We performed an overlapping PCR using the p2167 plasmid (kindly provided by W. Hammerschidt, Munich, Germany), in order to create the 30-bp and 69-bp deletions in the BNLF1 gene coding for LMP1 [41
]. The p2167 plasmid contains 9.5 kb of the EBV genome, as described [42
]. Two DNA fragments surrounding the deletions (respectively in position 1190-1229 and 1161-1229) were amplified by PCR with a high fidelity polymerase (Pfu, Promega, Fitschburg, WI, USA). The following primers were used,
First, two PCRs were performed with 50 ng of the p2167 plasmid in a total volume of 50 μl, with primers P1 + P2a and P3a + P4 for the 30-bp deletion or P1 + P2b and P3b + P4 for the 69-bp deletion. After 5 min of denaturation at 95°C, 10 cycles of 30 sec at 95°C, 30 sec at 55°C and 2 min at 72°C were carried out, followed by an elongation step for 5 min at 72°C. PCR products were purified by QIAEX purification kit (Qiagen, Hilden, Germany), quantified by the nanodrop system (Labtech, Uckfield, United-Kingdom) and equimolarly mixed to be submitted to a second PCR. After 10 cycles with the same program but without primers, allowing the first two PCR products to hybridize, external primers P1 and P4 were added for another 20 cycles. The final 1.6 kb PCR product containing the 30-bp or 69-bp deletion was reintroduced into the original p2167 plasmid in the NheI and Bst1107I restriction sites and subcloned into the Sfi-I sites of the pRT-LMP1 vector (kindly provided by J. Feuillard, Limoges, France) with the In-Fusion HD cloning kit (Clontech, Mountain View, CA, USA). This vector includes the EBNA1 and hygromycin B resistance genes, that favour maintenance of the vector as an episome in the cells, and allow selection of transfected cells respectively. A bidirectional tetracyclin-inducible promoter drives the expression of LMP1 and NGFRt . The pcDNA-WT-LMP1 plasmid is a kind gift from F. Grässer (Hambourg, Germany).
Cell culture, transfection and induction
293-HEK cells (human embryonic kidney cell line) were cultured in DMEM supplemented with heat inactivated fetal bovine serum 10% and antibiotics (100 units/ml penicillin/streptomycin and 1 μg/ml ofloxacin). KMH2 cells (EBV-negative Hodgkin Lymphoma-derived cell line obtained from DSMZ, Braunschweig, Germany) were cultured in RPMI supplemented with 10% heat inactivated fetal bovine serum and antibiotics (100 units/ml penicillin/streptomycin, 1 μg/ml ofloxacin), at 37°C in a 5% CO2 atmosphere. For transient transfection of pcDNA-WT-LMP1 in 293-HEK cells, 5.105 cells were plated in a 35-mm petri dish and transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in the ratio of 10 μl transfection reagent for 4 μg of DNA, following the manufacturer’s instructions. Establishment of KMH2 cell lines stably transfected with the three pRT-LMP1 plasmids (pRT-WT-LMP1, pRT-del30-LMP1 and pRT-del69-LMP1) was performed with the Amaxa electroporation system (Amaxa, Cologne, Germany), according to the manufacturer’s instructions. Five millions KMH2 cells were electroporated with 5 μg of DNA, using the Nucleofactor kit-T (Amaxa) and the T-001 program. Cells were then resuspended in 5 ml of fresh RPMI with 20% FBS, supplemented with 1X non-essential amino-acids, 10 mM Hepes and 1 mM sodium pyruvate. Hygromycin was introduced in the medium 48 h after the transfection at 200 μg/ml. After three weeks of selection, LMP1 expression was assessed by flow cytometry after inducing the cells with 1 μg/ml doxycyclin (Clontech) for 24 h, as described elsewhere .
Five millions KMH2-pRT-LMP1 cells were grown in presence or not of doxycyclin for 24 h. Cells were lysed in a buffer composed of 50 mM Tris pH 6.8, 2% SDS, 10% glycerol and protease inhibitors. Lysates were sonicated and total proteins were quantified with BCA kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Fifty micrograms of total proteins were separated on a 9% SDS-PAGE gel, transferred on a PVDF membrane and revealed by western-blot analysis with an anti-LMP1 antibody (monoclonal CS.1-4, Dako, Glostrup, Denmark, 1:100) and a rabbit polyclonal anti-actin (Sigma, St Louis, MO, USA; 1:400). Secondary HRP-conjugated anti-mouse (1:20000), anti-rabbit antibodies (1:10000) were purchased from Invitrogen). The signal was developed by Supersignal West Pico Chemiluminescent Substrate (Pierce). The intensity of protein bands was quantified with Adobe Photophop CS3 software.
