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View metadata,citationandsimilarpapersatcore.ac.uk This article isprotected rights by All copyright. reserved. 10.1002/hep.30235 doi: lead todifferences version between this andthe ofPleasecite article Version Record. this as been and throughtypesetting, proof thecopyediting, pagination This article undergone has for and buthas accepted been not review publication fullpeer Center ofGastroenterology Division Liver for Disease, and Hepatolog Pathology, InternalItaly.Pavia, andUniversity Medicine Therapeutics, of Infec 1 Mantovani *Stefania DeClaudio Vito Opocher Soldani Mele Mantovani Stefania in Deficient HEP Articletype : Original MISS ANDREALOMBARDI (Orcid ID : 0000 Division ofInfectiousDivision DiseasesImmunology and

Accepted Articlehepatocellular carcinoma - 18 t 1 ious Di ,

Angelo Sangiovanni - 6 0197.R1 , 8 , Camillo

natural killer natural Marcello University Hospitals s eases

3 , Laura, Rubbia Porta ,

Maestri Fondazione IRCCS Fondazione Policlinico San Matteo *

and , 7

Barbara Oliviero ,

cell Paolo Barbara Oliviero tothis contributed equally work. 4 9 , , Stefano NKp30 Giorgio Rossi

, Geneva, Switzerland. Pedrazzoli - Brandt - mediated function function mediated Bernuzzi - 3 0002 ,

Francesco 7 * , 5 1 , - Silvia Chiellino , 0383

Matteo Andrea

10 , , - Armando Rossello 9579) Department of Negro

Donadon Lombardi 4

CRC “A. M.andCRC A.Migliavacca”

and altered and 3, 1 7 2 , ,

Roberto Santambrogio 6 Mario UMondelli 3 reading process, whichmay reading process, , , Pavia, Italy , Pavia, 2,3

Division of Division Guido Torzilli , Medical SciencesMedical and Stefania Varchetta 11 y , NCR

Sophie Clément and and

. 5 3 splice variants variants splice 3 Clinical

Liver 2 Department of 6 , provided byAIRUniversitadeglistudidiMilano Cristiana

1, 2 .

8 1 , , Dalila Enrico 3 , brought toyouby CORE This article isprotected rights by All copyright. reserved. Negro [email protected], Rubbia [email protected], [email protected] santipaolocarlo.it Chielli Porta Camillo [email protected] Matteo Donadon Sangiovanni Stefania Varchetta Dalila Mele [email protected], Angelo [email protected], [email protected], AndreaLombardi [email protected], E University Hospitals Italy,Pharmacy, Pisa, University of IRCCSImmunology, SanMatteo,Italy Fondazione Policlinico Pavia, 10 9 Surgery IRCCSFondazione Policlinico SanMatteo,Italy Pavia, Clinical andResearchItaly Humanitas Center, University, Milan, Italy of Milan, Milan, Transplant Unit DepartmentSurgery, ofGeneral - Immunohematological

Accepted mails: Article

no f rancesco. , San Paolo Hospital, UniversitySchoolItaly. ofMedicine,, San Paolo Hospital, ofMilan Milan,

Stefania [email protected], Mantovani

[email protected] [email protected]

[email protected] , Fondazione IRCCS , Fondazione Cà Granda University OspedaleMaggiore Policlinico,

n , Enrico Opocher, Enrico matteo.donadon@ [email protected] , Geneva, Switzerland. , Geneva, . 6 DepartmentHepatobiliary and Surgery, General of and Transfusional and Service Transplantation Centre of , Stefano Bernuzzi , Cristiana Soldani Sophie ClémentSophie [email protected], ClaudioDe Vito Fondazione IRCCS Policlinico San Fondazione , Mario

[email protected] , Roberto Santambrogio cancercenter.humanitas.it 12 , Paolo Pedrazzoli [email protected], Division of Division Gastroenterologyand - Brandt laura.rubbia

U Mondelli mario.mondelli@un U Mondelli

[email protected]

cristiana.soldani@humanitas , Giorgio Rossi . 8 Division of Division

Barbara Oliviero

roberto.santambrogio@asst , Marcello Maestri - , GuidoTorzilli [email protected], .

[email protected] 7 Medical Oncology,Medical Gastrointestinal Matteo,Italy. Pavia, , , Armando Rossello 11 Hepatology,

Department of ipv.it. Humanitas research

Francesco

.it Silvia Silvia

- ,

This article isprotected rights by All copyright. reserved. Accepted Acknowledgements Giovani Financial Support: naturalgroup killer d;BAT3,2 member immunoreceptorITIMIgand with domains; cell immunoglobulin andmucin naturalNCR, cytotoxicityreceptor immunosorbentLiver assay;BCLC, Barcelona for Clinic Cancer;alpha AFP, liver Article quantitative realHepatocellular qPCR, carcinoma; healthycontrols; NK, HBV, naturalkiller; B hepatitis hepatitis virus;HCC, virus;HCV, C Abbreviations +390382526450.Mail: 502636; fax: IRCCSFondazione Policlinico SanMatteo, Piazzale Golgi 19,27100Pavia. InfettivologiaImmunologia,e ScienzeInfettive, di Mediche Dipartimento e Malattie author Corresponding Keywords: - infiltrating lymphocytes; TIL, tumor R icercatori, GR

Innate immunity : B7 - H6, B7H6, homolog mononuclear blood peripheral 6;PBMC, HC, cells; : This work AL wasNuovo AL aided by the . - Prof. Mario U. 2011 , B7 -

02349273 was supported by - - domain containing H6 ;

ADAM, A Disinte , NKp30 isoform. [email protected].

Mondelli. Address: S.C. Malattie Infettive Address: Malattie S.C. IIMondelli. HLA

to BO - infiltratingenzyme lymphocytes; ELISA,

PD – B . - - 1, programmed 1 1, receptor death

associated transcript 3. the Italian the Health, Ministry of - - Soldati CancerSoldati Foundation. time pol time - 3; FasL,Fas3; Tcell ligand;TIGIT,

grin And Metalloproteases

ymeraseLIL, reaction; chain

Tel. +39 0382 - fetoprotein; fetoprotein;

Bando ; NKG2D, ; Tim - linked -

- 3, T - This article isprotected rights by All copyright. reserved. NKp30 and majorl its theselarger findings ofa evidence nodule role Conclusions: size. provide in support of patientsaugmented HCC in form as asoluble Interestingly,responses. mediated knockdown expressing whichtissue may differencescytokine between milieu and inthe thesurrounding theneoplastic non consistent decreased with NKp30 T variant patients in advanced with tumor N containing increased proportion of frequenciescells of expressing cell phenotype was homolog 6 ( Inunderstood. study this transcribed variants intoseveral splice whosephysiological relevance is function The Abstract umor Accepted CR3 Article activating natural cytotoxicityreceptor NKp30

- immunostimulatorysplice variants infiltrating lymphocytes

and surveillance tumorimmune .

