Aberrant expression of B7-H4 correlates with poor prognosis and suppresses tumor-infiltration of CD8 + T lymphocytes in human cholangiocarcinoma

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1 ONCOLOGY REPORTS 36: , 2016 Aberrnt expression of B7-H4 correltes with poor prognosis nd suppresses tumor-infiltrtion of CD8 + T lymphocytes in humn cholngiocrcinom Xin Zho *, Fei Guo *, Zhonghu Li, Peng Jing, Xing Deng, Feng Tin, Xiowu Li nd Shugung Wng Institute of Heptobiliry Surgery, Southwest Hospitl, Third Militry Medicl University, Shpingb, Chongqing , P.R. Chin Received December 31, 2015; Accepted Februry 14, 2016 DOI: /or Abstrct. B7-H4, s member of the costimultory B7 fmily, serves criticl role in the negtive regultion of T cell-medited ntitumor immune responses. Cholngiocrcinom (CCA) hs poor prognosis due its invsiveness nd ssocited metstsis. The present study investigted the expression of B7-H4 in ptients with CCA nd its ssocition with ptient prognosis. The correltion between B7-H4 expression nd CD4 + nd CD8 + tumor-infiltrting lymphocytes ws lso investigted. The results demonstrted tht high B7-H4 expression ws detected in cncer tissues (54/110; 49.1%) compred with tht noted in chronic inflmmtory bile duct tissues (4/19; 21.1%). Furthermore, ll 8 biliry denom smples showed negtive stining. The expression of B7-H4 ws significntly ssocited with dverse clinicl nd pthologicl fetures including histologic grde (P<0.001), tumor sttus (P=0.025), lymph node metstsis (P=0.035) nd 6th Union for Interntionl Cncer Control stge (P=0.019). Kpln Meier survivl nlysis nd Cox regression nlysis indicted tht berrnt B7-H4 expression ws significnt independent predictor of poor overll survivl nd erly recurrence. In ddition, the present study demonstrted tht B7-H4 expression in tumor cells ws negtively correlted with the density of CD8 + T cells in the tumor strom. Co-culture ssys indicted tht knockdown of B7-H4 incresed CD8 + T cell-medited cytotoxicity in vitro, suggesting tht the expression of B7-H4 my serve role in shielding tumors from immune surveillnce by suppression of tumor-infiltrting CD8 + T lymphocytes in CCA. In conclusion, the present study showed tht berrnt expression of B7-H4 Correspondence to: Professor Shugung Wng, Institute of Heptobiliry Surgery, Southwest Hospitl, Third Militry Medicl University, 30 Gotnyn Street, Shpingb, Chongqing , P.R. Chin E-mil: sgwngxnyy@sin.com * Contributed eqully Key words: cholngiocrcinom, B7-H4, tumor immune escpe, tumor-infiltrting lymphocytes, prognosis ws correlted with poorer prognosis nd suppressed CD8 + tumor infiltrting lymphocytes in CCA. Introduction Cholngiocrcinom (CCA) is severe tumor originting from epithelil cells in the intrheptic nd extrheptic bile ducts nd is ssocited with poor prognosis (1,2). Surgicl resection is the predominnt tretment, however, the mjority of ptients re dignosed too lte to resect or present with metsttic disese (3). Additionlly, the lck of biomrkers nd effective non-surgicl therpeutic modlities limit current tretment options. To dte, no second-line therpy hs definitely demonstrted improved long-term survivl (4). Therefore, improving the understnding of the moleculr mechnisms of crcinogenesis is required to develop novel therpies for the tretment of CCA. The immunologicl response to cncer is n importnt protective mechnism ginst cncer. However, the escpe of tumors from immune surveillnce hs been ttributed to immune system dysfunction, leding to tumor progression, metstsis nd recurrence. There re number of strtegies tht enble tumor cells to escpe immune surveillnce, including dysfunctionl mjor histocomptibility complex clss I molecule, immunosuppressive fctors nd berrnt expression of costimultory molecules. While the regultory mechnisms by which costimultory molecules regulte the immune system hve received reserch focus, the B7 fmily hs not been investigted in detil. The costimultory B7 fmily re cell-surfce protein lignds, providing stimultory nd inhibitory signls to regulte the T cell response. The B7 fmily comprises seven members, B7.1 (CD80), B7.2 (CD86), B7-DC (CD273, PD-L2), B7-H1 (CD274, PD-L1), B7-H2 (ICOS-L), B7-H3 (CD276) nd B7-H4 (B7x, B7S1) (5). B7-H4 is new member of the B7 fmily, nd is type I-trnsmembrne protein nd functions vi glycosyl phosphte-dylinositol linkge, binding to currently unidentified receptor. B7-H4 is expressed t low levels in vrious peripherl tissues, including lung, colon, liver, kidney, pncres, smll bowel, brest nd uterus (6,7). Aberrnt expression of B7-H4 hs been observed in severl

