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Research Article

Transnuclear TRP1-Specific CD8 T Cells with High orLow Affinity TCRs Show Equivalent Antitumor Activity

Stephanie K. Dougan1, Michael Dougan1,3, Jun Kim1,2, Jacob A. Turner1,4, Souichi Ogata1,5,Hyun-Il Cho6, Rudolf Jaenisch1, Esteban Celis6, and Hidde L. Ploegh1

AbstractWe have generated, via somatic cell nuclear transfer, two independent lines of transnuclear mice, using

as nuclear donors CD8 T cells, sorted by tetramer staining, that recognize the endogenous melanomaantigen tyrosinase related protein 1 (TRP1). These two lines of nominally identical specificity differ greatlyin their affinity for antigen (TRP1high or TRP1low) as inferred from tetramer dissociation and peptideresponsiveness. Ex vivo–activated CD8 T cells from either TRP1high or TRP1low mice show cytolytic activityin three-dimensional tissue culture and in vivo, and slow the progression of subcutaneous B16 melanoma.Although na€�ve TRP1low CD8 T cells do not affect tumor growth, upon activation these cells functionindistinguishably from TRP1high cells in vivo, limiting tumor cell growth and increasing mouse survival.The antitumor effect of both TRP1high and TRP1low CD8 T cells is enhanced in RAG-deficient hosts.However, tumor outgrowth eventually occurs, likely due to T cell exhaustion. The TRP1 transnuclear miceare an excellent model for examining the functional attributes of T cells conferred by T cell receptor (TCR)affinity, and they may serve as a platform for screening immunomodulatory cancer therapies. CancerImmunol Res; 1(2); 99–111. �2013 AACR.

IntroductionInfiltration of CD8 T cells correlates positively with patient

outcomes, as shown for colorectal and other cancers (1, 2).Even in the absence of a successful endogenous antitumorimmune response, immunotherapy for cancer has begun todeliver on its promise (3, 4). The success of checkpoint block-ade therapies such as anti-CTLA-4 illustrates the importanceof the CD8 T cell response in tumor regression. At baseline,many human patients with cancer have antitumor CD8 T cellsthat are anergic or kept in check by CD4 regulatory T cells(Treg). CTLA-4 blockade can activate antitumor T cells andelicit clinical responses in 25% of patients. Anti-CTLA-4 workssynergistically with chemotherapy, radiotherapy, or othertherapies that generate tumor lysis and uptake of tumorantigens by dendritic cells (5). Blocking antibodies to PD-1and PDL1 (6, 7) produce similar outcomes. The endogenousT cell response to tumors correlates with overall survival in

patients with cancer (1), and therapies aimed at boosting theT cell response are now in widespread use (3, 8).

Many tumor antigens are mutated self-antigens or antigensthat are overexpressed. T cell receptors (TCR) on T cells thatrecognize such antigens are generally of low affinity (9), ashigh-affinity self-reactive cells are deleted in the thymus(10, 11) and suppressed in the tumor microenvironment(12). The type of CD8 T cell response necessary for optimaltumor rejection remains poorly characterized because of apaucity of animal models. Most of the available animal modelsmake use of TCR transgenic animals, the construction of whichis based on long-term T cell cultures from which the corre-sponding rearranged TCR segments are cloned and expressedas transgenes. Alternatively, tumor cell lines are engineeredto express antigens for which TCR transgenics happen to beavailable. By their very nature, populations of T cells harvestedfrom tumor-bearing animals or tumor-infiltrating lympho-cytes are necessarily heterogeneous, polyclonal, and variablefrom one experiment to the next, exactly the reason why TCRtransgenics are so widely used. Models of antitumor immunitytypically rely on overexpression of ovalbumin (OVA) in com-bination with OT-I transgenic T cells, known to be of very highaffinity. Pmel TCR transgenic mice contain CD8 T cells ofmore moderate affinity for the endogenous melanoma antigentyrosinase-related protein 1 (TRP1), but results from thesemice are difficult to compare with OVA/OT-I systems due todifferences in the antigen.

Somatic cell nuclear transfermay be used to clonemice fromthe nuclei of antigen-specific lymphocytes (13–16). Thesetransnuclear mice are derived from primary CD8 T cellsharvested in the course of a natural immune response, without

Authors' Affiliations: 1Whitehead Institute for Biomedical Research;2Massachusetts Institute of Technology, Cambridge; 3Department ofMed-icine, Massachusetts General Hospital, Boston, Massachusetts; 4Univer-sity of Cincinnati, Cincinnati, Ohio; 5Janssen Research and Development,Division of Janssen Pharmaceutica NV, Beerse, Belgium; and 6Departmentof Immunology, H. LeeMoffitt Cancer Center & Research Institute, Tampa,Florida

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

Corresponding Author:Hidde L. Ploegh, Whitehead Institute for Biomed-ical Research, 9 Cambridge Center, Rm 361, Cambridge, MA 02142.Phone: 617-324-2031; Fax: 617-452-3566; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-13-0047

�2013 American Association for Cancer Research.

CancerImmunology

Research

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any prolonged tissue culture to select for high-affinity T cellvariants in vitro. Transnuclear mice contain no genetic altera-tions other than the rearranged TCR genes, expressed fromtheir endogenous loci and under physiologic control. Finally,creation of transnuclear mice can be done rapidly, requiringonly 6 weeks from isolation of a selected lymphocyte popula-tion, accomplished through isolation of class I MHC tetramer-positive (tetramerþ) cells, to birth of chimeric mice. A keyadvantage of the transnuclear technology is the ability toclone multiple lines of mice with the same antigenic specificitybut with different TCR sequences and therefore differentaffinities (15). These mice can then be used as a ready sourceof lymphocytes of defined specificity for in vitro culture or foradoptive transfer experiments.

Most tumors are weakly immunogenic at best. The well-studied melanoma cell line B16 downregulates class I MHCand is poorly infiltrated by immune cells (17–19). CD8 T cellscan reject B16 in vivo, but only when mice are first exposedto some form of immune manipulation, such as vaccinationwith irradiated B16, engineered to secrete the cytokine gran-ulocyte macrophage colony-stimulating factor (GM-CSF;refs. 18, 20, 21). Because B16 does not induce a detectableCD8 T cell response under normal conditions, we used apeptide-based vaccine strategy to elicit a robust CD8 T cellresponse to themelanoma-specific antigen TRP1. This vaccineconsists of a palmitoylated version of the peptide to increaseits circulatory half-life, anti-CD40 antibody, and the TLR3ligand polyinosine-polycytidylic acid (poly-IC) and is referredto as TRIVAX. When administered to mice, it elicits a robustpopulation of tetramerþ CD8þ T cells that can reject estab-lished B16 tumors (22, 23).

We have used such vaccine-induced CD8 T cells as asource of donor nuclei to generate two independent lines ofTRP1-specific transnuclear mice. In doing so, we have gen-erated, for the first time, a pair of transnuclear mice withTCRs specific for the identical peptide–MHC combination.These TCRs are expressed under the control of their endog-enous promoters and represent cells directly harvested froman immunized mouse upon staining with the appropriateclass I MHC tetramers, without preselection in tissue cul-ture, and differ in their affinity for TRP1 by approximately100-fold. The T cells from these mice recognize an endog-enous tumor antigen and allow a direct assessment of therole of TCR affinity in antitumor CD8 T cell responses.The generation of antitumor transnuclear mice provides aunique set of tools for the fields of tumor immunology andCD8 T cell development.

