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The Journal of Immunology, 2008, 181, 6158 -6169
Copyright © 2008 by The American Association of Immunologists, Inc.
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Cross-Linking of B7-H1 on EBV-Transformed B Cells Induces Apoptosis through Reactive Oxygen Species Production, JNK Signaling Activation, and fasL Expression1

Yeong Seok Kim2,*, Ga Bin Park2,*, Hyun-Kyung Lee{dagger}, Hyunkeun Song*, In-Hak Choi{ddagger}, Wang Jae Lee§ and Dae Young Hur3,*

* Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, Busan, Republic of Korea; {dagger} Department of Internal Medicine, Inje University Busan Paik Hospital, Busan, Republic of Korea; {ddagger} Department of Microbiology, Inje University College of Medicine, Busan, Republic of Korea; and § Department of Anatomy and Cancer Immunology, Seoul National University College of Medicine, Seoul, Republic of Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
B7-H1 is a newly identified member of the B7 family with important regulatory functions in cell-mediated immune responses, and it is expressed in human immune cells and several tumors. We first observed that expression of surface B7-H1 on B cells was increased during the immortalization process by EBV, which is strongly related to both inflammation and tumorigenesis. Cross-linking of B7-H1 on EBV-transformed B cells using anti-B7-H1 Ab (clone 130002) induced reactive oxygen species (ROS) generation, mitochondrial disruption, release of apoptotic proteins from mitochondria, and subsequent apoptosis. Inhibition of caspases and ROS generation recovered B7-H1-mediated apoptosis and proteolytic activities of caspase-8, -9, and -3. We observed that B7-H1 stimulation induced both transcription and translation of fasL. ZB4, an antagonistic anti-fas Ab, and NOK-1, an antagonistic anti-fasL Ab, effectively blocked apoptosis without exerting any influence on ROS generation. N-acetylcysteine (NAC) completely blocked the induction of fasL mRNA and protein. We found that B7-H1 stimulation activated the phosphorylation of JNK and c-jun and down-regulated ERK1/2 and p-Akt. NAC blocked the activation of JNK and down-regulation of ERK, but both z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) and ZB4 did not inhibit JNK activation of B7-H1 stimulation. SP600125 blocked fasL induction and apoptosis but did not affect ROS generation after B7-H1 stimulation. Taken together, we concluded that B7-H1-mediated apoptosis on EBV-transformed B cells may be involved in the induction of fasL, which is evoked by ROS generation and JNK activation after cross-linking of B7-H1. These results provide a new concept for understanding reverse signaling through B7-H1 and another mechanism of tumor immunotherapy using anti-B7-H1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
B7-H1 (CD274, PD-L1) is a newly identified member of the B7 family with important regulatory functions in cell-mediated immune responses (1, 2). Although B7-H1 mRNA is widely distributed in normal tissues, expression of B7-H1 protein is reported in monocytes, dendritic cells, and macrophage-derived cells in the liver, lung, and tonsil (3). It has also been reported that the B7-H1 protein is expressed in placental syncytiotrophoblasts, extravillous cytotrophoblasts, and some epithelial cells (4, 5). Interestingly, the expression of B7-H1 in human CD19+ B cells is very low although B cells are activated with BCR ligation, but B7-H1 is abundantly expressed in murine T and B cells regardless of activation (6). Additionally, B7-H1 expression in monocytes, dendritic cells, and keratinocytes can be up-regulated after treatment with inflammatory cytokines such as IFN-{gamma} (1, 2, 7). B7-H1 interacts with PD-1 (programmed death-1),4 a member of the CD28 family found on activated T and B cells (2, 8). B7-H1 has dual functions of costimulation of naive T cells and inhibition of activated T cells. B7-H1-mediated signals are able to costimulate early T cell priming and differentiation (1, 9, 10). PD-1 on CTLs also induces anergy or apoptosis through interaction with B7-H1 (2, 3, 10). Due to an immunoregulatory role of B7-H1, several studies have attempted to modulate inflammatory diseases using neutralizing anti-B7-H1 Abs (9, 11, 12, 13). On the other hand, immunotherapy using selective blocking of B7-H1 or PD-1 is likely to be pertinent in tumor models as well as inflammation models because many human cancers significantly express B7-H1. B7-H1 is found to be highly expressed in tumors of the colon, breast, ovary, lung, brain, stomach, kidney, head, and neck (3, 14, 15, 16, 17). Tumor-associated B7-H1 has been found to promote apoptosis of tumor-specific CTLs, which results in tumor invasion and escape from immune surveillance (18). Great attention has been paid to the possible use of B7-H1 in antitumor immunotherapy, and several studies have reported that blockade of B7-H1 and PD-1 by mAbs enhances tumor regression and potentiates cancer therapeutic immunity (14, 19, 20, 21). However, most studies have focused on using PD-1 signals to induce inhibition of T cells, not to reverse signals to cells expressing B7-H1. B7-H1 has an intracellular domain consisting of one potential site for protein kinase phosphorylation and it shares 16% of its identity with B7-2. Thus, it is possible that B7-H1 can transmit signals just like B7-2 (22). Ligation of B7-H1 using mAb induces activation of naive T cells and apoptosis of activated T cells (11). Agonistic Ab against B7-DC, another ligand for PD-1, activates dendritic cells through a reverse signaling (23). These results support the possibility of downstream signals of B7-H1 as well as B7-DC, but this has not been extensively discussed.

EBV is a virus of the herpesvirus family and is the most common virus in humans. Most people are infected with EBV at young adolescent age, and EBV persistently infects in human B lymphocytes. EBV is also associated with pathogenesis of lymphoid and some epithelial tumors (24, 25). Thus, EBV is significantly related to both inflammation and tumors. In this study, we first observed that EBV-transformed B cells increased expression of B7-H1 on their surface. It is uncertain whether B7-H1 is involved in cellular responses and intracellular signaling of EBV-transformed B cells. A recent study reported that anti-B7-H1 Abs partly restore the proliferation of infiltrating CD4+CD25– T cells on non-Hodgkin lymphomas (26) and that a main source of B7-H1 is activated CD4+CD25+ regulatory T cells. A more recent study showed that blockade of the PD-1–PD-L pathway restores IFN-{gamma} production of infiltrating T cells in Hodgkin lymphomas, but it did not report on reverse signaling through PD-L (27). Thus, we investigated the association between B7-H1 on EBV-transformed B cells and cell responses. We observed whether cross-linking of B7-H1 on EBV-transformed B cells using anti-B7-H1 mAb would induce apoptosis and investigated some targets such as reactive oxygen species (ROS), mitochondrial membrane potential ({Delta}{psi}), and apoptotic molecules relevant to apoptosis after stimulation of B7-H1. The primary aim of this study was to better understand reverse signaling of B7-H1 and to provide a new mechanism of B7-H1 immunotherapy for tumors or EBV infections.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Preparation of EBV-infectious culture supernatant

EBV infection was induced using the cell-free supernatant. EBV stock supernatant was prepared from an EBV-transformed B95-8 marmoset cell line (a gift from Dr. B. G. Han, National Genome Research Institute, National Institute of Health, Seoul, Korea). Cells were grown in RPMI 1640 medium (HyClone) supplemented with streptomycin, glutamine, and 10% FBS (HyClone) at 37°C in 5% CO2. The infectious culture supernatant was harvested, centrifuged (1000 rpm for 10 min), and filtered using a 0.2-µm pore-sized filter (Corning) to remove cell debris as previously described (28). The supernatant was then used for determining dose titer and stored for the following experiments.

