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Keywords:

  • nasal natural killer/T cell lymphoma;
  • Epstein–Barr virus;
  • CD70;
  • CD27;
  • Epstein–Barr virus latent membrane protein 1

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Nasal natural killer (NK)/T cell lymphoma (NNKTL) is associated with Epstein–Barr virus (EBV). The present study analysed gene expression patterns of the NNKTL cell lines SNK6, SNK1 and SNT8, which are positive for EBV and latent membrane protein (LMP)-1, using a complementary DNA array analysis. We found that CD70 was specifically expressed in SNK6 and SNT8. Reverse transcription polymerase chain reaction and flow cytometric analyses confirmed that CD70 was expressed in all 3 NNKTL cell lines, but not in the other EBV-positive NK-cell lines. In vitro studies showed that NNKTL cell lines proliferated, in a dose-dependent fashion, in response to exogenous soluble CD27, which is the ligand for CD70. In NNKTL patients, we confirmed that the CD70 was expressed on the lymphoma cells in NNKTL tissues and that soluble CD27 was present in sera at higher levels as compared to healthy individuals. Finally, complement-dependent cytotoxicity assay showed that anti-CD70 antibody mediated effective complement-dependent killing of NNKTL cells and the affected target CD70 expression on the cells. These results suggest that CD70 acts as a functional receptor binding to soluble CD27, resulting in lymphoma progression and that immunotherapy using anti-CD70 antibody may be a potential candidate for treatment for NNKTL.

Nasal natural killer (NK)/T cell lymphoma (NNKTL), has distinct epidemiological, clinical, histological and aetiological features. NNKTL is clinically characterized progressive necrotic lesions in the nasal cavity and a poor prognosis caused by rapid progression (Harabuchi et al, 1996; Jaffe et al, 1996). Original cells of NNKTL are reported to be NK- or γ∂ T cell lineages, both of which express the NK-cell marker, CD56 (Harabuchi et al, 1996; Nagata et al, 2001). Regarding aetiological factors, since we first indicated the presence of Epstein–Barr virus (EBV) DNA, EBV-oncogenic proteins and clonotypic EBV genome in NNKTL, EBV is thought to play a role in lymphomagenesis (Harabuchi et al, 1990, 1996; Minarovits et al, 1994).

The biological characteristics of NNKTL have become gradually clearer following the establishment of the EBV-positive cell lines ‘SNK6’ and ‘SNT8’ from primary lesions (Nagata et al, 2001). We previously showed that NNKTL cells produce several cytokines and chemokines such as γ-interferon (IFNγ), interleukin (IL) 9, IL10 and IFNγ-inducible protein (IP)10, which play roles in the proliferation and invasion of the cells in an autocrine manner (Nagato et al, 2005; Takahara et al, 2006; Moriai et al, 2009). Furthermore, we have recently shown that environmental monocytes attracted by IP10 enhance proliferation of the NNKTL cells in a cell-to-cell contact manner (Ishii et al, 2012).

Histological characteristics of NNKTL include angiocentric and polymorphous lymphoreticular infiltrates, which are called polymorphic reticulosis (Harabuchi et al, 1996; Harris et al, 2000). The infiltrating cells in the NNKTL tissue contain many types of cells, including tumour cells as well as inflammatory cells, such as granulocytes, monocytes, macrophages, lymphocytes and plasma cells. Such inflammatory cell infiltration is likely to be caused by chemotactic effects of a wide variety of chemokines including IL8, Mig and IP10, which were reported to be produced by NNKTL cells (Ohshima et al, 2004; Moriai et al, 2009). The inflammatory cells, and the chemokines and cytokines that they produce, are likely to influence proliferation, survival and migration of the lymphoma cells.

In order to determine which genes are expressed specifically in NNKTL, the present study compared the gene expression profiles of NNKTL cell lines to those from the other cell lines using complementary DNA (cDNA). We found that CD70 was strongly expressed in NNKTL cell lines (SNK6 and SNT8) as compared to non-NNKTL cells (NK92) and peripheral blood mononuclear cells (PBMC) from healthy individuals.

CD70 is a member of the tumour necrosis factor (TNF) superfamily (Bowman et al, 1994; Hintzen et al, 1995). CD70 expression is normally restricted in normal cells to a small subset (10%) of activated B-cells, activated T cells and dendritic cells (Hintzen et al, 1994; Tesselaar et al, 2003). The only known receptor of CD70, CD27, is expressed on the surface of memory B-cells (Klein et al, 1998), most T cells (de Jong et al, 1991) and NK-cells (Sugita et al, 1992). Ligation of CD70 to its receptor CD27 induces a signal transduction pathway, resulting in activation and proliferation of B-cells and T cells (Garcia et al, 2004; Dang et al, 2011). With regard to haematopoietic malignancies, CD70 expression has been reported in 50% of B-cell chronic lymphocytic leukaemia, 33% of follicular lymphoma and 71% of diffuse large cell lymphoma (Davi et al, 1998; Lens et al, 1999), some cases of T cell lymphoma (Zambello et al, 2000) and a case of chronic active EBV infection-associated T cell lymphoma (Shaffer et al, 2011a). However, the functional role of CD70 on haematopoietic malignancies is not yet fully understood.

CD27 is known to be cleaved from in activated B-cells or in T cells after triggering of the T cell receptor (TCR)/CD3 complex (Hintzen et al, 1991; Bohnhorst et al, 2002), resulting in the formation of a soluble form of CD27 in serum (Loenen et al, 1992). Elevated serum levels of soluble CD27 have been reported in several autoimmune diseases (Font et al, 1996; Bohnhorst et al, 2002) and B-cell malignancies (van Oers et al, 1993). However, whether soluble CD27 binding to CD70 has any biological role in such diseases remains to be determined.