Materials and MethodsAnimal care

All animals were housed at the Whitehead Institute forBiomedical Research (Cambridge, MA) and were maintainedaccording to protocols approved by the Massachusetts Insti-tute of Technology (MIT) Committee on Animal Care. C57Bl/6,CD45.1 congenics, and OT-I transgenic mice were purchasedfrom The Jackson Laboratory. RAG2�/� (RAG2TN12) micewere purchased from Taconic.

Transnuclear mouse generationTransnuclear mice were generated as previously described

(14,15, 24). Briefly, CD8þ TRP1 tetramerþ cells were sorted byfluorescence-activated cell sorting (FACS) and used as a sourceof donor nuclei for somatic cell nuclear transfer (SCNT). Themitotic spindle was removed from mouse oocytes and replac-ed with donor nuclei. The nucleus-transplanted oocytes werethen activated in medium containing strontium and tricho-statin A (TSA), and allowed to develop in culture to the blasto-cyst stage. Because the live birth rate of SCNT blastocysts isclose to zero, SCNT blastocysts were instead used to derive em-bryonic stem cell lines. These embryonic stem cell lines werethen injected into wild-type B6xDBA F1 blastocysts and implant-ed into pseudopregnant females. The resulting chimeric pupswere mated to C57Bl/6 females to establish the 6.15 (TRP1low)and 6.17 (TRP1high) lines. All animals used were backcrossedfour to seven generations onto the C57BL/6 background.

Sequencing of the TCR genesTRP1high and TRP1low CD8 T cells were purified by positive

selection using CD8magnetic beads (Miltenyi Biotec) and usedas a source of RNA. 50-RACE was conducted according to themanufacturer's protocol (GeneRacer, #L1502-01; Invitrogen).

MHC tetramer production and peptide exchangeRecombinant protein expression, refolding of the H-2Db

complex with the SV9-P7� conditional ligand, and their subse-quent tetramerization were accomplished by following estab-lished protocols (25–27). The peptide exchange reaction wasinitiated by UV irradiation (360 nm), and the resulting MHCtetramers were used directly to stain freshly prepared spleno-cytes as described previously. TRP1-alteredpeptide ligandswereproduced by Fmoc-based solid-phase peptide synthesis by theMIT Center for Cancer Research (Cambridge, MA) biopoly-mers facility. All peptides were dissolved in dimethyl sulfoxide(10 mg/mL) and stored at �20�C until further use.

Flow cytometryCells were harvested from spleen, peripheral blood, or thymus.

Cell preparations were subjected to hypotonic lysis to removeerythrocytes, stained, and analyzed using a FACSCalibur (BD).Tetramers were prepared fresh from photocleavable stocks(25–27) or directly refolded with TRP1 heteroclytic peptide(TAPDNLGYM). All antibodies were from BD Pharmingen.

Cell culturingCells were cultured in RPMI-1640 medium supplemented

with 10% heat-inactivated FBS, 2 mmol/L L-glutamine, 100U/mL penicillin G sodium, 100 mg/mL streptomycin sulfate,1 mmol/L sodium pyruvate, 0.1 mmol/L nonessential aminoacids, and 0.1 mmol/L 2-ME. Bone marrow–derived dendriticcells (BMDC) were obtained by culturing of bone marrowaspirates in RPMI media supplemented with recombinantmurine GM-CSF (rmGM-CSF; Peprotech) and recombinantmurine interleukin-4 (rmIL-4; Peprotech). Fresh media wasadded to the BMDC cultures every 2 days for 6 days. BMDCswere used at day 6 to 8 of differentiation, and were washedbefore use. CD8 T cells were isolated from pooled spleen andlymph nodes of TRP1 transnuclear or wild-type mice using

Dougan et al.

Cancer Immunol Res; 1(2) August 2013 Cancer Immunology Research100




positive selection on anti-CD8 magnetic beads (Dynabeads;Invitrogen). For cocultures, BMDCs were pulsed with theindicated peptides for 30 minutes before the addition of CD8T cells at a ratio of 100,000 T cells to 50,000 BMDCs in round-bottomed 96-well culture plates. IL-2 and IFN-g productionwere measured by ELISA of 24-, 48-, or 72-hour culture super-natants as indicated (BD Pharmingen). CCL5 (RANTES) andMIP-1b were measured by cytokine bead array (BD).

3D B16 tumor culturesCD8 T cells from TRP1high, TRP1low, or OT-I transgenic mice

were isolated by positive selection on anti-CD8 Dynabeads(Invitrogen; 114.62D) and cultured for 18 hours with BMDCsplus either TRP1 heteroclytic peptide orOVApeptide (SIINFEKL)at 1 mmol/L concentration. Activated CD8 cells were then count-ed mixed with B16 cells at 20:1 ratio and resuspended in three-dimensional (3D) culture matrix cocktail as follows: Dulbecco'sPBS (D-PBS) containing 33% of Cultrex basement membraneextract (BME; Trevigen; 3445-005-01), 10% fetal calf serum (FCS),1 mg/mL fibrinogen (Calbiochem; 341578), 1.4 U/mL thrombin(MP Biomedicals; 02194083), 1.25 � 105 cells/mL of B16, and2.5 � 106 or 5.0 � 106 cells/mL of CD8þ T cells. Fifty-fivemicroliters of this cellþmatrix cocktail was cast in flat-bottomed96-well plates, incubated for 45 minutes to allow the matrixbecome solid and then overlaid with 100 mL of Dulbecco'smodified Eagle medium (DMEM)þ10% FCS medium on top ofthe solid gel matrix. Cultures were incubated for 4 days. Cellswere reisolated in ice-cold PBSþ5 mmol/L EDTA, washed, andresuspended in PBSþ0.5% bovine serum albumin and stainedusing anti-CD8 and the viability dye 7-amino-actinomycin D(7-AAD) (BD Pharmingen). B16 cells were quantified as a ratioof 7-AADþ to 7-AAD� cells to determine the dead:live ratio.

B16 tumor inoculationsB16 cells were a kind gift of Dr. Glenn Dranoff (Dana-Farber

Cancer Institute, Boston, MA). B16 cells were cultured to 90%confluency, trypsinized, washed in Hank's balanced salt solu-tion (HBSS), and resuspended inHBSS at 50,000 tumor cells per250 mL volume. Cells were injected subcutaneously in the leftflank of 6- to 12-week-old female C57BL/6 mice. In micereceiving no transferred T cells, palpable tumors arose 7 to10 days postinoculation. Tumor diameter was measured inthree dimensions, and mice were sacrificed when tumorvolume reached 2 cm3. For adoptive transfers, unless otherwisestated, mice received 300,000 CD8 T cells in 150 mL PBSadministered via tail vein injection within 24 hours of tumorinoculation.

Statistical analysisStatistical analysis of survival curves was conducted using

GraphPad Prism software. P values were determined using theMantel–Cox log-rank test. Delays in tumor growth wereobtained by comparison of median survival times.