2 420 zho et l: B7-H4 with poor prognosis nd suppressed CD8 + T lymphocytes in humn cca tumor types, including those of the brest, skin, lungs, colon, kidney, brin nd ovries (8). B7-H4 hs been demonstrted to effect the negtive regultion of T cell-medited immune responses by inhibiting T cell ctivtion, prolifertion, cytokine production nd cytotoxic ctivity (6). Furthermore, previous studies hve reported tht the expression levels of B7-H4 re correlted with clinicopthologicl prmeters, nd B7-H4 is currently considered to be prognostic mrker in vrious tumors. However, the expression levels of B7-H4 hve not been investigted in different tumor types, nd its correltion with clinicl outcomes remins controversil (8). In prticulr, the expression of B7-H4 in CCA nd its clinicl significnce hve not been nlyzed in detil. Further investigtion of the ssocition between B7-H4 nd CCA is required, nd my provide potentil moleculr trgets for improved methods of detection nd tretment. In the present study, the expression of B7-H4, nd its correltion with clinicopthologicl prmeters in CAA were investigted. The prognostic vlue of B7-H4 ws evluted using the Kpln-Meier estimtor nd Cox regression nlysis. The present study imed to exmine the tumor microenvironment by investigting the ssocition between B7-H4 protein nd the density of vrious T lymphocytes. Additionlly, to understnd the functionl role of B7-H4 in ntitumor T cell responses, we crried out co-culture with CD8 + T cytotoxic lymphocytes (CTLs) to identify its impct on the suppression of CTL ctivity. Together, the results suggest tht B7-H4 my represent novel prognostic predictor, in ddition to being potentil trget for ntitumor immunotherpy for ptients with CCA. Mterils nd methods CCA ptients nd clinicl smples. Tissues were obtined from 137 ptients who underwent surgery t the Southwest Hospitl (Chongqing, Chin) between 2005 nd Ptients who underwent pre opertive tretment, such s rdiotherpy nd/or chemotherpy, were excluded. A totl of 110 cncerous nd 28 lymph node metsttic smples from the ptients were collected. A totl of 19 chronic inflmmtory bile duct smples from ptients with heptolithisis nd 8 biliry denom smples were lso collected from Southwest Hospitl. All smples were obtined with informed consent from ll ptients, ccording to the protocols pproved by the Institutionl Review Bord of the Southwest Hospitl, Third Militry Medicl University. The pthologicl reports were reviewed nd the clinicl fetures of the 110 ptients with CCAs re presented in Tble I. Tumor-node-metstsis stges were ssigned ccording to the 6th Union for Interntionl Cncer Control. Overll survivl ws clculted from the dte of surgery to the dte of mortlity or lst contct. Recurrence free survivl ws computed from the dte of surgery to the dte of recurrence. Immunohistochemistry (IHC). Formlin fixed, prffin embedded resected tissue blocks were cut into 4 mm sections nd mounted on chrged glss slides, deprffinized nd rehydrted in grded series of ethnol. Endogenous peroxidse ctivity ws blocked with solution of 3% H 2 O 2 in methnol for 30 min. Following wshing in phosphte buffered sline (PBS), ntigen retrievl ws performed in citrte buffer (ph 6.0) t 120 C for 15 min. After cooling nd wshing 3 times with PBS (ph 7.4) for 5 min ech, sections were incubted with the primry ntibodies ginst B7-H4 (LLC ; dilution 1:200; Abbiotec, Sn Diego, CA, USA) (9), CD4 (TA802240S; dilution 1:150), CD8 (TA802079S; dilution 1:200) (both from OriGene Technologies, Inc., Rockville, MD, USA) in humid chmber t 