Generation of EBV-transformed B cells

To establish EBV-transformed B cells, PBMCs were obtained from the blood of six healthy human volunteers by Ficoll-Paque (Amersham Biosciences) gradient centrifugation. B cells were purified from PBMCs using a MACS B cell-negative depletion kit (Miltenyi Biotec). Purified cells were added to EBV stock supernatant in a culture flask, and after 2 h of incubation at 37°C, RPMI 1640 culture medium (HyClone) and 1 µg/ml of cyclosporine A (Sigma-Aldrich) were added (1 x 106 cells/ml). The cultures were incubated for 2–4 wk and observed every day until clumps of EBV-infected B cells were visible and the medium turned yellow (35). The phenotype was monitored by a FACSCalibur flow cytometry (BD Biosciences) and a confocal laser scanning microscopy (Carl Zeiss) using mouse anti-human anti-B7-H1 Ab (R&D Systems) and PE-conjugated anti-CD19 Ab (BD Pharmingen). This study was approved by the Institutional Bioethics Review Board at the Medical College of Inje University, and all donors gave informed consent for the study.

Cell culture and cross-linking of B7-H1 on EBV-transformed B cells

EBV-transformed cells (1 x 106 cells/ml) were harvested in a 1.5-ml tube and washed twice in cold PBS. Cells were resuspended in 100 µl of PBS and incubated with anti-B7-H1 mAb (clone 130002, 2 µg/ml, R&D Systems) or isotype control, MOPC21 (2 µg/ml, Sigma-Aldrich), at 37°C for 40 min. Cells were washed in PBS and resuspended in 100 µl of PBS and then incubated with goat anti-mouse IgG (2 µg/ml) for 15 min at 37°C. After cells were washed, they were further cultured in RPMI 1640 medium for 16 h at 37°C. To determine optimal conditions, experiments were performed at variable concentrations (1, 2, 5, and 10 µg/ml) and variable durations (10 min, and 2, 4, 8, and 16 h). To investigate the effects of caspase inhibitors, cells were pretreated with z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, 20 µM in DMSO, a broad-spectrum caspase inhibitor), z-DEVE-fmk (N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone, 20 µM in DMSO, a caspase-3 inhibitor), and z-IETD-fmk (N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone, 20 µM in DMSO, a caspase-8 inhibitor) for 2 h before B7-H1 stimulation. Control cells were incubated with DMSO for 2 h. To inhibit generation of ROS, cells were pretreated with N-acetylcysteine (NAC, 10 mM, an antioxidant; Sigma-Aldrich) for 1 h. To block fas-fasL interaction, antagonistic anti-fas Ab ZB4 (0.5 µg/ml, Abcam) or antagonistic anti-fasL Ab NOK-1 (10 µg/ml, Santa Cruz Biotechnology) was added 1 h before B7-H1 stimulation. To block the JNK cascade, SP600125 (25 µg/ml, Calbiochem) was pretreated for 1 h before B7-H1 stimulation. All chemicals and Abs except NOK-1 and SP600125 were removed from cell cultures before B7-H1 stimulation.

B7-H1 stimulation by PD-1 Ig fusion protein

Ninety-six-well plates were incubated with PD-1 Ig fusion protein (R&D Systems, IgG, 0.5, 1, and 2 µg/ml) or control IgG (R&D Systems, 2 µg/ml) for 18 h at 4°C. EBV-transformed B cells were cultured in the PD-1 Ig or control IgG-coated wells, and cells were harvested after 16 h. The number of annexin V-positive apoptotic cells, disrupted {Delta}{psi}, and generated ROS level were investigated using a FACSCalibur flow cytometer.

Annexin V/propidium iodide (PI) staining to assess apoptosis/necrosis

To investigate apoptosis after B7-H1 stimulation, cells were harvested and washed twice with PBS and resuspended in 100 µl of 1x binding buffer (10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2). Then, 2 µl of FITC-conjugated annexin V (BD Pharmingen) was added and cells were incubated at room temperature for 15 min in the dark with gentle vortexing. The cells were then washed twice with HEPES buffer and PI was added (5 µg/ml). Finally, cells were washed and 400 ml of 1x binding buffer was added to each tube and cells were analyzed using a FACSCalibur flow cytometer.

Detection of sub-G1 peak

To detect the sub-G1 peak, cellular DNA was stained with PI and quantified by flow cytometry. Cells were harvested after treatment, washed twice with PBS (2% FBS), fixed with 70% cold aqueous ethanol, and stored at 4°C for at least 24 h. Cell pellets were stained with PI staining solution containing RNase A (10 µg/ml) and PI (10 µg/ml) in PBS. The cell suspension was then incubated in the dark at room temperature for 30 min, and the DNA content was measured using a FACSCalibur flow cytometer.

Measurement of {Delta}{psi} and ROS levels

To determine whether B7-H1-induced apoptosis would be related to {Delta}{psi} disruption and ROS formation, we measured {Delta}{psi} and ROS levels of EBV-transformed B cells after cross-linking B7-H1. The ROS level was detected using 2',7'-dichlorofluorescin diacetate (DCFH-DA), a fluorescence probe, which could be converted to highly fluorescent dichlorofluorescein (DCF) in the presence of intracellular ROS. EBV-transformed B cells (1 x 106 cells/well) were pretreated with 10 µM DCFH-DA (Molecular Probes) to measure ROS levels. Cells were then stimulated with anti-B7-H1 Ab or MOPC. To measure {Delta}{psi}, cells were collected and further incubated in PBS containing 20 µM 3,3'-dihexyloxacarbocyanine iodide (DiOC6, Molecular Probes) for 15 min. Cells were harvested, and then ROS levels and {Delta}{psi} were determined using a FACSCalibur flow cytometer.