As described above, given that CD70 is more highly expressed in normal tissues and is widespread in various malignancies, CD70 has been known to be an attractive target for immunotherapies. Investigations to exploit CD70 as a cancer target have led to the identification of potential antibody-based clinical candidates. Both unconjugated antibodies and antibody-drug conjugates targeting CD70 have been tested in animal models of human cancers (Israel et al, 2005; McEarchern et al, 2007; Grewal, 2008). However, there has been no report regarding anti-CD70 antibody therapy for NNKTL.

In the present study, we found that CD70 was specifically expressed in NNKL cell lines and that it played a role in cell growth by binding to soluble CD27. Moreover, we confirmed that the lymphoma cells expressed CD70 in the NNKTL tissues and that soluble CD27 was present at higher levels in sera. Finally, we showed in vitro that the anti-CD70 antibody could mediate effective complement-dependent killing of NNKTL cells and the effects target CD70 expressed on the cells. These data suggest that CD70 may play a role in lymphoma proliferation by binding to soluble CD27 and that immunotherapy using anti-CD70 antibody may be a potential candidate for the treatment of NNKTL.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Patients

Twenty-one Japanese patients with NNKTL, 16 male and five female, 35–55 years of age, were analysed in this study. Of 21 patients, all were subjects for immunohistological study in lymphoma tissues and eight were subjects for measurement of soluble CD27 in sera taken at the time of diagnosis. The diagnosis was carried out between 2003 and 2011 according to the World Health Organization classification of haematological malignancies (Harris et al, 2000). All patients signed informed consent forms for this study, which was approved by the Institutional Review Board.

Cell lines

The features of the cell lines used in this study are listed in Table 1. SNK1, SNK6, and SNT8 were EBV-positive cell lines established from primary lesions of patients with NNKTL (Nagata et al, 2001) and were kindly provided by Dr. Shimizu (Tokyo Medical and Dental University, Tokyo, Japan). KAI3 originated from a patient with severe chronic active EBV infection (Tsuge et al, 1999). YT, which originated from a patient with acute lymphoblastic lymphoma (ALL; Yodoi et al, 1985), was kindly provided by Dr. Eva Klein (Karolinska Institutet, Stockholm, Sweden). NK92 was established from patients with NK-cell leukaemia (Gong et al, 1994). Raji was an EBV-positive cell line established from Burkitt lymphoma (Epstein et al, 1966). Jurkat human T cell lymphoma cell line, established from the peripheral blood (Schneider et al, 1977), and MOLT4 T cell ALL cell line (Minowada et al, 1972) were purchased from the American Type Culture Collection (Manassas, VA, USA). PEER was a human lymphoid cell line from of T-leukaemia (Ravid et al, 1980). KAI3 and PEER were purchased from the Health Science Research Resources Bank (Osaka, Japan).

Table 1. Cell lines used in this study
Cell lineDiseasePhenotypeEBVLMP-1Reference
  1. EBV, Epstein-Barr Virus; NK, natural killer. [Correction added on 17 December 2012, after first online publication: This table has been corrected by adding the first column ‘Cell lines’.]

SNK1Nasal NK/T-cell lymphomaNK++Nagata et al (2001)
SNK6Nasal NK/T-cell lymphomaNK++Nagata et al (2001)
SNT8Nasal NK/T-cell lymphomadgT++Nagata et al (2001)
KAI3Severe chronic active EB virus infection hypersensitivity to mosquito biteNK++ Tsuge et al (1999)
YTAcute lymphoblastic lymphomaNK++Yodoi et al (1985)
NK92Non-Hodgkin's lymphoma with large granular lymphocytesNKGong et al (1994)
JurkatAcute lymphoblastic lymphomaTSchneider et al (1977)
MOLT4Acute lymphoblastic lymphomaTMinowada et al (1972)
PEERAcute lymphoblastic lymphomaTRavid et al (1980)
RajiBurkitt lymphoma (CD70 positive control)B++Epstein et al (1966)

Cell culture

SNK1, SNK6 and SNT8 cells were cultured in RPMI 1640 medium (Life Technologies Inc., Gaithersburg, MD, USA) supplemented with 10% heat-inactivated human serum and 700 U/ml recombinant human IL2 (Takeda Pharmaceutical Company Limited, Osaka, Japan). KAI3 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml recombinant human IL2. NK92 cells were cultured in MEM (Life Technologies Inc.) supplemented with 12·5% horse serum, 12·5% FBS, 50 U/ml penicillin, 50 mg/ml streptomycin and 200 U/ml recombinant human IL2. YT, Jurkat, MOLT4, PEER and Raji were cultured in RPMI 1640 medium supplemented with 10% FBS and 50 U/ml penicillin, 50 mg/ml streptomycin. All cell lines were incubated at 37°C in an atmosphere containing 5% CO2. PBMC from healthy volunteers were isolated by centrifugation using Ficoll-Hypaque (Amersham Pharmacia Biotech, Buckinghamshire, UK).

cDNA array analysis

The procedure was performed as described previously (Nagato et al, 2005). Total RNA was extracted from SNK6, SNT8 and NK92 cells and from peripheral mononuclear cells of healthy volunteers. 32P-labelled cDNA probes were synthesized using the Atlas Pure Total RNA Labeling System (Clontech, Palo Alto, CA, USA). Total RNA from the PBMC of two healthy volunteers was mixed and used as a pooled sample. These probes were hybridized to the ATLAS Human Cancer 1.2 Array (Clontech) according to the manufacturer's protocols. The arrays were then exposed to a phosphorimaging screen at room temperature for 30 min and scanned using a BAS2000 phosphorimager (Fuji Photo Film, Tokyo, Japan). A grid was applied to the images of the hybridization spots, and the spot intensities were quantified using BAStation version 1.31 (Fuji Photo Film). Background signals were defined as the average of the hybridization signals produced by negative controls on the array. All hybridization signals were normalized by the mean of the internal control signals.