ResultsGeneration of high and low affinity TRP1 transnuclearmiceVaccination with palmitoylated TRP1 peptide, anti-CD40

antibody, and the TLR3 ligand poly-IC yields a robust

population of anti-TRP1 CD8 T cells relatively quickly andprovides a source of TRP1-specific lymphocytes for SCNT.SCNT is most successful using hybrid mouse strains as asource of donor nuclei. To this end, B6� 129 F1 male agoutimice were injected intravenously with palmitoylated peptideTAPDNLGYM, anti-CD40, and poly-IC. The TRP1 peptidesequence used differed from native TRP1 in the ninthposition (A to M substitution) to increase its binding toH-2Db. This heteroclitic peptide was used to construct TRP1tetramers, and robust TRP1 tetramerþ populations wereobserved in the spleens of immunized mice at 6 and 8 dayspostimmunization (Fig. 1A). These CD8þ TRP1 tetramerþ

populations were sorted by FACS and used as a source ofdonor nuclei for 2 separate SCNT experiments. A total of105 nuclear transfers were conducted, yielding five indepen-dent embryonic stem cell lines (Fig. 1B). Of these, two re-sulted in agouti chimeric mice containing TRP1 tetramerþ

CD8þ T cells in peripheral blood (Fig. 1C).Both lines of TRP1 transnuclear mice gave germline trans-

mission, and the F1 progeny showed skewing of the CD4:CD8ratio, with a large percentage of TRP1 tetramerþ cells atbaseline (Fig. 1D). Tetramerþ CD8 T cells were sorted fromeach mouse line and subjected to 50-RACE to sequence theTCR-a and -b genes (Supplementary Fig. S1). The 6.15mice hadconsistently fewer TRP1 tetramerþ cells than their 6.17 coun-terparts, and the tetramer staining was dimmer, as judged by areduction in mean fluorescent intensity. TCR, CD3, and CD8surface expression were normal on 6.15 cells (data not shown),suggesting that the lower intensity tetramer staining could bedue to weaker binding of tetramers to the TCR. To measureTCR avidity, splenocytes from 6.15 or 6.17mice were incubatedwith TRP1 tetramer at saturating conditions, washed, andincubated for various periods of time to allow dissociation ofthe TCR–peptide–MHC complexes (Fig. 2A and B). The 6.15cells rapidly lost tetramer staining, such that by 30 minutes allcells were tetramer-negative (tetramer�) and indistinguish-able from background. The 6.17 cells lost fluorescence overtime, but a significant fraction remained tetramerþ even afterovernight incubation. Thus, CD8 T cells from 6.17 mice showslower tetramer dissociation than do 6.15 cells (t1/2¼ 41 vs. 4.9minutes); we therefore reclassified these transnuclear mouselines as TRP1high and TRP1low, respectively.

The degree to which cultured CD8 T cells can producecytokines in response to properly presented serial dilutionsof peptide correlates with antitumor function (28). Wecultured isolated CD8 T cells from TRP1 transnuclear micewith BMDCs pulsed with different amounts of TRP1 peptide,either the heteroclitic variant used for tetramer staining orthe native TRP1 sequence. TRP1low T cells were unable torespond to the heteroclitic TRP1 peptide at concentrationsless than 100 pg/mL, whereas TRP1high T cells secreted IFN-gin response to as little as 1 pg/mL peptide under the sameconditions (Fig. 2C). Both TRP1high and TRP1low cells pro-duced IFN-g in response to native TRP1 peptide, but higherconcentrations of peptide were required (Fig. 2D). In addi-tion to IFN-g , we also measured chemokine production.TRP1low cells produced RANTES and MIP-1a even whenexposed to concentrations of peptide (Fig. 2E) that yielded

TRP1 Transnuclear Mice with Antimelanoma T Cells

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negligible IL-2 or IFN-g production, confirming previousstudies showing a hierarchy of effector functions based onTCR affinity (29).

TRP-altered peptide ligands assess fine specificity ofTCR

A panel of TRP1-altered peptide variants was generated(Fig. 3A) that differed from the heteroclitic reference peptide

by single amino acid substitutions. No substitutions weremade in anchor residue positions. Sorted CD8 cells fromTRP1high and TRP1low mice were incubated with BMDCspulsed with the altered peptide ligands to assess IL-2 andIFN-g production (Fig. 3B). TRP1high T cells were more pro-miscuous than TRP1low T cells. One peptide (A1) generatedsimilar levels of both IL-2 and IFN-g from both TRP1high andTRP1low CD8 cells, showing that the lower cytokine production

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Figure 1. TRP1 transnuclear micewere derived from TRP1-specificCD8 T cells. A, C57BL/6 � 129 F1agouti male mice were immunizedwith TRIVAX (palmitoylatedTAPDNLGYM 10 mg/mouse, anti-CD40 30mg/mouse, andpoly-IC 15mg/mouse) intravenously.Splenocytes were harvested 6 or8 days postimmunization andstained with anti-CD8 and TRP1tetramer. Gated cells were sortedby FACS and used as nucleusdonors for SCNT. B, success rateof nuclear transfer. C, embryonicstem (ES) cell lines 6.15 and 6.17were injected into wild-typeblastocysts to generate chimericmice as shown. Germlinetransmission was achieved bycrossing chimeras to C57BL/6females. D, peripheral bloodmononuclear cells were harvestedfrom germline transmitted 6.15or 6.17mice and stainedwith TRP1tetramer, anti-CD4, and anti-CD8.The calculated CD4:CD8 ratio isshown below each plot. Note thelower intensity tetramer staining inthe 6.15 mouse, representative ofmore than 100 mice analyzed.

Dougan et al.





typically observed from cells from TRP1low mice is not anintrinsic limit on the ability of these cells to produce cytokine.

Minor differences between TRP1high and TRP1low cellsduring thymic development and in the naïve stateWecompared the surface profile of na€�veCD8 cells fromage-

matched TRP1 transnuclear mice and found that TRP1low cellsshow higher expression of NK1.1 and KLRG1 than TRP1high or

wild-type control cells (Fig. 4A). No differences were seen inTim3 or CD25 staining, suggesting that natural killer (NK)receptor upregulation is not a function of global activationstate, but rather an intrinsic difference established by theclonotypic TCRs.

CD5 expression correlates with intensity of TCR signalingand is tightly regulated during T cell development (30).TRP1low cells also expressed lower levels of CD5 in both the

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Figure 2. TRP1 transnuclear lines differ in TCR affinity. A, splenocytes from TRP1low (6.15) and TRP1high (6.17) transnuclear mice were stained withTRP1 tetramer to saturation, washed, and incubated at room temperature for various times before fixation and analysis by flow cytometry. Time 0 is shown.B, percentage tetramerþ cells as defined by the gates shown in (A) were calculated for each time point and normalized to time 0. Representative of 3independent experiments. C, CD8 T cells isolated from TRP1low or TRP1high mice were cocultured with BMDCs pulsed with TAPDNLGYM at the indicatedconcentrations. IFN-g was measured by ELISA of 48-hour culture supernatants. Representative of 5 independent experiments. D, CD8 T cells werecocultured as in (C) using BMDCs pulsed with the native TRP1 peptide (TAPDNLGYA). Representative of 3 independent experiments. E, CD8 T cells werecultured as in (C). RANTES and MIP-1a were analyzed by cytokine bead array using 24-hour culture supernatants; IL-2 was measured by ELISA of thesame supernatants. Representative of 3 independent experiments.






periphery and during thymic development, whereas TRP1high

cells showed a peak of CD5 expression in CD4þCD8þ thymo-cytes (Fig. 4B). TRP1high cells show a decrease in CD4þCD8þ

thymocytes and an increase in TCR b and CD3e expression atthe CD4þCD8þ stage, indicative of accelerated thymic devel-opment, as is typical of mice with prerearranged TCR genes.TRP1low cells did not show accelerated thymic development.Overall, thymic development in both TRP1 transnuclearmousestrains is near normal, and the T cells produced have a na€�vephenotype.