- HCC 3 (Tim 3 B7 - H6

cells significantly down

have have - ) 3).

skewed toward a defective/

in patients with hepatocellular with carcinomain patients ,

igand in HCC development inHCC andevolution.igand Moreover, suggesting a rolesuggesting afor further ce

, B7

we investigated the role ofwe NKp30 investigatedand majorligand the its role lls expressing lls - H6

inf NKp30 and natural killer group killer d (NKG2D)and natural NKp30 2member

expression expression showed

luence - NKp30 mediated function. function. mediated , resulting in , resulting

d T a prevalent inhibitory profile, isoform a prevalent NKp30 . - -

positive , modulated NKp30, modulated - NKp30 The

cell immunoglobulin andmucincell immunoglobulin and was reduced in this ’ , particularly thosewithadvanced staging or natural cytotoxicityreceptor exhausted immune pro immune exhausted an

ligand ininhibiting NKp30

function increa NK cells had

is critical for natural killer deficient Of note,there were significant sed

. HCC E

expression of the ofexpression the xposure ofNKxposure cells toB7 that was preven

NKp30 (HCC) a reduc

tissue andtissue . - file with decreasedfile mediated functionality.mediated Peripheral NK blood ed expression ofexpression ed still incompletelystill - - - simultaneously domain 3 (NCR3) mediated ted by inhibitory (NK) (NK) B7 - neoplastic neoplastic siRNA cell

gene is -

- an This article isprotected rights by All copyright. reserved. on tumorcells NKp30 with interferon onNK cells results in andexpression enhance tumorsusceptibility cell lysis toNK stressed cells, including Interestingly, cell surface associated transcript 3( the cy N advanced liver disease inHCV on the mRNA encoding the immunosuppressive activity cytotoxicitycytokine andsecretion, Th1respectively, NKp30 whilethe NKp30 that aregenerated by a transcribed into Ig(NKp30 (NKp46) and ortwo NKp44) (NCR2) both MHC DNAM activating necessaryNK triggering signals thatare for cell of tumor targetsreduction throughofincrease occurs signals inhibitory anda in simultaneous recognitiongermline of Natural (NK) play killer cells ainnate responses to significant immune rolein Introduction

AcceptedCR3 Article the prognosis and evolution of the prognosis and evolution tokine

splice variants a -

1, NKG2D, and the natural and1, NKG2D, cytotoxicityre

and NKp46 and NKp30 - like and non protein protein - defined microenvironment . B7

three - H6 is not expressedH6 isnot by selectively butis human tissues normal expressed on B7 6( homolog

b (NCR1)

major major

byNK cells delineate isoforms lternati - BAT3 -

encoded ligands ofpositive both solid and transformedboth solid MHC molecules. The NKp30 isoforms trigger

whichI are type glycoproteins transmembrane comprising one ve splicing and that haveveand splicing that ) , -

infected patients areconsidered immunostimulatory as they the c

isoform compared toisoform isoform compared different ing B7 human cytomegalovirus protein pp65tegument ( - NKP30a, NKp30b andNKP30a, NKp30c NKp30b

IL H6), a member of the B7a ofthereceptorsH6), member family of

( - 9 like extracellular domains - , 10 10 release. malignancies s ) .

functionally distinct subsets and is governed is functionally subsetsand distinct by The NCR

( 5 NKp30 - ceptors 8 ) blood

. and negative family NKp30 includes

Moreover, Furthermore, t different biological functions.different biological

and

receptor cells

(NCR) ( 1 - - 3 γ ( might 14 ) . (IFN , which can up , which can - Activating 16 a the

, whose ligands comprise, whose ligands ) or signaling . The interactionB7 of recognizes

be associated with ( he relative abundancerelative he of expression ofexpression different  b 4 ) )

. can can ) production and tumor production The NCR3 gene NCR3 The c of the NKp30of protein the

isoform negatively impact receptors include

induce receptors. Lysisreceptors.

(NCR3) -

the regulateB7 cancer cells via

shows an HLA

and ( , NKp44 11 T - – he 13 the is B - ) . H6 - - H6

This article isprotected rights by All copyright. reserved. Supplementary M&M todetectqPCR NKp30isoforms determination and surgical liver resections pairedwere infiltrating bloodandtissue lymphocytes undergoing from obtained patients C Materials Methods. and and evolution. provide ofama evidenceNKp30 its role and insupport of patients,HCC particularlythose withadvanced nodule size. staging orlarger was theserum andin reducedof tissue simultaneouslyaugmentedas inHCC asolubleform functionalityaltered ofexpression and that In we study NKp30 this investigated have the and (HCV) chronic C virusinfections hepatitis carcinomamay(HCC) which thesetting in develop advancedB of(HBV) chronichepatitis inflammation procee aforementionedNKp30 thatthe escape cellscell from oftumor mechanism NK H6 as proteins soluble evidencecells that malignant indicates infection immunotherapy cell killing, suggesting that

Acceptedomplete Article

peripheral and s

technical details closely linked to carcinogenesisclosely linked

( 17

) redirected functionalredirected viaNKp30 ligation, analysis . tumor

In B7 addition, ding to tumor development. totumor Oneding such exampleishepatocellular .

that blockNKp30 activity Liver cirrhotic samplesfrom and HCC retrospecti patients were for HCC HCC for -

infiltrating

are Materials reportedandMethods. inSupplementary the NKp30 - . Primary HCC cell cultures. Primary cell HCC wereestablished as described B7H6 axis represent axis B7H6 animportantinchronic pathway (SupplementaryTable 1) - H6 canalso

NKp30 – N can alsocan B7 CR3 - such H6 +

- splice variants

B7 NK cellsNK - ( axis 2 mediated killing bypass theby NKB7 surveillance releasing as as 0 be , -

) H6 axis in patients in with H6 axis HCC. . suggesting may thatthis be an immune

induced can for be exploited cancer epatitis B virus(HBV) have

jor ligand development inHCC and prepared as a by stressas protein . Moreover,

deficient

( 19 ) . Itclearfrom the is RNA e NKp30 B7 for phenotype for

( - 18 xtraction andxtraction H6 expression These findings ) . -

mediated Current We show We viral viral

Briefly, vely ed

in -

This article isprotected rights by All copyright. reserved. death receptor 1(PD nostatisticallytime, differences significant were of CD69+ reduction inthe prop NK cells, c domain shown). C expression patientsHCC andH peripheral the investigatedWe the expressing exhaustedPeripheral an cells NKphenotype show Results. correlations. examine used testto comparetwogroups Pearson was ofdata. usedto than more pairedwere The as orunpaired comparison ttestappropriate. Dunn’swas used multiple matchedWilcoxon analysisStatistical was6software.GraphPad Prism performed non usingthe The M&M B7 examined as describedSupplementary in M&M ) .

Accepted Article -

gat H6 expressing cells and B7expressing and H6 cells There according differences etiology wereinNKp30expression no toliver disease (not . ing

- containing molecule

he f strategy shown in shown strategy (Fig. NK cells ompared toH were NK cells A requency p 1F

instead value ) -

pairs signedpairs testorMann rank C and of theCD56 NK frequency ofcir - ortion of NKG2D+ ortion are enriched in t enrichedare in 1), NKG2A, FasNKG2A,1),Ig ligand(FasL),cell T immunoreceptor with and .

