Polyxeni P Doumba 1,2, Marilena Nikolopoulou 2, Ilias P Gomatos 2, Manousos M Konstadoulakis 2 and John Koskinas 1*

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1 Doum et l. BMC Gstroenterology 2013, 13:17 RESEARCH ARTICLE Open Access Co-culture of primry humn tumor heptocytes from ptients with heptocellulr crcinom with utologous peripherl lood mononucler cells: study of their in vitro immunologicl interctions Polyxeni P Doum 1,2, Mrilen Nikolopoulou 2, Ilis P Gomtos 2, Mnousos M Konstdoulkis 2 nd John Koskins 1* Astrct Bckground: Mny studies hve suggested tht the immune response my ply crucil role in the progression of heptocellulr crcinom (HCC). Therefore, our im ws to estlish (i) functionl culture of primry humn tumor heptocytes nd non-tumor from ptients with heptocellulr crcinom (HCC) nd (ii) co-culture system of HCC nd non-hcc heptocytes with utologous peripherl lood mononucler cells (PBMCs) in order to study in vitro cell-to-cell interctions. Methods: Tumor (HCC) nd non-tumor (non-hcc) heptocytes were isolted from the liver resection specimens of 11 ptients operted for HCC, while PBMCs were retrieved immeditely prior to surgery. Four iopsies were otined from ptients with no liver disese who hd surgery for non mlignnt tumor (norml heptocytes). Heptocytes were either cultured lone (monoculture) or co-cultured with PBMCs. Flow cytometry mesurements for MHC clss II expression, poptosis, necrosis nd viility (7AAD) were performed 24 h, 48 h nd 72 h in co-culture nd monocultures. Results: HCC nd non-hcc heptocytes exhiited incresed MHC-II expression t 48h nd 72h in co-culture with PBMCs s compred to monoculture, with MHC II-expressing HCC heptocytes showing incresed viility t 72 h. PBMCs showed incresed MHC-II expression (ctivtion) in co-culture with HCC s compred to non-hcc heptocytes t ll time points. Moreover, CD8+ T cells hd significntly incresed poptosis nd necrosis t 48h in co-culture with HCC heptocytes s compred to monocultures. Interestingly, MHC-II expression on oth HCC nd non-hcc heptocytes in co-culture ws positively correlted with the respective ctivted CD8+ T cells. Conclusions: We hve estlished n in vitro co-culture model to study interctions etween utologous PBMCs nd primry HCC nd non-hcc heptocytes. This direct interction leds to incresed ntigen presenting ility of HCC heptocytes, ctivtion of PBMCs with concomitnt poptosis of ctivted CD8+ T cells. Although, prtilly effective immune response ginst HCC exists, still tumor heptocytes mnge to escpe. Keywords: HCC/non-HCC-Heptocyte-PBMCs co-cultures, CD8+ T lymphocyte ctivtion, Apoptosis, MHC-II expression * Correspondence: koskinsj@yhoo.gr 1 2nd Deprtment of Internl Medicine, Medicl School of Athens, University of Athens, Hippokrtion Hospitl, 114 Vs. Sofis Avenue, Athens , Greece Full list of uthor informtion is ville t the end of the rticle 2013 Doum et l.; licensee BioMed Centrl Ltd. This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

2 Doum et l. BMC Gstroenterology 2013, 13:17 Pge 2 of 12 Bckground The effective immune response ginst heptocellulr crcinom is of gret importnce in the progression nd clernce of the tumor. Mny studies hve shown tht the immune response ginst HCC is restrined nd not effective [1-3]. Cytotoxic T lymphocytes (CTLs) ply centrl role in induction of nti-tumor immunity. Indeed, high frequency of cytotoxic CD8+ T cells infiltrting cncer tissue cn e fvorle prognostic indictor in HCC [4]. However, the progression of tumors despite the presence of infiltrting or peripherl cytotoxic CD8+ T cells suggests tht immunologicl tolernce is induced, t lest in prt, y tumors. Tumor-specific immune responses were oserved in significnt proportion of ptients with HCC. However, spontneous clernce of estlished tumors y endogenous immune mechnisms is rre [5]. The fct tht mny tumor-ssocited ntigens (TAA) re norml selfconstituents, the immune responses tht my generte re generlly wek. Moreover, the pressure of the host immune system my led to the development of vrious mechnisms y which the tumor cells escpe from immune ttck [6]. The induction of MHC-II expression on heptocytes would it e possile mechnism of inducing the defective immune response ginst mny liver diseses including HCC. Severl studies hve reported tht heptocytes express MHC II molecules nd re le to ctivte CD4+ nd CD8+ T cells [7-9]. Although, in vitro nd in vivo studies hve demonstrted tht heptom cells my express high levels of oth clss I nd clss II molecules [10-12], little is known out their potentil immunogenicity [13-15]. Currently, there is no evidence on the immunologic outcome of the direct interction etween mlignnt humn heptocytes nd circulting T lymphocytes in n in vitro co-culture system. The existing liver sinusoid rrier, s well s the complex liver microcircultion hinders exmintion of the direct interction etween these two cell popultions in vivo. Therefore, the estlishment of humn experimentl in vitro co-culture system for the study of the interction etween HCC heptocytes nd PBMCs is of gret importnce since ll the evidence concerning the nti-tumor response to HCC is sed on niml models. Here we report the estlishment of functionl coculture system of HCC nd non-hcc heptocytes, isolted from the trnsected liver specimens of ptients sumitted to ntomic liver resection for primry HCC, with utologous PBMCs in order to study their direct effect. In ddition, these two