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Tumor Biology and Immunology

NFATc1 Promotes Antitumoral Effector Functionsand Memory CD8þ T-cell Differentiation duringNon–Small Cell Lung Cancer DevelopmentLisanne Heim1, Juliane Friedrich1, Marina Engelhardt1, Denis I. Trufa2,Carol I. Geppert3, Ralf J. Rieker3, Horia Sirbu2, and Susetta Finotto1

Abstract

Nuclear factor of activated T cells 1 (NFATc1) is a transcriptionfactor activated by T-cell receptor (TCR) and Ca2þ signaling thataffects T-cell activation and effector function.Upon tumor antigenchallenge, TCR and calcium-release–activated channels areinduced, promoting NFAT dephosphorylation and translocationinto the nucleus. In this study, we report a progressive decrease ofNFATc1 in lung tumor tissue and in tumor-infiltrating lympho-cytes (TIL) of patients suffering from advanced-stage non–smallcell lung cancer (NSCLC). Mice harboring conditionally inacti-vated NFATc1 in T cells (NFATc1DCD4) showed increased lungtumor growth associated with impaired T-cell activation andfunction. Furthermore, in the absence of NFATc1, reducedIL2 influenced the development of memory CD8þ T cells. Wefound a reduction of effector memory and CD103þ tissue-resi-dent memory (TRM) T cells in the lung of tumor-bearingNFATc1DCD4 mice, underlining an impaired cytotoxic T-cell

response and a reduced TRM tissue-homing capacity. InCD4þICOSþ T cells, programmed cell death 1 (PD-1) wasinduced in the draining lymph nodes of these mice and asso-ciated with lung tumor cell growth. Targeting PD-1 resulted inNFATc1 induction in CD4þ and CD8þ T cells in tumor-bearingmice and was associated with increased antitumor cytotoxicfunctions. This study reveals a role of NFATc1 in the activationand cytotoxic functions of T cells, in the development ofmemory CD8þ T-cell subsets, and in the regulation of T-cellexhaustion. These data underline the indispensability ofNFATc1 for successful antitumor immune responses in patientswith NSCLC.

Significance: Themultifaceted role of NFATc1 in the activationand function of T cells during lung cancer developmentmakes it acritical participant in antitumor immune responses in patientswith NSCLC. Cancer Res; 78(13); 3619–33. �2018 AACR.

IntroductionNuclear factor of activated T cells is a transcription factor family

that consists of fivemembers, among which, NFAT1–4 (NFATc1–NFATc4) are regulated by Ca2þ–calcineurin signaling. NAFTc1and NFATc2 are the main isoforms expressed in T cells critical inregulating early gene transcription in response to T-cell receptor(TCR)-mediated signals (1–3). In resting T cells, NFAT transcrip-tion factors are located in the cytosol in an inactive, phosphor-ylated state. Upon TCR stimulation, NFAT is activated by calciumflux released from the endoplasmic reticulum and the extracel-lular environment through store-operated channels. Induced

intracytoplasmic calcium promotes calmodulin, a calcium sensorprotein, which activates the phosphatase calcineurin. Calcineurindephosphorylates NFAT, resulting in its translocation into thenucleus, where it cooperates with other transcription factorspromoting cell-specific gene transcription (3, 4).

The expression of NFATc1 in T cells is initiated by two distinctpromoters, resulting, along with alternative splice/polyadenyla-tion events, in six isoforms: NFATc1aA-C andNFATc1bA-C (5, 6).NFATc1aA has been shown to be strongly induced following TCRstimulation and ismaintainedby apositive autoregulation,whichenables a massive synthesis and facilitates T cells to exert effectorfunctions and escape from apoptosis (5, 7–9). Cytotoxic effectorfunctions are mediated by CD8þ T cells via the secretion ofinflammatory cytokines (10, 11). A recent study described thatNFATc1-deficient cytotoxic T cells showed reduced cytotoxicityagainst tumor cells. Furthermore, transcriptome analysis demon-strates diminished RNA levels of numerous genes in NFATc1�/�

CD8þT cells, including Il2 (9). IL2 is oneof thefirstmolecules thathas been shown to be induced by NFATc1 (12) and is importantfor T-cell activation, proliferation, and survival (13, 14). Further-more, IL2 influences the generation of memory CD8þ T cellsclassified into central memory (TCM), effector memory (TEM),and tissue-residentmemory (TRM)CD8þT cells that differ in theirtissue-homing capacity and effector functions (13, 15–17). Inmost of the established tumors, tumor-infiltrating lymphocytes(TIL) are found to be exhausted, leading to cancer immuneevasion. One remarkable feature of functionally exhausted T cells

1Department of Molecular Pneumology, University Hospital, Friedrich-Alexander-Universit€at Erlangen-N€urnberg, Erlangen, Germany. 2Department ofThoracic Surgery, University Hospital, Friedrich-Alexander-Universit€at Erlan-gen-N€urnberg, Erlangen, Germany. 3Institute of Pathology, University Hospital,Friedrich-Alexander-Universit€at Erlangen-N€urnberg, Erlangen, Germany.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Susetta Finotto, Laboratories of Cellular and MolecularLung Immunology, Department of Molecular Pneumology, Friedrich-Alexander-Universit€at Erlangen-N€urnberg, Hartmannstraße 14, Erlangen 91052, Germany.Phone: 4991-3185-42454; Fax: 4991-3185-35981; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-17-3297

�2018 American Association for Cancer Research.

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is the expression of inhibitory checkpoint receptors that desen-sitize TCR signaling, leading to functional impairment of T-cellactivation. On the basis of these findings, cancer immunothera-pies have been developed that reactivate exhausted TILs by block-ing inhibitory checkpoint receptors (18–20). Programmed celldeath (PD)-1 is one of the most successful checkpoint target indifferent cancer types including non–small cell lung cancer(NSCLC). Nevertheless, only approximately 30% of patients areresponsive to this therapy, and there is a need to find alternateregulators to improve current approaches (19–23). NFATc1 hasbeen shown to induce the expression of PD-1 upon T-cell acti-vation (24). But there is still a contradiction regarding the influ-enceofaPD-1 antibody treatment onNFATc1 inT cells. Triggeringof PD-1 by its ligand PD-L1 expressed on tumor cells induces theinhibition of the PI3K/Akt pathway. Akt normally negativelyregulates the glycogen-synthase kinase 3 (GSK3), which inhibitsNFATc1 (3, 25, 26). Blocking PD-1 by aPD-1 antibodies restoresTCR signaling and ensures the activation of Akt, which inhibitsGSK3 and promotes the activation of NFATc1, which inducesT-cell activation and effector functions (7, 9, 25). Thus, NFATc1could be a key regulator in aPD-1 anticancer immunotherapy.

Here we demonstrate an important function of NFATc1for successful T-cell–mediated antitumoral immune responses inthe setting of NSCLC. Targeted deletion of Nfatc1 in T cells(NFATc1DCD4) induced increased lung tumor growth in miceassociated with impaired T-cell activation and function. In theabsence of NFATc1, reduced IL2 influenced the developmentof memory CD8þ T cells. Consistently, we found a reduction ofTEM and CD103þ TRM T cells in the lung of tumor-bearingNFATc1DCD4 mice underlining an impaired cytotoxic T-cellresponse and a reduced TRM tissue-homing capacity. PD-1 wasfound coexpressed and accumulated with CD4þICOSþ T-cellsin the draining lymph nodes (dLN) of NFATc1DCD4 mice andassociated with lung tumor cell growth. Moreover, targeting PD-1inducedsignificantlyNFATc1inTcellsaccompaniedbyincreasedantitumor cytotoxic functions. Thus, this study revealed a role ofNFATc1notonly in theactivationandcytotoxic functionsofTcells,butalsointhedevelopmentofmemoryCD8þT-cellsubsetsandtheregulation of T-cell exhaustion underlining the indispensability ofNFATc1 for successful antitumor immune responses in NSCLC.

Patients and MethodsHuman subjects and study population

This studywas performed at the Friedrich-Alexander-Universityof Erlangen in Germany (Erlangen, Germany) and was approvedby the ethics review board of the University of Erlangen (Re-No:56_12B; DRKS-ID: DRKS00005376). Fifty-eight patients thatsuffered from NSCLC underwent surgery and gave their approvalto be enrolled in this study in an informed written consent. Thepatient studies were conducted in accordance with the ethicalguidelines of the Declaration of Helsinki.

The diagnosis of lung cancer was based on pathologic con-firmation. The histologic types of lung cancer were classifiedaccording to the classification of the World Health Organiza-tion (WHO), formulated in 2004. The staging of lung cancerwas based on the Cancer TNM Staging Manual, formulated bythe International Association for the Study of Lung Cancer(IASLC) in 2010. During surgery, lung tissue samples weretaken from the tumoral area (TU: solid tumor tissue), theperitumoral area (PT: up to 2 cm away from the solid tumor)

and from the tumor-free control area (CTR: >5 cm away fromthe solid tumor). This cohort of patients with NSCLC wasdescribed previously (27).