TRP1 transnuclear CD8 T cells are cytotoxic in vitroTo determine whether TRP1 cells could be cytotoxic, we activ-

ated TRP1high and TRP1low CD8 T cells with anti-CD3/CD28–coated beads for 7 days and cultured with TRP1 A1 peptide-pulsed GFPþ B cells mixed with unlabeled B cells at a 1:1ratio. Both high and low affinity TRP1 cells displayed specifickilling in a dose-dependent fashion, with TRP1low cells slightlyoutperforming TRP1high cells (Fig. 5A). To determine whetherTRP1 transnuclear cells could exert cytotoxic function againsttumor cells, we established 3D cocultures using B16 cells andCD8 T cells seeded together in Matrigel. B16, whether grownin culture or explanted from a mouse, expresses TRP1 proteinas evidenced by immunoblotting (Fig. 5B). Coculture ofB16 and TRP1 transnuclear cells showed an increase in theratio of dead/live tumor cells in groups that received eitherTRP1low or TRP1high CD8 T cells. These results show thatboth lines of TRP1 transnuclear cells can be cytotoxic under3D culture conditions (Fig. 5C). Again, TRP1low cells tended toshow greater cytotoxicity than TRP1high cells although thesedifferences did not reach statistical significance.

TRP1 transnuclear cells delay tumor growth in vivoTo assess their tumor-protective function in vivo, na€�ve CD8

T cells isolated fromTRP1high or TRP1lowmicewere transferredinto wild-type recipients simultaneously with subcutaneousinoculation with B16 melanoma. The B16 dose of 50,000 cellswas empirically determined such that tumors would firstbecome palpable at day 7 to 10 post-inoculation and 50% ofuntreated mice would require euthanasia by 21 days post-inoculation. Under these conditions, administration of na€�veTRP1high cells showed a modest 2-day difference in mediansurvival, whereas TRP1low cells had no discernible effect ontumor growth (Fig. 6A). Administration of multiple doses ofna€�ve CD8 T cells (at days 0, 3, and 5 post-tumor inoculation)gave similar results to single dose trials, with TRP1high cellsconferring a 2-day survival benefit and TRP1low cells having nodiscernible effect (data not shown).

We next analyzed the effect of ex vivo activation of the TRP1cells before adoptive transfer. CD8 T cells from TRP1high orTRP1lowmice were culturedwith BMDCs and TRP1 peptide for18 hours and transferred into wild-type hosts at day 1 post-inoculation with B16. Under these conditions, both TRP1high

and TRP1low cells conferred a 4-day increase in mean survival(Fig. 6B). Thus TRP1low cells acquire antitumor propertiesequal to their high-affinity counterparts upon ex vivo activa-tion. As further confirmation, CD8 T cells from the sameTRP1low donormousewere adoptively transferred either na€�ve,or after culturewith peptide-pulsed BMDCs (Fig. 6C). Althoughactivation of polyclonal CD8 T cells has a modest effect,possibly due to the selective expansion of TRP1-specific cellsfrom the polyclonal pool, activated TRP1low cells conferred anadditional 5-day increase in median survival when comparedwith polyclonal activated CD8 T cells (Fig. 6C).

Studies using transgenic CD8 T cells from Pmel mice haveshown that decreasing the number of transferred CD8 T cellsleads to increased proliferation rates and increased antitumoreffects of the transferred cells (31). In Fig. 6A–C, we adminis-tered a constant dose of 3 million TRP1 cells per mouse.

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Figure 3. Fine specificity mapping of TRP transnuclear TCR specificities.A, an altered peptide library (as shown) was generated by substitution ofnon-MHC contact residues. B, CD8 T cells isolated from TRP1high orTRP1low mice were cocultured with BMDCs pulsed with the indicatedpeptides at 100 ng/mL. IL-2 was measured by ELISA of 24-hour culturesupernatants. IFN-g was measured by ELISA of 72-hour culturesupernatants. Representative of 2 independent experiments. Error barsare SD of triplicate samples.

Dougan et al.





Peptide-activated TRP1high cells retained their antitumoractivity at lower doses, and showed optimal antitumor effectat a dose of 300,000 to 500,000 cells (Fig. 6D); the 300,000 T celldose was used for all subsequent experiments. To assesspossible synergy between TRP1high and TRP1low cells, purifiedCD8 T cells from each transnuclear mouse line were peptide-activated ex vivo and transferred into wild-type hosts, either

alone or mixed at a 1:1 ratio. The total number of transferredT cells remained constant. The combination of TRP1high plusTRP1low cells did not confer additional survival benefit beyondwhat could be seen with either population alone (Fig. 6E).

The cumulative effect of activated TRP1 transnuclear cellsagainst subcutaneous B16 caused a delay, but did not preventtumor growth. This eventual failure to control the tumor could

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Figure 4. Surface profiling and thymic development of TRP1 transnuclear CD8 cells. A, pooled spleen and lymph node cells from 6-week-old maleTRP1high, TRP1low, and C57BL/6 mice were stained with the indicated antibodies. Plots shown are gated on CD8þ cells. B, thymocytes from 6-week-oldmale TRP1high, TRP1low, and C57BL/6 mice were stained with the indicated antibodies. Histogram plots are gated on CD4þCD8þ double-positive cells(top rows) or on CD8þ single-positive cells (bottom rows). Numbers indicate median fluorescent intensities of histograms. TRP1low cells show negligibletetramer staining. To confirm genotype, naïve CD8 T cells from the same mice were stimulated with antigen-presenting cells (APCs) and TRP1 A1 peptide intissue culture, and activation was assessed by CD69 expression. %CD69þ:TRP1high ¼ 81% (no peptide ¼ 0.5%); TRP1low ¼ 35% (no peptide ¼ 0.3%).






be due to several factors: (i) incomplete activation of the TRP1transnuclear cells ex vivo; (ii) failure to traffic to the tumor;(iii) immunosuppression in the tumor microenvironment; or(iv) loss of the TRP1 epitope. To address some of these issues,we first cultured TRP1high cells with anti-CD3/CD28–coated

beads and found that 40% of mice survived (Fig. 6F). We nexttransferred na€�ve TRP1high or TRP1low cells into RAG-deficientmice to induce homeostatic proliferation of the transferred Tcells and to observe TRP1 transnuclear CD8 T cell function inthe absence of CD4þ Tregs. When challenged with B16, theRAG-deficient mice reconstituted with either TRP1high orTRP1low cells showed a 7-day delay in tumor growth (Fig.7A). The tumors harvested from RAG-deficient mice in Fig.7 weremelanotic, showing thatmelanin productionwas intact.Immunoblotting for TRP1 protein showed no decrease inoverall TRP1 levels (data not shown). Amore likely explanationfor the eventual failure of CD8 T cells to control tumor growthis exhaustion. In RAG-deficient hosts, which lack endogenousT cells, the tumor-infiltrating CD8 T cells are derived entirelyfrom the transferred TRP1 transnuclear population. BothTRP1high and TRP1low cells can migrate to the tumor, yet evenwhen they accumulate to the extent seen in RAG-deficienthosts, they eventually fail to control tumor outgrowth. Surfaceexpression of PD-1 and LAG3 was increased on TRP1 trans-nuclear cells harvested from end-stage tumors (Fig. 7B), con-sistent with exhaustion (32, 33).