types of cells interct with ech other leding to increse MHC-II expression on oth HCC nd non-hcc heptocytes with concomitnt ctivtion of CD8+ T cells, which finlly ecome poptotic. Methods Ptients Eleven ptients, 10 mles nd 1 femle, ge etween yers with cirrhosis nd erly stge HCC hve een sumitted to ntomic liver resection with curtive intent; 5 ptients hd chronic HBV infection, 2 lcoholic liver disese, 1 NASH nd in 3 ptients the reson of liver dmge could not e defined (cryptogenic). All ptients hve een operted in the Surgicl Unit of Heptopncretoiliry Diseses of the 1st Deprtment of Propedeutic Surgery of the University of Athens, etween Decemer 2005 nd Decemer Consent hs een retrieved from our ptients, while the protocol hs een pproved y the Ethicl nd Scientific Committee of our Hospitl. Liver specimens (from tumor nd non-tumor regions) nd preopertive peripherl lood smples were otined from ll our ptients. Also, four iopsies were otined from ptients with no liver dmge who hd surgery for non mlignnt tumors (helthy, norml heptocytes). Isoltion nd culture of PBMCs Prior to liver tissue resection 10 to 15 ml of utologous peripherl lood ws collected in EDTA tues nd PBMCs were isolted y using Histopque g/ml (Sigm Chemicls, Germny), s previously descried [16]. The resulted cell pellet (PBMCs) ws resuspended in culture medium contining DMEM-HAM F12 (Biochrom co, Germny) supplemented with 1% FBS (Gico BRL, USA), 0.25 mg/ml humn insulin (Sigm Chemicls, Germny), 100 U/ml Pen/Strep, 50 μg/ml gentmycin (Sigm Chemicls, Germny), 20 mm HEPES (Gico BRL, USA) nd 1.3 mm L-Glutmine (Biochrom co, Germny) nd the yield of PBMCs ws determined y Neuuer enumertion. The ove culture medium ws chosen fter experiments tht were performed for oth heptocytes nd PBMCs, in order to find the optiml co-culture conditions. Therefore, heptocytes nd PBMCs were mintined in monocultures using different FBS concentrtions 0%, 1% nd 5% in DMEM-F12 supplemented with 0.25 mg/ml humn insulin, 100 U/ml Pen/Strep, 50 μg/ml gentmycin, 20 mm HEPES nd 1.3 mm L-Glutmine for 7 dys. The viility of oth heptocytes nd PBMCs fter 7 dys in culture ws high, lwys exceeding 75%. The optimum FBS concentrtion for the culture medium ws the 1%. PBMCs were then plted in 60-cm culture dish until the dy of the co-culture experiment in 1% culture medium. Isoltion nd culture of HCC nd non-hcc heptocytes Tumor liver specimens were otined from the primry tumor nd non-tumor surrounding liver prenchym nd trnsferred in ice-cold DMEM-F12 supplemented with 10% FBS. Heptocyte isoltion ws performed sed on previous isoltion protocol with modifictions [17-19].

3 Doum et l. BMC Gstroenterology 2013, 13:17 Pge 3 of 12 Liver specimens were treted with pre-wrmed 0.1% Collgense Type IV (Sigm Chemicls, Germny) nd filtered through 70 μm cell striner (BD, USA). The pellet contining the liver cells ws resuspended in icecold DMEM-F12 with 20%FBS nd triple centrifugtion t 81 g for 12 min t 4 C ws performed in order to seprte the purified heptocyte popultion (pellet) from the non-prenchyml cells (superntnt) [20]. The pellet contined the purified heptocytes, s further confirmed y light microscopy nd y flow cytometric nlysis of the two heptocyte specific mrkers Hep-Pr 1 nd lumin descried in the next prgrphs. Next, heptocytes remined in culture for few hours in 60-mm dishes without collgen I in order to chieve mximum heptocyte purity, y removing possile remining nonprenchyml cells. Then, we crefully removed the superntnt contining the heptocytes nd tumor nd non-tumor heptocytes were plced in 24-well pltes coted with 5 μg/cm 2 rt til collgen type I (BD, USA) in 10% FBS culture medium nd llowed to dhere for 48 h. 16 hours prior to co-culture experiment the heptocyte medium ws removed nd heptocytes were wshed twice with 1 PBS in order to remove the possile remining intrheptic lymphocytes. Then, 1% FBS culture medium ws dded to the heptocytes. The sme procedure ws performed for the norml liver iopsies. Co-cultures of HCC nd Non-HCC Heptocytes with PBMCs After initil plting the culture medium ws crefully spirted. The rtio of PBMCs (effector cells) to heptocytes in co-cultures ws 5:1. The rtio tht ws chosen ws sed on dt from the literture. Most studies tht involved co-culture of rt lymphocytes with utologous heptocytes used the rtio of 10:1 [21-23]. We thought tht rtio of 5:1 would e more relistic to seek interctions etween HCC nd non-hcc heptocytes with utologous peripherl immune cells. PBMCs were collected nd wshed y centrifugtion. The cell pellet ws resuspended in 1% FBS medium nd the pproprite numer of mononucler cells ws dded in ech well on top of the dhered heptocytes. PBMCs with HCC-heptocytes s well s PBMCs with non-hcc heptocytes were cultured for 24 h, 48 h nd 72 h. As control cultures tumor, non-tumor heptocytes nd PBMCs were mintined in monocultures. The sme procedure ws performed for the four norml heptocyte PBMCs co-cultures. Expression of Hep Pr-1 on heptocytes y flow cytometry After isoltion, norml, HCC nd non-hcc heptocytes were stined for the heptocyte specific mrker Hep Pr-1 in order to verify the purity of the heptocyte popultion. Heptocytes were fixed with 4% prformldehyde (PFA) (Sigm, chemicls, Germny) nd centrifuged in 1 PBS (Cmrex co, USA). Heptocytes were then permeilized with 0.1% Triton X-100 (Sigm, chemicls, Germny). Hep Pr-1 ntiody (Dko Cytomtion DK) (1:10 dilution) ws incuted with Zenon