Protein extraction and Western blot analysesFor protein extraction, lung tissue samples were lysed in RIPA

buffer (Thermo Fisher Scientific) with added inhibitor cocktail(Roche Diagnostics), followed by homogenization using theSpeedMill PLUS (Analytik Jena) and innuSPEED lysis Tube P(Analytik Jena). After centrifugation (5minutes, 3,000 rpm, 4�C),supernatants were incubated on ice for 45 minutes, followed bycentrifugation (1 � 5 minutes, 3,000 rpm, 4�C; 1 � 45 minutes,full speed, 4�C). Finally, protein concentration was calculatedafter using Bradford Assay (Protein Assay Dye Reagent Concen-tration, Bio-Rad). Western blot analysis to detect NFATc1 (1:250;sc-1149, Santa Cruz Biotechnology), pNFATc1 (1:250; sc-32978,Santa Cruz Biotechnology), and b-actin (1:500, sc-1616, SantaCruz Biotechnology) was performed as described previously (28)with 50 mg of total lung protein. Quantification of total NATc1and pNFATc1 was performed using the AlphaView Software forFluorChem Systems (Biozym Scientific).

Double immunohistochemistry of NFATc1 and CD3 onparaffin-embedded lung tissue sections

IHC was performed on paraffin-embedded sections. Beforestaining, paraffin was removed from the slides by incubation at72�C for 30 minutes and treatment with Roti-Histol (Carl Roth)two times for 10minutes. The tissue sectionswere then rehydratedby immersion in ethanol series with descending concentrations(100%, 95%, 70%) for 3minutes each and in deionised water for1 minute, followed by blocking endogeneous peroxidase in 3%H2O2 (inmethanol) for 20minutes.Heat-induced antigen retriev-al was performed as described previously (27) using 1 mmol/LTris-EDTA wash buffer. Afterwards, slides were incubated withprimary antibody to CD3 (1:100, RBK024, Zytomed SystemsGmbH) overnight at 4�C. After different washing steps anddetection of the primary antibody following the manufacturer'sinstructions of the ZytoChem-Plus AP Polymer Kit (POLAP006,Zytomed Systems GmbH), the second antibody to NFATc1 (1:50,sc-7294, Santa Cruz Biotechnology) was applied for 2 hours atroom temperature. The second antibody was detected followingthe manufacturer's instructions of the Dako EnVision DetectionSystem Kit (K4065). Slides were covered with coverslips usingAquatex (108562,Merck).Negative controlswere not treatedwiththe primary antibodies and the other steps remain the same.Stained slides were scanned using the digital slide scanner (Scan150, 3D Histech Ltd) and NFATc1þCD3þ cells were quantifiedusing the Definiens Tissue Studio 4.1 software (Definiens) at theInstitute of Pathology, Friedrich-Alexander-Universit€at Erlangen-N€urnberg (FAU; Erlangen, Germany). Whole-slide images werevisualized by the CaseViewer (Version 2.0, 3D Histech Ltd).

Total cell isolation and flow cytometry analyses of human cellsFrozen human tissue samples were cut into small pieces using

scalpels (1–3mm2) followed by the preparation of single-cellsuspensions using the Tumor Dissociation Kit, human (MiltenyiBiotec) and the gentleMACSDissociator (Miltenyi Biotec) accord-ing to the manufacturer's instructions. The resulting single-cellsuspension was washed and ammonium-chloride-potassium(ACK) lysis buffer was applied as described previously (27). Forflow cytometry analyzes, total cells were incubated with the

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respective mix of surface antibodies dissolved in PBS and incu-bated for 30 minutes at 4�C. For the intracellular staining ofNFATc1, cells were fixed and permeabilized with fix/permsolution, consisting of 1% paraformaldehyde in 70% ethanolin accordance to the manufacturer's protocol (Biolegend).Antibodies used for flow cytometry are shown in Supplemen-tary Table S1. Flow cytometric analyses were performed byusing FACS Canto II (BD Biosciences). Datasets were analyzedby Cell Quest Pro version 4.02 (BD Biosciences) and Flow-Jov10.2 (FlowJo, LLC).

Cell linesThe human A549 cell line was purchased authenticated from

the ATCC bank. ATCC authenticates cell lines by Mycoplasma,bacterial, and fungal contamination testing, PCR and sequencingof selected genes, human virus testing, COI assay, and STR DNAprofiling.WedetectedMycoplasma contaminationusing theMyco-plasma Detection Kit (Absource Diagnostics GmbH), accordingto the manufacturer�s protocol (latest date: Aug 9, 2016). Onaverage, we perform ten cell passages between thawing and use inthe described experiments. Cells were cultured in F-12K Nut mixmedium (Gibco, Thermo Fisher Scientific), supplemented with10 % of FCS (PAA Laboratories), 1% of the antibiotics penicillinand streptavidin (pen/strep; PAA Laboratories) and 1% ofL-glutamine (L-Glu; Gibco) at 37�C and 5% of CO2.

The murine LL/2-luc-M38 (LL/2) cell line was purchasedand authenticated from Caliper Life Sciences (Bioware cell line,Caliper Life Sciences). All Caliper Life Sciences cell lines wereconfirmed to be pathogen-free by the IMPACT profile I (PCR) atthe University of Missouri Research Animal Diagnostic and Inves-tigative Laboratory. Furthermore, the luciferase expression iscoupled to a neomycin-resistant gene, which renders the cellresistant to Geneticin (G418). Therefore, we treated LL/2 cellswith G-418 solution (500 mg/mL, Sigma-Aldrich) to select lucif-erase-expressing cells. Three cell passages between thawing andusage in the described experiments were performed. LL/2 cellswere cultured in DMEM (Thermo Fisher Scientific), supplemen-tedwith 10%FCS, 1%pen/strep, and 1% L-Glu at 37�C and 5%ofCO2. Mycoplasma contamination was detected using the Myco-plasma Detection Kit (Absource Diagnostics GmbH), accordingto the manufacturer's protocol (latest date: August 9, 2016).

The murine CTLL2 cell line was kindly provided to us by PDDr. Ulrike Schleicher from the Institute of Microbiology, Univer-sity Hospital Erlangen (Erlangen, Germany). CTTL2 cells werecultured inRPMI1640medium (Gibco, Thermo Fisher Scientific),supplementedwith 10%of FCS, 1%pen/strep, 1%of L-Glu, and 4ng/mL IL2 (PeproTech GmBH) at 37�C and 5% of CO2.

RNA isolation and cDNA synthesisHuman lung tissue samples were homogenized using Precellys

Lysing Kits (Bertin Technologies) and the benchtop homogenizerMinilys (Bertin Technologies) as described in the manufacturer'sprotocol. Total RNA was isolated using peqGold RNA Pure(Peqlab) according to manufacturer's instructions. RNA wasreverse-transcribed into cDNA using the RevertAid First StrandcDNA Synthesis Kit (Fermentas) in accordance to the manufac-turer's instructions.

Quantitative real-time PCRQuantitative real-time PCR (qPCR) of synthesized cDNA

was performed by using iTaq Universal SYBR Green Supermix

(Bio-Rad) in a total volumeof20mL. Primerswerepurchased fromEurofins-MWG-Operon. Primer sequences formurine andhumanqPCR analyzes are depicted in Supplementary Table S2. Reactions(50 cycles, initial activation 98�C, 2 minutes, denaturation 95�C,5 minutes, hybridization/elongation 60�C, 10 minutes) wereperformed using the CFX-96 Real-Time PCR Detection System(Bio-Rad), and analyzed by the CFXManager Software (Bio-Rad).Relative quantification was performed using the 2�DDCt method,hypoxanthine-guanine-phosphoribosyltransferase (Hprt) wasused as internal standard.

siRNA transfection of A549 cells and apoptosis assayFor siRNA-mediated silencing of NFATc1, 3 � 105 cells were

incubated overnight in 6-well plates in antibiotic-free F-12K Nutmix medium containing 10 % FCS. For transfection, 25 nmol/LNFATc1-siRNA (GE Dharmacon) or 25 nmol/L nontargetingcontrol siRNA (siNT; GE Dharmacon) were applied together with4 mL DharmaFECT Transfection reagent 1 (GE Dharmacon) in2-mL antibiotic-free medium, supplemented with 10 % FCSaccording to the manufacturer's instructions. After 24-hour incu-bation, transfected cells were washed with PBS and then culturedin 2-mL antibiotic-free medium, supplemented with 10% FCS,and LEAF purified anti-human PD-L1 antibody (5 mg/mL; Biole-gend) or the respective LEAF purified mouse IgG2bk isotypecontrol (5 mg/mL; Biolegend) for 48 hours. For the induction ofPD-L1 on A549 cells, 3 � 105 cells were incubated overnight �IFNg (50 ng/mL; Biolegend) followed by siRNA-mediated silenc-ing of NFATc1 as described above and subsequent treatmentwith above described anti-human PD-L1 antibody or IgG2bkisotype control antibody for 24 hours. RNA was isolated andNFATC1-mRNA expression was analyzed via qPCR. Apoptosisassay was performed according to themanufacturer's instructionsby staining the cells with Annexin V (BD Biosciences) and pro-pidium iodide (BD Biosciences), followed by flow cytometryanalyses.