T ≤0.05 was deemedstatistically≤0.05 of of

cells from HCC cells patientswith significantly he frequenc C the immunomodulatoryT

NK cells - S 3 (Tim upplementary - H6 siRNA knock

- y culating NK cells patientsculating NKinHCC andH (Fig. bright

3) lower inpatientscompared HCC to lower with of peripheral NK of peripheral umor receptor (Fig.

and CD56 1D

f - ) ig. 1, ig. 1, infiltrating lymphocytes . 1E Moreover, thereastatistically significant was . NKp30 down .

w ) no - - down are and a significant inthe proportion increase as Whitney and ted dim -

cell immunoglobulin significantly higher in HCC patients’ HCC significantly in higher in HCCpatients in compared withcontrols. s

subsets (Fig found that in ignificant. cells carrying

the proportion of

also describedinSupplementary U test as well as parametric as U test aswell - regulation regulation

frequencies of .

1A NK . )

and were in comparable p30 - experiments using

and mucin

programmed NKp30 C receptor

H , according to At thesame C total -

parametric ( F - ig -

and its .

1B - This article isprotected rights by All copyright. reserved. patients production was significantl cytotoxic augmented our laboratory control group(not shown) H in chronic HCV without infection with the Fc IL15LILmatched overnight incubated orwithout were with co and subsequently of patients, HCC using an mediated cytolytic potent Altered receptor ofpatients expression NKp30 inHCC prompted usto patients. Deficient and theNKp46 subset NK cells intensity others found a lower compared to ITIMand domains (TIGIT) CV CV

Acceptedperipheral Article infection

( in TIL compared with LILin TILwith compared 2

1 compared to

, , 2

(MFI)

(Fig

potential NKp30 2  ) in HCV+ patients in HCV+ patients . R+ P8

H However NKp30 .

frequencyof total

2B C (8) and H MFI of the NKp30 receptorNKp30 of the . . W - - After IL15 After stimulation, 15 murine cell line15 murine inthe presenceofanti D) mediated

compared toHC e investigated further intrahepatic

- were were . H C mediated cytotoxicitymediated between

, the p , the There was

C ( Fig. 3A , which , ial and cytokineof production

similar ex vivo compared toHC, y reduced inperiphera roportion

functionality functionality NKp46 compare HCC wereHCC added disease as controls. ) NK cells in was maintained .

afrequency NKp30+ higher NKin cells of

in tumor andin tumor non (Fig redirected functional redirected functional NKp30 expression wasexpression NKp30 reduced disease inthe HCV+ V+

(SupplementaryFig 2A

. d toHC of NKp30+

were 2E). patients (Fig.patients 3A).

NKp30 and

In contrast,t in keeping withpreviously published significantly TIL , whereas

altered HCC in isoform NKp30 profile in HCC patients HCC in - compared to l blood NKl blood cells

mediated cytotoxicity significantly was NK - tumor NK cells ( patients with

NK cells cells (ADCC) peripheral andintrahepaticcells NK HC he p

higher NKp30 cell cell C andfluorescence themean - NKp30 mAb. NKp30 mAb. - roportion

E patients displayed LIL receptors ) e

in TIL assay. PBMC, TILassay.and PBMC, also xpressi - HCC

from both mediated mediated (Fig. 2A), as 2A), shownby(Fig. data not shown not data There wasThere nodifference

after of NKp46+cellsof NK - study the , patients chronic with

in HCC patientsin HCC compared with LIL compared with ng NK cellsng NK

P IL15 stimulation IFN atients with

the HCC HCC

- CD56 a NKp30 - γ

cultured data from lower and HCV+ ).

bright

and

This article isprotected rights by All copyright. reserved. Accepted correlationstatistically theNKp30 significant positive between isoform Cancer (BCLC) staging we classification, found Interestingly, ratio values were than inH immunostimulatory isolated end,To this w reduced investigatedalternatively whether variantsof spliced functionality Recent shown) Article (dataexpression notshown). No NKp30 comparedLIL withmatched degranulationTIL ability of weWhen analy own and other laboratories degranulation cytokine consistent is with and poor production, dichotomy byperipheral inchronic exhibited NK blood HCV cells i.e.increased infection, patients compared tonon (Fig .

- studies have shown thatstudies have the shown production were noted followingproduction were NK mediated degranulation in TILmediated in degranulation ) was increased PBMC NKp30 . C There was a trend toward a more profound reduction in IFN reduction towardThere atrend in was a profound more

(Fig. ,

when HCC patients wereanaly when HCC the e quantified the three major NKp30 isoforms (NKp30a, b and c)e thethreeisoforms i and quantified (NKp30a,NKp30 majorb z - of patients andH ed mediated mediated 4A lower in NK cells in fromlower prognosis

the intrahepatic compartment, wecompartment,the intrahepatic found thattheNKp30 ). NKp30 I n agreement thelowNKp30a with the n and btranscripts, in patients adva with in - HCC, HCV+HCC, cytokine

( a and 23, 2423, - and evolution - NK cellsNK testedbyADCC was reverse significantly reduced NK cellsNK (Fig. 3C ) . statistically significant C

NKp30

using qPCR. NKp30 isoform expression pattern expression NKp30 isoform production - NK despitebeingNKp30 cells, able toboost controls

b patients patients

in the isoforms was isoforms nced tumor (Fig.nced tumor z p30 ligation ( edBarcelonaLiver for according Clinic tothe

). IL15 torescue was unable stimulation ). of

(Fig. 3B). 3B). (Fig. setting The of relativeexpression the that the immunosuppressiv that

with HCC with HCC peripheral in NK blood cells the NCR3gene s ofcancerand infection

differences in IFNdifferences in signifi

Fig. 3D T h compared to 4C e

previous cantly lower in cantly lower NKp30 a/c ).

Notably, a therewas ).

isoform ratio and IFN ratio andisoform

might explain might  - affect

mediated secretion findings H - γ C - e mediated s (

NKp30c

(Fig. and TNFα the HCC HCC patients.HCC  n freshly

(

6 ac from functional for HCC - NK 8 4B )

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patient We We

cell ). our

 , not  bc

by s

This article isprotected rights by All copyright. reserved. wereexpression than onuninfectedcells ligand co To assess could expression NKp30 whether beinfluencedB7 by NKp30 receptor IL observed for compared withthe non reducedofIL certaincontent cytokines, including mRNA fibrosis and liv previously involved inthe necroinflammatory showntobe advanced to process leading non prompted examine usto 4F) tumor liver counterpart, suggesting a LIL of the immunostimulatorysignificantly was inTIL NKp30b lower isoforms freshlyLILmatchedand isolated TIL TIL profilea thedeficient switchedcouldefficiency explain NKp30 degranulation isoform of microenvironment in cytokines Inconsideration of NKp30 NK cells, evidence further theimmunost providing of insupport

Accepted- Article culture experimentsculture . - - -

neoplastic liver tissue This difference intissue cells.NK cells patients.NK inHCC . Asshownin a isoform

TIL er carcinogenesis - 18 and TGF 18 downregulated after exposure of PBMC after exposure of downregulated (Fig. 4D). 4D). (Fig. is downregulated is downregulated -

the inhibi the NK cells exhibited a reducedNK exhibited cells fig. - with hepatocellular carcinoma . whereasnostatistically tissue, neoplastic differences significant were

6A changes T . To this end,. Tothis we analy he carrying frequency NK cells of ,

- - B7 tory  specific NKp30 isoform profilespecific NKp30 cell isoform and NK functionality

mRNAs (Fig. To this end,To this we quantifiedNKp30 thethreeisoforms in major - influenc H6 expression wasexpression higherH6 on

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as well as inNKcell regulation ing the ing 5 t

). inhibitory NKp30 NCR3 splice variantsNCR3

z  ed mRNAsofselectedthatwere cytokines bc

to B7 ( ratio matched compared tothe non 2 Huh7.5 5 )

and n in

from HC - - 6, IL 6, H6 expressing HCCcells H6 expressing

the the

f HCV cell line expressing ig - 8 and IL and 8 imulatory of the function . - potential NKp30 receptorNKp30 and its

- H6 contact we performedH6 4 mediated mediated E to uninfectedcells Huh7.5 - infected infected ,

(9) the relativethe expression ( , we exploredwhether 3 2 - ) 10, inthe role .