Confocal microscopy to detect cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (endoG)

To detect intracellular molecules, cells were permeabilized with permeabilization buffer (0.1% saponin in PBS). Cells were incubated with primary Ab against cytochrome c (mouse IgG2b), AIF (mouse IgG2b), or endoG (goat polyclonal IgG) (Santa Cruz Biotechnology), washed three times with PBS, and incubated with FITC-conjugated goat anti-mouse IgG or FITC-conjugated rabbit anti-goat IgG for 30 min. The nucleus was stained with PI (BD Pharmingen) in PI binding buffer (RNase A 10 µg/ml) at room temperature for 10 min. After being washed three times with PBS, cells were mounted in a Dako fluorescent mounting medium. Fluorescence-stained cells were examined by confocal laser scanning microscopy (Carl Zeiss) at x400 original magnification, and images were acquired using confocal microscopy software release 3.0 (Carl Zeiss, 510 META).

RT-PCR

Total RNA was isolated using an RNeasy Mini kit (Qiagen). RNA was transcribed into cDNA using oligo(dT) primers (Bioneer) and reverse transcriptase. PCR amplification was performed using specific primer sets (Bioneer) for FasL (upstream primer, 5'-GGT CCA TGC CTC TGG AAT GG; downstream primer, 5'-CAC ATC TGC CCA GTA GTG CA, 250-bp product). For the control group, a specific primer set for β-actin (upstream primer, 5'-ATC CAC GAA ACT ACC TTC AA; downstream primer, 5'-ATC CAC ACG GAG TAC TTG C) was used, which yielded a 200-bp product. PCR (25 cycles; 20 s at 94°C, 10 s at 60°C, and 30 s at 72°C) was performed using AccuPower PCR PreMix (Bioneer). PCR products were analyzed by agarose gel electrophoresis and visualized with ethidium bromide under UV light using the multiple GelDOC system (Fujifilm). Data were analyzed using ImageJ 1.38 software (National Institutes of Health, Bethesda, MD).

Quantitative real-time RT-PCR

Quantitative real-time RT-PCR was performed using a single tube SYBR Green kit (Takara), iCycler thermal real-time PCR system (Bio-Rad), and specific primer sets (Bioneer) for Bcl-2 (upstream primer, 5'-GGA TTG TGG CCT TCT TTG AG; downstream primer, 5'-CAG CCA GGA GAA ATC AAA CAG, 209-bp product), Bax (upstream primer, 5'-CCA AGA AGC TGA GCG AGT GT; downstream primer, 5'-CAG CCC ATG ATG GTT CTG AT, 250-bp product), Bad (upstream primer, 5'-CGA GTG AGC AGG AAG ACT CC; downstream primer, 5'-CTG TGC TGC CCA GAG GTT, 299-bp product), and FasL and β-actin (same primers used in conventional RT-PCR). Only experiments were used where a distinct single peak was observed with a melting temperature different than that of the no template control. The relative gene expression compared with the calibrator (unstimulated cells) was determined by the Mx3000P software’s built-in algorithm using an adaptive baseline to determine the Ct. Experiments were performed in triplicate, and data were expressed as means ± SD and analyzed using Student’s t test.

Western blot analysis (immunoblot)

After stimulation, EBV-transformed B cells were pelleted and lysed in RIPA buffer (Elpis Biotech). The proteins (10 µg/sample) were immediately heated for 5 min at 100°C. Total cell lysates (5 x 106 cells/sample) were subjected to SDS-PAGE on gel containing 15% (w/v) acrylamide under reducing conditions. Separated proteins were transferred to nitrocellulose membranes, and then the membranes were blocked with 5% skim milk and commercial Western blot analysis was performed. Chemiluminescence was detected using an ECL kit (Amersham Life Science) and the multiple GelDOC system. The following primary Abs were used: caspase-8, caspase-3, caspase-9, β-actin, phospho-ERK1/2, ERK1/2, phospho-Akt (Ser473), Akt, phospho-JNK (Thr183/Tyr185) and JNK from Cell Signaling Technology. Additionally, phospho-c-jun and c-jun from Santa Cruz Biotechnology, poly(ADP-ribose) polymerase (PARP) from Calbiochem, and anti-phosphotyrosine from Upstate Biotechnology were used. Data were analyzed using ImageJ 1.38 software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
EBV infection induced B7-H1 expression on B cells

To evaluate the B7-H1 (CD274) expression on B cells during the immortalization process induced by EBV infection, EBV-infected B cells were harvested every week until EBV transformation was completed. Cell phenotype was then monitored by flow cytometry using FITC-conjugated anti-B7-H1 and PE-conjugated anti-CD19 Abs. B7-H1 molecules were not expressed on the surface of CD19+ resting B cells but were resident in the cytoplasm of CD19+ resting B cells (Fig. 1, top panels). The other lymphocytes (lymphocyte gating) with the exception of CD19+ cells did not express B7-H1 proteins either on the surface or in the cytoplasm (Fig. 1, top panels). B7-H1 was induced on the surface of EBV-infected B cells until 1 wk after EBV infection (Fig. 1, middle, left panel). Interestingly, intracellular B7-H1 slightly increased in the other CD19– lymphocytes after 1 wk (Fig. 1, middle, right panel). At 4 wk when the transformation process was completed and most surviving cells (EBV-transformed B cells) were CD19+ cells, all of the EBV-transformed B cells highly expressed B7-H1 molecules on the surface and in the cytoplasm (Fig. 1, bottom panels).


Figure 1
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  		FIGURE 1. Distribution of B7-H1 protein during B cell transformation by EBV. PBMCs purified from human blood were immortalized with a medium containing EBV particles. Double staining with FITC-conjugated anti-B7-H1, PE-conjugated anti-CD19 Abs, and FITC or PE-conjugated isotype Abs were performed and analyzed by flow cytometry as described in Materials and Methods. Each scattergram was acquired using lymphocyte gating. Scattergrams on the left represent surface expression and those on the right represent intracellular expression. Results are representative of five independent experiments.

 
Cross-linking of B7-H1 induces apoptosis on EBV-transformed B cells

We assessed cell responses of EBV-transformed B cells after stimulation of B7-H1, which was abundant on the surface of the cells. We first assessed cell proliferation using AlamaBlue (Serotec) after stimulation of B7-H1 on EBV-transformed B cells. When cross-linking B7-H1 on EBV-transformed B cells using both anti-B7-H1 Ab and secondary link Ab, we observed the inhibition of cell proliferation (data not shown). Next, we examined whether the inhibition of cell proliferation would be associated with the induction of apoptosis. EBV-transformed B cells were incubated with anti-B7-H1 Ab/MOPC (isotype control) and secondary Ab, and further incubated for 24 h. Control and stimulated cells were stained with FITC-labeled annexin V and PI and then analyzed by flow cytometry. Cross-linking of B7-H1 induced the apoptosis of EBV-transformed B cells in a dose-dependent (Fig. 2A) and time-dependent manner (Fig. 2B, top). The most effective concentration of anti-B7-H1 Ab was 2 µg/ml because high concentrations (5 and 10 µg/ml) seemed to induce necrosis (annexin V–/PI+). Annexin V-positive cells were slightly increased until 4 h after B7-H1 stimulation, and two-thirds of cells were annexin V-positive at 16 h (63.46%, Fig. 2B, top). After cell cycle analysis was performed using PI dye, we found that B7-H1 stimulation significantly induced sub-G1 arrest (from 6.00% to 41.32%, Fig. 2C).


