il2/anti-il2complexcombinedwithctla-4,but not pd-1 ......sion. mononuclear cells from lungs were...

Research Article

IL2/Anti-IL2ComplexCombinedwithCTLA-4,ButNot PD-1, Blockade Rescues Antitumor NK CellFunction by Regulatory T-cell ModulationPamela Caudana1, Nicolas Gonzalo N�u~nez1, Philippe De La Rochere1, Ana€�s Pinto1,Jordan Denizeau1, Ruby Alonso1, Leticia Laura Niborski1, Olivier Lantz1,2,3,Christine Sedlik1, and Eliane Piaggio1,3

Abstract

High-dose IL2 immunotherapy can induce long-lastingcancer regression but is toxic and insufficiently efficacious.Improvements are obtained with IL2/anti-IL2 complexes(IL2Cx), which redirect IL2 action to CD8þ T and naturalkiller (NK) cells. Here, we evaluated the efficacy of combiningIL2Cx with blockade of inhibitory immune pathways. In anautochthonous lung adenocarcinoma model, we show thatthe IL2Cx/anti–PD-1 combination increases CD8þ T-cell infil-tration of the lung and controls tumor growth. In the B16-OVAmodel, which is resistant to checkpoint inhibition, combina-

tion of IL2Cx with PD-1 or CTLA-4 pathway blockade reversesthat resistance. Both combinations work by reinvigoratingexhausted intratumoral CD8þ T cells and by increasing thebreadth of tumor-specific T-cell responses. However, only theIL2Cx/anti–CTLA-4 combination is able to rescue NK cellantitumor function by modulating intratumoral regulatoryT cells. Overall, association of IL2Cx with PD-1 or CTLA-4pathway blockade acts by different cellular mechanisms, pav-ing the way for the rational design of combinatorial antitumortherapies.

IntroductionAdministration of high-dose IL2 immunotherapy, which can

induce durable cancer regression, is approved by the FDA fortreatment of metastatic melanoma and renal carcinoma (1). IL2therapy can, however, be toxic and has low efficacy (5%–20% ofresponders) due to its effect on regulatory T cells (Tregs), whichexpress the high-affinity IL2 receptor (composed of subunitsIL2-Ra, IL2Rb, and IL2Rgc) and can block the antitumor immuneresponse (2). Antitumoral activity requires activation of CD8þ Tand natural killer (NK) cells, which respond to IL2 through theintermediate affinity IL2 receptor (composed of IL2Rb and IL2Rgcsubunits; ref. 3).

One strategy to avoid unwanted activation of Tregs is to use IL2/anti-IL2 complexes (IL2Cx). IL2Cx can redirect IL2 to Treg orto IL2Rbgc-expressing cells that can mount an antitumoralresponse (4). IL2Cx has a longer half-life than IL2, resultingin better pharmacodynamics (5). High-dose IL2 therapy can betoxic due to vascular leak syndrome. IL2-associated pulmonaryedema seems to be caused by interaction of IL2 with its receptors

(IL2-Ra, CD25) on lung endothelial cells (6). This side effect canbe abrogated by use of IL2Rbgc-directed IL2Cxs, which should notactivate CD25 on the endothelial cells (5). In murine models ofcancer, the IL2Rbgc-directed IL2Cx can boost the antitumorresponse by stimulating NK and CD8þ T cells, encouraging theclinical development of IL2Cx (7). Nevertheless, in experimentalmodels, administration of IL2Cx as monotherapy delays tumorgrowth only modestly (6–8).

Given the success of anticheckpoint therapies in cancer,combination of IL2Cx with anticheckpoint mAbs that targetCTLA-4 or PD-1/PD-L1 inhibitory pathways could improveresults. Indeed, single-agent administration of anti–CTLA-4 oranti–PD-L1 shows clinical efficacy in around 20% of patientswith tumors (9). Combination of anti–CTLA-4 and anti–PD-1increases both response rate and toxicities (10), although themajority of cancer patients still do not respond. Acquiredresistance to treatment has also been observed (11, 12). Effi-cient combination therapies are needed to increase the numberof responding patients.

In this study, we evaluated whether combining IL2Cx admin-istration with the blockade of inhibitory immune pathwaysrepresents an effective antitumor therapy. We tested two exper-imental models: B16-OVA, which is resistant to anticheckpointinhibition (13), and the KrasLSL-G12D/þp53flox/flox geneticallyengineered mouse lung adenocarcinoma model (14). In bothmodels, we observed that combination of IL2Cx with antic-heckpoint mAbs was more effective. We found that IL2Cx worksin a cell-intrinsic manner, boosting tumoral NK, reinvigoratingexhausted CD8þ T cells, and increasing the breadth of tumor-specific T-cell responses. We also found that combination ofIL2Cx with blockade of the PD-1 or CTLA-4 pathways increasestumor infiltration by CD8þ T cells. Anti–CTLA-4 contributes toefficacy by a CD8þ T and NK cell–extrinsic mechanism,

1Institut Curie, PSL Research University, INSERM U932, TransImm Team, Paris,France. 2Institut Curie, PSL Research University, Clinical Immunology Labora-tory, Paris, France. 3Centre d'Investigation Clinique Bioth�erapie CICBT 1428,Institut Curie, Paris, France.

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

Corresponding Author: Eliane Piaggio, Institut Curie, PSL Research University,INSERM U932, Institut Curie, 26, rue d'Ulm, F-75005 Paris, France. Phone: 33-667834895; Fax: 33-1-44-07-07-85; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-18-0697

�2019 American Association for Cancer Research.

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releasing effector CD8þ T and NK cells from Treg suppression.Consequently, the combined treatment of IL2Cx with CTLA-4blockade relies on the action of NK cells. These two combina-tions, which act by different cellular mechanisms, suggestavenues for the design of combinatory immunotherapiesadapted to the individual tumor microenvironment of eachpatient. Our preclinical results show the validity of combiningIL2Cx with anticheckpoint mAbs to fight cancer.

Materials and MethodsMice

C57BL/6 female mice (5–6 weeks old) were purchased fromThe Charles River Laboratories. KrasLSL-G12D/þp53flox/flox mice(referred to as KP mice) were kindly given by T. Jacks (ref. 14;NIH) and backcrossed on the C57BL/6 background formore than10 generations at CNRS Central Animal Facility TAAM (Orl�eans,France). Experiments with KP mice were performed with femalesof 8 to 12 weeks old. Experimental animal procedures wereapproved by the ethics committee of the Institut Curie CEEA-IC#118 (DAP n� 05302.03), in compliance with internationalguidelines.

Cell linesB16F10 melanoma cell line expressing OVA (B16-OVA) was

kindly givenbyK. Rock in 1999 andwas cultured inRPMI (Gibco)supplemented with 10% heat-inactivated fetal bovine serum(Biosera), 2 mmol/L L-glutamine (Life Technologies), b-mercap-toethanol, and 1% penicillin/streptomycin (Life Technologies).For all experiments, cells were thawed from the same stockgenerated in 2016. Cells were cultured during maximum 2 weeksbefore injection intomice. Cellswere not authenticated in the pastyear. Cells were checked for the absence of Mycoplasma by PCRreaction (GAT-Biotech).