T - signaling NK thanin cells here Huh7.5

wa the tumor s a

cells (Fig. . B7

- -

H6 , This article isprotected rights by All copyright. reserved. degree differentiation of cirrhotic liver immunohistochemistry Expression B7 Fig. suggesting thatNKp30 down serum serum. and patients’ Nostatistically HCC observed differences significant were (sB7 Furthermore, t orpMXneotransduced withpMXneo exposure control PBMC from HC to NK cells receptor carrying theNKp30 andwereafter exposure expression higher its of knockdown of flow cytometry evaluated on was alsoconfirmed after not shown) percentage and MFI incubated with HCV express

Accepted- Article H6 protein expression 6 - B7 H6) we performed experiments we H6) performed ) .

ing (Fig. 6

- H6 exposure H6 to the breast cell carcinoma lines MCF

. low level of the Representative d HepG2 cell HepG2 s E o assesscouldB7expression whetherbeinfluenced NKp30 by soluble

B7 and

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. was significantly lessexpressed

siRNA by

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7 of B7of a

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well

after - able toreduce - B7 - induced i s H6. A - ot plots showingot plots and NKp30+ -

C - regulation requir - reduced tumor in - , moderately mediated knockdown. H6 transfection with transfection ) .

D Incontrast, remained expression unchanged NKp46 - transfected transfected

strong supplementary 3.

- Fig.and NKp30 downregulation NKp30 H6 incub - CD8L was evaluated was

cirrhotic liver cirrhotic B7 er , ating from HC withheterologous PBMC HC’s -

expressing high

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expression B7 7/VC or MCF or 7/VC tissue of HCCpatients tissue of

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Surface B7 - B7 HCC tissue HCC s B7 in HCC - . - tion wastion show

differentiated tissue differentiated

B7 - and

- H6 H6

compared to - er NKp46+ NK cell NKNKp46+ in HepG2 cells H6 was also - , or

induced NKp30 downregulationinduced NKp30

level respectively

- biopsy

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of B7 - , H6, confirmed n specimens siRNA

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T (

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) -

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by the

This article isprotected rights by All copyright. reserved. cirrhotic patients BCLC compared staging classification, stage toearly tumors detected withintermediate patients inHCC detectable Based on advancedand tumor Soluble of cells HCC toMN8 modest and increase notalwaysB7 consistent in MN8 primaryHCC cell lines or sp downregulation onHCC sheddingB7 of of escape immune inwhichtumorcells impede NK via H6 protein result didnot from expression but we expression hepatocytes stainingB7 showed that B7 Interestingly, ecific inhibitor

Accepted Article - analysed other H6 expression H6 we

or to were unable todetect B7 post in situ

in solvent alone (DMSO) solvent alone by -

H6 as shown inf as shown

B7 patients - we found that transcriptional i mmunohistochemistry - -

H6 serum concentrationselevated in are H6 mRNA tissue levels HCC in expression ofexpression . s

. B7 Differences between HC, cirrhosis and remained HCC between highlyDifferences HC, cirrhosis significant

compared to ( 3 ’ 3 .

) s -

sera . H6 surfacemeasuredH6 was expression cytometry by flow on

and and To address mechanism whetherthis plays inB7 arole - H6 was localized mainly in patients we treatedHCCprimary cel ig after by ELISA .

poorly correlate parameters clinical with

7B significant differences mechanisms B7 expos . better differentiated

To -

H6 for 24h - differentiatedtumor investigate .

ure

in situ in As shown in As shownin HCC

to ADAM . reduc .

Schlecker et al As shown in As shown andaccording advanced tumor,classified tothe

were , we next next , we

ed transcripted level whether - s H6 surface expression

along caused by a decrease

- f cell ( HCC tissue HCC 10 ig the F ig .

investigate and

8A HCC cells recognition by met . with matched non with matched different levels of B7different of levels cytoplasm and S

7C . upplementaryfig

, demonstrated a novel a mechanismdemonstrated ADAM higher serum B7 serumhigher ),

(BCLC showed l lines withdifferentl lines ADAM patients suggest s . .

s Immunohistochemical d , and might have occurred, and have might -

whether 17 inhibitors LT4 17 inhibitors or - A) a ing

with intermediate trend toward trend on the on the in only

that decreasedB7that nd - gene transcription tumor zincin .

7 - sB7 to HC andto HC after e H6 levels wereH6 levels , therea was - - membrane of H6 H6 protein -

- H6 specimens mediated untreated untreated xposure xposure

lower was

This article isprotected rights by All copyright. reserved. suggesting anti exhausted thatinnate immunity toward isskewed NKp30 and activating NKG2D togeth receptors, analysis showed HCC ourdecreased expressing frequencies NK patients of cells with of NKG2Acirculating receptorsNK and incells intrahepatic there are activatingandreceptors inhibitory onNK cells reflect et al. of thembeingcategorized as BCLC patient selection, sincepatients our in ourstudy andcontrols. betweendiscrepancies patients Theseconsequence mighta of be which reduced could contribute whereas to surveillance, immune found nodifferences were proportionblood NKcells ofperipheral and ofCD56 disease remain progression mechanismsfor responsible altered various humancancers includingcarcinoma hepatocellular Functional deficiencies Discussion. C between etiology significant difference whenaccord were HCC notstratified

Accepted Article).

(2 2

NK anergy cell )

and Cariani et al. or tumor gradingor tumor sB7 cont

- roversial data roversial data H6 levels H6 levels s

in sB7 in which, however, which,

and of circulatiof ( 3 (data notshown) (data 5

) largely unexplored regarding frequenciesof NK the HCC nod HCC

were classifiedBCLC as - H6 levels H6 levels ng NKhave and cells intrahepatic had an overall moreahad advanced larger anproportion overall disease, - NK effector function cell B, C,D,whereas majoritypatients Cheung thevast in of ing to BCLC stage(not shown). toBCLCing u le does of alwayscorrelateexpression not withaltered when HCC patients w when HCC

size andsize serum alpha .

Moreover, Moreover, .

in HCC patientsin HCC P er withanincreaseder frequency revious studiesfound dim dim - A. there w phenotype patients inHCC

An altered N ( ( 21 2

cells carryingcells NKG 1 , , 2

, and their associationand with their

ere 2 f 2 ere 2 etoprotein valuesetoprotein , . , 3

Relevant statement, this to 25 - 5 tumor immune responsestumor immune stratifi ) positive correlation positive , .