DiscussionThe role of TCR affinity in the antitumor T cell response

remains a topic of debate. On the one hand, the endogenouspool of antitumorT cells is generally of low affinity, presumablybecause T cells bearing high-affinity TCRs thatmight recognizeantigens on tumors are deleted in the course of thymicdevelopment, as most such antigens represent self-antigensexpressed at supranormal levels. Tumor-specific CD8 T cellsexpress TCRs of much lower affinity than typical antiviral Tcells, as was recently quantified for 24 different human TCRs(9). The repertoire of antitumor TCRs is limited not only bythymic deletion of high-affinity self-reactive clones but also bynegative regulatory mechanisms in the tumor environment.However, high-affinity antitumor T cells can be generated inexperimental mouse models or can be expanded by ex vivoculturing of human samples. The increased use of chimericantigen receptors (34, 35), usually selected to display higheraffinities for their cognate antigens than those of typicalendogenous TCRs, demands a better understanding of thesesupraphysiologic binding affinities: how does increased TCRaffinity affect functional outcomes such as cytokine produc-tion, cytotoxicity, and memory formation? The role of TCRaffinity with respect to tumor destruction and long-termantitumor memory is therefore an important question and ata minimum would require the comparison of T cells thatrecognize the same antigen with different affinities.

Although intuitively the highest affinity T cells would seemto be the most efficacious at tumor regression, the situation islikely more complicated. In human cytomegalovirus (CMV)infection, a population of T cells with such low affinity that theyfail to bind MHC tetramers, still produces more IFN-g and ismore cytotoxic than higher affinity T cells from that sameindividual (36). Several lines of evidence suggest that a CD8 Tcell's fate is determined, or programmed, by its initial encoun-ter with antigen (37–39). Duration and strength of antigenic

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Figure 5. TRP1 transnuclear CD8 T cells are tumoricidal in vitro. A, CD8þ Tcells were harvested from the indicatedmice and cultured for 8 days withanti-CD3/CD28beadsand IL-2 (20U/mL). ActivatedTcellswerewashed,counted, and aliquoted into a 96-well round-bottomed plate at 50,000 Tcells per well. B cells were harvested from a wild-type or MHCII-GFPmouse. GFPþ B cells were incubated with 100 mmol/L TRP1 A1 peptidefor 2 hours, washed twice, counted andmixedwith non-GFP cells at a 1:1ratio. This B-cell mixture was then added to the plated T cells at theindicated ratios where E ¼ effector T cells and T ¼ total pooled B cells.After 48 hours of coculture, the cells were stained with anti-CD19 and theviability dye 7-AAD. B, lysates were prepared from cultured B16 orPanc02 cells, and from B16 tumors more than 1 cm in diameter resectedfrom tumor-bearing mice. Immunoblotting shows TRP1 proteinexpression. C, CD8 T cells were cultured for 18 hours with BMDCs andtheir cognate peptide to induce activated. Peptide-activated CD8 T cellswere counted, washed, and seeded with B16 cells in Matrigel at 10:1ratio. 3D cultures were incubated for 4 days; cells were reisolated andstained with anti-CD8 and the viability dye 7-AAD. B16 cells were gatedby FSC/SSC profile of CD8� cells. The ratio of 7-AADþ:7-AAD� cells isshown. Representative of 2 independent experiments. Error bars are SD.�, P ¼ 0.026; ��, P ¼ 0.005 versus OT-I.

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Figure 6. TRP1 CD8 T cells delay tumor growth in vivo. A, naïve CD8 T cells were harvested from TRP1high, TRP1low, or wild-type mice and transferredat 3 million cells per mouse into female C57BL/6 recipients. On the same day, 50,000 B16 tumor cells were inoculated subcutaneously.Tumor volume was measured over time, and mice were euthanized when tumor size reached 2 cm3. n ¼ 30 mice per group, pooled from 3independent experiments. ��, P ¼ 0.0001 versus polyclonal CD8. B, CD8 T cells were harvested from TRP1 transnuclear mice and cultured withBMDCs pulsed with TRP1 heteroclytic peptide for 18 hours. CD8 cells were harvested, washed, counted, and transferred to recipient C57BL/6mice at 3 million cells per mouse 1 day after inoculation with 50,000 B16 cells. n ¼ 30 mice per group, pooled from 3 independent experiments.�, P ¼ 0.0119; ��, P ¼ 0.0161 versus polyclonal CD8. C, CD8 T cells were harvested from TRP1low or C57BL/6 donor mice and transferredinto recipients either directly (naïve) or after 18 hours of culture with peptide-pulsed BMDCs (activated). Recipient mice were inoculated with B16cells on the same day as naïve cell transfer, or 1 day before activated cell transfer. ��, P ¼ 0.001 versus polyclonal CD8 naïve; ��, P ¼ 0.0005versus polyclonal CD8 activated. D, CD8 T cells were harvested from TRP1 transnuclear mice and cultured with BMDCs pulsed with TRP1heteroclytic peptide for 18 hours. CD8 cells were harvested, washed, counted, and transferred to recipient C57BL/6 mice at the indicated numberof cells per mouse 1 day after inoculation with 50,000 B16 cells. n ¼ 10 mice per group. �, P ¼ 0.0361; ��, P ¼ 0.012; ��, P ¼ 0.0045;��, P ¼ 0.0011; ��, P < 0.0001 versus polyclonal CD8. E, CD8 T cells were harvested from TRP1 transnuclear mice and cultured withBMDCs pulsed with TRP1 heteroclytic peptide for 18 hours. CD8 cells were harvested, washed, counted, and transferred to recipient C57BL/6 miceat 300,000 cells per mouse 1 day after inoculation with 50,000 B16 cells. n ¼ 10 mice per group. �, P ¼ 0.0047; ��, P ¼ 0.0008; ��, P ¼ 0.0004versus polyclonal CD8. F, CD8 T cells were harvested from C57BL/6 mice or TRP1high mice and cultured for 5 days with anti-CD3/CD28–coated beads. CD8 cells were harvested, washed, counted, and transferred into C57BL/6 recipient mice at 300,000 cells per mouse 1 dayafter inoculation with 50,000 B16 cells. ��, P ¼ 0.0021.






stimulation are inversely correlated with CD8 memory celldifferentiation during acute viral infection (38, 39). Low-affinityT cells can increase their functional avidity by upregulatingexpression of CD8. Conversely, high-affinity T cells downre-gulate CD8 expression to avoid antigen-induced cell death (39,40). Upon encountering abundant antigen, high-avidity CD8 Tcells cease proliferation and initiate apoptosis, although theycan still exert cytotoxic function before death (18).

