Leveraging TCR Affinity in Adoptive Immunotherapy against … · Ovalbumin (OVA)-specific OT-I TCR-transgenic (Tg) CD45.1þ and OVA-specific OT-3 mice on a B6 background have been

Research Article

Leveraging TCR Affinity in AdoptiveImmunotherapy against Shared Tumor/Self-AntigensAaron M. Miller1,2, Milad Bahmanof1, Dietmar Zehn3, Ezra E.W. Cohen2,and Stephen P. Schoenberger1,2

Abstract

Adoptive cellular therapy (ACT) using T-cell receptor(TCR)–engineered lymphocytes holds promise for eradica-tion of disseminated tumors but also an inherent risk ofpathologic autoimmunity if targeted antigens or antigenicmimics are expressed by normal tissues. We evaluatedwhether modulating TCR affinity could allow CD8þ T cellsto control tumor outgrowth without inducing concomitantautoimmunity in a preclinical murine model of ACT. RIP-mOVA mice express a membrane-bound form of chickenovalbumin (mOVA) as a self-antigen in kidney and pancre-as. Such mice were implanted with OVA-expressing ID8ovarian carcinoma cells and subsequently treated withCD8þ T lymphocytes (CTL) expressing either a high-affinity(OT-I) or low-affinity (OT-3) OVA-specific TCR. The effectson tumor growth versus organ-specific autoimmunity weresubsequently monitored. High-affinity OT-I CTLs under-went activation and proliferation in both tumor-draining

and pancreatic lymph nodes, leading to both rapid eradi-cation of ID8-OVA tumors and autoimmune diabetes in alltreated mice. Remarkably, the low-affinity OT-3 T cells wereactivated only by tumor-derived antigen and mediatedtransient regression of ID8-OVA tumors without concomi-tant autoimmunity. The OT-3 cells eventually upregulatedinhibitory receptors PD-1, TIM-3, and LAG-3 and becamefunctionally unresponsive, however, allowing the tumors intreated mice to reestablish progressive growth. Antibody-mediated blockade of the inhibitory receptors preventedexhaustion and allowed tumor clearance, but these micealso developed autoimmune diabetes. The findings revealthat low-affinity TCRs can mediate tumor regression andthat functional avidity can discriminate between tumor-derived and endogenous antigen, while highlighting therisks involved in immune-checkpoint blockade on endog-enous self-reactive T cells.

IntroductionAdoptive T-cell therapy (ACT) is a potent and flexible cancer

treatment modality that can induce complete and durable regres-sion of solid tumors (1, 2). Current cell therapy options includemodifying autologous T cells to express either tumor-specific T-cell receptors (TCRs) or chimeric antigen receptors (CARs), whichtarget surface proteins via a single-chain molecule containing anantigen-binding domain fused to transmembrane and cyto-plasmic domains required for surface expression and T-cell trig-gering (3, 4). Although target recognition by CARs occurs in anHLA-unrestricted manner, TCRs have the ability to recognizepeptides derived from exogenous, membrane-bound, and intra-cellular proteins, thus allowing a far greater repertoire of antigens

to be targeted. Pioneering studies by Rosenberg and colleagueshave demonstrated that ACTwith tumor-infiltrating lymphocytes(TILs) can be effective against a variety of solid malignancies,including metastatic melanoma, gastrointestinal, and humanpapillomavirus–induced cancers, and this extends to bulky inva-sive tumors at multiple sites involving liver, lung, soft tissue, andbrain (1, 5–8). These responses are durable in approximately 20%of treated patients who experience prolonged remissions of 5years or more. However, a major limitation to the widespreadapplication of TIL therapy is the difficulty in routinely generatinghumanT cells with antitumor activity outside of a limited numberof highly specialized laboratories.