ALEXA Fluor- 488 (Moleculr Proes, UK) prior to ddition to the cell suspension. Then, heptocytes suspension ws incuted with the Hep Pr-1/Zenon ALEXA Fluor-488 ntiody mixture. The cells were then immeditely nlyzed y using EPICS-XL MCL flow cytometer. Unstined cells were used for the determintion of the positive stined heptocyte popultion. Hep Pr-1 immunocytochemistry Cytospin preprtions of heptocyte s suspension were fixed with 4% PFA. Heptocytes were wshed with 6% BSA (Sigm, chemicls, Germny) /0.2% Tween-20 (Sigm, chemicls, Germny) nd 0.1% Triton X-100 ws used for the permeiliztion step. After wshing step, locking ws performed with hydrogen peroxidse (1:10 dilution). Heptocytes were incuted with Hep Pr-1 ntiody (1:5 dilution). The Envision Peroxidse Detection Kit (Dko Cytomtion, DK) ws used in order to detect Hep-Pr-1. Hemtoxylin ws used for stining the nuclei. Heptocytes were then oserved using microscope Zeiss Axiostr plus. Anlysis of MHC clss II expression nd viility of tumour nd non-tumour heptocytes y flow cytometry The superntnt from HCC non-hcc nd norml cocultures, contining the PBMCs popultion ws crefully removed. The dhered heptocytes were detched from the wells y trypsiniztion t 24 h, 48 h nd 72 h fter co-culture. In ddition, heptocytes were treted with Stem Pro Accutse (Life technologies, Gico, USA) in order to void the formtion of cell clusters. The sme procedure ws performed for the monocultures s well. The heptocyte pellets were triple stined with the HLA-DR, DP, DQ-PE (Beckmn Coulter, USA) (MHC clss II-PE) (surfce), 7-AAD (BD, USA), s well s with nti-humn lumin-fitc (Dko Cytomtion, DK) (intrcellulr). After isoltion, heptocytes were lso stined with nti- CD3 (Beckmn Coulter, USA) nd nti-cd14 (Beckmn Coulter, USA) monoclonl ntiodies to further verify their purity. MHC clss II expression nd 7-AAD mesurements were lso performed fter isoltion. Heptocytes were immeditely nlyzed y flow cytometry. Unstined heptocytes were used s controls in order to determine the utofluorescence of the cells nd for setting the flow cytometry protocol.

4 Doum et l. BMC Gstroenterology 2013, 13:17 Pge 4 of 12 Flow Cytometric nlysis of PBMCs CD8+ T cells were chrcterized from the totl PBMCs y stining them with the surfce mrker CD8-FITC (Beckmn Coulter, USA). The ctivtion of oth mononucler cells nd CD8+ T cells ws estimted y the surfce ctivtion mrker HLA-DR, DP, DQ [24]. Viility, poptosis nd necrosis of PBMCs nd CD8+ T lymphocytes ws determined y 7-AAD stining. The ove mesurements were performed y flow cytometry fter isoltion, efore co-culture experiment, 24 h, 48 h nd 72 h post co-culture. The sme mesurements took plce for the PBMCsmonoculturesswell. As mentioned efore the superntnts from tumor nd non-tumor co-cultures contined the PBMCs popultion. Mononucler cells were crefully spirted from ech well nd centrifuged. Cells were then triple stined with the monoclonl CD8-FITC ntiody, HLADR, DP, DQ-PE nd 7-AAD. Unstined PBMCs were used s controls in order to determine the utofluorescence of the cells. Sttisticl nlysis All continuous vriles re reported s the men (± SD). Pired comprisons etween qulittive vriles were performed using the Wilcoxon sign rnk test.. Dt were nlyzed using SPSS 13.0 softwre. Persons correltion coefficient ws used to detect ny positive or negtive correltions. P vlues <0.05 were considered sttisticlly significnt. Results Viility nd lumin expression of heptocytes The cell yield per grm of wet tissue fter Collgense IV tretment vried etween the different tissue smples due to different underlying disese condition nd ge. In verge the cell yield ws for the HCCheptocytes nd for the non-hcc heptocytes. Immeditely post-isoltion the verge viility ws 90% nd 92% for HCC nd non-hcc heptocytes, respectively. HCC nd non-hcc heptocytes expressed lumin protein t the level of 94% nd 92%, respectively s documented y mens of flow cytometry, which remin t the sme levels throughout the experiment. Helthy heptocytes exhiited incresed cell yield, viility nd lumin levels. The morphologicl chrcteristics of HCC nd non- HCC heptocytes remined grossly intct in culture conditions, with vile heptocytes presenting typicl nd undmged morphology s demonstrted y light microscopy (Figure 1). Freshly isolted heptocytes ttched to collgen-coted dishes fter initil plting nd formed semi-confluent monolyers within 48 h. Hep Pr-1 immunocytochemistry (Figure 1), nd flow cytometry pnels (Figure 2,) demonstrted heptocellulr purity t the level of 85% to 90% for oth HCC- nd non-hcc-cell cultures. The functionlity of HCC nd non-hcc heptocytes ws mesured y nti-humn lumin-fitc nd ws 92% nd 89%, respectively (Figure 2c,d). Negtive stining for CD14, CD3 further confirmed the sence of Kupffer cells nd mononuclers cells in the co-culture system (Figure 3,). MHC-II expression nd viility of HCC nd non-hcc heptocytes in co-culture with utologous PBMCs After isoltion, the MHC II expression on HCC heptocytes ws higher compred to non HCC heptocytes, lthough this did not rech sttisticl significnce (20% vs 15%). On the other hnd, MHC II expression on heptocytes derived from individuls with no liver disese hd significnt lower MHC II expression, rnged etween 3%-8%. HCC HEPATOCYTES Figure 1 ) Representtive photos of HCC heptocytes in culture 48 hours fter isoltion (mgnifiction x 20) nd ) HCC heptocytes stined with Hep Pr-1 fter isoltion (immunocytochemistry) (mgnifiction 20).

