice was associated withincreased expression of genes associated with ocularinflammation.

Mfs are required for elimination of intraocularLuc-E.G7 tumors in SPLNX miceTo determine the contribution of Mfs in the elimination of

Luc-E.G7 intraocular tumors in SPLNX B6 mice, clodronateliposomes were used to deplete Mfs in vivo according toestablished protocols (20). In comparison with SPLNX micethat received PBS liposomes, a significantly greater percentageof SPLNXmice given clodronate liposomes showedprogressiveintraocular tumor growth (Fig. 6A).To further evaluate the role of Mfs in the elimination of

intraocular tumors, we developed a system in which in vitro

primed tumor-specific OT-I CD8þ T-cell effectors controlledgrowth of established intraocular Luc-E.G7 tumors in SPLNXCD8�/�mice. As shown in Fig. 6B andC, Luc-E.G7 tumors grewprogressively in control and SPLNX CD8�/� mice reproducingour previous observations. Control (Fig. 6B) and SPLNX (Fig.6C) CD8�/�mice that were given OT-I CD8 T effectors 10 daysafter tumor challenge in the a.c. of the eye both showedreduced tumor burden in comparison with that in nontrans-ferred mice. However, the magnitude of tumor regression (foldchange in tumor burden between nontransferred and trans-ferred mice) was significantly greater in SPLNX mice than incontrols 5 days after transfer (day 15). Hence, despite anequivalent T-cell transfer, the tumoricidal activity of trans-ferred CD8þ T effectors was much greater in SPLNX mice.

To determine whether the interaction between CD8þ Teffectors and Mfs contributed to the observed increasedtumoricidal activity in OT-I effector–transferred SPLNXCD8�/� mice, SPLNX CD8�/� mice were given PBS or clo-dronate liposome treatments before CD8þ T-effector celltransfer given 10 days after intraocular Luc-E.G7 tumor chal-lenge. It is important to note that day 10 tumor burden waslower in mice given clodronate liposomes, and this was aconsistent observation inmultiple experiments. CD8 T effectortransfer at day 10 caused a significant reduction in tumorburden in SPLNX CD8�/� mice treated with PBS liposomes

Figure 5. Intraocular Luc-E.G7 tumor rejection inSPLNXH-2bmice is associatedwith IFNg- and-Fas/FasL-dependent activation of intratumoralmacrophages.Number of GR-1þ (A), GR-1þ NOS2þ (B), GR-1� Mfs (C), and Mfs NOS2þ (D) within whole intraocular tumor-bearing eyes of indicated (H-2b) mouse strains16 days following challenge with Luc-E.G7 in the a.c. of the eye. Data from B6, gld, and IFNgR1�/� mice were pooled from two independent experiments;lpr data are from a single experiment. Each symbol represents measurements from an individual intraocular tumor-bearing eye; bars, median. SPLNXmice were compared with mice that were not surgically manipulated (none) within mouse strains by the two-tailed Student t tests or Mann–Whitney testsdepending on the normality of data determined by a D'Agostino–Pearson omnibus normality test. �, P < 0.05; ��, P < 0.01; ns, not statistically significant P >0.05. E, percentage of NOS2�/�H-2bmice that were SPLNX or not surgically manipulated with intraocular Luc-E.G7 tumors.P value indicates comparison oftreatment groups by a log-rank test.






(Fig. 6E). In contrast, the same effector T-cell transfer did notcause tumor regression in SPLNX CD8�/� mice treated withclodronate liposomes (Fig. 6F) despite lower tumor burden at

the time of transfer. These data confirmed the critical role ofMfs at the effector phase of CD8 T cell–mediated eliminationof intraocular tumors in SPLNX mice.