HEK293-LTV and 3TZ cells were obtained from ATCC in2013 and were used upon a second passage. Cells were culturedin Dubeco's modified Eagle medium (DMEM; Gibco) supple-mented with 10% heat-inactivated fetal bovine serum (Bio-sera). Cells were not authenticated in the past year. Cells werechecked for the absence of mycoplasma by PCR reaction (GAT-Biotech).

Lentiviral particle production and titrationA second generation of lentivirus (LV) was produced as previ-

ously described (15). Briefly, LV particles were produced bytransfection of the HEK-293LTV cell line (Cell Biolabs, Inc.) withpAX2 (packaging plasmid; Addgene), pCMV-VSV-G (envelopeplasmid; Addgene), and luciferase and recombinase Cre-expres-sing plasmid. The latter was modified to express SIINFEKL andDBY epitopes fused to the C-terminal end of luciferase protein.Moreover, this luciferase–SIINFEKL–DBY sequence wasmodifiedto restrict SIINFEKL andDBY expression tononhematopoietic celllineage as previously described (15). Functional particles werequantified using a Cre activity readout system based on thereporter cell line 3TZ expressing b-galactosidase upon Cre-medi-ated recombination.

Tumor experimentsFor the transplantable tumor model, C57BL/6 females (5–6

weeks old) were injected subcutaneously in the flank with 0.5 �

106 B16-OVA cells in 200 mL of PBS. Tumor growthwasmeasuredusing a metric caliper 2 to 3 times a week.

Lung adenocarcinoma was induced in KP mice (8–12 weeksold) by intratracheal injection of 2 � 104 lentiviral particlesexpressing the fusion protein luciferase–SIINFEKL–DBY andCre-recombinase as previously described (15). In vivo tumorgrowth was measured by bioluminescence. Briefly, lentiviral-injected mice were shaved and anesthetized with Isoflurane(Sigma-Aldrich). D-luciferin substrate (150 mg/kg, Promega)dissolved in PBS was injected intraperitoneally (i.p.) 15 minutesbefore imaging in IVIS Spectrum (PerkinElmer). Photon fluxeswere transformed intopseudocolor images using the Living Imagesoftware (PerkinElmer).

In vivo treatmentsRecombinant human IL2 (rhIL2, Proleukine, Novartis), anti–

CTLA4 (clone 9H10; Bio X Cell), anti–PD-1 (clone RMP1-14;Bio X Cell), anti–PDL-1 (clone 10F9G2; Bio X Cell), anti-human IL2 (clone MAB602; Bio-Techne), and anti-human IL2(clone 5344.111; BD Biosciences) were purchased. IL2Cx wereprepared mixing 15,000 UI of rhIL2 with 4.5 mg of Ab (molarratio 2:1) and incubated for 30 minutes at 37�C. Treatment ofB16-OVA engrafted mice began when tumors became measur-able (at approximately day 10). Treatment of KrasLSL-G12D/

þp53flox/flox was started when luciferase signal achieved 1 �105 photons/second (between 12 and 14 weeks after viralinoculation). Where indicated, tumor-bearing mice receivedi.p. injections of IL2Cx for 5 consecutive days. AnticheckpointmAbs were injected i.p. at 200 mg/dose 3 times a week, for7 total doses with the B16-OVA tumor or 8 weekly cycles forKrasLSL-G12D/þp53flox/flox mice.

For depletion experiments, CD8þ cell depletion wasperformed using 200 mg anti-CD8 (clone 53.6.72; Bio X Cell)and NK cells depletion using 200 mg anti-NK (clone PK136; BioX Cell). Depleting mAbs were administered i.p. 1 or 2 daysprior to therapeutic treatment and 1 or 2 days after. Depletionwas maintained by twice-a-week injection of depleting Absuntil the end of the experiment. CD8þ T-cell and NK celldepletion was monitored in blood after 4 and 6 doses ofdepleting mAbs.

Preparation of cell suspensionsSubcutaneous tumors were collected in CO2 independent

medium (Gibco). Then, tumors were cut into small pieces andplaced into 2.5 mL of CO2 independent medium containing0.1 mg/mL DNase I (Roche) and 0.1 mg/mL Liberase TL(Roche) in C tubes (Miltenyi Biotec). After mechanical disso-ciation with gentleMACS Dissociator, samples were incubatedwith shaking at 37�C for 30 minutes and processed again withgentleMACS. The cell suspension was then filtered with a 100-mm cell strainer for direct tumor cell analysis and furtherseparated on a Percoll gradient (GE Healthcare Life Sciences)from 40% to 75% interface to recover mononuclear cells forimmune cell infiltrate analysis.

Inguinal tumor-draining lymph nodes (dLN) were collectedin CO2 independent medium. Single-cell suspensions wereobtained by mechanical disruption over a 40-mm cell strainer.

Lungs were perfused with 20 mL of cold PBS to removecirculating blood cells prior to preparation of single-cell suspen-sion. Mononuclear cells from lungs were obtained as describedabove for subcutaneous tumors.

Caudana et al.

Cancer Immunol Res; 7(3) March 2019 Cancer Immunology Research444




Detection of Ag-specific T cells by ELISPOTIFNg-producing antigen-specific CD4þ or CD8þ T cells were

measured by ELISPOT. Briefly, microplates (MAIPS4510; Milli-pore) were coated with anti-murine IFNg (Diaclone).PBMCs (0.3 � 106) were cultured overnight in the presenceof either control medium or class I–restricted OVA-I peptide(257–264, SIINFEKL, 10 mmol/L) or the class II–restricted DBYpeptide (NAGFNSNRANSSRSS, 40 mmol/L), or B16-OVA mel-anoma neoepitopes Pool 1 (10 mmol/L) or melanoma neoe-pitopes Pool 2 (10 mmol/L). Cells and peptides were resus-pended in complete medium RPMI (Gibco) supplementedwith 10% heat-inactivated fetal bovine serum (Biosera), 2mmol/L L-glutamine (Life Technologies), 1% penicillin/strep-tomycin (Life Technologies), and b-mercaptoethanol. Melano-ma neoepitope peptides were kindly given by G. Lynn, NIH.Peptide sequences are: M23 (TNGSFIRLL), M27 (GVELCPGN-KYEM), M30 (PSKPSFQEFVDWENVSPELNSTDQPFL), M47(AAIVGKQVL), M49 (IAMQNTTQL); NB02 (AALTFRRL) andNB08 (KILRMSPL).

Detection of IFNg-producing cells was performed with bioti-nylated anti-IFNg (matched pairs; Diaclone) followed by strep-tavidin–alkaline phosphatase (Mabtech) and revealed using theappropriate substrate (Bio-Rad). Spots were counted usingan ELISPOT Reader System ELR02 (AID). Results were expressedas number of cytokine-producing cells (SFC) per 1 � 106 oftotal cells.