4 C overnight. The sections were then wshed with PBS nd incubted with secondry polymeric peroxidse lbeled rbbit nti-mouse ntibody (Dko, Glostrup, Denmrk) for 1 h t 37 C. Subsequently, the nuclei were counterstined with hemtoxylin nd visulized with 3,3-diminobenzidine tetrhydrochloride (Dko). A negtive control ws performed using PBS insted of the primry ntibodies under the sme conditions. The sections were dehydrted, clered nd mounted. Evlution of IHC stining. Two independent investigtors (G.F. nd J.P.), who were blinded to the ptient clinicopthologicl dt, nlyzed the IHC imges. Expression of B7-H4 ws nlyzed in 10 different high-power fields (HPFs). IHC for B7-H4 showed cytoplsmic nd membrne stining. The intensity (I) of stining ws scored s negtive (0), wek (1), moderte (2), or strong (3). The proportion (P) of B7-H4 positive cells ws defined s: 0, no stining; 1, <10% tumor cells with stining; 2, 10-50% tumor cells with stining; 3, 50-80% tumor cells with stining; 4, >80% tumor cells with stining. Smples with IHC scores (P x I) 3 were considered s negtive nd with scores >3 were defined s positive. Additionlly, the expression levels of CD4/CD8-positive T lymphocytes in the tumor nest nd tumor strom were seprtely determined ccording to IHC stining. The evlution ws bsed on review of 10 different HPFs tht showed the highest level of lymphocytic infiltrtes for ech cse (low <10%, high 10%). CCA cell lines nd trnsfection. Two humn CCA cell lines (QBC939 nd RBE), were obtined from the Cell Bnk of the Chinese Acdemy of Sciences (Shnghi, Chin) (10,11), nd cultured in RPMI-1640 medium with 10% fetl bovine serum (FBS; GE Helthcre Life Sciences, Chlfont, UK). All cell lines were incubted t 37 C in humidified tmosphere contining 5% CO 2. Cells were trnsfected with lentivirl vectors encoding short hirpin RNA (Shnghi GenePhrm Co., Ltd., Shnghi, Chin) trgeting humn B7-H4 for B7-H4 knockdown or scrmbled shrna-nc (Shnghi GenePhrm Co., Ltd.) s the control. The QBC939 nd RBE cell lines were co-trnsfected with lentivirl vectors. The recombinnt vector ws nmed GFP&Puro-B7-H4-shRNA, ccording to the mnufcturer's recommendtions. For the QBC939 nd RBE cell lines, multiplicity of infection of 10 ws used to chieve >90% trnsfection. The cells were cultured for 72 h following trnsfection. Stbly trnsfected QBC939 nd RBE cells were selected using puromycin. The efficiency of knockdown ws detected using western blotting. Western blot nlysis. Seventy-two hours fter co-trnsfection with the lentivirl vectors, cell extrcts were prepred on ice using RIPA lysis buffer plus complete protese inhibitor

3 ONCOLOGY REPORTS 36: , Tble I. Correltion between B7-H4 expression nd the CCA ptient clinicl fetures. B7-H4 expression Clinicl prmeters Cses Positive (%) Negtive (%) χ P-vlue b Gender Men (44.1) 38 (55.9) Women (57.1) 18 (42.9) Age (yers) (46.4) 37 (53.6) > (53.7) 19 (46.3) Tumor loction Intrheptic 18 7 (38.9) 11 (61.1) Extrheptic (51.1) 45 (48.9) Tumor size (cm) (48.4) 47 (51.6) > (52.6) 9 (47.4) Tumor (T) sttus c T (26.1) 17 (73.9) T (48.8) 21 (51.2) T (53.3) 14 (46.7) T (75.0) 4 (25.0) Lymph node (N) metstsis c With (61.9) 16 (38.1) Without (41.2) 40 (58.8) Distnt metstsis (M) With (68.4) 6 (31.6) Without (45.1) 50 (54.9) UICC stge c I (31.9) 32 (68.1) II (64.5) 11 (35.5) III 8 5 (62.5) 3 (37.5) IV (58.3) 10 (41.7) According to the T clssifiction of the 6th UICC-TNM stging. b P-vlue is for t-test (continuous vribles) or Chi-squre or Fisher exct test (ctegoricl vribles). c P<0.05, sttisticl significnce. UICC, Interntionl Union Aginst Cncer; TNM, tumor-node-metstsis. cocktil (both from Beyotime, Chin). Protein quntittion ws mesured by BCA protein ssy kit (Thermo Scientific, Rockford, IL, USA) ccording to the mnufcturer's instructions. Ten microgrms of protein/lne were seprted on 8% crylmide gels by sodium dodecyl sulfte (SDS) gel electrophoresis nd trnsferred to polyvinylidene fluoride (PVDF) membrnes. After blockge of non-specific binding sites using 5% skim milk with Tris-buffered sline with Tween-20 (TBST) t 4 C