car-t cells inﬂict sequential killing of multiple tumor target cells · currently, it is known...

Research Article

CAR-T Cells Inflict Sequential Killing of MultipleTumor Target CellsAlexander J. Davenport1,2,3, Misty R. Jenkins1,2, Ryan S. Cross2,4, Carmen S. Yong1,2,H. Miles Prince5, David S. Ritchie1,2,3,6, Joseph A. Trapani1,2, Michael H. Kershaw1,2,Phillip K. Darcy1,2, and Paul J. Neeson1,2

Abstract

Adoptive therapy with chimeric antigen receptor (CAR) T cellsshows great promise clinically. However, there are importantaspects of CAR-T-cell biology that have not been explored, partic-ularly with respect to the kinetics of activation, immune synapseformation, and tumor cell killing.Moreover, the effects of signalingvia the endogenous T-cell receptor (TCR) orCARonkilling kineticsare unclear. To address these issues, we developed a novel trans-genic mouse (designated CAR.OT-I), in which CD8þ T cellscoexpressed the clonogenic OT-I TCR, recognizing the H-2Kb

–

presented ovalbumin peptide SIINFEKL, and an scFv specific forhumanHER2. PrimedCAR.OT-I T cellsweremixedwithSIINFEKL-pulsed or HER2-expressing tumor cells and visualized in real-timeusing time-lapse microscopy. We found that engagement via CAR

or TCR did not affect cell death kinetics, except that the time fromdegranulation to CAR-T-cell detachment was faster when CAR wasengaged. We showed, for the first time, that individual CAR.OT-Icells can kill multiple tumor cells ("serial killing"), irrespective ofthemodeof recognition.At loweffector:target ratios, the tumor cellkilling rate was similar via TCR or CAR ligation over the first 20hours of coincubation. However, from 20 to 50 hours, tumor celldeath mediated through CAR became attenuated due to CARdownregulation throughout the time course. Our study providesimportant insights intoCAR-T–tumor cell interactions, with impli-cations for single- or dual receptor–focused T-cell therapy. CancerImmunol Res; 3(5); 483–94. �2015 AACR.

See related commentary by June, p. 470

IntroductionChimeric antigen receptor (CAR) T cells are at the forefront

of cell-based therapies for the successful treatment of cancer.CAR-T cells redirected to CD19 (CAR19) have shown spectac-ular results in clinical trials for patients with chronic lympho-cytic leukemia (CLL) and acute lymphocytic leukemia (ALL;refs. 1–6). Although the findings for CAR-T-cell therapy insolid tumors have been less convincing, objective clinicalresponses have been observed (7, 8). Despite these exciting

advances, there are still important aspects of CAR-T-cell biol-ogy that remain unexplored.

Currently, it is known that CAR-T-cell binding to target cellsfollowing antigen recognition results in target cell death andrelease of effector cytokines IFNg , IL2, and TNF (9). Further-more, preclinical studies have demonstrated that perforinand Granzyme B were required for CAR-T-cell efficacy in vivo(10, 11). Nevertheless, relatively little is known about thekinetics of (CAR-T tumor cell) immune synapse formation,release of the cytotoxic effector molecules, and tumor cellapoptosis. It is unclear whether these fundamental steps oftumor killing by T cells are different following stimulation bythe T-cell receptor (TCR) or CAR. Furthermore, although it isknown that individual cytotoxic lymphocytes (NK and CD8þ Tcells) can sequentially kill multiple targets ("serial killing";refs. 12–15), this has not yet been described for CAR-T cells.Understanding the cell interactions and kinetics of CAR-T-cellkilling of tumor cells will provide important base informationagainst which new, improved CARs can be compared and leadto increasing the effectiveness of this approach.

In this study, we developed a model system to explorewhether a difference exists in recognition and killing of tumorcells following stimulation through the TCR or CAR. We usedtime-lapse live microscopy to record the kinetics of CAR-T-cellinteractions with tumor cells and tumor cell killing, and com-pared these parameters in cocultures in which CAR-T cells wereactivated via their TCR versus CAR. Using this strategy, werevealed important information regarding the kinetics ofCAR-T-cell interactions with tumor cells and investigatedwhether CAR-T cells have the capacity to mediate serial killingof target cells.

1Cancer Immunology Research, Peter MacCallum Cancer Center, EastMelbourne, Victoria, Australia. 2Sir Peter MacCallum Department ofOncology, University of Melbourne, Parkville,Victoria, Australia. 3TheACRF Translational Research Laboratory, Royal Melbourne Hospital,Parkville, Victoria, Australia. 4Differentiation and Transcription Labo-ratory, Peter MacCallum Cancer Center, East Melbourne, Victoria,Australia. 5Department of Cancer Medicine, Peter MacCallum CancerCenter, East Melbourne, Victoria, Australia. 6Department of ClinicalHaematology and Bone Marrow Transplantation, Royal MelbourneHospital, Parkville, Victoria, Australia.

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

Corrected online February 16, 2018.

A.J. Davenport and M.R. Jenkins share first authorship of this article.

P.K. Darcy and P.J. Neeson share senior authorship of this article.

Corresponding Authors: Paul J. Neeson and Phillip K. Darcy, Peter MacCallumCancer Centre, St. Andrews Place, East Melbourne, Melbourne 3002, Australia.Phone:þ61 3 96563657, Fax: 613-9656-1411; E-mail: [email protected];[email protected]

doi: 10.1158/2326-6066.CIR-15-0048

�2015 American Association for Cancer Research.