Luminescence assayFor luminescence analysis, 7 � 103 LL/2-luc-M38 (LL/2) cells

were cultured in a 96-well plate and incubated for 24 hours at37�C, 5% CO2 in DMEM supplemented with 10% FCS, 1% pen/strep, and 1% L-Glu. After 24 hours, supernatants were removed,cells were washed in PBS, and incubated with either 20% lymphnode–conditioned medium (LNCM) or lung-conditioned medi-um (LUCM). For preparation of LNCM or LUCM, total cells ofdLNs or lungs were cultured for 24 hours in the presence of aCD3(10 mg/mL; BD Biosciences) and aCD28 (1 mg/mL; Biolegend)antibodies or in the presence ofaPD-1mAbRMP1-14 (10 mg/mL;H€olzel Diagnostika) or the respective rat IgG2a mAb 2A3 isotypecontrol (10 mg/mL; H€olzel Diagnostika). Resulting supernatantwas used in LL/2 luminescence assay. After 24-hour incubation,LNCMor LUCMwas removed and LL/2 cells were treated with 15mg/mL luciferin (Promega) to detect the luminescence intensityusing the Centro XS3 LB 960 Microplate Luminometer (BertholdTechnologies). Respective cell numbers were calculated by using astandard curve.

Apoptosis analysis of LL/2-luc-M38 cellsFor apoptosis analysis, 7� 103 LL/2-luc-M38 (LL/2) cells were

seeded in 96-well plates in DMEM supplemented with 10% FCS,1% pen/strep, and 1% L-Glu. IncuCyte Caspase-3/7 Reagent(Essen BioScience) was used at a final concentration of 1

NFATc1 Promotes Antitumoral T-cell Functions in NSCLC

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mmol/L (1:5,000) and added directly to LL/2 cells in addition to30 ng/mL rmTNFa (ImmunoTools). Apoptotic cells (green objectcount, 1/mm2) were detected using the IncuCyte Live-CellAnalysis System (Essen BioScience) over a period of 72 hours,and the images were captured every 4 hours. Apoptosis has beenquantified as the number of green fluorescent caspase-3/7–activeobjects for each time point.

ELISAThe ELISA technique was utilized to analyze the cytokine

concentration in cell culture supernatants. ELISA was performedin accordancewith themanufacturer's instructions.Murine TNFa,IL2, IL10, and IFNg ELISA Sets were obtained from BD Bio-sciences, VEGFA, and IL7 ELISA Sets from R&D Systems.

MiceNFATc1DCD4 and NFATc1fl/fl littermate control mice are on a

C57BL/6 genetic background. These mice were together withC57BL/6 wild-type mice kept in-house at the local animal carefacility of the Friedrich-Alexander-University Erlangen-N€urnberg(Erlangen, Germany) under specific pathogen-free conditions. Allexperiments were performed in accordance with the German andEuropean laws for animal protection and were approved by thelocal ethics committees of the Regierung Unterfranken (Az 55.2-2532.1-36/13).

Murine model of lung adenocarcinoma and in vivo imagingFor tumor induction, LL/2-luc-M38 cells were cultured in

DMEM supplemented with 10% FCS, 1% pen/strep, and 1% L-Glu. A total of 1� 106 cells suspended in 200-mLDMEM (withoutsupplements) were injected into the tail vein of 6- to 8-week-oldfemalemice. At the indicated timepoints,micewereweighted andinjected intraperitoneally (i.p.) with luciferin (0.15 mg/g bodyweight; Promega). After 20 minutes, luciferase activity was mea-sured by the IVIS Spectrum In Vivo Imaging System (PerkinElmer).Luciferase activity wasmeasured by detecting luminescence inten-sity (photons per second).Mice were anesthetized with isofluraneduring the measurements. Lung tumor load analysis was per-formed in a logarithmic scale mode and the total flux (photonsper second) was determined as described previously (29). Block-ing of PD-1 in vivowas performed by injectingaPD-1mAb RMP1-14 intraperitoneally (H€olzel Diagnostika) or the respective ratIgG2a mAb 2A3 isotype control (H€olzel Diagnostika) at 150 mg/mouse every 3 days for maximal four injections starting at day 9postintravenous injectionof LL/2 cells. At day 20post intravenousinjection, total lung cells were isolated as described previously(30) and in vitro rechallenged with aPD-1 mAb RMP1-14 (10 mg/mL; H€olzel Diagnostika) or the respective rat IgG2a mAb 2A3isotype control (10 mg/mL; H€olzel Diagnostika) for 24 hoursfollowed by flow cytometry analyses.

Hematoxylin and eosin staining onmurine paraffin-embeddedlung sections

Lungs were removed, fixed in 10% formalin–PBS solution,dehydrated, and embedded in paraffin. Five-micrometer–thicklung sections from paraffin blocks were stained with hematoxylinand eosin for visualization of lung tumors.

Flow cytometry analyses of murine cellsSingle-cell suspensions from murine lungs were prepared as

described previously (30). For total cell isolation from spleen and

dLNs, the same protocol was used without collagenase/DNAseIdigestion. Total cells were incubated with the respective mix ofsurface antibodies dissolved in PBS and incubated for 30minutesat 4�C. For intracellular staining, cells were fixed and permeabi-lized with Fixation/Permeabilization concentrate/diluent inaccordance to the manufacturer's protocol (eBioscience). Intra-cellular staining of NFATc1 was performed as described in aprevious section. For cytokine immunofluorescence analysis, cellswere stimulated for 4 hours with ionomycin (1 mmol/L, Sigma-Aldrich) and PMA (50 ng/mL, Sigma-Aldrich) in the presence ofthe Golgi inhibitor monensin (2 mmol/L, eBioscience). Antibo-dies for intracellular staining were dissolved in permeabilizationbuffer (eBioscience) and incubated for 30 minutes at 4�C. Anti-bodies used forflow cytometry are shown in Supplementary TableS1. Flow cytometric analyses were performed by using FACSCa-libur and FACSCanto II (BD Biosciences). Datasets were analyzedby Cell Quest Pro version 4.02 (BD Biosciences) and Flow-Jov10.2 (FlowJo, LLC).

In vitro analysis of lung CD8þ T cellsSingle-cell suspensions from murine lungs were prepared as

described previously (30). CD8þ T cells were isolated from thelungs of na€�ve and tumor-bearing mice by magnetic cell separa-tion using the CD8a (Ly-2) MicroBeads Mouse Kit (MiltenyiBiotec) in accordance with the manufacturer's instructions. Thepurity of the isolated CD8þ T cells was confirmed by FACSanalysis. CD8þ T cells were then cultured in RPMI1640 medium(Gibco) containing 10% FCS, 1% pen/strep, and 1% L-Glu andstimulated with aCD3 (10 mg/mL; BD Biosciences) and aCD28(1 mg/mL; Biolegend) antibodies. After 48 hours, CD8þ T cellswere harvested and resuspended in 500 mL 75% cold EtOH addeddropwise. Cells were incubated for 24 hours at�20�C. Fixed cellswere thenwashed twicewith 1-mLwash buffer (PBSwith 1%FCS,0.09% NaN3 pH 7.2) and stained with PI (BD Biosciences)staining solution (1:85 in wash buffer), followed by flow cyto-metric analyses using FACSCanto II (BD Biosciences).

Statistical analysisStatistics were computed with GraphPad Prism7. The unpaired

t test was performed for parametric data containing nomore thantwo groups. Data are presented as mean � SEM and significancelevels indicated as follows: �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

ResultsLoss of NFATC1 in the tumoral region correlates with poorprognosis in patients with NSCLC

In this study, we started to investigate the role of NFATc1 inNSCLC by analyzing its mRNA expression in the tumoral (TU,solid tumor), peritumoral (PT, 2 cm around the solid tumor) andcontrol lung region (CTR, tumor-free control area) of our cohortof patients with lung adenocarcinoma (ADC) and lung squamouscell carcinoma (SCC), collectively grouped as NSCLC (Table 1).Here we found a significantly decreased expression of NFATC1mRNA in the TU region of patients with both ADC and SCC ascompared with the respective PT and CTR region (Fig. 1A).Accordingly, higher amounts of total NFATc1/b-actin proteinlevels were found in the CTR and PT region of patients with ADC,whereas in the TU region, total NFATc1/b-actin protein levelsdecreased (Fig. 1B; Supplementary Fig. S1A). In addition, weanalyzed the phosphorylation status of NFATc1 (pNFATc1) as

Heim et al.