An alternative approach to TIL-based ACT is the use of high-affinity TCRs against tumor-associated antigens (TAA) that can beexpressed in a patient's normal T cells prior to expansion andreinfusion (9–13). Such genetically modified T cells have thepotential to overcome the variability in the specificity, potency,and longevity of TIL. A major limitation of adoptive cell therapyutilizing such genetically modified cytotoxic T lymphocytes(CTLs) has been "on-target, off-tumor" toxicities associated whenthe transferred cells recognize antigens expressed on both tumorcells andnormal tissue (14–17). In thefirst clinical trial using suchengineered T cells targeting themelanoma TAAMART-1, objectiveresponses were seen in 30% of melanoma patients receivingMART-1–targeted T cells, although over 80% of patients(29/36) developed an erythematous skin rash, and others devel-oped anterior uveitis or hearing loss attributable to recognition oflow expression of MART-1 in normal melanocytes, retina, and

1Laboratory of Cellular Immunology, La Jolla Institute for Allergy and Immu-nology, La Jolla, California. 2Division of Hematology and Oncology, UCSDMoores Cancer Center, La Jolla, California. 3Division of Animal Physiology andImmunology, School of Life Sciences Weihenstephan, Technical University ofMunich, Freising, Germany.

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

Corresponding Author: Stephen P. Schoenberger, La Jolla Institute for Allergyand Immunology, 9420AthenaCircle, La Jolla, CA92037. Phone: 858-752-6500;Fax: 858-752-6985; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-18-0371

�2018 American Association for Cancer Research.

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inner ear (9). Similarly, a phase I trial, using genetically modifiedT cells targeting carcinoembryonic antigen (CEA) resulted insevere transient inflammatory colitis as a result of "on-target"effects of the engineered T cells against colonic tissue expressingCEA (15). Another trial with genetically modified T cells targetingthe cancer testis antigen (CTA) MAGE-A3, known to be expressedin a range of epithelial malignancies but not most normal tissues,resulted in severe neurologic toxicity from periventricular infil-trationof adoptively transferredCD3þCD8þT cells with resultantnecrotizing leukoencephalopathy and death in 2 of 9 treatedpatients due to previously unrecognized expression of MAGEantigens in the human brain (14).

To better understand the risks involved in ACT, we developeda new preclinical model to study the phenomenon of "on-target, off-tumor" toxicities in the context of ACT, whichfeatures adoptively transferred T cells specific for an antigenexpressed on both tumor and normal tissues. This system isbased on a well-characterized murine model (RIP-mOVA),which features steady-state surface expression of a model anti-gen (membrane-bound chicken ovalbumin or mOVA) onproximal tubular cells of the kidney and beta islets of thepancreas. Although these mice lack high-affinity OVA-specificCD8þ T cells in their peripheral repertoire due to central andperipheral tolerance mechanisms, adoptive transfer of high-affinity OT-I cells specific for the OVA257-264/H-2Kb complexrapidly leads to their activation with subsequent destruction ofbeta cells, and autoimmune diabetes. RIP-mOVA mice dopossess a low-affinity OVA-specific T-cell repertoire, which canbe elicited by vigorous vaccination and can occasionally causeautoimmune diabetes (18). OT-3 mice are transgenic for onesuch low-affinity OVA257-264/K

b–specific TCR that was isolated

from immunized RIP-mOVA mice (18). In contrast to OT-I,OT-3 T cells become neither activated nor anergic in response tomOVA expression in pancreas or kidney tubulointerstitiumfollowing adoptive transfer to RIP-mOVA mice, and insteadmaintain their potential for response. In this way, OT-3 T cellsare functionally analogous to the postthymic repertoire of self-reactive T cells that exists in the periphery of healthy humansubjects. This model is therefore distinct from previous exam-ples in which low-affinity T cells against a shared tumor/self-antigen are rendered profoundly anergic by expression of thetarget antigen on self-tissues (19).

Our objective in these studies was to determine whether TCRaffinity could discriminate between self and tumor tissues expres-sing the same antigen. To address this, we established tumors inRIP-mOVA mice using an ovarian cancer model modified toexpress membrane-bound OVA (ID8-mOVA) and monitoredtumor control versus autoimmune diabetes development follow-ing ACT with OT-I or OT-3 CD8þ T cells (Supplementary Fig. S1).We found that the low-affinity OT-3 T cells could be cross-primed and activated by antigen originating from the tumor,but not from normal self-tissues, and that these cells couldthence mediate the selective eradication of tumor cells withoutconcomitant autoimmune beta cell destruction. OT-3 T cellseventually became functionally unresponsive, and treatmentwith checkpoint blockade immunotherapy both restored theirfunctionality with respect to tumor control, but also led todiabetes induction. These findings demonstrate that TCR affin-ity can allow ACT to discriminate between tumor and self,while highlighting the risks that exist for the use of systemicimmune-checkpoint blockade immunotherapy.