CancerImmunologyResearch

www.aacrjournals.org 483

on February 18, 2021. © 2015 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst February 24, 2015; DOI: 10.1158/2326-6066.CIR-15-0048





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Materials and MethodsCell culture

MC57 cells (mouse fibrosarcoma cell line) or MC57 cellsretrovirally transduced with the HER2 antigen (MC57-HER2)were cultured in complete media, RPMI-1640 comprising 10%FCS, 1 mmol/L sodium pyruvate, 2 mmol/L glutamine,0.1 mmol/L nonessential amino acids, 100 U/mL penicillin–streptomycin (Invitrogen; Life Technologies), and 5 mmol/L 2ME.Tumor lines were incubated at 37�C with 5% CO2 and grown to80% confluence in T-175 tissue culture flasks before passage.These cell lines were originally obtained from the ATCC, activelypassaged for less than 6 months and were authenticated usingshort tandem repeat profiling. United Kingdom CoordinatingCommittee on Cancer Research (UKCCCR) guidelines for theuse of cell lines in cancer researchwere followed. Tumor lineswerealso verified to beMycoplasma negative by the Victorian InfectiousDiseases References Laboratory (Melbourne, Victoria) by PCRanalysis.

MiceOT-I transgenic mice (16) were bred at the Peter MacCallum

Cancer Centre (PMCC). A unique C57BL/6 transgenic mousemodel, expressing a second generation CAR (anti-human HER-2 scFv and signaling domains for CD28 and CD3z) under the Vavpromoter was previously generated at the PMCC (YongC; unpub-lished data). The anti-HER2 scFv CAR construct used in this studywas previously described in detail (17), and uses an anti-HER2scFv, which was a kind gift from Winfred Wels (18). These CARmice were then bred with OT-I mice to create CAR.OT-I mice,which expressed both the OT-I TCR and the CAR on CD8þ T cells.

Activation and differentiation of T cellsNa€�ve splenocytes were harvested from OT-I or CAR.OT-I

mice and activated in the presence of 10 nmol/L SIINFEKL and100 U/mL IL2 in complete media. Splenocytes were incubatedfor three days at 37�C, and viable T cells recovered followingdensity gradient centrifugation. T cells were washed in andresuspended in complete media at a density of 1� 106/mL with100 U/mL recombinant human IL2 for an additional 4 days.These activated effector CD8þ T cells (CAR.OT-I or OT-I) wereused for subsequent tumor killing studies and in microscopyanalysis and are referred to as CTL throughout this study.

Flow cytometryEffector CTLs were labeled with antibodies in FACS buffer

(Magnesium and Calcium free PBS þ 2% FCS) with anti-CD44BV785, anti-CD62L BV510, and anti-CD8 BV711 (BioLegend),anti-CD107a PE (BD Biosciences), anti–c-myc TAG AF488 (CellSignaling Technology) and the viability dye Fixable blue (Invitro-gen) for 30minutes at 4�C.GranzymeBwas detected using the BDIntracellular Staining Kit according to the manufacturer's instruc-tions by labeling with anti–Granzyme B-APC (Clone GB11; BDBiosciences) antibody. This antibody is anti-human but shown tobe cross-reactive with mouse Granzyme B (19). Cells were sub-sequently analyzed using the LSR II (BD Biosciences) and finalanalysis was performed using the FlowJo software (Tree Star).

T-cell proliferation assayProliferation of splenocytes from na€�ve to day 3 (OT-I or

CAR.OT-I) was detected by performing a cell trace violet (CTV;

Invitrogen) dilution assay. Splenocytes were stained with 2.5mmol/L CTV for 20 minutes at 37�C before washing andcoculturing with MC57 cells pulsed with 10 nmol/L SIINFEKL.Cells were harvested 3 days later, and labeled for CD8 (BV711-BioLegend) and viability with Fixable blue dye (Invitrogen).Stained cells were analyzed on a BD LSRII (BD Biosciences) andCTV dilution assessed on CD8þ cells. Control wells containedsplenocytes alone.

Quantification of receptor and tumor target antigen moleculedensity

A FACS-based assay was used to quantify the density of CAR.OT-I cell antigen receptors (OT-I and CAR), and tumor antigen(OVA or HER2) inmolecules per cell. Effector OT-I or CAR.OT-I Tcells were stained for CAR expression with an anti–c-myc tagAF488 antibody (Cell Signaling Technology) or an anti–Va2-PEantibody (BD Biosciences), recognizing the TCR subunit specificto OT-I T cells. Target cells were stained for their expression of H-2Kb (BioLegend), H-2Kb SIINFEKL (25D1.16 PE; BioLegend) orHER2 (Mouse anti-human HER2 primary and anti-mouse sec-ondary PE; BioLegend). A proprietary bead standard kit (SimplyCellular Beads, BANGSLABS laboratories) was stained using thesame antibodies to create a standard curve with a known numberof binding sites per bead. The mean fluorescence intensity (MFI)for each receptor and bead combination was recorded and com-pared with a standard curve, and a data reduction performed inMS EXCEL. The final receptor density per cell was recorded usingGraphPad Prism software.