Table 1. Clinical data of the NSCLC patient cohort analyzed in this study

Samplenumber

SampleID

Histologicclassification

Maximal tumordiameter (cm) Ta Nb Mc

TNMStadium Gender Age

Averagesmoking(P/Y)

1 1-MP SCC 1.3 1b 0 0 IA Male 80 402 2-MP SCC 5.1 1a 0 0 IA Male 57 403 3-MP ADC 5 2a 0 0 IB Male 79 604 4-MP SCC 2 1b 1 0 IIA Female 53 255 8-MP SCC 5.5 3 0 0 IIB Male 66 306 9-MP ADC 2.7 1b 2 0 IIIA Female 84 07 13-MP SCC 3 1b 0 0 IA Male 69 508 14-MP SCC 1.9 1a 0 0 IA Female 58 309 15-MP ADC 2.5 1b 0 0 IA Male 63 10010 16-MP ADC 4.6 3 0 0 IIB Female 70 1511 17-MP ADC 2.6 2 0 0 IB Male 74 7012 19-MP ADC 6.5 2b 0 0 IIA Female 55 3013 20-MP ADC 2.8 1b 0 0 IA Male 65 6014 21-MP SCC 2.5 1b 0 0 IA Male 41 1015 22-MP ADC 7 2b 1 0 IIB Male 68 8216 23-MP ADC 4.5 2a 0 0 IB Male 73 7517 26-MP ADC 1.3 1a 0 1 IV Female 52 5018 27-MP ADC 1.4 1a 0 0 IA Female 70 5019 28-MP ADC 1.2 1a 0 0 IA Male 76 6020 29-MP SCC 3.7 1b 0 0 IIB Male 74 10021 30-MP SCC 1.8 1a 0 0 IA Female 70 3022 32-MP ADC 4.4 2a 2 0 IIIA Female 60 3023 34-MP ADC 1.8 1 0 0 IA Female 51 4524 35-MP ADC 3 1b 0 0 IA Female 72 025 36-MP SCC 3.5 2a 1 0 IB Male 74 4026 37-MP SCC 3.3 2a 1 0 IIA Male 60 4527 40-MP ADC 1.8 1a 1 0 IIA Male 82 10028 41-MP SCC 1.3 3 1 0 IIIA Male 70 4229 42-MP SCC 1.5 2b 1 0 IIB Male 74 4030 43-MP ADC 4 2a 2 0 IIIA Female 72 031 44-MP ADC 1.5 1a 0 0 IA Male 53 7032 46-MP SCC 9.5 3 1 0 IIIA Male 60 3033 47-MP SCC 6 2b 0 0 IIA Male 64 8034 48-MP SCC 5 2b 0 0 IIA Male 55 2035 50-MP SCC 2.5 3 1 0 IIIA Male 70 036 51-MP ADC 2.4 1b 2 1 IV Male 62 9037 52-MP ADC # # # # # # # #38 53-MP ADC 2.25 1a 0 0 IA Male 62 1039 54-MP SCC 4.1 3 0 0 IIB Male 72 040 55-MP ADC 1.8 1a 2 0 IIIA Female 64 4041 56-MP ADC 4 2a 0 0 IB Female 67 042 57-MP ADC 3.8 2a 0 0 IB Female 35 1043 58-MP ADC 6.5 3 0 0 IIB Female 69 044 59-MP ADC 0.9 4 0 0 IIIA Male 70 #45 60-MP SCC 2.5 1b 1 0 IIA Male 71 #46 61-MP SCC 1.1 1a 0 0 IA Male 75 #47 62-MP ADC 3.5 1b 0 0 IA Female 80 #48 63-MP SCC 9 3 1 0 IIIA Male 69 #49 64-MP ADC 3.5 1b 0 0 IA Male 55 3550 65-MP SCC 2.8 1b 0 0 IA Female 76 #51 68-MP ADC 8 3 0 0 IIB Male 42 2252 69-MP ADC # # # # # # # #53 73-MP ADC 4.8 2a 0 0 IB Female 67 2254 74-MP ADC 3.2 2a 0 0 IB Female 58 #55 75-MP SCC 4.8 2a 1 0 IIA Male 54 3556 105-MP ADC 1.8 1a 0 0 IA Male 77 3557 106-MP ADC 1.2 1a 0 0 IA Male 79 6058 108-MP ADC 3.6 2a 0 0 IB Female 60 40

NOTE: #, no information available.aT-primary tumor: 0, No evidence of primary tumor; 1a, tumor 2 cm or less in greatest dimension; 1b, tumor more than 2 cm but not more than 3 cm in greatestdimension; 2a, tumormore than 3 cmbut notmore than 5 cm in greatest dimension; 2b, tumormore than 5 cmbut notmore than 7 cm in greatest dimension; 3, tumormore than 7 cm.bN-regional lymph nodes: 0, no regional lymph node metastasis; 1, metastasis in ipsilateral peribronchial and/or ipsilateral hilar lymph nodes and intrapulmonarynodes, including involvement by direct extension; 2, Metastasis in ipsilateral mediastinal and/or subcarinal lymph node(s).cM-distant metastasis: 0, no distant metastasis; 1, distant metastasis.





dephosphorylation of NFAT proteins leads to their activation interms of nuclear translocation (3). As a result, we found nodifferences in pNFATc1/b-Actin and pNFATc1/NFATc1 proteinlevels in the CTR, PT, and TU regions (Fig. 1B; Supplementary Fig.S1A). We next analyzed the NFATC1 mRNA expression in differ-ent disease stages according to the TNM classification describingthe size of the primary tumor, the degree of spread to regionallymph nodes, and the presence of distant metastasis. We foundthat, in the tumoral area,NFATC1mRNA expression decreased asthe tumor stadium progressed (Fig. 1C). Moreover, using IHCdouble staining for NFATc1 and CD3 on lung tissue arraysobtained from our cohort of patients, we demonstrated a down-regulation of NFATc1 in CD3þ T cells in the tumoral region ofadvanced NSCLC stages (Fig. 1D; Supplementary Fig. S1B). Fur-thermore,flowcytometry revealed an increase ofNFATc1 inCD8þ

T cells in the PT region as compared with the respective TU regionof patients with ADC (Fig. 1E), whereas in CD4þ T cells, nodifferences of NFATc1 could be observed (Supplementary Fig.S1C). In addition,NFATc1was found to be increased inCD8þPD-1þ T cells (Fig. 1F) in the CTR and PT region as compared with therespective TU region. No regional regulation was observed in thedistribution of NFATc1 in CD4þPD-1þ T cells (SupplementaryFig. S1D). Regarding CD4þ T cells, we observed a strong directcorrelation ofNFATC1 and CD4 expression in the PT region (Fig.1G), whereas no direct correlation was found in the CTR and TUregion (Supplementary Fig. S1E). Moreover, NFATC1 directlycorrelates with TBX21 expression (Fig. 1H), a gene that codes forTbet (T-box transcription factor TBX21), the main transcriptionfactor controlling IFNg production in T cells (31). In general, theseresults indicate a downregulation of NFATc1 in T cells in the

Figure 1.

Loss of NFATC1 correlates with poor prognosis of patients with NSCLC. A, qPCR based analysis of NFATC1 mRNA expression in lung tissue samples from thetumoral (TU), peritumoral (PT), and control (CTR) region of patients suffering from ADC (NCTR ¼ 30, NPT ¼ 25, NTU ¼ 26) or SCC (NCTR ¼ 17, NPT ¼ 18,NTU ¼ 18) collectively grouped as NSCLC (NCTR ¼ 47, NPT ¼ 43, NTU ¼ 44). B, Quantitative Western blot analysis of total NFATc1/b-actin, pNFATc1/b-actin,and pNFATc1/NFATc1 protein levels in the CTR, PT, and TU area of patients with ADC (NCTR ¼ 6, NPT ¼ 6, NTU ¼ 5). C, Expression analysis of NFATC1 mRNAin the TU region of patients with ADC (IA, NTU ¼ 7; IIA, NTU ¼ 2; IIIA, NTU ¼ 4), patients with SCC (IA, NTU ¼ 5; IIA, NTU ¼ 5; IIIA, NTU ¼ 4), and totalpatients with NSCLC (IA, NTU ¼ 12; IIA, NTU ¼ 7; IIIA, NTU ¼ 8) classified according to the TNM staging system. D, Double IHC for NFATc1 (brown) and CD3(blue) on lung tissue array in the TU region of patients with NSCLC classified according to the TNM staging system (IA, NTU ¼ 7; IIA, NTU ¼ 2; IIIA, NTU ¼ 1;magnification, �60). E and F, Flow cytometry analyses of NFATc1 in CD8þ T cells (%; E), and NFATc1 in PD-1þCD8þ T cells (%) in total lung cell suspensionof the CTR, PT, and TU region of patients with ADC (ADC: NCTR¼ 3, NPT¼ 3, NTU¼ 3; F).G andH, Correlation between lungNFATC1 and CD4 (NPT¼ 41) expression inthe PT region; TBX21 (NTU ¼ 38) expression in the TU region (G); IL2 (NTU ¼ 23), CD122 (NTU ¼ 36), and CD132 (NTU ¼ 35) expression in the TU region (H);IL7 (NCTR¼ 37),CD127 (NCTR¼41) expression in the CTR region andCD127 (NTU¼40) expression in the TU region (I) frompatientswith NSCLC (J).N values are givenper group. Data are shown as mean values � SEM using Student two-tailed t test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

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presence of NSCLC and at the advanced disease stage. Further-more, NFATc1þPD1þCD8þ T cells were found induced in theCTRand PT region of the lung of patients with ADC, confirming recentfindings that T cells in these patients are exhausted (32) and thusrepresent the target for the aPD-1-tumor immunotherapy.Because NFATc1 controls the IL2 promoter, we next correlatedNFATC1 with IL2 and its receptor subunits IL2Ra (CD25), IL2Rb(CD122) and IL2Rg (common cytokine receptor g , CD132;ref. 33). We found that in the TU region, NFATC1 expressiondirectly correlates with IL2 as well as CD122 and CD132 (Fig. 1I)but not with CD25 (Supplementary Fig. S1F). The IL2Ra chain ispresent on T effector as well as T regulatory cells. In contrast, theIL2Rb and IL2Rg chain are characteristic for memory T cells (34,35), indicating a potential role of NFATc1 in T-cell memory

expansion. In accordance to this, we found a direct correlationofNFATC1with the T memory cell markers IL7 in the CTR regionas well as CD127 (IL7Ra; ref. 36) in both the CTR as well as TUregion of patients with NSCLC (Fig. 1J).