5 Doum et l. BMC Gstroenterology 2013, 13:17 Pge 5 of 12 HCC heptocytes Non-HCC heptocytes 85% 90% count Hep-Pr 1-FITC c HCC heptocytes d Non-HCC heptocytes 92% 89% count Alumin-FITC Figure 2 Flow cytometry digrms presenting: ) percentge of HCC-tumor heptocytes nd ) percentge of non-hcc heptocytes stined positive for Hep Pr-1, using the secondry ntiody Zenon-488, fter isoltion c) percentge of HCC-tumor nd d) percentge of non-hcc heptocytes stined for humn lumin-fitc. In co-culture with utologous PBMCs, the expression of MHC clss II molecules on heptocytes ws further incresed compred to the single cultures t ll time points oth for HCC nd non-hcc heptocytes. For the HCC heptocytes the respective difference reched sttisticl significnce t 48 h in co-culture compred to monocultures (31.4 vs 23.8, p < 0.05), with further increse t 72 h (37.1 vs 28.1, p = 0.05) (Figure 4,, c). Non-HCC heptocytes lso exhiited significnt incresed MHC II expression 48 h post-co-culture with PBMCs in comprison to mono-culture (21.9 vs 15, p < 0.05) (Figure 4d). Moreover, the expression of MHC clss II molecules on HCC heptocytes ws higher t ll time points compred to non-hcc heptocytes oth in co-cultures nd monocultures. MHC II expression on heptocytes from individuls with no liver disese remined the sme for oth monocultures nd co-cultures with utologous PBMCs t ll time points (3-5%). Interestingly, the viility of totl non-hcc heptocytes in co-culture ws significntly lower t ll time points compred to the respective heptocytes in monocultures (24 h: 27.1 vs 63.8, p < 0.05, 48 h: 10.4

6 Doum et l. BMC Gstroenterology 2013, 13:17 Pge 6 of 12 HCC heptocytes HCC heptocytes 0% 0% CD3-FITC CD14-PE Figure 3 After isoltion tumor, non-tumor nd norml heptocytes were stined for CD3 nd CD14 mrkers nd nlyzed y flow cytometry in order to verify the purity of the heptocyte culture ws performed; representtive flow cytometric digrm of ) CD3-FITC nd ) CD14-PE negtive stining in tumor heptocytes. vs 62.6, p < 0.05 nd 72 h: 27.4 vs 55.8, p < 0.05). In contrst, the viility of totl HCC heptocytes hd not difference compred to the respective heptocytes in monocultures. The viility of MHC II-expressing HCC heptocytes fter 72 h in co-culture with PBMCs ws significntly higher compred to MHC II-expressing HCC heptocytes in monoculture (11.5 vs 8.2 p < 0.05). In contrst, MHC II-expressing non-tumor heptocytes showed non-significnt incresed viility in co-culture compred to their monocultures. Effects of HCC nd non-hcc heptocytes on PBMCs nd CD8+ T cells The medin expression of MHC II (ctivtion) on PBMCs from ptients with HCC fter isoltion ws 8%. PBMCs exhiited n increse of MHC II expression in co-culture with HCC s compred to non-hcc heptocytes t ll time points. This difference in mononucler cells ctivtion ws found to e sttisticlly significnt t 24 h (9.7 vs 7.4, p < 0.05) nd 48 h (9.7 vs 7.9 p < 0.05) (Figure 5,, c). Although, MHC II expression on PBMCs ws elevted in co-culture with tumor heptocytes compred to PBMCs monocultures, the respective differences did not rech sttisticl significnce (Figure 5c). In contrst, in non-hcc co-culture there ws no evidence of incresed MHC II expression on PBMCs t ll time points, t lest with the ctivtion mrkers tht we hve used. Also, no differences were oserved concerning the poptosis nd necrosis of ctivted nd totl peripherl mononucler cells in oth HCC nd non-hcc heptocyte co-cultures compred with PBMCs monocultures. Further, we exmined the ehvior of CD8+ T cell suset from the totl PBMCs popultion when co-cultured with HCC nd non-hcc heptocytes. Therefore, in the totl PBMCs popultion we performed doule stining for CD8+ nd MHC II expression (CD8 + MHC II+, phenotype of ctivted CD8 + T cells). CD8+ T cells showed n increse of MHC II expression t ll time points with mximum t 72 h in coculture with HCC heptocytes compred to PBMCs monoculture lthough sttisticlly ws not significnt (Figure 6, ). No significnt ctivtion of CD8+ T cells ws oserved in co-culture with non-hcc heptocytes (Figure 6). 7-AAD nlysis of CD8+ T cells co-cultured with HCC or non-hcc heptocytes reveled n incresed necrosis t 48 h (p < 0.05) s compred to control (PBMCs monocultures). The ctivted CD8+ T cells (MHC II+) lso showed incresed poptosis nd necrosis in co-culture with HCC heptocytes compred to controls especilly t 48 h (0.18 vs 0.06, p = nd 0.15 vs 0.05, p < 0.05) (Figure 7). This phenomenon ws not oserved in non-hcc co-cultures. In the co-cultures of heptocytes from individuls with no liver disese with their utologous PBMCs no chnges were oserved in ctivtion or in viility sttus of either PBMCs or CD8+ T cells.