Materials and MethodsMice

Mice were maintained under specific pathogen-free conditionsin accordance with guidelines of the Association for Assessmentand Accreditation of Laboratory Animal Care International. Thestudies described in this paper conform to the principles outlinedby the Animal Welfare Act and the National Institutes of Healthguidelines for the care and use of animals in biomedical research.C57BL/6J (B6; CD45.2þ), B6.SJL (CD45.1þ), RIP-mOVA, andRag1�/� mice were purchased from The Jackson Laboratory.Ovalbumin (OVA)-specific OT-I TCR-transgenic (Tg) CD45.1þ

and OVA-specific OT-3 mice on a B6 background have beenpreviously described. Six- to 12-week-old mice were used in allexperiments.

Cell linesID8-OVA cells were a kind gift from Dr. Dietmar Zehn

(Technical University Munich) in 2014. The B3Z T-cell hybrid-oma was a kind gift from Dr. Nilabh Shastri (University ofCalifornia, Berkley). All tumor cell lines were tested prior toin vivo use and found to be free of Mycoplasma. In addition,model antigen expression for OVA was confirmed by flowcytometry. Due to the potential for cross-contamination of celllines, ID8-OVA cells were reauthenticated by flow cytometry atleast every 3 months, and cells were maintained in culture forno more than 30 passages. Mycoplasma testing was repeated atleast every 6 months.

Flow cytometry, intracellular cytokine staining, and cytokineanalysis

Single-cell suspensions were prepared from spleens, inguinallymph nodes (LN), and tumors. Cells were stained with fluores-cent-labeled antibodies (BioLegend; BD- Bioscience Pharmingen;or eBiosciences) and analyzed by either FACSCalibur or LSR IIflow cytometer (BD). The following cloneswere used: CD4 (RM4-5), CD8 (5H1), CD11c (HL3), Cd11b (M170), CD25 (PC61.5),SIINFEKL/H-2Kb (25-D1.16), CD44 (IM7), CD62L (MEL-14),IFNg (XMG1.2), TNFa (MP6-XT22), FAS (2495), CD40 (3/23),andFoxp3 (FJK-16s). For intracellular cytokine staining, cellswereactivated withOVA peptide-pulsed splenocytes or with PMA (100ng/mL) plus ionomycin (500 ng/mL) for 4 hours in the presenceof GolgiPlugTM (BD Biosciences), processed with a Cytofix/CytopermTMkit (BDBiosciences), and stained as indicated.Gatesand quadrants were set based on isotype control staining unlessotherwise indicated.

Adoptive transfer experiments and CTV labelingT cells fromspleens and LNsofOT1-RagKOandOT-3 TCRalpha

KO mice were used for adoptive cell transfer experiments. CD8þ

T cells were isolated by negative selection of single-cell suspensionsfrom homogenized donor tissue using a MACS CD8 isolation kit(Miltenyi Biotec). Purity of isolated CD8þ T cells was confirmed byflow cytometry analysis and was routinely greater than 95%, withless than 0.1% CD4þ T cell contamination. Donor T cells wereobtained fromCD45.1mice, whereas recipient mice were CD45.2,thereby permitting in vivo isolation via this congenic marker. Forin vivo cell proliferation studies, T cells (25 � 106) were washedtwice inPBSand labeled in5nmol/LCTVfor8minutes at37�Conashaker followed by quenching with ice-cold media. Cells werewashed twice in PBS, and 5 � 106 cells were injected per mouse

TCR Affinity in ACT against Tumor/Self-Antigens

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via retro-orbital injection. Mice were sacrificed 3 days later, and thespleen and LN cell suspensions were analyzed via flow cytometry.For other adoptive transfer experiments, donor T cells were har-vested from the draining LN and spleens at the indicated timepoints for analysis.