Figure 6. Mfs are required for elimination of ocular Luc-E.G7 tumors in SPLNX mice. A, percentage of SPLNX B6 mice, treated by scon. injections withclodronate or PBS liposomes with intraocular Luc-E.G7 tumors. Liposome injections were initiated 3 days before tumor challenge and continued every3 to 4 days thereafter. P value indicates comparison of treatment groups by a log-rank test. Intraocular Luc-E.G7 tumor burden measured by BLI incontrol (B) or SPLNX (C) CD8�/� H-2b mice that received on day 10 (arrow) after tumor challenge in vitro primed CD8 T effector OT-I cells or were leftuntreated.D, summaryof fold change in intraocular Luc-E.G7 tumor burdenbetween nontransferredandCD8Teffector–transferred control or SPLNXCD8�/�

mice. Fold change calculated 15 days after tumor challenge for each transferred mouse as [(mean tumor burden of nontransferred mice within group)/(tumorburden of individual transferred mice)]. Each symbol represents measurements from an individual CD8 T effector–transferred mouse. P value indicatescomparison of treatment groups by a one-tailedMann–Whitney test. Intraocular Luc-E.G7 tumor burden in SPLNXCD8�/�H-2bmice, treated with PBS (E) orclodronate (F) liposomes, that received on day 10 after tumor challenge (arrow) in vitro primed CD8 T effector OT-I cells. G, summary of fold change inintraocular Luc-E.G7 tumor burden between nontransferred and CD8 T effector–transferred SPLNX CD8�/� mice treated with PBS or clodronateliposomes as calculated in D. Data were pooled from two independent experiments. P value indicates comparison of treatment groups by a one-tailedMann–Whitney test. In addition to scon. liposome treatments administered as in A, mice were also given i.v. liposome treatments on days 3 and 7 in experimentationas shown in E and F.

Miller et al.





DiscussionNiederkorn and colleagues (21) reported more than 30 years

ago that immune responses directed against tumors trans-planted in the a.c. of the eye were different from those inducedby transplantation of the same tumors in the skin. For example,P815 tumors that were rejected when placed in the skin ofBalb/C mice induced robust DTH and CTL responses directedagainst minor alloantigens expressed by tumors (2). In con-trast, P815 tumors transplanted in the a.c. of the eye grewprogressively, did not induce DTH responses, but interestingly,demonstrated tumor-specific CTL responses equivalent tothose observed in mice that rejected P815 in the skin (2). Theterm "anterior chamber–associated immune deviation"(ACAID) was used to describe this unique immune response.SPLNX terminated ACAID as DTH responses were restored

in mice injected with P815 in the a.c. and these intraoculartumors were eliminated (8). The logical interpretation of thesedata, that CD4 T cell–dependent DTH responses mediatedintraocular tumor rejection in SPLNX mice, was further sup-ported by the destructive nature of tumor elimination, whichcaused fulminant inflammation that culminated in ocularphthisis (8) In contrast, CD8 T cell–mediated rejection of otherintraocular tumors left the eye intact (22). However, we defin-itively demonstrate that CD4þ T-cell responses were notrequired for rejection of P815 tumors in SPLNX Balb/C J mice.Rather, CD8þ T cells were necessary. These data are consistentwith another study describing a P815 variant, P91, which wasspontaneously rejected in the a.c. of non–SPLNX DBA/2 mice(23, 24). In these studies, the administration of depleting anti-CD4 antibodies abrogated DTH responses to P91 antigensmeasured in the skin but failed to prevent phthisical rejectionof intraocular tumors that demonstrated histopathologic fea-tures of a DTH response in the eye (23, 24). In contrast,progressive intraocular P91 tumor growth was observed inmice given depleting anti-CD8 antibodies although DTHresponses in the skin were manifested (23, 24). Hence, CD8þ

T cells are also capable of inducing destructive inflammatoryresponses resembling DTH responses within the eye thateliminate intraocular tumors by inducing ocular phthisis.In our study, CD8þ T cells, Mfs, and IFNg were critical for

phthisical elimination of intraocular Luc-E.G7 tumors. Thesedata suggest that SPLNX restored the DTH response (25) withinintraocular tumors and support a model in which CD8þ T cellsexpressed IFNg to activate Mfs, which induced intraocularinflammation. In further support of that interpretation, genearray analysis indicated increased expression of IFNg , IFNg-inducible genes, and several other inflammatory genes in intra-ocular Luc-E.G7 tumor-bearing eyes of SPLNX mice.IFNg andMfswere also necessary for elimination of another