overnight, the membrnes were incubted for 2 h t 37 C with got polyclonl ffinity purified nti-humn B7-H4 ntibody (b130151; dilution 1:1,000; Abcm, Cmbridge, MA, USA). A GAPDH ntibody ( AP; dilution 1:1,000; Proteintech, Chin) ws used s n internl control. Following primry ntibody incubtion, the membrnes were wshed in TBST nd incubted with horserdish peroxidse (HRP) conjugted pproprite secondry ntibodies (Dko) for 1 h t 37 C. The membrnes were wshed with TBST followed by visuliztion using enhnced chemiluminescence (Millipore, Billeric, MA, USA) ccording to the mnufcturer's protocol. Genertion of CCA-specific CTLs. Peripherl blood mononucler cells (PBMCs) were isolted by density grdient centrifugtion using Histopque-1077 (Sigm-Aldrich, Munich, Germny) from 11 helthy donors. PBMCs were seeded into 6-well culture pltes contining 2 ml RPMI 1640 medium nd 10% FBS t finl concentrtion of 5-10x10 6 cells/well. Following 2 h of incubtion, non dherent cells were removed by gentle wshing with wrm medium. The non-dherent cells (effector lymphocytes) were cryopreserved in FBS supplemented with 10% dimethyl sulfoxide. The resultnt dherent cells contining dendritic cells (DCs) were cultured in medium supplemented with 500 U/ml recombinnt humn grnulocyte-mcrophge colony-stimulting fctor (GM-CSF)

4 422 zho et l: B7-H4 with poor prognosis nd suppressed CD8 + T lymphocytes in humn cca Figure 1. (A) B7-H4 expression in tissue smples. From left to right: negtive stining of B7-H4 in cncerous tissue; positive stining in cncerous tissue; negtive stining in n inflmmtory bile duct smple (epithelil cells); negtive stining in biliry denom smples; nd positive cytoplsmic stining in lymph node metsttic tissue smples. (B) Positive cytoplsmic stining of CD4 + T cells in the tumor strom (blck rrow) nd tumor nest (red rrow). (C) Positive stining of B7-H4 nd low-level stining of CD8 + cells in cncerous tissue. (D) Positive cytoplsmic stining of CD8 + T cells in the tumor strom (blck rrow) nd tumor nest (red rrow). (E) Negtive stining of B7-H4 nd high-level stining of CD8 + cells in cncerous tissue. nd 1,000 U/ml recombinnt humn interleukin 4 (IL-4) (both from PreproTech, Inc., Rocky Hill, NJ, USA) t 37 C in 5% CO 2 (12). Every 2 dys, one-hlf of the medium ws replced with fresh medium contining double concentrtion of GM-CSF nd IL-4 s indicted bove. Following 5 dys in culture, 10 ng/ml of recombinnt humn tumor necrosis fctor-α (TNF-α; PreproTech, Inc.) ws dded to the medium to induce phenotypic nd functionl mturtion of DCs (12). CCA cells were induced to poptotic tumor cells (ATCs) with 100 µg/ml mitomycin for 24 h (Beijing Solrbio Science & Technology Co., Ltd., Beijing, Chin), nd were presented by DCs to induce specific CTLs in vitro. Subsequently, the isolted non-dherent effector lymphocytes were co-cultured with the ATC-pulsed utologous DCs in 6-well plte in the presence of 10 ng/ml recombinnt humn interleukin-7 (IL-7; PreproTech, Inc.). Hlf the medium ws replced with complete medium supplemented with 30 IU/ml recombinnt humn interleukin 2 (IL-2; PreproTech, Inc.) every 3 dys. Following 7 dys in culture, the lymphocytes were re-stimulted with the ATC-pulsed utologous DCs in medium contining 10 ng/ml IL-7 nd 20 U/ml IL-2. On dy 10, following the fourth round of re-stimultion, the cells were hrvested nd tested using CCA-specific CTL ssy (13). CD8 + T-medited CTLs were purified by negtive depletion using CD8 + T cell isoltion kit (Miltenyi Biotec, Bergisch Gldbch, Germny). CTL cytotoxicity ssy. CTL ctivity ws evluted using the CytoTox 96 non-rdioctive cytotoxicity ssy (Promeg, Mdison, WI, USA) bsed on lctte dehydrogense (LDH) relese. After wshing, the trget cells were counted nd seeded into 96-well V-bottomed culture pltes. Vrying numbers of CTLs were dded to finl volume of 100 µl t the effector to trget (E/T) rtios of 2.5:1, 5:1, 10:1 nd 20:1 nd incubted for 4 h t 37 C. The superntnts were hrvested nd the ssy pltes were incubted for 30 min t room temperture, protected from light. The bsorbnce t 490 nm ws recorded within 