Reverse transcription (RT)-PCR analysis

Expressions of CD70, CD27 and Epstein–Barr virus-encoded small RNA (EBER)-1 were analysed by RT-PCR method as described previously (Nagato et al, 2005). Total RNA was extracted from all cell lines and from PBMC of healthy volunteers using the SV Total RNA Isolation System (Promega, Madison, WI, USA). The RNA was reverse-transcribed for 60 min at 37°C using Moloney murine leukaemia virus reverse transcriptase (GeneHunter, Nashville, TN, USA) with oligo (dT) primers (Applied Biosystems, Foster City, CA, USA) according to the manufacturers' protocols. The following primers were used for CD70 (sense, 5′-AATCACACAGGACCTCAGCAGGACC-3′; antisense, 5′-AGCAGATGGCCAGCGTCACC-3′), CD27 (sense, 5′-GCCAGGAACATTCCTCGTGA-3′; antisense, 5′-TTACAGTGCCGACAGCTCTCA-3′), EBER-1 (sense, 5′-AGGACCTACGCTGCCCTAGA-3′; antisense, 5′- AAAACATGCGGACCACCAGC-3′), β2-microglobulin (B2M; sense, 5′-TGTCTTTCAGCAAGGACTGG-3′; antisense, 5′-CCAGATTAACCACAACCATG-3′; Sigma Genosis Japan, Hokkaido, Japan). Hot-start PCR was performed in a 10 μl reaction mixture containing 4·95 μl H2O, 1 μl 10× PCR buffer (containing 15 mmol/l MgCl2), 1 μl of 2 mmol/l dNTP mixture, 1 μl of 5 μmol/l sense primer, 1 μl of 5 μmol/l antisense primer, 0·05 μl of 5 U/ml AmpliTaq Gold DNA polymerase (Applied Biosystems), and 1 μl of 100 ng/μl cDNA. The reaction was carried out as follows: initial denaturation at 94°C for 10 min followed by 30 (B2M), 32 (CD70 and CD27), or 35 (EBER-1) cycles of 1 min at 65°C (CD70), 60°C(CD27), 55°C (EBER-1) or 57°C (B2M), and 1 min at 72°C, followed by a final elongation step of 5 min at 72°C. The PCR products were separated by electrophoresis on 1% agarose gels and visualized by ethidium bromide staining.

Western blot analysis

Expression of latent membrane protein (LMP)-1 was analysed by western blotting method as described previously (Takahara et al, 2006). One million tumour cells were washed in phosphate buffered saline (PBS) and lysed in NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA, USA). The cell lysate was subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in a 4% to 12% NuPAGE bis-Tris SDS-PAGE gel (Invitrogen) under reducing condition and then transferred to an Immobilon-P (Millipore, Bedford, MA, USA) membrane. The membrane was then blocked in PBS containing 0·01% Tween 20 and 5% nonfat dry milk for 1 h at room temperature and incubated with anti-EBV LMP1 mouse monoclonal antibody CS1-4 (1:3000; Dako, Glostrup, Denmark) or anti-β actin mouse monoclonal antibody AC-15 (1:10 000; Sigma, St. Louis, MO, USA) overnight at 4°C. After washing, the membrane was incubated with horseradish peroxidase (HRP)-labelled sheep anti-mouse IgG and subjected to an enhanced chemiluminescence (ECL) assay using the ECL detection system (Amersham Pharmacia Biotech). Beta-actin (ACTB) was used as a control to ensure that the same amount of protein was loaded in each well.

Flow cytometric analysis

Cell surface expressions of CD70 and CD27 were analysed by flow cytometry as described previously (Nagato et al, 2005). Briefly, cells were washed in cold PBS, centrifuged, and resuspended in an appropriate volume of fluorescence-activated cell sorting staining buffer (PBS containing 0·1% NaN3 and 2% FBS). Cells were incubated with antibodies for 60 min in the dark at 4°C, and excess antibodies were removed by washing the cells twice in cold staining buffer. Detection of surface CD70 was carried out using phycoerythrin (PE)-conjugated mouse IgG3κ (10 μl; isotype control; BD Pharmingen, Franklin Lakes, NJ, USA) and PE-conjugated mouse anti-human CD70 (10 μl; BD Pharmingen). Detection of surface CD27 was carried out using fluorescein isothiocyanate (FITC)-conjugated mouse IgG1κ (10 μl; isotype control; BD Pharmingen) and FITC-conjugated mouse anti-human CD27 (10 μl; BD Pharmingen). Fluorescence-activated cell sorting (FACS) scanning and data analysis were carried out using the Becton Dickinson FACScan and accompanying CellQuest software (San Jose, CA, USA) according to the manufacturer's protocols.