Flow-cytometric analysisFor flow-cytometry analysis, mAbs specific for mouse CD152

(UC10-4F10-11), CD274 (MIH5), CD4 (RM4-5), CD8 (53-6.7), GITR (DTA-1), Ki67 (B56), KLRG1(2F1), and TCRb(H57-597) were purchased from BD Biosciences. mAbs againstmouse CD19 (6D5), CD25 (3C7), CD27 (LG.3A10), CD4(GK1.5), CD44 (IM7), CD45.2 (104), Foxp3 (MF-14), GzmB(GB11), ICOS (C398.4A), IFNg (XMG1.2), NK 1.1 (PK136),PD-1 (29F.1A12), Tbet (4B10), and TCRb (H57-597) werepurchased from BioLegend. mAb against mouse EOMES(Dan11mag) was purchased from eBioscience/Thermo Fisher,unconjugated gp100 (EP4863) was purchased from Abcam,and anti-rabbit IgG (H þ L)-A488 secondary Ab was purchasedfrom Thermo Fisher.

Surface staining was performed at 4�C with antibodiesresuspended in PBS with 2% FBS and 2 mmol/L EDTA. Forintracellular staining of cytokines, cells were restimulated withphorbol 12-myristate 13-acetate (PMA, 20 ng/mL) and 1 mg/mLof ionomycin (Sigma-Aldrich) for 4 hours at 37�C in thepresence of GolgiStop and GolgiPlug (BD Biosciences). Allintracellular staining was performed using intracellular Fixa-tion/Permeabilization buffer set (eBioscience/Thermo Fisher)according to the manufacturer's instructions. Live-cell detectionwas performed using the LIVE/DEAD Fixable Aqua Dead CellStain Kit (Life Technologies).

FACS data were acquired using an LSRFortessa flow cytometer(BD Biosciences) and analyzed using FlowJo software (version10x, Tree Star).

Statistical analysesStatistical analyses were performed in GraphPad Prism v7.

P values were calculated with one-way ANOVA and Kruskal–Wallis posttest.

ResultsIL2Cx with anti–PD-1 mAb controls established tumors in alung adenocarcinoma model

In patients with lung cancer, inhibition of the PD-1/PD-L1pathway has proven to be efficient and safe, leading to theapproval of these treatments (16, 17). Nevertheless, not allpatients respond, and combination with IL2Cx, which activatesNK and CD8þ T cells, could improve treatment efficacy. Thus,we evaluated the therapeutic effect of IL2Cx (made of IL2/a-IL2mAb MAB602) alone or combined with an anti–PD-1 blockingmAb in an autochthonous lung tumor model. With this aim,we used the spontaneous tumor model described by DuPageand colleagues (14), in which autochthonous lung tumorsare induced by lentiviral infection of a limited number ofsomatic cells in which oncogenesis is stimulated by recombi-nation of LoxP sites, leading to the activation of one oncogene(Kras) and deletion of one suppressor gene (p53; KP mice).This model not only recapitulates the most frequent genemutations found in human lung tumors, but also mimicsfeatures of the human situation, such as vasculature, tumorarchitecture, and speed of growth. We modified the lentivirusto induce tumor expression of luciferase and one class I(SIINFEKL)– and one class II (DBY)–restricted nominal anti-gen. To reduce expression of the nominal antigens after virusinoculation and unwanted priming, we used a lentivirus har-boring four tandem target sequences of the hematopoietic-specific mir142-3p after the luciferase-Ag cassette, to induceits degradation in hematopoietic cells (ref. 15; see Materialsand Methods).

KP mice infected with the lentivirus were monitored fortumor development by whole-body bioluminescence imaging.The first tumors were detected at 13 to 26 weeks after virusinoculation. Once mice reached a bioluminescence signal of 1� 105 photons/second, they were randomized to receive one ofthe following treatments: PBS, IL2Cx (made of 15,000 IU of IL2and anti-IL2; MAB602, 2:1 molar ratio), anti–PD-1 (200 mgtwice per week), or IL2Cx plus anti–PD-1, for at least 7 weeks,and tumor growth followed (Fig. 1A and B). Bioluminescencedetection usually correlated with tumor burden when evaluatedby hematoxylin and eosin staining, which also revealed thatIL2Cx-treated mice did not develop lung edema, previouslyassociated with high-dose IL2 therapy (ref. 6; Fig. 1B). Single-agent therapies showed little antitumor effect, whereas thecombination of IL2Cx with anti–PD-1 controlled tumors(Fig. 1C). No tumor regression was observed in the PBS group,whereas 33% of the mice showed tumor control or regression inthe IL2Cx group, 27% in the anti–PD-1 group, and 42% in thecombination group (Fig. 1C). Treatment efficacy was associatedwith more circulating antitumor CD8þ T cells (specific for theMHC class I–restricted peptide, SIINFEKL) and CD4þ T cells(specific for the MHC class II–restricted DBY epitope; Supple-mentary Fig. S1A) in treated groups than in the PBS group. Atthe end of the experiment, no changes were observed onthe proportions of CD4þ T effector (Teff) or NK cells in lungtissue (Supplementary Fig. S1B and S1C). IL2Cx administrationincreased the frequency and the cycling of CD8þ CD44High

T cells (Fig. 1D), an effect reinforced by combination with anti–PD-1. The frequency of Tregs also increased in mice receivingcombination therapy (Fig. 1E), but the CD8þCD44High/Tregratio was unaltered (Fig. 1F). Thus, the combination of IL2Cx

IL2Cx and Immune-Checkpoint Combinations in Cancer

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with anti–PD-1 increases CD8þ T-cell infiltration of the lungand controls cancer growth, rendering autochthonous lungtumors more sensitive to immune attack.

IL2Cx delays tumor growth in the B16F10-OVA melanomamouse model

To confirm our results in a different tumor type, we switched tothe transplantable syngeneic B16F10-OVA melanoma mousemodel. The IL2/a-IL2 mAb Mab602 complex can controlB16F10 melanoma growth (4, 5). We used an Ova-expressingtumor that allows us to follow the antitumor-specific T-cellresponse. For this, mice were inoculated with 0.5 � 106 B16-Ovamelanoma tumor cells. When tumors were palpable, mice wererandomized to different treatments: high-dose IL2 (200,000 IU),

IL2Cx (made of 15,000 IU of IL2 and anti-IL2 Mab602 at 2:1molar ratio), or PBS (Fig. 2A). As in ref. 6, both high-dose (HD)IL2 and the IL2Cx administered at a 13 times lower dose of IL2(LD)delayed tumor growth (Fig. 2B). IL2 actionwasmonitored inthe blood by measuring the increase of activated and highlycycling CD44High CD8þ T cells (Fig. 2C). ELISPOT analysis atday 6 after the beginning of the treatment showed that admin-istration of bothHD IL2 or LD IL2 in the formof IL2Cx induced oramplified tumor-specific T cells, as indicated by the increase ofIFNg-producing T cells upon ex vivo restimulation with an OVApeptide or with peptides containing tumor neoepitopes (Fig. 2D).IL2Cx, at lower doses, thus less toxic thanHD IL2, induced a delayin B16 tumor growth and an increase of tumor-specific IFNg-producing T cells.