1 h fter dding the stop solution. The corrected vlues were used in the following formul to compute percent cytotoxicity: Cytotoxicity = [(Experimentl - Effector Spontneous - Trget Spontneous)/(Trget Mximum - Trget Spontneous)] x 100%. Sttisticl nlysis. Dt were nlyzed using SPSS softwre, version 19.0 (IBM SPSS, Armonk, NY, USA). Group comprisons of continuous dt were mde using t-test on independent mens. For ctegoricl dt, Chi-squre nlysis or the Fisher's exct test ws used. The Kpln-Meier estimtor nd Cox nlysis were used for overll survivl nd recurrence-free survivl. P<0.05 ws considered to indicte sttisticlly significnt difference. Results Expression of B7-H4 s detected by IHC nlysis in CCA. Representtive IHC imges of B7-H4 re presented in Fig. 1. The expression of B7-H4 protein ws detected in 54/110 (49.1%) cncerous tissues, 15/28 (53.6%) lymph node metsttic tissues nd 4/19 (21.1%) chronic inflmmtory bile duct tissue smples (Tble II). Notbly, in the inflmmtory bile duct tissues, B7-H4 ws predominntly expressed in the infiltrting mononucler cells rther thn the epithelil cells of the bile duct, which is in line with its role during inflmmtory rections. In ddition, 8 biliry denom smples (Fig. 1) stined negtive for B7-H4. As shown in Tble II, positive stining of B7-H4 ws detected in cncerous nd lymph node metsttic smples, which ws significntly greter compred with the non-tumorous tissues. These dt indicted tht the high expression of B7-H4 ws specific to the CCA tissues.

5 ONCOLOGY REPORTS 36: , Tble II. Differences in B7-H4 expression in CCA nd chronic inflmmtory bile duct smples. B7-H4 expression Smple Cses Positive (%) Negtive (%) P-vlue Cncerous (49.1) 56 (50.9) Control Lymph node metsttic (53.6) 13 (46.4) Chronic inflmmtory bile duct 19 4 (21.1) 15 (78.9) P<0.05, sttisticl significnce, compred with the cncerous smple group. CCA, cholngiocrcinom. Tble III. Univrite nlysis of vrious clinicopthologicl prmeters in reltion to the survivl of ptients with CCA. Overll survivl Disese-free survivl Clinicl prmeters N (%) Medin (months) Log-rnk (P-vlue) Medin (months) Log-rnk (P-vlue) Gender Mle 57 (62.0) Femle 35 (38.0) Age (yers) (63.0) >60 34 (37.0) Tumor size (cm) 5 81 (88.0) 17.1 <0.001 b 17.2 <0.001 b >5 11 (12.0) Tumor (T) sttus T1 11 (12.0) <0.001 b <0.001 b T2 39 (42.4) T3 27 (29.3) T4 15 (16.3) Lymph node (N) metstsis With 37 (40.2) 8.4 <0.001 b b Without 55 (59.8) Distnt metstsis (M) With 17 (18.5) 8.2 <0.001 b 6.1 <0.001 b Without 55 (81.5) UICC stge I 39 (42.4) 23.2 <0.001 b 31.9 <0.001 b II 30 (32.6) III 6 (6.5) IV 17 (18.5) B7-H4 expression Positive 47 (51.1) b b Negtive 45 (48.9) According to the T clssifiction of the 6th UICC-TNM stging. b P<0.05, sttisticl significnce. CCA, cholngiocrcinom; UICC, Interntionl Union Aginst Cncer; TNM, tumor-node-metstsis. Expression of B7-H4 is significntly ssocited with clinicopthologicl fetures including tumor sttus, lymph node metstsis nd Interntionl Union Aginst Cncer (UICC) stge in CCA. As B7-H4 ws highly expressed in the cncer tissues of ptients with CCA, its expression ws investigted s to whether it correltes with clinicopthologicl prmeters in ptients with CCA. The clinicopthologicl fetures of the 110 cses of CCA were grouped by positive or negtive

6 424 zho et l: B7-H4 with poor prognosis nd suppressed CD8 + T lymphocytes in humn cca Tble IV. Correltion between B7-H4 expression nd the densities of TILs in the CCA tissue sections. CD4 + T cells CD8 + T cells In tumor nest In tumor strom In tumor nest In tumor strom B7-H expression Cses Low High P-vlue Low High P-vlue Low High P-vlue Low High P-vlue Positive Negtive P<0.05, sttisticl significnce. TILs, tumor-infiltrting lymphocytes; CCA, cholngiocrcinom. Tble V. Differences in Cox nlysis for overll survivl nd recurrence-free survivl of CCAs fter surgicl resection (n=110). Overll survivl Recurrence-free survivl Fctors HR (95% CI) P-vlue HR (95% CI) P-vlue Expression of B7-H4 (+/-) ( ) ( ) Gender (mle/femle) ( ) ( ) Age, yers ( 60, >60) ( ) ( ) Loction (hilr/distl) ( ) ( ) Histologic