3 Interestingly, phenotypic 4

, been demonstrated in been There w There 3

K cell phenotype K cell may 5 a inthe reduction ) . ed

Nevertheless, the

according to ere of Tim of 2D and

(Fig no (2 . 1 s

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3 2 , , 3 and 5

)

This article isprotected rights by All copyright. reserved. alternative splice variants cytokine production. cells displaying NKp30reduced a/c exhibit aisoform ratio tumor and b Indeed, c altered expres for thefirst thatperipheral time, andtumorinfiltrating NKp30 asexpression, In thepresent information currently is available patients, hasthe prognosis beenand gastrointestinal evolution within correlated tumor stromal of theNCR3gene h myeloid regulatory frequency, Tcell phenotype markerexhaustion inadvanced melanoma cancerand patients, colleagues da showed Silva studies whereas another Tim suggested study that Ndhlovu and colleagues showed thatTim Recent despite Acceptedompatible Article

, immunostimulatory

expression ofexpression studie focus of an increased proportion ofNKof an cells proportion increased early expressingmarker CD69 activation the PBMC of HCC patientsHCC displayedPBMCa of levelmRNA expression lower ofthe - derived suppressor cellsderived suppressor as well as other

(

3 with 8 ed ) s have reporteds have conflic

. sion of sion of study study

well as Functional on defective healthyand chronic notpatientswith donors diseases, as cancer. such as been shownto as been we show we the Moreover, consideringMoreover, the the major the

NKp30

inhibitory canc ,

NK cell isoforms NKp30 therea wasbiastoward an ed patients thatinHCC - ers, viral infection mediatedsecretion cytokinecytotoxicity. and also We showed, a NKp30 ltered ( - on 21 mediated mediated deficiency might be deficiency might

NKp30c isoform compared to HC compared to , ting data about Tim about datating

25 N profound

, dendritic cell function cell anddendritic an proportion increased of CR3 39 isoforms (NKp ) . - - 3 inhibits NK cell3 inhibits Apart thosemechanisms, from and 3 may

splice variant inliverand cancer. profilefunction tumor immune tumor immune ly influence ly that its blockade its reversesthat the

s relative relative that that

and and instead

prevalent was clearly increased Tim and inthose patients wit NK cells displayed reduced miscarriage 30a, NKp30b and30a, NKp30c) NKp30b

also - expression ofexpression thedifferent 3 function inNKcells - enhance IFNenhance

3 ed

NKp30 could surveillance associated an increased with –

mediated cytotoxicity a reduced NKp30 - inhibitory expressingNK cells displayed

function as anNKfunction ( 5 - - dependent function and dependent it 10 ) - . γ production γ production However,

in HCC patients.HCC in NKp30 isoform NKp30 alternative splicing .

Furthermore

NK h adv

- ( mediated

3 exhausted exhausted 6 NKp30 NKp30a no - , 3 anced

8 NCR3 NCR3 which is (3 ) ( -

. 3 cell 6 7 ) ) , .

, NK Both In

. This article isprotected rights by All copyright. reserved. a stress inflammatory during infections,B7 define sepsisand monocytes inviral ( of B7 Our dataemphasized therole clearly of B7 NK positive relative cellsNKp30 proportion of NKp30 point microenvironment affectingcell NKresponses. inthemRNA expression neoplastic 6 (9, 10) to convert the cytokines, as TGF such neoplastic may influenced by be compared with relative frequency ofNKp30+ cells density andof higher th compartment which in NKp30 compared withHC. frequency cells ofNKp30+ NK and densityrec a ofNKp30 lower patientsin HCC comparedwas withcontrols,inkeeping foundtobe reduced withalower interpretation,line withthis NKp30 aforementioned profile 4 , I , 0 )

Accepted. Article L The absenceB7 of . -

ed - 8) mRNA content and a concomitant8) and inhibitory mRNAcontent signal delivered by In line withthes H6 with NKp30 on NKH6 withcells NKp30 NK on leads efficient to in the liver compartment, -

isoform induced selfinduced molecule to peculiar characteristics of theneoplas tissue NCR3 NCR3

non

shed new lightshed new onthe analysis wasanalysis compatible with ,

as - neoplastic shown inprevious studies These findingsThese differ from intrahepatic thoseobtained somehow inthe splice variant profile of NK cellsspliceNK and variantprofile toaffect cytolytic of the NK behav

e findingse differences betweenmilieu inthecytokine the - - H6 transcripts andH6 innormal tissues cells, thepresence intumor on  , IL, - 15 and IL 15 and

- ( adjacentIn respect,this tissues. NKp30 18, 18, mediated NK cell degranulation celldespitemediatedreduced higher NK was , our data showing our datashowing consistent 4 1 ,

- tissue 4 mediated cytokinemediated production bycirculatingcells NK 2 ) role for the NKp30for receptorrole the inH . - Until now,Until hepatic B7 18, thataremicroenvironment enriched inaspecific ,

- support theexistence ofan

H6 in regulating NKp30H6 in expression. with

an profile inhibitory supportingepigeneticrole an for certain . Thus, the evidence gathered in this study the evidenceinthis gathered Thus, tic tissue in patients with HCC, inwhom inpatientstic tissue HCC, with

decreased NKp30 a reduced pro

cell an activation

e NKp30 receptor in receptor e NKp30 - H6 expression remain H6 expression - inflammatory cytokine (IL inflammatory eptor expression in HCC inHCC expression eptor , despite an increased , despite - mediated function.mediated neoplastic neoplastic

- inhibitory inhibitory mediated respons mediated CC - H6 as anexampleH6 of unaltered d target cell killing surveillance.I and non I neoplastic nteraction TGF ed The

- - es 

n the

io - , r

This article isprotected rights by All copyright. reserved. NK cellswith the presence were associated of itsligandB7 which In the proportion of short incubation withheterologous serum data inmelanoma patients slightly compatible with NKp30 also identified inasoluble formreceptorto prevent capable of and toNKp30 binding mechanisms might beinvolved. differentfrom t caused by not significantly so, accordingexpression to imm cirrhotics liver showing a compared controls liver tonormal (1 positivelyand expression its correlated withtheliver severity injuryclinical inthis setting of found tobemarkedlyenhanced onHBV Inexplored. former the in hepatocellularcarcinoma chronic liver failure poorly investigatedZouB7al. explored. et 8 stead )

Accepted Article. In al.observedaddition, FieglerB7 et elevated unohistochemistry.

down , -

increas mediated NK triggering.mediated cell a

competing role forcompetingrole B7 altered - modulation ofmodulation the ed hose of the surroundinghose of non the

a metzincin

NKp30+ NKNKp30+ cells gene transcription, B7 in poorly HCC. differentiated and anotherB7 showed study and - H6 surfaceH6 onprimary expression

study, B7 study, Notably, the tumor tumor ignificantly ( 3 3 ( - 18 ) mediated shedding since mechanism, . , The role differentiation status 4 NKp30 receptor NKp30 receptor

3 - Similarly membersB7 toother ofthe famil ) H6 - ; however, the H6 expression, assessedexpression, H6 by was immunohistochemistry, ( 4 3

in vitro

has ) since HCC . Importantly,. our results comparedand tumor tissue Release ofsB7 reduced B7reduced of - convincingly infected hepatocytes thehealthyinfected compared liver to containing cell

soluble B7soluble - tumor B7 tumor tissue exhibit tissue - , whereas cell H6 protein expression onHBV expression H6 protein neoplastic

expression andexpression function non - - H6 expression in HCC tissueH6 expression inHCC by Decreased - H6 expression at , the H6 mRNA levels in HCC H6 mRNA levels HCC in - - - neoplastic liver w - wereH6 mRNAnotsignificantly levels H6 was shown to be at least part showntobe H6 was in H6

been shown been tissue, suggestingtissue, other that ed HCC HCC ligand -

- free B free in HCC in HCC associatedB7

different - H6 as asurface/cytosolic H6 B7 cell lines cell

being 7 - - H6 H6 was todecrease unable H6 is in ovarian carcinomain ovarian ADAM

the not entirelyclear intensity of

expression expression less as poorly only , inagreement with transcriptional lev transcriptional - on tumor on H6 clearlyH6 was expressed - - specific inhibitors induced acute y, B7 tissue was not B7 - - H6 was associated -