Figure 2
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  		FIGURE 2. Apoptosis, ROS production, and disruption of {Delta}{psi} in EBV-transformed B cells after B7-H1 stimulation. A, EBV-transformed cells (4 wk) were incubated with anti-B7-H1 mAb (1, 2, 5, and 10 µg/ml) or MOPC21 (10 µg/ml) at 37°C for 40 min. Cells were washed and incubated with goat anti-mouse IgG (2 µg/ml) at 37°C for 15 min. Cells were harvested and further incubated in RPMI 1640 medium. Twenty-four hours later, cells were stained with FITC-conjugated annexin V and PI as described in Materials and Methods and analyzed by flow cytometry. The indicated number represents the cell proportion in each quadrant. Data are representative of the three independent experiments. B, EBV-transformed cells (4 wk) were incubated with anti-B7-H1 mAb (2 µg/ml) or MOPC21 (2 µg/ml) at 37°C for 40 min. Cells were washed and incubated with goat anti-mouse IgG (2 µg/ml) at 37°C for 15 min. Cells were harvested and further incubated in RPMI 1640 medium for 10 min or 2, 4, 8, or 16 h. Double staining for FITC-tagged annexin V and PI was performed to assess apoptosis. To measure ROS generation, cells were preincubated with DCFH-DA before being treated with anti-B7-H1 or MOPC Ab. Diminished DiOC6 fluorescence indicates {Delta}{psi} disruption. The indicated percentage is the cell proportion in each bar. The number in DCF histograms indicates the MFI. Results are representative of four independent experiments. C, EBV-transformed cells (4 wk) were incubated with anti-B7-H1 mAb (2 µg/ml, 40 min) or MOPC21 (2 µg/ml, 40 min) and goat anti-mouse IgG (2 µg/ml, 15 min). Cells were harvested and further incubated in RPMI 1640 medium for 16 h. To evaluate the peak level of sub-G1, cells were fixed with ethanol and stained with PI as described in Materials and Methods, and then analyzed by flow cytometry. The indicated percentage represents the fraction of sub-G1 cells relative to the total number of cells. Results are representative of three independent experiments. D, EBV-transformed cells (4 wk) were incubated in PD-1-Ig (0.5, 1, and 2 µg/ml) or control Ig (2 µg/ml)-coated wells at 37°C for 16 h. Cells were harvested and the numbers of annexin V-positive cells, ROS level (DCF), and {Delta}{psi} (DiOC6) were obtained as described in Materials and Methods. The indicated percentage is the cell proportion in each bar. The number in DCF histograms indicates the MFI. The thin-line histogram represents the control Ig-treated group. Results are representative of four independent experiments.

 
We next examined whether B7-H1-mediated apoptosis would be related to the disruption of {Delta}{psi} and ROS production following stimulation. ROS is thought to participate in a wide variety of cellular functions, including intracellular signaling, transcription activation, cell proliferation, and apoptosis (29, 30). Cells were preincubated with DCFH-DA to detect intracellular ROS, and DiOC6 staining was performed to investigate {Delta}{psi}, which is related to apoptosis and intracellular ROS generation. When we examined cells immediately after B7-H1 stimulation, a significant ROS generation was detected within 10 min, but both {Delta}{psi} disruption and apoptosis were barely detectable until 2 h after stimulation. Four hours after B7-H1 stimulation, both disruption of {Delta}{psi} and apoptosis dramatically increased (Fig. 2B, middle and bottom). When EBV-transformed B cells were stimulated (2 µg/ml, 16 h) with PD-1 as a physiologic ligand of B7-H1, PD-1-Ig fusion protein recapitulated apoptosis, mitochondrial disruption, and ROS generation induced by anti-B7-H1 Ab (Fig. 2D).

ROS and caspase participate in B7-H1-mediated apoptosis

In apoptosis, ROS is related to protease cascade, such as caspases, and lastly it is involved directly or indirectly in the mitochondria (31, 32, 33). Therefore, we next examined whether B7-H1-mediated apoptosis would be related to ROS and caspases following {Delta}{psi} disruption, because the caspase released from the mitochondria is an important mediator of apoptosis. EBV-transformed B cells were preincubated with z-VAD-fmk, a pan-caspase inhibitor, or NAC, an ROS inhibitor, before B7-H1 stimulation. As depicted in Fig. 3A, z-VAD-fmk pretreatment blocked B7-H1-mediated apoptosis (44.10% to 23.67%) and (blocked) {Delta}{psi} disruption (65.64% to 0.28%), but it did not alter ROS generation. NAC successfully blocked B7-H1-mediated apoptosis (44.10% to 19.12%) and {Delta}{psi} disruption (65.64% to 2.48%) as well as ROS generation (mean fluorescence intensity (MFI) 1695.58 to 11.77). We more specifically examined whether B7-H1-mediated apoptosis would be related to initiator caspase-8 and executor caspase-3. EBV-transformed B cells were preincubated with z-IETD-fmk, a caspase-8 inhibitor, or z-DEVD-fmk, a caspase-3 inhibitor, for 2 h before B7-H1 stimulation. As shown in Fig. 3B, both z-IETD-fmk and z-DEVD-fmk pretreatments effectively blocked B7-H1-mediated apoptosis (50.34% to 15.45%, 14.70%) and {Delta}{psi} disruption (53.52% to 2.73%, 0.34%), but they had no influence on ROS production. Taken together, these data indicate that caspases are deeply related to apoptosis but not ROS generation after B7-H1 stimulation. We next investigated proteolytic activities of caspases by immunoblot. In untreated and treated cells with isotype-controlled Abs, caspase-8 was present as a 57-kDa proenzyme (see Fig. 6A, first panel, columns 1 and 2), caspase-9 was present as a 47-kDa proenzyme (see Fig. 6A, second panel, columns 1 and 2), caspase-3 appeared as a 35-kDa proenzyme (see Fig. 6A, third panel, columns 1 and 2), and PARP as a substrate of caspase-3 was also present as a precursor protein (see Fig. 6A, fourth panel, columns 1 and 2). These bands represented full-length precursors. EBV-transformed B cells stimulated with anti-B7-H1 Ab displayed initial activation of caspase-8 with the appearance of a large cleavage fragment of 41–43 kDa, the cleavage of caspase-9 into 37-kDa fragments, the cleavage of caspase-3 into 17/19-kDa fragments, active products and the cleavage of PARP (see Fig. 6A, column 3). As depicted in column 4 of Fig. 6A, z-VAD-fmk, a broad-spectrum caspase inhibitor, completely inhibited activation of caspases-8, -9, and -3, as well as degradation of PARP after B7-H1 stimulation. NAC, an ROS inhibitor, also completely inhibited activation of caspases and degradation of PARP (see Fig. 6A, column 5).