7 Doum et l. BMC Gstroenterology 2013, 13:17 Pge 7 of 12 Alumin-FITC HCC +PBMCs co-culture HCC monoculture M2 35% 15% M2 M4 M4 HLA-DR-PE MHC-II expression (%) c p<0.05 P=0.05 HCC hep monoculture HCC hep coculture MHC-II expression (%) P<0.05 Non-HCC hep monoculture Non-HCC hep coculture 0 0 h 24 h 48 h 72 h time in culture (h) 0 0 h 24 h 48 h 72 h time in culture (h) Figure 4 Representtive flow cytometry sctterplot depicting the incresed expression of MHC II on HCC heptocytes (M region) ) 48 hours in co-culture with utologous PBMCs vs. ) 48 hours in monoculture. Representtive grph showing the percentge of MHC II expression on c) HCC nd d) non-hcc heptocytes in co-culture compred to heptocyte monocultures. Time point zero (0 h) depicts MHC II expression on heptocytes directly fter isoltion. We next exmined the correltion etween the MHC- II expression on HCC nd non-hcc heptocytes nd ctivtion of CD8+ T cells nd PBMCs. Indeed, we found tht the MHC-II expressing HCC heptocytes were positively correlted with the ctivted CD8+ T cells (CD8 + MHC-II+) fter 24 h nd 48 h in HCC co-culture (r = 0.947, p < nd r = 0.878, p = 0.021, respectively). In ddition, MHC-II expressing non-hcc heptocytes were lso positively correlted with the ctivted CD8+ T cells fter 24 h nd 72 h in co-culture (r = , p = nd r = 0.948, p = 0.014, respectively). Discussion We hve estlished functionl culture system for HCC nd non-hcc heptocytes originting from surgicl tissue specimens retrieved from HCC ptients undergoing heptectomy with intention to cure. 7-AAD nd humn lumin stining indicted high viility nd functionlity of heptocytes (HCC, non-hcc nd helthy) in vitro, respectively. Heptocyte purity fter isoltion with the isoltion protocol tht we used ws exceeding 95% nd ws demonstrted y positive Hep Pr-1 nd negtive CD3 nd CD14 immunostining. Dt from the literture indicte tht the outcome of interctions etween tumor heptocytes nd immune cells is criticl for cncer regression or progression. There is lck of pproprite models to ddress how humn immune system intercts with liver cncer cells. With the restriction of humn experimentl system, most knowledge on liver cncer immunity is derived from niml models. So, we hve estlished n in vitro co-culture system of tumor nd nontumor heptocytes with PBMCs isolted from peripherl lood drwn prior to resection in order to study their immunologicl interctions. With restrictions we elieve tht this in vitro system will provide to us informtion of wht hppens in vivo. There re mny scientific reports which hve shown tht heptocytes express MHC clss II molecules nd cn ct s ntigen presenting cells. Although, heptocytes cn ctivte CD4+ nd CD8+ T cells there re not le to sustin this ctivtion nd to crete n effective immune response [7-9]. In ddition, MHC molecules my e either expressed de novo or up-regulted on numer of tumor cells [25,26], ut ny reltion to n effective presenttion of tumor ntigen to T cells for stimultion nd immunologicl rejection of tumors is controversil. Although

8 Doum et l. BMC Gstroenterology 2013, 13:17 Pge 8 of 12 HCC+PBMCs coculture Non-HCC+PBMCscoculture Count 10% 6% c 20 HLADR-PE MHC-IIexpression (ctivtion) (%) P<0.05 P<0.05 PBMCs HCC COCUL PBMCs NON-HCC COCUL PBMCs monoculture 0 0h 24h 48h 72h Time in culture (h) Figure 5 Flow cytometric digrms showing totl PBMCs ctivtion (MHC-II expression) fter 48 h in co-culture with ) HCC nd ) non-hcc heptocytes. c) PBMCs exhiited significnt ctivtion t 24 h nd 48 h when co-cultured with HCC heptocytes compred to non-hcc heptocytes. Also, the ctivtion of PBMCs in HCC co-culture ws higher compred to PBMCs monoculture. Time point zero (0 h) depicts ctivtion of totl PBMCs fter isoltion (0 h). mny tumors express rejection ntigens s well s MHC molecules they void destruction y T cells [27-33]. Therefore, it is of gret importnce to study if primry humn neoplstic heptocytes derived from HCC tumors re le to express MHC clss II molecules nd hve the ility to ct s ntigen presenting cells when they re in contct with peripherl lood mononucler cells. If this is true, HCC heptocytes will e le to provide help to immune cells, which re lredy defective, oth in the periphery nd in the tumor microenvironment. Under norml conditions, heptocytes do not express MHC II molecules. However, heptocellulr MHC clss II expression hs een reported in ptients with heptitis [34,35], s well s in ptients with HCC [10,11]. Furthermore, tumor heptocytes from heptom cell lines express MHC clss II molecules nd could ct s ntigen presenting cells [15,35]. Recent experimentl evidence suggests tht the liver cts s site of primry T-cell ctivtion leding to functionl impirment nd premture deth of cytotoxic T cells [36]. In n experimentl niml study y