, though H6 , since a .

in el on

This article isprotected rights by All copyright. reserved. Accepted fashion, microenvironment and primes yearsfor interventions monoclonal antibodies cytotoxic Tlymphocyteactivity suchas inhibitors, withcheckpoint PD immuno axis Inprovides conclusion, our study surveillance.mechanism immune from thehost Articleshed from sheddases and pati patients with tissues. hypothesis regulatoryby effectcaused reduced ligand NKp30 expression agreement wit molecule intumorcells ents

NKp30 in HCC andin HCC reveals

proinflammatory , suggesting inhibitory a ligand thissoluble potential role for on Moreover, therapeutic approaches providing for insights immune new was

from neoplasticfrom cellslargeramounts ofsoluble are compatibleligand with larger comes from our dataof expression from B7 showingcomes decreased our - mediated NK cell NK activity mediated intermediate and advanced tumor to the only systemic treatmentthe only systemic h ourshowing dataclear ligand a modulat neoplastic neoplastic si observed gnificantlyelevatedsB7 of levels induces anti induces (4 as wellas e 4 several responses of ) , altered it couldit alsobe possible nodule in TIL compared to LILin TILan compared down be absent to due may to

as asolublemolecule for liver cancer for mechanisms

NK cellNK activity evidence s -

tumor NK cell responsescell inacytokinetumor NK or advanced macrophages located within the HCC macrophages . Our available for -

receptor interaction.supportofreceptor this Evidence in in support in support

data suggesting data stimulatory treatments

that can be exploited forcanexploited novel bethat . - compared withhealthy control

induced receptorinduced down It may envisaged be that HCC . Also, i to ( - . of 19 associate specific immune associate specificimmune

H6 wereinsera detected of patients with This mayrepresent evasion an ) . an alterationof

t is ofinterestt is that In observation, line withthis that B7

- ( 4 H6 is released by H6 is advanced HCC advanced 6 - ) H6 ligand in HCC H6 ligand inHCC .

- -

modulation, modulation, N 1/PD the the beyondrestoring K - dependent

p30 expression p30 expression s NKp30/B7 orafenib - L1 s

and cirrhotic - specific specific HCC HCC -

( higher

4 , being 5

which which ) and in - ,

H6 This article isprotected rights by All copyright. reserved. Accepted Article References 3) 2) 1) 8) 7) 6) 5) 4)

2015;146:234 NKp30 B,Mantovani MeleBarbarini S, D,Oliviero G,Varchetta MondelliMU. S, tumors. Nat Med2011;17:700 Alternatively isoforms spliced affect NKp30 gastrointestinal the prognosisstromal of I, Martins LyonneDelahaye S, S, Roux C, NF, Menard Rusakiewicz Transl Med 2015;7:283ra55. Clinical impact ofthe NKp30/B7 Minard S, Semeraro M,Rusakiewicz e115 pattern inrare patientsOncoimmunology melanoma favorable with evolution. 2016;5: isoforms andNKp46 metastaticpatients: transcripts in Unique melanoma NKp30 MessaoudeneFregni G,EnotD,JacquelotN,Nevesal. NKp30 M, et E,Germaud N, ligands.Immunol 20 Biol Cell Vivier S, E.NaturalKruse Ugolini PH, Matta J, cytotoxicityreceptors and their 2013;31:227 Immunolresponses:signals AnnuRev integration activation ofinhibition. forand LiuLong D, PetersonKim HS, ME EO, Immunol 2008;9:495 Lanier,LL. tightrope: natural Uponthe activation cell killer Nat. and inhibition. Nat RevCancer2016;16:7 youLanierMorvanLL.NK MG,cancer:can cells tricks. and teach innatenew cells

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risk neuroblastoma Sci patients. virus infection. Immunologyvirus infection. t L, al. t et This article isprotected rights by All copyright. reserved. Accepted Article 10) 9) 15) 14) 13) 12) 11)

6:766 protein inhuman expression oral squamouscellOral carcinoma. Med 2017; Pathol J Liu Zhao JinX,etWang al.TheB7 K,Xu H,Wenprognostic J, J, value of 2409. costimulatoryB7 molecule Tao H,Li J, etSun P, al. Wu L,Clinical significance X,WangJ, Yang ofnovel Exp Med 2009;206:1495humans. J B7 Brandt Kennedy Gao EC, BaratinB, Z,Fox M,Yi J, CS, etB7 al.The family member 2005;6:515 NKp30 activating byImmunol receptor human Nat cytomegalovirus. pp65of Arnon 2007;27:965 tumor cells and engagesImmunity theNKp30 cells. receptorkiller onnatural leukocyteHansen antigenHP, et al. Human Po 2015;6:189. IsoformsNKp30 Tissue. Maternal FrontiersImmunology Blood in andPlacental in al. FirstofAlternatively TrimesterLossand Pregnancy theExpression Spliced Shemesh A, TiroshD,Benshalom Sheiner E, natural 2015;6:1 cell Nat killer Commun subtypes. cytotoxicity functions distinct ofhuman receptor variantsorchestrate the splice LevySiewiera Gouilly Al H,G,C, Hocine Cartron J, J, gge vonTresckow Simhadri VR, vonStrandmann E, KS, B,Reiners S, Sasse - H6 isa

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0183 -

Daccak R,etal. Natural Inhibition of the 2017;14:2405 - H6 -

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virus infections. Gastroenterology 2009;137:1151 Natural hepatitis B dichotomy functional cell killer and inchronic chro Oliviero B,Varchetta S granulin natural activity killer inhepatocellularca LW,Lo Ng Cheung TT, Yip CW, Wong Cheung PF, CK, of et CM, al.Restoration carcinoma Immunol Clin 2008;129:428 patients. circulating NK andcellsrelativemechanism intrahepatic andinhepatocellular Zhang Z Cai L, tumorigenesis. Gastroenterol 2017;52:26 J associated hepatocellularcarcinoma: betweeninflammation, and infection, linkage TakedaTakai H, A, escape.immune Oncoimmunology 2015;4:e1001224. mediated downregulation of toovarian NKp30 contributes inNKcells carcinoma Pesce G Tabellini S, 2015;10:e0134568. in Hepatitis B Virus AggravatesDamageHepatocyteUp through Zou Y,BaoJ imm activating, co and inhibitory, Chester Fritsch C, K,H.E.Natural cellimmunomodulation: targeting killer Kohrt 37447 H6 inhumanglioma promotes tissues Oncotarget progression. tumor 2017;8:37435 Zhang W, Jiang T,Wu ZhangH, X, et al.High D,Wangexp Q, unotherapy. 601 2015;6: Immunol Front