Cell proliferation assay using MTS solution

The procedure was performed as described previously (Nagato et al, 2005; Moriai et al, 2009). Cells (1 × 105 cells per well) in 96-well plate were cultured in 200 μl RPMI 1640 medium containing 10% FBS, without IL2 for 1 d. Each well was treated with recombinant CD27 (0. 01, 0·1, 0·5, 1 μg/ml; Abnova, Taipei, Taiwan) for 48 h. For neutralizing assay, each well with recombinant CD27 (0·5 μg/ml) was cultured together with anti-human CD27 neutralizing antibody (0·1, 1, 10 μg/ml; R&D Systems, Inc. Minneapolis, MN, USA) for 48 h. To determine the number of viable cells, we used the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) -2-(4-sulphophenyl)-2H-tetrazolium solution (MTS; 20 μl) was added to each well, and then incubated for 2 or 1 h at 37°C under 5% CO2. The absorbance at 490 nm was measured using a microplate plate reader. Results were expressed as 100% of the average of untreated controls. Measurements were determined in duplicate and experiments were repeated at least three times. The results corresponded to mean ± standard deviation (SD).

Complement-dependent cytotoxicity assay

The procedure was performed as described previously (Israel et al, 2005). Cells (1 × 104 cells per well) in 96-well plate were cultured with mouse anti-human CD70 antibody (BD Pharmingen) at a final concentration of 10 μg/ml and active rabbit complement (Cedarlane, ON, Canada) or inactive rabbit complement at a final concentration of 5% rabbit serum in a final volume of 100 μl. Mouse IgG3 was used as an isotype Ig control. Heat-inactivated complement controls were generated by heating an aliquot to 55°C for 30 min and then returning the sample to the ice. After 3 h, the number of viable cells was determined in an aliquot from each well, using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega). MTS (20 μl) was added to each well, and then incubated for 1 h at 37°C under 5% CO2. The absorbance at 490 nm was measured using a microplate reader. Results were expressed as 100% of the average of untreated controls. Measurements were determined in duplicate and experiments were repeated at least three times. The results correspond to mean ± SD.

Enzyme-linked immunosorbent assay (ELISA)

Measurement of soluble CD27 in sera was performed using PeliKine compact human soluble CD27 ELISA kit (Sanquin, Amsterdam, the Netherlands). All sera were from diagnostic samples and frozen at −80°C. The polystyrene microtitre wells coated with monoclonal anti-CD27 antibody were blocked with blocking buffer (PBS containing 1% bovine serum albumin) for 1 h at room temperature to eliminate nonspecific binding by the primary antibody. After the plates were washed with PBS-Tween (PBS containing 0·05% Tween 20), appropriate diluted sera (100 μl) were incubated in the well plates for 1 h at room temperature. Soluble CD27 standards (100, 50, 25, 12·5, 6·25, 3·12, and 1·56 U/ml) were also incubated to generate the standard curve. After incubation, the plates were washed with PBS-Tween. Subsequently, a biotinylated second monoclonal CD27 antibody (100 μl) was added at a 1:100 dilution. The plates were then washed again, and 1:1000 diluted HRP-conjugated streptavidin was added for 30 min. After washing again, 100 μl substrate solution was added, and the plates were incubated for 20 min in the dark. The reaction was terminated by adding 50 μl of stop solution. The optical density of each well was determined at 450 nm using a microplate reader. For each assay, the soluble CD27 concentration of the sample was calculated using the standard curve.

Immunohistological staining

Two-colour immunostaining for CD70 and CD56 in lymphoma tissues was done as follows (Nagato et al, 2005). Formalin-fixed, paraffin-embedded specimens obtained from pretreatment biopsy samples were cut in 4 μm tissue sections on glass slides. The sections were deparaffinized in xylene and ethanol and then placed in 10 mmol/l citric acid buffer, pH 6·0. Antigen retrieval was carried out by microwave irradiation for 7 min at 750 W. The sections were then incubated with 3% hydrogen peroxide for 30 min. After washing with PBS three times for 3 min, prevention of nonspecific staining was by Protein Block Serum-Free (Dako) for 60 min and the sections were incubated overnight at 4°C with 1:20 mouse anti-human CD27 Ligand/TNFSF7 monoclonal antibody (R&D Systems). After washing with PBS, the sections were incubated for 30 min at room temperature with En-Vision + peroxidase-labelled dextran polymer (Dako). Immunoreactive CD70 was visualized by immersing the slides in freshly prepared diaminobenzidine tetrahydrochloride substrate solution (Dako) for 10 min. After washing out the substrate solution, microwave irradiation and prevention of nonspecific staining was carried out as described above. The sections were incubated overnight at 4°C with 1:50 (paraffin sections) mouse anti-human CD56 monoclonal antibody (Novocastra, Newcastle, UK), washed in Tris-buffered saline/0·1% (TBS) three times for 3 min, and then incubated at room temperature in EnVision/AP alkaline phosphatase-labelled dextran polymer for 30 min. Slides were washed again with TBS. Immunoreactive CD56 was visualized by immersing the slides in freshly prepared Fast Red substrate solution (Dako) for 10 min. Finally, the sections were counterstained with Lillie-Mayer's haematoxylin and mounted on glass slides. Specimens that contained over 25% of CD56-positive lymphoma cells stained with anti-human CD27 Ligand/TNFSF7 monoclonal antibody, were considered positive (Law et al, 2006).

Statistical analysis

Two group comparisons were tested using nonparametric test procedures, such as Mann–Whitney U-test and Willcoxon signed rank test. Statistical tests were based on a level of significance < 0·05.