Adoptive cell transfer therapy, as pioneered by Rosenbergand colleagues, selects for the highest affinity CD8 T cells andaims for maximal activation in vitro before infusion into thepatient. This strategy has been remarkably successful in alimited clinical population (41–43), and suggests that high-affinity, maximally activated CD8 T cells confer the maximalantitumor response. Likewise, comparison of two different T

cell lines specific for the same model antigen overexpressed inthe pancreas showed that the higher affinity clone 4 cells werebetter able to expand in the pancreatic environment and causetumor destruction, as measured by increased serum glucose(44) although low-affinity clone 1 cells were able to mediatetumor destruction when pairedwith antigen-specific CD4 cells(45). However, when the same clone 4 cells were injected intomice with hemagglutinin (HA)-expressing renal cell carcino-mas, the antitumor effects of high-affinity clone 4 cells werecurtailed by suppressive mechanisms in the tumor microen-vironment (46). Sequence analysis of TCRs fromTcells inducedby effective versus ineffective vaccine strategies shows thathigher affinity T cells were induced by the effective vaccines,although the KD values measured for each group differed byless than 2-fold (47).

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Figure 7. TRP1 transnuclear cellsmigrating to the tumor show an exhausted phenotype. A, RAG2�/�micewere inoculatedwith 100,000B16 cells 1 day beforetransfer of naïve CD8þ T cells from TRP1high or TRP1low transnuclear mice. Tumor volume was measured over time. ��, P ¼ 0.001. Representative of 3independent experiments. B, end-stage tumors frommice in (A) were harvested, digestedwith collagenase, and tumor-infiltrating lymphocyteswere analyzedby flow cytometry after staining with the indicated antibodies. n ¼ 5 mice per group.

Dougan et al.





On the other hand, several studies have suggested that anoptimal CD8 T cell response uses low- to moderate-affinityTCRs. Retroviral transduction of T cells with a panel of TCRsof known gradations in affinity showed that the highestaffinity TCRs were absent from tumor-infiltrating CD8 Tcells, although these TCRs could be found on tumor-infil-trating CD4 cells (48). A panel of NY-ESO1–specific T celllines engineered to express TCRs with affinities ranging fromlow to supraphysiologic showed that optimal cytotoxicityand calcium flux occurred using TCRs with intermediate KD

values of 1 to 5 mmol/L with a decline in functionality forhigher affinity TCRs (49, 50). A similar ceiling was observedusing vaccination with peptide mimetics with a range ofaffinities for a given T cell clone (51). A recent studyevaluated a large panel of human anti-melanoma cell linesfor peptide sensitivity, production of various cytokines, andTCR affinity as defined by the kinetics of multimer binding.Although peptide sensitivity and production of IL-2, TNF-a,and IFN-g correlated with tumor cell killing, multimerbinding failed to correlate with any of the functional para-meters measured (28), suggesting that TCR affinity alonecould not predict antitumor responses. This raises theinteresting possibility that attributes other than affinity, yetassociated with a particular TCR clonotype, may determinethe functional property of a T cell. Using tumors transducedwith altered peptide ligands for OT-I CD8 T cell recognition,low-affinity TCR interactions generated enhanced cytokineproduction during both primary and secondary responses,but the lower affinity T cells were more susceptible to TGF-b–mediated suppression (52). An interesting study com-pared the TCR affinity of the endogenous CD8 response toOVA-expressing glioma as a function of the vaccine injectionsite (53). Mice vaccinated at sites proximal to the tumorgenerated lower affinity CD8 T cells than mice vaccinated atmore distant sites, which could implicate suppressivemechanisms that specifically limit high-affinity CD8 primingin the tumor-draining lymph nodes (53).In the TRP1 transnuclear system, we show that an approx-

imately 10-fold difference in tetramer dissociation rates anda 100-fold difference in peptide responsiveness have almostno effect on the overall antitumor function of the transferredCD8 T cells. How do the low-affinity T cells manage toperform as well as their high-affinity counterparts? Althoughtetramer dissociation and IFN-g production showed cleardistinctions between the two TRP1-specific transnuclearlines, we observed much less of a difference when comparingcytotoxic function in a 3D culture model. Likewise, bothTRP1high and TRP1low cells produced the chemokinesRANTES and MIP-1a when stimulated with picomolar con-centrations of native TRP1 peptide, conditions under whichno IL-2 or IFN-g could be detected in supernatants from theTRP1low cultures. Because the concentration of antigenpresented in a tumor-bearing mouse is likely to be small,the fact that TRP1low cells produce monocyte-attractingchemokines in response to low doses of peptide could berelevant for how TRP1low CD8 T cells influence the tumormicroenvironment. Likewise, the increased IL-2 productionfrom TRP1high cells could enhance Treg recruitment or

proliferation in the tumor microenvironment, although suchan effect would be eliminated in RAG-deficient hosts, whichlack Tregs. Another likely possibility is increased activation-induced cell death of TRP1high cells in the tumor environ-ment, which could limit the efficacy of high-affinity CD8 Tcells (54, 55). Indeed, TRP1high tumor-infiltrating cells showreduced CD8 expression, indicative of a more activated state.

Here, we have generated a pair of mice with T cells specificfor the same MHC–peptide combination but differing in affi-nity by one to two orders of magnitude. The TCR a and TCR bchains are under the control of their endogenous promoters,and expressed at physiologic levels. Both TCRs were selectedby directly harvesting tetramerþ CD8 cells from a vaccinatedmouse without any selection in tissue culture, and thus arerepresentative of the normal pool of activated CD8 T cells thatarises during an immune response. As such, we anticipatethat the TRP1 transnuclear mice reported here may be usefulfor the studies of T cell development and T cell activation.

Although TRP1 is a self-antigen expressed in healthy mel-anocytes, we did not observe vitiligo in TRP1high or TRP1low

mice on a RAG-proficient background, suggesting a possiblerole for CD4þ Tregs. Tregs are absent in TRP1highRAG2�/�

mice, and these mice develop a mild spontaneous vitiligoaround 3 months of age, affecting primarily the periorbitalregion. The Tregs that arise in TRP1 transnuclear mice areconstrained to use a single TCR Vb8.2 or TCR Vb5, respect-ively, thus limiting the diversity of potential Tregs specificitiesin these mice. Nevertheless, the Tregs that develop are appar-ently adequate to prevent autoimmunity even in the presenceof a high fraction of autoreactive CD8 T cells.

TRP1 can serve as a tumor rejection antigen, and we show amodest antitumor effect of anti-TRP1 CD8 T cells as a mono-therapy. Although the effects are small, the delay in tumorgrowth and slight increase in overall survival parallels thehuman situation, where monotherapies such as anti-CTLA-4also show modest clinical benefit (56). A future challengefor tumor immunology will be to determine which therapiessynergize to achieve maximum clinical effect. CD8 T cellexhaustion is commonly observed in human patients, andmust be overcome for immunotherapy to succeed. The TRP1mice offer an excellent preclinical platform for testing theimmunomodulatory effects of potential new drugs or drugcombinations.

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

Authors' ContributionsConception and design: S.K. Dougan, M. Dougan, J. Kim, J.A. Turner,R. Jaenisch, E. Celis, H.L. PloeghDevelopment of methodology: S.K. Dougan, S. Ogata, H.-I. Cho, E. CelisAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.K. Dougan, M. Dougan, S. OgataAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.K. Dougan, M. Dougan, J. Kim, J.A. Turner, H.L.PloeghWriting, review, and/or revision of the manuscript: S.K. Dougan,M. Dougan, J. Kim, J.A. Turner, E. Celis, H.L. PloeghAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J. Kim, J.A. Turner, H.-I. ChoStudy supervision: R. Jaenisch






AcknowledgmentsThe authors thank Patti Wisniewski and Chad Araneo for cell sorting, and

John Jackson for mouse husbandry.