Ectopic ID8 tumormodel, intravital imaging (IVIS), and tumorvaccinations

Previously published methods were used for ectopic implan-tation of ID8 tumor cells (20). Briefly, while anesthetized, micewere inoculated s.c. with single-cell suspensions of 5 � 106

tumor cells suspended in 200 mL of PBS into the right flank.Three weeks later, the mice were monitored for tumor growthby tumor bioluminescence using an IVIS system and inresponse to treatment thereafter at the indicated time points.To monitor for tumor bioluminescence, mice received 300 mgof D-luciferin (Promega) by intraperitoneal injection 10 min-utes before imaging ID8-OVA-luc tumors using a Xenogen IVIS100 to maximum tumor radiance. Results are reported as thepercent change from baseline tumor radiance on the day oftreatment. For tumor vaccination studies, single-cell suspen-sions of tumor cells (107/mL) in PBS were subjected to 150 Gyof gamma ionizing radiation using an irradiator prior to s.c.flank inoculations of 5 � 106 cells into the right flank ofrecipient mice.

Antigen-specific immune response assaySpleen and LN cells were incubated for 5 hours with the

OVA257–264 (SIINFEKL) at 5 mg/mL final concentration in thepresence of Brefeldin A (BD Biosciences) directly ex vivo and werestained for surface expression of CD8 and CD3, fixed and per-meabilized using the Cytofix/Cytoperm kit (BD Biosciences) andstained for intracellular IFNg according to the manufacturer'sprotocol.

B3Z assaysB3Z is a lacZ-inducible CD8þ T-cell hybridoma expressing TCR

specific for OVA257–264 (SIINFEKL), presented on the murineH2Kb MHC class I molecule (21). ID8-OVA-luc, SAMBOK,or MECI cells were cocultured with B3Z for 3 hours at 37�C ina 5% CO2/air atmosphere, after which cells were washed, andb-galactosidase activity was detected by addition of substrate(CPRG, Sigma-Aldrich) and absorption readings at 600 nm usinga spectrophotometer plate reader.

Monoclonal antibodies for in vivo immune-checkpointstudies

The following mAbs were purchased from Bio X Cell:InVivoPlus anti-mouse PD-1 (J43; cat. #BP0033-2), InVivoPlusanti-mouse LAG-3 (C9B7W; cat. #BP0174), and InVivoPlus anti-mouse TIM-3 (RMT3-23; cat. #BP0115). A total of 250 mg of each

Figure 1.

Induction of autoimmune diabetes by OT-3 cytotoxic T lymphocytes. A, Systemic activation of 106 adoptively transferred OT-3 CD8þ T cells by intravenous(i.v.) cotransfer of 3,000 CFU of OVA-expressing Listeria monocytogenes (Lm-OVA) resulted in autoimmune destruction of beta islets in RIP-mOVA mice.B, Systemic activation of OT-3 CD8 T cells by i.v. cotransfer of 106 CFU of Lm-OVA deficient for Act A (Lm-OVA Act A�/�) is sufficient to result inautoimmune diabetes. C, Autoimmune diabetes induced by adoptively transferred OT-3 CD8þ T cotransferred with 3,000 CFU of Lm-OVA cells is dose-dependent, requiring 105 OT-3 T cells to develop diabetes at 100% penetrance of the phenotype.

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antibody was transferred intravenously in 200 mL of PBS intorecipient mice.

Statistical analysisStatistical analysis was performed using Prism 5 (GraphPad).

One-way ANOVA and unpaired two-tailed Student t tests wereconducted and considered statistically significant at P values �0.05 (�), 0.01 (��) and 0.001 (��).

ResultsInduction of autoimmune diabetes by OT-3 CTLs

We first sought to determine the quantitative threshold fordiabetes induction by fully activated OT-3 in RIP-mOVA mice inthe setting of ACT. Na€�ve OT-3 CD8þ T cells were harvested fromdonor mice by negative selection of homogenized splenocytes,and graded doses (103, 105, or 106) were transferred to recipientRIP-mOVA mice, which were subsequently infected by an OVA-expressing strain of wild-type Listeria monocytogenes (Lm-OVA) byintravenous injection. Mice were then monitored for diabetesinduction (blood glucose > 200 mg/dL) daily after transfer.Despite the lower functional avidity for the OVA antigenexpressed by the pancreatic beta cells, systemic activation of106 OT-3 cells by Lm-OVA resulted in autoimmune destructionof beta islets in all RIP-mOVAmice (Fig. 1A). In addition, 106OT-3 cells were also activated by an attenuated strain of Lm-OVA(ActA�) that is unable to spread laterally via actin polymerization,demonstrating that a single priming stimulus, and not continuedactivation by proliferating Lm-OVA within reticuloendothelialcells, is sufficient to result in autoimmune diabetes (Fig. 1B).Finally, this phenomenonwas dose-dependent, requiring 105OT-3 cells to develop diabetes at 100% penetrance of the phenotype

(Fig. 1C). These data indicate that OT-3 cells can be activated andinduce autoimmunity in RIP-mOVA mice reproducibly with theappropriate activating stimulus.