grde (G1-2/G3) ( ) ( ) P<0.05, sttisticl significnce. CCAs, cholngiocrcinoms; HR, hzrd rtio; CI, confidence intervl. B7-H4 expression. As shown in Tble I, B7-H4 expression in CCA tissues ws significntly ssocited with tumor sttus (P=0.025), lymph node metstsis (P=0.035) nd UICC stge (P=0.019), however, not with gender, ge, tumor loction nd size. The dt indicte tht the cses of CCA positive for B7-H4 exhibited more extensive metsttic behvior. Expression of B7-H4 indictes poorer prognosis in ptients with CCA. The results indicted tht the expression of B7-H4 correltes with dverse pthologicl fetures, which re ssocited with ptient prognosis. First, the correltion of B7-H4 expression with disese prognosis ws nlyzed. The results indicted tht the expression of B7-H4 is ssocited with poorer outcome following surgery. The medin overll survivl time of ptients negtive for B7-H4 ws 19.5 months, which is longer thn the 12.6 months observed in ptients positive for the expression of B7-H4 (P=0.015; Tble III). In ddition, the medin disese-free survivl time of ptients negtive for B7-H4 expression ws longer thn tht of ptients with positive B7-H4 expression (16.7 vs months; P=0.046; Tble III). Kpln-Meier nlysis indicted tht the expression of B7-H4 is ssocited with reduced survivl (log-rnk P=0.015; Fig. 2A) nd time to recurrence (log-rnk P=0.046; Fig. 2B) in the 110 cses. To further vlidte these observtions, multivrite Cox nlysis ws used, which showed tht B7-H4 expression ws n independent indictor of poorer overll survivl nd erly recurrence [hzrd rtio (HR)=1.786; 95% confidence intervl (CI), ; P=0.017; nd HR=2.062; 95% CI, ; P=0.014; Tble V]. Tken together, the dt suggest tht berrnt expression of B7-H4 my be risk fctor for poorer prognosis in ptients with CCA following surgicl resection. Expression of B7-H4 in tumor cells is inversely ssocited with the density of CD8 + T cells in the tumor strom. Previous studies hve shown tht B7-H4 is ble to directly or indirectly modulte immune infiltrte cells, which re comprised predominntly of CD4 + helper T cells nd CD8 + cytotoxic T cells (8,14-16). The presence of tumor-infiltrting lymphocytes (TILs) within the tumor strom or nest is considered n indictor of the host immune response to the tumor (17). Therefore, whether the expression of B7-H4 correltes with CD4 + nd CD8 + TILs ws investigted in the 110 CCA tissues. As shown in Tble IV, levels of B7-H4 expression in the tumor cells ws inversely correlted with the density of CD8 + T cells in the tumor strom (P=0.0004), however, ws not correlted with the density of CD8 + T cells in the tumor nest (P=0.776). In ddition, there ws no significnt ssocition between B7-H4 expression nd the CD4 + T cells in the tumor strom or nest (P=0.567 nd P=0.822, respectively). Therefore, these dt provide further evidence of the potentil role of B7-H4 in the suppression of cellulr immune surveillnce in ptients with CAA, nd in prticulr tumor infiltrting CD8 + T cells. Knockdown of B7-H4 increses CD8 + T-medited cytotoxicity (CTL) in CCA cell lines. Considering the bove observtions, which indicted negtive correltion between B7-H4 expression nd the density of CD8 + T cells in the tumor strom, the impct of B7-H4 on CD8 + T cells ws further investi-

7 ONCOLOGY REPORTS 36: , Figure 2. Assocition of tumor B7-H4 expression with cncer-specific survivl in 110 ptients with CCA. (A) Overll survivl of ptients in ssocition with B7-H3 protein expression. The medin survivl time of ptients ws 19.5 months (B7-H4-negtive) compred with 12.6 months in B7-H4-positive ptients. (B) Cncer recurrence in ptients by B7-H4 protein expression level. The medin time to recurrence ws 16.7 months in B7-H4-negtive ptients compred with 10.9 months in B7-H4-positive ptients. Figure 3. (A) B7-H4 protein levels in QBC939 nd RBE cells mesured by western blot nlysis. (B nd C) Vlidtion of B7-H4 protein levels following shrna trnsfection by western blotting. (D) The B7-H4 shrna trnsfected cells were exmined by immunofluorescence (green, shrna with fluorescent