Influence of siRNA-mediated knockdown ofNFATC1 in humanlung tumor cells in response to PD-L1 inhibitors

Likewise PD-1, which is highly expressed on TILs in manycancer types, its ligand PD-L1 is commonly upregulated on thetumor cell surface of different human tumors including lungcancer (37, 38). On the basis of this notion, we analyzed whetherthere is a relation between NFATc1 and PDL-1 in NSCLC devel-opment. We found a direct correlation between NFATc1 and PD-L1 in the TU region of patients suffering from NSCLC (Fig. 2A).

Figure 2.

Influence of siRNA-mediated knockdown of NFATC1 in tumor cells on the response to PD-L1 inhibitors. A, Correlation between NFATC1 and PD-L1 (NTU ¼ 24)mRNA expression in the lung TU region of patients with NSCLC. B, Flow cytometry analysis of NFATc1þPD-L1þEpCAMþ cells (%) in total lung cell suspensionof the CTR, PT, and TU region of patientswith ADC (NCTR¼ 3, NPT¼ 3, NTU¼ 3). C, Experimental design. A549 cells were cultured overnight, followed by transfectionwith a NFATC1-directed siRNA (siNFATc1) or a nontargeting siRNA (siNT). Twenty-four hours later, A549 cells were incubated with 5 mg/mL aPD-L1 antibodyor IgG2b isotype control, followed by another 48-hour incubation time and subsequent analysis ofNFATC1mRNA expression (D), PD-L1þPD-1� aswell as PD-L1�PD-1þ A549 cells (%; E), A549 cell number (F), and AnnVþPIþ and AnnV�PIþ apoptotic A549 cells (IgG2b: NsiNT¼ 6, NsiNFATc1¼ 6; aPD-L1: NsiNT¼ 6, NsiNFATc1¼ 6; G).H,Experimental design. A549 cells were cultured with � 50 ng/mL IFNg for 24 hours, followed by transfection with siNFATc1 or siNT. Twenty-four hours later,A549 cells were incubated with 5 mg/mL aPD-L1 antibody or IgG2b isotype control, followed by another 24-hour incubation time and subsequent analysisof NFATC1 mRNA expression (I), PD-L1þPD-1� as well as PD-L1�PD-1þ A549 cells (%; J), and A549 cell number (unstimulated, US: NsiNT ¼ 3, NsiNFATc1 ¼ 3; IFNg :NsiNT ¼ 3, NsiNFATc1 ¼ 3; IFNgþIgG2b: NsiNT ¼ 3, NsiNFATc1 ¼ 3; IFNgþaPD-L1: NsiNT ¼ 3, NsiNFATc1 ¼ 3; K). N values are given per group. Data are shown as meanvalues � SEM using Student two-tailed t test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.





Furthermore, lung cancer is of epithelial cell origin and thus, weanalyzedNFATc1 in epithelial cells of the lung of these patients byusing an epithelial-specific antibody (anti-EpCAM). This analysisshowed decreased NFATc1þPDL-1þEpCAMþ cells in the TUregion of patients with ADC, whereas in the CTR region, amodestproportion of EpCAMþ cells expressing both NFATc1 and PD-L1could be detected (Fig. 2B). We next analyzed the influence ofNFATc1 on the response to PD-L1 inhibitors in lung tumor cells.To this aim, we knocked down the expression of NFATC1 in thehuman lung adenocarcinoma cell line A549 using NFATC1-directed siRNA (siNFATc1) followed by treatment with anaPD-L1 antibody (aPD-L1; Fig. 2C). In this experimental setting,we achieved a specific NFATC1-knockdown as compared withnontargeting siRNA (siNT) after both aPD-1 and IgG2b isotypecontrol treatment (Fig. 2D). Moreover, aPD-L1 antibody treat-ment of A549 successfully blocked PD-L1, indicated by adecreased proportion of PD-L1þPD-1� A549 cells, whereassiRNA-mediated NFATC1 knockdown did not influence PD-L1on A549 (Fig. 2E, left). Notably, we only detected a low propor-tion of PD-1þPD-L1� A549 cells (Fig. 2E, right). To get a firstindication whether the loss ofNFATC1 in tumor cells effects theirgrowth, we counted A549 cells and found that NFATC1 knock-down with or without aPD-L1 antibody treatment did not affectthe overall numbers of living A549 (Fig. 2F). Furthermore, anal-ysis of apoptosis induction using Annexin (Ann)V/PI stainingrevealed a slight increase of AnnVþPIþ late apoptotic A549 cellsafter siRNA-mediated NFATC1-knockdown in combination withPD-L1 blockade compared with A549 cells transfected with siNTand treated with IgG2b (Fig. 2G). In addition, aPD-L1 antibodytreatment of A549 cells, in which NFATC1 was knocked down,induced a slight expansion of AnnV�PIþ dead A549 cells ascompared with the IgG2b treatment (Fig. 2G). As we detectedonly a modest expression of PD-L1 by A549 cells (Fig. 2E), wesubsequently stimulated them with IFNg followed by siRNA-mediated NFATC1-knockdown and aPD-1 antibody treatment(Fig. 2H). IFNg has been shown to induce PD-L1 in human airwayepithelial cells (39). Furthermore, stimulation of A549 cells withIFNg mimics host immune responses as this cytokine is predom-inantly produced by T helper 1 (Th1) cells as well as cytotoxicCD8þ T cells to mediate antitumoral immune responses (40). Inthis experimental setting, downregulation of NFATc1 mRNA wasobserved by siRNA-mediated NFATC1 knockdown and main-tained after aPDL-1 antibody treatment (Fig. 2I). While stimu-lation of A549 cells with IFNg induced a strong increase inPD-L1þPD-1� A549 cells (Fig. 2J), siRNA-mediated NFATC1knockdown did not influence the growth of A549 tumor cellsneither in combination with aPD-L1 nor with IgG2b (Fig. 2K).Regarding PD-1 expression, again we only detected a low pro-portion of PD-1þPD-L1� A549 cells (Fig. 2J). Altogether, NFATc1expressed in tumor cells has no influence, neither on tumor cellgrowth, nor on their response to aPD-L1 antibody treatment.

Targeted deletion of Nfatc1 in T cells causes severe lung tumordevelopment

As we demonstrated that NFATc1 expression in tumor cells hasno influence on the tumor cell growth and response to aPD-L1antibody treatment (Fig. 2), we started to investigate the role ofNFATc1 in T cells in a murine model of lung adenocarcinoma, acondition that closely resembles the human condition in NSCLC.Lung tumor development was induced by intravenous injectionof LL/2-luc-M38 (LL/2) cells followed by the analysis of tumor

growth via an in vivo bioluminescence-based imaging system(Fig. 3A). Here we observed a downregulation of NFATc1a inCD4þ T cells isolated from the lung of mice suffering from lungadenocarcinoma (LL/2) as compared with those isolated fromna€�ve control mice (na€�ve; Fig. 3B), indicating that NFATc1amediates anti-tumoral effector functions in T cells. On the basisof this observation, we went on and analyzed lung adenocar-cinoma development in mice with a conditional inactivation ofNFATc1 in CD4þ and CD8þ T cells (NFATc1DCD4) comparedwith control littermates (NFATc1fl/fl). Here, we found thattargeted deletion of Nfatc1 in T cells resulted in increased lungtumor growth as shown by bioluminescence in vivo imaging(Fig. 3C) and histologic analysis indicated increased infiltrationof tumor cells into the lung (Fig. 3D). Furthermore, we coulddemonstrate that exon 3 of Nfatc1 was successfully deleted in Tcells of NFATc1DCD4 mice as shown by decreased Nfatc1ex3mRNA expression in isolated lung CD8þ T cells (Fig. 3E) andsplenic CD4þ T cells (Fig. 3F).

NFATc1 influences tissue homeostasis and differentiation ofmemory CD8þ T cells

NFATc1 is highly expressed in peripheral T cells and importantfor their activation and cytotoxic function (5, 7). Thus, we nextanalyzed whether the increased tumor growth in NFATc1DCD4

mice could possibly be explained by reduced antitumoral T-cell–mediated immune responses. As effector CD8þ T cells haveantitumor functions (10, 11), we next analyzed the number ofCD8þ TILs in the lungs of NFATc1fl/fl and NFATc1DCD4 mice. Wefound a reduced proportion CD8þ T cells in the lungs ofNFATc1DCD4 mice (Fig. 4A). In NFATc1DCD4 mice bearing lungtumor, these CD8þ T cells are characterized by a decreasedexpression of CD25 (IL2Ra; Fig. 4B) as well as by a diminishedproliferative capacity indicated by a reduced proportion of CD8þ

T cells in the G2–M phase of the cell cycle (Fig. 4C). Furthermore,while we found no differences in the IL2 production by lungCD8þ T cells, we could show a strong decrease in IL2 produced byCD8þ T cells in the spleen of NFATc1DCD4 tumor-bearing mice(Fig. 4D). A reduced production of IL2 by CD8þ T cells is acharacteristic feature of T-cell exhaustion (41). Moreover, CD8þ

memory T cells relay on autocrine IL2 production, which enablesoptimal secondary population expansion and promotes a robustresponse in advanced tumor-bearing states (13).Memory CD8þ Tcells are heterogeneous with respect to phenotypic markers,effector functions, and tissue-homing capabilities and are classi-fied into central memory T cells (TCM), effector memory T cells(TEM), and tissue-resident memory T cells (TRM). TCM cellsreside in secondary lymphoid organs and provide protectionfrom a systemic challenge while TEM cells are highly reactive forimmediate protection and circulate between lymphoid organsand peripheral tissues or inflammatory sites (15). In the spleen oftumor-bearing NFATc1DCD4 mice, we found an increase in TCMcell populations and a trend toward reduction of TEM cells(Fig. 4E). Furthermore, in the tumor-bearing lung of NFATc1DCD4

mice TEM cells were significantly decreased (Fig. 4F) underliningan impaired cytotoxic T-cell response in these mice. TRM cellsoccupy tissues and mucosal sites such as the lung and are char-acterized by the lack of recirculation via the bloodstream, which isimportant for long-term regional immunity. The surfacemoleculeintegrin aE CD103 is linked to TRM cells and promotes theirlocalization to epithelia (16). Furthermore, NFATc1 has beenshown to induce the expression of the Itgae gene encoding CD103