9 Doum et l. BMC Gstroenterology 2013, 13:17 Pge 9 of 12 HCC+PBMCs coculture CD8-FITC HLADR-PE MHC-II expression (ctivtion) (%) ctivted CD8+ T cells HCC COCUL ctivted CD8+ T cells Non- HCC COCUL ctivted CD8+ T cells monoculture 0 0 h 24 h 48 h 72 h time in culture Figure 6 ) Representtive flow cytometric digrm showing the incresed expression of MHC clss II on CD8+ T cells (ctivted) t 72 h HCC co-culture, ) CD8+ T cells exhiited higher ctivtion t ll time points compred to control PBMCs culture with pek t 72 h. Also, the ctivtion of CD8+ T cells in oth HCC nd non-hcc cocultures ws higher compred to CD8+ T cells ctivtion fter isoltion (0h). Bertolino et l. [7], heptocytes were cple of inducing in vitro ctivtion nd prolifertion of nive CD8+ T cells, in the sence of CD4+ T cell co-stimultion, cler indiction tht they possess the ility of ntigen presenttion. Indeed, ccording to our results, HCC heptocytes exhiited high expression of MHC II on their surfce compred to non-hcc heptocytes, expression tht is further enhnced upon co-culture with utologous PBMCs t ll time points. Incresed MHC-II expression ws lso oserved in the non-hcc heptocytes in coculture with PBMCs. However it hs to e ddressed tht the non HCC heptocytes re not relly helthy s they hve derived from the HCC ptients with underlying virl or other etiology chronic liver disese. In contrst, heptocytes derived from individuls with no liver disese hd miniml MHC clss II expression t the eginning, which remined t the sme low levels t ll time points in the monoculture or co-culture with PBMCs system. With the respect to the effect of heptocytes on PBMCs our experiments showed tht the expression of MHC II on totl PBMCs, which indictes n ctivtion sttus, ws sttisticlly higher in the presence of HCC thn non-hcc cells, prticulrly fter 24 h nd 48 h of incution. Furthermore, CD8+ T cell ctivtion ws clerly seen fter 72 h in the HCC co-culture system thn in the non-hcc system. No effect on MHC II expression on PBMCs nd CD8+ T cells ws oserved in the non-hcc co-culture system. We next exmine if the MHC-II expression on the tumor nd non-tumor heptocytes in co-culture hd ny correltion with the ctivtion sttus of the CD8+ T cells or the totl PBMCs. Interestingly, we found tht the expression of MHC-II on HCC heptocytes ws positively correlted with the ctivted CD8+ T cells 24 h nd 48 h post-coculture. The sme correltion ws lso true for the non-hcc heptocytes ut t different time points (24 h nd 72 h post-coculture).therefore, oth MHC-II

10 Doum et l. BMC Gstroenterology 2013, 13:17 Pge 10 of 12 HCC coculture HCC monoculture 0.48% 0.05% Figure 7 Flow cytometric digrms showing the % of ctivted CD8+ T cells (CD8+ MHC II) tht re poptotic (D region) nd necrotic (E region) in ) HCC co-cultures nd ) in control PBMCs cultures. expression on HCC nd non-hcc heptocytes in coculture ws directly linked to the ctivted CD8+ T cells, leding to effective immune response. In greement to these results is the fct tht γ- interferon (IFN-gmm) produced y T lymphocytes tht infiltrte the liver during the course of chronic heptitis induces MHC II expression nd my endow the heptocytes with the cpcity to perform ccessory, ntigen-presenting, cell functions [35]. There is possiility tht similr effect could lso hppen in our in vitro co-culture system. In this respect IFN-γ could possily hve dul effect on HCC heptocytes in co-culture; destruction of tumor cells nd overexpression of MHC II on them. This my lso true for the non-hcc virus infected heptocytes. However, the viility of MHC II-expressing HCC cells ws significntly higher in the co-culture system s compred to the monocultures t 72 h, oservtion tht ws not evident in non-hcc cells. In ddition to the ove oservtion, the totl viility of HCC heptocytes in co-culture remins unchnged, while the viility of the respective non-hcc heptocytes ws significntly lower t ll time points compred to the respective monoculture. Therefore, this further enhnced the ide tht HCC heptocytes mnged to survive nd escpe from the immune ttck in contrst to the respective non-hcc tht exhiited lower viility. As possile exmintion is the fct tht incresed necrosis t 48 h in the totl CD8+ T lymphocytes in the HCC co-culture compred to PBMCs monoculture ws seen. More importntly, t 48 h, the ctivted CD8+ T cells hd undergone significnt poptosis nd necrosis compred to controls. However, the fctors nd the mechnisms involved could e eyond the elimintion of the ctivted CD8+ T cells. Indeed, severl studies hve shown tht tumor cells expressing decoy molecules, such s DcR3, cn escpe FsL-dependent immune-cytotoxic ttck. Thus, DcR3 is considered to e one of the immune evsion systems for tumor progression [37]. Also, it hs een reported tht the numer of regultory T cells in the peripherl lood nd in the tumor microenvironment is incresed nd tht my exerts n inhiitory effect in the function of the immune cells, including CD8+ T cells, [38], offering n dditionl escpe mechnism to HCC heptocytes. The elimintion of CD8+ T cells here is not due to deth y neglect or lck of secondry ctivtion signls (cytokines etc.), s it ws referred in other studies [7]. In our co-culture system, other immune cells re present which could provide CD8+ T cells with secondry signls nd support CD8+ T cell ctivtion something tht is verified from the incresed ctivtion of PBMCs popultion. Conclusions In conclusion we hve estlished, for the first time, n in vitro model to study the interction etween utologous PBMCs nd CD8+ T cells with primry HCC nd non-hcc heptocytes. Our results reveled very interesting counterction; n incresed expression of MHC II on oth HCC nd non- HCC heptocytes nd ctivtion of PBMCs followed y n enhnced viility of MHC-II-expressing HCC cells with concomitnt poptosis of the ctivted CD8+ T cells. Further experiments should tke plce in order to understnd the mechnisms of this interction nd enefit from this