RNA extraction and RT-PCR
Total RNA was extracted from 5.106 KMH2-pRT-LMP1 cells with TRI-Reagent (Euromedex, Souffelweyersheim, France), quantified with a nanodrop ND1000 instrument system (Labtech) and treated with Turbo-DNase (Ambion, Austin, TX, USA). cDNA was synthesized from 1 μg total RNA with random hexamers, using the superscript III (Invitrogen) in a total volume of 20 μl. PCRs were then performed on 1 μl of cDNA, with 0.25U of the Phusion polymerase (Thermo-Scientific, Waltham, MA, USA) in the provided buffer, in presence of 200 μM dNTP and 200 μM of specific primers. Primer sequences are: LMP1-forward, 5′-CCTCATAGCCCTAGCGACTC-3′ ; LMP1-reverse, 5′-GTCGTCATCATCTCCACCGG-3′ ; EBNA1-forward, 5′-CCGCAGATGACCCAGGAGAA-3′ ; EBNA1-reverse, 5′-TGGAAACCAGGGAGGCAAAT-3′ ; β-actin-forward, 5′-CGTGATGGTGGGCATGGG-3′ β-actin-reverse, 5′-CTGGGTCATCTTCTCGCG-3′. After a denaturation step of 3 min at 94°C, the following cycle was applied to the samples : 30 sec at 94°C, 30 sec at 60°C (for EBNA1, TH was 56°C), 30 sec at 72°C, repeated 26 times for the actin amplification and 30 times for LMP1 and EBNA1 amplifications. A final elongation step was performed for 5 min at 72°C. PCR products were migrated on a 2% agarose gel in 1X TAE buffer and visualized with ethidium bromide staining (Invitrogen).
LMP1 staining and intracellular cytokine detection
KMH2 cells were either treated by doxycyclin (1 μg/ml) to induce LMP1 expression or stimulated with 50 ng/ml PMA (Sigma) and 1 μg/ml ionomycin (Sigma) to be used as positive control for cytokine expression during 24 h. Throughout the last 4 h of doxycyclin or PMA-ionomycin induction, cells were treated with 10 μg/ml brefeldin A (BioLegend, San Diego, CA, USA) and 2 μM monensin (Sigma) to block secretion of cytokines. Cells were collected and fixed with fixation buffer (BioLegend) for 20 min at room temperature. Cells were then permeabilized by two centrifugations (for 8 min at 350 g) with permeabilization wash buffer (BioLegend) allowing sequential double immunolabeling of LMP1 and cytokines of interest. LMP1 staining was performed primarily as follows: cells were saturated for 30 min in 100 μl RPMI containing 10% human plasma (kindly provided by EFS Rhône-Alpes, Grenoble, France) and 0.1% triton X-100, incubated for 30 min with the anti-LMP1 antibody (1:100) and washed twice with 5 ml of RPMI. Cells were then incubated for 30 min with an Alexa A488 or A594-conjugated anti-mouse antibody (Invitrogen, 1:2500) and washed twice with 5 ml of RPMI. An anti-mouse secondary antibody free Fab sites was finally satured with a mouse hybridoma supernatant (clone D3210, kindly provided by E. Drouet, UVHCI, Grenoble, France). In a second step, intracellular cytokine staining was carried out by resuspending cells in 100 μl of permeabilization wash buffer supplemented with the anti-cytokine antibody and incubated for 20 min at room temperature, protected from light. Cells were then washed twice with 2 ml of permeabilization wash buffer and finally resuspended in cell staining buffer (BioLegend) for flow cytometry analysis on a MACSQuant VYB (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany). Anti-cytokine antibodies used were APC-conjugated anti-human TNF-α (clone Mab11) or its mouse isotype control (clone MOPC21), PE-conjugated anti-human IL-6 (clone MQ2-13A5), anti-human IL-8 (E8N1), anti-human TNF-β (clone 359-81-11), anti-human TGF-β (TW4-2 F8), anti-human IL-1α (364-3B3-14), rat isotype control (clone RTK2071) and mouse isotype control (clone MOPC21) from BioLegend, PE-conjugated anti-human IFN-γ (clone 4S.B3), anti-human IL-9 (MH9A3) and anti-human RANTES/CCL5 (clone 2D5) from BD Pharmingen (San Diego, CA, USA), or FITC-conjugated anti-human IL1-RA (CRM17) and its mouse isotype control (P126.96.36.199.1) from eBioscience (San Diego, CA, USA).
Cell cycle and proliferation
In order to determine the percentage of cells in the different phases of cell cycle, we used the EdU-click 555 (Jena Bioscience, Jena, Germany) combined with LMP1 and DAPI staining. EdU is a nucleoside analogue to thymidine incorporated into DNA, it reveals the proliferating cells in S phase. DAPI measures the total quantity of DNA in the cells and the LMP1 labelling enables to select only the LMP1-expressing cells for our study. Cells were induced with doxycyclin for 24 h and, during the last 150 min, 10 μM EdU was added in the culture medium. Cells were, then, fixed with fixation buffer and permeabilized with permeabilization wash buffer (BioLegend). For EdU revelation, cells were resuspended in the cocktail-click containing TAMRA-azide, incubated for 30 min at room temperature protected from light and washed twice with 6 ml PBS. LMP1 staining was performed as described previously for intracellular cytokine detection and cells were finally incubated in PBS with 1 g/L glucose, DAPI 1:1000 and 100 μg/ml RNAseA until flow cytometry analysis.
Means, standard deviations and p-values were calculated with the GraphPad InStat3 software (San Diego, CA, USA). All bar graphs represent means as per at least three independent experiments for cytokine detection and at least five independent experiments for cell cycle studies. Error bars represent standard deviations. To assess statistical differences, means were compared through a one-way ANOVA test followed by a Tukey post-test for multiple comparison significance test between the analyzed groups. When specified, for single comparison of a population with the control cell group, the ANOVA test was followed by a Dunnett post-test. * stands for p < 0.05, ** for p < 0.01 and *** for p < 0.001.