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(9). Consistently, we found a decreased proportion of CD103þ

TRM cells in the lung of tumor-bearing NFATc1DCD4 mice (Fig.4G), indicating an impaired tissue-homing capacity by reducedCD103. Furthermore, in a recent study, a greater density ofCD103þ TRM cells has been linked to an enhanced cytotoxicityof CD8þ T cells inNSCLC (42). Thus, a reduction inCD103þ TRMcell populations is accompanied by decreased cytotoxicity ofCD8þ T cells, a situation that we found in the lungs of tumor-bearing NFATc1DCD4 mice.

In general, IL2 signals during different phases of an immuneresponse are key inoptimizingCD8þ effector aswell asmemory T-cell functions. A loss of NFATc1 affects the production ofIL2 as well as of CD103, which influences antitumor effectorfunctions of CD8þ T cells and the tissue-homing capacity of TRMcells.

Cytokines secreted from NFATc1DCD4 dLNs support tumor cellgrowth

Aswe foundadecreased expressionofNFATc1a in lungCD4þTcells of tumor-bearing wild-type mice (Fig. 3B), we analyzed theinfluence of the conditional inactivation of NFATc1 in T cells onCD4þ T-cell properties in NFATc1DCD4 mice. While we could notdetect a difference in the proportion of lung CD4þ T-cell popula-tions neither in na€�ve nor in tumor-bearing NFATc1DCD4 mice(Supplementary Fig. S2A), we found that in the absence ofNFATc1, lung CD4þ T cells express a lower amount of theactivation marker CD25 (IL2Ra; Fig. 5A). Furthermore, lung

CD4þ T cells of tumor-bearing NFATc1DCD4 mice, but not ofna€�ve mice, are characterized by a reduced production of IL2 (Fig.5B; Supplementary Fig. S2B), important for T-cell proliferation,survival, and activation (14). The observed reduction of IL2production by lung CD4þ T cells was even more dramaticallyreduced by CD4þ T cells in the spleen of tumor-bearingNFATc1DCD4 mice (Fig. 5B). In addition, reduced CD4þ T-cellproportions in the spleen and the draining lymph nodes (dLN) ofna€�ve and tumor-bearing NFATc1DCD4mice (Fig. 5C; Supplemen-tary Fig. S2C) indicate an impaired CD4þ T-cell development insecondary lymphoid organs. Moreover, we detected an increaseof PD1 on ICOSþCD4þ T cells in the dLNs of tumor-bearingNFATc1DCD4 mice (Fig. 5D). We hypothesized that thePD1þICOSþCD4þ T-cell population represents lymphocytesthat are inhibited in their cytotoxic properties by T-cell exhaus-tion, which dampened antitumoral immune responses andpromotes lung tumor growth. Therefore, we analyzed whethersoluble factors present in the supernatants of cells isolatedfrom the dLNs of tumor-bearing NFATc1DCD4 mice (¼lymphnode-conditioned medium, LNCM) would influence the sur-vival of LL/2 lung tumor cells (Fig. 5E). We could show that,LNCM of tumor-bearing NFATc1DCD4 mice significantlyincreased LL/2 cell numbers as compared with LNCM oftumor-bearing NFATc1fl/fl control littermates (Fig. 5F). In addi-tion, we analyzed the influence of supernatants of total lungcells (¼lung-conditioned medium, LUCM), but could notobserve any effect on LL/2 cell proliferation (Supplementary

Figure 3.

Targeted deletion of Nfatc1 in T cells causes severe lung tumor development. A, Experimental design for the induction of lung adenocarcinoma developmentin mice. Tumor growth was induced via intravenous (i.v.) injection of LL/2-luc-M38 (LL/2) lung carcinoma cells. Tumor load was measured using an in vivobioluminescence–based imaging system. The experiment endedonday 16 to 21.B,qPCR-based analysis ofNfatc1alphamRNAexpression in isolated lungCD4þTcellsof na€�ve and tumor-bearingwild-typemice (LL/2; Nna€�ve¼ 5, NLL/2¼ 5). C,Representative in vivo images of lung tumor load analysis in NFATc1fl/fl control littermates(NFATc1fl/flþ LL/2) andNFATc1DCD4 (NFATc1DCD4þ LL/2)mice (left). Radiance (photonflux/second) of the last dayof three independent experiments is summarized(right), (NNFATc1fl/flþLL/2¼ 13, NNFATc1DCD4þLL/2¼ 14). D, Representative hematoxylin and eosin staining of the lung of a NFATc1fl/fl and NFATc1DCD4 mouse. E, qPCR-based analysis of exon 3ofNfatc1 (Nfatc1ex3) in isolated lungCD8þT cells of tumor-bearingNFATc1fl/fl andNFATc1DCD4mice (NNFATc1fl/flþLL/2¼4, NNFATc1DCD4þLL/2¼3). F, qPCR-based analysis of Nfatc1ex3 mRNA expression in isolated CD4þ T cells from the spleen of lung tumor-bearing NFATc1fl/fl and NFATc1DCD4 mice(NNFATc1fl/flþLL/2¼ 3, NNFATc1DCD4þLL/2¼ 2).N values are given per group. Data are shown asmean values� SEMusing Student two-tailed t test. � ,P<0.05; �� ,P<0.01;�� , P < 0.001.





Fig. S2D and S2E). Thus, LNCM but not LUCM, of NFATc1DCD4

mice contain soluble factors that promote LL/2 lung tumor cellsurvival. To start to identify this factor, we analyzed the pres-ence of different cytokines and growth factors. While VEGFA,IFNg , and IL10 were not found to be differentially regulated(Supplementary Fig. S2F–S2H), we observed a decrease ofTNFa, IL2, and IL7 in LNCM of NFATc1DCD4 mice (Fig. 5G–

I). Moreover, we could show that TNFa, an antitumoral cyto-toxic molecule, induces apoptosis in LL/2 cells (Fig. 5J). Insummary, during lung adenocarcinoma development, the lossof NFATc1 in CD4þ T cells resulted in decreased CD4þ T-cellactivation and IL2 production in both the lung and secondarylymphoid organs. Furthermore, we could show that NFATc1

upregulates antitumor cytokines in the dLNs of tumor-bearingNFATc1DCD4 mice.

aPD-1 antibody treatment induced NFATc1 in CD4þ and CD8þ

T cellsIn our cohort of patients, we observed an increase of NFATc1 in

CD8þPD-1þ T cells in the CTR and PT region (Fig. 1F) confirmingrecent findings that T cells in these patients are exhausted (32)probably because of chronic activation via the tumor antigen.They thus represent the target for aPD1-tumor immunotherapy.To analyze whether a blockade of PD-1 influences NFATc1 inT cells, we continued treating lung tumor–bearing B6 wild-type(WT) mice with aPD-1 antibodies (aPD-1; Fig. 6A). In terms of

Figure 4.

NFATc1 influences tissue homeostasis and differentiation of memory CD8þ T cells. A, Flow cytometry analyses of CD8þ T cells (%) in total lung cell suspension ofna€�ve and lung tumor–bearing NFATc1fl/fl and NFATc1DCD4 mice (NNFATc1fl/fl ¼ 12, NNFATc1DCD4 ¼ 11; NNFATc1fl/flþLL/2 ¼ 11, NNFATc1DCD4þLL/2 ¼ 13). B, Flow cytometryanalyses of CD25 expressing CD8þ T cells (%) in total lung cell suspension of lung tumor–bearing NFATc1fl/fl and NFATc1DCD4 mice (NNFATc1fl/flþLL/2 ¼ 8,NNFATc1DCD4þLL/2¼ 11).C, PI proliferation analysis of CD8þ T cells isolated from the lungs of na€�ve and tumor-bearing NFATc1fl/fl andNFATc1DCD4mice (NNFATc1fl/fl¼ 3,NNFATc1DCD4 ¼ 3; NNFATc1fl/flþLL/2 ¼ 4, NNFATc1DCD4þLL/2 ¼ 4). D, Flow cytometry analysis of IL2 production by CD8þ T cells (%) in total cell suspension of thelung and the spleenof tumor-bearingNFATc1fl/fl andNFATc1DCD4mice (lung: NNFATc1fl/flþLL/2¼8, NNFATc1DCD4þLL/2¼ 11; spleen:NNFATc1fl/flþLL/2¼4, NNFATc1DCD4þLL/2¼5). E–G, Flow cytometry analysis of CD8þ memory T cells in the spleen and the lung of tumor-bearing NFATc1fl/fl and NFATc1DCD4 mice. Percentage ofCCR7þCD62LþCD8þ (%) central memory T cells (TCM) and CCR7�CD62L�CD8þ (%) effector memory T cells (TEM) in total cell suspension of the spleen(NNFATc1fl/flþLL/2 ¼ 3, NNFATc1DCD4þLL/2 ¼ 3; E). Percentage of Ly6CþKLRG1þCD62L�CD8þ (%) TEM cells (F) and CD103þCD8þ (%) tissue-resident memoryT cells (TRM; G) in total cell suspension of the lung (NNFATc1fl/flþLL/2 ¼ 4, NNFATc1DCD4þLL/2 ¼ 5). N values are given per group. Data are shown as meanvalues � SEM using Student two-tailed t test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