11 Doum et l. BMC Gstroenterology 2013, 13:17 Pge 11 of 12 ility of tumor heptocytes to ct s ntigen presenting cells nd to ctivte the immune cells. Arevitions PBS: Phosphte uffer sline; 7-AAD: 7-ctinomycin-D; FBS: Fetl ovine serum; HCC: Heptocellulr crcinom; NASH: Non- lcoholic stetoheptitis; Pen/Strep: Penicillin/streptomycin. Competing interests The uthors declre tht they hve no competing interests. Authors contriution JK (Associte Professor of Internl Medicine-Heptology), hs designed the protocol, supervising in ll the steps of the study nd contriuted to the finl form of the mnuscript. MMK (Associte Professor of Surgery), hs contriuted to the collection of the surgicl humn liver specimens, the design of the protocol nd supervising in ll the steps of the study. PPD, (Biochemist, PhD student), hs done the reserch work nd hs written the pper. MN, (Biologist, PhD), hs contriuted in the l work. IPG, (MD, PhD), hs contriuted prtilly in the collection of surgicl liver specimens nd hs done the sttisticl nlysis. All uthors hve contriuted significntly nd gree with the content of the mnuscript. Acknowledgements We thnk the Hellenic Society for the Study of the Liver Diseses for their finncil support nd Professor John Brmis for his scientific nd finncil support throughout the experimentl project. Author detils 1 2nd Deprtment of Internl Medicine, Medicl School of Athens, University of Athens, Hippokrtion Hospitl, 114 Vs. Sofis Avenue, Athens , Greece. 2 1st Deprtment of Propedeutic Surgery, Lortory of Surgicl Reserch, Medicl School of Athens, Hippokrtion Hospitl, Athens, Greece. Received: 20 July 2012 Accepted: 11 Jnury 2013 Pulished: 18 Jnury 2013 References 1. Korngy F, Ormndy LA, Bleck JS, Klempnuer J, Wilkens L, Mnns MP, Greten TF: Spontneous tumor-specific humorl nd cellulr immune responses to NY-ESO-1 in heptocellulr crcinom. Clin Cncer Res 2004, 10: Mizukoshi E, Nkmoto Y, Mrukw Y, Ari K, Ymshit T, Tsuji H, Kuzushim K, Tkiguchi M, Kneko S: Cytotoxic T cell responses to humn telomerse reverse trnscriptse in ptients with heptocellulr crcinom. Heptology 2006, 43: Liu Y, Dley S, Evdokimov VN, Zdoinski DD, Potter DM, Butterfield LH: Hierrchy of lph fetoprotein (AFP)-specific T cell responses in sujects with AFP-positive heptocellulr cncer. J Immunol 2006, 177: Wd Y, Nkshim O, Kutmi R, Ymmoto O, Kojiro M: Clinicopthologicl study on heptocellulr crcinom with lymphocytic infiltrtion. Heptology 1998, 27: Png YL, Zhng HG, Peng JR, Png XW, Yu S, Xing Q, Yu X, Gong L, Yin YH, Zhng Y, Chen WF: The immunosuppressive tumor microenvironment in heptocellulr crcinom. Cncer Immunol Immunother 2009, 58: Zou W: Immunosuppressive networks in the tumor environment nd their therpeutic relevnce. Nt Rev Cncer 2005, 5: Bertolino P, Trescol-Biémont MC, Rourdin-Come C: Heptocytes induce functionl ctivtion of nive CD8+ T lymphocytes ut fil to promote survivl. Eur J Immunol 1998, 28: Bertolino P, Trescol-Biémont MC, Thoms J, Fzeks de St Groth B, Pihlgren M, Mrvel J, Rourdin-Come C: Deth y neglect s deletionl mechnism of peripherl tolernce. Int Immunol 1999, 11: Herkel J, Jgemnn B, Wiegrd C, Lzro JFG, Lueth S, Knzler S, Blessing M, Schmitt E, Lohse AW: MHC Clss II expressing heptocytes function s ntigen-presenting cells nd ctivte specific CD4 T lymphocytes. Heptology 2003, 37: Pterson AC, Sciot R, Kew M, Clle CF, Dusheiko GM, Desmet VJ: HLA expression in humn heptocellulr crcinom cells. Br J Cncer 1988, 57: Sung CH, Hu C, Hsu HC Ng AK, Chou CK, Ting LP, Su TS, Hn SH, Chnget CM: Expression of clss I nd clss II Mjor histocomptiility ntigens on humn heptocellulr crcinom. J Clin Invest 1989, 83: Krief PB, Azzrone B, Boucheix C: Different levels of interferon-γ induction of clss I nd clss II mjor histocomptiility ntigens in humn heptom cell lines. In Virl Heptitis nd Liver Disese. Edited y Aln R. New York: Liss Inc; 1988: Hsu HC, Sheu JC, Lin YH: Prognostic histologic fetures in restricted smll heptocellulr crcinom (HCC) I Twin: comprison with resected lrge HCC. Cncer 1985, 56: Tkgi S, Chen K, Schwrz R, Shunzuro I, Heermn RB, Whiteside TL: Functionl nd phenotypic nlysis of tumor-infiltrting lymphocytes isolted from humn primry nd metsttic liver tumors nd cultured in recominnt interleukin-2. Cncer 1989, 63: Proli M, Perrone A, Bonvit MS, Brn V: Immunology of heptocellulr crcinom. Itl J Gstroenterol 1991, 23: Serti E, Doum PP, Thyphronitis G, Tsitour P, Ktsrou