Figure 3
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  		FIGURE 3. Effects of caspase and ROS inhibitions on B7-H1-mediated apoptosis of EBV-transformed B cells. EBV-transformed B cells were preincubated with (A) z-VAD-fmk (20 µM, 2 h), NAC (10 mM, 1 h), (B) z-IETD-fmk (20 µM, 2 h), or z-DEVD-fmk (20 µM, 2 h). Cells were then washed with PBS and incubated with anti-B7-H1 mAb (2 µg/ml, 40 min) or MOPC21 (2 µg/ml, 40 min) and goat anti-mouse IgG (2 µg/ml, 15 min). Cells were harvested and further incubated in RPMI 1640 medium. Sixteen hours later, the number of annexin V-positive cells, ROS level (DCF), and {Delta}{psi} (DiOC6) were obtained as described in Materials and Methods. The thin-line histogram represents the isotype control (MOPC). The indicated percentage is the cell proportion in each bar. The number in the DCF histograms indicates the MFI. Results are representative of four independent experiments.

 

Figure 6
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  		FIGURE 6. Caspase activities and phosphorylations of signaling proteins of EBV-transformed B cells after B7-H1 stimulation. EBV-transformed B cells were pretreated with z-VAD-fmk (20 µM, 2 h), antagonistic anti-fas Ab ZB4 (0.5 µg/ml, 1 h), or NAC (10 mM, 1 h). Cells were washed and incubated with anti-B7-H1 mAb (2 µg/ml, 40 min) or MOPC21 (2 µg/ml, 40 min) and goat anti-mouse IgG (2 µg/ml, 15 min). Cells were further incubated in RPMI 1640 medium for 16 h and then total cell lysates were collected. Immunoblot was performed as described in Materials and Methods to investigate (A) proteolytic activities of caspase-8, -9, or -3 and (B) JNK and c-jun as substrates of JNK, and ERK1/2 and Akt as substrates of ERK1/2, bid, or Bcl-xL. β-actin served as an internal control. Number under box indicates ratio between densities of marked band and β-actin. Results are representative of four independent experiments.

 
Cross-linking of B7-H1 induces fas ligand expression through ROS production

Because caspase-8, an initiator caspase linked with fas, was involved in B7-H1-mediated apoptosis, we examined surface fas (CD95) and fasL (CD178) expression after B7-H1 stimulation by flow cytometric analysis. We found that fasL was scarcely expressed on unstimulated EBV-transformed B cells although B7-H1 stimulation clearly induced surface expression of fasL (MFI of 9.05–78.92; Fig. 4A). In contrast to fasL, fas molecules were constitutively expressed and the MFI was increased (MFI of 273.38–696.59) after stimulation of B7-H1 (Fig. 4A). To confirm the induction of fasL on EBV-transformed B cells after stimulation of B7-H1, quantitative real-time RT-PCR for fasL was performed (Fig. 4C). After B7-H1 stimulation, fasL mRNA was increased (1.21 ± 0.366 to 18.913 ± 5.177, p = 0.03095) and NAC pretreatment blocked an increase in fasL mRNA (18.913 ± 5.177 to 1.385 ± 0.113, p = 0.02745). z-VAD-fmk pretreatment slightly blocked an increase in fasL transcription, but the change was not statistically significant (18.913 ± 5.177 to 12.523 ± 3.738, p = 0.05938).


Figure 4
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  		FIGURE 4. Involvement of fas-fasL interaction in B7-H1-mediated apoptosis. Antagonistic anti-fas Ab ZB4 (0.5 µg/ml, 1 h), antagonistic anti-fasL Ab NOK-1 (10 µg/ml, 1 h), z-VAD-fmk (20 µM, 2 h), or NAC (10 mM, 1 h) was added before B7-H1 stimulation. Cells other than those in the NOK-1 group were washed and incubated with anti-B7-H1 mAb (2 µg/ml, 40 min) or MOPC21 (2 µg/ml, 40 min) and goat anti-mouse IgG (2 µg/ml, 15 min). Cells were harvested and further incubated in RPMI 1640 medium for 16 h. A, Surface fas and fasL expression levels were observed by flow cytometry using PE-conjugated anti-fas and fasL Ab. The thin-line histogram represents the isotype control, and each isotype control is identical because the isotype of Abs used in experiments is identical (IgG1). The number indicates the MFI. Results are representative of three experiments. B, The numbers of annexin V-positive cells, ROS level (DCF), {Delta}{psi} (DiOC6), and surface fasL were obtained as described in Materials and Methods. The thin-line histogram represents the isotype control (MOPC). The indicated percentage is the cell proportion of each bar. The number in DCF and fasL histograms indicates the MFI. Results are representative of five independent experiments. C, Total RNA was extracted from cell lysates, cDNA was synthesized, and quantitative real-time RT-PCR for fasL, bax, bad, bcl-2, and β-actin mRNA was performed as described in Materials and Methods. Experiments were performed in triplicate, and data were analyzed using Student’s t test (*, p < 0.05) and are expressed as mean ± SD.

 
Apoptosis of EBV-transformed B cells after B7-H1 stimulation is dependent on fas-fasL interaction

It is feasible that induced fasL after B7-H1 stimulation can trigger apoptosis of adjacent cells in a fas/fasL mechanism. To check whether constitutive fas on cell surface would interact with fasL that was induced after B7-H1 stimulation, cells were pretreated with ZB4, an antagonistic anti-fas Ab, and NOK-1, an antagonistic anti-fasL Ab. NOK-1 was not washed out and further incubated in immobilized anti-B7-H1 Ab or MOPC. ROS is also able to activate the expression of fas ligand (34) or to directly disrupt the {Delta}{psi} without fas-fasL interaction (29, 30). Hence, we performed flow cytometric analysis using FITC-labeled annexin V to estimate apoptosis, DCFH-DA to determine ROS level, DiOC6 to measure {Delta}{psi}, and FITC-labeled anti-fasL Ab to check changes in fasL expression. As shown in Fig. 4B, NAC pretreatment completely blocked the induction of surface fasL (MFI of 50.48 to 11.53), apoptosis (55.19% to 8.59%), and {Delta}{psi} disruption (46.59% to 1.37%) as well as ROS production (MFI of 1366.97 to 22.35). Both ZB4 and NOK-1 effectively blocked B7-H1-mediated apoptosis and {Delta}{psi} disruption (Fig. 4B, columns 4 and 5). Moreover, ZB4 pretreatment completely blocked proteolytic activities of caspases-8, -9, and -3 after B7-H1 stimulation (see Fig. 6A, column 6). However, both ZB4 and NOK-1 did not block ROS generation and induction of surface fasL after B7-H1 stimulation (Fig. 4B, columns 4 and 5). Additionally, quantitative real-time RT-PCR for fasL showed that ZB4 pretreatment did not significantly block increase in fasL transcription after B7-H1 stimulation (18.913 ± 5.177 to 15.099 ± 3.549, p = 0.06318, Fig. 4C, top).