Chromium release assayTumor cell targets were labeled with 100 mCi 51Cr � 1 mmol/L

SIINFEKL, at 37�C in T-cell media, before washing and seeding at1� 105/mL. CTLswere added at various effector:target (E:T) ratiosand incubated at 37�C for 4 hours. Chromium release was readusing the Wallac Wizard 1470 (PerkinElmer), and the percentageof lysis calculated using the following formula: [(experimentalcounts/minute � spontaneous counts/minute)/(total counts/minute� spontaneous counts/minute)]�100. Results are shownas the percentage of cytotoxicity (mean� SEM), using pooled datafrom five experiments.

xCELLigence killing assaysMC57 or MC57-HER2 cells (2 � 104/well) were left to adhere

for 16 hours to xCELLigence E-plates (Cat 67710; ACEA Biosci-ence). On the day of assay, peptide was added (�1 mmol/LSIINFEKL (MC57-OVA257) before washing and adding CTLs(d7 OT-I or CAR.OT-I) at an E:T ratio of 10:1 and 5:1 for 8 hours(Fig. 4), or an E:T ratio of 1:1 and 1:4 for 50 hours (Fig. 5). Anelectrical current was applied to each coculture (20, 21) in thexCELLigence assay. Adherent cells (MC57) are resistant; however,as the effector cells kill the MC57 cells and they detach fromthe plate, the electrical resistance in the coculture decreases.Electrical resistance as arbitrary units (AU) over time (minutes)was monitored and the rate of killing detected by the slope of theresistance curve. E-plates were read every 2minutes and data werenormalized to starting resistance, using the formula: x¼ resistancereading þ10 and y ¼ (xbasereading/xbasereading)/(xn time point/xbasereading), where y is the final AU plotted as fold change against timein GraphPad Prism software to create kinetic curves.

Davenport et al.

Cancer Immunol Res; 3(5) May 2015 Cancer Immunology Research484




Perforin protein detection by Western blot analysisTotal cell extracts were prepared from OT-I or CAR.OT-I CTLs

using lysis buffer (25mmol/LHepes, 0.25mol/LNaCl, 2.5mmol/L EDTA, 0.1% Triton X-100), in combination with completeProtease Inhibitor Cocktail (Roche diagnostics). SDSPAGE wasperformed on cell extracts under reducing conditions, using Bis-Tris 4% to 12% Bis-Tris gel (Invitrogen). Following transfer ontopolyvinylidene difluoride (PVDF) membrane (Amersham GEHealthcare), aWestern blot analysis was performed using primaryantibodies for rat anti-human perforin (clone P1-8; ref. 22) andmouse anti-human b-actin (Sigma-Aldrich), overnight at 4�C.PVDF membranes were probed using horseradish peroxidase–conjugated anti-rat IgG or anti-mouse IgG secondary antibodies(Dako) followed by the chemiluminescence developer, ECL (GEHealthcare). Protein bandswere imagedusing Image Lab software(Bio-Rad).

CD107a degranulation assayTumor cells (MC57, MC57-OVA257, and MC57-HER2) were

cocultured at 1 � 105/mL in a 96-well plate, with 1 � 106/mL ofCTLs, in the presence of 0.5 mg of anti-CD107a PE antibody (BDBiosciences), at 37�C, 5%CO2. Cells were harvested at 30, 60, and120 minutes, labeled with anti-CD8a antibody and stained withviability dye as described above. FACS lismode fileswere analyzedusing FlowJo software (Tree star).

Generation of tumor targets expressing human HER2MC57 fibrosarcoma cells were transduced with a retrovirus

encoding human HER2 and GFP (MC57-HER2), as describedpreviously (23). GFP- and HER2-positive cells were then purifiedby FACS using the FACS ARIA (BD Biosciences). HER2 wasdetected by indirect staining with a mouse anti-human HER2antibody (9G6.10; NeoMarker) and a secondary anti-mouseantibody conjugated to PE (donkey anti-mouse).

Time-lapse live video microscopyInteractions between CTLs and tumor cells were assessed by

time-lapse live microscopy, using a protocol previously pub-lished by Lopez and colleagues (15) and M.R. Jenkins (sub-mitted for publication) (see Supplementary Methods fordetails). Image analysis was performed using Leica LAS AF Litesoftware or MetaMorph Imaging Series 7 software (UniversalImaging).

Statistical analysisStatistical analyses were performed using GraphPad Prism 6

software. Statistical tests applied include Student t test andANOVA. Asterisks within figures refer to statistical differencebetween test and control groups, P values and the number ofreplicate experiments performed to derive the data are indicatedin the legends for Figs. 2, 4, 5, and 7.

ResultsExpression of CAR in OT-I cells does not affect stimulationthrough the TCR

OT-I T cells become activated and differentiate into effector andmemory cells following engagement of antigen-presenting cellsdisplaying SIINFEKL (OVA257) in the context of H-2Kb (16, 24).We used well-established protocols to induce activation anddifferentiation of na€�ve CAR.OT-I T cells to effector CTLs. CAR.

OT-I cells expressed both the OT-I TCR and a second-generationCAR (scFv-CD28-z) recognizing the HER2 antigen (11). We firstcompared the effect of stimulation through either the TCR or CARon target cell recognition and effector function, by examiningwhether the presence of the CAR in OT-I T cells affected signalingand subsequent activation through the TCR. Na€�ve splenocytesisolated from CAR.OT-I or OT-I transgenic mice and activatedwith OVA257 over 7 days displayed equivalent levels of activationmarkers CD44 and CD62L (Fig. 1A and B), and equivalentproliferative capacity, as measured by CTV dilution (Fig. 1C).There was no statistically significant difference in either theproliferation or division index for OT-I CTLs, compared withCAR.OT-I CTLs, in response to SIINFEKL-pulsed syngeneic sple-nocytes. These data indicate that the expression of CAR did notdisrupt TCR-mediated activation of CAR.OT-I CTLs.