K, Fok P, Konstndoulkis MM, Koskins J, Mvromr P, Georgopoulou U: Modultion of IL-2 expression fter uptke of heptitis C virus non-enveloped cpsid-like prticles: the role of p38 kinse. Cell Mol Life Sci 2011, 68: Thornton AJ, Hm J, Kunkel SL: Kupffer Cell-derived cytokines induce the synthesis of leukocyte chemotctic peptide, interleukin-8, in humn heptom nd primry heptocyte cultures. Heptology 1992, 15: Gehring AJ, Sun D, Kennedy PTF, Nolte- t Hoen E, Lim SG, Wsser S, Selden C, Mini MK, Dvis DM, Nssl M, Bertoletti A: The level of virl ntigen presented y heptocytes influences CD8 T-cell function. J Virol 2007, 81: Wigmore SJ, Feron KCH, Mingy JP, Li PBS, Ross JA: Interleukin-8 cn medite cute-phse protein production y isolted humn heptocytes. Am J Physiol Endocrinol Met 1997, 273: Fok P, Pourchet A, Hernndez-Alcoce R, Doum PP, Pisss G, Kouvtsis V, Dlgiorgou G, Kzzi D, Mrconi P, Foschini M, Mnservigi R, Konstdoulkis MM, Koskins J, Epstein AL, Mvromr P: Novel tumourspecific promoters for trnscriptionl trgeting of heptocellulr crcinom y herpes simplex virus vectors. J Gene Med 2010, 12: Wu J, Wng C, Liu Q, Yng T, Zhng Q, Peng J, Go Y, Sun H, Kku T, Liu K: Protective effect of JBP485 on concnvlin A-induced heptocyte toxicity in primry cultured rt heptocytes. Eur J Phrmcol 2008, 589: Jing X, Gregory SH, Wing EJ: Immune CD8+ T lymphocytes lyse Listeri monocytogenes-infected heptocytes y clssicl MHC clss I-restricted mechnism. J Immunol 1997, 158: Slgdo FJ, Lojo J, Fernández-Alonso CM, Viñuel J, Cordero OJ, Nogueir M: Interleukin-dependent modultion of HLA-DR expression on CD4nd CD8 ctivted T cells. Immunol Cell Biol 2002, 80: Rmdori G, Moeius U, Dienes HP, Meuer S, Meyer zum Büschenfelde KH: Lymphocytes from heptic inflmmtory infiltrte kill rt heptocytes in primry culture. Comprison with peripherl lood lymphocytes. Virchows Arch B Cell Pthol Incl Mol Pthol 1990, 59: Ntli PG, Gicomini P, Bigotir A, Imi K, Nicotr MR, Ng AK, Ferrone S: Heterogeneity in the expression of HLA nd tumour ssocited ntigens y surgiclly removed nd cultured rest crcinom cells. Cncer Res 1983, 43: Goodenow RS, Vogel JM, Linsk RL: Histocomptiility ntigens on murine tumors. Science 1985, 230: Festenstein H, Grrido F: MHC ntigens nd mlignncy. Nture 1986, 322: Btemn WJ, Jenkinson E, Owen J: T-cell immunity to murine Moloney srcom virus-induced tumors: L3T4 T cells re necessry for resistnce to primry srcom growth, ut Lyt-2 cells re required for resistnce to secondry tumour cell chllenge. Immunology 1987, 61: Greenerg P: Requirements for ntigen-specific induction nd expression of CD4 nd CD8 T-cell responses to tumors. InCellulr Immunity nd the Immunotherpy of Cncer. Edited y Lotze M, Finn O. New York: Wiley-Liss; 1990: Hmmerling GJ, Klr D, Pulm W, Momurg F, Moldenhuer G: The influence of mjor histocomptility complex clss I ntigens on tumor growth nd metstsis. Biochim Biophys Act 1987, 907: Tnk K, Hyshi H, Hmd C, Khoury G, Jy G: Expression of mjor histocomptiility complex clss I ntigens s strtegy for the

12 Doum et l. BMC Gstroenterology 2013, 13:17 Pge 12 of 12 potentition of immune recognition of tumor cells. Proc Ntl Acd Sci USA 1986, 83: Wllich R, Buluc M, Hmmerling G, Ktzv S, Segl S, Feldmn M: Arogtion of metsttic properties of tumour cells y the novo expression of H-2K ntigens following H-2 gene trnsfection. Nture 1985, 315: Gops J, Roger-Zismn B, Hmmerling GJ, Segl S: The reltionship etween MHC gene expression nd metstsis. Adv Cncer Res 1989, 53: Dienes HP, Hutteroth T, Hess G, Meuer SC: Immunoelectron microscopic oservtions on the inflmmtory infiltrtes nd HLA ntigens in heptitis B nd non-a, non-b. Heptology 1987, 7: Frnco A, Brn V, Ntli P, Blsno C, Musc A, Blsno F: Expression of clss I nd clss II mjor histocomptiility complex ntigens on humn heptocytes. Heptology 1988, 8: Bowen DG, Zen M, Holz L, Dvis T, McCughn GW, Bertolino P: The site of primry T cell ctivtion is determinnt of the lnce etween intrheptic tolernce nd immunity. J Clin Invest 2004, 114: Pitti RM, Mrsters SA, Lwrence DA, Roy M, Kischkel FC, Dowd P, Hung A, Donhue CJ, Sherwood SW, Bldwin DT, Godowski PJ, Wood WI, Gurney AL, Hilln KJ, Cohen RL, Goddrd AD, Botstein D, Ashkenzi A: Genomic mplifiction of decoy receptor for Fs lignd in lung nd colon cncer. Nture 1998, 396: Unitt E, Rushrook SM, Mrshll A, Dvies S, Gis P, Morris LS, Colemn N, Alexnder GJM: Compromised lymphocytes infiltrte heptocellulr crcinom: the role of T-regultory cells. Heptology 2005, 41: doi: / x Cite this rticle s: Doum et l.: Co-culture of primry humn tumor heptocytes from ptients with heptocellulr crcinom with utologous peripherl lood mononucler cells: study of their in vitro immunologicl interctions. BMC Gstroenterology :17. Sumit your next mnuscript to BioMed Centrl nd tke full dvntge of: Convenient online sumission Thorough peer review No spce constrints or color figure chrges Immedite puliction on cceptnce Inclusion in PuMed, CAS, Scopus nd Google Scholr Reserch which is freely ville for redistriution Sumit your mnuscript t