protein). (E) A CytoTox 96 Non-Rdioctive Cytotoxicity Assy ws used to mesure cell deth in the QBC939 peptide-loded B7-H4-shRNA trnsfected cells compred with the sh-control trnsfected nd untrnsfected cells in co-culture with different rtios of QBC939-specific T cells. (F) A CytoTox 96 Non- Rdioctive Cytotoxicity Assy mesured cell deth in the QBCRBE peptide-loded B7-H4-shRNA-trnsfected cells compred with sh-control-trnsfected nd untrnsfected cells in co-culture with different rtios ofrbe-specific T cells. * P<0.05. gted in vitro. Western blot nlysis showed tht B7-H4 ws expressed in QBC939 nd RBE cells (Fig. 3A). Notbly, the protein expression levels of B7-H4 were significntly greter in QBC939 cells compred with RBE cells (Fig. 3A). Knockdown of B7-H4 ws performed in QBC939 nd RBE cells using B7-H4-shRNA lentivirl trnsfection (Fig. 3B nd C). By culturing with CD8 + cytotoxic T cells, the cytotoxicity of CD8 + T cells ws mrkedly improved by the knockdown of B7-H4 in QBC939 nd RBE cells (Fig. 3E nd F). These dt indicte tht knockdown of B7-H4 in tumors increses CD8 + T cell medited cytotoxicity in vitro. Discussion Epidemiologicl studies hve demonstrted tht the incidence of CCA hs been incresing in recent yers (18). However, ptients with CCA hve poor prognosis, with medin survivl of <24 months, due to lte dignosis nd the limited efficcy of non-surgicl therpies (19). B7-H4, s negtive regultor of T cell responses, hs been observed to be expressed in vriety of humn tumors. Numerous studies tht focus on the clinicl significnce of B7-H4 hve been reported (20-23). However, there re limited studies regrding

8 426 zho et l: B7-H4 with poor prognosis nd suppressed CD8 + T lymphocytes in humn cca the expression of B7-H4 in CCA, nd its functionl relevnce hs not been reported in detil. The present study, to the best of our knowledge, is the first demonstrtion tht the expression of B7-H4 is low in noncncerous nd high in CCA tissues nd lymph node metstses. These results re in ccordnce with previous studies in which the expression of B7-H4 hs been observed in gstric nd lung cncer tissues, with positive rte of 44.9 nd 40.7% (24,25). Furthermore, ovrin, brest nd esophgel squmous crcinom hve demonstrted higher expression of B7-H4 in 93.5, 94.8 nd 95.5% of cses, respectively (2,20,26). In ddition, the present study observed tht positive expression of B7-H4 is ssocited with tumor sttus, lymph node metstsis nd tumor stge in CCA. These dt re in ccordnce with the ssocition between the expression of B7-H4 nd clinicopthologicl fctors ssocited with the prognosis of tumor ptients reported in previous studies (20-23). In contrst to the present study, Tringler et l observed no significnt ssocition between B7-H4 expression nd grde, stge or other clinicopthologicl fetures in brest cncer (2). This discrepncy my be explined by the tumor heterogeneity between CCA nd brest cncer. Therefore, the present study demonstrtes tht B7-H4 expression is ssocited with dvnced CCA, nd indictes more ggressive biologicl potentil. Due to the forementioned results, the present study further nlyzed the ssocition between B7-H4 nd the prognosis of ptients with tumors. The results suggest tht B7-H4 is n independent fctor in the prognosis of ptients with CCA. The presence of T-lymphocytes within the tumor microenvironment is considered n importnt component of the ntitumor immune response nd reflects the process of cncer immunoediting in solid tumors (27). In the present study, the immune responses ginst cncer cells were investigted using IHC of TILs in CCA. TILs in the tumor microenvironment re predominntly CD4 + nd CD8 + T cells, which re considered to be the effector cells in the Th2 nd Th1 ntitumor immune responses, respectively. A subset of CD4 + TILs ws selected to exmine the T-helper popultion, nd subset of CD8 + TILs ws selected to specificlly exmine the cytotoxic T cell popultion (17,28). The CD8 + TILs serve vitl role in the killing of tumor cells. Previous studies hve shown tht incresed B7-H4 expression is involved in shping the tumor