Activated OT-I and CAR.OT-I cells display similar levels ofcytotoxic granule proteins

Although the presence of the CAR had no adverse effect on theactivation and differentiation of CAR.OT-I effector cells (compar-ison of cytotoxicity will be described below), it was important toexamine expression of the key cytotoxic granule proteins, perforinand Granzyme B. Perforin (Supplementary Fig. S1A) and gran-zyme B (Supplementary Fig. S1B) were equivalently expressed byOT-I and CAR.OT-I CTL. We next compared the functional abilityof CAR.OT-I CTL with exocytose cytotoxic granules in responseto either TCR or CAR target cell antigens. There was no statis-tically significant difference in the level of CD107a exposure onthe surface of CAR.OT-I cells following activation with eitherMC57-OVA257 or MC57-HER2 tumor targets. CAR.OT-I CTLs(Supplementary Fig. S1C) or OT-I CTLs (Supplementary Fig.S1D) degranulated over a 2-hour period in response to TCR(MC57-OVA257) or CAR stimulation (MC57-HER2), but not inresponse to control MC57 cells. Finally, we compared the effectof OVA257 peptide dose in the initial activation culture, on thefunctional capacity of the generated effector T cells. We showedthat T-cell CD69 and Granzyme B expression levels followedthe same trend from days 4 to 7, despite being stimulated byaccessory cells cultured in limiting concentrations of OVA257

(Supplementary Fig. S2A–S2D). In addition, OVA257 dose didnot affect the maturation status of the activated effector cells, asmeasured by the percentage of CD44þ and the percentage ofCD62Lþ CD8þ T cells in the culture from days 4 to 7 (Sup-plementary Fig. S2E–S2H).

CAR and TCR on CAR.OT-I cells are differentially expressedBefore comparing the kinetics of target cell engagement and

cytotoxicity following activation via CARor TCR, it was importantto confirm thatCAR.OT-I cells coexpressedCARand TCRVa2, andto compare the relative expression of both antigen receptors, asdifferences have a significant impact on their responses to cognateantigen.We demonstrated that CAR.OT-I CTLs expressed both theCAR and OT-I TCR, although CAR expression levels variedbetween cells (Fig. 2A). CAR.OT-I mouse T cells expressed signif-icantly lower levels of CAR than TCRVa2 (P ¼ 0.003, Fig. 2B).The level of TCRVa2 expressed was similar between OT-I andCAR.OT-I CTL (Fig. 2B). We next compared the level of CAR onCAR.OT-I CTLs with that on transduced mature T cells. Wild-typeC57BL/6 splenocytes were retrovirally transduced with the samesecond-generation CAR as that expressed by CAR.OT-I cells. In

Individual CAR-T Cells Kill Multiple Tumor Targets

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these transduced C57BL/6 cells, CAR density (8,797 � 4,203molecules/cell) was moderately increased compared with that ofCAR.OT-I cells (3,040 � 660 molecules/cell), although this dif-ference was not statistically significant (Fig. 2B).

The TCR and CAR target antigen expression levels were alsoassessed on MC57-HER2 tumor cells. We found that MC57-HER2 cells uniformly expressed both human HER2 and H-2Kb

(Fig. 2C and D). Furthermore, when pulsed with OVA257, theOT-I TCR antigen OVA257/H-2Kb were similarly displayed onboth MC57-HER2 and MC57 cells (Fig. 2D and E). Finally,the number of tumor target antigen molecules was not sta-tistically different for HER2 and OVA257 /H-2Kb (Fig. 2F).

Taken together, our data indicated that CAR.OT-I cell TCRexpression levels were significantly higher than that of theCAR molecules. In subsequent experiments, we comparedeffector cell killing of tumor cells, and explored whether thedifference in CAR.OT-I cell antigen receptor levels influencedthe functional outcomes of T-cell activation and the kineticsof tumor cell killing.

Cytotoxic T cells can effectively kill tumor targets via CAR orTCR ligation

Havingdefined theparameters of ourmodel,wenext comparedthe kinetics of attachment, recognition, and cytotoxicity of tumor

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Figure 1.The presence of a CAR does not affect TCR-induced effector phenotype and proliferation. OT-I or CAR.OT-I splenocytes were pulsed with SIINFEKL for3 days. Following enrichment of viable cells on day 3, OT-I and CAR.OT-I cells were assessed for the T-cell differentiation markers CD44 and CD62L at theindicated time points by FACS. A, representative density plots for CD44 and CD62L expression on CD8þ cells at days 0, 3, and 6. B, pooled data (mean� SEM, fromthree separate experiments) for the percentage of CD8þCD44þ and the percentage of CD8þCD62Lþ cells over the 7-day culture period. C, to measure proliferation,na€�ve OT-I and CAR.OT-I T cells were labeled with CTV and cell division of SIINFEKL-pulsed T cells assessed over 3 days. Data are representative of two independentexperiments, and overlay histograms for SIINFEKL-pulsed (black line) and nonpulsed control T cells are shown (dotted line).

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Figure 2.Expression of CAR, endogenous TCR, and target-cell receptors. A, CAR.OT-I andOT-I TCR expressionswere analyzed by labeling day 7 activated T cells with both anFITC-conjugated anti–c-myc tag antibody and PE-conjugated TCR Va2 antibody. Cells were gated on TCR Va2, and expression of CAR via c-myc tag isshown in a representative overlay histogram for the isotype control (dotted black line) and c-myc tag (thick black line). B, antigen receptor quantification oneffector CAR.OT-I, OT-I and transduced wild-type T cells (C57BL/6 CAR) was performed using a flow cytometry–based assay with Simply Cellular Beads, aproprietary kit from BANGSLABS. Antibody directed to c-myc Tag (FITC) was used to determine the expression of CAR on transgenic CAR.OT-I T cells andtransduced wild-type C57BL/6 T cells. Antibody specific for Va2 TCR were used to determine the expression level of OT-I TCR on OT-I and CAR.OT-I T ce lls.Data are representative of two separate experiments. C, to assess tumor antigen expression, MC57-HER2 target cells were labeled with an anti-human HER2. MC57-HER2 (D)orMC57 (E)werepulsedwithOVA257, followedby stainingwithH-2Kbantibody (left), or 25-D1.16 antibody (H-2Kb-OVA257; right). Cellswere analyzedbyflow cytometry and overlay histograms are shown for the isotype control (gray line) and the test mAb (black line). The isotype control for H-2Kb was mouse IgG2a(msIgG2a), and for 25-D1.16 was mouse IgG1 (msIgG1). F, cells were either unpulsed or pulsed with OVA257 and stained for H-2kb, H-2kb OVA257 (25-D1.16) or hHER2,and the number of molecules for each antigen was calculated by comparison with a standard curve.