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

NNKTL cell lines express CD70

To investigate the genes expressed specifically in NNKTL cell lines, we initially used a cDNA microarray to compare the gene expression patterns among NNKTL cell lines (SNK6 and SNT8), non-NNKTL cells (NK92), and PBMC from healthy individuals. The analysis showed that CD70 mRNA was expressed at much higher levels in SNK6 and SNT8 than NK92 and PBMC (Fig 1A). Compared with PBMC, the expression was 6·43-fold higher in SNK6 and 2·12-fold higher in SNT8; compared with NK92, the expression was 8·71-fold higher in SNK6 and 2·87-fold higher in SNT8.

image

Figure 1. Expressions of CD70 (A, B, D), EBV-encoded small nuclear early region type-1 (EBER-1; B) and latent membrane protein-1 (LMP-1; C) of nasal NK/T cell lymphoma (NNKTL) cell lines. (A) cDNA array profiles of peripheral blood mononuclear cells (PBMC) from healthy volunteers and from SNK6, SNT8, and NK92 cells. CD70 mRNA was expressed at much higher levels in SNK6 and SNT8 than NK92 and PBMC. (B) RT-PCR profiles of CD70 and EBER-1 in PBMC and various cell lines. The expression of CD70 mRNA was detected in SNK6, SNT8, SNK1, KAI3 and Raji, but not in YT, NK92, Jurkat, MOLT4 or PEER. EBER-1 mRNA was detected in SNK6, SNT8, SNK1, KAI3, YT, NK92 and Raji, but not in Jurkat, MOLT4 or PEER. As an internal control, B2M cDNA was co-amplified in each sample. (C) Western blot profiles of LMP-1 in EBER-1-positive cell lines. LMP-1 expression was detected in SNK6, SNT8, SNK1, KAI3 and Raji. However, the expression was not detected in YT and NK92, even though they were positive for EBER-1. As an internal control, β-actin protein (ACTB) was co-loaded in each sample. (D) Histograms of CD70 expression in various cell lines. Surface expression of CD70 was detected on SNK6, SNT8, SNK1, KAI3 and Raji cells, but not expressed in YT, NK92, Jurkat, MOLT4 or PEER. Cells were stained with a phycoerythrin-conjugated mouse anti-human CD70 antibody (thick lines) and with isotype control antibody (filled histograms).

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To confirm that NNKTL cell lines (SNK6, SNT8 and SNK1) specifically express CD70 mRNA, various cell lines were analysed by RT-PCR. CD70 mRNA expression was detected in SNK6, SNT8, SNK1, KAI3 and Raji, but it was not detected in YT, NK92, Jurkat, MOLT4 or PEER (Fig 1B).

Next, flow cytometric analysis was performed to confirm whether NNKTL cell lines express CD70 protein (Fig 1D). The analysis revealed that CD70 was expressed on SNK6, SNT8, SNK1, KAI3 and Raji cells, but not expressed in YT or NK92.

NNKTL cell lines express EBER-1 and LMP-1

We investigated also EBV features such as expressions of EBER-1 and LMP-1 in various cell lines. EBER-1 mRNA was detected in SNK6, SNT8, SNK1, KAI3, YT, NK92 and Raji (Fig 1B). These results corresponded to previously reported data (Tsuge et al, 1999; Nagata et al, 2001; Matsuo & Drexler, 2003). NK92, which had initially been described as EBV-negative (Gong et al, 1994), was positive for EBER-1 as reported previously (Matsuo & Drexler, 2003).

We also investigated whether these cell lines express LMP-1 by Western blot analysis. LMP-1 expression was detected in EBER-1-positive cell lines, SNK6, SNT8, SNK1, KAI3 and Raji (Fig 1C). However, LMP-1 expression was not detected in YT or NK92, even though they were positive for EBER-1.

These results indicated that the NNKTL cell lines (SNK6, SNT8 and SNK1) specifically expressed CD70 and these cell lines also showed LMP-1 expression. Given that CD70 expression was also detected in KAI3 and Raji cells, which are LMP1-positive, but not expressed in YT and NK92 cells (EBV-positive but LMP-1-negative), it was suggested that CD70 expression was restricted by LMP-1 expression.

NNKTL cell lines do not express CD27

We next investigated whether CD27, which is the only receptor of CD70, is expressed in NNKTL cell lines by RT-PCR. CD27 mRNA expression was not detected in SNK6, SNT8, SNK1, as well as KAI3, YT and NK92, but it was detected in PBMC, Jurkat, MOLT4, PEER and Raji (Fig 2A). Flow cytometric analysis confirmed that CD27 was not expressed on SNK6, SNT8, SNK1, KAI3, YT and NK92, but it was detected in PBMC, Jurkat, MOLT4, PEER and Raji (Fig 2B).

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Figure 2. Expression of in various cell lines. (A) RT-PCR profile of CD27 in PBMC and various cell lines. The expression of CD27 mRNA was not detected in SNK6, SNT8, SNK1, as well as KAI3, YT and NK92, but it was detected in PBMC, Jurkat, MOLT4, PEER and Raji. As an internal control, B2M cDNA was co-amplified in each sample. (B) Histograms of CD27 expression in various cell lines. Surface CD27 was not expressed on SNK6, SNT8, SNK1, as well as KAI3, YT and NK92, but it was expressed in PBMC, Jurkat, MOLT4, PEER and Raji. Cells were stained with a FITC-conjugated mouse anti-human CD27 antibody (thick lines) and with isotype control antibody (filled histograms).

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CD70 acts for growth factor by binding to soluble CD27 in NNKTL cell lines

CD27 is cleaved from membrane-bound CD27, resulting in the formation of a soluble form of CD27. Recently, it is reported that soluble CD27 conjugates CD70 expressed on the cell surface and then induces IgG production from antigen-primed B-cells (Dang et al, 2011), suggesting that CD70 operates as a receptor to induce a signal transduction pathway in these cells, as previously shown in some B-cell lines (Lens et al, 1999).