Cytochrome c, AIF, or endoG is released into the cytoplasm after stimulation of B7-H1 on EBV-transformed B cells

When the permeabilization of the outer mitochondrial membrane is evoked, proapoptotic proteins, such as cytochrome c, endoG, and AIF, are released from the mitochondria (35). Cytochrome c activates effector caspases to degrade DNA. EndoG or AIF is translocated into the nucleus and then is involved in apoptosis (36). Therefore, we needed to examine the translocation of several proapoptotic proteins in mitochondria. To investigate the cytosolic distribution of proapoptotic proteins released from the mitochondria, we performed confocal microscopy using anti-cytochrome c, anti-AIF, and anti-endoG and fluorescence-conjugated secondary Abs. When stimulated with MOPC, EBV-transformed B cells showed localization of cytochrome c, AIF, and endoG within small mitochondrion-like granules as untreated controls (Fig. 5, columns 1 and 2). In contrast to untreated and isotype controls, stimulation with anti-B7-H1 Ab caused the translocation of cytochrome c, AIF, and endoG from the mitochondria to the cytosol (Fig. 5, column 3). AIF and endoG were even translocated into the nucleus (Fig. 5, middle and bottom panels, column 3). Consistent with previous results, z-VAD-fmk, NAC, and ZB4 almost completely blocked their release from the mitochondria (Fig. 5, columns 4 and 5).


Figure 5
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  		FIGURE 5. Subcellular distribution of cytochrome c, AIF, and endoG in EBV-transformed B cells after B7-H1 stimulation. EBV-transformed B cells were pretreated with z-VAD-fmk (20 µM, 2 h), antagonistic anti-fas Ab ZB4 (0.5 µg/ml, 1 h), or NAC (10 µM, 1 h). Cells were washed and incubated with anti-B7-H1 mAb (2 µg/ml, 40 min) or MOPC21 (2 µg/ml, 40 min) and goat anti-mouse IgG (2 µg/ml, 15 min). Cells were harvested and further incubated in RPMI 1640 medium for 16 h. Cells were permeabilized with 0.1% saponin in PBS. Intracellular staining was performed using anti-cytochrome c (mouse IgG2b), AIF (mouse IgG2b), or endoG (goat polyclonal IgG) Ab, and FITC-conjugated goat anti-mouse IgG or FITC-conjugated rabbit anti-goat IgG. The nucleus was stained with PI. Cells were observed under a confocal microscope (x400 magnification). The procedure is described in detail in Materials and Methods. Green fluorescence indicates cytochrome c, AIF, or endoG, respectively, and red fluorescence indicates nucleus.

 
Bax, bad, bid, bcl-2, Bcl-xL, JNK, and ERK were involved in B7-H1-mediated apoptosis of EBV-transformed B cells

To elucidate the mechanism of fasL induction, mitochondrial membrane disruption, and subsequent apoptosis after B7-H1 stimulation, we investigated some candidates for signaling molecules by quantitative real-time RT-PCR and immunoblot. Although bax, bad, and bcl-2 mRNA were constitutively transcribed in EBV-transformed B cells, cross-linking of B7-H1 significantly increased the transcription of bax (fold induction of 4.722 ± 0.507) and bad (fold induction of 6.975 ± 0.483), which are known to be proapoptotic genes (Fig. 4C, middle panels). In contrast to bax and bad, the transcription of bcl-2, an antiapoptotic gene, was weakly decreased after B7-H1 stimulation but it was statistically significant (1.269 ± 0.177 to 0.053 ± 0.011, p = 0.00615, Fig. 4C, bottom panel). Consistent with previous results, NAC, z-VAD-fmk, and ZB4 almost completely blocked the altered transcription of bad and bax after B7-H1 stimulation. Decreased bcl-2 transcription was also recovered by pretreatment with z-VAD-fmk (p = 0.02239), NAC (p = 0.01875), and ZB4 (p = 0.01249). Immunoblot for bid, a member of the proapoptotic bcl-2 family, showed that B7-H1 stimulation induced bid truncation, which was restored by pretreatment of z-VAD-fmk, NAC, and ZB4 (Fig. 6B, Bid). Bcl-2, another prosurvival protein, was slightly down-regulated after B7-H1 stimulation, and z-VAD-fmk, NAC and ZB4 sufficiently blocked down-regulation of bcl-2 (Fig. 6B, Bcl-xL). Because these apoptosis-associated genes were regulated by kinase activities in upstream cell signaling, including the JNK and ERK pathways, we studied phosphorylation of kinase by B7-H1 stimulation using immunoblot. Cross-linking of B7-H1 up-regulated the phosphorylated form of JNK and its major substrate c-jun (Fig. 6B, p-JNK and p-c-jun), whereas the phosphorylated forms of ERK1/2 and Akt were decreased (Fig. 6B, p-ERK1/2 and p-Akt). NAC almost completely blocked phophorylation of JNK, ERK, and Akt via B7-H1 stimulation (Fig. 6B, column 5). However, both z-VAD-fmk and ZB4 inhibited phosphorylation of ERK and Akt by B7-H1 stimulation on EBV-transformed B cells, but they did not restore the phosphorylated form of JNK and its major substrate c-jun (Fig. 6B, columns 4 and 6).

Cross-linking of B7-H1 induces fasL expression through ROS generation and the JNK pathway

Cross-linking of B7-H1 quickly evoked ROS generation on EBV-transformed B cells 10 min later (Fig. 2B). As depicted in Figs. 6B and 4, abolishment of ROS generation by NAC was able to block both up-regulation of JNK and induction of fasL expression after B7-H1 stimulation. To determine whether the JNK pathway was involved in fasL expression, SP600125, a JNK inhibitor, was administered before B7-H1 stimulation. RT-PCR for fasL revealed that fasL transcription had gradually increased since 30 min after B7-H1 stimulation and significantly increased at 60 min (Fig. 7A, right). Both SP600125 and NAC completely abrogated increase of fasL transcription by cross-linking of B7-H1 within 2 h (Fig. 7A, left and middle). To investigate the effect of SP600125 on B7-H1-mediated apoptosis, ROS generation, and fasL expression, flow cytometric analysis was performed. SP600125 as well as NAC effectively blocked apoptosis (65.29% to 12.30%) and fasL expression (MFI of 27.44 to 4.45) after B7-H1 stimulation (Fig. 7B). However, SP600125 rarely affected ROS generation by B7-H1 (Fig. 7B, middle).