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Combination of IL2Cx with PD-1mAb limits tumor growth in aninducible spontaneous model oflung cancer. KrasG12D p53flox/flox

mice were intratracheallyinoculated with a lentivirus codingfor luciferase, OVA-SIINFEKL, andDBY peptides. Tumor growth wasmonitored by recording emittedbioluminescence. Whenquantification of the average totalflux reached 1� 105 photons/second, mice were randomized forthe treatments shown in A.B, Bioluminescence quantificationin mice over time for tworepresentative responder (R1 andR2) and two nonresponder (NR1and NR2) mice treated with IL2Cxplus-PD-1 mAb (right pictures) andhematoxylin and eosin staining ofthe lower lung lobe of thecorresponding mice (left pictures).Black arrows indicate the tumorpresence in the lung lobe.C,Quantification of tumor size overtime expressed as a percentage ofchange from baseline. The pie chartindicates the percentage of miceshowing tumor regression(sustained or diminution of tumorsize) from baseline (thick lines), pertreatment group. PBS, n¼ 11;IL2Cx, n¼ 7; anti–PD-1, n¼ 7;IL2Cx/anti–PD-1, n¼ 11.D, Percentages of lung-infiltratingCD8þ CD44High T cells (left) andKi67þCD8þCD44High T cells (right)among CD45þ cells. E, Percentagesof lung-infiltrating Tregs, defined asTCRbþCD4þ FoxP3þ cells. F, Ratioof CD8/Tregs among lunghematopoietic cells. CD8/Treg ratiowas calculated as a frequency ofCD8þCD44High T cells amongCD45þ cells/frequency of totalTregs among CD45þ cells. P valueswere calculated using one-wayANOVA and Kruskal–Wallis test.� , P < 0.05; �� , P < 0.01. See alsoSupplementary Fig. S1.

Caudana et al.





We considered the effect that dissociation of IL2 from theIL2Cx in vivo might have on Tregs and cancer growth. Most ofthe studies evaluating the role of Tregs in cancer have assessedthe impact of depleting (18) but not of increasing Tregs in thecancer context (19). Thus, we studied the consequences ofadministering LD IL2 (15,000 IU of IL2, same amount of IL2present in the complex), an IL2Cx that redirects IL2 action toTregs (mAb 5344), and the IL2Cx that redirects IL2 action toCD8þ T and NK cells (mAb MAB602). In tumor-free mice, LDIL2 did not cause changes in circulating Tregs and CD8þ T cells.Both IL2Cx variants induced an increase of Tregs and enhancedtheir cycling. Only the IL2Cx that redirect IL2 action to CD8þ Tand NK cells increased the proportion and the cycling of

circulating CD8þ CD44High T cells (Supplementary Fig. S2A–S2C). In tumor-bearing mice, neither LD IL2 nor the IL2/anti-IL2 mAb MAB602 Cx accelerated tumor growth (Fig. 2E–H).Thus, an unwanted boosting effect of free IL2 on Tregs duringcancer appears unlikely. These data indicate that although IL2/anti-IL2 MAB602 Cx boosts circulating Tregs (SupplementaryFig. S2C), it also delays tumor growth (Fig. 2H).

IL2Cx enhances therapeutic efficacy of immune-checkpoint-blocking mAbs

IL2Cx and checkpoint-blocking mAbs each alone have lim-ited efficacy (Fig. 2; ref. 13). We observed a synergistic effectwhen both were used in combination in mice with palpable

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Figure 2.

IL2Cx therapy delaysmelanomagrowth and increases tumor-specific CD8þ T-cell responses.A, Schematic representation ofmouse treatment strategy. Micewith palpable tumors were (i.p.)treated for five consecutive dayswith IL2 (200,000 IU), IL2Cx (madeof 15,000 IU of IL2 and a 2:1 molarratio of anti-IL2 MAB602), or PBS.B, Tumor growth kinetics. Data,mean� SEM of one representativeexperiment with n¼ 12 (PBS),n¼ 10 (HD IL2), and n¼ 13 (IL2Cx).C, Blood from tumor-bearing miceanalyzed at day 6 after beginning ofthe treatments. Shown arerepresentative dot plots of assessedparameters and numbers arepercentages of cells in the indicatedgates. D, IFNg ELISPOT oncirculating T cells specific ofOVA-SIINFKEL peptide and B16F10melanoma neoepitope pools (Pool1: M30þM23þM49); Pool 2:NB02þ NB08þM27þM47). Eachdot represents a pool of twomice.Tables show the number ofresponder and nonrespondermice (R/NR) according to thethreshold value (dashed line;SIINFEKL¼ 70 SFC; Pool 1¼ 1.667SFC and Pool 2¼ 12.5 SFC) definedfrom ROC curves. ROC curves werecalculated using data from allELISPOT experiments analyzed(n¼ 67). SFC, spot-forming cells.E–H,Mice harboring B16F10-OVAmelanoma tumors were treated asdescribed in E or G. Tumor growthkinetics are shown in F and H. Data,mean� SEM of n¼ 3 or 4 pergroup. P values were calculatedusing one-way ANOVA andKruskal–Wallis test. NS, notsignificant; � , P < 0.05; �� , P < 0.01.See also Supplementary Fig. S2.






B16-OVA tumors (Fig. 3A–C). Figure 3D and E, depicting theresults of five pooled experiments, show that monotherapywith anti–CTLA-4 was inefficient; blockade of the PD-1 path-way alone and IL2Cx alone both conferred only intermediatecontrol of tumor growth. The combination treatments weremore efficient, as reflected by a significant delay in the tumordevelopment (Fig. 3D). The maximum reduction of the meantumor volume (measured at the end of the experiment,between days 16 and 20) was obtained with the combinatorialtreatments, reaching a 6-fold diminution for the IL2Cx withanti–CTLA-4 and 4-fold with PD-1 blockade relative to the PBSgroup (Fig. 3E). Mice receiving either single or combinedtreatments showed no signs of toxicity, such as hair or weightloss. Overall, these results show the greater antitumor effect ofthe combination therapies over the IL2Cx or inhibitory check-point blockade monotherapies.

IL2Cx together with anti-CKP increases tumor infiltration byCD8þ T cells

Given that IL2Cx, anti–CTLA-4, or anti–PD-1/anti–PD-L1 maybe acting by different mechanisms, we explored the contributionof different immune cell subsets to the efficacy of the combinedtreatments by flow cytometry analysis of tumor infiltrate. Toverify the interpretation of our results, we assessed the relativecontribution of blood circulating and tumor-resident lympho-cytes among cells retrieved from tumor cell suspensions. Regard-less of the treatment, more than 98% of the analyzed hemato-poietic cells (total CD45þ cells) are tumor tissue-infiltrating cells(Supplementary Fig. S3).

First, we analyzed the effect of the treatments on the tumormicroenvironment. Tumors from PBS-treated mice were littleinfiltrated by hematopoietic cells (mean � SD of 8.5% � 2.7%of CD45þ cells among live cells, n ¼ 10), as previously

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Figure 3.