In the present study, in order to investigate whether CD70 acts as a growth factor by binding to soluble CD27 in NNKTL cell lines, we performed MTS assays on NNKTL cell lines under culture conditions with exogenous soluble CD27. As shown in Fig 3A, exogenous soluble CD27 enhanced cell growth in a dose-dependent manner in NNKTL cell lines (SNK6, SNK1) and Raji cells. The exogenous soluble CD27-dependent cell growth showed a statistically significant level at a dose of 1 μg/ml of soluble CD27 (P < 0·05). However, exogenous soluble CD27 did not affect cell growth of the CD70-negative cell line NK92.

image

Figure 3. Effects of exogenous soluble CD27 (A) and anti-CD27 neutralizing antibody (B) on the cell growth in nasal NK/T cell lymphoma (NNKTL) cell lines SNK6 and SNK1. Cell growth was measured by cell proliferation assay with MTS solution and the results were expressed as the percentage of untreated controls. All assessments were carried out in duplicate in at least five independent experiments, and results represent the means ± standard deviation. (A) Cell growth under culture conditions with exogenous soluble CD27. Exogenous soluble CD27 enhanced cell growth in a dose-dependent manner in SNK6, SNK1 and Raji cells. The soluble CD27-dependent cell growth was statistically significant at a dose of 1 μg/ml of soluble CD27 (P < 0·05; black bar). However, exogenous soluble CD27 did not affect cell growth of the CD70-negative cell line NK92. Concentration of the exogenous soluble CD27 was 0 μg/ml (white bar; untreated control), 0·01 μg/ml (light grey bar), 0·1 μg/ml (grey bar), 0·5 μg/ml (dark grey bar), and 1·0 μg/ml (black bar). (B) Cell growth under culture conditions with exogenous soluble CD27 (0·5 μg/ml) and anti-CD27 neutralizing antibody. The exogenous soluble CD27-dependent cell growth (black bar) was inhibited by anti-CD27 neutralizing antibody in a dose-dependent manner and it was completely inhibited by 10 μg/ml of the antibody (P < 0·05; light grey bar). However, exogenous soluble CD27 as well as anti-CD27 neutralizing antibodies did not affect cell growth of the CD70-negative cell line NK92. Concentrations of soluble CD27 and the anti-CD27 neutralizing antibody was 0 μg/ml (white bar; untreated control), 0·1 μg/ml (black bar), 1·0 μg/ml (dark grey bar), and 10 μg/ml (light grey bar).

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We next examined the effect of anti-CD27 neutralizing antibody on cell growth under culture conditions with exogenous soluble CD27. As shown in Fig 3B, 0·5 μg/ml of exogenous soluble CD27 induced cell growth of NNKTL cell lines (SNK6, SNK1), but the soluble CD27-dependent cell growth was inhibited by anti-CD27 neutralizing antibodies in a dose-dependent manner and it was completely inhibited by the concentration of 10 μg/ml of the antibody. However, exogenous soluble CD27 as well as anti-CD27 neutralizing antibodies did not affect cell growth of the CD70-negative cell line NK92.

CD70 is expressed in lymphoma tissues and soluble CD27 is detected in sera from patients with NNKTL

Next, we performed double immunohistological staining for expressions of CD70 and CD56 on the biopsy samples from 21 patients with NNKTL, to confirm whether CD70 is expressed on the lymphoma cells in the patients' tissues. CD70 expression of CD56-positive lymphoma cells was detected in the tissues from 7 (33%) of 21 patients. Representative immunohistological feature is shown in Fig 4A: A certain number of atypical large lymphoma cells co-expressed with CD70 and CD56 (arrow) are seen together with some small sized CD56-positive lymphoma cells without CD70 expression (white arrow head) and CD70-positive small lymphocytes (black arrow head) in the lymphoma tissue.

image

Figure 4. Representative immunohistological feature of CD70 expression in the lymphoma tissue (A) and soluble CD27 levels in sera from patients with nasal NK/T cell lymphoma (NNKTL) (B). (A) A certain number of atypical large lymphoma cells co-expressed with CD70 and CD56 (arrow) are seen together with some small sized CD56-positive lymphoma cells without CD70 expression (white arrow head) and CD70-positive small lymphocytes (black arrow head) in the lymphoma tissue. CD70 was stained brown and CD56 was stained red. Original magnification = ×400. (B) The serum level of soluble CD27 in NNKTL patients (n = 8) was significantly higher that the level in healthy individuals (n = 7) (median: 25–75 percentile = 3930: 3352–4894 U/ml vs. 2966: 1768–3351 U/ml; P = 0·018).

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We also performed ELISA for measurement of soluble CD27 in sera from eight patients with NNKTL patients and seven healthy individuals, to confirm whether soluble CD27 was present in patients' sera. As shown in Fig 4B, the serum level of soluble CD27 in NNKTL patients was significantly higher than that of healthy individuals (median: 25–75 percentile = 3930: 3352–4894 U/ml vs. 2966: 1768–3351 U/ml; P = 0·018).