immunogenic tumor cell line, Ad5E1, transplanted in the a.c.(20, 26–29) However, there are several differences betweenelimination of intraocular Ad5E1 and intraocular P815 or Luc-E.G7 that bear noting. Rejection of intraocular Ad5E1 tumorswas spontaneous and did not require SPLNX. In addition,Ad5E1 tumor rejection did not induce ocular phthisis, andrequiredCD4þ but not CD8þT cells (26–28). The cellular targetof IFNg was also different between the experimental models.IFNg targeted Ad5E1 tumors directly as intraocular Ad5E1

tumors were rejected in IFNgR1�/� mice (28) and in vitroaddition of IFNg induced Ad5E1 apoptosis via increasedexpression of TRAIL (27). As Ad5E1 is MHC class II negative,these data suggest that cross-presentation of Ad5E1 tumorantigens to CD4þ T cells by intratumoral Mfs promoted T-cellexpression of IFNg that induced tumor cell apoptosis viaTRAIL interactions. IFNg also appeared to limit tumor vascu-larization by inducing Ad5E1 expression of antiangiogenicchemokines (28). In contrast, rejection of P815 and Luc-E.G7in SPLNX mice required that IFNg-targeted immune cells,including Mfs, within the tumor.

The extent of Mf activation may determine whether intra-ocular tumors are eliminated in a sight-preserving or blinding(phthisical) fashion. For example, Coursey and colleaguesrecently generated Ad5E1 variants that were rejected in anonphthisical (clone 4) or phthisical (clone 2.1) manner by aprocess that required Mfs and IFNg (30). Phthisical rejectionwas dictated by tumor sensitivity to TNFa, as clone 2.1 tumorswere rejected without phthisis in TNFa-deficient mice, where-as TNF receptor 1–deficientmice rejected clone 2.1 tumors in aphthisical manner. These data suggested that a second signalreleased from necrotic tumor cells in combination with IFNginduced stronger Mf activation, thereby causing greater intra-ocular inflammation that eliminated intraocular tumors bydestroying the eye.

Phthisical rejection of intraocular Luc-E.G7 or P815 tumorsin SPLNX mice was also associated with Mf activation thatrequired two signals (IFNg and Fas/FasL interactions) pre-sumably delivered by CD8þ T cells that were also indispens-able. Interestingly, FasL expression by CD8þ T cells was notnecessary for regression of Luc-E.G7 intraocular tumors. How-ever, Mfs (31, 32) and CD8þ T cells express both Fas and FasL.Therefore, Fas expression by CD8þ T cells could engage FasLon tumor-associated Mfs to induce their activation. AlthoughFas/FasL interactions have traditionally been considered proa-poptotic, accumulating evidence indicates that Fas/FasL inter-actions induceMf activation (31–34) without apoptosis induc-tion, which supports this model.

We do not fully understand how splenectomy restoredtumoricidal activity of CD8þ T cells and Mfs within the eye.One potential explanation is that SPLNX influences tumor-specific CD8þ T-cell numbers within intraocular tumors. Con-sistentwith that notion, Boonmanandcolleaguesdemonstratedthat another tumor cell line (Ad5E1 plus EJ-ras) transplanted inthe a.c. of the eye formedprogressively growing tumors in 40%ofB6mice, whereas the remainingmice spontaneously eliminatedtumors by a CD8 T cell–dependent process that culminated inphthisis (35, 36). Although tumor growth in both progressor andregressor mice induced expansion of tumor-specific CD8þ Tcells in draining lymph nodes, only regressor mice showedsystemic dissemination of these effector CD8þ T cells. Hence,these data could suggest that SPLNX restored tumor-specificCD8 T-cell migration into intraocular Luc-E.G7 tumors.