Grant SupportS.K. Dougan was funded by the Cancer Research Institute and by the Bushrod

H. Campbell and Adah F. Hall Charity Fund and Harold Whitworth PierceCharitable Trust. S. Ogatawas funded by Janssen PharmaceuticaNV. E. Celis, H.L.Ploegh, and R. Jaenisch are funded by grants from the NIH. S.K. Dougan and H.L.

Ploegh are funded by Janssen Pharmaceuticals, Inc. and by the AACR-PancreaticCancer Action Network.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 28, 2013; revised June 12, 2013; accepted June 13, 2013;published OnlineFirst July 3, 2013.

References1. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A,Mlecnik B, Lagorce-

Pages C, et al. Type, density, and location of immune cells withinhuman colorectal tumors predict clinical outcome. Science 2006;313:1960–4.

2. Hodi FS, Butler M, Oble DA, Seiden MV, Haluska FG, Kruse A, et al.Immunologic and clinical effects of antibody blockade of cytotoxic Tlymphocyte-associated antigen 4 in previously vaccinated cancerpatients. Proc Natl Acad Sci U S A 2008;105:3005–10.

3. Dougan M, Dranoff G. Immune therapy for cancer. Annu Rev Immunol2009;27:83–117.

4. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, HaanenJB, et al. Improved survival with ipilimumab in patients with metastaticmelanoma. N Engl J Med 2010;363:711–23.

5. Korman AJ, Peggs KS, Allison JP. Checkpoint blockade in cancerimmunotherapy. Adv Immunol 2006;90:297–339.

6. Brahmer JR, Tykodi SS, Chow LQ, HwuWJ, Topalian SL, Hwu P, et al.Safety and activity of anti-PD-L1 antibody in patients with advancedcancer. N Engl J Med 2012;366:2455–65.

7. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDer-mott DF, et al. Safety, activity, and immune correlates of anti-PD-1antibody in cancer. N Engl J Med 2012;366:2443–54.

8. Pardoll DM. The blockade of immune checkpoints in cancer immu-notherapy. Nat Rev Cancer 2012;12:252–64.

9. Aleksic M, Liddy N, Molloy PE, Pumphrey N, Vuidepot A, Chang KM,et al. Different affinity windows for virus and cancer-specific T cellreceptors: implications for therapeutic strategies. Eur J Immunol2012;42:3174–9.

10. Bouneaud C, Kourilsky P, Bousso P. Impact of negative selection onthe T cell repertoire reactive to a self-peptide: a large fraction of T cellclones escapes clonal deletion. Immunity 2000;13:829–40.

11. Nugent CT, Morgan DJ, Biggs JA, Ko A, Pilip IM, Pamer EG, et al.Characterization of CD8þ T lymphocytes that persist after peripheraltolerance to a self antigen expressed in the pancreas. J Immunol2000;164:191–200.

12. Willimsky G, Blankenstein T. Sporadic immunogenic tumours avoiddestruction by inducing T cell tolerance. Nature 2005;437:141–6.

13. Dougan SK, Ogata S, Hu CC, Grotenbreg GM, Guillen E, Jaenisch R,et al. IgG1þ ovalbumin-specific B-cell transnuclear mice show classswitch recombination in rare allelically included B cells. Proc Natl AcadSci U S A 2012;109:13739–44.

14. Kirak O, Frickel EM, Grotenbreg GM, Suh H, Jaenisch R, Ploegh HL.Transnuclearmicewith pre-defined T cell receptor specificities againstToxoplasma gondii obtained via SCNT. J Vis Exp 2010:2168.

15. Kirak O, Frickel EM, Grotenbreg GM, Suh H, Jaenisch R, Ploegh HL.Transnuclear mice with predefined T cell receptor specificities againstToxoplasma gondii obtained via SCNT. Science 2010;328:243–8.

16. Sehrawat S, Kirak O, Koenig PA, IsaacsonMK, Marques S, Bozkurt G,et al. CD8(þ) T cells frommice transnuclear for a TCR that recognizes asingle H-2K(b)-restricted MHV68 epitope derived from gB-ORF8 helpcontrol infection. Cell Rep 2012;1:461–71.

17. Curran MA, Montalvo W, Yagita H, Allison JP. PD-1 and CTLA-4combination blockade expands infiltrating T cells and reduces regu-latory T and myeloid cells within B16 melanoma tumors. Proc NatlAcad Sci U S A 2010;107:4275–80.

18. Quezada SA, Peggs KS, Curran MA, Allison JP. CTLA4 blockade andGM-CSF combination immunotherapy alters the intratumor balance ofeffector and regulatory T cells. J Clin Invest 2006;116:1935–45.

19. Taniguchi K, Karre K, Klein G. Lung colonization and metastasis bydisseminated B16 melanoma cells: H-2 associated control at the levelof the host and the tumor cell. Int J Cancer 1985;36:503–10.

20. Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H, Brose K, et al.Vaccination with irradiated tumor cells engineered to secrete murinegranulocyte-macrophage colony-stimulating factor stimulates potent,specific, and long-lasting anti-tumor immunity. Proc Natl Acad SciU S A 1993;90:3539–43.

21. Dougan M, Dougan S, Slisz J, Firestone B, VannemanM, Draganov D,et al. IAP inhibitors enhance co-stimulation to promote tumor immu-nity. J Exp Med 2010;207:2195–206.

22. Cho HI, Celis E. Optimized peptide vaccines eliciting extensive CD8 Tcell responses with therapeutic antitumor effects. Cancer Res 2009;69:9012–9.

23. Cho HI, Lee YR, Celis E. Interferon gamma limits the effectiveness ofmelanoma peptide vaccines. Blood 2011;117:135–44.

24. Hochedlinger K, Jaenisch R. Monoclonal mice generated by nucle-ar transfer from mature B and T donor cells. Nature 2002;415:1035–8.

25. Frickel EM, Sahoo N, Hopp J, Gubbels MJ, Craver MP, Knoll LJ, et al.Parasite stage-specific recognition of endogenous Toxoplasma gon-dii-derived CD8þ T cell epitopes. J Infect Dis 2008;198:1625–33.

26. Gredmark-Russ S, Cheung EJ, Isaacson MK, Ploegh HL, GrotenbregGM. The CD8 T cell response against murine gammaherpesvirus 68 isdirected toward a broad repertoire of epitopes from both early and lateantigens. J Virol 2008;82:12205–12.

27. Grotenbreg GM, Roan NR, Guillen E, Meijers R, Wang JH, Bell GW,et al. Discovery of CD8þ T cell epitopes in Chlamydia trachomatisinfection through use of caged class I MHC tetramers. Proc Natl AcadSci U S A 2008;105:3831–6.

28. Wilde S, SommermeyerD, LeisegangM, Frankenberger B,Mosetter B,Uckert W, et al. Human antitumor CD8þ T cells producing Th1 poly-cytokines show superior antigen sensitivity and tumor recognition.J Immunol 2012;189:598–605.