Anti-CD70 antibody directs complement-mediated lysis of NNKTL cells

Finally, we performed complement-dependent cytotoxicity assay, to determine whether anti-CD70 antibodies induce killing of CD70-positive NNKTL cells in the presence or absence of complement. As shown in Fig 5, either anti-CD70 antibody alone did not significantly affect cell growth of NNKTL cells (SNK6, SNK1, SNT8) as well as Raji cells. However, in the presence of active (but not inactive) rabbit complement, the anti-CD70 antibody significantly inhibited cell growth of these cells (P < 0·05). To confirm that the effects target for CD70 expressed on the cells, CD70-negative NK-cell lymphoma cells (NK92) were subjected to complement-dependent cytotoxicity assay. Administration of anti-CD70 antibody did not inhibit cell growth of NK92 even in the presence of active complement. These results indicate that the anti-CD70 antibody can mediate effective complement-dependent killing of NNKTL cells and the effects target for CD70 expressed on the cells.

image

Figure 5. Effects of anti-CD70 antibody (CD70Ab) in the presence of active complement on the cell growth in NK/T cell lymphoma (NNKTL) cell lines SNK6, SNK1 and SNT8. Cells were cultured alone (white bar; untreated control), with mouse anti-human CD70 antibody (10 μg/ml; light grey bar), with anti-CD70 antibody at the presence of inactive complement (5% rabbit serum after heating an aliquot to 55°C for 30 min; dark grey bar), or with anti-CD70 antibody at the presence of active complement (5% rabbit serum; black bar). After 3 h, cell growth was measured by cell proliferation assay with MTS solution. The results were expressed as the percentage of untreated controls. All assessments were carried out in duplicate in at six independent experiments, and results represent the means ± SD. In the presence of active complement, the anti-CD70 antibody significantly inhibited cell growth of SNK6, SNK1, SNT8 and Raji cells (P < 0·05; black bar). However, anti-CD70 antibody alone (light grey bar) and the antibody with inactive complement (dark grey bar) did not significantly affect cell growth of these cells. Administration of anti-CD70 antibody did not inhibit cell growth of NK92 even in the presence of active complement.

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Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In the present study, analysis of gene expression using cDNA array revealed that CD70 mRNA is expressed at a much higher level in NNKTL cells (SNK6 and SNT8) than in PBMC of healthy volunteers and NK92 non-NNKTL cells (Fig 1). RT-PCR analysis confirmed that CD70 mRNA was expressed in SNK6, SNT8, KAI3 and Raji cells, but not in YT and NK92, Jurkat, MOLT4 and PEER cells (Fig 2). It is notable, in EBV-positive cell lines, that CD70 expression was detected in KAI3 and Raji cells, which were also LMP-1-positive, however, the expression never detected in YT and NK92 cells, which were LMP-1-negative. In the literature, Burkitt lymphoma lines containing the most stringent form of EBV latency (type I), in which EBV-determined nuclear antigen-1 (EBNA-1) is the only viral protein produced, do not express CD70 (Israel et al, 2001). However, Burkitt lymphoma lines with type III latency, EBV-immortalized lymphoblastoid cell lines, nasopharyngeal carcinomas and Hodgkin lymphoma and chronic active EBV infection-associated T cell lymphoma, all of which are LMP-1-positive, were reported to express CD70 (Herbst et al, 1996; Israel et al, 2001; Shaffer et al, 2011a). In vitro study previously showed that CD70, which was absent from the parental line, was expressed in virtually all LMP-1-transfected epitherial cells (Niedobitek et al, 1992). It is suggested, on the basis of the data, that LMP-1 may induce CD70 expression in EBV-positive malignancies.

Aside from EBV-positive malignancies, CD70 expression has been reported on 50% of B-cell chronic lymphocytic leukaemia cases, 33% of follicular lymphomas and 71% of diffuse large cell lymphomas (Davi et al, 1998; Lens et al, 1999) and some cases of T cell lymphoma (Zambello et al, 2000). However, the functional role of CD70 in malignancy is not fully understood. Several reports described its role in tumour immunity, but they were not uniform. It was reported that deliberate expression of CD70 on tumour cells stimulated both NK and T cell immunity in lymphoma and glioma models (Kelly et al, 2002). On the contrary, CD70 expression was reported to protect the tumour from lysis by CD27-expressing cytotoxic T cells in some glioblastomas (Wischhusen et al, 2002). On the other hand, Lens et al (1999) demonstrated that some B-cell leukaemia cell lines, which expressed CD70 as well as CD27, could proliferate vigorously in response to anti-CD70 monoclonal antibody due to an agonistic signal delivered via CD70, suggesting that CD70 can operate as receptor inducing a signal transduction pathway, thus contributing to the progression of these B-cell malignancies.

The present study clearly showed that NNKTL cell lines (SNK6, SNK1 and SNT8) that were positive for CD70, but negative for CD27 (Figs 1 and 2), could proliferate vigorously in response to exogenous soluble CD27 dose-dependently, but CD70-negative NK cell lines could not (Fig 3A). We further showed that the exogenous soluble CD27-dependent cell growth of NNKTL cell lines was inhibited completely by administration with anti-CD27 neutralizing antibody (Fig 3B). These findings suggest that CD70 acts as a functional receptor that binds to soluble CD27, inducing cell growth. In NNKTL patients, we showed that CD70 was expressed on CD56-positive lymphoma cells in NNKTL tissues (Fig 4A). We further found that soluble CD27 was present in sera at significantly higher level as compared to healthy individuals (Fig 4B). The findings observed in patients' samples support that soluble CD27 also acts as a growth factor for NNKTL cells in vivo.

Elevated serum levels of soluble CD27 have been reported in diseases characterized by abnormalities in B-cell differentiation and activation including autoimmune diseases (Font et al, 1996; Bohnhorst et al, 2002) and B-cell malignancies (van Oers et al, 1993). However, the biological role of soluble CD27 has not been extensively studied. It was initially thought that soluble CD27 would compete with the membrane-bound CD27 receptor for binding to CD70, thus blocking the CD27–CD70 pathway (Hintzen et al, 1991). Such reaction thereby results in creating a feedback mechanism that would hamper immune responses. On the contrary, soluble CD27 has been recently demonstrated as an enhancer of immune responses; soluble CD27 induced IgG production from antigen-primed B-cells (Dang et al, 2011) as well as expression of CD40LG and APRIL for B-cell activation (Ho et al, 2008). This is the first report to show that soluble CD27 binding to CD70 has a biological role for cell growth of NNKTL cells.