Combination of IL2Cx with anti-CKPenhances therapeutic efficacyagainst established B16F10-OVAmelanoma tumors. A, Schematicrepresentation of mouse treatmentstrategy. Mice with palpable tumorswere treated (i.p.) with IL2Cx (grayarrows tips) for 5 days, anti–PD-1/anti–PD-L1 or anti–CTLA4 (200 mg,gray thin arrows) three times perweek or PBS. IL2Cx were made asdescribed in Fig. 2. Mice received atotal of 7 doses of anti–PD-1/anti–PD-L1 or anti–CTLA4. B and C,Tumor growth kinetics and survival(Kaplan–Meier curves) from onerepresentative experiment out of 5(n¼ 6mice per group). D, Tumorgrowth kinetics from 5 pooledindependent experiments. Data arerepresented as mean� SEM ofn¼ 5–6mice per group perexperiment including a total of 30mice. E, Tumor volume at endpointfrom experiments shown in D.P values were calculated usingone-way ANOVA and Kruskal–Wallis test (D and E). NS, notsignificant; � , P < 0.05; �� , P < 0.01;�� , P < 0.001; �� , P < 0.0001. Seealso Supplementary Fig. S3.

Caudana et al.





described for this nonimmunogenic tumor (ref. 17; Supple-mentary Fig. S4A). Compared with the PBS group, treatmentwith IL2Cx alone or PD-1 pathway blockade alone did notinduce a significant increase in the tumor infiltrate [mean � SDof 12.3% � 3.4% (n ¼ 10), and 11.2% � 2.8% (n ¼ 9)of CD45þ cells among live cells, respectively]. Other treatmentspromoted a significant recruitment of immune cells [mean �SEM of 13.1%� 5.2% of CD45þ cells among live cells for anti–CTLA-4, P < 0.05 (n¼ 10); 17.7%� 7.0% for IL2Cx/anti–CTLA-4, P < 0.001(n ¼ 10); and 18.6% � 5.6% for IL2Cx/PD-1blockade, P < 0.0001 (n ¼ 10)].

Then, we evaluated the impact of the different therapies onCD8þ T cells, which mediate antitumor immunity. Comparedwith the PBS-treated group, all therapies except PD-1 blockadealone induced a significant increase in the percentage of CD8þ

TILs among the CD45þ infiltrate, the vast majority beingCD44High, in accordance with the finding that only effector cellsreach the tumor (Supplementary Fig. S4A and S4B). Moreover,onlymice receiving the combination therapies showed significanthigher absolute numbers of CD8þ CD44High TILs (Fig. 4A). Tostudy CD8þ T-cell function, we analyzed T-cell division and IFNgproduction. We observed that compared withmice receiving PBS,IL2Cx alone and the combination therapies induced an increase inthe division of CD8þ CD44High TILs (Fig. 4B) and in circulatingOVA-specific CD8þ T cells, aswell as in tumor neoepitope-specificT cells (Fig. 4C).

We observed that in the B16-OVA model, a high proportionof CD8þ CD44High TILs expressed PD-1 (Supplementary Fig.S4C) and that the tumor cells expressed its ligand PD-L1,though at different amounts depending on the treatment (Sup-plementary Fig. S4D). During chronic viral inflammation,increased expression of PD-1 has been associated with a stateof T-cell exhaustion, characterized by lower effector cytokineproduction and lower replication. In the cancer setting,exhausted CD8þ T cells coexpress the transcription factoreomesodermin (Eomes) along with PD-1 (20). Using thesemarkers, we observed that the percentage of TILs with anexhausted phenotype (Eomesþ PD-1þ CD44High CD8þ) wasnot significantly different among the various groups of treat-ment (Fig. 4D; Supplementary Fig. S4E). Notwithstanding, ithas been reported that during treatment with anti–PD-1,exhausted cells may undergo a reinvigoration process markedby an upregulation of Ki67 and an increase in granzyme B(GzmB) production (20, 21). As shown in Fig. 4E, upon ex vivostimulation, most of the CD8þ TILs from mice treated with IL2alone or in combinatory treatments significantly increased Ki67and GzmB expression, whereas CD8þ TILs from mice treatedwith PBS, anti–CTLA-4 alone, or PD-1 pathway blockade alonemaintained a pool of cells unable to divide and produce GzmB(Fig. 4F).

Altogether, these results suggest that tumor growth controlduring IL2Cx administration is associated with a significantincrease of the CD8þ T-cell tumor infiltrate (which is higher incombination with anticheckpoints Abs), an increase in magni-tude and breadth of tumor-specific T-cell responses and thereinvigoration of exhausted CD8þ TILs.

Therapy with IL2Cx/anti–CTLA-4 combination is associatedwith higher CD8þ cell/Treg ratio

The antitumor effect of anti–CTLA-4 in mice may be in partdue to Treg depletion (22). On the other hand, IL2 can boost

Treg numbers and function (23). We analyzed the effect ofthe combinations on Tregs. As observed in Fig. 5A and B,IL2Cx administration was not associated with an increasein intratumoral Treg frequencies. Blockade of the PD-1 path-way alone or in combination with IL2Cx did not affectTreg proportions. However, administration of the anti–CTLA-4 alone induced a decrease of intratumoral CD4þ

FoxP3þ Treg proportions, which was more pronounced inassociation with the IL2Cx. The proportion of CD4þ Teffcells was not significantly affected by the different treatments(Supplementary Fig. S5A).

Therapeutic efficacy of anticancer immunotherapies is associ-ated with increased CD8/Treg ratios (22, 24). In our model, onlyadministration of anti–CTLA-4 induced an increase of the tumor-al CD8þ/Treg ratio compared with untreated mice. This increasewas greatest when anti–CTLA-4 was combined with IL2Cx(Fig. 5C).

To better understand where CTLA-4–mediated Treg deple-tion was occurring, we also quantified Treg proportions in thetumor-draining lymph nodes. As observed in Fig. 5A and B,Treg frequencies were only reduced in the tumor. Selectivedepletion of a specific T-cell population may be due to thedifferential expression density of the target molecule (25). Toexplore this hypothesis, we quantified the median fluorescenceintensity (MFI) value of CTLA-4 on the T cells in the differentorgans (Fig. 5D). Tregs from the tumor of PBS-treated miceexpressed the highest MFI values of CTLA-4, compared withCD4þ Teff and CD8þ T cells, which expressed less CTLA-4.Upon IL2Cx administration, CTLA-4 MFI was not significantlymodified, and anti–CTLA-4 administration induced a reductionon its target expression on Tregs, probably due to internaliza-tion or to blockade of the target CTLA-4 molecule. T cells in thedraining lymph nodes barely expressed the CTLA-4 molecule,validating our hypothesis that CTLA-4 mAb-selective depletionof tumor-infiltrating Tregs is likely due to the high expression ofits target uniquely on Tregs in this location. We extended thisanalysis to other immune checkpoints and showed (Fig. 5D;quantified in Supplementary Fig. S5B–S5E) that Tregs in thetumor showed the highest MFI not only of CTLA-4, but also ofICOS, and GITR, compared with Treg in lymph nodes, and toCD4þ Teff and CD8þ T cells in all organs. Conversely, CD8þ Tcells in the tumor expressed the most PD-1. As previouslydescribed (23, 26), CD25 expression on Tregs increased uponIL2 administration; this increase serves as a biomarker of IL2 invivo activity.