- epithelin precursor. Oncoimmunologyepithelin precursor. 2015;4:e1016706 , Pan X, Lu Y, LiaoLu Y, , Pan X,et al.NKP30 X, S,Wang , Zhou L, Wang H, Fu J, ZhangL, WangFu et. al.Functional, Zhou J, H, S, impair

- Inuzuka T,Marusawa basis ofhepatitis H.Genetic virus Related AcuteRelated , Cantoni C, Patrizi O, C, , Cantoni Coltrini D,RampinelliF, et al. , PaudiceG, Zaramella E,Michelone D,et M,Mavilio al. - stimulatory receptor signalingstimulatoryreceptor cancer for - On - Chronic Liver Failure.Chronic PLo rcinoma by antibodyrcinoma treatment with against - 38. -

Regulation of Interleukin of Regulation

- 37. - 1160

- B7 - H6 Interaction H6

S OneS - ression of B7 nic hepatitis C hepatitis C nic 32 Expression Expression 32 ment in B7 - - H6 - - - This article isprotected rights by All copyright. reserved. Accepted Article 26) 25) 24) 32) 31) 30) 29) 28) 27)

cells. Nat Immunolcells. Nat 2008;9:503 Vivi BatallerLiver R,Brenner fibrosis.DA. Gastroenterology 2013;144:512 microenvironmentpathogenesis of hepatocellular theand carcinoma. in treatment Massague TGFbetaCancer. J. in Cell 2008;134:215 111. responseimmune Cancer signatureofthe liver microenvironment. Cell 2006;10:99 metastases, prognosis recurrence, and carcinoma inhepatocellular a based unique on HL,BudhuForgues QH, Jia Zanetti A, He M,Ye P, 2016;76:2394 accumulationactivity and of tumorinfiltrating lymphocytes. Cancer Res DependentIL18 signalingcarcinoma hepatocellular restricts growth by enhancing the Yang GJ, Markowitz1 MichelottiGA, FuC P, J, LettImmunol 2002;84:163 interleukin Ban CS, IthninChia K, H,Singh etof R,Mokhtar H,Krishnan S al.Expression hepatocellular viathecarcinoma NKp30Hepato receptor. et natural al.Myeloid inhibit cells derivedsuppressor cellswith killer inpatients Hoechst B,Voigtlaender T dependent manner cells are cytotoxicity toward polarized inaninterferonhepatitis inchronicC YetAhlenstielRotman KohRH, EdlichB,JJ, G,Titerence C, al.Natural Feld killer Hernandez

erBaratin1 E,T Tomasello& M,Walzer E,Functions UgoliniS. ofnatural killer - 18, interferon - Gea V, Toffanin S Gea V, Toffanin – 2405. . Gastroenterology 2010;138:325

- gamma and interleukin - 72. , Ormandy L, Gamrekelashvili J, ZhaoL,, Ormandy J, F, Gamrekelashvili Wedemeyer H, -

- 10 , Friedman of Role the SL,Llovet JM. 27.

J Clin Invest Clin J 2005;115:209 hen R, Sui J et hen al. R,Sui J -

10 inhepatocellularcarcinoma. – -

KA etvenous al.Prediction of 35. 230.

logy 2009;50:799 Inflammation – 218.

- 807 - alfa -

- – This article isprotected rights by All copyright. reserved. Accepted Article 35) 34) 33) 40) 39) 38) 37) 36)

mechanism ofalertingLife cells NK against Mol tumors.Cell Sci 2011;68:3531 LN,Kaifu T,EscaliereB, M.B7E, Baratin Vivier Gastinel Gastroenterol 2006;12:3275 analysis dendritic inpatients hepatocellular with of cells carcinoma. J World 2014;2:410 NK IP,da Silva A,Jimenez Gallois gamma togalectin inresponse production Tim LenvikGleason McCullar V,O'Brien et Felicesal. MK, Cooley M, SA, TR, MS, Blood 2012;119:3734 marks huma LC,Ndhlovu Lopez curative treatments. Oncoimmunology 2016;5:e1154249. phenotypiccharacterization as ofHCV anoutcome predictor M,BariliCariani E, E,Pilli V,Porro Biasini E,O cancer. Oncoimmunology2016;6:e1264562. contributes toNKcell and predicts exhaustion prognosiswithliver ofpatients a poor C, Sun killer cell Metalloprotease SchleckerArnoldFiegler Rose N, A,Altevogt E, P, Ormandy LAOrmandy - - cell exhaustion inadvanced exhaustion cell by Tim melanoma 3 isaninducible humannatural receptor cell killer thatenhances interfero

Xu J, - activating2014;74:3429 NKp30. receptor Cancer Res - 22. n natural killer celln naturaland cell suppresses maturation killer

Huang Q, ,

Farber A

- mediated tumorcellB7mediated shedding of - Verges S, Barbour JD, Jones RB, Jha AR, Long JonesBarbour AR, RB, JD, al.Tim Verges Jha et S, BR, –

43. Huang M, ,

Cantz T Cantz

- 82. -

Baranda S, Khan S, AndersonS, Baranda AC et al.Reversal Khan of S, ,

Wen H, Wen Petrykowska S -

9. Blood 2012;119:3064 9. Zhang C, etC, Zhang al.High expression NKG2A

livani Aet al. livani ,

Wedemeyer H - - 3 blockade. Cancer Immunol3 blockade. Res. Cancer John S, Moldenhauer S, G,John et al. - H6, the li

- H6/NKp30 interac - linked HCC after linked HCC - gandnatural the of mediated cytotoxicity. Natural cells killer - 40. ,

et al. Direct ex vivo et al.Direct ex –

n tion: a tion: –

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3 This article isprotected rights by All copyright. reserved. F NKp30 lymphocytes NKp30patients and Figure 1. Legends. Author namesdesignate bold shared co in Acceptedrequencies Article 43) 42) 41) 46) 45) 44)

+ crosstalkcells. betweenandHepatology macrophages naturalkiller 2013;57:2358 Sorafenib anticancercellular perpetuates by effector functions modulating the MF, ReisingerSprinzl F,PuschnikA,Ringelh advanced Medcarcinoma. hepatocellular2008;359:378 N Engl J MazzaferroLlovet Ricci S, JM, Hilgard P,GaneE,Blanc et V, JF al. Hepatology 2017;68:157 GretenB. TF,Sangro Targets for immun recognition byNK Blood cells. 2013;122:684 of the activatingB7 NKp30 ligand Fiegler Arnold S, A,Oehme A,Rölle I, Textor Breuhahn N, al. Downregulation Ket Escape Virol 2016;9 byNatural Elimination Killer J Cells. 6B of Activating DownregulatesLigands during Expression Lytic InfectionTo Levi Schmiedel D,Tai J, Bloodconditions. 2013;122:394 B7 BaratinMattaL, J, Farnarier M,Chiche Piperoglou Forel J, et C, C, al.

NK cellsNK and NKp30

P - H6, a ligand forH6, the cell naturalkiller eripheral NK cells of HCC eripheral of cells NK

of circulatingof Tim .