target cells by CAR.OT-I cells through engagement of either TCRor CAR, by live-cell microscopy. We used a well-characterizedmethod (M.R. Jenkins; submitted for publication; refs. 15, 25)to precisely pinpoint the time taken for a CAR-T cell to deliver alethal hit to a target cell, after binding of the CAR-HER2receptor. In this experiment, either OT-I or CAR.OT-I cells werelabeled with the Ca2þ indicator fluo-4 AM and cocultured withMC57, MC57-OVA257, or MC57-HER2 target cells, in the pres-ence of 100 mmol/L propidium iodide (PI). The formation ofperforin pores on the target cell was inferred by the strong PIbinding and fluorescence within the cytosol of the target cellemanating from the region of the immune synapse (termed the"PI blush," Fig. 3A–C). Montages of single-cell conjugates areshown for control OT-I cells cocultured with MC57-OVA257

(Fig. 3A; Supplementary Movie 1), and CAR.OT-I cells cocul-tured with either MC57-OVA257 (Fig. 3B, Movie 2), or MC57-HER2 cells (Fig. 3C; Supplementary Movie 3). In each corre-sponding image, we analyzed the fold change in fluorescenceintensity of fluo-4-AM within the killer cell, and the fold changein PI fluorescence in the target cell, in real time (right for Fig.3A–C). Thus, upon antigen engagement, the Ca2þ flux (greenfluorescence) preceded tumor cell uptake of PI (red fluores-cence). In contrast, when there was no effector cell recognition(control MC57 cells), both green and red fluorescenceremained at basal levels (data not shown). When the same

effector cells were cocultured with tumor cells that did notexpress cognate antigen, degranulation and cell killing were notobserved (data not shown).

We analyzed the kinetics ofmany single-cell conjugates by live-cellmicroscopy,measuring the time taken for each stage of killing:(i) effector cell "binding" (scanning by the effector cell); (ii)"recognition" (antigen recognition indicated by Ca2þ flux ineffector cell); (iii) delivery of the "lethal hit" (visualized whenPI binds cytosolic RNAafter entering throughperforin pores in thetarget cell membrane; ref. 15); and (iv) "target cell rounding,"signifying loss of cell adhesion and commitment to cell death.Using the same coculture combinations as in Fig. 3, we subse-quently characterized the kinetics of individual effector cell–tumor cell killing interactions (Fig. 4). There was no statisticallysignificant difference in the time interval for "Ca2þ flux to lethalhit delivery" (Fig. 4A), "lethal hit delivery to tumor cell rounding"(Fig. 4B), and "lethal hit delivery to tumor cell detachment"(Fig. 4C) following recognition of the target cell via the TCR ortheCAR.However, we found thatwhenCAR.OT-I cells recognizedtumor antigen via the CAR (vs. the TCR), the time intervalfor "Ca2þflux to tumor cell detachment"was significantly reduced(Fig. 4D). Interestingly, effector CAR.OT-I cells respondingvia CAR stimulation displayed a more uniform response forthis parameter, indicating a more consistent duration of synapse(Fig. 4D).

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Figure 3.T cells can effectively kill tumor targets following specific activation of TCR or CAR. A, time-lapse live microscopy was used to show Fluo-4-AM labeled OT-I orCAR.OT-I T cell cytotoxicity of target cells in the presence of 100 mmol/L PI. OT-I CTLswere coculturedwithMC57-OVA257 cells; or B, CAR.OT-I CTLswere coculturedwith MC57-OVA257 cells or C, MC57-HER2 cells. Images were acquired every 15 seconds, and shown are overlays of the Fluo-4 (green), PI (red), andBrightfield channels. Fluorescence images are shown at indicated time points denoting binding of effector and target, recognition (effector cell calcium flux-brightgreen), delivery of perforin (tumor cell PI blush), and finally cell rounding of the target (loss of target membrane integrity, blebbing morphology and PIþ).Images are representative of a minimum of three experiments each and are selected to depict effector cell–tumor cell interactions. Images are representativeof n¼ 32, n¼ 23, and n¼ 62 individual conjugates, respectively. Time stamps are displayed in the bottom left of the image and scale bars, 10 mm. Each correspondingfold change in fluorescence intensity of PI (red) and Fluo4-AM (green) are shown in the right for the images.

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CAR.OT-I T cells can similarly kill targets throughCAR and TCRstimulation in short-term but not long-term assays

Our data suggested that despite equivalent activation,CAR.OT-I CTLs engaging targets via CARs remained in synapsefor shorter times. However, a cytotoxicity assay performed usingCAR.OT-I T cells cocultured for 4 hours with MC57-OVA257 orMC57-HER2 showed no statistical difference in killing by CAR.OT-I cells targeting the TCR (MC57-OVA257) or CAR (MC57-HER2; Fig. 5A). Predictably, there wasminimal cytotoxicity in theabsence of antigen (Fig. 5A, diamonds). Although 51Cr-releaseassays are suitable for determining overall cytotoxic function, theydo not provide direct information on the rate of target cell death.To address this, we used an xCELLigence assay, which measureskilling by recording the reduction in resistance to an electricalcurrent passed through the adherent target cells over time (20, 21).At each time point, arbitrary resistance values are normalized tothe starting timepoint and expressed as fold change and the rate ofkilling is reflected by the gradient of the curve.We observed that attwo different E:T ratios (10:1 and 5:1), the rate of killing ofMC57-OVA257 or MC57-HER2 cells by CAR.OT-I effector cells wasequivalent for up to 8 hours (Fig. 5B and C).