Overall, compared with anti–PD-1 action, additionalimmune modulation is achieved by anti–CTLA-4, which spe-cifically depletes intratumoral Tregs and tips the effector/reg-ulatory balance in the tumor in favor of CD8þ T cells, animbalance further amplified when anti–CTLA-4 is combinedwith IL2Cx.

IL2Cx with anti–CTLA-4 enhances NK cell activation andincreases NK cell/Treg ratio

NK cells, which are highly cytotoxic and produce proinflam-matory cytokines, are effectors of antitumor immune responses.Thus, we investigated the effect of the combinatorial therapies ontumor-infiltrating NK cells, which are known to be sensitive toIL2 (27). Mice were treated as described in Fig. 3A and sacrificed 6days after the beginning of the treatment. As observed in Fig. 6Aand B (and Supplementary Fig. S6A), the IL2Cx/anti–CTLA-4






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Figure 4.

Combination of IL2Cx with anti-CKP reinvigorates tumor-infiltrating CD8þ T cells. Mice harboring B16F10-OVAmelanoma tumors were treated as describedin Fig. 3A. Tumors and blood were analyzed 6 days after the beginning of the treatment. A,Quantification of effector CD8þ TILs expressed as numberof CD8þ CD44High cells per gram of tumor. B, Frequency (%) of Ki67þ cells among tumor-infiltrating CD8þ CD44High cells. C, IFNg ELISPOT of circulating T cellsagainst OVA-SIINFEKL peptide (top) and B16-F10 melanoma neoepitopes Pool1 (M30þM23þM49; middle) and Pool 2 (NB02þNB08þM27þM47; bottom),analyzed as in Fig. 2. SFC, spot-forming cells. D, Representative dot plots showing the expression of Eomes and PD-1 among infiltrating CD8þ CD44High

T cells (top), and of Ki67 and Granzyme B (GzmB) among Eomesþ PD-1þ cells (bottom). Numbers indicate the frequency (%) of cells in each quadrant.E, Frequency (%) of GzmBþKi67þ T cells among PD-1þEomesþCD8þCD44High T cells infiltrating the tumor. F, Frequency (%) of GzmB�Ki67� T cells, amongPD-1þEomesþCD8þCD44High T cells infiltrating the tumor. Data are a pool of three (A–C) or two (D–F) independent experiments. P values were calculated usingone-way ANOVA and Kruskal–Wallis test or Student t test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001. See also Supplementary Figs. S3 and S4.

Caudana et al.





combination induced the largest increase in the number of NKcells and in the proportion of IFNg-producing tumor-infiltratingNK cells. Otherwise, IL2Cx alone and combined with the check-point blockers enhanced NK division and activation, as reflected

by the significant increase in the proportion of Ki67þ NK cells, aswell as by the increment in the expression of KLRG1 and thestimulatory receptor NKG2D by these cells (Fig. 6C and D;Supplementary Fig. S6B–S6D).

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IL2Cx alone or in combination with anti–CTLA-4 increases the CD8/Treg ratio in the tumor. C57BL/6mice harboring B16F10-OVA tumors were treated asdescribed in Fig. 3A. Flow-cytometry analysis of tumor-infiltrating T cells was done 6 days after the beginning of the treatment. A, Representative dot plots ofFoxp3 and CD4þ expression on CD45þ cells from the tumor and tumor-draining lymph nodes (dLN). Numbers indicate the frequency (%) of Tregs in theindicated gates. B, Frequency (%) of Tregs among CD45þ cells in the tumor (left) and in dLN (right) from A. C, Ratio of CD8þ/Tregs in the tumor, calculated asfrequency of total CD8þT cells among CD45þ cells/frequency of total Tregs among CD45þ cells. D,Quantification of CTLA-4 median fluorescence intensity (MFI)in the indicated TILs (left). Heat map of the MFI of CTLA4, CD25, ICOS, GITR, and PD-1 on Treg, Teff, and CD8þ T cells in the tumor and dLNs (right). Data are fromthree (A–C) or one (D) independent experiments. P values were calculated using one-way ANOVA and Kruskal–Wallis test. �� , P < 0.01; �� , P < 0.0001. See alsoSupplementary Fig. S5.






We also analyzed the impact of the treatments on NK matu-ration, which is modulated by IL2 (27, 28). For that, based onthe expression of CD27 and CD11b, we subdivided NK cells intothree subsets with different maturation status (CD27þCD11b–

!DP !CD27–CD11bþ; ref. 29). None of the treatmentsmodified the percentage of immature CD27þ CD11b� NK cells,but administration of IL2Cx alone and in both combinationssignificantly increased the more mature CD27þ CD11bþ NKcells, at the expense of the more terminally differentiated/senes-cent CD27– CD11bþ NK cells (ref. 30; Fig. 6E). Given that bothNK and Tregs can be boosted by IL2 (26), that CTLA-4 mAb candeplete Tregs (22), and that Tregs suppress NK cells (31, 32), weinvestigated the impact of the treatments on the NK cell/Tregratio. The only treatment that significantly increased the NKcell/Treg ratio was the combination IL2Cx plus anti–CTLA-4

(Fig. 6F). Overall, these data indicate that intratumoral NK cellsare sensitive to IL2Cx,which induces their accumulation, division,maturation, and activation, and it is the combination of IL2Cxwith anti–CTLA-4 that maximizes this effect. In view of theseresults and on our previous observation of the anti–CTLA-4-mediated depletion on Tregs, the increase in the NK cell/Tregratio observed with the IL2Cx/anti–CTLA-4 combination can beexplained by the fact that IL2-boosting effect on NK cells ispotentiated in the absence of Tregs, which both compete for IL2and suppress NK cells (28, 32, 33).

IL2Cx plus anti–CTLA-4 therapeutic effect depends on NK andCD8þ T cells

Our results suggested that the additive effects of the twocombinations could involve different cellular effectors. Thus, to

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IL2Cx in combination withanti–CTLA-4 boosts NK cells andincreases the NK cell/Treg ratio inthe tumor. Mice harboring B16F10-OVAmelanoma tumors weretreated as described in Fig. 3A.Flow-cytometry analysis of tumor-infiltrating NK cells was made 6days after beginning of thetreatment. A,Quantification of NKcells infiltrating the tumorexpressed as number of cells pergram of tumor. B, Frequency (%) ofIFNg-producing cells among totalNK cells infiltrating the tumor.C, Frequency (%) of Ki67þ cellsamong tumor-infiltrating NK cells.D, Frequency (%) of KLRG1þ cellsamong total tumor-infiltrating NKcells. E,Maturation status oftumor-infiltrating NK cells. Arepresentative dot plot of CD27 andCD11b staining on total NK cells isshown (panel). Percentages of NKcells with the indicated maturationstatus (CD11b�CD27þ! DP!CD11bþCD27�) are shown.F, Ratio NK cells/Tregs in the tumor,calculated as the frequency of totalNK cells/frequency of total Tregamong CD45þ cells. Data are fromtwo independent experiments.P values were calculated usingone-way ANOVA andKruskal–Wallis test. � , P < 0.05;�� , P < 0.01; �� , P < 0.001. See alsoSupplementary Fig. S6.