It is also well appreciated that intraocular tumor growthinduces the generation of tumor antigen–specific CD8þ Tregsthat mediate suppression of DTH responses to tumor antigens(7). SPLNX prevents the generation of these CD8þ Tregs,suggesting that the splenic microenvironment is critical for






their generation (37). Therefore, it is possible that a splenicCD8þTreg infiltrates intraocular tumors to limitMf activationby CD8þ T-cell effectors. On the basis of the data presentedherein, a mechanism of CD8 Treg immunosuppression couldinvolve inhibiting IFNg production by CD8 T-cell effectors orinterfering with Fas/FasL interactions between CD8þ T cellsand Mfs within the tumor microenvironment. We favor thelater mechanism, as the reduction in IFNg mRNA in controlversus SPLNX mice was modest, and we previously demon-strated that primed CD8þ CTLs that infiltrated establishedintraocular tumors were not impaired in IFNg production (10).Gregory and colleagues (38, 39) demonstrated that tumorsengineered to express a membrane-only form of FasL werespontaneously rejected when placed in the a.c. of the eye,whereas tumors that expressed both membrane and solubleFasL formed progressively growing intraocular tumors thatmetastasized. Hence, a CD8 Treg that expressed soluble FasLcould limit intraocular inflammation by binding CD8 T-cell–expressed Fas, thereby preventing Mf activation.

The role of Mfs in tumor growth is complex as we demon-strate that intraocular Luc-E.G7 tumors were smaller in B6mice depleted of Mfs by clodronate liposome injections (Fig.6). These data suggest that Mfs contributed to intraoculartumor growth, which is consistent with the observations of Lyand colleagues (40). Therefore, tumor regression, which alsorequired Mfs, may be due to the conversion of intratumoralMfs from a protumor to an antitumor phenotype. However, itis important to note that direct tumoricidal activity by Mfsmay not be required to eliminate intraocular tumors as phthi-sis can result fromdestruction of the ciliary body of the eye (36).Therefore, SPLNX may remove an immunosuppressive mech-anism that normally protects the ciliary body from destructiveinflammatory mediators expressed by Mfs.

In conclusion, SPLNX promotes indirect elimination ofintraocular tumors by CD8þ T cells that is associated with

IFNg- and Fas/FasL-dependent activation of intratumoralMfs. Therefore, mechanisms that maintain ocular IP mayinterfere with the interaction between CD8þ T cells and Mfsto limit the immunosurveillance of intraocular tumors.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K.C. McKennaDevelopment of methodology: K.M. Beatty, S.H. Thorne, K.C. McKennaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.R. Miller, J.B. Mandell, S.A.K. Harvey, M.J. Rizzo,D.M. Previte, K.C. McKennaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):M.R. Miller, J.B. Mandell, K.M. Beatty, S.A.K. Harvey,D.M. Previte, S.H. Thorne, K.C. McKennaWriting, review, and/or revision of the manuscript: M.R. Miller, J.B.Mandell, S.A.K. Harvey, K.C. McKennaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases):M.R. Miller, D.M. Previte, S.H. Thorne,K.C. McKennaStudy supervision: K.C. McKenna

AcknowledgmentsThe authors thank Nancy Zurowski for excellent technical assistance in flow

cytometry; Robert Hendricks, Walter Storkus, and Pawel Kalinski for criticalreview of this article; Richard Bilonick for statistical analysis; Joseph Brown andJessamee Mellon (University of Texas Southwestern Medical School) for prep-aration of liposomes; and Rachel Sikorski for transduction of E.G7-OVA toexpress firefly luciferase. The authors regret to announce Rachel's untimelydeath and dedicate this article to her memory.

Grant SupportThis work was supported by NIH grants R01 EY018355 (to K.C. McKenna,

Principal Investigator), P30-EY08098, and P30-CA047904, The Eye and EarFoundation of Pittsburgh, and by an unrestricted grant fromResearch to PreventBlindness, Inc.

Received May 13, 2014; revised September 5, 2014; accepted September 14,2014; published OnlineFirst September 23, 2014.

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2014;2:1175-1185. Published OnlineFirst September 23, 2014.Cancer Immunol Res Maxine R. Miller, Jonathan B. Mandell, Kelly M. Beatty, et al. Fas/FasL-Dependent Activation of Intratumoral Macrophages

- andγ T Cells That Is Associated with IFN+by CD8Splenectomy Promotes Indirect Elimination of Intraocular Tumors

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