ID8-OVA tumors induce proliferation of adoptively transferredOT-3 CTLs via cross-presentation

Although OT-3 cells can induce autoimmune diabetes in RIP-mOvA after priming with a strong stimulus such as Lm-OVAinfection, it was not known if a progressively growing tumorcould also do so. To address this, OT-3 cells were labeled withthe viable dye CTV (which allows visualization of cellulardivision by its portioning into daughter cells) and adoptivelytransferred to RIP-mOVA harboring ID8-OVA tumors. The micewere euthanized 96 hours following transfer, and the trans-ferred cells were analyzed within the tumor-draining LN forproliferation using their congenic marker (CD45.1). The OT-3T cells within RIP-mOVA mice bearing ID8-OVA tumors bothproliferated (Fig. 2A) and acquired effector functionality(Fig. 2B), whereas those transferred to RIP-mOVA mice lackingtumors did neither. These results indicate that na€�ve OT-3 cellscan be activated to proliferate by cross-presentation of OVAantigen in the tumor-draining LN.

OT-I and OT-3 CTLs have different thresholds of antitumorreactivity versus autoimmune diabetes

Having established that OT-3 T cells cause autoimmunediabetes when systemically activated by a strong inflammatorystimulus (Lm-OVA), and that ID8-OVA tumors engraft in RIP-mOVA mice and are able to cross-prime adoptively transferredOT-3 T cells, we sought to compare whether OT-3 versus OT-ICD8 T cells could mediate control of ID8-OVA tumors without

Figure 2.

ID8-OVA tumors induce proliferation of adoptively transferred OT-3 CD8 T cells by cross-presentation. A, RIP-mOVA mice bearing 3-week-old ID8-OVAtumors elicited proliferation of adoptively transferredOT-3 CD8þ T cells that were identifiable by the congenicmarker CD45.1 and labeledwith cell-trace violet (CTV)within the tumor-draining LN 72 hours after transfer, whereas mice lacking tumors did not demonstrate proliferation. B, Adoptively transferred OT-3 CD8þ

T cells into tumor-bearing mice produced Granzyme B 72 hours after transfer, consistent with functional activation.






simultaneous off-tumor, on-target autoimmune destruction ofpancreatic beta islets. To address this, 5 � 106 OT-I or OT-3CD8 T cells were transferred to ID8-OVA tumor–bearing RIP-mOVA mice, which were then monitored simultaneously forchanges in tumor size by CCD and diabetes induction by bloodglucose analysis. As shown in Fig. 3, although the high-affinityOT-I T cells were able to mediate destruction of ID8-OVAtumors, they did so at the expense of autoimmune destructionof the beta islets, as demonstrated by development of diabetes(Fig. 3A). OT-3 cells, in contrast, were able to achieve partialdestruction of the incipient tumor, but without concomitantautoimmunity. This effect was transient, however, and despitethe partial destruction of the ID8 tumor by OT-3 ACT, thetumors eventually resumed progressive growth in all treatedmice, suggesting that the transferred T cells had become inef-fective or functionally silenced (Fig. 3B).

OT-3 CTLs upregulate inhibitory receptors in response to ID8-OVA tumors and become exhausted

We next investigated the functional status of OT-3 T cells frommicewhose tumors had reinitiated progressive growth. To accom-plish this, OT-3 T cells were isolated from tumor-draining LNs oftreated mice and tested for their ability to produce effectorcytokines in response to restimulation by their cognate peptide(SIINFEKL) alongside those primed by the same ID8-OVA tumoroffered as an irradiated cellular vaccine. As shown in Fig. 4,whereas the OT-3 T cells were clearly detectable in the tumor-draining LN, they were unable to produce IFNg in response toOVA257-264 (SIINFEKL) peptide, although those primed by theID8 vaccinewere able to do so. TheOT-3 cells from tumor-bearingmice were nonetheless able to produce IFNg following treatmentwith PMA/ionomycin, indicating that the functional defect was inTCR-mediated activation (Fig. 4A). These findings demonstrate

Figure 3.