Caudana et al.





evaluate the relative contribution of NK and CD8þ T cells on thetherapeutic efficacy of the combined treatments, we performedAb-mediated selective depletion (Fig. 7A).We verified the efficacyof depletion by periodic immunomonitoring of CD8þ T and NKcells in the blood (Supplementary Fig. S7). Figure 7B–D illustratesthe effect of NK or CD8þ T-cell depletion on the efficacy ofthe combination treatments. In mice receiving the IL2Cx/anti–CTLA-4 combo, both depletion of CD8þ T or of NK cells abro-gated treatment efficacy (Fig. 7B and C). In mice receiving theIL2Cx/anti–PD-1/anti–PD-L1 combo, depletion of CD8þ T cellscompletely abrogated the therapeutic effect of the treatment.However, although NK cell depletion induced a nonstatistically

significant diminution in the efficacy of the combo, the treatmentstill delayed tumor growth (Fig. 7B–D). Overall, these resultsindicate that both combinations work through CD8þ T-cellactivation, but only the IL2Cx/anti–CTLA-4 combination isdependent on NK cells to be effective.

DiscussionSuccessful anticancer immunotherapies aimed at inducing

effective antitumor T-cell responses have delivered impres-sive clinical results, but only in some patients and in sometumor types (34, 35). Combinatory strategies represent

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Figure 7.

Depletion of CD8þ T or NK cells onB16F10-OVA tumor-bearing micetreated with IL2Cx and antibodies tocheckpoint pathways. A, Schematic ofthe treatment strategy for B16F10-OVAtumor–bearing mice. NK cell and CD8T-cell depletionwas started oncetumors were palpable and continuedduring the experiment. B, Tumorgrowth kinetics of individual mice.Dashed curves represent the tumorgrowth of mice under NK cell or CD8þ

T-cell depletion. C and D, Tumorgrowth kinetics represented as a meanSEM of n¼ 7–8 per group from onerepresentative experiment out of two.P values were calculated usingone-way ANOVA and Kruskal–Wallistest. NS, not significant; � , P < 0.05;�� , P < 0.01; See also SupplementaryFig. S7.






powerful therapeutic tools (36). Thus, we evaluated theefficacy of combining a T-cell growth factor, IL2, formulatedas an IL2/anti-IL2 complex, with clinically approved immune-checkpoint inhibitors. In an autochthonous lung adenocarci-noma model, we show that the IL2Cx/anti–PD-1 combinationdurably controls tumor growth and induces lung infiltration byactivated CD8þ T cells. Also, in a transplantable melanomamodel refractory to monotherapy with immune-checkpointblockers we found that IL2Cx synergizes with anti–CTLA-4and anti–PD-1/anti–PDL-1, improving the clinical outcomeof the monotherapies. In addition, we show that combinationof IL2Cx with PD-1 or CTLA-4 pathway blockade acts bydifferent cellular mechanisms, differentially targeting CD8þ

T, NK, and Tregs.High-dose IL2 was the first immunotherapy assessed for the

treatment of human cancer, achieving similar objectiveresponses in metastatic melanoma as anti-CKP treatment(15%; refs. 37, 38). However, fewer than 10% of eligiblepatients receive this potentially curative treatment, likely dueto its associated toxicity and need for hospitalization (3, 39), aswell as concern about the expansion of Tregs. Consequently,different approaches have been tried to bring IL2 back to theclinics, trying to bypass Treg activation and to improve IL2pharmacodynamics; and some of these approaches are underclinical evaluation. These strategies include, among others, amutated IL2 superkine with increased affinity for IL2Rbgc, amutein IL2 fused to tumor-specific Abs (40), an IL2 bound toreleasable polyethylene glycol chains (41); and IL2Cx (4) thathave longer half-life and improved pharmacodynamics. Ourresults, showing that administration of either the IL2Cx thatactivates effector cells, low doses of IL2 (that could be releasedupon dissociation from anti-IL2), or even an IL2Cx designed topreferentially stimulate Tregs (42–44), did not affect tumorgrowth, casting doubt on a potential unwanted stimulation ofTregs. Consequently, IL2-based therapeutic approaches in gen-eral, and IL2Cx, especially in combination with immune-check-point blockers, may be useful in addition to the existing arsenalof antitumor immunotherapies.

Few studies have evaluated the efficacy of immunomodulatorytreatments in genetic mouse models. In a model of spontaneousskin melanoma (NrasQ61K Ink4a–/–), IL2Cxs have shown someefficacy as monotherapy (7) and greater efficacy when combinedwith an epigenetic modulator (8). In another model of autoch-thonous melanoma (Braf/Pten model), antitumor effectsrequired amultidrug therapy including a tumor antigen–targetingantibody, IL2, anti–PD-1, and a T-cell vaccine, which activatedinnate and adaptive immune responses (45). Finally, anotherstudy using a geneticmodel of lung adenocarcinoma showed thatthe KP-OVA lung adenocarcinoma model, which was non–T-cellinfiltrated, was refractory to an anti–PD-1 plus anti–CTLA-4combination therapy, and only combination of these two mAbswith immunogenic drugs oxaliplatin and cyclophosphamideinduced antitumor CD8þ T cells and controlled tumor progres-sion (46). In light of these results underscoring the elevatedresistance of this tumor model to checkpoint blockade therapy,our data showing that IL2Cx increases the sensibility of autoch-thonous lung tumors to anti–PD-1provide proof of concept of theefficacy of this combined therapy for lung tumors. The lack ofobserved toxicity, plus the obtained mechanistic insight on themechanism of action of this therapeutic combination thatincreases tumor infiltration by CD8þ T cells and induces systemic

tumor-specific CD4þ and CD8þ T-cell responses, provides arationale for the design of combination therapies that shouldincrease the response rate of patients to immune-checkpointtherapy.

Blockade of the inhibitory PD-1/PD-L1 pathway has beenreported to reinvigorate chronically stimulated exhaustedCD8þ T cells during lymphocytic choriomeningitis virus(LCMV) infection (47, 48). Similar results were later observedduring cancer immunotherapy in mice models and in patientstreated with mAbs to PD-1 (49, 50). The benefits of combiningIL2 with PD-1/PD-L1 blockade were explored in the LCMVmodel, in which IL2 synergized with anti–PD-L1 treatment,improving CD8þ T-cell viral-specific responses and decreasingviral load (21). Our results show that IL2 (in the form of IL2Cx)can synergize with immune-checkpoint blockade to rescueCD8þ T cells function in cancer. Indeed, IL2Cx increased thebreadth of CD8þ T-cell responses, including reactivities againsttumor neoepitopes, boosted CD8þ T-cell division, and reinvi-gorated exhausted CD8þ TILs. This action was reinforced by anincrease in tumor infiltration by CD45þ cells and CD8þ acti-vated T cells when IL2Cx was combined with blockade ofimmune checkpoints.