Figure 7
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  		FIGURE 7. Involvement of JNK in expression of fasL of EBV-transformed B cells after B7-H1 stimulation. EBV-transformed B cells were pretreated with SP600125 (25 µM, 1 h) or NAC (10 mM, 1 h). Cells were washed and incubated with anti-B7-H1 mAb (2 µg/ml, 40 min) or MOPC21 (2 µg/ml, 40 min) and goat anti-mouse IgG (2 µg/ml, 15 min). A, Cells were further incubated in the absence or presence of SP600125 (25 µg/ml) for 0, 5, 10, 30, 60, or 120 min. Cells were harvested and RT-PCR for fasL and β-actin was performed as described in Materials and Methods. For normalization, β-actin was amplified. The density of bands was obtained by ImageJ software, and the number under the bands represents the relative ratio of fasL/β-actin. Results are representative of three independent experiments. B, Cells were further incubated in the absence or presence of SP600125 (25 µg/ml) for 16 h. The number of annexin V-positive cells, the ROS level (DCF), and surface fasL were obtained as described in Materials and Methods. A thin-line histogram represents the isotype control (MOPC). The indicated percentage is the cell proportion in each bar. The number in DCF and fasL histograms indicates the MFI. Results are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
B7-H1 has been shown to be transcribed in most normal cells as well as immune cells, but the expression of B7-H1 protein is confirmed only in some phagocyte-like cells such as monocytes, macrophages, and Kupffer cells (1, 2, 3). In accord with previous reports, we found that B7-H1 protein was not expressed on the surface of resting B cells from healthy human blood (Fig. 1). However, intracellular staining analyzed by flow cytometry revealed that B7-H1 protein was distributed in the cytosol of resting B cells. During the EBV transformation process of EBV-infected B cells, such as tumorigenesis, cytosolic B7-H1 was promptly moved onto the surface of B cells. Expression of B7-H1 protein is also found in several tumors (3, 14, 15, 16, 17). We investigated whether tumor B cell lines such as Ramos, Raji, and IM9 could express B7-H1 protein (supplemental Fig. 1A).5 Interestingly, Ramos is EBV-negative, and Raji and IM9 are EBV-positive. Expression of B7-H1 on Ramos was much less than Raji and IM9. However, it has not yet been determined how cytosolic B7-H1 could be translocated onto the cell surface, although we considered that EBV genes or proteins such as EBV-encoded nuclear Ags, latent membrane proteins, and Epstein-Barr encoded early RNA transcripts might be involved in the process (31). Because the EBV transformation process is similar to the activation process of B cells induced by peptide Ag, we presumed that some activation process triggered both induction and translocation of B7-H1. This presumption was supported by some experiments. We investigated the induction of B7-H1 expression in resting B cells purified from human blood following treatment with sCD40L, anti-IgM, LPS, and ionomycin (supplemental Fig. 1B). Interestingly, another polyclonal activator, PMA, rarely induced B7-H1 expression, although it increased activation markers (e.g., CD38, data not shown). Additionally, it is possible that cytokines such as IFN-{gamma}, which is related to B7-H1 transcription, may also be involved in the process (1, 2, 7). However, further studies are required to answer this question. When B7-H1 on EBV-transformed B cells was cross-linked using anti-B7-H1 mAb and secondary bridging Ab, we investigated the inhibition of cell proliferation (data not shown) and the induction of apoptosis in dose- and time-dependent manners (Fig. 2). To exclude the effect of the Fc receptor, Fc receptor-blocking agents (Miltenyi Biotec) were applied, which revealed the same results, regardless of the absence or presence of Fc receptor-blocking agents (data not shown). As depicted in Fig. 2B, B7-H1-mediated apoptosis was accompanied by not only the generation of ROS but also the disruption of {Delta}{psi}. NAC, an ROS inhibitor, and z-VAD-fmk, a broad caspase inhibitor, were able to inhibit apoptosis after B7-H1 stimulation, and NAC effectively restored {Delta}{psi} disruption as well as ROS generation, but z-VAD-fmk did not block ROS generation after B7-H1 stimulation. Additionally, ROS generation was detected immediately after B7-H1 stimulation (Fig. 2B, 10 min), but {Delta}{psi} disruption was observed much later (Fig. 2B, 4 h). The results support the conclusion that ROS generation after B7-H1 stimulation is independent of caspase activity although {Delta}{psi} disruption is deeply associated with caspases and that ROS generation might precede {Delta}{psi} disruption.

To determine the direct relationship between mitochondrial events and (this) apoptosis, we investigated whether proapoptotic molecules that were released from the mitochondria and caspases were directly involved in apoptosis after B7-H1 stimulation. We showed that proapoptotic molecules, such as cytochrome c, AIF, and endoG, were released from the mitochondria into the cytosol after stimulation of B7-H1 (35, 36). In this study, both z-VAD-fmk and NAC effectively blocked the release of proapoptotic molecules (Fig. 5). Additionally, z-DEVD-fmk, an executor caspase-3 inhibitor, restored B7-H1-mediated apoptosis (Fig. 3B), and cleavage of procaspase-9, procaspase-3, and PARP were found after B7-H1 stimulation (Fig. 6A). Thus, we concluded that B7-H1-mediated apoptosis resulted from activation of caspases, although we did not have enough clues to explain direct relationships between cytochrome c and activation of caspases. Translocation into the nuclei of both AIF and endoG, known as executors of caspase-independent apoptosis (36), was investigated (Fig. 5), but we presumed that involvement of AIF and endoG was minimal because the efficiency of z-DEVD-fmk to block apoptosis was almost equal to those of NAC, z-VAD-fmk, and z-IETD-fmk (Fig. 5). z-IETD-fmk, an initiator caspase-8 inhibitor, also restored B7-H1-mediated apoptosis as well as z-DEVD-fmk and z-VAD-fmk. This result suggested that activation of other caspases and {Delta}{psi} disruption might be triggered by fas, a death receptor linked to caspase-8 (37). As expected, cross-linking of B7-H1 increased transcription and translation of fasL on EBV-transformed B cells (Fig. 4). After bid, which is known to link caspase-8 and bax (37, 38), was truncated to activate bax and bad (Fig. 6B), compensatory transcription of bax and bad was significantly increased after B7-H1 stimulation (Fig. 4C). These results provided some evidence that fas-fasL interaction was essential to B7-H1-mediated apoptosis. Both antagonistic anti-fas ZB4 and antagonistic anti-fasL NOK-1 blocked apoptosis and {Delta}{psi} disruption but did not influence ROS generation or fasL expression after B7-H1 stimulation (Fig. 4B). z-VAD-fmk also did not sufficiently block B7-H1-mediated transcription of fasL (Fig. 4C). Interestingly, NAC completely inhibited both surface expression and transcription of fasL as well as apoptosis after cross-linking of B7-H1 (Fig. 4, B and C). The results strongly suggested that induction of fasL after B7-H1 stimulation was related to ROS generation.