29. Laugel B, van den Berg HA, Gostick E, Cole DK,Wooldridge L, BoulterJ, et al. Different T cell receptor affinity thresholds and CD8 coreceptordependence govern cytotoxic T lymphocyte activation and tetramerbinding properties. J Biol Chem 2007;282:23799–810.

30. Azzam HS, Grinberg A, Lui K, Shen H, Shores EW, Love PE. CD5expression is developmentally regulated by T cell receptor (TCR)signals and TCR avidity. J Exp Med 1998;188:2301–11.

31. Rizzuto GA, Merghoub T, Hirschhorn-Cymerman D, Liu C, LesokhinAM, Sahawneh D, et al. Self-antigen-specific CD8þ T cell precursorfrequency determines the quality of the antitumor immune response.J Exp Med 2009;206:849–66.

32. WooSR, TurnisME,GoldbergMV, Bankoti J, SelbyM,Nirschl CJ, et al.Immune inhibitory molecules LAG-3 and PD-1 synergistically regulateT cell function to promote tumoral immune escape. Cancer Res2012;72:917–27.

33. Wherry EJ, Ha SJ, Kaech SM, Haining WN, Sarkar S, Kalia V, et al.Molecular signature of CD8þ T cell exhaustion during chronic viralinfection. Immunity 2007;27:670–84.

34. Scholler J, Brady TL, Binder-Scholl G, HwangWT, Plesa G, Hege KM,et al. Decade-long safety and function of retroviral-modified chimericantigen receptor T cells. Sci Transl Med 2012;4:132ra53.

35. Lanitis E, Poussin M, Klattenhoff A, Song D, Sandaltzopoulos R, JuneC, et al. Chimeric antigen receptor T cells with dissociated signaling

Dougan et al.





domains exhibit focused antitumor activity with reduced potential fortoxicity in vivo. Cancer Immunol Res. 2013Apr 7. [Epub ahead of print].

36. KhanN, CobboldM, Cummerson J,Moss PA. Persistent viral infectionin humans can drive high frequency low-affinity T cell expansions.Immunology 2010;131:537–48.

37. Kalia V, Sarkar S, Ahmed R. CD8 T cell memory differentiation duringacute and chronic viral infections. Adv Exp Med Biol 2010;684:79–95.

38. Sarkar S, Kalia V, HainingWN, Konieczny BT, Subramaniam S, AhmedR. Functional and genomic profiling of effector CD8 T cell subsets withdistinct memory fates. J Exp Med 2008;205:625–40.

39. Sarkar S, Teichgraber V, Kalia V, Polley A, Masopust D, Harrington LE,et al. Strength of stimulus and clonal competition impact the rate ofmemory CD8 T cell differentiation. J Immunol 2007;179:6704–14.

40. Feinerman O, Veiga J, Dorfman JR, Germain RN, Altan-Bonnet G.Variability and robustness in T cell activation from regulated hetero-geneity in protein levels. Science 2008;321:1081–4.

41. MackensenA,MeidenbauerN, Vogl S, LaumerM,Berger J, AndreesenR. Phase I study of adoptive T cell therapy using antigen-specificCD8þ

T cells for the treatment of patients with metastatic melanoma. J ClinOncol 2006;24:5060–9.

42. Yee C, Thompson JA, Byrd D, Riddell SR, Roche P, Celis E, et al.Adoptive T cell therapy using antigen-specific CD8þ T cell clones forthe treatment of patients with metastatic melanoma: in vivo persis-tence, migration, and antitumor effect of transferred T cells. Proc NatlAcad Sci U S A 2002;99:16168–73.

43. Yee C, Thompson JA, Roche P, Byrd DR, Lee PP, Piepkorn M, et al.Melanocyte destruction after antigen-specific immunotherapy of mel-anoma: direct evidence of t cell-mediated vitiligo. J Exp Med 2000;192:1637–44.

44. Bos R, Marquardt KL, Cheung J, Sherman LA. Functional differencesbetween low- and high-affinity CD8(þ) T cells in the tumor environ-ment. Oncoimmunology 2012;1:1239–47.

45. Lyman MA, Nugent CT, Marquardt KL, Biggs JA, Pamer EG, ShermanLA. The fate of low affinity tumor-specific CD8þ T cells in tumor-bearing mice. J Immunol 2005;174:2563–72.

46. Janicki CN, Jenkinson SR, Williams NA, Morgan DJ. Loss of CTLfunction among high-avidity tumor-specific CD8þ T cells followingtumor infiltration. Cancer Res 2008;68:2993–3000.

47. Jordan KR, Buhrman JD, Sprague J, Moore BL, Gao D, Kappler JW,et al. TCR hypervariable regions expressed by T cells that respond toeffective tumor vaccines. Cancer Immunol Immunother 2012;61:1627–38.

48. Chervin AS, Stone JD, Soto CM, Engels B, Schreiber H, Roy EJ,et al. Design of T cell receptor libraries with diverse binding prop-erties to examine adoptive T cell responses. Gene Ther 2013;20:634–44.

49. Irving M, Zoete V, Hebeisen M, Schmid D, Baumgartner P, Guil-laume P, et al. Interplay between T cell receptor binding kinetics andthe level of cognate peptide presented by major histocompatibilitycomplexes governs CD8þ T cell responsiveness. J Biol Chem 2012;287:23068–78.

50. Schmid DA, Irving MB, Posevitz V, Hebeisen M, Posevitz-Fejfar A,Sarria JC, et al. Evidence for a TCR affinity threshold delimitingmaximal CD8 T cell function. J Immunol 2010;184:4936–46.

51. McMahan RH, McWilliams JA, Jordan KR, Dow SW, Wilson DB,Slansky JE. Relating TCR–peptide–MHC affinity to immuno-genicity for the design of tumor vaccines. J Clin Invest 2006;116:2543–51.

52. O'Sullivan JA, Zloza A, Kohlhapp FJ, Moore TV, Lacek AT, Dulin NO,et al. Primingwith very low-affinity peptide ligands gives rise to CD8(þ)T cell effectorswith enhanced functionbutwith greater susceptibility totransforming growth factor (TGF)beta-mediated suppression. CancerImmunol Immunother 2011;60:1543–51.

53. Ohlfest JR, Andersen BM, Litterman AJ, Xia J, Pennell CA, Swier LE,et al. Vaccine injection sitematters: qualitative andquantitative defectsin CD8 T cells primed as a function of proximity to the tumor in amurineglioma model. J Immunol 2012;190:613–20.

54. Tabbekh M, Franciszkiewicz K, Haouas H, Lecluse Y, Benihoud K,Raman C, et al. Rescue of tumor-infiltrating lymphocytes from acti-vation-induced cell death enhances the antitumor CTL response inCD5-deficient mice. J Immunol 2011;187:102–9.

55. Kim EY, Teh SJ, Yang J, Chow MT, Teh HS. TNFR2-deficient memoryCD8 T cells provide superior protection against tumor cell growth.J Immunol 2009;183:6051–7.

56. Vanneman M, Dranoff G. Combining immunotherapy and targetedtherapies in cancer treatment. Nat Rev Cancer 2012;12:237–51.






2013;1:99-111. Published OnlineFirst July 2, 2013.Cancer Immunol Res Stephanie K. Dougan, Michael Dougan, Jun Kim, et al. TCRs Show Equivalent Antitumor ActivityTransnuclear TRP1-Specific CD8 T Cells with High or Low Affinity

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