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survival (Fig. 6B) and weight change (Fig. 6C), we could notobserve any differences between WT mice treated with aPD-1 orthe respective IgG2a isotype control (IgG2a). Analysis of lungtumor load revealed a trend toward reduced tumor developmentin aPD-1–treated WT mice at day 15 postinjection (p.i.), whichwas declined at day 18 p.i. (Fig. 6D). As we could not observe adifference in lung cancer development after aPD-1 antibodytreatment, we rechallenged total cells from the lungs of thesemice with aPD-1 or IgG2a in vitro for 24 hours (Fig. 6A). Rechal-lenging the lung cells in vitro with aPD-1 antibodies induced astrong increase of NFATc1 in both CD4þ and CD8þ T cells fromabout 70%up to 90% (Fig. 6E and F; Supplementary Fig. S3A andS3B). Afterwards, we applied supernatants of the rechallengedtotal lung cells (LUCM) in a cytotoxicity assay with LL/2 tumorcells used to induce lung tumor growth in mice (Fig. 6G). As aresult, we observed a reduction in LL/2 cell growth induced byLUCMof total lung cells obtained frommice bearing tumor and invitro rechallenged with aPD-1 as compared with IgG2a (Fig. 6H).Collectively, these data demonstrate a strong inductionofNFATc1

in T cells byaPD-1 antibody treatment,which seems to inhibit LL/2 lung tumor growth via a soluble released factor. These are verysupportive and challenging results in T cells as a new therapeuticavenue for NSCLC.

DiscussionInour current study,wedemonstrated an important functionof

NFATc1 for successful T-cell–mediated antitumoral immuneresponses in the setting of NSCLC. In our human patient cohort,we found a downregulation ofNFATc1 in T cells in the presence ofNSCLC and at advanced disease stages, indicating that a loss ofNFATc1 in T cells is associated with poor prognosis. Upon T-cellactivation, NFATc1 regulates the expression of numerous genescontrolling the activity and fate of T cells. Among them IL2, whichhas been shown to be important for T-cell activation, prolifera-tion, and survival as well as for CD8þ effector and memory T-cellgeneration (9, 14, 17). Accordingly, we found a direct correlationbetween NFATc1 and IL2 as well as its receptor subunits CD122

Figure 5.

Cytokines secreted from NFATc1DCD4 dLNs support tumor cell growth. A, Flow cytometry analysis of CD25-expressing CD4þ T cells (%) in total lung cell suspensionof lung tumor-bearing NFATc1fl/fl and NFATc1DCD4 mice (NNFATc1fl/flþLL/2 ¼ 8, NNFATc1DCD4þLL/2 ¼ 11). B, Flow cytometry analysis of IL2 production by CD4þ Tcells (%) in total cell suspension of the lung and the spleen of tumor-bearing NFATc1fl/fl andNFATc1DCD4mice (lung: NNFATc1fl/flþLL/2¼ 8, NNFATc1DCD4þLL/2¼ 11; spleen:NNFATc1fl/flþLL/2 ¼ 4, NNFATc1DCD4þLL/2 ¼ 5). C, Flow cytometry analyses of CD4þ T-cell population (%) in total cell suspension of the spleen and the draininglymph nodes (dLN) of tumor-bearingNFATc1fl/fl andNFATc1DCD4mice (spleen: NNFATc1fl/flþLL/2¼ 4, NNFATc1DCD4þLL/2¼ 5; dLN: NNFATc1fl/flþLL/2¼ 8, NNFATc1DCD4þLL/2¼10).D, Flow cytometry analysis of PD-1 (%) gated on CD4þICOSþ T cells in total cell suspension of dLNs of tumor-bearing NFATc1fl/fl and NFATc1DCD4mice (NNFATc1fl/

flþLL/2 ¼ 8, NNFATc1DCD4þLL/2 ¼ 9). E, Experimental design. Cytotoxicity assay of LL/2 cells incubated with lymph node–conditioned medium (LNCM)from dLNs of tumor-bearing mice. Total cells of dLNs were cultured for 24 hours in the presence of aCD3 and aCD28 antibodies and resulting supernatant (SN) wasdefined as LNCM. LL/2 cells were incubated with 20% LNCM for 24 hours, followed by luminescence analysis to determine LL/2 cell numbers. F, Resultsof LL/2 cytotoxicity assay. LL/2 cellswere incubatedwith 20%LNCMof tumor-bearingNFATc1fl/fl andNFATc1DCD4mice. RPMImediumwasused as a negative control,supernatant of CTLL2 cells as a positive control (NNFATc1fl/flþLL/2¼ 7, NNFATc1DCD4þLL/2¼ 10, NRPMI¼ 8, NCTLL2¼ 7).G–I, ELISA analysis of TNFa (G), IL2 (H), and IL7 (I)levels in LNCM of lung tumor–bearing NFATc1fl/fl and NFATc1DCD4mice (TNFa: NNFATc1fl/flþLL/2¼ 6, NNFATc1DCD4þLL/2¼ 10; IL2: NNFATc1fl/flþLL/2¼ 3, NNFATc1DCD4þLL/2¼6; IL7: NNFATc1fl/flþLL/2¼ 3, NNFATc1DCD4þLL/2¼ 5). J, LL/2 cellswere cultured in the presence or absence (CTR) of TNFa (30 ng/mL) for 0–48 hours and treatedwith theIncuCyte Caspase-3/7 reagent to detect apoptotic cells (green object count, 1/mm2) using the IncuCyte Live Cell Analysis System (NCTR¼ 3, NTNFa¼ 3).N values aregiven per group. Data are shown as mean values � SEM using Student two-tailed t test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.





and CD132, but not CD25. While immunosuppressive Treg cellsare characterized by the expression of all three IL2-R subunits,CD122 and CD132 are characteristic for memory T cells and NKcells (34, 35). Among fate markers for memory T cells, the IL7Rgenes are NFATc1 targets (9). According to that, NFATc1 corre-lates with the IL7Ra chain as well as with IL7 implicated inCD8þ memory T-cell differentiation and homeostasis (9, 36).In addition, NFATc1 directly correlates with Tbet, the maintranscription factor controlling IFNg that is relevant for thecytotoxic antitumor activity of TIL (31), which was founddownregulated in the CD8þ T cells isolated from the tumoralregion of our cohort of patients with NSCLC (27). Thus, in thetumoral and control region of patients with NSCLC, NFATc1

correlates with factors that are characteristic for cytotoxic andmemory T-cell responses and seems to be important for anti-tumoral immunity.

In our murine model of lung adenocarcinoma, we found adecreased expression of NFATc1a in lung CD4þ T cells of tumor-bearing wild-typemice. Moreover, targeted deletion of NFATc1 inT cells (NFATc1DCD4) resulted in increased lung tumor growth,which was associated with an impairment of CD4þ and CD8þ Tcells consistent with the importance of NFATc1 for T-cell activa-tion and cytotoxic T-cell functions (5, 7). We further detectedreduced CD8þ T-cell proportions as well as decreased prolifera-tion of CD8þ T cells in the tumor-bearing lung of NFATc1DCD4

mice. The production of IL2 by lung CD8þ T cells was not

Figure 6.

aPD-1 antibody treatment induced NFATc1 in CD4þ and CD8þ T cells. A, Experimental design. Lung tumor growth was induced via intravenous injection ofLL/2-luc-M38 (LL/2) lung carcinoma cells in C57BL/6 wild-type (WT) mice. At day 9, 12, 15, and 18 p.i., mice were treated intraperitoneally (i.p.) with aPD-1 antibodyor IgG2a isotype control and tumor load was analyzed via an in vivo bioluminescence–based imaging system. At day 20 p.i., total lung cells were in vitrorechallengedwithaPD-1 and IgG2a antibodies for 24 hours.B-D,Overall survival rate (%), weight change (%;C), and radiance (photon flux/second;D) of C57BL/6WTmice in vivo treated with aPD-1 or IgG2a antibodies (day 9–18 NIgG2a ¼ 5, NaPD-1 ¼ 5; day 18–20 NIgG2a ¼ 4, NaPD-1 ¼ 4). E and F, Total lung cell suspension ofC57BL/6 WT mice in vivo treated with aPD-1 or IgG2a antibodies was in vitro rechallenged with aPD-1 or IgG2a antibodies or left unstimulated (US). Flowcytometry analyses of NFATc1 in CD4þ T cells (%; E) and NFATc1 in CD8þ T cells (%; USIn vitro: NIgG2a¼ 3, NaPD-1 ¼ 3; IgG2aIn vitro: NIgG2a¼ 3, NaPD-1 ¼ 3; aPD-1In vitro:NIgG2a ¼ 3, NaPD-1 ¼ 3; F). G, Experimental design. Cytotoxicity assay of LL/2 cells incubated with supernatant from total lung cells (¼ lung-conditioned medium,LUCM) of C57BL/6 WT mice in vivo treated with aPD-1 or IgG2a and in vitro rechallenged with aPD-1 or IgG2a antibodies or left unstimulated (US). LL/2 cellswere incubated with 20% LUCM for 24 hours, followed by luminescence analysis to determine LL/2 cell numbers. H, Results of LL/2 cytotoxicity assay.Supernatant of CTLL2 cellswas used as apositive control (USIn vitro: NIgG2a¼4,NaPD-1¼4; IgG2aIn vitro: NIgG2a¼4,NaPD-1¼4;aPD-1In vitro: NIgG2a¼4,NaPD-1¼4; CTLL2:N ¼ 3). N values are given per group. Data are shown as mean values � SEM using Student two-tailed t test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