CAR.OT-I T cells stimulated through the CARs are capable ofserial killing

The capacity of CTLs to serially kill target cells is important foreradicating large tumor burdens. However, the capability of CARreceptors to sequentially kill multiple tumor targets ("serial kill-ing") has not been previously investigated. Hence, we examinedthis issue by using CAR.OT-I cocultured with either MC57-HER2or MC57-OVA257 targets and live time-lapse microscopy. Serialkilling of tumor targets by CAR.OT-I effector cells was observedfollowing engagement through the TCR (Fig. 6A; SupplementaryMovie 4) or CAR (Fig. 6B; Supplementary Movie 5). Individualeffector CAR.OT-I cells sequentially delivered a lethal hit to two(Fig. 6A) or three (Fig. 6B) tumor targets. Interestingly, CAR.OT-Icells mediated efficient tumor cell killing of adjacent tumorcells almost immediately after the first hit (Fig. 6B, right; Supple-mentary Movie 5). As previously described in mouse naturalkiller cells (25), we did not see repetitive repriming of Ca2þ fluxwithin a single CTL between these rapid killing events. We furtherexamined the videos of effector–target cell interactions, andassessed the frequency of serial killing events (Fig. 6C). CAR.OT-I CTL engaged in serial killing ofMC57-OVA257 (22.52%) and

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Figure 4.CAR.OT-I cells stimulated via theCARs induce more rapid detachmentfrom tumor cells. Fluo4-AM labeledCTLs (OT-I or CAR.OT-I) were addedto either MC57-OVA257 or MC57-HER2target cells in the presence of 100mmol/L PI and images acquired bytime-lapse live microscopy. Datarepresent individual T-cell–tumor cellinteractions for either OT.I CTLsrecognizing tumor targets via the TCR,or CAR.OT-I CTLs ligated via the TCRor CAR. Time was recorded betweenCa2þ flux and lethal hit (A), lethal hit totumor cell rounding (B), lethal hit toeffector cell detachment from thetumor cell (C), and Ca2þ flux to CTLdetachment from the tumor cell (D).All statistics were defined using theStudent t test, where �, P < 0.01 and�� , P < 0.0001; n.s., not statisticallysignificant.






MC57-HER2 (21.74%), and this was equivalent to that observedwhen OT-I cells were cocultured with MC57-OVA257 (23.53%).

To explore the serial killing, we previously observed by video, a51Cr-release assay using target:effector (T:E) ratios of 1:1 through32:1 over 18 hours was used. This showed CAR.OT-I CTLs killedeither MC57-OVA257 or MC57-HER2 at equivalent levels across awide range of T:E ratios. Furthermore, the amount of CAR.OT-ICTLs killing of tumor cells (MC57-OVA257 or MC57-HER2) wassignificantly different compared with MC57 cells for T:E of 1:1thru to 8:1 (Fig. 7A). To assess whether the difference in CAR andTCR antigen receptor levels might have long-term consequencesfor tumor cell killing (whether single or serial killing), we per-formed an xCELLigence assay over 50 hours. A lower E:T ratio wasused than was used previously to reduce the rate of target celldeath and so potentially reveal differences in effector cell–tumorkilling kinetics. TCR-ligated CAR.OT-I CTLs killed tumor cells at aconsistent rate over the extended time period at an E:T ratio of 1:1(Fig. 7B), as indicated by the continual reduction in impedance.The rate of killing by CAR-ligated CAR.OT-I CTLs was similar forthe first 20 hours of the assay, but slowed markedly beyond thistime point at both E:T ratios (Fig. 7B). By measuring the slope ofthe two curves, we were able to compare the rates of target celldeath from 20 to 50 hours. Pooled data from three independentexperiments showed a significantly higher CAR.OT-I CTL killingof MC57-OVA257 compared with that of MC57-HER2 (Fig. 7C).To explore the mechanism(s) leading to this observed differencein killing rate, we recovered the CAR.OT-I CTLs from the cocultureat 20 and 50 hours and examined their viability (Fig. 7D) andantigen receptor (TCR and CAR) expression levels (Fig. 7E and F).CAR.OT-I CTL viability remained equivalent irrespective ofthe antigen stimulus and the time point (Fig. 7D). Interestingly,

when ligated by the CARs, CAR.OT-I CAR expression was down-regulated by 20 hours, and this persisted through to 50hours (Fig.7E). In contrast, when ligated via the TCRs, CAR.OT-I endogenousTCR expression levels were unchanged at 20 hours, and althoughTCR levels decreased by 50 hours, they were clearly detectableabove the level of the negative control. In summary, persistentendogenous TCR, but not CAR, expression provides a potentialexplanation for the difference in long-term CAR.OT-I CTL cyto-toxic function following activation via H-2Kb-OVA257 versusHER2 antigen.