CTLA-4 blockade during cancer is associated with increasedCD8þ T cell/Treg ratios and tumor rejection. Vaccination ofB16-bearing mice with a GM-CSF–transduced tumor cell vac-cine (GVAX) induced T-cell activation and infiltration, an effectpotentiated by CTLA-4 blockade (24). Additional blockade ofthe PD-1/PD-L1 pathway allowed further tumor-specific T-cellactivation (51). The antitumoral activity of anti–CTLA-4required binding of the Ab to Fcg receptors, which mediatethe reduction of Tregs at the tumor site (22, 52). In thepreclinical cancer setting, one study reported CTLA-4 blockadecombined with IL2 administration (53). Although this workused IL2 (and not IL2Cx) and different doses and treatmenttiming (beginning of treatment at day 3 after tumor implan-tation) than our study, they observed, as we did, a synergisticeffect of the combination on tumor growth delay and onthe increase in tumor-infiltrating CD8þ T and NK cells. How-ever, they did not observe Treg depletion when an mAb toa-CTLA-4 was combined with IL2, indicating that CTLA-4mAb–mediated Treg depletion can be differently modulatedwhen administered with plain IL2 or with IL2 in the form ofIL2Cx. Given our results suggesting that CTLA-4 mAb–mediat-ed depletion correlates with the high expression of CTLA-4 onTregs, it could be interesting to compare CTLA-4 kinetics ofexpression on intratumoral Tregs during these different thera-peutic interventions.

The combination of radiation with CTLA-4 and PD-L1blockade in both mice and human works by an increase inthe CD8þ T cell/Treg ratio induced by CTLA-4 blockade, andby reversion of T-cell exhaustion by PD-L1 blockade; radiationcontributed to enhanced diversity of the tumor T-cell reper-toire (20). Here, we observed that (i) the IL2Cx/anti–PD-1/PD-L1 combination worked through increasing tumor infil-tration by reinvigorated CD8þ T cells; and (ii) that the IL2Cx/anti–CTLA-4 therapy was the only combination dependentupon CD8þ T and NK cells, as depletion of either of these cellsdisabled the antitumoral effect of this combination. Indeed,we report that only the IL2Cx/anti–CTLA-4 combinationinduced an increased NK cell/Treg ratio in the tumor, whichwas due to the diminution of tumor-infiltrating Tregs (likely

Caudana et al.





mediated by anti–CTLA-4), and the accumulation of NK cells(likely mediated by the exogenously administrated IL2Cx andthe increased availability of endogenous IL2 secondary todecreased consumption by Tregs). During homeostasis, deple-tion of Tregs using an anti-CD25 can boost NK cell prolifer-ation and cytotoxicity in vivo (31). Gasteiger and collea-gues (27) proposed that Tregs can suppress mature NK cellfunction via TGFb and restrain NK cell maturation by IL2consumption. These two works support our hypothesis thatCTLA-4 mAb–mediated Treg depletion in the tumor facilitatesNK cell accumulation and maturation.

B16F10 melanoma tumors recruit CD27þCD11bþ NK cellswith low proliferative capacity, reduced KLRG1 and NKG2Dexpression, and low IFNg and GM-SCF production (54), sug-gesting reduced effector functions. Our results showed thatIL2Cx alone or combined with immune-checkpoint blockersalso increased the frequency of CD27þCD11bþ NK cells, whichrepresented the predominant NK subset in the tumor. How-ever, the phenotype of NK cells was modified by the treat-ments, as KLRG1 and NKG2D expression was increased, andproliferation and IFNg production was enhanced, likely over-coming the inhibitory phenotype induced by the tumordescribed by these authors. Overall, these results suggest thatdepletion of Tregs with concomitant IL2-mediated activation ofNK cells could complement immune-checkpoint blockade forcancer therapy.

In conclusion, our data suggest that IL2-Cx acts on T andNK cells by boosting their expansion and effector function.Blockade of the PD-1/PD-L1 pathway reinforces IL2Cx-inducedreinvigoration of exhausted CD8þ T cells. Anti–CTLA-4 also actsby depleting Tregs, which in turn affects CD8þ T and NK cells,increasing the CD8þ T cell/Treg and NK cell/Treg ratiosand unleashing the effector cells from Treg suppression. Althoughactivation of CD8þ T cells accounts for most of the efficacy ofthe IL2Cx/PD-1 pathway blockade combination treatment,CD8þ T-cell reinvigoration by the IL2Cx/anti–CTLA-4 combina-tion is not sufficient to control tumor progression, as thelatter combination was the only one dependent on NK cellactivation. Thus, our results clarify the cellular and molecularconsequences that follow from the combination of IL2Cx with

CTLA-4 or PD-1/PD-L1 blockade, and should help refine com-binatory cancer immunotherapies.

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

Authors' ContributionsConception and design: P. Caudana, N.G. N�u~nez, R. Alonso, L.L. Niborski,E. PiaggioDevelopment of methodology: O. Lantz, C. Sedlik, E. PiaggioAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N.G. N�u~nez, P. De La Rochere, A. Pinto, O. Lantz,C. Sedlik, E. PiaggioAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):N.G. N�u~nez, P. De La Rochere, A. Pinto, J. Denizeau,E. PiaggioWriting, review, and/or revision of the manuscript: P. Caudana, R Alonso,L.L. Niborski, O. Lantz, C. Sedlik, E. PiaggioAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P. De La Rochere, J. Denizeau, R. Alonso,E. PiaggioStudy supervision: E. Piaggio

AcknowledgmentsThis work was supported by the Fondation Chercher et Trouver-2016,

Institute Curie, Institut National de la Sant�e et de la Recherche M�edicale,Association pour la Recherche sur le Cancer (ARC PJA 20131200444); theAgence Nationale pour la Recherche (ANR-16-CE18-0002-02 and ANR-10-IDEX-0001-02 PSL); Labex DCBIOL (ANR-10-IDEX-0001-02 PSL and ANR-11-LABX0043), SIRIC INCa-DGOS-Inserm_12554, Center of Clinical Investi-gation (CIC IGR-Curie 1428). N.G. N�u~nez was supported by a fellowship fromLigue Nationale Contre le Cancer and P. Caudana by the International PhDprogram of Institut Curie.

We thank Virginie Dangles-Marie, Celine Daviaud, Isabelle Grandjean,Mikael Garcial, and Cedrik Pauchard, the mouse facility technicians and flowcytometry core at Institut Curie. We thank Sebastian Amigorena and ClotildeTh�ery for discussions and critical review of the manuscript and NathalieAmzallag for technical assistance.

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

Received October 1, 2018; revised December 21, 2018; accepted January 11,2019; published first January 16, 2019.

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2019;7:443-457. Published OnlineFirst January 16, 2019.Cancer Immunol Res Pamela Caudana, Nicolas Gonzalo Núñez, Philippe De La Rochere, et al. T-cell ModulationBlockade Rescues Antitumor NK Cell Function by Regulatory IL2/Anti-IL2 Complex Combined with CTLA-4, But Not PD-1,

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http://cancerimmunolres.aacrjournals.org/lookup/doi/10.1158/2326-6066.CIR-18-0697

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il2/anti-il2complexcombinedwithctla-4,but not pd-1 ......sion. mononuclear cells from lungs were...

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