Although this study detected significantly higher levels of soluble CD27 in sera of NNKTL patients, we cannot explain completely how soluble CD27 increased in sera of NNKTL patients. Soluble CD27 is cleaved from the extracellular portion of the surface CD27 receptor presenting in a wide variety of immune cells, such as most T cells (de Jong et al, 1991), memory B-cells (Klein et al, 1998) and NK-cells (Sugita et al, 1992). The microenvironmental feature of NNKTL is characterized as polymorphous lymphoreticular infiltrates containing many types of immune cells, such as granulocytes, monocytes, macrophages, T and B-cells and plasma cells (Harabuchi et al, 1996; Harris et al, 2000). Such infiltrating immune cells are caused by the chemotactic effects of chemokines, including IL8, Mig and IP10, secreted by NNKTL cells (Ohshima et al, 2004; Moriai et al, 2009) and are likely to release soluble CD27. Recently, matrix metalloproteases (MMPs) were reported to induce cleavage of soluble CD27 (Kato et al, 2007). Interestingly, we recently found, on the cDNA array and RT-PCR analyses, that NNKTL (SNK6) cells produce high levels of MMPs (data not shown). In addition, some cytokines, such as IL2, IL9, IL10 and IFNγ, which are secreted by NNKTL cells (Nagato et al, 2005; Takahara et al, 2006), may possibly induce CD27 cleavage. In the NNKTL tissue microenvironment, a positive feedback loop of interaction between lymphoma cells and infiltrating immune cells may contribute to lymphoma progression, i.e. NNKTL cells produce a wide variety of cytokines, chemokines and MMPs, which induce chemotactic reaction as well as soluble CD27 release of such immune cells, resulting in proliferation of lymphoma cells by binding of soluble CD27–CD70 on the NNKTL cells. We have already shown that microenvironmental monocytes, attracted by IP10, which is secreted by NNKTL cells (Moriai et al, 2009), enhance proliferation of NNKTL cells by cell contact-dependent interaction through membrane-bound IL15 (Ishii et al, 2012).

Recently other roles of the CD27–CD70 pathway on tumour progression have been reported. Claus et al (2012) demonstrated that CD70 expression in tumours is a negative prognostic factor and correlates with increased regulatory T cell accumulation, which promotes tumour progression by CD70 on the tumour cell, stimulating CD27 on the regulatory T cells. Schurch et al (2012) showed that stimulation of the TNF family receptor, CD27, on leukaemic stem-like cells of chronic myeloid leukaemia, where CD27 is a receptor for the CD70 ligand, is involved in leukaemic stem-like cell proliferation. Further studies that knockdown CD70 in NNKTL cells will be needed to define the mechanism via which CD70 engagement on NNTKL cells by soluble CD27 may stimulate cell proliferation.

The restricted expression pattern of CD70 in normal tissues and its widespread expression in various malignancies makes it an attractive target for cytotoxic T cell therapy as well as antibody-based therapy. Recently, Shaffer et al (2011b) demonstrated that CD70-specific T cells killed CD70-positive lymphoma cell lines by IFNγ and IL2 secretion and that adoptively transferred CD70-specific T cells induced sustained regression of established murine xenografts, suggesting that CD70-specific T cells may be a promising immunotherapeutic approach for CD70-positive malignancies. In the present study, we clearly showed in vitro that the anti-CD70 antibody can mediate effective complement-dependent killing of NNKTL cells and the effects target for CD70 expressed on the cells (Fig 5). Israel et al (2005) also showed that an anti-CD70 antibody mediated complement-dependent killing of CD70-positive Burkitt lymphoma cells in vitro. They further showed in severe combined immunodeficiency (SCID) mice that anti-CD70 antibody strikingly inhibited the growth of CD70-positive Burkitt lymphoma cells (Israel et al, 2005). McEarchern et al (2007) showed that the administration of an engineered anti-CD70 monoclonal antibody significantly prolonged the survival of SCID mice bearing CD70-disseminated human non-Hodgkin lymphoma xenografts. Although further experiences in animal models will be needed before clinical use, it is suggested, on the basis of our in vitro data together with these previous results in SCID mice, that the administration with anti-CD70 antibodies may be useful for the treatment of NNKTL patients.

In conclusion, this study clearly showed that NNKTL cells expressed CD70 possibly due to the ability of LMP-1, but did not express its only receptor, CD27. We further showed that CD70 played a role for cell growth by binding to soluble CD27. In NNKTL patients, we confirmed that the lymphoma cells expressed CD70 in the tissues and that soluble CD27 was present at higher levels in sera. Finally, we showed in vitro that the anti-CD70 antibody could mediate effective complement-dependent killing of NNKTL cells and the effects target for CD70 expressed on the cells. We conclude that NNKTL expresses CD70, which plays a role in lymphoma proliferation by binding to soluble CD27, and that immunotherapy using anti-CD70 antibody may be a potential candidate for treatment for NNKTL

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Dr. Shimizu N (Tokyo medical and dental university) and Prof. Klein E (Karolinska Institutet) for generously providing cell lines. Ministry of Education, Science, Sports, and Culture of Japan grant in-aid 19791180 (M. Takahara) and 20390438 (Y. Harabuchi).

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  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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