DiscussionCAR-T cells have evoked exciting clinical responses in CLL

and ALL (1, 2, 4–6). A hallmark of many tumors is the loss of T-cell recognition molecules (MHC I), and therefore the use ofCAR-T targeting tumor antigens may be of considerable clinicalbenefit. However, responses to CAR-T therapy in patients withsolid tumors have been modest to date (8), and this is likelydue to several factors, including the effect of the suppressivetumor microenvironment and low frequency of CAR-T cellstrafficking to the tumor site. Despite the enormous progress inCAR-T cell therapy, there are fundamental aspects of CAR-T-cellbiology that remain unstudied. Pivotal to the CAR-T-cell anti-tumor effect is their ability to form an effective immunesynapse with a tumor target. Similarly, the respective role ofthe CAR versus that of the endogenous TCR in formation of thesynapse and the subsequent activation of T cells remainsunknown. This study addressed these issues in a model systemin which individual CAR-T cells (CAR.OT-I) coexpressed twoantigen receptors (OT.I TCR and anti-HER2 CAR), and the

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Figure 5.Equivalent level of killing of targetcells by CAR.OT-I cells stimulatedthrough the TCRs or CARs. A,activated CAR.OT-I cells wereincubated with 51Cr-labeledMC57 (opendiamonds),MC57-OVA257

(open circles), or MC57–HER-2 cells(open squares) for 4 hours at theindicated E:T ratios, before measuringCr-release. Data represent thepercentage of tumor cell lysis (mean� SEM) and are pooled from twoseparate experiments. � , P < 0.05using a Student t test, comparingcontrol and test tumor cells. The rateof killing was measured using anxCELLigence assay. B and C, activatedCAR.OT-I T cells were incubatedat an E:T ratio of 10:1 (B) or 5:1 (C) withMC57-HER-2 (dashed black) or MC57-OVA257 (black), MC57 alone (dottedgray) is included as a control. The dataare presented as the averageresistance over time for duplicatewells, which were then converted tofold change. Graphs show the rate ofdeath over 8 hours (500 minutes),in which the gradient reflects the rateof killing. Data are representative oftwo independent experiments.

Davenport et al.





tumor cells were capable of displaying the cognate antigen forboth antigen receptors.

In our methodology, CAR.OT-I cells expressed TCR at higherlevels than CAR, although cognate antigen levels onMC57 tumorcells were equivalent, this created an equivalent comparison. Thekinetics ofCAR.OT-I cell activation, delivery of the "lethal hit" andtumor cell killing were comparable when binding to targetsoccurred via the TCR or CAR. Calcium flux into the effector cellis a marker of antigen recognition, and surprisingly, the timeinterval from Ca2þ flux until CTL detachment from the tumor cellwas reduced when CAR.OT-I cells were ligated via the CAR.Nevertheless, both were equally effective in killing tumor cellsin the short term. In long-term assays, CAR.OT-I cells displayedequivalent rates of killing when recognition occurred via TCR orCAR, for the first 20 hours. However TCR-stimulated CAR.OT-Icells were more effective killers between 20 and 50 hours. Wesubsequently showed that CAR.OT-I cells downregulate CARs to a

higher degree thanTCRs in response to cognate antigen long-term.This provides the most likely explanation for loss of cytotoxicfunction in the long-term assays. Our results are also consistentwith that observed previously, in which CAR expression wasdownregulated following long-term in vitro culture (26). Inter-estingly, this study demonstrated that CAR expression could bereinduced by further activation through TCR signaling, and itwould be intriguing to determine whether this was the case inCAR.OT-I cells. Regarding the endogenous TCR, prior studieshave shown that effector T cells require only 10 to 100 pep-tide–MHC complexes on target cells for their activation andinduction of cytotoxicity (27–29). Although our results showthat the CAR.OT-I CTLs display reduced endogenous TCR expres-sion by 50 hours, this is above the threshold required for T-cellactivation. It is also possible that with the high avidity, CAR-TCTLs become exhausted more rapidly, and therefore studies onthe formation of long-term memory cells will be of critical

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Figure 6.Individual CAR.OT-I T cells can kill multiple tumor cellswhen activated via the TCR or CAR. Effector Fluo-4-AM-labeled CAR.OT-I cellswere added to adherentMC57-HER-2 or MC57-OVA257 cells in the presence of 100 mmol/L PI and images were acquired every 15 seconds. Shown are overlays of Fluo-4 (green), PI (red),and cell structure (bright field). A and B, montage images show one CAR OT-I cell serial killing MC57-HER-2 (A) or MC57-OVA257 (B), where white arrowsindicate delivery of the lethal hit for each killing event. Serial-killing eventswere observed inmultiple experiments for each test condition. Time stamps are displayedfor recognition and delivery of the lethal hit to serial targets (min:sec). In addition, the fold change in both PI and Fluo-4 AM fluorescence intensity (right)are shown for corresponding montages (A and B). C, the frequency of killing events that were single (gray) or serial (black) is shown for OT-I CTLs ligated via theirTCRs (left), CAR.OT-I CTLs ligated via their TCRs (middle), and CAR.OT-I cells activated via their TCRs (right); n ¼ the number of individual events examined.






importance. As CAR-T-cell persistence after adoptive transfer iscritical for a clinical response (1, 2, 4–6, 30), the pathway toeffective CAR-T-cell memory, and the effect of TCR versus CARengagement on this, will be further explored both in vitro and invivo using our model system. Adoptive therapy with CAR-T cellsuses autologous polyclonal T cells expressing an endogenousTCRs and CARs, and recently, dual-specific T cells (31) have alsobeen used with success. In this context, our study suggests thatpatient CAR-T cells may respond differently in vivowhen activatedvia their CARs or TCRs.

Effector-to-target ratios, avidity of interactions, and ability ofCTLs to penetrate tumors may all affect the success of cell-medi-ated therapy. The ability of adoptively transferred CAR-T cells toundertake serial killing of multiple tumor targets is likely to be akey requirement for effective therapy, as CAR-T cells will initiallybe "outnumbered" by tumor cells. As has been shown withbispecific T-cell engagers (32), we also now show that individual

T cells redirected to tumor antigens by CAR expression are capableof killing multiple tumor targets. This serial-killing capacity alongwith the ability of CAR-T cells to proliferate in vitro (9) and in vivo(CAR19 cells in CLL and ALL; refs. 4, 33) likely underpins thetherapeutic success of comparatively small CAR-T-cell doses in theface of high tumor burden. Our work establishes the means bywhich to measure the potency of CAR-mediated T-cell activationagainst the internal control of endogenous TCR-mediated activa-tion, and a means by which to measure enhanced synapseformation and/or serial killing by the addition of immune-poten-tiating therapies such as lenalidomide (34) or checkpoint block-ade inhibitors (35). The combination therapies are likely toenhance CAR-T-cell proliferation and serial killing, resulting inmaximal tumor cell death and improved clinical benefit.