OT-1 and OT-3 CD8 T cells havedifferent thresholds of antitumorreactivity versus autoimmunediabetes. A, Adoptive cell transferof 5 � 106 na€�ve OT-I CD8þ T cells,prepared by negative selection fromdonor transgenic OT-I micesplenocytes and LNs, mediatesdestruction of ID8-OVA tumors asreflected by decreased tumorbioluminescence. However, allrecipient mice develop autoimmunediabetes. ACT of OT-3 CD8þ T cells,in contrast, mediates partialdestruction of the incipient tumorwithout concomitant autoimmunity.B, ID8-OVA tumors that are transientlycontrolled byACTofOT-3CD8þTcellsdemonstrate progressive tumoroutgrowth by 21 days post celltransfer.

Miller et al.





Figure 4.

OT-3 CD8 T cells upregulate inhibitoryreceptors in response to ID8-OVAtumors and become functionallyexhausted. A, 5 � 106 OT-3 CD8þ

T cells adoptively transferred to micebearing mature ID8-OVA tumors andharvested from the tumor-draining LN15 days after transfer are unable toproduce IFNg in response OVA257–264 (SIINFEKL) peptide restimulation,whereas OT-3 CD8þ T cells primed byan ID8 tumor cell vaccine that lackspersistence of the tumor cells in vivodemonstrate successfully SIINFEKLantigen-specific restimulation.B,OT-3CD8þ T cells adoptively transferred tomice bearing ID8-OVA tumors andharvested from the tumor-draining LN15 days after transfer demonstrateupregulation of the inhibitorycoreceptors PD-1, Tim-3, and Lag-3,whereas mice bearing ID8-WT tumorsor mice vaccinated with irradiatedID8-OVA cells did not upregulateexhaustion markers.






that the growing tumor is able to induce a state of unresponsive-ness, termed "exhaustion," in the respondingOT-3 T cells. Accord-ingly, the OT-3 cells adoptively transferred to mice bearing ID8-OVA tumors versus ID8-WT tumors, or mice vaccinated withirradiated ID8-OVA cells, demonstrated upregulation of theinhibitory coreceptors PD-1, Tim-3, and Lag-3 (Fig. 4B).

Immune-checkpoint inhibitors reverse OT-3 CTL exhaustionbut induce autoimmune diabetes

Inhibitory receptors on immune cells are pivotal regulators ofimmune escape in cancer (22). We have shown that adoptivelytransferred OT-3 cells demonstrate extensive coexpression of PD-1, LAG-3, and TIM-3, and we have documented functionalexhaustion in SIINFEKL antigen restimulation experiments fol-lowing adoptive cell transfer into RIP-mOVA mice bearing ID8-Ova tumors. T-cell exhaustion is a plausible mechanism for thetransient tumor control elicitedbyOT-3 cells in recipientmice.Wetherefore next sought to determine if coadministration ofimmune-checkpoint inhibitors along with OT-3 cells could pre-clude exhaustion and potentiate the antitumor reactivity of theadoptively transferred T cells in RIP-mOVA mice bearing ID8-OVA tumors. In these experiments, 250 mg of anti–PD-1, anti-Lag3, anti-Tim3, or a combination of all 3 immune-checkpointantibodies, was administered to the contralateral orbital sinusalong with adoptive cell transfer of 5 � 106 OT-3 cell into ID8-OVA tumor–bearing RIP-mOVA mice. Coadministration ofimmune-checkpoint inhibitors did not demonstrate statistical-ly significant changes in tumor killing versus controls (Fig. 5A).However, linear regression analysis of all treated animals fromall groups demonstrated that tumor killing did correlate withblood glucose concentration in treated mice (Fig. 5B). Thehighest blood glucose and tumor killing responses were allobserved in the combinatorial immune-checkpoint cohort thatreceived OT-3 cells in addition to PD-1, Lag-3, and Tim-3inhibitors. In this group, half of the treated mice developedautoimmune diabetes with a trend toward greater tumor killing(Fig. 6). In addition, OVA antigen restimulation of splenocytesharvested from treated mice showed higher responses in thediabetic versus nondiabetic mice in this group. These datasuggest that combinatorial ICB can preclude functional exhaus-

tion in adoptively transferred OT-3 cells with a trend towardenhanced tumor killing but at the expense of developingautoimmune diabetes.