microenvironment by modulting the infiltrtion of CD3 + nd CD8 + TILs in brest cncer (14), however, the subtypes of the T lymphocytes were not further nlyzed. The ssocition between B7-H4 nd CD4 +, CD8 + TILs hs not been reported in CCA. The present study demonstrted tht the expression of B7-H4 is inversely correlted with the density of CD8 +, not with CD4 + TILs in the tumor strom. Notbly, CD4 + nd CD8 + TILs exhibit low expression in the tumor nest regrdless of the expression levels of B7-H4. This phenomenon my be due to the immunosuppression of the locl tumor nest. These dt suggest tht B7-H4 my reduce the totl number of infiltrting lymphocytes in tumor strom, in prticulr, CD8 + TILs rther thn CD4 + TILs, by inhibiting their recruitment or survivl in the tumor microenvironment. The present study indicted tht B7-H4 cts s negtive regultor of T cells, by inhibiting the infiltrtion of the CD8 + TILs. However, the functionlity of the immune cells is of greter importnce compred to the frequencies of immune infiltrtes (28,30). Therefore, the impct of cncer ssocited B7-H4 on CCA-specific CD8 + T cytotoxicity ws further explored in vitro. Sic et l (6) reported tht B7-H4 inhibits T cell prolifertion nd cytotoxicity ginst llogeneic ntigens in vitro. Additionlly, it hs been demonstrted in lung cncer tht blockde of B7-H4 using neutrlizing monoclonl ntibodies promotes the poptosis of T cells, nd inhibits CTL-medited cytotoxicity (29). In contrst to inhibiting the function of B7-H4 using B7-H4 Ig, the present study knocked down the expression of B7-H4 using lentivirl vectors encoding shrna. This enbles the direct observtion of the reduction of B7-H4 in tumor cells, nd the screening for stble tumor cell lines which express low levels of B7-H4. Following co-culture with CCA cells trnsfected with lentivirl vectors, CD8 + T cell medited cytotoxicity ws mesured. Following the reduction in the expression of B7-H4, the CD8 + T cell medited cytotoxicity ws incresed. Therefore, the present study suggests tht it my be possible to increse CD8 + T cell-medited cytotoxicity using tretments to reduce or block B7-H4. B7-H4 my contribute to the inhibition of CD8 + T cell medited cytotoxicity, however, the mechnism hs not been investigted in detil. Previous studies hve suggested vrious possible explntions for the suppression of cytotoxicity. B7-H4 hs been reported to interfere with T cell ctivtion, t lest in prt, through signling pthwys downstrem of CD28, including protein kinse B, extrcellulr signl-regulted kinse nd c-jun N-terminl kinse (30). As cell-surfce protein, B7-H4 is extensively N-glycosylted, which ppers to regulte surrounding T cell function (31). Alterntively, B7-H4 my prtilly contribute to the production of cytokines such s TGF-β nd IL-6 (16,32). These cytokines re ble to induce CD8 + CTLs to differentite into non-cytotoxic IL-17 producing cells (33). The results of the present study provide evidence tht B7-H4 my serve n importnt role in shielding tumors from immune surveillnce by reducing the number nd cytotoxic bility of CD8 + TILs. In conclusion, the present study showed tht B7-H4 is overexpressed in CCA nd is ssocited with multiple ggressive tumor fetures nd poor prognosis. The berrnt expression of B7-H4 observed in CCA cells my significntly suppress the number nd cytotoxicity of CD8 + T cells in the tumor microenvironment. However, the precise role of B7-H4 in T cell regultion nd the underlying mechnisms remin to be fully elucidted. Further studies re required to explore the specific role of B7-H4 in CCA. The present study indictes tht B7-H4 my be promising new trget for the dignosis nd tretment of ptients with CCA. Acknowledgements The present study ws supported by the Ntionl Nturl Science Foundtion of Chin (nos nd ). We thnk Spndidos Publictions for providing English Lnguge editing Service. References 1. Lee BS, Ch BH, Prk EC nd Roh J: Risk fctors for perihilr cholngiocrcinom: A hospitl-bsed cse-control study. Liver Int 35: , 2015.

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