Heim et al.




influenced, while splenic CD8þ T cells were characterized by astrongly decreased IL2 secretion. IL2 contributes to the primary aswell as secondary expansion of CD8þ T cells. As the magnitude ofT-cell expansion defines the number ofmemory CD8þ T cells, IL2influences memory cell generation. Furthermore, at the memorystate, CD8þ T-cell frequencies can be strengthened by adminis-tration of IL2 (13, 17). Consistently, we found a reduction of TEMcell populations in the spleen and the lung of tumor-bearingNFATc1DCD4 mice underlining an impaired cytotoxic T-cellresponse in these mice. Interestingly, splenic TCM cells oftumor-bearing NFATc1DCD4 mice were significantly increased.The capacity to secrete IL2 has been associated with TCM cellsrather than TEM cells (15). Thus, even though we detected anincreased TCM cell population in the spleen of tumor-bearingNFATc1DCD4 mice, we suggest that these cells are ineffective insecreting IL2 as indicated by a reduced IL2 production by splenicCD8þ T cells. This reduced IL2 production could inhibit thedevelopment of splenic TEM cells as IL2 signals are able to rescueCD8þ T cells from cell death and provide an increase in CD8þ

memory T-cell counts (17). Furthermore, as TEM cells circulatebetween lymphoid organs and peripheral tissue, a decreasedsplenic TEM cell population is accompanied by a reduction inlung TEM cells in tumor-bearing NFATc1DCD4. A recent studydescribed increased TCM and decreased TEM cell populations inthe spleen of Listeria monocytogenes–infected NFATc1DCD4 (9),which is similar to the results we obtained in the murine lungadenocarcinoma model. Beside TEM and TCM cell populations,we also analyzed the proportion of TRM cells, a unique tissue-resident memory T-cell subset important for enhanced regionalimmunity. TRM cells in various nonlymphoid tissues are charac-terized by the integrin aE CD103, which promotes the localiza-tion to epithelia (16). In the tumor-bearing lung of NFATc1DCD4

mice, we found a strong decrease of CD103þ TRM cells consistentwith the finding that NFATc1 has been shown to induce theexpression of the Itgae gene encoding CD103 (9). These resultsindicate that the tissue-homing capacity of lung TRM cells oftumor-bearing NFATc1DCD4 mice is impaired. In addition, in arecent study a greater density of CD103þ TRM cells has beenlinked to an enhanced cytotoxicity of CD8þ T cells and waspredictive for a better survival outcome of patients with lungcancer (42). Thus, a reduction in CD103þ TRM cell populations isaccompanied by a decreased function and cytotoxicity of CD8þ Tcells, a situation that we found in the lungs of tumor-bearingNFATc1DCD4 mice mainly promoted by decreased IL2.

Furthermore, we found decreasedCD4þ T-cell proportions thatproduced reduced amounts of IL2 in secondary lymphoid organsof tumor-bearing NFATc1DCD4 mice, indicating impaired T-cellactivation and effector functions also in terms of CD4þ T cells.Interestingly, in the dLN of tumor-bearing NFATc1DCD4 mice, wefound a strongly increased proportion of PD-1 on ICOSþCD4þ Tcells. High expression of PD-1 is a key feature of T-cell exhaustion,a state of T-cell dysfunction defined by inhibited cytotoxic prop-erties (18, 41). Furthermore, ICOS has been described to becrucial for the expansion and suppressive activity of Foxp3þ Tregcells as well as for IL10 production (43). Thus, we hypothesizedthat the PD1þICOSþCD4þ T-cell population represents highlysuppressive lymphocytes affecting T-cell effector functions andinhibiting antitumoral immune responses while promotingtumor growth. This was supported by the fact that lymphnode–conditioned medium (LNCM) of tumor-bearingNFATc1DCD4mice induced LL/2 tumor cell survival. Consistently,

we found a decreased production of TNFa. TNFa is a pleiotropiccytokine with pro- and antitumoral functions as it regulatesdiverse events like proliferation, invasion, and apoptosis (44).We could show that TNFa induced apoptosis in LL/2 lung tumorcells, underlining its ability to suppress tumor cell proliferationand induce tumor regression in our murine model of lungadenocarcinoma. Moreover, reduced proportion of IL7 in LNCMconfirms impaired memory T-cell responses in the absence ofNFATc1.

Immune checkpoint blockade with aPD-1 or aPD-L1 hasproven to be a highly promising treatment of human cancers,including lung cancer (19, 22, 23). As lung cancer is of epithelialcell origin and NFATc1 has been described to be expressed inmouse lung epithelial cells (45), we analyzed whether there is acorrelation of NFATc1 and PD-L1 in EpCAMþ epithelial cells inthe different regions of patients with NSCLC. We found a directcorrelation between NFATc1 and PDL-1 in the TU region ofpatients with NSCLC, whereas NFATc1þPDL-1þEpCAMþ cellsdecreased, indicating that epithelial cells expressing NFATc1together with PD-L1 are not present in the TU region of the lung.Furthermore, usingNFATC1-directed siRNA followed by aPD-L1antibody treatment of the human lung adenocarcinoma cell lineA549,we could show that,NFATc1 expressed in tumor cells hasnoinfluence on tumor cell growth and their response to aPD-L1antibody treatment. After this observation, we focused onNFATc1and PD-1 expression in T cells in the setting of NSCLC. We foundthat NFATc1 is induced in CD8þPD-1þ T cells of the CTR and PTregion of the lung of patients with ADC. In the tumor microen-vironment, tumor antigens induce a durative activation of T cells,which probably contributes to T-cell exhaustion via PD-1 andpromotes the impairment of effector functions (41, 46). AsNFATc1 is important for T-cell activation (3, 9), and has beenshown to induce the expression of PD-1, (24) the NFATc1þPD-1þCD8þ T cells could represent exhausted T cells that are targetsfor aPD1-tumor immunotherapy. Indeed, we could show thatrechallenged total lung cells of tumor-bearingwild-typemicewithaPD-1 antibodies induced a strong increase of NFATc1 fromabout 70% up to 90% in T cells. This strong increase could beexplained by the PD-1 signaling pathway: triggering of PD-1 by itsligand PD-L1 expressed on tumor cells induces the inhibition ofthe PI3K/Akt pathway. Akt normally negatively regulates theGSK3, which inhibits NFATc1 (3, 25, 26). Thus, the blockade ofPD-1 restores TCR signaling, ensured the activation of Akt, whichinhibits GSK3 and promotes the activation of NFATc1, whichinduces T-cell activation and effector functions (7, 9, 25).Although preliminary, this is a very promising avenue: to chal-lenge T cells of patients with a-PD-1 antibodies ex vivo to induceNFATc1 and promote T-cell activation followed by the transfer ofthese cells back into the patients to boost antitumoral T-cellresponses. In fact, supernatants of lung cells rechallenged witha-PD-1 antibodies inhibited LL/2 lung tumor cell growth sup-porting the importance of NFATc1 for T-cell–mediated cytotoxicimmune responses.

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

Authors' ContributionsConception and design: L. Heim, S. FinottoDevelopment of methodology: L. Heim, J. Friedrich, M. Engelhardt, S. Finotto





Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Heim, J. Friedrich, D.I. Trufa, C.I. Geppert,R.J. Rieker, H. Sirbu, S. FinottoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Heim, S. FinottoWriting, review, and/or revision of the manuscript: L. Heim, S. FinottoAdministrative, technical, ormaterial support (i.e., reportingororganizingdata,constructing databases): L. Heim, D.I. Trufa, C.I. Geppert, R.J. Rieker, S. FinottoStudy supervision: S. FinottoOther (collection of human samples and cell isolation): D.I. Trufa

AcknowledgmentsThis work was supported by the SFB643 (Project B12) in Erlangen and by the

Department of Molecular Pneumology. We thank the whole team atthe Department of Molecular Pneumology, especially Sonja Trump, Susanne

Mittler, and Adriana Geiger for their technical help, as well as the team at theDepartment of Thoracic Surgery and the Institute of Pathology at the UniversityHospital, Friedrich-Alexander-Universit€at Erlangen-N€urnberg, for their helpand support to this work. Furthermore, we thank Dr. Paolo Ceppi at theInterdisciplinary Center for Clinical Research at the Friedrich-Alexander-Uni-versit€at Erlangen-N€urnberg for giving us the opportunity to perform apoptosisanalysis using the IncuCyte live-cell analysis system.

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 October 24, 2017; revised March 12, 2018; accepted April 20, 2018;published first April 24, 2018.

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