DiscussionThe current landscape of clinical cancer immunotherapy

involves strategies aimed at both uncoupling a patient's T cellsfrom negative regulation and the adoptive transfer of those whichhave been expanded in vitro and in some settings, engineered toacquire antitumor specificity (23–25). In each of these settings,the risk of pathologic autoimmunity must be balanced with thetherapeutic potential inherent in trying to direct powerful effec-tors exclusively toward the tumor and its antigens. The modelsystem established and described in this work represents an effortto explore these dichotomies in the context of varying TCR affinityin a controlled preclinical setting to better understand their causesand, thereby, to inform clinical practice.

Our results clearly show that T cells possessing an antigenreceptor of sufficiently low functional avidity against a normalself-antigen to escape central tolerance can, nonetheless, be acti-vated when the antigen in question is expressed on a growingtumor. In many key aspects, the OT-3 T cells in RIP-mOVA micecan be considered as a surrogate for the postthymic selectionperipheral repertoire of potentially self-reactive T cells, which existin a state of "immunologic ignorance" with respect to theircognate antigen, undergoing neither activationnor tolerance untilsome crucial aspect of their regulation is disrupted to enable theirfull activation. In the present study, this transition could be(unsurprisingly) induced by a strong inflammatory challengewith an OVA-expressing bacterial pathogen, or by a progressivelygrowing tumor, with the latter leading to both proliferation andacquisition of effector functionality, producing a transient reduc-tion in tumor volume. Remarkably, activation of the OT-3 T cellsunder these conditions did not lead to autoimmune diabetes,despite OVA expression on beta cells, suggesting that a window ofopportunity exists for the selective targeting of a tumor by self-reactive T cells without concomitant recognition of a self-tissuesharing expression of the same antigen. These results do notexclude clinically subapparent toxicity, because elevated serum

Figure 5.

Cotransfer of OT-3 CD8þ T cells with the immune-checkpoint inhibitors anti–PD-1, anti-TIM3, and anti-LAG3 elicits autoimmune diabetes that correlates withantitumor responses. A, Intravenous coadministration of the immune-checkpoint inhibitors anti–PD-1, anti-TIM3, and anti-LAG3 (250 mg) with 5 � 106 OT-3 CD8þ

T cells into RIP-mOVA mice bearing 3-week-old ID8-OVA tumors did not demonstrate statistically significant changes in tumor killing by tumor bioluminescenceby day 7 after ACT as single agents or in combination versus mice that received only OT-3 CD8þ T cells. B, Linear regression analysis of all treatmentconditions demonstrated that tumor killing correlates with blood glucose concentration in treated mice.

Miller et al.





glucose concentration is typically detectable once 90% of insulin-producing cells are destroyed. The basis for this selective recog-nition of tumor over self is unclear, but could result from differ-ences in the level of antigen presented by the 2 target cellpopulations or the local regulatory environment which may

govern access and functional recognition of the OVA antigen in2 sites. It is worth noting that, in light of the previously statedignorance ofOVA as an antigen by theOT-3 cells, theOVA257–264/Kb epitope is functionally equivalent to a foreign antigen for theOT-3 T cells, albeit one they recognize with low affinity. In the

Figure 6.

Combinatorial immune-checkpoint blockade with anti–PD-1, anti-TIM3, and anti-LAG3 reverses OT-3 CD8þ T-cell exhaustion with a trend to enhanced tumorkilling but induces autoimmune diabetes. A, Intravenous coadministration of anti–PD-1, anti-TIM3, and anti-LAG3 (250 mg) with 5 � 106 OT-3 CD8þ T cellsinto RIP-mOVA mice bearing 3-week-old ID8-OVA tumors induced diabetes in half of the treated mice. B, There is a nonstatistically significant trend towardgreater tumor killing in mice that developed autoimmune diabetes. C, SIINFEKL restimulation of splenocytes harvested from treatedmice showed higher responsesin the diabetic mice versus nondiabetic mice in this group.






context of their selective recognition of the ID8-OVA tumor cells,OVA257–264/K

b functions as a tumor-specific neoantigen. In thisregard, our findings suggest that such low-affinity T cells, whetherinduced by vaccination or provided through adoptive transfer,can be an effective therapeutic force for eradicating establishedtumors.