We next evaluated whether JNK, ERK, and Akt were involved in B7-H1-mediated apoptosis. The activation of JNK as well as p38 MAPK is generally associated with promotion of apoptosis (39). Extrinsic stimuli, such as stress, NO, cisplantin, and TRAIL, activate the JNK pathway, which results in apoptosis (39, 40). Caspase-3 can also amplify activation of JNK, and the activation of JNK can promote the up-regulation of fasL expression. On the other hand, ERK and Akt have generally been considered a survival signaling pathway, although some evidence exists that the ERK pathway mediates apoptosis (38). ERK and Akt inhibit apoptosis through phosphorylation of bad, which has proapoptotic functions. ERK activates B-Raf to inhibit the activation of caspases, and Akt is able to inhibit the release of cytochrome c from the mitochondria. In this study, we investigated whether B7-H1 stimulation induced the up-regulation of JNK and the down-regulation of ERK and Akt (Fig. 6B). NAC completely blocked the modification of JNK, ERK, and Akt after B7-H1 stimulation. However, z-VAD-fmk and ZB4 could recover the down-regulation of ERK and Akt, but they could not inhibit the activation of JNK. The results suggested that ERK and Akt were involved in the activity of caspases following fas-fasL interaction and that ROS was closely connected with JNK as well as ERK and Akt. Recent evidence indicated that the accumulated ROS causes sustained JNK activation and leads to apoptosis (41). Furthermore, it was reported that berberine induces apoptosis via the induction of fasL expression through the JNK/p38 MAPK pathway, which is induced by intracellular ROS (34). Thus, we investigated whether fasL expression was connected with ROS or JNK. As shown in Fig. 4, B and C, ROS inhibition by NAC blocked the up-regulation of fasL expression after B7-H1 stimulation. SP600125, a JNK inhibitor, also reversed both transcription and expression of fasL, and it efficiently restored apoptosis after B7-H1 stimulation without interference of ROS generation (Fig. 7). Cross-linking of B7-H1 evoked generation of ROS on EBV-transformed B cells within 10 min (Fig. 2B), but we detected a significant transcription of fasL after 60 min (Fig. 7A). Thus, we concluded that ROS generation by B7-H1 stimulation subsequently induced the activation of JNK, which promoted the expression of fasL, resulting in apoptosis of adjacent fas-positive cells. However, the molecular connection between JNK and fasL in our experiments was still unclear. It has recently been reported that rituximab (chimeric anti-CD20 Ab) sensitizes the non-Hodgkin lymphoma cell line to fas-induced apoptosis in association with modulation of p38 MAP kinase and NF-{kappa}B (42). Further molecular studies should be conducted to elucidate how EBV-transformed B cells would be sensitized to fas-induced apoptosis following cross-linking of B7-H1.

EBV-transformed cells constitutively expressed fas on their surface (Fig. 4A). FasL induced by B7-H1 stimulation could interact with fas on adjacent EBV-transformed B cells that were subjected to apoptosis. Fas, a death receptor, is strongly expressed on human normal cells such as T cells. T cells also express PD-1, a natural ligand of B7-H1 (1, 2). Thus, it is possible that fasL on EBV-transformed B cells induced by B7-H1/PD-1 interaction can give rise to apoptosis of adjacent T cells. This hypothesis provides another possibility that EBV-infected cells and EBV-related lymphomas can escape from CTLs (fas+) through fasL induced by B7-H1 stimulation when in contact with CTLs. Actually, when PD-1-overexpressed murine cells were cocultured with EBV-transformed B cells, we found that apoptosis of both cells occurred, although there were some limitations in the human-mouse model (data not shown). However, we could not rule out the possibility that B7-H1 directly induced PD-1-positive cells via PD-1 signaling. Further studies are necessary to elucidate which EBV-transformed B cells and CTLs cause apoptosis or which PD-1/B7-H1 mechanism and fas/fasL mechanism are dominant when they are in contact with each other. It may be dependent on different ratios of cells and different conditions. On the other hand, it was very interesting that B7-H1 stimulation using anti-B7-H1 Ab instead of PD-1 induced fasL expression. Hirano et al. reported that blockade of B7-H1 and PD-1 by mAbs potentiates cancer-therapeutic immunity (19). They reported that B7-H1-expressing tumor affects PD-1-expressing CTLs, but they did not investigate the effects of anti-B7-H1 Ab on the tumor cell itself. In this study, we showed that cross-linking of B7-H1 using anti-B7-H1 Ab induced fasL expression on EBV-transformed B cells. We conclude that the administration of anti-B7-H1 Ab may not only abrogate the interaction between B7-H1 on EBV-infected cells and PD-1 on T cells, but also may induce the apoptosis of EBV-infected cells through reverse signaling of B7-H1. Thus, our results provide a novel explanation for immunotherapy using anti-B7-H1 Ab on EBV-infected cells or EBV-related lymphomas.


   Acknowledgments
 
We thank Dr. Jae Seung Kang (Department of Anatomy and Cancer Immunology, Seoul National University College of Medicine) for critically reviewing the revised manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Science Research Center/Engineering Research Center (SRC/ERC) program of the Ministry of Science and Technology (MOST)/Korea Science and Engineering Foundation (KOSEF) (Grant R11-2005-017-02002-0), the Medical Reserve Corp (MRC) program of KOSEF funded by the Korean government (MOST, Grant R13-2007-023-00000-0), the Korea Research Foundation Grant funded by the Korean government (Ministry of Education and Human Resources Development (MOEHRD), KRF-2005-042-E00055), and Inje University Research Grant. Back

2 Y.S.K. and G.B.P. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Dae Young Hur, Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, 633-165 Kaekum-2-dong, Jin-gu, Busan 614-735, Republic of Korea. E-mail address: dyhur{at}inje.ac.kr Back

4 Abbreviations used in this paper: PD-1, programmed death-1; AIF, apoptosis-inducing factor; DCF, dichlorofluorescein; DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate; {Delta}{psi}, mitochondrial membrane potential; DiOC6, 3,3'-dihexyloxacarbocyanine iodide; endoG, endonuclease G; MFI, mean fluorescence intensity; NAC, N-acetylcysteine; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; ROS, reactive oxygen species; z-DEVD-fmk, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone; z-IETD-fmk, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone; z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Back

5 The online version of this article contains supplemental material. Back

Received for publication March 31, 2008. Accepted for publication September 2, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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