F requenc - expressing expressing ies

of circulating cells NK - - MFI 3 - -

166. SchafferF, Mandelboim O.Human DovratS, Herpesvirus (D) NK tumor in cells enriched are

(B , NKG2D

- C) C) - 404. patients - in HCC patientsin HCC (n= H6 by HDAC inhibitors impairs tumorcell HDAC inhibitors H6 by -

first authorshi first

(E) - otherapyof liver cancer. Journalof

activating NKp30, receptor ininflam show an show ,

and CD56 - an M, AckermannanK, Hartmann Det al. 93. CD69

bright

exhausted phenotype p.

and CD56 (F)

55 ) and expressing NK cellsexpressing NK 0:9608 - infiltrating infiltrating HC dim - - 90. 9617.

subsets (A) (n=

Sorafenib in Sorafenib in 3 Induction of

9 )

. in HCC in

matory , in - 68.

This article isprotected rights by All copyright. reserved. signed test rank values. maximum represent un NKp30 IL15 and degranulation Figure data. values. median plots values,are box and TIL NKp30 NKp30 receptor comparedLIL withmatched i Figure 2. were values. whiskers andmaximum are minimum respect patientsHCC (n= n TIL n

AcceptedIL15 orstimulated Article

HCV+ patients (n=11)

- used tocompare data. used stimulated PBMC ofHCCstimulated patients (n=

- i - - The 3 vely NK cells - mediated degranulation NKpositive cells NK cellsNK .

median values, box plots are plots 25%medianbox and values, 75% percentiles, whiskers and are minimum Increased proportion of of proportion Increased Deficient

). Wilcoxon matchedWilcoxon

Middle bars represent median values, box plots represent are medianbox values, bars Middle 75% 25%percentiles, and in were used tocomparewere used data. density un of HCCpatients. of 14, n=47

with The Mann stimulated or IL15 orstimulated NKp30 - stimulated LIL stimulated in ( the C in LILin and TIL. matched

. ). and

(B), NKp30 -

- mediated CD56 25% and 75% percentiles, whiskers areand andmaximum 75%25% percentiles, minimum ( ( Whitney A D -

), ) pairs signedtest rankpairs n=44 (C) and NKp30 ,

with a R bright

epresentative dot plots plots epresentative dot The frequencyof NKp30 , and matched TIL and matched

- respect U stimulated stimulated function and CD56 -

relative increase mediated cytokinemediated production test, unpaired ttesttest, unpaired

receptor 11 i The Mann vely ) - ,

mediated cytokine (D) production in HC (n= HC dim PBMC of ) and HC(n=

HCC patients (E) subsets

- or total positive NKpositive cells - ,

NK cellsNK

F paired ttest were - 15

Whitney

requency of of NKp30+ NK cells ( cells of NK NKp30+ showing the NK cellsNK ) and HCV+ patients (n=11)) and patients HCV+

HCC patientsHCC (n= or

the 10, n=38 18 (n= . ) U

Wilcoxon matchedWilcoxon . (A), in TIL in TIL 13 Middle barsMiddle represent test

NKp30

) in unstimulated orin unstimulated frequenc .

NKp30

and and Middle barsMiddle or and used to compare (n=

( the unpaired ttest n=23

- NKp30 density n=34 30 expressing - B ies ) mediated , ) ) and ) H , was lowerwas

of C

- (n=

pairs pairs . LIL 29 )

This article isprotected rights by All copyright. reserved. was HepG2 cells NKp30 cells transfect culture NKp30+cells NK B7(A), Figure comparison surrounding TGF Figure from patients. 19HCC the NKp30 IL dependence between staging ( classification. patients. ( isoforms (A) and NKp30 FigureAltered isoform NKp30 4.balancein

Accepted Article- 15 stimulation, in 19 HCC patients. 19HCC in 15 stimulation,

used to compare data. -  d

mRNA expression - 6 5 expression expression

H6 expression with uninfected or HCV with uninfected . NKp30 . HCCand in non Cytokine profile . C ), NKp30c isoform expression in HCC patients), NKp30c inHCC with expression isoform . 

, tissue ac, ac, siRN ed

 with B7 bc and bc

is A (B) (n=11) ( F

down control NKp30 a/c isoform ratio NKp30 )

and NKp30 and after exposure of D

o n uninfectedHCV or - ),  H6  . The

- on ab r The Pearson correlation coefficientTheexamine correlation was Pearson usedto ac, regulated - - transfected HepG2 cells and mediumalone transfected cells HepG2

HCC tissues tissues HCC and or controland or  atios (F) were determined atios were (F) determined Wilcoxon matched Wilcoxon bc and bc - infected Huh7.5 cellinfected Huh7.5line. MFI

after Theisoforms of relative expression (E) NKp30 and 

(C) PBMC from PBMC from ab ratios (B)ab ratios compared co in - siRNA. The frequencyNKp30siRNA. - - - and NKp30 and infected Huh7.5 cellinfected Huh7.5line. HC neoplastic culture with culture with HCC patients - PBMC (n=11) pairs used signed testwas rank with matched non 10 in PBMC of 15 HC and 33 HCC 33HCC HC and PBMC of15 HC to siRNA HC to in LILin and TIL matched

tissue. - mediated IFNγproduction upon mediated

( D a . ), B7), B7 R stratified

elative expression of NKp30

IL - culture H6 positive - - H6 expre H6 6, IL 6, - B7 - neoplastic neoplastic . (B) d

- The p accordingBCLC to

8, IL 8, - alone alone H6 + cells ,

F ssion ssion - requency of transfected - HCC aired t 10, IL 10, or - NK cellsNK

( for co on E

cell line. )

- - test HepG2 18 and 18 and This article isprotected rights by All copyright. reserved. patients dependence between sB7 data presentedas m accordingBCLC to stage stage BCLC patients with Figure statistically significant (ns). neoplastic magnification on B7 differentiated ***P<0.001 as m according to Immunohistochemical Figure

Accepted Article- cirrhotic H6 , ean values ±SEM. *P<0.05,

and matchedcontrol negative 8 7 .

. . B7

S specimens oluble oluble . samples

degree ofdifferentiation degree - WD ) H6 HCC . ean values ± ean values

** ( C

- protein protein *P HCC= well differentiated ) B7

, (n=34)

mRNA B7 mRNA <0. and

- (n=11) H6 analysis of 001 The compare Comparison testwas Dunn's Multiple usedto data

HCC tissue classified tissue HCC as -

(n= H6 protein expression isreduced on protein , PD

. SEM  . Thecorrelation Pearson

87 B The

. - -

) HCC= poorly differentiated H6 expression on (A) , .

cirrhotic patients The matched Wilcoxon B7 correlates with clinical parameterscorrelates clinical with and ,

S -

(B) or haematoxylin compared H6 protein expression expression H6 protein erum Dunn's Multiple Comparison testwasDunn's used Multiple and nodulesize maximum

B7 HCC - H6 H6 to cirrhotic liver

WD HCC tissues tissues HCC (

n=18) (n= concentrations neoplastic neoplastic - coefficient examine was usedto - - pairs signed rank test wasrankpairs used. signedtest eosin , MD 39 ) , MD

and - HCC immunohistochemistry staining on

and PD -

HCC= moderately along withmatched non HC HCC HCC tissue in s

(n= ( in n=

(n=48) or

- HCC patientsHCC 5) tissue 28 HCC

sAFP . ).

( HCC patients. B Data are presented .

) ( Data are stratified , 1

is (C) Representative 00x 00x higher in ,

in HCC in HCC to compare

stratified

Not - ,

(A)

This article isprotected rights by All copyright. reserved. Accepted Article