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

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(black line), or MC57–HER-2 targets(dashed black line) at an E:T ratio of 1:1.The rate of tumor cell killing wasmeasured using an xCELLigenceassay, and graphs show the rate ofdeath over 50 hours, with the gradientreflecting the rate of killing. The dataare the average resistance over timefor duplicate wells, which wasconverted to fold change. Data arerepresentative of three separateexperiments and collated in C. D–F,CAR.OT-I cells were cocultured withtumor cells as in B. D, CAR.OT-I cellswere harvested 20 and 50 hours laterand their viability checked by FACSusing fixable yellow. E and F, theharvested cells were labeled with ananti-myc PE antibody (E) or a TCRVa2PE antibody (F) to detect the level ofCAR or endogenous TCR expression,respectively. FACS data are presentedas MFI for each experimentalcondition, and are the mean oftriplicate wells (representative oftwo separate experiments).


Davenport et al.




Authors' ContributionsConception anddesign:A.J. Davenport,M.R. Jenkins, H.M. Prince, D.S. Ritchie,J.A. Trapani, M.H. Kershaw, P.K. Darcy, P.J. NeesonDevelopment of methodology: A.J. Davenport, M.R. Jenkins, R.S. Cross,C.S. Yong, D.S. Ritchie, J.A. Trapani, P.K. Darcy, P.J. NeesonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.J. Davenport, M.R. Jenkins, R.S. Cross, P.K. Darcy,P.J. NeesonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.J. Davenport, M.R. Jenkins, R.S. Cross, H.M. Prince,D.S. Ritchie, J.A. Trapani, M.H. Kershaw, P.K. Darcy, P.J. NeesonWriting, review, and/or revision of the manuscript: A.J. Davenport,M.R. Jenkins, R.S. Cross, C.S. Yong, H.M. Prince, D.S. Ritchie, J.A. Trapani,M.H. Kershaw, P.K. Darcy, P.J. NeesonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.J. Davenport, D.S. Ritchie, P.K. DarcyStudy supervision: M.R. Jenkins, D.S. Ritchie, J.A. Trapani, M.H. Kershaw,P.K. Darcy, P.J. Neeson

AcknowledgmentsThe authors acknowledge the assistance of the PMCC Experimental

Animal facility technicians for animal care, the PMCC FACS Core for cell

sorting, and the Microscopy Core facility at PMCC for technical support. Theauthors thank Dr. Hideo Yagita (Juntendo University, Tokyo) for theGranzyme B antibody p1-8.

Grant SupportThis work was funded by a program grant from the National Health

and Medical Research Council (NHMRC). A.J. Davenport was supported bya scholarship from the Fight Cancer Foundation, and M.R. Jenkins issupported by a National Health and Medical Research Council of Australia(NHMRC)/RG Menzies postdoctoral training fellowship and an NHMRCNew Investigator Project grant. P.K. Darcy and M.H. Kershaw were supportedby NHMRC Senior Research Fellowships (#1041828 and 1058388,respectively).

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 February 14, 2015; accepted February 17, 2015; publishedOnlineFirst February 24, 2015.

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Correction

Correction: CAR-T Cells Inflict SequentialKilling of Multiple Tumor Target CellsThe article by Davenport and colleagues that was published in the May 2015 issue ofCancer Immunology Research (1) contained errors in Fig. 2D and E. In both panels, theauthors used an incorrect isotype control for theH-2Kb antibody (msIgG1, instead ofmsIgG2a). The authors also used the incorrect FACS file as the test overlay for theH-2Kb antibody in both panels. The authors have repeated the experiments usingthe correct isotype controls, and the corrected figures incorporating these new dataare below. These corrections do not alter the main conclusions drawn from thisstudy. The authors sincerely regret these errors. Both the HTML and the PDF versionsof the article online have been changed to reflect the content of this correction.

Reference1. Davenport AJ, Jenkins MR, Cross RS, Yong CS, Prince HM, Ritchie DS, et al. CAR-T cells inflict

sequential killing of multiple tumor target cells. Cancer Immunol Res 2015;3:483–94.

Published online February 16, 2018.doi: 10.1158/2326-6066.CIR-18-0027�2018 American Association for Cancer Research.

Figure 2.

MC57-HER2 (D) or MC57 (E) were pulsed with OVA257, followed by staining with H-2Kb antibody (left), or 25-D1.16antibody (H-2Kb-OVA257; right). Cells were analyzed by flow cytometry and overlay histograms are shown forthe isotype control (gray line) and the test mAb (black line). The isotype control for H-2Kb was mouse IgG2a(msIgG2a), and for 25-D1.16 was mouse IgG1 (msIgG1).

CancerImmunologyResearch

Cancer Immunol Res; 6(3) March 2018370

http://crossmark.crossref.org/dialog/?doi=10.1158/2326-6066.CIR-18-0027&domain=pdf&date_stamp=2018-2-15

2015;3:483-494. Published OnlineFirst February 24, 2015.Cancer Immunol Res Alexander J. Davenport, Misty R. Jenkins, Ryan S. Cross, et al. CellsCAR-T Cells Inflict Sequential Killing of Multiple Tumor Target

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