Although the low-affinity OT-3 T cells could be selectivelyactivated by tumor-derived OVA and mediate some degree oftumor killing, the effect was temporary, and the populationeventually underwent functional silencing accompanied by coor-dinated upregulation of immune-checkpoint molecules PD-1,TIM-3, and LAG-3. In this regard, the low-affinity T cells behavedas numerous reports have demonstrated for the endogenoustumor-reactive T-cell repertoire (26). Attempts to overcome this"exhausted" state through antibody-mediated blockade of therelevant receptors restored antigen-specific production of theeffector cytokine IFNg , by OT-3 T cells, but the ensuing responseinvariably led to diabetes induction in the majority of treatedanimals in the current study. These findings demonstrate that thismodel system can allow an unprecedented degree of insight intothe hazards implicit in systemic blockade of immune-checkpointpathways when a self-reactive T-cell population has been activat-ed, with autoimmune diabetes being the primary immune-medi-ated adverse event (irAE) observed (27).

A number of studies have demonstrated the existence of opti-mal TCR "affinity windows" for key parameters of T-cell function,including proliferation, antigen sensitivity, and polyfunctionality(28), above which signaling can render cells hyporesponsive tolow amount of antigen and/ormediate dangerous cross-reactivityto self MHC (29–31). These findings add to the growing consen-sus that TCRs with low to moderate affinities may possess impor-tant functional features necessary for sustained signaling tophysiologic concentrations of antigen (32, 33). This notion issupported by studies by Sherman and colleagues, who showedthat vaccination against an antigen shared between tumor andself-tissue can induce low-avidity antigen-specific CD8þ T cells toreject tumor cells with high expression of target antigen yet remaintolerant of antigen-expressing pancreatic beta cells (34). In adop-tive cell transfer experiments conducted by this group, low-avidityantigen-specific T cells that were tolerized by cross-presentedtumor antigen could subsequently eradicate tumors if antigen-specific CD4þ T cells were cotransferred into tumor-bearing miceharboring the target antigen, lending further evidence that such

"affinity-tuned" cytotoxic T cells can be exploited therapeutically(35, 36).

This study reports that under conditions of steady-state pro-gressive growth, a low-affinity TCR can preferentially be primed toand recognize antigen on tumor without causing autoimmunepathology to a normal self-tissue sharing its expression, whereas ahigh-affinity TCR was unable to discriminate between the 2 typesof target cells. This suggests that, whether due to the amount ofantigen produced or the homeostatic state of the tissue in ques-tion, the tumor environment is distinct from that of a stable self-tissue in terms of the magnitude and context of antigen (cross-)presentation and that only a low-affinity TCR was capable ofresponding to this. This finding suggests that further investi-gation of low-affinity TCRs could further elucidate the factorsthat govern the functionality in the setting of ACT againstshared tumor/self-antigens and in doing so augment the pre-cision and efficacy of ACT.

Disclosure of Potential Conflicts of InterestE.E.W. Cohen is a consultant/advisory board member for Pfizer, Merck,

AstraZeneca, Bristol-Myers Squibb, EMD Serono, and Amgen. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: A.M. Miller, E.E.W. Cohen, S.P. SchoenbergerDevelopment of methodology: A.M. Miller, M. BahmanofAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.M. Miller, M. BahmanofAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.M. Miller, E.E.W. Cohen, S.P. SchoenbergerWriting, review, and/or revision of the manuscript: A.M. Miller, D. Zehn,E.E.W. Cohen, S.P. SchoenbergerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.M. Miller, M. Bahmanof, S.P. SchoenbergerStudy supervision: S.P. Schoenberger

AcknowledgmentsThis work was supported by 1R21CA179362 and 1R01AI107010 from the

NIH to S.P. Schoenberger and from a gift from the Kimmelman FamilyFoundation.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 6, 2018; revised September 2, 2018; accepted November 21,2018; published first November 27, 2018.

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2019;7:40-49. Published OnlineFirst November 27, 2018.Cancer Immunol Res Aaron M. Miller, Milad Bahmanof, Dietmar Zehn, et al. Shared Tumor/Self-AntigensLeveraging TCR Affinity in Adoptive Immunotherapy against

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